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WO2005120511A1 - Methods for treating mast cell disorders - Google Patents

Methods for treating mast cell disorders

Info

Publication number
WO2005120511A1
WO2005120511A1 PCT/US2005/019558 US2005019558W WO2005120511A1 WO 2005120511 A1 WO2005120511 A1 WO 2005120511A1 US 2005019558 W US2005019558 W US 2005019558W WO 2005120511 A1 WO2005120511 A1 WO 2005120511A1
Authority
WO
WIPO (PCT)
Prior art keywords
quinazolin
methyl
purin
ylamino
phenyl
Prior art date
Application number
PCT/US2005/019558
Other languages
French (fr)
Inventor
Joel S. Hayflick
Noah Pefaur
Kamal D. Puri
William Tino
Toshi Kawakami
Yuko Kawakami
Original Assignee
Icos Corporation
La Jolla Institute For Allergy And Immunology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Icos Corporation, La Jolla Institute For Allergy And Immunology filed Critical Icos Corporation
Priority to EP05756523A priority Critical patent/EP1750715A1/en
Priority to CA002569406A priority patent/CA2569406A1/en
Priority to JP2007515614A priority patent/JP2008501707A/en
Publication of WO2005120511A1 publication Critical patent/WO2005120511A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to methods and compounds for modulating mast cell activity by inhibiting PI3K ⁇ .
  • mast cell activity includes, but is not limited to, mast cell degranulation, mast cell migration, mast cell proliferation, and the expression and secretion of cytokines, chemokines, and growth factors by mast cells.
  • the methods and compounds of the invention may be used to treat or prevent conditions associated with such mast cell activity that is undesirable.
  • Phosphoinositide 3-kinase is a signaling enzyme that plays key roles in many cellular activities, including cellular growth, remodeling, and apoptosis [Wymann and Pirola, Biochem Biophys Acta. 1998;1436:127-150; Anderson et al, J Biol Chem. 1999;274:9907-9910; Rameh et al, JBiol Chem. 1999;274:8347-8350; Cantrell, JCell Sci. 2001;114:1439-1445; Coelho and Leevers, J Cell Sci. 2000;113:2927-2934; Vanhaesebroeck et al, Ann. RevBiochem.
  • PI3K also plays roles in many other cellular processes, such as malignant transformation, growth factor signaling, inflammation, and immunity. See Rameh et al, J. Biol Chem, 274:8347-8350 (1999) for a review. Such diverse activities may be attributed at least in part to PDK's lipid and protein kinase activity.
  • Class I PI3-kinases are the most extensively investigated class and contain two subunits, one of which plays primarily a regulatory/adaptor role (p85 ⁇ , ⁇ , p55 ⁇ or plOl isoform) and the other that maintains the catalytic role of the enzyme (pi 10a, ⁇ , ⁇ , or ⁇ isoform) [Wymann and Pirola, supra; Anderson et al, supra; Rameh et al, supra; Cantrell, supra; Coelho and Leevers, supra; Vanhaesebroeck et al, supra.] Identification of the pi lO ⁇ isoform of PI3-kinase is described in Chantry et al [JBiol Chem, 272:19236-41 (1997)].
  • PI3Ks achieve intracellular signaling at least in part by catalyzing the addition of a phosphate group to the inositol ring of phosphoinositides [Wymann, et al, supra].
  • One target of these phosphorylated products is the serine/threonine protein kinase B (P B or Akt).
  • Akt subsequently phosphorylates several downstream targets, including the Bcl-2 family member Bad and caspase-9, thereby inhibiting their pro-apoptotic functions [Datta et a , Cell 91:231-41 (1997); Cardone et /.,
  • Akt has also been shown to phosphorylate the forkhead transcription factor FKHR [Tang et al, J Biol. Chem., 274:16741-6 (1999)].
  • FKHR forkhead transcription factor
  • many other members of the apoptotic machinery as well as transcription factors contain the Akt consensus phosphorylation site [Datta et al, supra], Phosphorylation of Akt has been widely used as an indirect measure of
  • PI3 -kinase activity in multiple cell types, including endothelial cells, [Shiojima, et al. , Circ. Res., 90: 1243-1250 (2002); Kandel et al. , Exp. Cell Res., 253:210-229 (1999); Cantley et al, Science 296:1655-1657 (2002)].
  • PI3K activity is required for growth factor mediated survival of various cell types [Fantl et al, Ann. Rev. Biochem. 62:453-81 (1993); Datta et al, Genes & Dev. 13:2905-27 (1999)].
  • PI3K nonselective phosphoinositide 3-kinase
  • LY294002 and wortmannin have been shown to enhance destruction of tumor vasculature in irradiated endothelial cells [Edwards et al, Cancer Res. 62:4671-77 (2002)] and partially inhibit mast cell degranulation [Tkaczyk et al, JBiol Chem. 278:48474-84 (2003)].
  • LY294002 and wortmannin do not distinguish among the four members of class I PI3Ks, however.
  • the IC 50 values of wortmannin against each of the various class I PI3Ks are in the range of 1-10 nM.
  • the IC 50 values for LY294002 against each of these PI3Ks is about 1 ⁇ M [Fruman, et al,
  • mast cells play diverse and significant roles. For example, mast cells are involved in mediatmg first line immune responses of the innate immune system seen in response to allergens or parasitic or bacterial infections.
  • Mast cells also contribute to activation and recruitment of other inflammatory cells, such as neutrophils and T cells, to bring about second line immune responses required for an adaptive immune response.
  • CD34 + mast cells circulate in the blood as committed precursor cells and fully mature in specific tissue sites.
  • Mast cell development and maturation requires mast-cell growth factor, also known as stem cell factor (SCF), steel, or KIT ligand [Gurish et al., J. Exp. Med. 194:F1-F5 (2001)].
  • SCF stem cell factor
  • KIT ligand KIT ligand
  • the interaction of KIT receptor with its ligand drives mast cell proliferation and differentiation (Feger et al, Int. Arch. Allergy Immunol. 127:110-14 (2002)].
  • Mast cells are activated through crosslinking of the high affinity Fc ⁇ RI
  • IgE receptor on the cell surface by antigen-bound IgE and to a lesser extent through crosslinking of the Fc ⁇ RI receptor by IgG [Tkaczyk et al., Lit Arch Allergy Immunol. 133:305-15 (2004)].
  • Activation through Fc ⁇ RI is typically seen in acute allergic reactions and other types of hypersensitivity reactions, leading to the stimulation of additional immune cells and a full blown immune response.
  • Mast cells contain metachromatic granules which store a variety of inflammatory mediators that are released upon mast cell activation.
  • mediators include: histamine and serotonin; prostaglandin D2; proteolytic enzymes, such as tryptase that can destroy tissue or cleave complement components or coagulation components; heparin or chondroitin sulfate, which are anticoagulants; chemotactic factors, such as eosinophil chemotactic factor of anaphylaxis (an important regulator of eosinophil function) and neutrophil chemotactic factor.
  • these mediators are released into the cellular environment causing acute and immediate immune responses such as vascular permeability and recruitment of lymphocytes.
  • mast cell activity is necessary and desirable in healthy individuals. Unwanted mast cell activity, or excessive proliferation of otherwise normal or abnormal mast cells, may be a component of a wide variety of disease states and/or their symptoms, however.
  • mast cell activity and/or proliferation it is often desirable, from a therapeutic or preventative standpoint, to reduce or eliminate mast cell activity and/or proliferation.
  • numerous immune mediated diseases involve the release by mast cells of cytokines, chemokines and other factors. Cytokines, chemokines, and other factors recruit additional immune cells such as lymphocytes, including neutrophils and T cells, to sites of inflammation. This may lead to numerous immune mediated diseases.
  • mast cell activity such as degranulation and tryptase protein have recently been localized to cerebrospinal fluid of patients with multiple sclerosis, an autoimmune disease typically thought to be mediated by T cell activity [Rozniecki et al., Ann Neurol 37:63-66 (1995)].
  • mast cell deficient mice (W/W v ) induced to develop an experimental model of multiple sclerosis demonstrate delayed development of MS-like symptoms [Secor et al., J. Exp. Med. 191:813-22 (2000)].
  • Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation of the joints and the presence of inflammatory cells in the synovial fluid of the joints, leading to a painful and debilitating disease.
  • Mice lacking mast cells show resistance to induction of arthritis-like symptoms after infusion of antibodies to a cytoplasmic enzyme [Lee et al., Science 297:1689-1693 (2002)].
  • mast cells accumulate in the extremities of mice affected by collagen-induced arthritis and degranulate during the disease [Woolley et al., Arthritis Res. 2:65-74 (2000)], indicating that mast cells may play a role in mediatmg inflammation and recruiting additional cells to the joints of patients suffering from rheumatoid arthritis.
  • a significant population of mast cells resides in the skin. Bullous pemphigoid, an autoimmune disease of the skin exhibiting autoantibodies to cell junction proteins has also been shown to depend on mast cell activation. [Chen et al., JClin Invest. 108:1151-58 (2001)].
  • W/W v mice deficient in mast cells did not develop bullous pemphigoid, although autoantibodies and complement proteins were present in the skin similar to normal mice, W/W v mice lacked neutrophil recruitment to the skin. Also, evidence of mast cell mediators have been detected in patients with bullous pemphigoid [Wintroub et al., New Eng. J. Med. 298:417-21 (2001)].
  • Other immune disorders in which mast cells are thought to play a role include Sjogren's syndrome [Konttinen et al., Rheumatol Int. 19:141-7 (2000)], chronic urticaria [Napoli et al., Curr Allergy Asthma Rep.
  • mast cell disease encompasses a heterogeneous group of clinical disorders characterized by the proliferation and accumulation of mast cells in a variety of tissues, most often the skin, but also in the skeletal, hematopoietic, gastrointestinal, cardiopulmonary, and central nervous systems.
  • Mastocytosis is characterized by excess proliferation of mast cells, distributed in a predictable pattern throughout the skin (e.g., cutaneous mastocytosis and urticaria pigmentosa), bone marrow, gastrointestinal tract, lymph nodes, liver and spleen [Brockow et al, CWT. Opin. Allergy Clin. Immunol 1:449-54 (2001)].
  • Mastocytosis is classified as either familial or sporadic, the latter being further subdivided into either cutaneous or systemic.
  • Systemic mastocytosis is still further classified into indolent (chronic) mastocytosis and aggressive mastocytosis, as well as mast cell leukemia.
  • CM hematologic disorder
  • AHD hematologic disorder
  • Cutaneous mastocytosis (CM) demonstrates typical clinical and histological skin lesions and absence of definitive signs (criteria) of systemic involvement.
  • Most patients with CM are children and present with maculopapular cutaneous mastocytosis (for example, urticaria pigmentosa, UP).
  • Other less frequent forms of CM are diffuse cutaneous mastocytosis (DCM) and mastocytoma of skin.
  • SM Systemic mastocytosis
  • MC pathologic mast cells
  • Mast cell leukemia is a 'high-grade' leukemic disease defined by increased numbers of MC in bone marrow smears (greater than or equal to 20%) and peripheral blood, absence of skin lesions, multiorgan failure, and a short survival. In typical cases, circulating MC amount to greater than or equal to 10% of leukocytes (classical form of MCL).
  • Mast cell sarcoma is a unifocal tumor that consists of atypical MC and shows a destructive growth without (primary) systemic involvement. This high-grade malignant MC disease has to be distinguished from a localized benign mastocytoma in either extracutaneous organs (extracutaneous mastocytoma) or skin. Additionally, mutations in the KIT receptor leading to mast cell hyper- proliferation have been found in patients with acute myeloid leukemia (AML)
  • AML acute myeloid leukemia
  • treatment regimens for mast cell related conditions typically employ non-specific treatment regimens developed for other proliferative or immune disorders (e.g., histamine receptor blockers, prostaglandin blockers, steroids), resulting in incomplete treatment, treatments which are not effective, or treatments that cause numerous immunosuppressive side-effects.
  • a significant drawback to many therapies that may be used to treat conditions associated with undesirable mast cell activity is the non-specific inhibition of many cellular tyrosine kinases in mast cells and other cell types that are targeted by the treatment.
  • kinase inhibitor therapeutics for treating mast cell proliferative disorders were originally developed to inhibit kinases such as platelet-derived growth factor receptor (PDGF-R), vascular endothelial growth factor receptor (VEGFR), or the Bcr/Abl mutation. These potential therapeutics proved ineffective at treating all forms of mastocytosis.
  • PDGF-R platelet-derived growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • Bcr/Abl mutation the Bcr/Abl mutation
  • the present invention provides methods for effectively treating or preventing a condition, and/or a symptom of a condition, associated with or caused, at least in part, by undesirable mast cell activity .
  • the methods of the invention are particularly useful in treating or preventing conditions (or symptoms associated with conditions) mediated by immunoglobulin receptor cross-linking on mast cells.
  • the invention provides a method for inhibiting an activity of mast cells, comprising administering to an individual a selective inhibitor of phosphoinositide 3 -kinase delta (PI3K ⁇ ) in an amount effective to inhibit mast cell activity.
  • PI3K ⁇ phosphoinositide 3 -kinase delta
  • the mast cell activity being inhibited is mast cell migration, mast cell proliferation, mast cell degranulation, or expression of or secretion of cytokines, chemokines, or growth factors from mast cells.
  • the cytokine is TNF- ⁇ .
  • the cytokine is LL-6.
  • the chemokine being inhibited is eotaxin, MlPl- ⁇ , MlPl- ⁇ , MDC-1, MCP-1, or lymphotactin.
  • the invention provides methods of reducing or preventing lymphocyte infiltration to a site of inflammation in a condition associated with undesirable mast cell activity comprising the step of administering to an individual a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) in an ⁇ amount effective to reduce or prevent lymphocyte infiltration to said site of inflammation in an amount effective to reduce lymphocyte recruitment signaling by mast cells in said individual.
  • PI3K ⁇ phosphoinositide 3-kinase delta
  • a condition associated with undesirable mast cell activity is any condition caused by or involving the underlying effects of any undesirable mast cell activity.
  • the invention provides methods for treating or preventing a condition associated with undesirable mast cell activity in an individual, comprising the step of administering a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) in an amount effective to treat or prevent a condition associated with undesirable mast cell activity.
  • a selective inhibitor of phosphoinositide 3-kinase delta PI3K ⁇
  • the selective PI3K ⁇ inhibitor inhibits mast cell activity. Examples of such mast cell activity include mast cell migration, mast cell proliferation, mast cell degranulation, or expression or secretion of cytokines, chemokines, or growth factors from mast cells.
  • the methods of the invention encompass treating or preventing conditions (or symptoms associated with conditions) mediated by immunoglobulin receptor cross-linking on mast cells
  • Mediated by immunoglobulin crosslinking or “Ig-mediated” refers to the ability of Ig bound to receptors on mast cells to initiate, or facilitate, a condition associated with undesirable mast cell activity.
  • Immunoglobulins which activate mast cells include IgG and IgE.
  • the condition associated with undesirable mast cell activity is an IgE-mediated condition.
  • the condition is an IgG-mediated condition.
  • the condition is mediated by other stimuli such cytokines, chemokines or other growth factors.
  • Conditions, and symptoms of conditions amenable to treatment or prevention by methods according to the invention include, but are not limited to, asthma, allergic reactions, or autoimmune diseases.
  • the allergic reaction is type I hypersensitivity, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, or allergic asthma.
  • Type I hypersensitivity reactions are reactions in which antigens (allergens) combine with specific IgE antibodies that are bound to membrane receptors on tissue mast cells and blood basophils.
  • the antigen-antibody reaction causes the rapid release of potent vasoactive and inflammatory mediators, (e.g., histamine, tryptase, leukotrienes and prostaglandins) and later release of proinflammatory cytokines (e.g., interleukin-4 and interleukin-13).
  • the mediators produce vasodilation, increased capillary permeability, glandular hypersecretion, smooth muscle spasm, and tissue infiltration of other inflammatory cells.
  • Exemplary type I hypersensitivity disorders include allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergic asthma, some cases of urticaria and GI food reactions, and systemic anaphylaxis.
  • the condition is an autoimmune disease.
  • the autoimmune disease contemplated by the invention may be multiple sclerosis, rheumatoid arthritis, bullous pemphigoid, Sjogren's syndrome, chronic urticaria, thyroid eye disease, vasculitis, and peritonitis.
  • the invention provides for a method of the invention wherein the
  • PI3K ⁇ selective inhibitor is administered in an amount effective to inhibit Akt phosphorylation in said mast cells.
  • selective PI3K ⁇ inhibitor and variants thereof such as “PI3K ⁇ selective inhibitor” and “selective inhibitor of PI3K ⁇ ” as used herein refer to a compound that inhibits the PI3K ⁇ isozyme more effectively than other isozymes of the PI3K family.
  • a "selective PI3K ⁇ inhibitor” compound is understood to be more selective for PI3K ⁇ than compounds conventionally and generically designated PI3K inhibitors, e.g., wortmannin or LY294002.
  • wortmannin and LY294002 are deemed “nonselective PI3K inhibitors.”
  • the relative efficacies of compounds as inhibitors of an enzyme activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined extent and then comparing the results. Typically, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or "IC50.”
  • IC50 determinations can be accomplished using . conventional techniques known in the art. In general, an IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study.
  • the experimentally obtained values of enzyme activity then are plotted against the inhibitor concentrations used.
  • concentration of the inhibitor that shows 50% enzyme activity is taken as the IC50 va e -
  • other inhibitory concentrations can be defined through appropriate determinations of activity. For example, in some settings it can be desirable to establish a 90% inhibitory concentration, i.e., IC90, etc.
  • a "selective PI3K ⁇ inhibitor” alternatively can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC50) with respect to PI3K ⁇ that is at least 10-fold, in another aspect at least 20-fold, and in another aspect at least 30-fold, lower than the IC50 value with respect to any or all of the other Class I PI3K family members.
  • IC50 inhibitory concentration
  • the term selective PI3K ⁇ inhibitor can be understood to refer to a compound that exhibits an IC50 with respect to PI3K ⁇ that is at least 50-fold, in another aspect at least 100-fold, in an additional aspect at least 200-fold, and in yet another aspect at least 500-fold, lower than the IC50 with respect to any or all of the other PI3K Class I family members.
  • the term selective PI3K ⁇ inhibitor refers to an oligonucleotide that negatively regulates pi lO ⁇ expression at least 10-fold, in another aspect at least 20-fold, and in a further aspect at least 30-fold, lower than any or all of the other Class I PI3K family catalytic subunits (i.e., pi 10a, pi lO ⁇ , and pi lO ⁇ ).
  • a PI3K ⁇ selective inhibitor is administered to an individual in an amount such that the inhibitor retains its PI3K ⁇ selectivity, as described above. Ranges may be expressed herein as from “about” or “approximately” one particular value and/or to "about” or “approximately” another particular value.
  • Another embodiment includes from the one particular value and/or to the other particular value.
  • values are expressed as approximations, by use of the antecedents such as "about” or "at least about,” it will be understood that the particular value forms another embodiment.
  • Any selective inhibitor of PI3K ⁇ activity including but not limited to small molecule inhibitors, peptide inhibitors, non-peptide inhibitors, naturally occurring inhibitors, and synthetic inhibitors, may be used. Suitable PI3K ⁇ selective inhibitors have been described in U.S. Patent Publication 2002/161014 to Sadhu et al, the entire disclosure of which is hereby incorporated herein by reference.
  • compounds of any type that selectively negatively regulate p 11 O ⁇ expression i. e.
  • the invention provides for the use of antisense oligonucleotides which negatively regulate pi lO ⁇ expression via hybridization to messenger RNA (mR A) encoding pi 10 ⁇ .
  • oligonucleotides that decrease pi lO ⁇ expression may be used in the methods of the invention.
  • the methods of the invention may be applied to cell populations in vivo or ex vivo. "In vivo" means within a living individual, as within an animal or human.
  • the methods of the invention may be used therapeutically in an individual, as described infra.
  • the methods may also be used prophylactically.
  • "Ex vz ' vo" means outside of a living individual.
  • Examples of ex vivo cell populations include in vitro cell cultures and biological samples including but not limited to fluid or tissue samples obtained from individuals. Such samples may be obtained by methods well known in the art.
  • Exemplary biological fluid samples include blood, cerebrospinal fluid, and saliva.
  • Exemplary tissue samples include tumors samples and biopsies of tissue.
  • the invention may be used for a variety of purposes, including therapeutic and experimental purposes.
  • the invention may be used ex vivo to determine the optimal schedule and/or dosing of administration of a PI3K ⁇ selective inhibitor for a given indication, cell type, individual, and other parameters. Information gleaned from such use may be used for experimental purposes or in the clinic to set protocols for in vivo treatment. Other ex vivo uses for which the invention may be suited are described below or will become apparent to those skilled in the art.
  • Mast cell activity refers to those biological activities carried out by mast cells which may be modulated by the compounds useful in the methods of the invention.
  • Examples of these activities include cell migration, proliferation, activation, degranulation, expression of or secretion of chemokines, cytokines or other growth factors, and modulation of cell signaling pathways, for example, modulation of AKT phosphorylation.
  • modulation of mast cell activity may be used herein.
  • Modulation of mast cell activity as used herein refers to the reduction, inhibition, prevention, promotion or increase of one of the above listed activities of mast cells upon administration of a selective PI3K ⁇ inhibitor.
  • a selective PI3K ⁇ inhibitor may inhibit the enzyme itself, may inhibit any downstream signaling effect of the PI3K ⁇ enzyme, or inhibit any further downstream activity of a mast cell.
  • mast cell activity means mast cell activity that deviates from the normal, proper, or expected course.
  • undesirable mast cell degranulation may include degranulation during allergic reaction causing hypersensitivity of the individual, while undesirable migration may include movement of mast cells into or out of tissue sites having unfavorable biological effects.
  • Undesirable mast cell proliferation may include cell proliferation mediated by, or resulting in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both.
  • "Inhibiting undesirable mast cell activity” means to slow or stop the rate at which undesirable mast cell activity takes place. This may result either from a decreased rate of mast cell receptor activation, decreased inflammatory mediator or growth factor release, decreased cellular replication, or an increased rate of cell death. Cell death may occur by any mechanism, including apoptosis and mitotic catastrophe. Use of the methods in accordance with the present invention may result in partial or complete inhibition of undesirable mast cell activity,.
  • Preventing undesirable mast cell activity means that the methods of the present invention may be used prophylactically to prevent or inhibit undesirable mast cell activity before it occurs, or to prevent or inhibit the recurrence thereof.
  • the invention may be used in vivo or ex vivo where no undesirable cell activity has been identified or where no undesirable cell activity is ongoing, but where undesirable cell activity is suspected or expected, respectively.
  • the invention may also be used in all its embodiments wherever undesirable cell activity has been previously treated to prevent or inhibit recurrence of the same.
  • a “therapeutically effective amount” or “amount effective” means an amount effective to inhibit or reverse development of, to alleviate the existing symptoms of, to prolong survival of, or to cure the individual being treated.
  • the "therapeutic index” is the dose ratio between toxic or undesired effect and therapeutic, or desired, effects, and is expressed as the ratio of LD 50 to ED 50 , which are defined below.
  • An increase in the therapeutic index refers to a reduction in the amount of therapeutic necessary to reach a desired effect or to increase the effectiveness of the therapeutic administered.
  • the treatment methods of the invention are useful in the fields of human medicine and veterinary medicine.
  • the individual to be treated may be a mammal, preferably human, or another animal.
  • individuals include but are not limited to farm animals including cows, sheep, pigs, horses, and goats; companion animals such as dogs and cats; exotic and/or zoo animals; laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducks, and geese.
  • the invention further provides methods wherein the selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) s is administered in a plurality of doses.
  • a plurality of doses includes administration of the inhibitor or other agent in more than one dose.
  • the invention further provides that the selective PI3K ⁇ inhibitor is administered in a regimen which includes administering one or more additional therapeutic compounds commonly utilized in treatment of a condition associated with undesirable mast cell activity, including at least one immunomodulatory agent or other agent as appropriate to the condition or symptom being treated or prevented.
  • the invention provides a method of reducing or preventing mast cell activity in an individual having a condition associated with undesirable mast cell activity, comprising administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) and an immunomodulatory agent.
  • the combination therapy may be administered in a single composition or each agent, such as the inhibitor and immunomodulatory agent, may be administered as a separate composition.
  • each agent may be administered in a plurality of doses as necessary.
  • the invention provides a method of reducing or preventing lymphocyte infiltration to a site of inflammation in an individual having a condition associated with undesirable mast cell activity, comprising administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) and a immunomodulatory agent.
  • Immunomodulatory agents contemplated by the invention include glucocorticoids or corticosteroids, immunosuppressants, antihistamines, aminosalicylates, steroid hormones, non-steroidal anti-inflammatory drugs (NSAIDs), .
  • glucocorticoids are chosen from the group consisting of cortisone, dexamethosone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and budesonide.
  • NSALDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as Vioxx ® (rofecoxib) and Celebrex® (celecoxib), and salicylate.
  • Suitable analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hygrochloride.
  • immunosuppressants include azathioprine (6-mercaptopurine (6-MP)), cyclophosphamide, cyclosporine, methotrexate, or penicillamine. Also contemplated are Xolair® (omalizumab), leukotriene antagonists, or other drugs commonly used for allergy or asthma. Methods of the invention may include administering formulations comprising an inhibitor of the invention with a particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • methods of the invention may comprise administering an inhibitor with one or more of TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • compositions in accordance with the invention may also include other known angiopoietins, for example Ang- 1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, and/or vascular endothelial growth factor (VEGF).
  • angiopoietins for example Ang- 1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, and/or vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • Growth factors for use in pharmaceutical compositions of the invention include angiogenin, bone morpho genie protein- 1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein- 14, bone morphogenic protein- 15; bone morphogenic protein receptor IA, bone morphogenic protein receptor LB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor ⁇ , cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2 ⁇ , cytokine-induced neutrophil chemotactic factor 2 ⁇ , ⁇ endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial- derived neutrophil attractant,
  • Immunomodulatory agents used for treatment can be administered in a plurality of doses. It is contemplated that the agents are administered in the combination methods according to the invention at a low dose, that is, at a dose lower than conventionally used in clinical situations where the agent or therapy is administered alone, because the PI3K ⁇ selective nature of the inliibitors of the invention increases the therapeutic index (i.e., the specificity) of the inventive combination therapies. Lowering the dose of the agent or therapy administered to an individual decreases the incidence of adverse effects associated with higher dosages, and can thereby improve the quality of life of a patient undergoing treatment. Further benefits include improved patient compliance, and a reduction in the number of hospitalizations needed for the treatment of adverse effects.
  • methods of the invention are advantageous in that they permit treatment at higher doses of the PI3K ⁇ selective inhibitor(s) than nonselective inhibitors such as LY294002 and wortmannin, further maximizing the therapeutic efficacy of the inventive methods
  • methods may include administering an inhibitor with one or more other agents which either enhance the activity of the inhibitor or compliment its activity or use in treatment. Such additional factors and/or agents may produce a synergistic effect with an inhibitor of the invention, or to minimize side effects.
  • Methods of the invention contemplate use of a selective PI3K ⁇ inhibitor compound having formula (I) or pharmaceutically acceptable salts and solvates thereof:
  • A is an optionally substituted monocyclic or bicyclic ring system containing at least two nitrogen atoms, and at least one ring of the system is aromatic;
  • X is selected from the group consisting of C(R D )2, CFTjCHRb, and
  • R a is selected from the group consisting of hydrogen, Ci .galkyl, C3.. gcycloalkyl, C3_gheterocycloalkyl, C ⁇ _3alkyleneN(R c )2, aryl, arylCi ⁇ alkyl, C ⁇ _ 3alkylenearyl, heteroaryl, heteroarylCi _3alkyl, and Ci _3alkyleneheteroaryl; or two R a groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
  • R b is selected from the group consisting of hydrogen, Ci _6alkyl, heteroCi _3alkyl, Ci ⁇ alkyleneheteroCi ⁇ alkyl, arylheteroCi _3alkyl, aryl, heteroaryl, arylC ⁇ _3alkyl, heteroarylC ⁇ _3alkyl, C ⁇ _3alkylenearyl, and C
  • R c is selected from the group consisting of hydrogen, C ⁇ . ⁇ alkyl, C3_ gcycloalkyl, aryl, and heteroaryl; and,
  • alkyl is defined as straight chained and branched hydrocarbon groups containing the indicated number of carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups.
  • the hydrocarbon group can contain up to 16 carbon atoms, for example, one to eight carbon atoms.
  • alkyl includes "bridged alkyl,” i.e., a C6-C 16 bicyclic or polycyclic hydrocarbon group, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl.
  • cycloalkyl is defined as a cyclic C 3 -C 8 hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.
  • alkenyl is defined identically as “alkyl,” except for containing a carbon-carbon double bond.
  • Cycloalkenyl is defined similarly to cycloalkyl, except a carbon-carbon double bond is present in the ring.
  • alkylene is defined as an alkyl group having a substituent.
  • C 1-3 alkylenearyl refers to an alkyl group containing one to three carbon atoms, and substituted with an aryl group.
  • heteroC 1-3 alkyl is defined as a C 1-3 alkyl group further containing a heteroatom selected from O, S, and NR a .
  • arylheteroC salkyl refers to an aryl group having a heteroC ⁇ -3 alkyl substituent.
  • halo or halogen is defined herein to include fluorine, bromine, chlorine, and iodine.
  • aryl alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, e.g., phenyl or naphthyl.
  • an "aryl” group can be unsubstituted or substituted, for example, with one or more, and in particular one to three, halo, alkyl, phenyl, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, and amino.
  • exemplary aryl groups include phenyl, naphthyl, biphenyl, tetrahydronaphthyl, chlorophenyl, fluorophenyl, aminophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, carboxyphenyl, and the like.
  • arylC 1-3 alkyl and “heteroarylC 1-3 alkyl” are defined as an aryl or heteroaryl group having a C 1-3 alkyl substituent.
  • heteroaryl is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, like halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, and amino.
  • heteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.
  • Het is defined as monocyclic, bicyclic, and tricyclic groups containing one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur.
  • Het groups include 1,3-dioxolane, 2-pyrazoline, pyrazolidine, pyrrolidine, piperazine, a pyrroline, 2H-pyran, 4H-pyran, morpholine, thiopholine, piperidine, 1,4-dithiane, and 1,4-dioxane.
  • methods of the invention contemplate use of a PI3K ⁇ selective inhibitor compound having formula (II) or pharmaceutically acceptable salts and solvates thereof:
  • R 8 is selected from the group consisting of hydrogen, Ci _6alkyl, halo,
  • X 1 is selected from the group consisting of CH (i.e., a carbon atom having a hydrogen atom attached thereto) and nitrogen;
  • R a is selected from the group consisting of hydrogen, Ci .galkyl, C3_ gcycloalkyl, C3_gheterocycloalkyl, C ⁇ _3alkyleneN(R c )2, aryl, arylCj_3alkyl, C _ 3alkylenearyl, heteroaryl, heteroarylC 1.3 alkyl, and Ci _3alkyleneheteroaryl; or two R a groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
  • R c is selected from the group consisting of hydrogen, Ci .galkyl, C3., gcycloalkyl, aryl, and heteroaryl; and,
  • methods of the invention include use of a selective inhibitor of PI3K ⁇ compound having formula (III) or pharmaceutically acceptable salts and solvates thereof:
  • R a is selected from the group consisting of hydrogen, Ci _6alkyl, C3.. gcycloalkyl, C3_gheterocycloalkyl, C ⁇ _3alkyleneN(R c )2, aryl, arylC 1.3 alkyl, C 3alkylenearyl, heteroaryl, heteroarylCi _3alkyl, and Ci _3alkyleneheteroaryl; or two R a groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
  • methods of the invention embrace use of a PI3K ⁇ selective inhibitor selected from the group consisting of 2-(6-aminopurin-9-ylmethyl)- 3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-6-bromo-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-6-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one; 2-(6-amino
  • the methods can be practiced using a racemic mixture of the compounds or a specific enantiomer.
  • the S-enantiomer of the above compounds is utilized. Therefore, the present invention includes all possible stereoisomers and geometric isomers of the aforementioned compounds.
  • “Pharmaceutically acceptable salts” means any salts that are physiologically acceptable insofar as they are compatible with other ingredients of the formulation and not deleterious to the recipient thereof. Some specific preferred examples are: acetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, oxalate.
  • the invention contemplates an article of manufacture comprising a phosphoinositide 3-kinase delta (PI3K ⁇ ) selective inhibitor and a label indicating a method of use according to any one of the methods of the invention.
  • the invention provides for use of a composition comprising at least one PI3K ⁇ selective inhibitor in the manufacture of a medicament for treating or preventing a condition associated with undesirable mast cell activity.
  • Administration of prodrugs are also contemplated.
  • prodrug refers to compounds that are rapidly transformed in vivo by hydrolysis to, for example, a compound having a structural formula described herein. Prodrug design is discussed generally in Hardma et al.
  • prodrugs which, following a biotransformation, become more physiologically active in their altered state.
  • Prodrugs therefore, encompass pharmacologically inactive compounds that are converted to biologically active metabolites.
  • prodrugs can be converted into a pharmacologically active form through hydrolysis of, for example, an ester or amide linkage, thereby introducing or exposing a functional group on the resultant product.
  • the prodrugs can be designed to react with an endogenous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life.
  • prodrugs can be designed to undergo covalent modification on a functional group with, for example, glucuronic acid, sulfate, glutathione, amino acids, or acetate.
  • the resulting conjugate can be inactivated and excreted in the urine, or rendered more potent than the parent compound.
  • High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and released back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound.
  • PI3K ⁇ selective inhibitors include compounds that selectively negatively regulate pllO ⁇ mRNA expression more effectively than they do other isozymes of the PI3K family, and that possess acceptable pharmacological properties.
  • Polynucleotides encoding human pi lO ⁇ are disclosed, for example, in Genbank Accession Nos. AR255866, NM 005026, U86453, U57843 and Y10055, the entire disclosures of which are incorporated herein by reference. See also, Vanhaesebroeck et al, Proc. Natl. Acad. Sci. 94:4330-4335 (1997), the entire disclosure of which is incorporated herein by reference.
  • mRNA messenger RNA
  • antisense oligonucleotides at least 5 to about 50 nucleotides in length, including all lengths (measured in number of nucleotides) in between, which specifically hybridize to mRNA encoding pi lO ⁇ and inhibit mRNA expression, and as a result pi 1 O ⁇ protein expression, are contemplated for use in the methods of the invention.
  • Antisense oligonucleotides include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e. ' , make the oligonucleotide more resistant to nuclease degradation, particularly in vivo.
  • antisense oligonucleotides that are perfectly complementary to a region in the target polynucleotide possess the highest degree of specific inhibition antisense oligonucleotides that are not perfectly complementary, i.e., those which include a limited number of mismatches with respect to a region in the target polynucleotide, also retain high degrees of hybridization specificity and therefore also can inhibit expression of the target mRNA.
  • the invention contemplates methods using antisense oligonucleotides that are perfectly complementary to a target region in a polynucleotide encoding pi lO ⁇ , as well as methods that utilize antisense oligonucleotides that are not perfectly complementary (i.e., include mismatches) to a target region in the target polynucleotide to the extent that the mismatches do not preclude specific hybridization to the target region in the target polynucleotide.
  • Methods for designing and optimizing antisense nucleotides are described in Lima et al, (JBiol Chem ;272:626-38. 1997), Kurreck et al, (Nucleic Acids Res. ;30:1911-8. 2002) and U.S. Patent No. 6,277,981 , which are incorporated herein by reference.
  • antisense compounds are described in International Patent Publication WO 01/05958, which is incorporated herein by reference.
  • the invention further contemplates methods utilizing ribozyme inhibitors which, as is known in the art, include a nucleotide region which specifically hybridizes to a target polynucleotide and an enzymatic moiety that digests the target polynucleotide. Specificity of ribozyme inhibition is related to the length the antisense region and the degree of complementarity of the antisense region to the target region in the target polynucleotide.
  • ribozyme inhibitors comprising antisense regions from 5 to about 50 nucleotides in length, including all nucleotide lengths in between, that are perfectly complementary, as well as antisense regions that include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target p 11 O ⁇ -encoding polynucleotide.
  • Ribozymes useful in methods of the invention include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo, to the extent that the modifications do not alter the ability of the ribozyme to specifically hybridize to the target region or diminish enzymatic activity of the molecule. Because ribozymes are enzymatic, a single molecule is able to direct digestion of multiple target molecules thereby offering the advantage of being effective at lower concentrations than non-enzymatic antisense oligonucleotides.
  • RNAi technology for inhibiting pi 1 O ⁇ expression.
  • the invention provides double-stranded RNA (dsRNA) wherein one strand is complementary to a target region in a target pi 1 O ⁇ -encoding polynucleotide.
  • dsRNA molecules of this type are less than 30 nucleotides in length and referred to in the art as short interfering RNA (siRNA).
  • dsRNA molecules longer than 30 nucleotides in length and in certain aspects of the invention, these longer dsRNA molecules can be about 30 nucleotides in length up to 200 nucleotides in length and longer, and including all length dsRNA molecules in between.
  • complementarity of one strand in the dsRNA molecule can be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target pi 1 O ⁇ -encoding polynucleotide.
  • dsRNA molecules include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo.
  • dsRNA double stranded
  • siRNA short-interfering RNA
  • Circular RNA lasso inhibitors are highly structured molecules that are inherently more resistant to degradation and therefore do not, in general, include or require modified internucleotide linkage or modified nucleotides.
  • the circular lasso structure includes a region that is capable of hybridizing to a target region in a target polynucleotide, the hybridizing region in the lasso being of a length typical for other RNA inhibiting technologies.
  • the hybridizing region in the lasso may be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target pi lO ⁇ -encoding polynucleotide.
  • RNA lassos are circular and form tight topological linkage with the target region, inhibitors of this type are generally not displaced by helicase action unlike typical antisense oligonucleotides, and therefore can be utilized as dosages lower than typical antisense oligonucleotides. Preparation and use of RNA lassos is described in U.S.
  • the inhibitors of the invention may be covalently or noncovalently associated with a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Patents 4,289,872 and 5,229,490; PCT Publication WO 93/21259 published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide.
  • a carrier molecule such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Patents 4,289,872 and 5,229,490; PCT Publication WO 93/21259 published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide.
  • carriers for use in the pharmaceutical compositions of the invention include carbohydrate-based polymers, such as trehalose, mannitol, xylitol, sucrose, lactose, sorbitol, dextrans, such as cyclodextran, cellulose, and cellulose derivatives. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Other carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Patent Nos: 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337.
  • Still other useful carrier polymers known in the art include monomethoxy-polyethylene glycol, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.
  • Derivatization with bifunctional agents is useful for cross-linking a compound of the invention to a support matrix or to a carrier.
  • One such carrier is polyethylene glycol (PEG).
  • the PEG group may be of any convenient molecular weight and may be straight chain or branched.
  • the average molecular weight of the PEG can range from about 2 kDa to about 100 kDa, in another aspect from about 5 kDa to about 50 kDa, and in a further aspect from about 5 kDa to about 10 kDa.
  • the PEG groups will generally be attached to the compounds of the invention via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, haloacetyl, maleimido or hydrazino group) to a reactive group on the target inhibitor compound (e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimido or hydrazino group).
  • a reactive group on the PEG moiety e.g., an aldehyde, amino, ester, thiol, haloacetyl, maleimido or hydrazino group
  • target inhibitor compound e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimid
  • Cross-linking agents can include, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-l,8-octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos.
  • compositions of the invention may also include compounds derivatized to include one or more antibody Fc regions.
  • Fc regions of antibodies comprise monomeric polypeptides that may be in dimeric or multimeric forms linked by disulfide bonds or by non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of Fc molecules can be from one to four depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, IgGA2) of antibody from which the Fc region is derived.
  • the term "Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms of Fc molecules, with the Fc region being a wild type structure or a derivatized structure.
  • the pharmaceutical compositions of the invention may also include the salvage receptor binding domain of an Fc molecule as described in WO 96/32478, as well as other Fc molecules described in WO 97/34631.
  • Such derivatized moieties preferably improve one or more characteristics of the inhibitor compounds of the invention, including for example, biological activity, solubility, absorption, biological half life, and the like.
  • derivatized moieties result in compounds that have the same, or essentially the same, characteristics and/or properties of the compound that is not derivatized.
  • the moieties may alternatively eliminate or attenuate any undesirable side effect of the compounds and the like.
  • Compounds that compete with an inhibitor compound described herein for binding to PI3K ⁇ are also contemplated for use in the invention. Methods of identifying compounds which competitively bind with PI3K ⁇ , with respect to the compounds specifically provided herein, are well known in the art.
  • inhibitor as used herein embraces compounds disclosed, compounds that compete with disclosed compounds for PI3K ⁇ binding, and in each case, conjugates and derivatives thereof.
  • Methods include administration of an inhibitor to an individual in need, by itself, or in combination as described herein, and in each case optionally including one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants/flavoring, carriers, excipients, buffers, stabilizers, solubilizers, other materials well known in the art and combinations thereof.
  • any pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents known in the art that serve as pharmaceutical vehicles, excipients, or media may be used.
  • exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma, methyl- and propylhydroxybenzoate, talc, alginates, carbohydrates, especially mannitol, ⁇ -lactose, anhydrous lactose, cellulose, sucrose, dextrose, sorbitol, modified dextrans, gum acacia, and starch.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present inhibitor compounds. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
  • Pharmaceutically acceptable fillers can include, for example, lactose, microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, calcium sulfate, dextrose, mannitol, and/or sucrose.
  • Inorganic salts including calcium triphosphate, magnesium carbonate, and sodium chloride may also be used as fillers in the pharmaceutical compositions.
  • Amino acids may be used, such as use in a buffer formulation of the pharmaceutical compositions.
  • Disintegrants may be included in solid dosage formulations of the inhibitors. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge, corn starch, potato starch, and bentonite may all be used as disintegrants in the pharmaceutical compositions. Other disintegrants include insoluble cationic exchange resins.
  • Powdered gums including powdered gums such as agar, Karaya or tragacanth may be used as disintegrants and as binders. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin.
  • crystalline cellulose examples include crystalline cellulose, cellulose derivatives such as methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC), acacia, corn starch, and/or gelatins
  • Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
  • An antifriction agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process.
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, talc, and waxes.
  • Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. Suitable glidants include starch, talc, pyrogenic silica and hydrated silicoaluminate. To aid dissolution of the therapeutic into the aqueous environment, a surfactant might be added as a wetting agent. Natural or synthetic surfactants may be used.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents such as benzalkonium chloride and benzethonium chloride may be used.
  • Nonionic detergents that can be used in the pharmaceutical formulations include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the pharmaceutical compositions of the invention either alone or as a mixture in different ratios.
  • Controlled release formulation may be desirable.
  • the inhibitors of the invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums.
  • Slowly degenerating matrices may also be incorporated into the pharmaceutical formulations, e.g., alginates, polysaccharides.
  • Another form of controlled release is a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push the inhibitor compound out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect. Colorants and flavoring agents may also be included in the pharmaceutical compositions.
  • the inhibitors of the invention may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • the therapeutic agent can also be administered in a film coated tablet.
  • Nonenteric materials for use in coating the pharmaceutical compositions include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy- methyl cellulose, povidone and polyethylene glycols.
  • Enteric materials for use in coating the pharmaceutical compositions include esters of phthalic acid. A mix of materials might be used to provide the optimum film coating.
  • Film coating manufacturing may be carried out in a pan coater, in a fluidized bed, or by compression coating.
  • Compositions can be administered in solid, semi-solid, liquid or gaseous form, or may be in dried powder, such as lyophilized form.
  • the pharmaceutical compositions can be packaged in forms convenient for delivery, including, for example, capsules, sachets, cachets, gelatins, papers, tablets, capsules, ointments, granules, solutions, inhalants, aerosols, suppositories, pellets, pills, troches,' lozenges or other forms known in the art.
  • the type of packaging will generally depend on the desired route of administration.
  • Implantable sustained release formulations are also contemplated, as are transdermal formulations.
  • Such pharmaceutical compositions may be for administration for injection, or for oral, nasal, transdermal or other forms of administration, including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intratracheal, intrathecal, intraocular, retrobulbar, intrapuhnonary (e.g., aerosolized drugs) or subcutaneous injection (including depot administration for long term release e.g., embedded under the splenic capsule, brain, or in the cornea); by sublingual, anal, vaginal, or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea.
  • intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intratracheal, intrathecal, intraocular, retrobulbar, intrapuhnonary (e.g., aerosolized drugs) or subcutaneous injection including depot administration for long term release e.g., embedded under the splenic capsule, brain, or in
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • the methods of the invention involve administering effective amounts of an inhibitor of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers, as described above.
  • a chosen route of administration may dictate the physical form of the compound being delivered.
  • the invention provides methods for oral administration of a pharmaceutical composition of the invention. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges, and cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673).
  • Liposomal encapsulation may include liposomes that are derivatized with various polymers (e.g., U.S. Patent No. 5,013,556).
  • the formulation will include a compound of the invention and inert ingredients which protect against degradation in the stomach and which pe ⁇ nit release of the biologically active material in the intestine.
  • the inhibitors can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the capsules could be prepared by compression.
  • pulmonary delivery of the present inhibitors in accordance with the invention is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn H nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and or carriers useful in therapy.
  • the inhibitors of the invention are most advantageously prepared in particulate form with an average particle size of less than 10 ⁇ m (or microns), for example, 0.5 to 5 ⁇ m, for most effective delivery to the distal lung.
  • Formulations suitable for use with a nebulizer will typically comprise the inventive compound dissolved in water at a concentration range of about 0.1 to 100 mg of inhibitor per mL of solution, 1 to 50 mg of inhibitor per mL of solution, or 5 to 25 mg of inhibitor per mL of solution.
  • the formulation may also include a buffer.
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the inhibitor caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive inhibitors suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1 ,2-tetrafluoroethane, or combinations thereof.
  • Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent or diluent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • a bulking agent or diluent such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the inhibitor to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery may include dextran or cyclodextran
  • Toxicity and therapeutic efficacy of the PI3K ⁇ selective compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Additionally, this information can be determined in cell cultures or experimental animals additionally treated with other therapies such as radiation, chemotherapeutic agents, and antiangiogenic agents.
  • the pharmaceutical compositions are generally provided in doses ranging from 1 pg compound/kg body weight to 1000 mg/kg, 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg to 50 mg/kg, and 1 to 20 mg/kg, given in daily doses or in equivalent doses at longer or shorter intervals, e.g., every other day, twice weekly, weekly, or twice or three times daily.
  • the inhibitor compositions may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product.
  • Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient.
  • the frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration.
  • the optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents.
  • a suitable dose may be calculated according to body weight, body surface area or organ size.
  • FIG. 1 shows a count of inflammatory cell infiltration (total cells and differential cellular component) of BAL fluids isolated from OVA- sensitized/challenged mice treated with p 11 O ⁇ inhibitor IC87114 or control in BAL fluid.
  • Macrophage Mac
  • Lym lymphocyte
  • Neu neutrophils
  • Es eosinophils
  • Figure 2 depicts total lung inflammation, defined as the average of the peribronchial and perivascular inflammation scores, in OVA-sensitized/challenged mice treated with IC87114 or control. Bars represent mean ⁇ SEM from 6 independent experiments. #, p ⁇ 0.05 vs. SAL+SAL; *, p ⁇ 0.05 vs. OVA+SAL.
  • Figure 3 shows airway responsiveness to aerosolized methacholine measured in unrestrained, conscious OVA-sensitized/challenged mice treated with IC87114 or control, at 72 h after the last challenge. Readings of breathing parameters were taken for 3 min after each nebulization during which Penh values were determined. Data represent mean ⁇ SEM from 6 independent experiments. #, p ⁇ 0.05 vs. SAL+SAL; *, p ⁇ 0.05 vs. OVA+SAL.
  • EXAMPLES The following examples are provided to illustrate the invention, but are not intended to limit the scope thereof.
  • EXAMPLE 1 SELECTIVE PI3K ⁇ INHIBITOR INHIBITS MAST CELL DEGRANULATION
  • Mast cells are centrally important in allergic inflammation of the airways. Mast cell activation is mediated by antigen/IgE cross-linking of the Fc ⁇ RI receptor, complement components and cytokines activation, leading to a spontaneous release of preformed histamine and proteases from secretory granules. These activated cells then transcribe, translate and secrete proinflammatory cytokines and chemokines which lead to increased inflammation.
  • Non-specific PI3-kinase inhibitors such as wortmannin have been shown to inhibit mast cell degranulation from rat basophilic leukemia cells (RBL- 2H3) (Kitani et al., Biochem Biophys Res Commun. 183:48-54, 1992).
  • RBL- 2H3 rat basophilic leukemia cells
  • PI3-kinase subunits in regulating mast cell degranulation remains unclear [Tkaczyk et al, JBiol Chem. 278:48474-84 (2003); Windmiller et al, JBiol Chem. 278:11874-8 (2003)].
  • Bone marrow cells from mouse femur were cultured in mouse IL-3- containing RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies, Carlsbad, CA), 2 raM glutamine, and 50 ⁇ M 2-ME in humidified 95% air/5% CO 2 at 37°C [Saito, H., F. et al., J. Immunol. 138:3927-3934 (1987)].
  • BMMC bone marrow-derived mast cells
  • DNP antidinitrophenyl
  • Tyrode buffer 112 mM NaCl, 2.7 mM KC1, 0.4 mM NaH 2 PO 4 , 1.6 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes [pH 7.5], 0.05% gelatin, 0.1% glucose), resuspended in Tyrode buffer to 1 x 10 cells/ml and incubated with pi 10-delta inhibitor or vehicle control (0.3% DMSO) for 30 minutes.
  • DNP antidinitrophenyl
  • DNP-HSA human serum albumin
  • results of the ELISA indicate that mast cells pretreated with IC87114 (5 ⁇ M) reduce the amount of histamine in the cell supernatant by approximately 40% compared to vehicle control, and increase the amount of histamine detected in the cell pellet accordingly.
  • This demonstrates that inhibition of pi lO ⁇ kinase reduces the amount of histamine released from mast cell storage granules which has utility as an effective therapy for decreasing histamine release from mast cell activation mediated through Fc ⁇ RI.
  • BMMCs were sensitized by an overnight incubation with 0.5-1 ⁇ g/ml antidinitrophenyl (DNP) IgE mAb, washed once in BMMC medium in the absence of IL-3 and resuspended in the same medium at 1 x 10 7 cells/ml.
  • DNP antidinitrophenyl
  • cytokines include TNF- ⁇ , IL-6, IL-2, and granulocyte/monocyte-colony stimulating factor (GM-CSF). Results show that stimulation with 10 ng/ml antigen and addition of only 2 ⁇ M IC87114 to mast cells decreases the amount of TNF- ⁇ secreted by mast cells to undetectable levels.
  • IL-6 100 ng/ml of antigen plus 3 ⁇ M inhibitor inhibits TNF- ⁇ production from activated mast cells by 85%.
  • Assessment of IL-6 levels after addition of the p 11 O ⁇ kinase inhibitor demonstrates similar effects.
  • Addition of IC87411 to activated mast cells (10 ng/ml antigen) showed a dose dependent reduction in IL-6 production, from 6000 pg/ml with no inhibitor, reduced to approximately 2000 pg/ml IL-6 in the presence of 50 ⁇ M inhibitor.
  • Bone marrow mast cells isolated from pi lO ⁇ knockout mice were also assessed for IL-6 secretion levels after activation by antigen (100 ng/ml).
  • pi lO ⁇ inhibitor up to 10 ⁇ M had no effect on levels of IL-6 in knockout cells, indicating that the effect of the inhibitor is through inhibition of the protein kinase pi lO ⁇ isoform.
  • GM-CSF secretion by activated mast cells was also inhibited by approximately 45% after addition of the p 110 ⁇ inhibitor.
  • Levels of the inflammatory cytokine IL-2 were also measured from antigen stimulated BMMC.
  • BMMC activated with 10 ng/ml antigen showed a dose response inhibition of IL-2 secretion upon addition of p 110 ⁇ inhibitor, with levels decreased from control values of approximately 10 pg/ml to ⁇ 4 pg/ml IL-2 in the presence of 20 ⁇ M inhibitor, a 2.5 fold reduction in IL-2 secretion.
  • Chemokines play a significant role in attracting cells to the site of inflammation thereby promoting the inflammatory response.
  • Antigen activated BM mast cells were assessed for chemokine production in the presence and absence of pi lO ⁇ inhibitor using Multi-Analyte Profile Technology (Rules-Based Medicine, Inc. Austin, TX).
  • pi lO ⁇ inhibitor (5 ⁇ M) on chemokine production from activated mast cells resulted in approximately 57% inhibition of eotaxin levels, approximately 37% inhibition of lymphotactin, approximately 60% inhibition of macrophage-derived chemokine (MDC) secretion, and approximately 35% inhibition in the levels of macrophage-inflammatory proteins MlP-l ⁇ and MlP-l ⁇ .
  • MDC macrophage-derived chemokine
  • Akt activation has previously been associated with secretion of cytokines from activated rat basophilic leukemia cells or cells isolated from tyrosine kinase knockout mice [Kitaura et al, J. Exp. Med. 192:729-39 (2000)]. Broad inhibition of class la PI3Ks in certain cell types, such as endothelium, using LY294002 has been shown to not only to reduce phosphorylation of Akt in response to TNF- ⁇ , but also in non-cytokine stimulated cells, as these lipid kinases are essential for both cell motility and survival. [See Madge et al, J. Biol. Chem., 275:15458-15465 (2000)]. Because Akt activation occurs through several different biochemical pathways, it was necessary to determine the role PI3K pi lO ⁇ played in triggering Akt phosphorylation in activated mast cells. To assess the effects of PI3K inhibitor on downstream signaling,
  • BMMC were stimulated with a dose range of antigen and incubated with IC87114 as above with 0, 2 ⁇ M, 5 ⁇ M, 20 ⁇ M or 50 ⁇ M inhibitor.
  • Cells were lysed as described above and phsophoserine or phosphotyrosine levels for several pi lO ⁇ downstream effectors were measured by Western blot.
  • Antibodies used in the Western analysis include: Akt pS473 ; 3-phos ⁇ hoinositide-dependent kinase (PDKl) pS241 ; pERK; pJNK; Lyn pY396 and Bruton's tyrosine kinase (Btk) pY223.
  • Antibodies were obtained from Cell Signaling Technology (Beverly, MA), Upstate Biotechnology (Lake Placid, NY), Zymed, and Santa Cruz Biotechnology.. Western blot analysis demonstrated that pi lO ⁇ inhibitor as low as 5 ⁇ M decreased Akt phosphorylation after 10 minutes of antigen stimulation and significantly inhibited phosphorylation after 30 minutes of antigen stimulation. The 50 ⁇ M inhibitor dose again significantly inhibited S243 Akt phosphorylation after 2 minutes of antigen stimulation, and completely abolished phosphorylation at 10 or 30 minutes of antigen stimulation.
  • pi lO ⁇ selective inhibitor regulation of Akt which is involved in mast cell cytokine secretion, will provide an effective therapeutic method for modulating ongoing inflammation in an already developed mast cell disorder by reducing cellular infiltrate to the site of inflammation, or may act preventatively in disorders such as allergy or asthma to prevent the onset of an immune reaction.
  • EXAMPLE 4 ALLERGEN-INDUCED AIRWAY INFLAMMATION LEADS TO INCREASED ACTIVITY OF PllO ⁇ IN LUNG TISSUE PI3K activity is stimulated after antigen challenge in a murine model of allergic asthma, and administration of wortmannin or LY294002, two broad- spectrum inhibitors of PI3Ks, attenuate inflammation and airway hyperresponsiveness (AHR) (Ezeamuzie et al., Am JRespir Crit Care Med 164:1633-39, 2001; Kwak et al., J. Clin. Invest 111 : 1083-92, 2003).
  • AHR airway hyperresponsiveness
  • pi lO ⁇ was reported to be essential for allergen-IgE-induced mast cell degranulation and vascular permeability (Ali et al., Nature 431:1007-11, 2004).
  • OVA ovalbumin
  • mice On days 21, 22, and 23 after the initial sensitization, the mice were challenged for 30 min with an aerosol of 3% (wt/vol) OVA in saline (or with saline as a control) using an ultrasonic nebulizer (NE-U12, Omron, Japan).
  • IC87114 0.1 or 1 mg/kg body weight/day
  • DMSO vehicle control
  • IC87114 0.1 or 1 mg/kg body weight/day
  • DMSO vehicle control
  • PIP3 levels increased from approximately 15 pmol/ ml pre-challenge to approximately 75 pmol/ml at 1 hr, approximately 100 pmol/ml at 24 hr, and approximately 160 pmol/ml at 48 hrs and 72 hrs, indicating that class I PI3K activity increased approximately 4.6- , 6.1-, 9.5-, and 9.6-fold, respectively, after OVA inhalation, compared with the pre- challenge period. In contrast, no significant changes in PI3K activity were observed after saline inhalation. Activation of these kinases has been linked to phosphorylation of Ser- 473 of Akt, an event crucial for Akt enzymatic activation (Alessi et al., Curr.
  • Akt levels were measured by Western blot. Protein extracts from lung tissue homogenates (30 ⁇ g/lane) were electrophoresed in polyacrylamide gels (Invitrogen Life Technologies, Carlsbad, CA), transferred electrophoretically to a PVDF membrane (Immobilon-P; Millipore, Billerica, MA), and incubated overnight at 4°C with anti phoshposerine antibodies as described above. Consistent with increased PI3K activity, levels of p-Akt protein in the lung tissues were also increased at 72 h after OVA inhalation, as detected by Western blot, compared with levels in the control animals that received saline inhalation. No significant changes in total Akt protein levels were observed.
  • the level of phosphorylation was similar to saline control levels indicating that pi lO ⁇ contributed significantly to the overall class I PI3K activity in allergen-induced Akt activation. While PLP3 formation and Akt phosphorylation indicated that the PI3K family plays a role in allergy and asthma, administration of the pi lO ⁇ specific inhibitor described herein demonstrates that the important PI3K for allergy and asthma is pi lO ⁇ .
  • mice were sacrificed with an overdose of pentobarbital- Na (100 mg/kg of body weight/administered intraperitoneally). Blood was drawn by puncture of the vena cava and centrifuged. Serum was shock frozen in liquid nitrogen and stored at -70°C for IgE measurements. BAL was performed as described (Kwak et al., supra). Briefly, the chest cavity was exposed to allow for expansion, after which the trachea was carefully intubated and the catheter secured with ligatures. Pre-warmed 0.9% NaCl solution was slowly infused into the lungs and withdrawn. Total BAL cells were counted using a hemocytometer.
  • Differential cell counts were obtained from BAL cells spun down onto slides with a cytocentrifuge (Shannon Scientific Ltd., Cheshire, United Kingdom) and treated with Diff-Quik solution (Dade Diagnostics of Puerto Rico Inc. Aguada, Puerto Rico). Two independent, blinded investigators counted the cells using a microscope. Approximately 400 cells, in each of four different random locations were counted. The mean number from the two investigators was used to estimate the cell differentials. For cytokine and leukotriene measurements, supernatants of BAL were shock frozen in liquid nitrogen and stored at -70°C until use.
  • OVA inhalation significantly increased the absolute numbers of eosinophils, lymphocytes, and neutrophils, as compared with saline control ( Figure 1).
  • Intratracheal administration of IC87114 reduced the number of eosinophils, lymphocytes, and neutrophils detected in BAL fluids at 72 hours post challenge by 79.8%, 63.5%, and 80%, respectively, compared with mice treated with vehicle control.
  • mice were sacrificed and the lungs and trachea were filled intratracheally with a fixative (0.8% formalin, 4% acetic acid) using a ligature around the trachea.
  • Lungs were removed and lung tissues were fixed with 10% (vol/vol) neutral buffered formalin.
  • the specimens were dehydrated and embedded in paraffin.
  • 4 ⁇ m sections of fixed embedded tissues were cut on a Leica model 2165 rotary microtome (Leica Microsystems Nussloch GmbH,, Nussloch, Germany), placed on glass slides, deparaffinized, and stained with hematoxylin 2, eosin-Y (Richard- Allan Scientific, Kalamazoo, MI) and periodic acid-Schiff (PAS).
  • OVA- exposed mice showed numerous inflammatory cells in the peribronchiolar zone and accumulation of mucus and cellular debris within the lumen of the bronchioles.
  • IC87114-treated mice showed substantial attenuation in the eosinophil-rich leukocyte infiltration in the peribronchiolar regions and in the amount of debris present in the lumen.
  • representative sections of each group were stained with periodic acid-Schiff (PAS) for detection of goblet cells.
  • PAS periodic acid-Schiff
  • EXAMPLE 6 EFFECTS OF PllO ⁇ INHIBITOR ON CYTOKINES AND CHEMOKINES IN ALLERGEN INDUCED AIRWAY INFLAMMATION Eosinophil accumulation and subsequent activation in bronchial tissues is known to play a critical role in the pathogenesis of allergic airway inflammation (Busse et al., NEnglJMed 344:35062, 2001; Humbles et al., Science 305:1776-79, 2004).
  • Eosinophil transmigration into the airways is a multistep process that is orchestrated by Th2 cytokines (IL-4, IL-5, and IL-13), and coordinated by specific chemokines such as eotaxin in combination with adhesion molecules such as VCAM- 1 and VLA-4 (10, 11).
  • Th2 cytokines IL-4, IL-5, and IL-13
  • specific chemokines such as eotaxin in combination with adhesion molecules such as VCAM- 1 and VLA-4 (10, 11).
  • IL-13 is a potent inducer of eotaxin expression in airway epithelial cells (Tigani et al., Eur J Pharmacol 433:217-23, 2001) Given the essential role of Th2 cytokines in evoking allergic inflammatory responses, the concentrations of IL-4, IL-5, and IL-13 were measured from BAL fluid as well as in the lung tissue of OVA-challenged mice that received either pi 10 ⁇ inhibitor or vehicle control.
  • IL-l ⁇ , TNF- ⁇ , IL-4, IL-5, IL-13, and RANTES were quantified in the supematants of BAL fluids by enzyme immunoassays according to the manufacturer's protocol (IL-l ⁇ , TNF- ⁇ , IL-4, and IL-5; Endogen, Inc., Woburn, MA; IL-13 and RANTES; R&D Systems, Inc., Minneapolis, MN).
  • the lower detection limit for IL-l ⁇ , TNF ⁇ , IL-4, IL-5, IL-13, and RANTES in these assays was 3, 10, 5, 5, 1.5, and 2 pg/ml, respectively.
  • BAL fluids were also increased significantly (p ⁇ 0.05) at 72 h after OVA inhalation compared with the levels after saline inhalation, from approximately 100 pg/ml TNF ⁇ and 15 pg/ml IL-l ⁇ in control animals up to approximately 280 pg/ml TNF ⁇ and 30 pg/ml IL-1 ⁇ .
  • IC87114 reduced the increased levels of these proinflammatory cytokines by more than 50%, down to approximately 140 pg/ml TNF ⁇ and 15 pg/ml IL-l ⁇ .
  • One of the responses to these cytokines is the induction of leukocyte- endothelial adhesion molecules.
  • ICAM-1 and VCAM-1 proteins in the lung tissue were increased significantly (p ⁇ 0.05) at 72 h after OVA inhalation and these levels were substantially reduced by the administration of IC87114.
  • Western blot analysis revealed that protein levels of the chemokines eotaxin and RANTES in the lung tissue were increased significantly (p ⁇ 0.05) at 72 h after OVA inhalation compared with the saline control.
  • Administration of IC87114 reduced the increased levels of these chemokines by more than 50%.
  • enzyme immunoassays revealed that increased levels of RANTES in BAL fluids at 72 10. h after OVA inhalation were also significantly (p ⁇ 0.05) reduced by IC87114 treatment. Effect ofIC87114 on serum IgE levels' and LTC4 release in BAL fluid IL-4 and IL-13 are important in directing B cell growth, differentiation, and secretion of IgE (Emson et 1., JExp Med 188:399-404, 1998). The biological 15 activities of IgE are mediated through high affinity IgE receptors (Fc ⁇ RI) on mast cells and basophils.
  • Fc ⁇ RI high affinity IgE receptors
  • TMBS 30 teframethylbenzidine substrate
  • TMBS 30 teframethylbenzidine substrate
  • the plates were read at 450 nm on a microplate reader (Molecular Dynamics, Sunnyvale, CA).
  • Total serum IgE was measured by capture ELISA in a manner similar to the detection of OVA-specific IgE.
  • a biotinylated rat anti-mouse IgE (PharMingen) was used to detect captured IgE in place of the biotinylated OVA.
  • Substantial elevation in total IgE and OVA-specific IgE was observed in serum from OVA-challenged mice (approx. 12 ng/ml total IgE, approx.
  • LTC 4 Levels of LTC 4 were quantified in the supematants of BAL fluids by enzyme immunoassay according to the manufacturer's protocol (Cayman Chemical Co., Ann Arbor, MI). The lower detection limit for LTC 4 in this assay was 10 pg/ml.
  • the BAL fluid levels of the LTC 4 were 3.1-fold higher in the OVA- sensitized/challenged mice (approximately 40 pg/ml) than in the mice receiving saline only (approximately 13 pg/ml) (p ⁇ 0.05 compared to saline).
  • IC87114 (0.1 and 1 mg/kg), significantly (p ⁇ 0.05) inhibited LTC 4 levels by 37 and 50%, respectively decreasing LTC 4 levels to approximately 26 pg ml and 21 pg/ml, respectively.
  • the amounts of LTC 4 in the BAL fluid of OVA-sensitized/challenged mice treated with vehicle control were not significantly different from those of the saline control group.
  • Start of an inspiration is determined by extrapolating from a straight line drawn from two levels of the rising inspiratory phase of the box pressure signal.
  • Time of inspiration (TI) is defined as the time from the start of inspiration to the end of inspiration; time of expiration (TE) as the time from the end of inspiration to the start of the next inspiration.
  • the maximum box pressure signal occurring during one breath in a negative or positive direction is defined as peak inspiratory pressure (PIP) or peak expiratory pressure (PEP), respectively. Recordings of every 10 breaths are extrapolated to define the respiratory rate in breaths per minute.
  • the relaxation time (Tr) is defined as the time of pressure decay to 36% of the total expiratory pressure signal (area under the box pressure signal in expiration).
  • Airway responsiveness was substantially increased in the OVA- challenged group in response to methacholine inhalation as compared with the saline- challenged group (Figure 3).
  • Administration of IC87114 to OVA-sensitized mice prior to OVA challenge showed a significant (p ⁇ 0.05) attenuation in Penh measured at all methacholine levels tested suggesting a role for pi lO ⁇ in immune-mediated events leading to airway hyperresponsiveness in vivo.
  • These results are contemplated to be associated with reduction in Th2 cytokine production, tissue eosinophilia, and mast cell activation, following pllO ⁇ inhibition.
  • Allergic airway inflammation and AHR development involve multiple inflammatory cells and a wide array of mediators.
  • EXAMPLE 8 EFFECTS OF PllO ⁇ INHIBITORS ON RAT MAST CELL DEGRANULATION Mast cells and basophils express Fc ⁇ RI, the high affinity receptor for
  • IgE rat basophil leukemia cells
  • RBL-2H3 rat basophil leukemia cells
  • Cells were then plated in a 24 well plate at a concentration of 4 x 10 5 cells/ml, and cultured with 25 ⁇ l of 1 mCi/ml 3 H-labeled serotonin (0.5 ⁇ Ci/ml final) and 1 ⁇ g/ml anti-DNP IgE overnight at 37°C. Cell media was aspirated from the wells and cells washed twice by adding 500 ⁇ l of PBS to the well and inverting the plate onto a stack of paper towels!
  • a final volume of 200 ⁇ l PBS was added and cells equilibrate ⁇ 2 min in a 37°C water bath, and 10 ⁇ l DNP- albumin was added to each well (10 ng/ml final) and incubated for the 10 to 30 min at 37°C.
  • the reaction was stopped by transferring the buffer from each well into a liquid scintillation vial.
  • Wells were washed two times using 500 ⁇ l of 1% Triton X- 100 in PBS incubated 10 min at room temperature, and the liquid transferred to the vial for measuring.
  • pi lO ⁇ inhibitors were effective at reducing the levels of mast cell degranulation mediated by IgE crosslinking.
  • Mast cell degranulation plays a significant role in mediating allergic reactions and other type I hypersensitivity responses in vivo.
  • an animal model of dermal hypersensitivity was used. To sensitize, the shaved dorsal skin of Lewis rats was injected with either saline or anti-DNP monoclonal IgE (1.25 to 25 ng in 50 ⁇ l per site) intradermally.
  • Ketotifen (10 mg/kg) was intraperitoneally injected 30 minutes before antigen challenge. Blood samples were drawn immediately after the wheal size measurements and plasma concentration of IC87114 was determined by determined liquid-liquid extraction by liquid chromatography/mass spectroscopy as described previously (Puri et al., Blood 103:3448-3456, 2004). Type I hypersensitivity responses showed a dose dependent response to IC87114, decreasing to approximately 70% of control at a dose of 20 mg/kg, to approximately 55% at a dose of 60 mg/kg. The positive control ketotifen (10 mg/kg) decreased sensitivity responses to approximately 35% of the control response. These results indicate that pi lO ⁇ plays a role in mediating type I hypersensitivity reactions in sensitized animals, indicating that administration of pi lO ⁇ inhibitors may reduce or prevent type I sensitivity reactions
  • EXAMPLE 10 EFFECTS OF PllO ⁇ INHIBITOR ON HUMAN MAST CELL DEGRANULATION To determine the effect of pi lO ⁇ inhibitors on human mast cells, cells were isolated from human cord blood, differentiated to mast cell lineage and assayed for degranulation and histamine release in the presence of p 11 O ⁇ inhibitors .
  • CD34 + human cord blood cells were isolated and differentiated using stem cell factor and IL-4 following the protocol set out in Hsieh et al. (JExp Med. 193:123-33, 2001), in the presence and absence of methylcellulose (Iida et al, Blood 97:1016-22, 2001). Cells were cultured for approximately 5 weeks and were harvested by centrifugation at 1000 rpm for 3 minutes.
  • D-PBS Dulbecco's- phosphate buffered saline

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Abstract

The present invention provides methods of inhibiting mast cell activity by administering a selective inhibitor of phosphoinositide 3-kinase delta (P13Kδ). The invention also provides methods for treating or preventing a condition associated with undesirable mast cell activity in an individual comprising administering an effective amount of a selective P13Kδ inhibitor.

Description

METHODS FOR TREATING MAST CELL DISORDERS This application claims the priority benefit of U.S. Provisional Patent Application No. 60/576,947, filed June 4, 2004, incorporated herein by reference.
FIELD OF THE INVENTION The present invention relates to methods and compounds for modulating mast cell activity by inhibiting PI3Kδ. Such mast cell activity includes, but is not limited to, mast cell degranulation, mast cell migration, mast cell proliferation, and the expression and secretion of cytokines, chemokines, and growth factors by mast cells. As such, the methods and compounds of the invention may be used to treat or prevent conditions associated with such mast cell activity that is undesirable.
BACKGROUND OF THE INVENTION Phosphoinositide 3-kinase (PI3K) is a signaling enzyme that plays key roles in many cellular activities, including cellular growth, remodeling, and apoptosis [Wymann and Pirola, Biochem Biophys Acta. 1998;1436:127-150; Anderson et al, J Biol Chem. 1999;274:9907-9910; Rameh et al, JBiol Chem. 1999;274:8347-8350; Cantrell, JCell Sci. 2001;114:1439-1445; Coelho and Leevers, J Cell Sci. 2000;113:2927-2934; Vanhaesebroeck et al, Ann. RevBiochem. 2001;70:535-602; Northcott et al, Circ Res.9\ : 60-369 (2002); Yang et al., Am JPhysiol Heart Circ Physiol 280:H2144-H2152 (2001); Ko alavilas, et al, JApplPhysiol. 91:1819-1827 (2001)], PI3K also plays roles in many other cellular processes, such as malignant transformation, growth factor signaling, inflammation, and immunity. See Rameh et al, J. Biol Chem, 274:8347-8350 (1999) for a review. Such diverse activities may be attributed at least in part to PDK's lipid and protein kinase activity. Cloning of the catalytic subunits of PI3 -kinase led to organizing the multi-gene family into three main classes based on their substrate specificity, sequence homology and regulation. Class I PI3-kinases are the most extensively investigated class and contain two subunits, one of which plays primarily a regulatory/adaptor role (p85α, β, p55γ or plOl isoform) and the other that maintains the catalytic role of the enzyme (pi 10a, β, δ, or γ isoform) [Wymann and Pirola, supra; Anderson et al, supra; Rameh et al, supra; Cantrell, supra; Coelho and Leevers, supra; Vanhaesebroeck et al, supra.] Identification of the pi lOδ isoform of PI3-kinase is described in Chantry et al [JBiol Chem, 272:19236-41 (1997)]. It was observed that the human pi lOδ isoform is expressed in a tissue-restricted fashion. It is expressed at high levels in lymphocytes and lymphoid tissues. Details concerning the pi lOδ isoform also can be found in U.S. Patent Nos. 5,858,753; 5,822,910; and 5,985,589, each incorporated herein by reference. See also, Vanhaesebroeck et al, Proc Natl Acad Sci USA, 94:4330-5 (1997), and International Publication No WO 97/46688. PI3Ks achieve intracellular signaling at least in part by catalyzing the addition of a phosphate group to the inositol ring of phosphoinositides [Wymann, et al, supra]. One target of these phosphorylated products is the serine/threonine protein kinase B (P B or Akt). Akt subsequently phosphorylates several downstream targets, including the Bcl-2 family member Bad and caspase-9, thereby inhibiting their pro-apoptotic functions [Datta et a , Cell 91:231-41 (1997); Cardone et /.,
Science 282:1318-21 (1998)]. Akt has also been shown to phosphorylate the forkhead transcription factor FKHR [Tang et al, J Biol. Chem., 274:16741-6 (1999)]. In addition, many other members of the apoptotic machinery as well as transcription factors contain the Akt consensus phosphorylation site [Datta et al, supra], Phosphorylation of Akt has been widely used as an indirect measure of
Class I PI3 -kinase activity in multiple cell types, including endothelial cells, [Shiojima, et al. , Circ. Res., 90: 1243-1250 (2002); Kandel et al. , Exp. Cell Res., 253:210-229 (1999); Cantley et al, Science 296:1655-1657 (2002)]. PI3K activity is required for growth factor mediated survival of various cell types [Fantl et al, Ann. Rev. Biochem. 62:453-81 (1993); Datta et al, Genes & Dev. 13:2905-27 (1999)]. The nonselective phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, have been shown to enhance destruction of tumor vasculature in irradiated endothelial cells [Edwards et al, Cancer Res. 62:4671-77 (2002)] and partially inhibit mast cell degranulation [Tkaczyk et al, JBiol Chem. 278:48474-84 (2003)]. LY294002 and wortmannin do not distinguish among the four members of class I PI3Ks, however. For example, the IC50 values of wortmannin against each of the various class I PI3Ks are in the range of 1-10 nM. Similarly, the IC50 values for LY294002 against each of these PI3Ks is about 1 μM [Fruman, et al,
Ann. Rev. Biochem. 67:481-507 (1998)]. These inhibitors are not only nonselective with respect to class I PBKs, but are also potent inhibitors of DNA dependent protein kinase, FRAP-mTOR, smooth muscle myosin light chain kinase, and casein kinase 2 [Hartley, et al, Cell 82:849-56 (1995); Davies, et al, Biochem. J. 351 :95-105 (2000); Brunn, et al, EMBO J. 15:5256-67 (1996)]. Because pi 10a, pi lOβ, pi lOγ, and pi lOδ isoforms are expressed differentially by a wide variety of cell types, the administration of nonselective PI3K inhibitors such as LY294002 and wortmannin often affect cell types that may not be targeted for treatment. Therefore, the effective therapeutic dose of such nonselective inhibitors would be expected to clinically unusable because otherwise non-targetedcell types will likely be affected, especially when such nonselective inhibitors are combined with other immunomodulatory therapies. Mast cells play diverse and significant roles. For example, mast cells are involved in mediatmg first line immune responses of the innate immune system seen in response to allergens or parasitic or bacterial infections. Mast cells also contribute to activation and recruitment of other inflammatory cells, such as neutrophils and T cells, to bring about second line immune responses required for an adaptive immune response. CD34+ mast cells circulate in the blood as committed precursor cells and fully mature in specific tissue sites. Mast cell development and maturation requires mast-cell growth factor, also known as stem cell factor (SCF), steel, or KIT ligand [Gurish et al., J. Exp. Med. 194:F1-F5 (2001)]. The interaction of KIT receptor with its ligand drives mast cell proliferation and differentiation (Feger et al, Int. Arch. Allergy Immunol. 127:110-14 (2002)]. Mast cells are activated through crosslinking of the high affinity FcεRI
IgE receptor on the cell surface by antigen-bound IgE, and to a lesser extent through crosslinking of the FcγRI receptor by IgG [Tkaczyk et al., Lit Arch Allergy Immunol. 133:305-15 (2004)]. Activation through FcεRI is typically seen in acute allergic reactions and other types of hypersensitivity reactions, leading to the stimulation of additional immune cells and a full blown immune response. Mast cells contain metachromatic granules which store a variety of inflammatory mediators that are released upon mast cell activation. These mediators include: histamine and serotonin; prostaglandin D2; proteolytic enzymes, such as tryptase that can destroy tissue or cleave complement components or coagulation components; heparin or chondroitin sulfate, which are anticoagulants; chemotactic factors, such as eosinophil chemotactic factor of anaphylaxis (an important regulator of eosinophil function) and neutrophil chemotactic factor. During mast cell activation, these mediators are released into the cellular environment causing acute and immediate immune responses such as vascular permeability and recruitment of lymphocytes. For example, tryptase levels rise within 1 hour and remain elevated for 4-6 hours, while histamine levels peak at approximately 5 minutes and decline rapidly within fifteen minutes. Histamine release causes dilation of blood vessels leading to fluid leak into the surrounding tissues, causing many initial symptoms of allergic reactions. Release of histamine and other mediators also leads to airway constriction, edema, vascular congestion and inflammatory cell recruitment characteristic of allergic reactions and asthma [Djukanovic et al., Clin Exp Allergy. 26 Suppl 3:44-51 (1996)]. Mast cell activity is necessary and desirable in healthy individuals. Unwanted mast cell activity, or excessive proliferation of otherwise normal or abnormal mast cells, may be a component of a wide variety of disease states and/or their symptoms, however. In such instances, it is often desirable, from a therapeutic or preventative standpoint, to reduce or eliminate mast cell activity and/or proliferation. For example, numerous immune mediated diseases involve the release by mast cells of cytokines, chemokines and other factors. Cytokines, chemokines, and other factors recruit additional immune cells such as lymphocytes, including neutrophils and T cells, to sites of inflammation. This may lead to numerous immune mediated diseases. For example, mast cell activity such as degranulation and tryptase protein have recently been localized to cerebrospinal fluid of patients with multiple sclerosis, an autoimmune disease typically thought to be mediated by T cell activity [Rozniecki et al., Ann Neurol 37:63-66 (1995)]. Additionally, mast cell deficient mice (W/Wv) induced to develop an experimental model of multiple sclerosis demonstrate delayed development of MS-like symptoms [Secor et al., J. Exp. Med. 191:813-22 (2000)]. Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation of the joints and the presence of inflammatory cells in the synovial fluid of the joints, leading to a painful and debilitating disease. Mice lacking mast cells show resistance to induction of arthritis-like symptoms after infusion of antibodies to a cytoplasmic enzyme [Lee et al., Science 297:1689-1693 (2002)]. Mast cells accumulate in the extremities of mice affected by collagen-induced arthritis and degranulate during the disease [Woolley et al., Arthritis Res. 2:65-74 (2000)], indicating that mast cells may play a role in mediatmg inflammation and recruiting additional cells to the joints of patients suffering from rheumatoid arthritis. A significant population of mast cells resides in the skin. Bullous pemphigoid, an autoimmune disease of the skin exhibiting autoantibodies to cell junction proteins has also been shown to depend on mast cell activation. [Chen et al., JClin Invest. 108:1151-58 (2001)]. W/Wv mice deficient in mast cells did not develop bullous pemphigoid, although autoantibodies and complement proteins were present in the skin similar to normal mice, W/Wv mice lacked neutrophil recruitment to the skin. Also, evidence of mast cell mediators have been detected in patients with bullous pemphigoid [Wintroub et al., New Eng. J. Med. 298:417-21 (2001)]. Other immune disorders in which mast cells are thought to play a role include Sjogren's syndrome [Konttinen et al., Rheumatol Int. 19:141-7 (2000)], chronic urticaria [Napoli et al., Curr Allergy Asthma Rep. 1:329-36 (2001)], thyroid eye disease [Ludgate et al., Clin Exp Immunol. 2002 127:193-8. (2002)], vasculitis [Kiely et al., J Immunol. 159:5100-6 (1997)] and peritonitis [Malavyn et al., Nature 381:77-80 (1996)]. Another broad category of mast cell associated condition or disorder is termed mast cell disease, or mastocytosis. Mastocytosis encompasses a heterogeneous group of clinical disorders characterized by the proliferation and accumulation of mast cells in a variety of tissues, most often the skin, but also in the skeletal, hematopoietic, gastrointestinal, cardiopulmonary, and central nervous systems. Mastocytosis is characterized by excess proliferation of mast cells, distributed in a predictable pattern throughout the skin (e.g., cutaneous mastocytosis and urticaria pigmentosa), bone marrow, gastrointestinal tract, lymph nodes, liver and spleen [Brockow et al, CWT. Opin. Allergy Clin. Immunol 1:449-54 (2001)]. Mastocytosis is classified as either familial or sporadic, the latter being further subdivided into either cutaneous or systemic. Systemic mastocytosis is still further classified into indolent (chronic) mastocytosis and aggressive mastocytosis, as well as mast cell leukemia. Types of mastocytosis also emerge which have an associated hematologic disorder (AHD) (Brockow et al, supra). Cutaneous mastocytosis (CM) demonstrates typical clinical and histological skin lesions and absence of definitive signs (criteria) of systemic involvement. Most patients with CM are children and present with maculopapular cutaneous mastocytosis (for example, urticaria pigmentosa, UP). Other less frequent forms of CM are diffuse cutaneous mastocytosis (DCM) and mastocytoma of skin. Systemic mastocytosis (SM) is commonly seen in adults and defined by multifocal histological lesions in the bone marrow (almost ubiquitously expressed, a major criteria of diagnosis) or other extracutaneous organs together with cytological and biochemical signs (minor criteria) of systemic disease (SM-criteria). Aggressive systemic mastocytosis is characterized by impaired organ-function due to infiltration of the bone marrow, liver, spleen, Gl-tract, or skeletal system, by pathologic mast cells (MC). Mast cell leukemia is a 'high-grade' leukemic disease defined by increased numbers of MC in bone marrow smears (greater than or equal to 20%) and peripheral blood, absence of skin lesions, multiorgan failure, and a short survival. In typical cases, circulating MC amount to greater than or equal to 10% of leukocytes (classical form of MCL). Mast cell sarcoma is a unifocal tumor that consists of atypical MC and shows a destructive growth without (primary) systemic involvement. This high-grade malignant MC disease has to be distinguished from a localized benign mastocytoma in either extracutaneous organs (extracutaneous mastocytoma) or skin. Additionally, mutations in the KIT receptor leading to mast cell hyper- proliferation have been found in patients with acute myeloid leukemia (AML)
[Beghini et al., Cancer Genet Cytogenet. 119:26-31 (2000)], chronic myelogenous leukemia [Cairoli et al, HematolJ. 5:273-5 (2004)], chronic myelomonocytic leukemia [Sotlar et al., LeukRes. 26:979-84 (2002)], germ cell tumors [Sakuma et al., Cancer Sci. 94:486-91 (2003)], and gastrointestinal stromal tumors (GISTs) [Heinrich et al, J. Clin. Oncol. 20:1692-1703. (2002)]. While some specific treatments for conditions associated with undesirable mast cell activity, for example allergy and asthma, have been developed, treatment regimens for mast cell related conditions typically employ non-specific treatment regimens developed for other proliferative or immune disorders (e.g., histamine receptor blockers, prostaglandin blockers, steroids), resulting in incomplete treatment, treatments which are not effective, or treatments that cause numerous immunosuppressive side-effects. Further, a significant drawback to many therapies that may be used to treat conditions associated with undesirable mast cell activity is the non-specific inhibition of many cellular tyrosine kinases in mast cells and other cell types that are targeted by the treatment. For instance, two kinase inhibitor therapeutics for treating mast cell proliferative disorders were originally developed to inhibit kinases such as platelet-derived growth factor receptor (PDGF-R), vascular endothelial growth factor receptor (VEGFR), or the Bcr/Abl mutation. These potential therapeutics proved ineffective at treating all forms of mastocytosis. Thus, there remains a need in the art to develop effective therapeutics that more specifically target kinases involved in mediating mast cell activity. Therefore, an important and significant goal is to develop and make available safer and more effective methods of treating and preventing disorders associated with allergy and mast cell related disorders, and to provide therapies which facilitate clinical management and continued patient compliance. The present invention addresses this and other needs.
SUMMARY OF THE INVENTION The present invention provides methods for effectively treating or preventing a condition, and/or a symptom of a condition, associated with or caused, at least in part, by undesirable mast cell activity . The methods of the invention are particularly useful in treating or preventing conditions (or symptoms associated with conditions) mediated by immunoglobulin receptor cross-linking on mast cells. In one embodiment, the invention provides a method for inhibiting an activity of mast cells, comprising administering to an individual a selective inhibitor of phosphoinositide 3 -kinase delta (PI3Kδ) in an amount effective to inhibit mast cell activity. In one aspect, the mast cell activity being inhibited is mast cell migration, mast cell proliferation, mast cell degranulation, or expression of or secretion of cytokines, chemokines, or growth factors from mast cells. In a further aspect, the cytokine is TNF-α. In a related aspect, the cytokine is LL-6. In a related aspect, the chemokine being inhibited is eotaxin, MlPl-α, MlPl-β, MDC-1, MCP-1, or lymphotactin. In a related embodiment, the invention provides methods of reducing or preventing lymphocyte infiltration to a site of inflammation in a condition associated with undesirable mast cell activity comprising the step of administering to an individual a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in an ■ amount effective to reduce or prevent lymphocyte infiltration to said site of inflammation in an amount effective to reduce lymphocyte recruitment signaling by mast cells in said individual. A condition associated with undesirable mast cell activity is any condition caused by or involving the underlying effects of any undesirable mast cell activity. In another embodiment, the invention provides methods for treating or preventing a condition associated with undesirable mast cell activity in an individual, comprising the step of administering a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in an amount effective to treat or prevent a condition associated with undesirable mast cell activity. In one aspect, in order to treat or prevent a condition associated with undesirable mast cell activity, the selective PI3Kδ inhibitor inhibits mast cell activity. Examples of such mast cell activity include mast cell migration, mast cell proliferation, mast cell degranulation, or expression or secretion of cytokines, chemokines, or growth factors from mast cells. The methods of the invention encompass treating or preventing conditions (or symptoms associated with conditions) mediated by immunoglobulin receptor cross-linking on mast cells "Mediated by immunoglobulin crosslinking" or "Ig-mediated" refers to the ability of Ig bound to receptors on mast cells to initiate, or facilitate, a condition associated with undesirable mast cell activity. Immunoglobulins which activate mast cells include IgG and IgE. In one embodiment, the condition associated with undesirable mast cell activity is an IgE-mediated condition. In an alternative embodiment, the condition is an IgG-mediated condition. In a further embodiment, the condition is mediated by other stimuli such cytokines, chemokines or other growth factors. Conditions, and symptoms of conditions amenable to treatment or prevention by methods according to the invention include, but are not limited to, asthma, allergic reactions, or autoimmune diseases. In one aspect, the allergic reaction is type I hypersensitivity, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, or allergic asthma. Type I hypersensitivity reactions are reactions in which antigens (allergens) combine with specific IgE antibodies that are bound to membrane receptors on tissue mast cells and blood basophils. The antigen-antibody reaction causes the rapid release of potent vasoactive and inflammatory mediators, (e.g., histamine, tryptase, leukotrienes and prostaglandins) and later release of proinflammatory cytokines (e.g., interleukin-4 and interleukin-13). The mediators produce vasodilation, increased capillary permeability, glandular hypersecretion, smooth muscle spasm, and tissue infiltration of other inflammatory cells. Exemplary type I hypersensitivity disorders include allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergic asthma, some cases of urticaria and GI food reactions, and systemic anaphylaxis. In another aspect, the condition is an autoimmune disease. The autoimmune disease contemplated by the invention may be multiple sclerosis, rheumatoid arthritis, bullous pemphigoid, Sjogren's syndrome, chronic urticaria, thyroid eye disease, vasculitis, and peritonitis. The invention provides for a method of the invention wherein the
PI3Kδ selective inhibitor is administered in an amount effective to inhibit Akt phosphorylation in said mast cells. The terms "selective PI3Kδ inhibitor," and variants thereof such as "PI3Kδ selective inhibitor" and "selective inhibitor of PI3Kδ" as used herein refer to a compound that inhibits the PI3Kδ isozyme more effectively than other isozymes of the PI3K family. A "selective PI3Kδ inhibitor" compound is understood to be more selective for PI3Kδ than compounds conventionally and generically designated PI3K inhibitors, e.g., wortmannin or LY294002. Concomitantly, wortmannin and LY294002 are deemed "nonselective PI3K inhibitors." The relative efficacies of compounds as inhibitors of an enzyme activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined extent and then comparing the results. Typically, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or "IC50." IC50 determinations can be accomplished using . conventional techniques known in the art. In general, an IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study. The experimentally obtained values of enzyme activity then are plotted against the inhibitor concentrations used. The concentration of the inhibitor that shows 50% enzyme activity (as compared to the activity in the absence of any inhibitor) is taken as the IC50 va e- Analogously, other inhibitory concentrations can be defined through appropriate determinations of activity. For example, in some settings it can be desirable to establish a 90% inhibitory concentration, i.e., IC90, etc.
Accordingly, a "selective PI3Kδ inhibitor" alternatively can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC50) with respect to PI3Kδ that is at least 10-fold, in another aspect at least 20-fold, and in another aspect at least 30-fold, lower than the IC50 value with respect to any or all of the other Class I PI3K family members. In an alternative embodiment of the invention, the term selective PI3Kδ inhibitor can be understood to refer to a compound that exhibits an IC50 with respect to PI3Kδ that is at least 50-fold, in another aspect at least 100-fold, in an additional aspect at least 200-fold, and in yet another aspect at least 500-fold, lower than the IC50 with respect to any or all of the other PI3K Class I family members. In yet a further embodiment, the term selective PI3Kδ inhibitor refers to an oligonucleotide that negatively regulates pi lOδ expression at least 10-fold, in another aspect at least 20-fold, and in a further aspect at least 30-fold, lower than any or all of the other Class I PI3K family catalytic subunits (i.e., pi 10a, pi lOβ, and pi lOγ). A PI3Kδ selective inhibitor is administered to an individual in an amount such that the inhibitor retains its PI3Kδ selectivity, as described above. Ranges may be expressed herein as from "about" or "approximately" one particular value and/or to "about" or "approximately" another particular value.
When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedents such as "about" or "at least about," it will be understood that the particular value forms another embodiment. Any selective inhibitor of PI3Kδ activity, including but not limited to small molecule inhibitors, peptide inhibitors, non-peptide inhibitors, naturally occurring inhibitors, and synthetic inhibitors, may be used. Suitable PI3Kδ selective inhibitors have been described in U.S. Patent Publication 2002/161014 to Sadhu et al, the entire disclosure of which is hereby incorporated herein by reference. In another aspect, compounds of any type that selectively negatively regulate p 11 Oδ expression (i. e. , more effectively than other isozymes of the PI3K family) and that possess acceptable pharmacological properties can be used as selective PI3Kδ inhibitors in the methods of the invention. Accordingly, in one embodiment the invention provides for the use of antisense oligonucleotides which negatively regulate pi lOδ expression via hybridization to messenger RNA (mR A) encoding pi 10δ. In one aspect, oligonucleotides that decrease pi lOδ expression may be used in the methods of the invention. The methods of the invention may be applied to cell populations in vivo or ex vivo. "In vivo" means within a living individual, as within an animal or human. In this context, the methods of the invention may be used therapeutically in an individual, as described infra. The methods may also be used prophylactically. , "Ex vz'vo" means outside of a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples including but not limited to fluid or tissue samples obtained from individuals. Such samples may be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, and saliva. Exemplary tissue samples include tumors samples and biopsies of tissue. In this context, the invention may be used for a variety of purposes, including therapeutic and experimental purposes. For example, the invention may be used ex vivo to determine the optimal schedule and/or dosing of administration of a PI3Kδ selective inhibitor for a given indication, cell type, individual, and other parameters. Information gleaned from such use may be used for experimental purposes or in the clinic to set protocols for in vivo treatment. Other ex vivo uses for which the invention may be suited are described below or will become apparent to those skilled in the art. "Mast cell activity" as used herein refers to those biological activities carried out by mast cells which may be modulated by the compounds useful in the methods of the invention. Examples of these activities include cell migration, proliferation, activation, degranulation, expression of or secretion of chemokines, cytokines or other growth factors, and modulation of cell signaling pathways, for example, modulation of AKT phosphorylation. Alternatively, "modulation of mast cell activity" may be used herein. "Modulation" of mast cell activity as used herein refers to the reduction, inhibition, prevention, promotion or increase of one of the above listed activities of mast cells upon administration of a selective PI3Kδ inhibitor. A selective PI3Kδ inhibitor may inhibit the enzyme itself, may inhibit any downstream signaling effect of the PI3Kδ enzyme, or inhibit any further downstream activity of a mast cell. It may alternatively act prophylactically to prevent a particular activity of a mast cell as described above, such as degranulation or cellular migration. An inhibitor may promote or increase one mast cell activity in the course of reducing, inhibiting or preventing another mast cell activity. For example, production of a first cytokine, chemokine or growth factor may be increased upon reduction or inhibition of another second cytokine, chemokine or growth factor. "Undesirable mast cell activity" means mast cell activity that deviates from the normal, proper, or expected course. For example, undesirable mast cell degranulation may include degranulation during allergic reaction causing hypersensitivity of the individual, while undesirable migration may include movement of mast cells into or out of tissue sites having unfavorable biological effects. Undesirable mast cell proliferation may include cell proliferation mediated by, or resulting in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. . "Inhibiting undesirable mast cell activity" means to slow or stop the rate at which undesirable mast cell activity takes place. This may result either from a decreased rate of mast cell receptor activation, decreased inflammatory mediator or growth factor release, decreased cellular replication, or an increased rate of cell death. Cell death may occur by any mechanism, including apoptosis and mitotic catastrophe. Use of the methods in accordance with the present invention may result in partial or complete inhibition of undesirable mast cell activity,. "Preventing undesirable mast cell activity" means that the methods of the present invention may be used prophylactically to prevent or inhibit undesirable mast cell activity before it occurs, or to prevent or inhibit the recurrence thereof. Thus, in all embodiments, the invention may be used in vivo or ex vivo where no undesirable cell activity has been identified or where no undesirable cell activity is ongoing, but where undesirable cell activity is suspected or expected, respectively. Moreover, the invention may also be used in all its embodiments wherever undesirable cell activity has been previously treated to prevent or inhibit recurrence of the same. As used herein, a "therapeutically effective amount" or "amount effective" means an amount effective to inhibit or reverse development of, to alleviate the existing symptoms of, to prolong survival of, or to cure the individual being treated. As used herein, the "therapeutic index" is the dose ratio between toxic or undesired effect and therapeutic, or desired, effects, and is expressed as the ratio of LD50 to ED50, which are defined below. An increase in the therapeutic index, as used herein, refers to a reduction in the amount of therapeutic necessary to reach a desired effect or to increase the effectiveness of the therapeutic administered. It will be appreciated that the treatment methods of the invention are useful in the fields of human medicine and veterinary medicine. Thus, the individual to be treated may be a mammal, preferably human, or another animal. For veterinary purposes, individuals include but are not limited to farm animals including cows, sheep, pigs, horses, and goats; companion animals such as dogs and cats; exotic and/or zoo animals; laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducks, and geese. The invention further provides methods wherein the selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) s is administered in a plurality of doses. A plurality of doses includes administration of the inhibitor or other agent in more than one dose. The invention further provides that the selective PI3Kδ inhibitor is administered in a regimen which includes administering one or more additional therapeutic compounds commonly utilized in treatment of a condition associated with undesirable mast cell activity, including at least one immunomodulatory agent or other agent as appropriate to the condition or symptom being treated or prevented. In one embodiment, the invention provides a method of reducing or preventing mast cell activity in an individual having a condition associated with undesirable mast cell activity, comprising administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) and an immunomodulatory agent. It is contemplated that the combination therapy may be administered in a single composition or each agent, such as the inhibitor and immunomodulatory agent, may be administered as a separate composition. It is further contemplated that each agent may be administered in a plurality of doses as necessary. In a related embodiment, the invention provides a method of reducing or preventing lymphocyte infiltration to a site of inflammation in an individual having a condition associated with undesirable mast cell activity, comprising administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) and a immunomodulatory agent. Immunomodulatory agents contemplated by the invention include glucocorticoids or corticosteroids, immunosuppressants, antihistamines, aminosalicylates, steroid hormones, non-steroidal anti-inflammatory drugs (NSAIDs), . sympathomimetics, and analgesics. Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethosone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and budesonide. Exemplary NSALDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as Vioxx ® (rofecoxib) and Celebrex® (celecoxib), and salicylate. Suitable analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hygrochloride. Exemplary immunosuppressants include azathioprine (6-mercaptopurine (6-MP)), cyclophosphamide, cyclosporine, methotrexate, or penicillamine. Also contemplated are Xolair® (omalizumab), leukotriene antagonists, or other drugs commonly used for allergy or asthma. Methods of the invention may include administering formulations comprising an inhibitor of the invention with a particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. More specifically and without limitation, methods of the invention may comprise administering an inhibitor with one or more of TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Pharmaceutical compositions in accordance with the invention may also include other known angiopoietins, for example Ang- 1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, and/or vascular endothelial growth factor (VEGF). Growth factors for use in pharmaceutical compositions of the invention include angiogenin, bone morpho genie protein- 1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein- 14, bone morphogenic protein- 15; bone morphogenic protein receptor IA, bone morphogenic protein receptor LB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor α, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, β endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial- derived neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acidic, fibroblast growth factor basic, glial cell line-derived neutrophic factor receptor αl, glial cell line-derived neutrophic factor receptor α2, growth related protein, growth related protein α, growth related protein β, growth related protein γ, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor α, platelet derived growth factor receptor β, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor α, transforming growth factor β, transforming growth factor βl, transforming growth factor βl.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor βl , transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof. ' Immunomodulatory agents used for treatment can be administered in a plurality of doses. It is contemplated that the agents are administered in the combination methods according to the invention at a low dose, that is, at a dose lower than conventionally used in clinical situations where the agent or therapy is administered alone, because the PI3Kδ selective nature of the inliibitors of the invention increases the therapeutic index (i.e., the specificity) of the inventive combination therapies. Lowering the dose of the agent or therapy administered to an individual decreases the incidence of adverse effects associated with higher dosages, and can thereby improve the quality of life of a patient undergoing treatment. Further benefits include improved patient compliance, and a reduction in the number of hospitalizations needed for the treatment of adverse effects. Additionally, the specificity of the methods of the invention are advantageous in that they permit treatment at higher doses of the PI3Kδ selective inhibitor(s) than nonselective inhibitors such as LY294002 and wortmannin, further maximizing the therapeutic efficacy of the inventive methods In another aspect, methods may include administering an inhibitor with one or more other agents which either enhance the activity of the inhibitor or compliment its activity or use in treatment. Such additional factors and/or agents may produce a synergistic effect with an inhibitor of the invention, or to minimize side effects. Methods of the invention contemplate use of a selective PI3Kδ inhibitor compound having formula (I) or pharmaceutically acceptable salts and solvates thereof:
Figure imgf000018_0001
( I ) wherein A is an optionally substituted monocyclic or bicyclic ring system containing at least two nitrogen atoms, and at least one ring of the system is aromatic;
X is selected from the group consisting of C(RD)2, CFTjCHRb, and
CH=C(Rb); Y is selected from the group consisting of null, S, SO, SO2, NH, O, C(=O), OC(=O), C(=O)O, and NHC(=O)CH2S;
R! and R^, independently, are selected from the group consisting of hydrogen, C1.βalkyl, aryl, heteroaryl, halo, NHC(:=O)Cι _3alkyleneN(Ra)2, NO2, ORa, CF3, OCF3, N(Ra)2, CN, OC(=O)R C(=O)Ra, C(=O)ORa, arylORb, Het, NRaC(=O)Cι_3alkyleneC(=O)ORa, arylOCι„ alkyleneN(Ra)2, arylOC(=O)Ra, C
Figure imgf000018_0002
4alkyleneC(=O)ORa C(=O)NR SO2Ra, C1.4alkyleneN(R )2, C2- 6alkenyleneN(Ra)2, C(=O)NRaCι _4alkyleneORa, C(=O)NRaCι_4alkyleneHet, OC2. 4alkyleneN(Ra)2, OC1 _4alkyleneCH(ORD)CH2N(Ra)2, OCι _4alkyleneHet, OC2. 4alkyleneORa, OC2-4alkyleneNRaC(=O)OR , NRaC1. alkyleneN(Ra)2, NRaC(=O)Ra NRaC(=O)N(Ra)2, N(SO2Cι.4alkyl)2, NR (SO2Cι_4alkyl), SO2N(Ra)2, OSO2CF3, Cι _ alkylenearyl, C^alkyleneHet, Cι.6alkyleneORb, Cι _ 3alkyleneN(Ra)2,
Figure imgf000019_0001
C3.8cycloalkyl, C3_ gheterocycloalkyl, arylOCι_3alkyleneN(Ra)2, arylOC(=O)Rb, NHC(0)Cι_ 3alkyleneC3_gheterocycloalkyl, NHC(=O)C \ .3 alkyleneHet, OC \ _4alkyleneOC \ _
4alkyleneC(=O)OR , C(=O)Cι. alkyleneHet, and NHC(=O)haloC1_6alkyl; or R! and R^ are taken together to form a 3- or 4-membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;
R3 is selected from the group consisting of optionally substituted hydrogen, Cι_6atkyl, C3_8cycloalkyl, C3_8heterocycloalkyl, Cι_4alkylenecycloalkyl, C2-6 lkenyl, C^alkylenearyl, arylC^alkyl, C(=O)Ra, aryl, heteroaryl, C(=O)ORa, C(=O)N(R )2, C(=S)N(Ra)2, SO2Ra SO2N(Ra)2, S(=O)R , S(=O)N(Ra)2, C(=O)NRaCι.4alkyleneORa, C(=O)NRaC1_4alkyleneHet, C(=O)Cι_4alkylenearyl, C(=O)Cι _4alkyleneheteroaryl, Ci _4alkylenearyl optionally substituted with one or more of halo, SO2N(R )2, N(Ra)2, C(=O)ORa, NRaSO2CF3, CN, NO2, C(=O)Ra, ORa, Cι_4alkyleneN(Ra)2, and OCι_ alkyleneN(Ra)2, C^alkyleneheteroaryl, Cι_ 4alkyleneHet, C \ _4alkyleneC(=O)C \ _4alkylenearyl, C \ _4alkyleneC(=O)C 1 _
4alkyleneheteroaryl, Cι _4alkyieneC(=O)Het, Cι_4alkyleneC(=O)N(Ra)2, C _
4alkyleneORa, Cι _4alkyleneNRaC(=O)Ra, Cι.4alkyleneOCι.4alkyleneORa, C\_
4alkyleneN(Ra)2, Cμ alkyleneC(=O)ORa, and C^alkyleneOC alkyleneC(=O)ORa;
Ra is selected from the group consisting of hydrogen, Ci .galkyl, C3.. gcycloalkyl, C3_gheterocycloalkyl, Cι _3alkyleneN(Rc)2, aryl, arylCi^alkyl, Cι_ 3alkylenearyl, heteroaryl, heteroarylCi _3alkyl, and Ci _3alkyleneheteroaryl; or two Ra groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom; Rb is selected from the group consisting of hydrogen, Ci _6alkyl, heteroCi _3alkyl, Ci^alkyleneheteroCi^alkyl, arylheteroCi _3alkyl, aryl, heteroaryl, arylCι _3alkyl, heteroarylCι _3alkyl, Cι _3alkylenearyl, and C^alkyleneheteroaryl;
Rc is selected from the group consisting of hydrogen, C^.^alkyl, C3_ gcycloalkyl, aryl, and heteroaryl; and,
Het is a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Cι_4alkyl or C(=O)ORa. As used herein, the term "alkyl" is defined as straight chained and branched hydrocarbon groups containing the indicated number of carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups. The hydrocarbon group can contain up to 16 carbon atoms, for example, one to eight carbon atoms. The term "alkyl" includes "bridged alkyl," i.e., a C6-C16 bicyclic or polycyclic hydrocarbon group, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl. The term "cycloalkyl" is defined as a cyclic C3-C8 hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl. The term "alkenyl" is defined identically as "alkyl," except for containing a carbon-carbon double bond. "Cycloalkenyl" is defined similarly to cycloalkyl, except a carbon-carbon double bond is present in the ring. The term "alkylene" is defined as an alkyl group having a substituent. For example, the term "C1-3alkylenearyl" refers to an alkyl group containing one to three carbon atoms, and substituted with an aryl group. The term "heteroC1-3alkyl" is defined as a C1-3alkyl group further containing a heteroatom selected from O, S, and NRa. For example, -CH OCH3 or -CH2CH2SCH3. The term "arylheteroC salkyl" refers to an aryl group having a heteroCι-3alkyl substituent. The term "halo" or "halogen" is defined herein to include fluorine, bromine, chlorine, and iodine. The term "aryl," alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwise indicated, an "aryl" group can be unsubstituted or substituted, for example, with one or more, and in particular one to three, halo, alkyl, phenyl, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, and amino. Exemplary aryl groups include phenyl, naphthyl, biphenyl, tetrahydronaphthyl, chlorophenyl, fluorophenyl, aminophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, carboxyphenyl, and the like. The terms "arylC1-3alkyl" and "heteroarylC1-3alkyl" are defined as an aryl or heteroaryl group having a C1-3alkyl substituent. The term "heteroaryl" is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, like halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, and amino. Examples of heteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl. The term "Het" is defined as monocyclic, bicyclic, and tricyclic groups containing one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. A "Het" group also can contain an oxo group (=O) attached to the ring. Nonlimiting examples of Het groups include 1,3-dioxolane, 2-pyrazoline, pyrazolidine, pyrrolidine, piperazine, a pyrroline, 2H-pyran, 4H-pyran, morpholine, thiopholine, piperidine, 1,4-dithiane, and 1,4-dioxane. Alternatively, methods of the invention contemplate use of a PI3Kδ selective inhibitor compound having formula (II) or pharmaceutically acceptable salts and solvates thereof:
Figure imgf000022_0001
wherein R4, R5, R6, and R7, independently, are selected from the group consisting of hydrogen, Ci .galkyl, aryl, heteroaryl, halo, NHC(=O)Cι _ 3alkyleneN(Ra)2, NO2, ORa, CF3, OCF3, N(Ra)2, CN, OC(=O)Ra, C(=O)Ra,
C(=O)ORa, arylOR , Het,
Figure imgf000022_0002
arylOCi.
3alkyleneN(Ra)2, arylOC(=O)Ra, C1.4alkyleneC(=O)OR OCι_
4alkyleneC(=O)OR , Cι_4alkyleneOCι.4alkyleneC(=O)ORa, C(=O)NRaSO2Ra,
Cι_4alkyleneN(Ra)2, C2-6alkenyleneN(Ra)2, C(=O)NRaC1.4alkyleneORa, C(=O)NRaC1.4alkyleneHet, OC2.4 lkyleneN(Ra)23 OC
4alkyleneCH(ORb)CH2N(Ra)2, OCι_4alkyleneHet, OC2-4alkyleneORa, OC2- 4alkyleneNRaC(=O)ORa, NRaC1_4alkyleneN(Ra)2, NRaC(=O)R , NR C(=O)N(Ra)2, N(SO2Ci.4alkyl)2, NRa(SO2Cι.4alkyl), SO2N(Ra)2, OSO2CF3, Cι_3alkylenearyl, Cμ4alkyleneHet, Cι_6alkyleneORb, Cμ 3alkyleneN(Ra)2, C(=O)N(Ra)2, NHC(=O)Cι_3alkylenearyl, C3.8cycloalkyl, C3_ gheterocycloalkyl, arylOC1.3alkyleneN(Ra)2, arylOC(=O)R , NHC(=O)Cι _ 3alkyleneC3_gheterocycloalkyl, NHC(=O)C .3 alkyleneHet, OC \ _4alkyleneOC \ _
4alkyleneC(=O)ORb, C(=O)Cι,4alkyleneHet, and NHC(==O)haloCι _6alkyl; R8 is selected from the group consisting of hydrogen, Ci _6alkyl, halo,
CN, C(=O)Ra, and C(=O)ORa; X1 is selected from the group consisting of CH (i.e., a carbon atom having a hydrogen atom attached thereto) and nitrogen; Ra is selected from the group consisting of hydrogen, Ci .galkyl, C3_ gcycloalkyl, C3_gheterocycloalkyl, Cι_3alkyleneN(Rc)2, aryl, arylCj_3alkyl, C _ 3alkylenearyl, heteroaryl, heteroarylC 1.3 alkyl, and Ci _3alkyleneheteroaryl; or two Ra groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom; Rc is selected from the group consisting of hydrogen, Ci .galkyl, C3., gcycloalkyl, aryl, and heteroaryl; and,
Het is a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with C\ .4 alkyl or C(=O)ORa. In yet another embodiment, methods of the invention include use of a selective inhibitor of PI3Kδ compound having formula (III) or pharmaceutically acceptable salts and solvates thereof:
Figure imgf000024_0001
(in) wherein R9, R10, R11, and R12, independently, are selected from the group consisting of hydrogen, Ci.galkyl, aryl, heteroaryl, halo, NHC(=O)Cι _
3alkyleneN(Ra)2, NO2, ORa, CF3, OCF3, N(Ra)2, CN, OC(=O)Ra, C(=O)Ra,
C(=O)ORa, arylOR , Het,
Figure imgf000024_0002
aiylOCi.
3alkyleneN(Ra)2, arylOC(=O)Ra, Cι_4alkyleneC(=O)0R , OCι_
4alkyleneC(=O)ORa, Ci.4alkyleneOCi_4alkyleneC(=O)ORa, C(=O)NRaSO2Ra,
Cι.4alkyleneN(Ra)2, C2_6alkenyleneN(Ra)2, C(=O)NRaCι _4alkyleneORa,
C(=O)NRaCι_4alkyleneHet, OC2.4alkyleneN(R )2, OCι_
4alkyleneCH(ORb)CH2N(R )2, OCι_4alkyleneHet, OC2-4alkyleneORa, OC2-
4alkyleneNRaC(=O)ORa, NRaC1.4alkyleneN(Ra)2, NRaC(=O)Ra,
NRaC(=O)N(Ra)2, N(SO2Cι.4alkyl)2, NRa(SO2Cι.4alkyl), SO2N(Ra)2,
OSO2CF3, Cι_3alkylenearyl, C1_ alkyleneHet, Cι_6alkyleneOR , Cμ
3alkyleneN(Ra)2, C(=O)N(Ra)2, NHC(=O)Cι _3alkylenearyl, C3_gcycloalkyl, C3_ gheterocycloalkyl, arylOCι_3alkyleneN(Ra)2, arylOC(=O)R , NHC(=O)Cι_ 3alkyleneC3_gheterocycloalkyl, NHC(=O)Cι _3alkyleneHet, OCι_4alkyleneOCι_
Figure imgf000024_0003
R13 is selected from the group consisting of hydrogen, Ci _6alkyl, halo, CN, C(=O)Ra, and C(=O)ORa;
Ra is selected from the group consisting of hydrogen, Ci _6alkyl, C3.. gcycloalkyl, C3_gheterocycloalkyl, Cι _3alkyleneN(Rc)2, aryl, arylC 1.3 alkyl, C 3alkylenearyl, heteroaryl, heteroarylCi _3alkyl, and Ci _3alkyleneheteroaryl; or two Ra groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
Rc is selected from the group consisting of hydrogen, C\ .galkyl, C3_ gcycloalkyl, aryl, and heteroaryl; and, Het is a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Cj _4alkyl or C(=O)ORa. More specifically, methods of the invention embrace use of a PI3Kδ selective inhibitor selected from the group consisting of 2-(6-aminopurin-9-ylmethyl)- 3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-6-bromo-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-6-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-5-chloro-3-(2-chloro-phenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-8-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-biphenyl-2-yl-5-chloro-3H-quinazolin-4-one; 5-chloro-2-(9H-purin-6- ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one; 5-chloro-3-(2-fluorophenyl)-2- (9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)- 5-chloro-3-(2-fluorophenyl)-3H-quinazolin-4-one; 3-biphenyl-2-yl-5-chloro-2-(9H- purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 5-chloro-3-(2-methoxyphenyl)-2- (9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 3-(2-chlorophenyl)-5-fluoro-2- (9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 3-(2-chlorophenyl)-6,7- dimethoxy-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 6-bromo-3-(2- chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 3-(2- chlorophenyl)-8-trifluoromethyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-benzo[g]quinazolin-4- one; 6-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 8-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-7-fluoro-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-7-nitro-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-6-hydroxy-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin- 4-one; 5-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin- 4-one; 3-(2-chlorophenyl)-5-methyl-2-(9H-purin-6-yl-sulfanylmethyl)-3H- quinazolin-4-one; 3-(2-chlorophenyl)-6,7-difluoro-2-(9H-purin-6-yl-sulfanylmethyl)- 3H-quinazolin-4-one; 3-(2-chlorophenyl)-6-fluoro-2-(9H-purin-6-yl-sulfanylmethyl)- 3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-(2-isopropylρhenyl)-5-methyl- 3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H- quinazolin-4-one; 3-(2-fluorophenyl)~5-methyl-2-(9H-purin-6-yl-sulfanylmethyl)- 3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-5-chloro-3-o-tolyl-3H- quinazolin-4-one; 2-(6-aminopurin-9~ylmethyl)-5-chloro-3-(2-methoxy-phenyl)-3H- quinazolin-4-one; 2-(2-amino-9H-purin-6-ylsulfanylmethyl)-3-cyclopropyl-5-methyl- 3H-quinazolin-4-one; 3-cyclopropylmethyl-5-methyl-2-(9H-purin-6- ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3- cyclopropylmethyl-5-methyl-3H-quinazolin-4-one; 2-(2-amino-9H-purin-6- ylsulfanylmethyl)-3-cyclopropylmethyl-5-methyl-3H-quinazolin-4-one; 5-methyl-3- phenethyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(2-amino-9H- purin-6-ylsulfanylmethyl)-5-methyl-3-phenethyl-3H-quinazolin-4-one; 3- cyclopentyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(6- aminopurin-9-ylmethyl)-3-cyclopentyl-5-methyl-3H-quinazolin-4-one; 3-(2- chloropyridin-3-yl)-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-(2-chloropyridin-3-yl)-5-methyl-3H-quinazolin-4- one; 3-methyl-4-[5-methyl-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3- yl]-benzoic acid; 3-cyclopropyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-cyclopropyl-5-methyl-3H- quinazolin-4-one; 5-methyl-3-(4-nitrobenzyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 3-cyclohexyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-cyclohexyl-5-methyl-3H- quinazolin-4-one; 2-(2-amino-9H-purin-6-ylsulfanylmethyl)-3-cyclo-hexyl-5-methyl- 3H-quinazolin-4-one; 5-methyl-3-(E-2-phenylcyclopropyl)-2-(9H-purin-6- ylsulfanylmethyl)-3H-quinazolin-4-one; 3-(2-chlorophenyl)-5-fluoro-2-[(9H-purin-6- ylamino)methyl]-3H-quinazolin-4-one; 2-[(2-amino-9H-purin-6-ylamino)methyl]-3- (2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one; 5-methyl-2-[(9H-purin-6- ylamino)methyl]-3-o-tolyl-3H-quinazolin-4-one; 2-[(2-amino-9H-purin-6- ylamino)methyl]-5-methyl-3-o-tolyl-5H-quinazolin-4-one; 2-[(2-fluoro-9H-purin-6- ylamino)methyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one; (2-chlorophenyl)- dimethylamino-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 5-(2- benzyloxyethoxy)-3 -(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 6-aminopurine-9-carboxylic acid 3-(2-chlorophenyl)-5-fluoro-4- oxo-3,4-dihydro-quinazolin-2-ylmethyl ester; N-[3-(2-chlorophenyl)-5-fluoro-4-oxo- 3 ,4-dihydro-quinazolin-2-ylmethyl]-2-(9H-purin-6-ylsulfanyl)-acetamide; 2-[ 1 -(2- fluoro-9H-purin-6-ylamino)ethyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5- methyl-2-[l-(9H-purin-6-ylamino)ethyl]-3-σ-tolyl-3H-quinazolin-4-one; 2-(6- dimethylaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5-methyl- 2-(2-methyl-6-oxo-l,6-dihydro-purin-7-ylmethyl)-3- -tolyl-3H-quinazolin-4-one; 5- methyl-2-(2-methyl-6-oxo-l,6-dihydro-purin-9-ylmethyl)-3-o-tolyl-3H-quinazolin-4- one; 2-(amino-dimethylaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4- one; 2-(2-amino-9H-purin-6-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4- one; 2-(4-amino-l ,3,5-triazin-2-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin- 4-one; 5-methyl-2-(7-methyl-7H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin- 4-one; 5-methyl-2-(2-oxo-l,2-dihydro-pyrimidin-4-ylsulfanylmethyl)-3-o-tolyl-3H- quinazolin-4-one; 5-methyl-2-purin-7-ylmethyl-3-o-tolyl-3H-quinazolin-4-one; 5- methyl-2-purin-9-ylmethyl-3-o-tolyl-3H-quinazolin-4-one; 5-methyl-2-(9-methyl- 9H-purin-6-ylsulfanylmethyl)-3-σ-tolyl-3H-quinazolin-4-one; 2-(2,6-Diamino- pyrimidin-4-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5-methyl-2- (5-methyl-[l,2,4]triazolo[l,5-α]pyrimidin-7-ylsulfanylmethyl)-3-o-tolyl-3H- quinazolin-4-one; 5-methyl-2-(2-methylsulfanyl-9H-purin-6-ylsulfanylmethyl)-3-o- tolyl-3H-quinazolin-4-one; 2-(2-hydroxy-9H-purin-6-ylsulfanylmethyl)-5-methyl-3- o-tolyl-3H-quinazolin-4-one; 5-methyl-2-(l-methyl-lH-imidazol-2- ylsulfanylmethyl)-3 -o-tolyl-3H-quinazolin-4-one; 5-methyl-3 -o-tolyl-2-( 1 H- [l,2,4]triazol-3-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(2-amino-6-chloro-purin- 9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(6-aminopurin-7-ylmethyl)- 5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(7-amino-l,2,3-triazolo[4,5-^]pyrimidin- 3 -yl-methyl)-5 -methyl-3 -ø-tolyl-3H-quinazolin-4-one; 2-(7-amino- 1,2,3 -triazolo [4,5- ]pyrimidin- 1 -yl-methyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(6-amino-9H- purin-2-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(2-amino-6- ethylamino-pyrimidin-4-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(3-amino-5-methylsulfanyl-l,2,4-triazol-l-yl-methyl)-5-methyl-3-σ-tolyl-3H- quinazolin-4-one; 2-(5-amino-3-methylsulfanyl- 1 ,2,4-triazol- 1 -ylmethyl)-5-methyl- 3-o-tolyl-3H-quinazolin-4-one; 5-methyl-2-(6-methylaminopurin-9-ylmethyl)-3-o- tolyl-3H-quinazolin-4-one; 2-(6-benzylaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl- 3H-quinazolin-4-one; 2-(2,6-diaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H- quinazolin-4-one; 5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3-σ-tolyl-3H- quinazolin-4-one; 3-isobutyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; N-{2-[5-Methyl-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H- quinazolin-3-yl]-phenyl} -acetamide; 5-methyl-3-(E-2-methyl-cyclohexyl)-2-(9H- purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-[5-methyl-4-oxo-2-(9H-purin-6- ylsulfanylmethyl)-4H-quinazolin-3-yl]-benzoic acid; 3-{2-[(2- dimethylaminoethyl)methylamino]phenyl}-5-methyl-2-(9H-purin-6- ylsulfanylmethyl)-3H-quin-azolin-4-one; 3-(2-chlorophenyl)-5-methoxy-2-(9H- purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 3 -(2-chlorophenyl)-5 -(2-morpholin- 4-yl-ethylamino)-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 3-benzyl- 5-methoxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin- 9-ylmethyl)-3-(2-benzyloxyphenyl)-5-methyl-3H-quinazolin-4-one; 2-(6- aminopurin-9-ylmethyl)-3-(2-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one; 2-(l- (2-amino-9H-purin-6-ylamino)ethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5- methyl-2-[l-(9H-purin-6-ylamino)propyl]-3-o-tolyl-3H-quinazolin-4-one; 2-(l-(2- fluoro-9H-purin-6-ylamino)propyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(l-(2- amino-9H-purin-6-ylamino)propyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(2- benzyloxy-l-(9H-purin-6-ylamino)ethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2- (6-aminopurin-9-ylmethyl)-5-methyl-3- {2-(2-(l -methylpyrrolidin-2-yl)-ethoxy)- phenyl}-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-(2-(3-dimethylamino- propoxy)-phenyl)-5-methyl-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-5- methyl-3 -(2-prop-2- ynylox yphenyl)-3H-quinazolin-4-one; 2- {2-( 1 -(6-aminopurin-9- ylmethyl)-5-methyl-4-oxo-4H-quinazolin-3-yl]-phenoxy}-acetamide; 2-[(6- aminopurin-9-yl)methyl]-5-methyl-3-o-tolyl-3-hydroquinazolin-4-one; 3-(3,5- difluorophenyl)-5-methyl-2-[(purin-6-ylamino)methyl]-3-hydroquinazolin-4-one; 3- (2,6-dichlorophenyl)-5-methyl-2-[(purin-6-ylamino)methyl]-3-hydroquinazolin-4- one; 3-(2-Fluoro-phenyl)-2-[ 1 -(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3- hydroquinazolin-4-one ; 2-[l-(6-aminopurin-9-yl)ethyl]-3-(3,5-difluorophenyl)-5- methyl-3-hydroquinazolin-4-one; 2-[l-(7-Amino-[l,2,3]triazolo[4,5-d]pyrimidin-3- yl)-ethyl]-3-(3,5-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one; 5-chloro-3-(3,5- difluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 3-phenyl- 2-[ 1 -(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 5-fluoro-3-phenyl-2-[ 1 - (9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 3-(2,6-difluoro-phenyl)-5- methyl-2- [ 1 -(9H-purin-6-ylamino)-propyl] -3H-quinazolin-4-one; 6-fluoro-3 -phenyl- 2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 3-(3,5-difluoro-phenyl)-5- methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5-fluoro-3-phenyl-2- [ 1 -(9H-purin-6-ylamino)-ethyl] -3H-quinazolin-4-one; 3 -(2,3 -difluoro-phenyl)-5 - methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5-methyl-3-phenyl-2- [ 1 -(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 3-(3-chloro-phenyl)-5-methyl- 2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[(9H- purin-6-ylamino)-methyl]-3H-quinazolin-4-one; 2-[(2-amino-9H-purin-6-ylamino)- methyl]-3-(3,5-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one; 3- {2-[(2- diethylamino-ethyl)-methyl-amino]-phenyl}-5-methyl-2-[(9H-purin-6-ylamino)- methyl]-3H-quinazolin-4-one; 5-chloro-3-(2-fluoro-phenyl)-2-[(9H-purin-6-ylamino)- methyl]-3H-quinazolin-4-one; 5-chloro-2-[(9H-purin-6-ylamino)-methyl]-3-o-tolyl- 3H-quinazolin-4-one; 5-chloro-3-(2-chloro-phenyl)-2-[(9H-purin-6-ylamino)- methyl]-3H-quinazolin-4-one; 6-fluoro-3-(3-fluoro-phenyl)-2-[l-(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5- chloro-3-(3-fluoro-phenyl)-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[l-(9H-purin- 6-ylamino)-propyl] -3H-quinazolin-4-one; 2- [ 1 -(2-fluoro-9H-purin-6-ylamino)-ethyl] - 5-methyl-3-phenyl-3H-quinazolin-4-one; 3-(2,6-difluoro-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- ethyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H-quinazolin-4-one; 3-(2,6-difluoro- phenyl)-2-[l-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3H-quinazolin-4-one; 3- (2,6-difluoro-phenyl)-5-methyl-2-[ 1 -(7H-pyrrolo [2,3- ]pyrimidin-4-ylamino)-ethyl]- 3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6-ylamino)-propyl]-5-methyl-3- phenyl-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[l-(ZH-pyrrolo[2,3-c jpyrimidin-4- ylamino)-propyl]-3H-quinazolin-4-one; 2-[l-(2-fluoro-9/z-purin-6-ylamino)-propyl]- 5 -methyl-3 -phenyl-3 /z-quinazolin-4-one; 5 -methyl-3 -phenyl-2-[ 1 -(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5- methyl-3-phenyl-3H-quinazolin-4-one; 2-[2-benzyloxy-l-(9H-purin-6-ylamino)- ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- 2-benzyloxy-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[2-benzyloxy-l-(7H- pyrrolo[2,3- ]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[2-benzyloxy-l-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H- quinazolin-4-one; 3-(4-fluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]- 3H-quinazolin-4-one; 2- [ 1 -(2-amino-9H-purin-6~ylammo)-ethyl]-3 -(4-fluoro-phenyl)- 5-methyl-3H-quinazolin-4-one; 3-(4-fluoro-phenyl)-2-[ 1 -(2-fluoro-9H-purin-6- ylamino)-ethyl]-5-methyl-3H-quinazolin-4-one;3-(4-fluoro-phenyl)-5-methyl-2-[l- ( 7H-pyrrolo [2,3 - ]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one; 5 -methyl-3 - phenyl-2-[l-(7H-pyrrolo[2,3- ]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one; 3- (3-fluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-ρurin-6-ylamino)-ethyl]-3-(3-fluoro-phenyl)-5-methyl-3H- quinazolin-4-one; 3-(3-fluoro-phenyl)-5-methyl-2-[l-(7H-pyrrolo[2,3-c ]pyrimidin-4- ylamino)-ethyl]-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[l-(9H-purin-6-yl)- pyrrolidin-2-yl]-3H-quinazolin-4-one; 2-[2-hydroxy-l-(9H-purin-6-ylamino)-ethyl]- 5-methyl-3-phenyl-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[phenyl-(9H-purin-6- ylamino)-methyl]-3H-quinazolin-4-one; 2-[(2-amino-9H-purin-6-ylamino)-phenyl- methyl] -5 -methyl-3 -phenyl-3H-quinazolin-4-one; 2-[(2-fluoro-9H-purin-6-ylamino)- phenyl-methyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 5-methyl-3-phenyl-2- [phenyl-(7H-pyrrolo[2,3- ]pyrimidin-4-ylamino)-methyl]-3H-quinazolin-4-one; 5- fluoro-3-phenyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-ethyl]-5-fluoro-3-phenyl-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-phenyl-3H-quinazolin-4-one; [5-(5- methyl-4-oxό-3-phenyl-3,4-dihydro-quinazolin-2-yl)-5-(9H-purin-6-ylamino)- pentyl]-carbamic acid benzyl ester; [5-(2-amino-9H-purin-6-ylamino)-5-(5-methyl-4- oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-pentyl]-carbamic acid benzyl ester; [4-(5- methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-4-(9H-purin-6-ylamino)-butyl]- carbamic acid benzyl ester; [4-(2-amino-9H-purin-6-ylamino)-4-(5-methyl-4-oxo-3- phenyl-3,4-dihydro-quinazolin-2-yl)-butyl]-carbamic acid benzyl ester; 3-phenyl-2- [l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[5-amino-l-(9H-purin-6- ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one); 2-[5-amino-l-(2-amino- 9H-purin-6-ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-Dimethyl-phenyl)-5-methyl-3H-quinazolin- 4-one; 3-(256-dimethyl-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 5-morpholin-4-ylmethyl-3-phenyl-2-[ 1 -(9H-purin-6-ylamino)- ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)~ethyl]-5-morpholin- 4ylmethyl-3-phenyl-3H-quinazolin-4-one; 2-[4-amino- 1 -(2-amino-9H-purin-6- ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 6-fluoro-3-phenyl-2-[l- (9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-97J-purin-6- ylamino)-ethyl]-6-fluoro-3-phenyl-3H-quinazolin-4-one; 2-[2-tert-butoxy-l-(9H- purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 3-(3-methyl- phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-ethyl]-3-(3-methyl-phenyl)-5-methyl-3H-quinazolin-4- one; 3-(3-chloro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin- 4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-chloro-phenyl)-5-methyl-3H- quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6-ylamino)-2-hydroxy-ethyl]-5-methyl-3- phenyl-3H~quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-fluoro- phenyl)-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6- difluoro-phenyl)-3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6-ylamino)-propyl]- 5 -fluoro-3 -phenyl-3H-quinazolin-4-one; 5 -chloro-3 -(3 -fluoro-phenyl)-2-[ 1 -(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- ethyl]-5-chloro-3-(3-fluoro-phenyl)-3H-quinazolin-4-one; 3-phenyl-2-[l-(9H-purin-6- ylamino)-ethyl]-5-trifluoromethyl-3H-quinazolin-4-one; 3-(2,6-difluoro-phenyl)-5- methyl-2-[l-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 3-(2,6-difluoro- phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-propyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H- quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)- 5-methyl-3H-quinazolin-4-one; 3-(3,5-dichloro-phenyl)-5-methyl-2-[l-(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 3-(2,6-dichloro-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l -(2-amino-9H-purin-6-ylamino)- ethyl]-3-(2,6-dichloro-phenyl)-5-methyl-3H-quinazolin-4-one; 5 -chloro-3 -phenyl-2- [l-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6- ylamino)-propyl]-5-chloro-3-phenyl-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[l- (9H-purin-6-ylamino)-butyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6- ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-ρurin-6- ylamino)-ethyl]-3-(3,5-dichloro-phenyl)-5-methyl-3H-quinazolin-4-one; 5-methyl-3- (3-morpholin-4-ylmethyl-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4- one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-(3-morpholin-4-ylmethyl- phenyl)-3H-quinazolin-4-one; 2-[l-(5-bromo-7H-pyrrolo[2,3-c Ipyrimidin-4- ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 5-methyl-2-[l-(5-methyl- 7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4-one; 2-[l- (5-fluoro-7H-pyrrolo[2,3-J]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H- quinazolin-4-one; 2-[2-hydroxy-l-(9H-purin-6-ylamino)-ethyl]-3-phenyl-3H- quinazolin-4-one; 3-(3,5-difluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)- propyl]-3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6-ylamino)-propyl]-3-(3,5- difluoro-phenyl)-5 -methyl-3H-quinazolin-4-one; 3 -(3 ,5 -difluoro-phenyl)-2- [ 1 -(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(5-bromo-7H-pyrrolo[2,3- J]pyrimidin-4-ylamino)-ethyl] -3 -(3 -fluoro-phenyl)-5 -methyl-3H-quinazolin-4-one; 3 - (3-fluoro-phenyl)-5-methyl-2-[l-(5-methyl-7H-pyrrolo[2,3-cT]pyrimidin-4-ylamino)- ethyl]-3H-quinazolin-4-one; 3-phenyl-2-[l-(9H-purin-6-ylamino)-propyl]-3H- quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3,5-difluoro-phenyl)- 3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-propyl]-3-phenyl-3H- quinazolin-4-one; 6,7-difluoro-3-phenyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 6-fluoro-3-(3-fluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 2-[4-diethylamino- 1 -(9Η-purin-6-ylamino)-butyl]-5-methyl-3- phenyl-3H-quinazolin-4-one; 5-fluoro-3-phenyl-2-[l-(9H-purin-6-ylamino)-propyl]- 3H-quinazolin-4-one; 3 -phenyl-2- [ 1 -(9H-purin-6-ylamino)-ethyl] -3H-quinazolin-4- one; 6-fluoro-3-phenyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 3- (3,5-difluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4- one; 5-fluoro-2-[l-(2-fluoro-9H-purin-6-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4- one; 3-(3-fluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5- chloro-3-(3,5-difluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4- one; 3-(2,6-difluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 3-(2,6-difluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 5-Methyl-3-phenyl-2-[3,3,3-trifluoro-l-(9H-purin-6-ylamino)- propyl]-3H-quinazolin-4-one; 3-(3-hydroxy-phenyl)-5-methyl-2-[ 1 -(9Η-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 3-(3-methoxy-phenyl)-5-methyl-2-{l-[9H- purin-6-ylamino]-ethyl}-3H-quinazolin-4-one; 3-[3-(2-dimethylamino-ethoxy)- phenyl]-5-methyl-2- { 1 -[9H-purin-6-ylamino]-ethyl} -3H-quinazolin-4-one; 3-(3- cyclopropylmethoxy-phenyl)-5-methyl-2-{l-[9H-purin-6-ylamino]-ethyl}-3H- quinazolin-4-one; 5-methyl-3-(3-prop-2-ynyloxy-phenyl)- 2- {l-[9H-purin-6- ylamino]-ethyl} -3H-quinazolin-4-one; 2- {l-[2-amino-9H-purin-6-ylamino]ethyl}-3- (3-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one; 2- { 1 -[2-amino-9H-purin-6- ylamino]ethyl} -3-(3-methoxyphenyl)-5-methyl-3H-quinazolin-4-one; 2- { 1 -[2-amino- 9H-purin-6-ylamino]ethyl}-3-(3-cyclopropylmethoxy-phenyl)-5-methyl-3H- quinazolin-4-one; 2-{l-[2-amino-9H-purin-6-ylamino]ethyl}-5-methyl-3-(3-prop-2- ynyloxy-phenyl)-3H-quinazolin-4-one; 3-(3-ethynyl-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 3-{5-methyl-4-oxo-2-[l-(9H-purin-6- ylamino)-ethyl]-4H-quinazolin-3-yl} -benzonitrile; 3- {5-methyl-4-oxo-2- { 1 -[9H- purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl} -benzamide; 3-(3-acetyl-phenyl)-5- methyl-2-{l-[9H-purin-6-ylamino]-ethyl}-3H-quinazolin-4-one; 2-(3-(5-methyl-4- oxo-2-{l-[9H-purin-6-ylamino]-ethyl}-4H-quiriazolin-3-yl-phenoxy acetamide; 5- methyl-2-{l-[9H-purin-6-ylamino]-ethyl}-3-[3-(tetrahydropuran-4-yloxy)-phenyl]- 3H-quinazolin-4-one; 3-[3-(2-methoxy-ethoxy)-phenyl]-5-methyl-2-[l-(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 6-fluoro-2-[l-(9H-purin-6-ylamino)ethyl]-3-[3- (tetrahydro-pyran-4-yloxy)-phenyl]-3H-quinazolin-4-one; 3-[3-(3-dimethylamino- propoxy)-phenyl]-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-ethynyl-phenyl)-5-methyl-3H- quinazolin-4-one; 3-{2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-4-oxo-4H- quinazolin-3-yl} -benzonitrile; 3-{2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5- methyl-4-oxo-4H-quinazolin-3-yl}-benzamide; 3-{2-[l-(2-amino-9H-purin-6- ylamino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl}-benzamide; 5-methyl-3-(3- morpholin-4-yl-phenyl)-2- [ 1 -(9H-purin-6-ylamino)-ethyl] -3H-quinazolin-4-one; 2- [ 1 - (2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-(3-morpholin-4-yl-phenyl)-3H- quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-[3-(2-methoxy- ethoxy)-phenyl]-5-methyl-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- ethyl]-3-[3-(2-dimethylamino-ethoxy)-phenyl]-5-methyl-3H-quinazolin-4-one; 2-[l- (2-amino-9H-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4- one; 5-chloro-3-(3,5-difluoro-phenyl)-2-[ 1 -(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-(3,5- difluoro-phenyl)-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5- chloro-3 -(3, 5 -difluoro-phenyl)-3H-quinazolin-4-one; 3-(3,5-difluoro-phenyl)-6- fluoro-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5-chloro-3-(2,6- difluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-propyl}-5-chloro-3-(2,6-difluoro-phenyl)-3H-quinazolin- 4-one; 5-methyl-3-phenyl-2-[l-(9H-purin-6-yloxy)-ethyl]-3H-quinazolin-4-one; and mixtures thereof.. Where a stereocenter is present, the methods can be practiced using a racemic mixture of the compounds or a specific enantiomer. In preferred embodiments, the S-enantiomer of the above compounds is utilized. Therefore, the present invention includes all possible stereoisomers and geometric isomers of the aforementioned compounds. "Pharmaceutically acceptable salts" means any salts that are physiologically acceptable insofar as they are compatible with other ingredients of the formulation and not deleterious to the recipient thereof. Some specific preferred examples are: acetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, oxalate. In one embodiment, the invention contemplates an article of manufacture comprising a phosphoinositide 3-kinase delta (PI3Kδ) selective inhibitor and a label indicating a method of use according to any one of the methods of the invention. In a related embodiment, the invention provides for use of a composition comprising at least one PI3Kδ selective inhibitor in the manufacture of a medicament for treating or preventing a condition associated with undesirable mast cell activity. Administration of prodrugs are also contemplated. The term "prodrug" as used herein refers to compounds that are rapidly transformed in vivo by hydrolysis to, for example, a compound having a structural formula described herein. Prodrug design is discussed generally in Hardma et al. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion is provided in Higuchi et al., Prodrugs as Novel Delivery Systems, Vol. 14, ASCD Symposium Series, and in Roche (ed.), Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987). Briefly, administration of a drug is followed by elimination from the body or some biotransformation whereby biological activity of the drug is reduced or eliminated. Alternatively, a biotransformation process can lead to a metabolic by-product, which is itself more active or equally active as compared to the drug initially administered. Increased understanding of these biotransformation processes permits the design of so-called "prodrugs," which, following a biotransformation, become more physiologically active in their altered state. Prodrugs, therefore, encompass pharmacologically inactive compounds that are converted to biologically active metabolites. To illustrate, prodrugs can be converted into a pharmacologically active form through hydrolysis of, for example, an ester or amide linkage, thereby introducing or exposing a functional group on the resultant product. The prodrugs can be designed to react with an endogenous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life. Alternatively, prodrugs can be designed to undergo covalent modification on a functional group with, for example, glucuronic acid, sulfate, glutathione, amino acids, or acetate. The resulting conjugate can be inactivated and excreted in the urine, or rendered more potent than the parent compound. High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and released back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound. Additionally, compounds that selectively negatively regulate pllOδ mRNA expression more effectively than they do other isozymes of the PI3K family, and that possess acceptable pharmacological properties are contemplated for use as PI3Kδ selective inhibitors in the methods of the invention. Polynucleotides encoding human pi lOδ are disclosed, for example, in Genbank Accession Nos. AR255866, NM 005026, U86453, U57843 and Y10055, the entire disclosures of which are incorporated herein by reference. See also, Vanhaesebroeck et al, Proc. Natl. Acad. Sci. 94:4330-4335 (1997), the entire disclosure of which is incorporated herein by reference. Representative polynucleotides encoding mouse pi lOδ are disclosed, for example, in Genbank Accession Nos. BC035203, AK040867, U86587, and NM_008840, and a polynucleotide encoding rat pi lOδ is disclosed in Genbank Accession No. XM_345606, in each case the entire disclosures of which are incorporated herein by reference. In one embodiment, the invention provides methods using antisense oligonucleotides which negatively regulate pi lOδ expression via hybridization to messenger RNA (mRNA) encoding pi lOδ. In one aspect, antisense oligonucleotides at least 5 to about 50 nucleotides in length, including all lengths (measured in number of nucleotides) in between, which specifically hybridize to mRNA encoding pi lOδ and inhibit mRNA expression, and as a result pi 1 Oδ protein expression, are contemplated for use in the methods of the invention. Antisense oligonucleotides include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e.', make the oligonucleotide more resistant to nuclease degradation, particularly in vivo. It is understood in the art that, while antisense oligonucleotides that are perfectly complementary to a region in the target polynucleotide possess the highest degree of specific inhibition, antisense oligonucleotides that are not perfectly complementary, i.e., those which include a limited number of mismatches with respect to a region in the target polynucleotide, also retain high degrees of hybridization specificity and therefore also can inhibit expression of the target mRNA. Accordingly, the invention contemplates methods using antisense oligonucleotides that are perfectly complementary to a target region in a polynucleotide encoding pi lOδ, as well as methods that utilize antisense oligonucleotides that are not perfectly complementary (i.e., include mismatches) to a target region in the target polynucleotide to the extent that the mismatches do not preclude specific hybridization to the target region in the target polynucleotide. Methods for designing and optimizing antisense nucleotides are described in Lima et al, (JBiol Chem ;272:626-38. 1997), Kurreck et al, (Nucleic Acids Res. ;30:1911-8. 2002) and U.S. Patent No. 6,277,981 , which are incorporated herein by reference.
Exemplary antisense compounds are described in International Patent Publication WO 01/05958, which is incorporated herein by reference. The invention further contemplates methods utilizing ribozyme inhibitors which, as is known in the art, include a nucleotide region which specifically hybridizes to a target polynucleotide and an enzymatic moiety that digests the target polynucleotide. Specificity of ribozyme inhibition is related to the length the antisense region and the degree of complementarity of the antisense region to the target region in the target polynucleotide. The methods of the invention therefore contemplate ribozyme inhibitors comprising antisense regions from 5 to about 50 nucleotides in length, including all nucleotide lengths in between, that are perfectly complementary, as well as antisense regions that include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target p 11 Oδ-encoding polynucleotide. Ribozymes useful in methods of the invention include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo, to the extent that the modifications do not alter the ability of the ribozyme to specifically hybridize to the target region or diminish enzymatic activity of the molecule. Because ribozymes are enzymatic, a single molecule is able to direct digestion of multiple target molecules thereby offering the advantage of being effective at lower concentrations than non-enzymatic antisense oligonucleotides. Preparation and use of ribozyme technology is described in U.S. Patent Nos. 6,696,250, 6,410,224, 5,225,347, the entire disclosures of which are incorporated herein by reference. The invention also contemplates use of methods in which RNAi technology is utilized for inhibiting pi 1 Oδ expression. In one aspect, the invention provides double-stranded RNA (dsRNA) wherein one strand is complementary to a target region in a target pi 1 Oδ-encoding polynucleotide. In general, dsRNA molecules of this type are less than 30 nucleotides in length and referred to in the art as short interfering RNA (siRNA). The invention also contemplates, however, use of dsRNA molecules longer than 30 nucleotides in length, and in certain aspects of the invention, these longer dsRNA molecules can be about 30 nucleotides in length up to 200 nucleotides in length and longer, and including all length dsRNA molecules in between. As with other RNA inhibitors, complementarity of one strand in the dsRNA molecule can be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target pi 1 Oδ-encoding polynucleotide. As with other RNA inhibition technologies, dsRNA molecules include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo. Preparation and use of RNAi using double stranded (dsRNA) (Fire et al, Nature 391: 806-811. 1998) or short-interfering RNA (siRNA) sequences (Yu et al, Proc Natl Acad Sci USA. 99:6047-52. 2002) compounds are further described in U.S. Patent Application No. 20040023390, the entire disclosure of which is incorporated herein by reference. The invention further contemplates methods wherein inhibition of pi 1 Oδ is effected using RNA lasso technology. Circular RNA lasso inhibitors are highly structured molecules that are inherently more resistant to degradation and therefore do not, in general, include or require modified internucleotide linkage or modified nucleotides. The circular lasso structure includes a region that is capable of hybridizing to a target region in a target polynucleotide, the hybridizing region in the lasso being of a length typical for other RNA inhibiting technologies. As with other RNA inhibiting technologies, the hybridizing region in the lasso may be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target pi lOδ-encoding polynucleotide. Because RNA lassos are circular and form tight topological linkage with the target region, inhibitors of this type are generally not displaced by helicase action unlike typical antisense oligonucleotides, and therefore can be utilized as dosages lower than typical antisense oligonucleotides. Preparation and use of RNA lassos is described in U.S. Patent 6,369,038, the entire disclosure of which is incorporated herein by reference. The inhibitors of the invention may be covalently or noncovalently associated with a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Patents 4,289,872 and 5,229,490; PCT Publication WO 93/21259 published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide. Specific examples of carriers for use in the pharmaceutical compositions of the invention include carbohydrate-based polymers, such as trehalose, mannitol, xylitol, sucrose, lactose, sorbitol, dextrans, such as cyclodextran, cellulose, and cellulose derivatives. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Other carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Patent Nos: 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. Still other useful carrier polymers known in the art include monomethoxy-polyethylene glycol, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers. Derivatization with bifunctional agents is useful for cross-linking a compound of the invention to a support matrix or to a carrier. One such carrier is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be straight chain or branched. The average molecular weight of the PEG can range from about 2 kDa to about 100 kDa, in another aspect from about 5 kDa to about 50 kDa, and in a further aspect from about 5 kDa to about 10 kDa. The PEG groups will generally be attached to the compounds of the invention via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, haloacetyl, maleimido or hydrazino group) to a reactive group on the target inhibitor compound (e.g., an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido or hydrazino group). Cross-linking agents can include, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-l,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 may be employed for inhibitor immobilization. The pharmaceutical compositions of the invention may also include compounds derivatized to include one or more antibody Fc regions. Fc regions of antibodies comprise monomeric polypeptides that may be in dimeric or multimeric forms linked by disulfide bonds or by non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of Fc molecules can be from one to four depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, IgGA2) of antibody from which the Fc region is derived. The term "Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms of Fc molecules, with the Fc region being a wild type structure or a derivatized structure. The pharmaceutical compositions of the invention may also include the salvage receptor binding domain of an Fc molecule as described in WO 96/32478, as well as other Fc molecules described in WO 97/34631. Such derivatized moieties preferably improve one or more characteristics of the inhibitor compounds of the invention, including for example, biological activity, solubility, absorption, biological half life, and the like. Alternatively, derivatized moieties result in compounds that have the same, or essentially the same, characteristics and/or properties of the compound that is not derivatized. The moieties may alternatively eliminate or attenuate any undesirable side effect of the compounds and the like. Compounds that compete with an inhibitor compound described herein for binding to PI3Kδ are also contemplated for use in the invention. Methods of identifying compounds which competitively bind with PI3Kδ, with respect to the compounds specifically provided herein, are well known in the art. In view of the disclosures above, therefore, the term "inhibitor" as used herein embraces compounds disclosed, compounds that compete with disclosed compounds for PI3Kδ binding, and in each case, conjugates and derivatives thereof. Methods include administration of an inhibitor to an individual in need, by itself, or in combination as described herein, and in each case optionally including one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants/flavoring, carriers, excipients, buffers, stabilizers, solubilizers, other materials well known in the art and combinations thereof. Any pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents known in the art that serve as pharmaceutical vehicles, excipients, or media may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma, methyl- and propylhydroxybenzoate, talc, alginates, carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, dextrose, sorbitol, modified dextrans, gum acacia, and starch. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present inhibitor compounds. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference. Pharmaceutically acceptable fillers can include, for example, lactose, microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, calcium sulfate, dextrose, mannitol, and/or sucrose. Inorganic salts including calcium triphosphate, magnesium carbonate, and sodium chloride may also be used as fillers in the pharmaceutical compositions. Amino acids may be used, such as use in a buffer formulation of the pharmaceutical compositions. Disintegrants may be included in solid dosage formulations of the inhibitors. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge, corn starch, potato starch, and bentonite may all be used as disintegrants in the pharmaceutical compositions. Other disintegrants include insoluble cationic exchange resins. Powdered gums including powdered gums such as agar, Karaya or tragacanth may be used as disintegrants and as binders. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include crystalline cellulose, cellulose derivatives such as methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC), acacia, corn starch, and/or gelatins Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic. An antifriction agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, talc, and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. Suitable glidants include starch, talc, pyrogenic silica and hydrated silicoaluminate. To aid dissolution of the therapeutic into the aqueous environment, a surfactant might be added as a wetting agent. Natural or synthetic surfactants may be used. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents such as benzalkonium chloride and benzethonium chloride may be used. Nonionic detergents that can be used in the pharmaceutical formulations include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the pharmaceutical compositions of the invention either alone or as a mixture in different ratios. Controlled release formulation may be desirable. The inhibitors of the invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the pharmaceutical formulations, e.g., alginates, polysaccharides. Another form of controlled release is a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push the inhibitor compound out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect. Colorants and flavoring agents may also be included in the pharmaceutical compositions. For example, the inhibitors of the invention may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. The therapeutic agent can also be administered in a film coated tablet. Nonenteric materials for use in coating the pharmaceutical compositions include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy- methyl cellulose, povidone and polyethylene glycols. Enteric materials for use in coating the pharmaceutical compositions include esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating manufacturing may be carried out in a pan coater, in a fluidized bed, or by compression coating. Compositions can be administered in solid, semi-solid, liquid or gaseous form, or may be in dried powder, such as lyophilized form. The pharmaceutical compositions can be packaged in forms convenient for delivery, including, for example, capsules, sachets, cachets, gelatins, papers, tablets, capsules, ointments, granules, solutions, inhalants, aerosols, suppositories, pellets, pills, troches,' lozenges or other forms known in the art. The type of packaging will generally depend on the desired route of administration. Implantable sustained release formulations are also contemplated, as are transdermal formulations. Methods of the invention contemplate administration of inhibitor compounds by various routes. Such pharmaceutical compositions may be for administration for injection, or for oral, nasal, transdermal or other forms of administration, including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intratracheal, intrathecal, intraocular, retrobulbar, intrapuhnonary (e.g., aerosolized drugs) or subcutaneous injection (including depot administration for long term release e.g., embedded under the splenic capsule, brain, or in the cornea); by sublingual, anal, vaginal, or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time. In general, the methods of the invention involve administering effective amounts of an inhibitor of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers, as described above. As is understood in the art, a chosen route of administration may dictate the physical form of the compound being delivered. In one aspect, the invention provides methods for oral administration of a pharmaceutical composition of the invention. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, and cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may include liposomes that are derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). In general, the formulation will include a compound of the invention and inert ingredients which protect against degradation in the stomach and which peπnit release of the biologically active material in the intestine. The inhibitors can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The capsules could be prepared by compression. Also contemplated herein is pulmonary delivery of the present inhibitors in accordance with the invention. According to this aspect of the invention, the inhibitor is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn H nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts. All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and or carriers useful in therapy. When used in pulmonary administration methods, the inhibitors of the invention are most advantageously prepared in particulate form with an average particle size of less than 10 μm (or microns), for example, 0.5 to 5μm, for most effective delivery to the distal lung. Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration range of about 0.1 to 100 mg of inhibitor per mL of solution, 1 to 50 mg of inhibitor per mL of solution, or 5 to 25 mg of inhibitor per mL of solution. The formulation may also include a buffer. The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the inhibitor caused by atomization of the solution in forming the aerosol. Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive inhibitors suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1 ,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent or diluent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the inhibitor to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery may include dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated. Toxicity and therapeutic efficacy of the PI3Kδ selective compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Additionally, this information can be determined in cell cultures or experimental animals additionally treated with other therapies such as radiation, chemotherapeutic agents, and antiangiogenic agents. In practice of the methods of the invention, the pharmaceutical compositions are generally provided in doses ranging from 1 pg compound/kg body weight to 1000 mg/kg, 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg to 50 mg/kg, and 1 to 20 mg/kg, given in daily doses or in equivalent doses at longer or shorter intervals, e.g., every other day, twice weekly, weekly, or twice or three times daily. The inhibitor compositions may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in the human clinical trials discussed above. Appropriate dosages may be ascertained through use of established assays for determining blood levels dosages in conjunction with appropriate physician, considering various factors which modify the action of drugs, e.g. the drug's specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a count of inflammatory cell infiltration (total cells and differential cellular component) of BAL fluids isolated from OVA- sensitized/challenged mice treated with p 11 Oδ inhibitor IC87114 or control in BAL fluid. Macrophage (Mac), lymphocyte (Lym), neutrophils (Neu), and eosinophils (Eos) were counted. Bars represent mean ± SEM from 6 independent experiments. #, p < 0.05 vs. SAL+SAL; *, p < 0.05 vs. OVA+SAL. Figure 2 depicts total lung inflammation, defined as the average of the peribronchial and perivascular inflammation scores, in OVA-sensitized/challenged mice treated with IC87114 or control. Bars represent mean ± SEM from 6 independent experiments. #, p < 0.05 vs. SAL+SAL; *, p < 0.05 vs. OVA+SAL. Figure 3 shows airway responsiveness to aerosolized methacholine measured in unrestrained, conscious OVA-sensitized/challenged mice treated with IC87114 or control, at 72 h after the last challenge. Readings of breathing parameters were taken for 3 min after each nebulization during which Penh values were determined. Data represent mean ± SEM from 6 independent experiments. #, p < 0.05 vs. SAL+SAL; *, p < 0.05 vs. OVA+SAL.
EXAMPLES The following examples are provided to illustrate the invention, but are not intended to limit the scope thereof. EXAMPLE 1 SELECTIVE PI3Kδ INHIBITOR INHIBITS MAST CELL DEGRANULATION Mast cells are centrally important in allergic inflammation of the airways. Mast cell activation is mediated by antigen/IgE cross-linking of the FcεRI receptor, complement components and cytokines activation, leading to a spontaneous release of preformed histamine and proteases from secretory granules. These activated cells then transcribe, translate and secrete proinflammatory cytokines and chemokines which lead to increased inflammation. Non-specific PI3-kinase inhibitors such as wortmannin have been shown to inhibit mast cell degranulation from rat basophilic leukemia cells (RBL- 2H3) (Kitani et al., Biochem Biophys Res Commun. 183:48-54, 1992). However, the role for specific PI3-kinase subunits in regulating mast cell degranulation remains unclear [Tkaczyk et al, JBiol Chem. 278:48474-84 (2003); Windmiller et al, JBiol Chem. 278:11874-8 (2003)]. To determine the role of PI3 -kinase pi lOδ subunit in regulating mast cell activity, the ability of specific pi lOδ inhibitors to regulate human or mouse bone marrow derived mast cell degranulation was assessed. Bone marrow cells from mouse femur were cultured in mouse IL-3- containing RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies, Carlsbad, CA), 2 raM glutamine, and 50 μM 2-ME in humidified 95% air/5% CO2 at 37°C [Saito, H., F. et al., J. Immunol. 138:3927-3934 (1987)]. More than 95% pure mast cells (bone marrow-derived mast cells (BMMC)) were obtained after 5 wk of culture. BMMCs were sensitized by an overnight incubation with 0.5-1 μg/ml antidinitrophenyl (DNP) IgE mAb, washed once in Tyrode buffer (112 mM NaCl, 2.7 mM KC1, 0.4 mM NaH2PO4, 1.6 mM CaCl2, 1 mM MgCl2, 10 mM Hepes [pH 7.5], 0.05% gelatin, 0.1% glucose), resuspended in Tyrode buffer to 1 x 10 cells/ml and incubated with pi 10-delta inhibitor or vehicle control (0.3% DMSO) for 30 minutes. Cells were then stimulated by polyvalent antigen, 0.1-100 ng/ml DNP conjugates of human serum albumin (DNP-HSA) for 45 minutes. Cell suspensions were briefly centrifuged to separate Tyrode solution from cell pellets. Histamine secreted into the Tyrode solution during 45-min stimulation was measured by enzyme immunoassay (Cayman Chemical Company, Ann Arbor, MI). Percent inhibition of histamine release by an inhibitor was expressed by the following formula: (l-(% histamine release in the presence of an inhibitor)/(%> histamine release from control cells)) x 100%. Results of the ELISA indicate that mast cells pretreated with IC87114 (5 μM) reduce the amount of histamine in the cell supernatant by approximately 40% compared to vehicle control, and increase the amount of histamine detected in the cell pellet accordingly. This demonstrates that inhibition of pi lOδ kinase reduces the amount of histamine released from mast cell storage granules which has utility as an effective therapy for decreasing histamine release from mast cell activation mediated through FcεRI.
EXAMPLE 2 SELECTIVE PllOδ KINASE INHIBITOR REDUCES INFLAMMATORY CYTOKINE SECRETION BY MAST CELLS A significant effect of FcεRI crosslinking of mast cells is the induction of numerous inflammatory cytokines such as IL-2, IL-3, IL-4, IL-6, IL-13, GM-CSF, and TNF-α. Both TNF- and IL-2 are known to play a role in late phase hypersensitivity reactions like those in asthma and allergy [Galli et al, Fundamental Immunology, 4th Ed., Paul ed., Lippincott-Raven Publishers, Philadelphia, PA, (1998); p. 1127-1174]. To determine the effect pi lOδ kinase inhibitors have on the downstream effects of mast cell activation, the ability of a selective pi lOδ inhibitor to modulate the secretion of inflammatory cytokines after mast cell activation was examined. BMMCs were sensitized by an overnight incubation with 0.5-1 μg/ml antidinitrophenyl (DNP) IgE mAb, washed once in BMMC medium in the absence of IL-3 and resuspended in the same medium at 1 x 107 cells/ml. Cells were incubated with pi lOδ inhibitor or vehicle control (0.3% DMSO) for 30 minutes and then stimulated by polyvalent antigen, 1-100 ng/ml DNP conjugates of human serum albumin (DNP-HSA) for 20 hours. The cell supernatant was collected and the levels of inflammatory cytokines assessed by ELISA (BD PharMingen or Endogen). The cytokines measured include TNF-α, IL-6, IL-2, and granulocyte/monocyte-colony stimulating factor (GM-CSF). Results show that stimulation with 10 ng/ml antigen and addition of only 2 μM IC87114 to mast cells decreases the amount of TNF-α secreted by mast cells to undetectable levels. Additionally, 100 ng/ml of antigen plus 3 μM inhibitor inhibits TNF-α production from activated mast cells by 85%. Assessment of IL-6 levels after addition of the p 11 Oδ kinase inhibitor demonstrates similar effects. Addition of IC87411 to activated mast cells (10 ng/ml antigen) showed a dose dependent reduction in IL-6 production, from 6000 pg/ml with no inhibitor, reduced to approximately 2000 pg/ml IL-6 in the presence of 50 μM inhibitor. Bone marrow mast cells isolated from pi lOδ knockout mice were also assessed for IL-6 secretion levels after activation by antigen (100 ng/ml). Addition of pi lOδ inhibitor (up to 10 μM) had no effect on levels of IL-6 in knockout cells, indicating that the effect of the inhibitor is through inhibition of the protein kinase pi lOδ isoform. GM-CSF secretion by activated mast cells was also inhibited by approximately 45% after addition of the p 110δ inhibitor. Levels of the inflammatory cytokine IL-2 were also measured from antigen stimulated BMMC. BMMC activated with 10 ng/ml antigen showed a dose response inhibition of IL-2 secretion upon addition of p 110δ inhibitor, with levels decreased from control values of approximately 10 pg/ml to < 4 pg/ml IL-2 in the presence of 20 μM inhibitor, a 2.5 fold reduction in IL-2 secretion. Chemokines play a significant role in attracting cells to the site of inflammation thereby promoting the inflammatory response. Antigen activated BM mast cells were assessed for chemokine production in the presence and absence of pi lOδ inhibitor using Multi-Analyte Profile Technology (Rules-Based Medicine, Inc. Austin, TX). The effects of pi lOδ inhibitor (5 μM) on chemokine production from activated mast cells resulted in approximately 57% inhibition of eotaxin levels, approximately 37% inhibition of lymphotactin, approximately 60% inhibition of macrophage-derived chemokine (MDC) secretion, and approximately 35% inhibition in the levels of macrophage-inflammatory proteins MlP-lα and MlP-lβ. These results demonstrate that specific inhibition of pi lOδ reduces the amount of inflammatory cytokine and chemokine secreted as a result of antigen cross linking of FcεRI on the surface of mast cells. The ability to target and inhibit pi lOδ activation in these stimulated mast cells provides a method for an improved, specific method for treatment of mast cell related disorders, and a useful therapeutic for downregulating aberrant mast cell activation.
EXAMPLE 3 PllOδ INHIBITOR SPECIFICALLY INHIBITS AKT PHOSPHORYLATION IN ANTIGEN ACTIVATED MAST CELLS PI3K pi lOδ is involved in a complex signaling pathway leading to phospholipase C activation and influx of calcium into the cell. Akt is a downstream target to PI3 kinases that gets phosphorylated at serine 243 in response to PI3K activation. The phosphorylation event is essential to the kinase activity of Akt. Akt activation has previously been associated with secretion of cytokines from activated rat basophilic leukemia cells or cells isolated from tyrosine kinase knockout mice [Kitaura et al, J. Exp. Med. 192:729-39 (2000)]. Broad inhibition of class la PI3Ks in certain cell types, such as endothelium, using LY294002 has been shown to not only to reduce phosphorylation of Akt in response to TNF-α, but also in non-cytokine stimulated cells, as these lipid kinases are essential for both cell motility and survival. [See Madge et al, J. Biol. Chem., 275:15458-15465 (2000)]. Because Akt activation occurs through several different biochemical pathways, it was necessary to determine the role PI3K pi lOδ played in triggering Akt phosphorylation in activated mast cells. To assess the effects of PI3K inhibitor on downstream signaling,
BMMC were stimulated with a dose range of antigen and incubated with IC87114 as above with 0, 2 μM, 5 μM, 20 μM or 50 μM inhibitor. Cells were lysed as described above and phsophoserine or phosphotyrosine levels for several pi lOδ downstream effectors were measured by Western blot. Antibodies used in the Western analysis include: Akt pS473 ; 3-phosρhoinositide-dependent kinase (PDKl) pS241 ; pERK; pJNK; Lyn pY396 and Bruton's tyrosine kinase (Btk) pY223. Antibodies were obtained from Cell Signaling Technology (Beverly, MA), Upstate Biotechnology (Lake Placid, NY), Zymed, and Santa Cruz Biotechnology.. Western blot analysis demonstrated that pi lOδ inhibitor as low as 5 μM decreased Akt phosphorylation after 10 minutes of antigen stimulation and significantly inhibited phosphorylation after 30 minutes of antigen stimulation. The 50 μM inhibitor dose again significantly inhibited S243 Akt phosphorylation after 2 minutes of antigen stimulation, and completely abolished phosphorylation at 10 or 30 minutes of antigen stimulation. Analysis of other downstream effectors showed that antigen/IgE stimulation induced phosphorylation of PDKl, ERK, JNK, Lyn and Btk, but addition of pi lOδ inhibitor did not affect the phosphorylation levels of any of these molecules. Additionally, overall tyrosine or serine phosphorylation for the cell was not affected by the p 11 Oδ inhibitor. Protein levels of all molecules tested remained constant indicating that the decrease in Akt phosphorylation was specifically due to inhibition of pi lOδ activity. These results indicate that selective inhibition of PI3K pi lOδ in activated mast cells specifically decreases the levels of Akt phosphorylation without affecting phosphorylation levels of other related downstream tyrosine or serine kinases. This indicates that pi lOδ selective inhibitor regulation of Akt, which is involved in mast cell cytokine secretion, will provide an effective therapeutic method for modulating ongoing inflammation in an already developed mast cell disorder by reducing cellular infiltrate to the site of inflammation, or may act preventatively in disorders such as allergy or asthma to prevent the onset of an immune reaction.
EXAMPLE 4 ALLERGEN-INDUCED AIRWAY INFLAMMATION LEADS TO INCREASED ACTIVITY OF PllOδ IN LUNG TISSUE PI3K activity is stimulated after antigen challenge in a murine model of allergic asthma, and administration of wortmannin or LY294002, two broad- spectrum inhibitors of PI3Ks, attenuate inflammation and airway hyperresponsiveness (AHR) (Ezeamuzie et al., Am JRespir Crit Care Med 164:1633-39, 2001; Kwak et al., J. Clin. Invest 111 : 1083-92, 2003). However, these inhibitors do not distinguish among the four class I PI3Ks (Davies et al., Biochem J. 351 :95-105, 2000; Sadhu et al., J. Immunol 170:2647-54, 2003) and also broadly impact multiple cell types that express these kinases. Previous studies have indicated a crucial role for pi lOδ in B and T cell antigen receptor signaling and activation (Okkenhaug et al., Science 297:1031-34, 2002; Clayton et al., J Exp Med 196:753-63, 2002) and, neutrophil migration and activation (Puri et al., Blood 103:3448-56, 2004; Sadhu et al., Biochem Biophys Res Commun 308:764-69, 2003). In addition, pi lOδ was reported to be essential for allergen-IgE-induced mast cell degranulation and vascular permeability (Ali et al., Nature 431:1007-11, 2004). This study did not determine if blockade of activity of this leukocyte and endothelial cell specific PI3K isoform, pi lOδ alone would be sufficient to attenuate allergic inflammation and AHR. To examine the role of p 11 Oδ in the pathogenesis of asthma the effects of administration of a p 11 Oδ inhibitor was investigated in animal models of asthma. Female BALB/c mice, 8-10 weeks of age and free of murine-specific pathogens, were obtained from the Korean Research Institute of Chemistry Technology (Daejon, Korea). Mice were housed throughout the experiments in a laminar flow cabinet, and were maintained on standard laboratory chow ad libitum. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the Chonbuk National University Medical School. Mice were sensitized on days 1 and 14 by intraperitoneal injection of 20 μg ovalbumin (OVA) (Sigma- Aldrich, St. Louis, MO) emulsified in 1 mg of aluminum hydroxide (Pierce Chemical Co., Rockford, IL) in a total volume of 200 μl, as previously described (Kwak et al., supra). On days 21, 22, and 23 after the initial sensitization, the mice were challenged for 30 min with an aerosol of 3% (wt/vol) OVA in saline (or with saline as a control) using an ultrasonic nebulizer (NE-U12, Omron, Japan). IC87114 (0.1 or 1 mg/kg body weight/day) or DMSO (vehicle control) diluted with 0.9% NaCl, was administered in a volume of 50 μl by intratracheal instillation two times to each animal, once on day 21 (1 h before the first airway challenge with OVA) and the second time on day 23 (3 h after the last airway challenge with OVA). All statistical comparisons were performed using one-way ANOVA followed by the Fisher's test. Significant differences between groups were determined using the unpaired Student's t test. Statistical significance was set at p < 0.05. In OVA-exposed mice, class I PI3K activity was measured using protein extracts from lung tissue homogenates prepared in the presence of protease inhibitors, as described previously (Kwak et al., supra). Protein concentrations were determined using the Bradford reagent (Bio-Rad, Hercules, CA) and PI3K activity in the tissue extracts was quantified by PLP3 competition enzyme immunoassays according to the manufacturer's protocol (Echelon, Inc., Salt Lake City, UT). PIP3 levels increased from approximately 15 pmol/ ml pre-challenge to approximately 75 pmol/ml at 1 hr, approximately 100 pmol/ml at 24 hr, and approximately 160 pmol/ml at 48 hrs and 72 hrs, indicating that class I PI3K activity increased approximately 4.6- , 6.1-, 9.5-, and 9.6-fold, respectively, after OVA inhalation, compared with the pre- challenge period. In contrast, no significant changes in PI3K activity were observed after saline inhalation. Activation of these kinases has been linked to phosphorylation of Ser- 473 of Akt, an event crucial for Akt enzymatic activation (Alessi et al., Curr. Biol. 7:261-69, 1997). Akt levels were measured by Western blot. Protein extracts from lung tissue homogenates (30 μg/lane) were electrophoresed in polyacrylamide gels (Invitrogen Life Technologies, Carlsbad, CA), transferred electrophoretically to a PVDF membrane (Immobilon-P; Millipore, Billerica, MA), and incubated overnight at 4°C with anti phoshposerine antibodies as described above. Consistent with increased PI3K activity, levels of p-Akt protein in the lung tissues were also increased at 72 h after OVA inhalation, as detected by Western blot, compared with levels in the control animals that received saline inhalation. No significant changes in total Akt protein levels were observed. Intratracheal administration of a selective pi 1 Oδ inhibitor, IC87114, blocked Akt phosphorylation. The level of phosphorylation was similar to saline control levels indicating that pi lOδ contributed significantly to the overall class I PI3K activity in allergen-induced Akt activation. While PLP3 formation and Akt phosphorylation indicated that the PI3K family plays a role in allergy and asthma, administration of the pi lOδ specific inhibitor described herein demonstrates that the important PI3K for allergy and asthma is pi lOδ.
EXAMPLE 5 PllOδ CONTRIBUTES TO OVA-INDUCED EOSINOPHIL RECRUITMENT IN BAL FLUID The experiments above established that pi lOδ mediates allergen- induced Akt activation. To determine the effect of pi lOδ kinase in eosinophil recruitment following allergen challenge, the number of inflammatory cells in the lung was assessed in OVA-challenged animals. Mice were administered ovalbumin as described in Example 4 above and broncliialveolar lavage (BAL) fluid was collected 72 h after the last OVA aerosol challenge, and total leukocyte and differential cell counts were performed. At 72 hours after the last challenge, mice were sacrificed with an overdose of pentobarbital- Na (100 mg/kg of body weight/administered intraperitoneally). Blood was drawn by puncture of the vena cava and centrifuged. Serum was shock frozen in liquid nitrogen and stored at -70°C for IgE measurements. BAL was performed as described (Kwak et al., supra). Briefly, the chest cavity was exposed to allow for expansion, after which the trachea was carefully intubated and the catheter secured with ligatures. Pre-warmed 0.9% NaCl solution was slowly infused into the lungs and withdrawn. Total BAL cells were counted using a hemocytometer. Differential cell counts were obtained from BAL cells spun down onto slides with a cytocentrifuge (Shannon Scientific Ltd., Cheshire, United Kingdom) and treated with Diff-Quik solution (Dade Diagnostics of Puerto Rico Inc. Aguada, Puerto Rico). Two independent, blinded investigators counted the cells using a microscope. Approximately 400 cells, in each of four different random locations were counted. The mean number from the two investigators was used to estimate the cell differentials. For cytokine and leukotriene measurements, supernatants of BAL were shock frozen in liquid nitrogen and stored at -70°C until use. OVA inhalation significantly (p < 0.05) increased the absolute numbers of eosinophils, lymphocytes, and neutrophils, as compared with saline control (Figure 1). Intratracheal administration of IC87114 (0.1 mg/kg) reduced the number of eosinophils, lymphocytes, and neutrophils detected in BAL fluids at 72 hours post challenge by 79.8%, 63.5%, and 80%, respectively, compared with mice treated with vehicle control. In contrast, the number of macrophages was not affected by IC87114. No eosinophils were found in unsensitized control animals, whereas many eosinophils were present in BAL fluids of allergen-treated mice. Reduction in the total cell number recovered in BAL fluid in IC87114-treated mice as compared with vehicle control, was mainly due to a significant (p < 0.05) reduction in eosinophils in the IC87114-treated mice. These results indicate that pi lOδ activity contributed to eosinophil recruitment during the allergic inflammatory response. To determine if pi lOδ affects OVA-induced tissue eosinophilia, mucus production, and airway inflammation, lung tissue was extracted and histological analysis performed. Lung tissues were collected 72 h after the last OVA challenge. Mice were sacrificed and the lungs and trachea were filled intratracheally with a fixative (0.8% formalin, 4% acetic acid) using a ligature around the trachea. Lungs were removed and lung tissues were fixed with 10% (vol/vol) neutral buffered formalin. The specimens were dehydrated and embedded in paraffin. For histological examination, 4 μm sections of fixed embedded tissues were cut on a Leica model 2165 rotary microtome (Leica Microsystems Nussloch GmbH,, Nussloch, Germany), placed on glass slides, deparaffinized, and stained with hematoxylin 2, eosin-Y (Richard- Allan Scientific, Kalamazoo, MI) and periodic acid-Schiff (PAS). Three independent blinded investigators graded the inflammation score. The degree of peribronchial and perivascular inflammation was evaluated on a subjective scale of 0 to 3, as described in Kwak et al (supra). A value of 0 was adjudged when no inflammation was detectable, a value of 1 for occasional cuffing with inflammatory cells, a value of 2 for most bronchi or vessels surrounded by thin layer (one to five cells) of inflammatory cells, and a value of 3 when most bronchi or vessels were surrounded by a thick layer (more than five cells) of inflammatory cells. Histological analyses revealed typical pathologic features of asthnia- like inflammation in the OVA-exposed mice. In contrast to the saline controls, OVA- exposed mice showed numerous inflammatory cells in the peribronchiolar zone and accumulation of mucus and cellular debris within the lumen of the bronchioles. In contrast, IC87114-treated mice showed substantial attenuation in the eosinophil-rich leukocyte infiltration in the peribronchiolar regions and in the amount of debris present in the lumen. In addition, representative sections of each group were stained with periodic acid-Schiff (PAS) for detection of goblet cells. As compared to the control, OVA-exposed mice exhibited severe goblet cell hyperplasia in the airways, which was markedly reduced by IC87114-treatment. The inflammation scores of peribronchial, perivascular regions as well as total lung were increased significantly (p < 0.05) at 72 h after OVA inhalation compared with scores after saline inhalation (Figure 2). The increased lung inflammation observed after OVA inhalation was reduced by more than 50% by administration of the pi lOδ inhibitor. These results indicate that p 11 Oδ inhibitor significantly reduces allergen-induced leukocyte influx, airway inflammation, and goblet cell hyperplasia.
EXAMPLE 6 EFFECTS OF PllOδ INHIBITOR ON CYTOKINES AND CHEMOKINES IN ALLERGEN INDUCED AIRWAY INFLAMMATION Eosinophil accumulation and subsequent activation in bronchial tissues is known to play a critical role in the pathogenesis of allergic airway inflammation (Busse et al., NEnglJMed 344:35062, 2001; Humbles et al., Science 305:1776-79, 2004). Eosinophil transmigration into the airways is a multistep process that is orchestrated by Th2 cytokines (IL-4, IL-5, and IL-13), and coordinated by specific chemokines such as eotaxin in combination with adhesion molecules such as VCAM- 1 and VLA-4 (10, 11). IL-13 is a potent inducer of eotaxin expression in airway epithelial cells (Tigani et al., Eur J Pharmacol 433:217-23, 2001) Given the essential role of Th2 cytokines in evoking allergic inflammatory responses, the concentrations of IL-4, IL-5, and IL-13 were measured from BAL fluid as well as in the lung tissue of OVA-challenged mice that received either pi 10δ inhibitor or vehicle control.
Effects ofIC87114 on Cytokine protein levels Levels of IL-lβ, TNF-α, IL-4, IL-5, IL-13, and RANTES were quantified in the supematants of BAL fluids by enzyme immunoassays according to the manufacturer's protocol (IL-lβ, TNF-α, IL-4, and IL-5; Endogen, Inc., Woburn, MA; IL-13 and RANTES; R&D Systems, Inc., Minneapolis, MN). The lower detection limit for IL-lβ, TNFα, IL-4, IL-5, IL-13, and RANTES in these assays was 3, 10, 5, 5, 1.5, and 2 pg/ml, respectively. OVA-challenge induced significant (p < 0.05) increases in the concentrations of all three IL-4, IL-5, IL-13 cytokines detected by protein-blotting in . lung tissues as well as by ELISA of BAL fluids compared with the levels detected after saline challenge. The increased levels of these cytokines in both lung tissue and in BAL fluids were significantly (p < 0.05) reduced by IC87114. Enzyme immunoassays revealed that levels of IL- 1 β and TNFα in
BAL fluids were also increased significantly (p < 0.05) at 72 h after OVA inhalation compared with the levels after saline inhalation, from approximately 100 pg/ml TNFα and 15 pg/ml IL-lβ in control animals up to approximately 280 pg/ml TNFα and 30 pg/ml IL-1 β. IC87114 reduced the increased levels of these proinflammatory cytokines by more than 50%, down to approximately 140 pg/ml TNFα and 15 pg/ml IL-lβ. One of the responses to these cytokines is the induction of leukocyte- endothelial adhesion molecules. Indeed, levels of ICAM-1 and VCAM-1 proteins in the lung tissue were increased significantly (p < 0.05) at 72 h after OVA inhalation and these levels were substantially reduced by the administration of IC87114. Effects ofIC87114 on eotaxin and RANTES protein levels in lung tissues and BAL fluids of OVA sensitized and —challenged mice 5 Western blot analysis revealed that protein levels of the chemokines eotaxin and RANTES in the lung tissue were increased significantly (p < 0.05) at 72 h after OVA inhalation compared with the saline control. Administration of IC87114 reduced the increased levels of these chemokines by more than 50%. In addition, enzyme immunoassays revealed that increased levels of RANTES in BAL fluids at 72 10. h after OVA inhalation were also significantly (p < 0.05) reduced by IC87114 treatment. Effect ofIC87114 on serum IgE levels' and LTC4 release in BAL fluid IL-4 and IL-13 are important in directing B cell growth, differentiation, and secretion of IgE (Emson et 1., JExp Med 188:399-404, 1998). The biological 15 activities of IgE are mediated through high affinity IgE receptors (FcεRI) on mast cells and basophils. Cross-linking of FcεRI initiates multiple signaling cascades leading to cellular degranulation and activation (Nadler et al., Adv Immunol 76:325- . 55, 2000: Kawakami et al., Nat Rev Immunol 2:77386, 2002). In order to determine whether IC87114 could modify OVA-specific 20 Th2 response in vivo in mice, circulating IgE antibody levels were analyzed 72 hours following OVA challenge. OVA-specific IgE levels were measured by capture ELISA as previously described (MacLean et al., J Immunol 165:6568-75, 2000). Briefly, microtiter plates were coated with 2 μg/ml of purified monoclonal anti-mouse IgE
25 (BD PharMingen, San Diego, CA). After blocking with PBS-10% FCS, appropriate dilutions of serum samples in PBS-10% FCS were added to the plate and incubated for 2 h at room temperature. After washing with PBS-Tween, biotinylated OVA (10 μg/ml) and HRP-conjugated streptavidin were added to the wells and incubated for 1 hour. The plates were washed followed by addition of the HRP substrate, 3,3'5,5'-
30 teframethylbenzidine substrate (TMBS, Sigma Chemical Co.). After incubation for 30 min in the dark at room temperature, the plates were read at 450 nm on a microplate reader (Molecular Dynamics, Sunnyvale, CA). Total serum IgE was measured by capture ELISA in a manner similar to the detection of OVA-specific IgE. A biotinylated rat anti-mouse IgE (PharMingen) was used to detect captured IgE in place of the biotinylated OVA. Substantial elevation in total IgE and OVA-specific IgE was observed in serum from OVA-challenged mice (approx. 12 ng/ml total IgE, approx. 3 ng/ml OVA-Specific IgE) as compared with untreated mice (approx. 3 ng/ml total IgE, less than 1 ng/ml OVA-specific IgE). IC87114 significantly (p < 0.05) lowered total circulating IgE levels in a dose-dependent manner (approx. 6ng/ml total IgE and less than 1.5 ng/ml OVA-specific IgE). In agreement with the inhibitory effect on total IgE level, IC87114 at 0.1 and 1 mg/kg significantly (p < 0.05) reduced OVA-specific IgE levels by 63 and 72%, respectively. BAL fluid was obtained 72 h after the last OVA challenge and assayed for LTC4. Levels of LTC4 were quantified in the supematants of BAL fluids by enzyme immunoassay according to the manufacturer's protocol (Cayman Chemical Co., Ann Arbor, MI). The lower detection limit for LTC4 in this assay was 10 pg/ml. The BAL fluid levels of the LTC4 were 3.1-fold higher in the OVA- sensitized/challenged mice (approximately 40 pg/ml) than in the mice receiving saline only (approximately 13 pg/ml) (p < 0.05 compared to saline). IC87114 (0.1 and 1 mg/kg), significantly (p < 0.05) inhibited LTC4 levels by 37 and 50%, respectively decreasing LTC4 levels to approximately 26 pg ml and 21 pg/ml, respectively. The amounts of LTC4 in the BAL fluid of OVA-sensitized/challenged mice treated with vehicle control were not significantly different from those of the saline control group.
EXAMPLE 7 INHIBITION OF PllOδ ATTENUATED AIRWAY HYPERRESPONSIVENESS (AHR) The results above indicate that administration of pi lOδ specific inhibitor effectively inhibited many side effects associated with airway inflammation. To determine the effect of IC87114 on the development of AHR in an experimental animal model, OV A-treated mice were induced with AHR and treated with p 110δ inhibitor. Sensitized BALB/c mice challenged with 3% OVA aerosol for 30 min daily for 3 consecutive days developed AHR to inhaled methacholine. Three days after the last ovalbumin challenge, airway responsiveness was measured in unrestrained conscious animals using whole body plethysmography (All Medicus Co., Seoul, Korea), as described previously (Kwak et al., supra). Briefly, in whole body plethysmography, respiratory function is measured in conscious, freely moving mice in chambers which allow animals to move freely within the chamber while respiratory function is measured. Each chamber is connected to a bias flow regulator to supply a smooth, constant flow of fresh air during testing. A transducer attached to each chamber detects pressure changes that occur as the animal breathes. Inspiration and expiration are recorded by establishing start-inspiration and end-inspiration as the box pressure/time curve crosses the zero point. Start of an inspiration is determined by extrapolating from a straight line drawn from two levels of the rising inspiratory phase of the box pressure signal. Time of inspiration (TI) is defined as the time from the start of inspiration to the end of inspiration; time of expiration (TE) as the time from the end of inspiration to the start of the next inspiration. The maximum box pressure signal occurring during one breath in a negative or positive direction is defined as peak inspiratory pressure (PIP) or peak expiratory pressure (PEP), respectively. Recordings of every 10 breaths are extrapolated to define the respiratory rate in breaths per minute. The relaxation time (Tr) is defined as the time of pressure decay to 36% of the total expiratory pressure signal (area under the box pressure signal in expiration). This may thus serve as a correlate to the time constant (RC) of the decay of the volume signal to 36% of the peak volume in passive expiration. During bronchoconstriction, the main alteration in the signal occurs during early expiration and leads to changes in the waveform of the box pressure signal. Other parameters measured include Tidal Volume (ml), Respiratory Rate (breaths per minutes), Minute Volume (tidal volume multiplied by respiratory rate, ml/min), Inspiratory Time (sec), Expiratory Time (sec), Peak Inspiratory Flow (ml/sec), and Peak Expiratory Flow (ml/sec). Airway responsiveness was assessed as percent increase of Penh
(Helmann et al., Am JRespir Crit Care Med 156:766-75, 1997; Chong et al., J Pharmacol Toxicol Methods 39:163-68, 1998). Readings were obtained at baseline and after exposure to aerosolized saline or methacholine (2.5 to 50 mg/ml). Data were collected and averaged for 3 min after each nebulization. Enhanced pause (Penh), calculated as: (expiratory time/relaxation time-1) x (peak expiratory flow/peak inspiratory flow), according to the manufacturers' protocol. Results are expressed as the percentage increase of Penh following challenge with each concentration of methacholine, where the baseline Penh (after saline challenge) is expressed as 100%. Airway responsiveness was substantially increased in the OVA- challenged group in response to methacholine inhalation as compared with the saline- challenged group (Figure 3). Administration of IC87114 to OVA-sensitized mice prior to OVA challenge showed a significant (p < 0.05) attenuation in Penh measured at all methacholine levels tested suggesting a role for pi lOδ in immune-mediated events leading to airway hyperresponsiveness in vivo. These results are contemplated to be associated with reduction in Th2 cytokine production, tissue eosinophilia, and mast cell activation, following pllOδ inhibition. Allergic airway inflammation and AHR development involve multiple inflammatory cells and a wide array of mediators. The results above demonstrate that pi lOδ inhibition effectively reduced OVA-induced Th2 cytokine production, pulmonary eosinophilia, serum IgE levels, goblet cell hyperplasia, and AHR in a mouse asthma model. These findings indicate that pi lOδ plays an important role in the pathogenesis of allergic asthma and that specific inhibitors of pi lOδ are useful therapeutics for the treatment of asthma and airway hyperresponsiveness.
EXAMPLE 8 EFFECTS OF PllOδ INHIBITORS ON RAT MAST CELL DEGRANULATION Mast cells and basophils express FcεRI, the high affinity receptor for
IgE, and play a central role for IgE-associated immediate hypersensitivity reactions and allergic disorders. Cross-linking of FcεRI-bound IgE with multivalent antigen initiates the activation of mast cells and basophils, resulting in the degranulation from these cells. To determine the effects of pi lOδ inhibitors on mast cell/basophil degranulation, the release of serotonin by degranulation of rat basophil leukemia cells (RBL-2H3) was measured in the presence of pi lOδ-specific inhibitors. RBL-2H3 cells are grown in monolayer cultures in media. RBL cells were grown to confluence in 25 ml Eagle minimum essential medium containing 16% FCS (EMEM-16) in a 75-cm2 tissue culture flask. Medium was removed via aspiration and cells washed with 3 ml trypsin-EDTA to remove trypsin inhibitor present in serum. Trypsin-EDTA was then added 37°C for 5 min to remove the adherent cells. Cells are removed from the flask, washed in EMEM-16 and collected by centrifugation at 1000 rpm for 5 minutes. Cells were washed a second time and cell pellet resuspended in EMEM-16. Cells were then plated in a 24 well plate at a concentration of 4 x 105 cells/ml, and cultured with 25 μl of 1 mCi/ml 3H-labeled serotonin (0.5 μCi/ml final) and 1 μg/ml anti-DNP IgE overnight at 37°C. Cell media was aspirated from the wells and cells washed twice by adding 500 μl of PBS to the well and inverting the plate onto a stack of paper towels! A final volume of 200 μl PBS was added and cells equilibrate <2 min in a 37°C water bath, and 10 μl DNP- albumin was added to each well (10 ng/ml final) and incubated for the 10 to 30 min at 37°C. The reaction was stopped by transferring the buffer from each well into a liquid scintillation vial. Wells were washed two times using 500 μl of 1% Triton X- 100 in PBS incubated 10 min at room temperature, and the liquid transferred to the vial for measuring. 10 ml of liquid scintillation cocktail is added to the sample vials and the level of radioactivity counted in a liquid scintillation counter, calculating the percentage of serotonin released by the cells. In samples cultured with either pi lOδ inhibitor IC87114 or a second pi lOδ-specific inhibitor was added over a range of doses to determine the efficacy of each dose. Measurement of radioactivity levels in control and pi lOδ-treated cells demonstrated that at IC87114 at a concentration of 2.5 micromolar showed 50% inhibition of serotonin release. These results indicate that pi 10δ specific inhibitors are effective at reducing mast cell degranulation mediated by Fc-receptor cross-linking, providing a means for reducing these affects during allergic reactions.
EXAMPLE 9 EFFECTS OF PI 1 Oδ INHIBITORS ON TYPE I HYPERSENSITIVITY RESPONSES
As shown above, pi lOδ inhibitors were effective at reducing the levels of mast cell degranulation mediated by IgE crosslinking. Mast cell degranulation plays a significant role in mediating allergic reactions and other type I hypersensitivity responses in vivo. To examine the effects of pi lOδ inhibitors on mast cell degranulation in vivo, an animal model of dermal hypersensitivity was used. To sensitize, the shaved dorsal skin of Lewis rats was injected with either saline or anti-DNP monoclonal IgE (1.25 to 25 ng in 50 μl per site) intradermally. Forty-eight hours after IgE sensitization, 1 ml saline solution containing the antigen (1 ng/ml DNP-BSA) and 0.5% Evan's Blue dye was intravenously injected, and 30 min later the rats were euthanized. Skin edema was assessed by measuring the size of the wheal at the site of injection using a caliper. The skin around the wheal was also excised and the amounts of extravasated dye were measured as previously described (inagaki et al., 1986). A single dose of IC87114 (20 or 60 mg/kg) or PEG-400 (used as vehicle control) was administered orally to the rats 1 hour before antigen administration. Ketotifen (10 mg/kg) was intraperitoneally injected 30 minutes before antigen challenge. Blood samples were drawn immediately after the wheal size measurements and plasma concentration of IC87114 was determined by determined liquid-liquid extraction by liquid chromatography/mass spectroscopy as described previously (Puri et al., Blood 103:3448-3456, 2004). Type I hypersensitivity responses showed a dose dependent response to IC87114, decreasing to approximately 70% of control at a dose of 20 mg/kg, to approximately 55% at a dose of 60 mg/kg. The positive control ketotifen (10 mg/kg) decreased sensitivity responses to approximately 35% of the control response. These results indicate that pi lOδ plays a role in mediating type I hypersensitivity reactions in sensitized animals, indicating that administration of pi lOδ inhibitors may reduce or prevent type I sensitivity reactions
EXAMPLE 10 EFFECTS OF PllOδ INHIBITOR ON HUMAN MAST CELL DEGRANULATION To determine the effect of pi lOδ inhibitors on human mast cells, cells were isolated from human cord blood, differentiated to mast cell lineage and assayed for degranulation and histamine release in the presence of p 11 Oδ inhibitors . CD34+ human cord blood cells were isolated and differentiated using stem cell factor and IL-4 following the protocol set out in Hsieh et al. (JExp Med. 193:123-33, 2001), in the presence and absence of methylcellulose (Iida et al, Blood 97:1016-22, 2001). Cells were cultured for approximately 5 weeks and were harvested by centrifugation at 1000 rpm for 3 minutes. Cells were resuspended in fresh medium (RPMI, IMDM or methylcellulose) at 1 x 106 cell/ml containing IgE at 10 μg/ml. Cells were primed for 5-days before testing for activation by anti-IgE cross-linking. To assess mast cell activation by IgE cross-linking, cells were harvested by centrifugation 2 minutes at 1000 rpm, and washed twice in Dulbecco's- phosphate buffered saline (D-PBS). Cells were then resuspended at a concentration of 2.5 x 105 cells/ml in D-PBS. To 95 μl of cells, 5 μl of either 200 μM IC87114 or ketoprofen in 3% DMSO, and cells were incubated at 37° C for 30 minutes. After 30 minutes, 2 μl of a 1:20 dilution of anti-IgE for a final concentration of 1:1000 IgE, and cells incubated in the presence of anti-IgE for 45 minutes. Cells were then centrifuged as above and the supernatant collected. An equivalent amount of D-PBS was added to the cells and the cells were freeze-thawed 3 -times to lyse the cells. The lysed cells were then centrifuged 4 minutes at 10,000 rpm and analyzed for histamine release by histamine ELISA. Histamine was measured in both the cell supernatant and the cell pellet. In this experiment IC87114 pi lOδ inhibitor reduced histamine release by approximately 18 % at a 10 μM concentration (based on normalization of total control release of 100%). These results show that pi lOδ exhibits an effect on human mast cells as well as in murine mast cells, indicating that pi lOδ inhibitors are useful regulators of histamine release and other mast cell activity in the treatment of patient with diseases or conditions mediated by aberrant mast cell activity. Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the invention.

Claims

WHAT IS CLAIMED IS: 1. A method for inhibiting an activity of mast cells comprising the step of administering to an individual a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in an amount effective to inhibit said activity.
2. The method of claim 1 wherein the mast cell activity being inhibited is mast cell migration.
3. The method of claim 1 wherein the mast cell activity being inhibited is mast cell degranulation.
4. The method of claim 1 wherein the mast cell activity being inhibited is mast cell proliferation.
5. The method of claim 1 wherein the mast cell activity being inhibited is secretion of a cytokine, chemokine or growth factor.
6. The method of claim 1 wherein the mast cell activity being inhibited is expression of a cytokine, chemokine or growth factor.
7. The method of claim 5 or 6 wherein the cytokine is TNF-α.
8. The method of claim 5 or 6 wherein the cytokine is IL-6.
9. The method of claim 5 or 6 wherein the chemokine is eotaxin, MlP-lα, MJP-lβ, MDC-1, MCP-1 or lymphotactin.
10. A method of reducing lymphocyte infiltration to a site of inflammation in a condition associated with undesirable mast cell activity comprising the step of administering to an individual a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in an amount effective to reduce or prevent lymphocyte infiltration to said site of inflammation in an amount effective to reduce lymphocyte recruitment signaling by mast cells in said individual.
11. A method for treating or preventing a condition associated with undesirable mast cell activity in an individual, comprising the step of administering a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in an amount effective for treating or preventing a condition associated with undesirable mast cell activity.
.
12. The method of claim 11 wherein the mast cell activity inhibited is mast cell migration, mast cell proliferation, mast cell degranulation, or expression or secretion of cytokines, chemokines or growth factors from mast cells.
13. The method of claim 11 wherein the condition is an IgE-mediated condition.
14. The method of claim 11 wherein the condition is asthma, an allergic reaction, or an autoimmune disease.
15. The method of claim 14 wherein the allergic reaction is type I hypersensitivity, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, or allergic asthma.
16. The method of claim 14 wherein the autoimmune disease is bullous pemphigoid or chronic urticaria.
17. The method of any one of claims 11-16, wherein the PI3Kδ selective inhibitor is administered in an amount effective to inhibit Akt phosphorylation in said mast cells.
18. The method of any one of claims 11-16, further comprising administering an immunomodulatory agent to the individual.
19. The method of claim 18, wherein the immunomodulatory agent is administered over a plurality of doses.
20. The method of claim 18, wherein the immunomodulatory agent is a glucocorticoid or corticosteroid, an immunosuppressant, an antihistamine, an aminosalicylate, a steroid hormone, a non-steroidal anti-inflammatory drug (NSALO), a sympathomimetic, or an analgesic.
21. The method of claim 18, wherein the immunosuppressant is an azathioprine, cyclosporine, cyclophosphamide, methotrexate, or penicillamine.
22. The method of claim 18, wherein the glucocorticoid is cortisone, dexamethosone, hydrocortisone, methylpredmsolone, predmsolone, prednisone, or budesonide.
23. The method of any one of claims 11-16, wherein the PI3Kδ selective inhibitor is administered over a plurality of doses.
24. A method of reducing or preventing mast cell proliferation in an individual having a condition associated with undesirable mast cell activity, comprising: administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) and an immunomodulatory agent.
25. A method of reducing or preventing lymphocyte infiltration to a site of inflammation in an individual having a condition associated with undesirable mast cell activity, comprising: administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) and a immunomodulatory agent.
26. A method of increasing the therapeutic index of an immunomodulatory agent administered to an individual to treat a condition associated with undesirable mast cell activity, comprising: administering to said individual a combination therapy comprising an immunomodulatory agent and an amount of a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) effective to increase the therapeutic index of the immunomodulatory agent.
27. The method of any one of claims 24-26 wherein the condition is asthma, an allergic reaction, or an autoimmune disease.
28. The method of any one of claims 24-26 wherein the allergic reaction is type I hypersensitivity, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, or allergic asthma.
29 The method of any one of claims 24-26 wherein the autoimmune disease is bullous pemphigoid, or chronic urticaria.
30. The method of any one of claims 1-16 or 24-26 wherein the selective PI3Kδ inhibitor is a compound having formula (I) or pharmaceutically acceptable salts and solvates thereof:
Figure imgf000069_0001
( I ) wherein A is an optionally substituted monocyclic or bicyclic ring system containing at least two nitrogen atoms, and at least one ring of the system is aromatic;
X is selected from the group consisting of C(Rb)2, CH2CHRb, and
CH= (R ); Y is selected from the group consisting of null, S, SO, SO2, NH, O, C(=O), OC(=O), C(=O)O, and HC(=O)CH2S;
R! and R2, independently, are selected from the group consisting of hydrogen, Cι_6alkyl, aryl, heteroaryl, halo,
Figure imgf000069_0002
NO2, OR , CF3, OCF3, N(Ra)2, CN, OC(=O)R , C(=O)Ra, C(=O)ORa, arylOR , Het, NRaC(=O)C1. 3alkyleneC(=O)ORa, arylOCι_3alkyleneN(R )2, arylOC(=O)Ra, Cχ_ alkyleneC(=O)ORa, OCι_4alkyleneC(=O)ORa, Ci^alkyleneOCj.
Figure imgf000069_0003
6alkenyleneN(Ra)2, C(=O)NRaCι_ alkyleneOR , C(=O)NRaC1.4alkyleneHet, OC2. 4alkyleneN(Ra)2, OC1.4alkyleneCH(ORb)CH2N(Ra)2, OCi^alkyleneHet, OC2_ 4alkyleneORa, OC2-4alkyleneNRaC(=O)OR NRaC1.4alkyleneN(Ra)2, NRaC(=O)Ra, NRaC(=O)N(Ra)2, N(SO2Ci_4alkyl)2, NRa(SO2Cι _4alkyl), SO2N(Ra)2, OSO2CF3, Ci _3alkylenearyl, Cι_4alkyleneHet, Cι_6alkyleneORb, Ci _ 3alkyleneN(Ra)2, C(=O)N(Ra) , NHC(=O)Cι_3alkylenearyl, C3_gcycloalkyl, C3_ gheterocycloalkyl, arylOCι_3alkyleneN(Ra)2, arylOC(=O)Rb, NHC(=O)C1. 3 alkyleneC3_gheterocycloalkyl, NHC(=O)C 1.3 alkyleneHet, OC 1 _4alkyleneOC 1 _ 4alkyleneC(=O)ORb, C(=O)Cι _4alkyleneHet, andNHC(=O)haloC!.6alkyl; or R! and R^ are taken together to form a 3- or 4-membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;
R3 is selected from the group consisting of optionally substituted hydrogen, Cj.βalkyl, C3_gcycloalkyl, C3_gheterocycloalkyl, Cχ_4alkylenecycloalkyl, C2- galkenyl, C^alkylenearyl; arylCi _3alkyl, C(=O)Ra, aryl, heteroaryl, C(=O)ORa, C(=O)N(R )2, C(=S)N(Ra)2, SO2Ra SO2N(R )2, S(=O)Ra, S(=O)N(R )2, C(=O)NRaCi.4alkyleneORa, C(=O)NRaCi.4alkyleneHet, C(=O)Ci.4alkylenearyl, C(=O)C _4alkyleneheteroaryl, Cχ_4alkylenearyl optionally substituted with one or more of halo, SO2N(Ra)2, N(Ra)2, C(=O)ORa, NRaSO CF3, CN, NO2, C(=O)Ra,
ORa, Cχ_4alkyleneN(Ra)2, and OCχ_4alkyleneN(Ra)2, Cχ_4alkyleήeheteroaryl, Cχ_ 4alkyleneHet, C \ _4alkyleneC(=O)C 1 _4alkylenearyl, C \ _4alkyleneC(=O)C \ _
4alkyleneheteroaryl, Cχ_4alkyleneC(=O)Het, Cι_4alkyleneC(=O)N(Ra)2, Cχ_
4alkyleneORa, C1_4alkyleneNRaC(=O)Ra, Cι.4alkyleneOCι.4alkyleneORa, Cχ_
4alkyleneN(Ra)2, Cι_4alkyleneC(=O)ORa, and Cι_4alkyleneOC1. alkyleneC(=O)ORa;
Ra is selected from the group consisting of hydrogen, Cχ_6alkyl, C3_ gcycloalkyl, C3_gheterocycloalkyl, Cχ_3alkyleneN(Rc)2, aryl, arylCχ_3alkyl, Cχ_ 3alkylenearyl, heteroaryl, heteroarylCχ_3alkyl, and Cχ_3alkyleneheteroaryl; or two Ra groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
Rb is selected from the group consisting of hydrogen, Cj.galkyl, heteroCχ_ 3alkyl, Cι_3alkyleneheteroCχ_3alkyl, arylheteroCχ_3alkyl, aryl, heteroaryl, arylCi _ 3alkyl, heteroarylCχ_3alkyl, Cχ_3alkylenearyl, and Cχ_3alkyleneheteroaryl;
Rc is selected from the group consisting of hydrogen, Cχ_6alkyl, C-$_ gcycloalkyl, aryl, and heteroaryl; and, Het is a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Cχ_4alkyl or C(=O)ORa.
31. The method of any one of claims 1 - 16 or 24-26 wherein the selective PI3Kδ inhibitor is selected from the group consisting of: 2-(6-aminopurin-9- ylmethyl)-3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one; 2-(6-aminopurin- o-ylmethyl)-6-bromo-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-6-chloro-3-(2-chlόrophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-5-chloro-3-(2-chloro-phenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-chlorophenyl)-5-methyl-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-8-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-biphenyl-2-yl-5-chloro-3H-quinazolin-4-one; 5-chloro-2-(9H-purin-6- ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one; 5-chloro-3-(2-fluorophenyl)-2-(9H- purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-5- chloro-3-(2-fluorophenyl)-3H-quinazolin-4-one; 3-biphenyl-2-yl-5-chloro-2-(9H- purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 5-chloro-3-(2-methoxyphenyl)-2- (9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 3-(2-chlorophenyl)-5-fluoro-2- (9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 3-(2-chlorophenyl)-6,7- dimethoxy-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 6-bromo-3-(2- chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4-one; 3-(2- chlorophenyl)-8-trifluoromethyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-benzo[g]quinazolin-4- one; 6-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 8-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-7-fluoro-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-7-nitro-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 3-(2-chlorophenyl)-6-hydroxy-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin- 4-one; 5-chloro-3-(2-chlorophenyl)-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin- 4-one; 3-(2-chlorophenyl)-5-methyl-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin- 4-one; 3-(2-chlorophenyl)-6,7-difluoro-2-(9H-purin-6-yl-sulfanylmethyl)-3H- quinazolin-4-one; 3-(2-chlorophenyl)-6-fluoro-2-(9H-purin-6-yl-sulfanylmethyl)-3H- quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-(2-isopropylphenyl)-5-methyl-3H- quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4- one; 3-(2-fluorophenyl)-5-methyl-2-(9H-purin-6-yl-sulfanylmethyl)-3H-quinazolin-4- one; 2-(6-aminopurin-9-ylmethyl)-5-chloro-3-o-tolyl-3H-quinazolin-4-one; 2-(6- aminopurin-9-ylmethyl)-5-chloro-3-(2-methoxy-phenyl)-3H-quinazolin-4-one; 2-(2- amino-9H-purin-6-ylsulfanylmethyl)-3-cycloproρyl-5-methyl-3H-quinazolin-4-one; 3-cyclopropylmethyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4- one; 2-(6-aminopurin-9-ylmethyl)-3-cyclopropylmethyl-5-methyl-3H-quinazolin-4- one; 2-(2-amino-9H-purin-6-ylsulfanylmethyl)-3-cyclopropylmethyl-5-methyl-3H- quinazolin-4-one; 5-methyl-3-phenethyl-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 2-(2-amino-9H-purin-6-ylsulfanylmethyl)-5-methyl-3-phenethyl- 3H-quinazolin-4-one; 3-cyclopentyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazόlin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-cyclopentyl-5-methyl-3H- quinazolin-4-one; 3-(2-chloropyridin-3 -yl)-5-methyl-2-(9H-purin-6- ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3-(2- chloropyridin-3-yl)-5-methyl-3H-quinazolin-4-one; 3-methyl-4-[5-methyl-4-oxo-2- (9H-purin-6-ylsulfanylmethyl)-4H-quinazolin-3-yl]-benzoic acid; 3-cyclopropyl-5- methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H-qumazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-cyclopropyl-5-methyl-3H-quinazolin-4-one; 5 -methyl-3 -(4-nitrobenzyl)- 2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 3-cyclohexyl-5-methyl-2-(9H- purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3- cyclohexyl-5-methyl-3H-quinazolin-4-one; 2-(2-amino-9H-purin-6- ylsulfanylmethyl)-3 -cyclo-hexyl-5 -methyl-3H-quinazolin-4-one; 5 -methyl-3 -(E-2- phenylcyclopropyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 3-(2- chlorophenyl)-5-fluoro-2-[(9H-purin-6-ylamino)methyl]-3H-quinazolin-4-one; 2-[(2- amino-9H-purin-6-ylamino)methyl]-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4- one; 5-methyl-2-[(9H-purin-6-ylamino)methyl]-3-o-tolyl-3H-quinazolin-4-one; 2-[(2- amino-9H-purin-6-ylamino)methyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-[(2- fluoro-9H-purin-6-ylamino)methyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one; (2- chlorophenyl)-dimethylamino-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 5-(2-benzyloxyethoxy)-3-(2-chlorophenyl)-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 6-aminopurine-9-carboxylic acid 3-(2-chlorophenyl)-5-fluoro-4- oxo-3,4-dihydro-quinazolin-2-ylmethyl ester; N-[3-(2-chlorophenyl)-5-fluoro-4-oxo- 3,4-dihydro-quinazol'in-2-ylmethyl]-2-(9H-purin-6-ylsulfanyl)-acetamide; 2-[l-(2- fluoro-9H-purin-6-ylamino)ethyl]-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5-methyl- 2-[l-(9H-purin-6-ylamino)ethyl]-3-o-tolyl-3H-quinazolin-4-one; 2-(6- dimethylaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5-methyl- 2-(2-methyl-6-oxo- 1 ,6-dihydro-purin-7-ylmethyl)-3-σ-tolyl-3H-quinazolin-4-one; 5- methyl-2-(2-methyl-6-oxo- 1 ,6-dihydro-purin-9-ylmethyl)-3 -ø-tolyl-3H-quinazolin-4- one; 2-(ammo-dimethylaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4- one; 2-(2-amino-9H-purin-6-ylsulfanylmethyl)-5 -methyl-3 -o-tolyl-3H-quinazolin-4- one; 2-(4-amino-l,3,5-triazin-2-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin- 4-one; 5-methyl-2-(7-methyl-7H-ρurin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin- 4-one; 5-methyl-2-(2-oxo-l,2-dihydro-pyrimidin-4-ylsulfanylmethyl)-3-o-tolyl-3H- quinazolin-4-one; 5 -methyl-2-purin-7-ylmethyl-3 -o-tolyl-3H-quinazolin-4-one; 5 - methyl-2-purin-9-ylmethyl-3 -o-tolyl-3H-quinazolin-4-one; 5 -methyl-2-(9-methyl-9H- purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4-one; 2-(2,6-Diamino-pyrimidin- 4-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5-methyl-2-(5-methyl- [l,2,4]triazolo[l,5-α]pyrimidin-7-ylsulfanylmethyl)-3-σ-tolyl-3H-quinazolin-4-one; 5- methyl-2-(2-methylsulfanyl-9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H-quinazolin-4- one; 2-(2-hydroxy-9H-purin-6-ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin- 4-one; 5-methyl-2-(l-methyl-lH-imidazol-2-ylsulfanylmethyl)-3-o-tolyl-3H- quinazolin-4-one; 5-methyl-3-o-tolyl-2-(lH-[l,2,4]triazol-3-ylsulfanylmethyl)-3H- quinazolin-4-one; 2-(2-amino-6-chloro-purin-9-ylmethyl)-5 -methyl-3 -o-tolyl-3H- quinazolin-4-one; 2-(6-aminopurin-7-ylmethyl)-5 -methyl-3 -o-tolyl-3H-quinazolin-4- one; 2-(7-amino-l,2,3-triazolo[4,5-(i]pyrimidin-3-yl-methyl)-5-methyl-3-o-tolyl-3H- quinazolin-4-one; 2-(7-amino-l,2,3-triazolo[4,5-(f|pyrimidin-l-yl-methyl)-5-methyl- 3-o-tolyl-3H-quinazolin-4-one; 2-(6-amino-9H-purin-2-ylsulfanylmethyl)-5-methyl- 3-o-tolyl-3H-quinazolin-4-one; 2-(2-amino-6-ethylamino-pyrimidin-4- ylsulfanylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(3-amino-5- methylsulfanyl-l,2,4-triazol-l-yl-methyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2- (5-amino-3-methylsulfanyl-l,2,4-triazol-l-ylmethyl)-5-methyl-3-o-tolyl-3H- quinazolin-4-one; 5-methyl-2-(6-methylaminopurin-9-ylmethyl)-3-o-tolyl-3H- quinazolin-4-one; 2-(6-benzylaminopurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H- quinazolin-4-one; 2-(2,6-diaminoρurin-9-ylmethyl)-5-methyl-3-o-tolyl-3H- quinazolin-4-one; 5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3-o-tolyl-3H- quinazolin-4-one; 3-isobutyl-5-methyl-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; N-{2-[5-Methyl-4-oxo-2-(9H-purin-6-ylsulfanylmethyl)-4H- quinazolin-3-yl]-phenyl}-acetamide; 5-methyl-3-(E-2-methyl-cyclohexyl)-2-(9H- purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2- [5 -methyl-4-oxo-2-(9H-purin-6- ylsulfanylmethyl)-4H-quinazolin-3-yl]-benzoic acid; 3-{2-[(2- dimethylaminoethyl)methylamino]phenyl}-5-methyl-2-(9H-purin-6- ylsulfanylmethyl)-3H-quin-azolin-4-one; 3-(2-chlorophenyl)-5-methoxy-2-(9H-purin- 6-ylsulfanylmethyl)-3H-quinazolin-4-one; 3-(2-chlorophenyl)-5-(2-morpholin-4-yl- ethylamino)-2-(9H-purin-6-ylsulfanylmethyl)-3H- quinazolin-4-one; 3-benzyl-5 - methoxy-2-(9H-purin-6-ylsulfanylmethyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-benzyloxyphenyl)-5-methyl-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one; 2-(l-(2-amino-9H- purin-6-ylamino)ethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 5-methyl-2-[l-(9H- purin-6-ylamino)propyl]-3-o-tolyl-3H-quinazolin-4-one; 2-(l-(2-fluoro-9H-purin-6- ylamino)propyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(l-(2-amino-9H-purin-6- ylamino)propyl)-5-methyl-3-o-tolyl-3H-quinazoIin-4-one; 2-(2-benzyloxy-l-(9H- purin-6-ylamino)ethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-5-methyl-3-{2-(2-(l-methylpyrrolidin-2-yl)-ethoxy)-phenyl}-3H- quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-3 -(2-(3 -dimethylamino-propoxy)- phenyl)-5-methyl-3H-quinazolin-4-one; 2-(6-aminopurin-9-ylmethyl)-5-methyl-3-(2- prop-2-ynyloxyphenyl)-3H-quinazolin-4-one; 2- {2-(l -(6-aminopurin-9-ylmethyl)-5- methyli4-oxo-4H-quinazolin-3-yl]-phenoxy}-acetamide; 5-chloro-3-(3,5-difluoro- phenyl)-2-[ 1 -(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 3-phenyl-2-[ 1 -(9H- purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 5-fluoro-3-phenyl-2-[l-(9H-purin-6- ylamino)-propyl]-3H-quinazolin-4-one; 3-(2,6-difluoro-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 6-fluoro-3-phenyl-2-[l-(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 3-(3,5-difluoro-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5-fluoro-3-phenyl-2-[ 1 -(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 3-(2,3-difluoro-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5 -methyl-3 -phenyl-2-[l -(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 3-(3-chloro-phenyl)-5-methyl-2-[l-(9H-purin- 6-ylamino)-ethyl]-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[(9H-purin-6-ylamino)- methyl]-3H-quinazolin-4-one; 2-[(2-amino-9H-purin-6-ylamino)-methyl]-3-(3,5- difluoro-phenyl)-5 -methyl-3H-quinazolin-4-one; 3 - {2-[(2] -diethylamino-ethyl)- methyl-amino]-phenyl}-5-methyl-2-[(9H-purin-6-ylamino)-methyl]-3H-quinazolin-4- one; 5-chloro-3-(2-fluoro-phenyl)-2-[(9H-purin-6-ylamino)-methyl]-3H-quinazolin-4- one; 5-chloro-2-[(9H-purin-6-ylamino)-methyl]-3-o-tolyl-3H-quinazolin-4-one; 5- chloro-3-(2-chloro-phenyl)-2-[(9H-purin-6-ylamino)-methyl]-3H-quinazolin-4-one; 6-fluoro-3-(3-fluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5-chloro-3-(3-fluoro-phenyl)-3H- quinazolin-4-one; 5-methyl-3-phenyl-2-[l-(9H-purin-6-ylamino)-propyl]-3H- quinazolin-4-one; 2-[l-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H- quinazolin-4-one; 3 -(2,6-difluoro-phenyl)-5 -methyl-2- [ 1 -(9H-purin-6-ylamino)- ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6- difluoro-phenyl)-5-methyl-3H-quinazolin-4-one; 3-(2,6-difluoro-phenyl)-2-[l-(2- fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3H-quinazolin-4-one; 3-(2,6-difluoro- phenyl)-5-methyl-2-[l-(7H-pyrrolo[2,3-(i]pyrimidin-4-ylamino)-ethyl]-3H- quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-propyl]-5-methyl-3-phenyl- 3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[l-(7H-pyrrolo[2,3-^pyrimidin-4- ylamino)-propyl]-3H-quinazolin-4-one; 2-[ 1 -(2-fluoro-9A-purin-6-ylamino)-propyl]- 5-methyl-3 -phenyl-3 A-quinazolin-4-one; 5-methyl-3-phenyl-2-[ 1 -(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 2-[ 1 -(2-amino-9/J-purin-6-ylamino)-ethyl]-5- methyl-3-phenyl-3H-quinazolin-4-one; 2-[2-benzyloxy-l-(9Hr-purin-6-ylamino)- ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- 2-benzyloxy-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[2-benzyloxy-l-(7H- pyrrolo[2,3-./]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[2-benzyloxy-l-(2-fluoro-9H-purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H- quinazolin-4-one; 3-(4-fluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]- 3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(4-fluoro-phenyl)- 5-methyl-3H-quinazolin-4-one; 3-(4-fluoro-phenyl)-2-[l-(2-fluoro-9H-purin-6- ylamino)-ethyl]-5-methyl-3H-qUinazolin-4-one;3-(4-fluoro-phenyl)-5-methyl-2-[l- (7H-pyrrolo[2,3-^]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one; 5-methyl-3- phenyl-2-[l-(7H-pyrrolo[2,3- ]pyrimidin-4-ylamino)-ethyl]-3H-quinazolin-4-one; 3- (3-fluoro-phenyl)-5-methyl-2-[l-(9Hr-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-fluoro-phenyl)-5-methyl-3H- quinazolin-4-one; 3-(3-fluoro-phenyl)-5-methyl-2-[l-(7H-pyrrolo[2,3-( ]pyrimidin-4- ylamino)-ethyl]-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[ 1 -(9H-purin-6-yl)- pyrrolidin-2-yl]-3H-quinazolin-4-one; 2-[2-hydroxy-l-(9H-purin-6-ylamino)-ethy ]- 5-methyl-3-phenyl-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[phenyl-(9H-purin-6- ylamino)-methyl]-3H-quinazolin-4-one; 2-[(2-amino-9H-purin-6-ylamino)-phenyl- methyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[(2-fluoro-9H-purin-6-ylamino)- phenyl-methyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 5 -methyl-3 -phenyl-2- [phenyl-(7H-pyrrolo[2,3-cT]pyrimidin-4-ylamino)-methyl]-3H-quinazolin-4-one; 5- fluoro-3-phenyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-ethyl]-5-fluoro-3-phenyl-3H-quinazolin-4-one; 2-[l-(2- amino-9Hr-purin-6-ylamino)-ethyl]-5-chloro-3-phenyl-3H-quinazolin-4-one; [5-(5- methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-5-(9H-purin-6-ylamino)- pentyl]-carbamic acid benzyl ester; [5-(2-amino-9H-purin-6-ylamino)-5-(5-methyl-4- oxo-3-phenyl-3,4-dihydro-quinazolin-2-yl)-pentyl}-carbamic acid benzyl ester; [4-(5- methyl-4-oxo-3-phenyl-3,4-dihydro-quinazolm-2-yl)-4-(9H-purin-6-ylamino)-butyl]- carbamic acid benzyl ester; [4-(2-amino-9H-purin-6-ylamino)-4-(5-methyl-4-oxo-3- phenyl-3,4-dihydro-quinazolin-2-yl)-butyl]-carbamic acid benzyl ester; 3-phenyl-2- [l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[5-amino-l-(9H-purin-6- ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one); 2-[5-amino-l-(2-amino- 9H-purin-6-ylamino)-pentyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[ 1 -(2- amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-Dimethyl-phenyl)-5-methyl-3H-quinazolin- 4-one; 3-(2,6-dimethyl-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 5-morpholin-4-ylmethyl-3-phenyl-2-[l-(9H-purin-6-ylamino)- ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5-morpholin- 4ylmethyl-3-phenyl-3H-quinazolin-4-one; 2-[4-amino- 1 -(2-amino-9H-purin-6- ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 6-fluoro-3-phenyl-2-[l- (9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6- ylamino)-ethyl]-6-fluoro-3-phenyl-3H-quinazolin-4-one; 2-[2-tert-butoxy-l-(9H- purin-6-ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 3-(3-methyl- phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-ethyl]-3-(3-methyl-phenyl)-5-methyl-3H-quinazolin-4- one; 3-(3-chloro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin- 4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-chloro-phenyl)-5-methyl-3H- quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-2-hydroxy-ethyl]-5-methyl-3- ρhenyl-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-fluoro- phenyl)-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6- difluoro-phenyl)-3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6-ylammo)-propylJ- 5-fluoro-3-phenyl-3H-quinazolin-4-one; 5-chloro-3-(3-fluoro-phenyl)-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- ethyl] -5 -chloro-3 -(3 -fluoro-phenyl)-3H-quinazolin-4-one; 3 -phenyl-2- [ 1 -(9H-purin-6- ylamino)-ethyl]-5-trifluoromethyl-3H-quinazolin-4-one; 3-(2,6-difluoro-phenyl)-5- methyl-2-[l-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 3-(2,6-difluoro- phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3iJ-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-propyl]-3-(2,6-difluoro-phenyl)-5-methyl-3H- quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(2,6-difluoro-phenyl)- 5 -methyl-3H-quinazolin-4-one; 3 -(3 ,5-dichloro-phenyl)-5 -methyl-2- [ 1 -(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 3-(2,6-dichloro-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- ethyl]-3-(2,6-dichloro-phenyl)-5-methyl-3H-quinazohn-4-one; 5-chloro-3-phenyl-2- [l-(9H-purin-6-ylamino) ropyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6- ylamino)-propyl]-5-chloro-3-phenyl-3H-quinazolin-4-one; 5-methyl-3-phenyl-2-[l- (9H-purin-6-ylamino)-butyl]-3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6- ylamino)-butyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6- ylamino)-ethyl]-3-(3,5-dichloro-phenyl)-5-methyl-3H-quinazolin-4-one; 5-methyl-3- (3-mo holin-4-ylmethyl-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4- one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-3-(3-mo holin-4-ylmethyl- phenyl)-3H-quinazolin-4-one; 2-[l-(5-bromo-7H-pyrrolo[2,3- ]pyrimidin-4- ylamino)-ethyl]-5-methyl-3-phenyl-3H-quinazolin-4-one; 5-methyl-2-[l-(5-methyl- 7H-pyιτolo[2,3- ]pyrimidin-4-ylamino)-ethyl]-3τphenyl-3H-quinazolin-4-one; 2-[ 1 - (5-fluoro-7H-pyrrolo[2,3- ]pyrimidin-4-ylamino)-ethyl]-5-methyl-3-phenyl-3H- quinazolin-4-one; 2-[2-hydroxy- 1 -(9H-purin-6-ylamino)-ethyl]-3-phenyl-3H- quinazolin-4-one; 3-(3,5-difluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)- propyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-propyl]-3-(3,5- difluoro-phenyl)-5-methyl-3H-quinazolin-4-one; 3-(3,5-difluoro-phenyl)-2-[l-(9H- purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(5-bromo-7H-pyrrolo[2,3- J]pyrimidin-4-ylamino)-ethyl]-3-(3-fluoro-ρhenyl)-5-methyl-3H-quinazolin-4-one; 3- (3-fluoro-phenyl)-5-methyl-2-[l-(5-methyl-7H-pyrrolo[2,3-cTlpyrimidin-4-ylamino)- ethyl]-3H-quinazolin-4-one; 3-phenyl-2-[l-(9H-purin-6-ylamino)-propyl]-3H- quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3,5-difluoro-phenyl)- 3H-quinazolin-4-one; 2-[ 1 -(2-amino-9H-purin-6-ylamino)-propyl]-3-phenyl-3H- quinazolin-4-one; 6,7-difluoro-3-phenyl-2-[ 1 -(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 6-fluoro-3-(3-fluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 2-[4-diethylamino-l-(9H-purin-6-ylamino)-butyl]-5-methyl-3- phenyl-3H-quinazolin-4-one; 5-fluoro-3-phenyl-2-[l-(9H-purin-6-ylamino)-propyl]- 3H-quinazolin-4-one; 3-phenyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4- one; 6-fluoro-3-phenyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 3- (3,5-difluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4- one; 5-fluoro-2-[ 1 -(2-fluoro-9H-purin-6-ylamino)-ethyl]-3-phenyl-3H-quinazolin-4- one; 3-(3-fluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5- chloro-3-(3,5-difluoro-phenyl)-2-El-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4- one; 3-(2,6-difluoro-phenyl)-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 3-(2,6-difluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 5-Methyl-3-phenyl-2-[3,3,3-trifluoro-l-(9H-purin-6-ylamiήo)- propyl]-3H-quinazolin-4-one; 3 -(3 -hydroxy-pheny l)-5 -methyl-2- [1 -(9Η-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 3-(3-methoxy-phenyl)-5-methyl-2-{l-[9H- purin-6-ylamino]-ethyl}-3H-quinazolin-4-one; 3-[3-(2-dimethylamino-ethoxy)- phenyl]-5-methyl-2- { 1 -[9H-purin-6-ylamino]-ethyl} -3H-quinazolin-4-one; 3-(3- cyclopropylmethoxy-phenyl)-5-methyl-2- { 1 -[9H-purin-6-ylamino] -ethyl} -3H- quinazolin-4-one; 5-methyl-3-(3-prop-2-ynyloxy-phenyl)- 2- { 1 -[9H-purin-6- ylamino]-ethyl} -3H-quinazolin-4-one; 2- { 1 -[2-amino-9H-purin-6-ylamino]ethyl} -3 - (3-hydroxyphenyl)-5-methyl-3H-quinazolin-4-one; 2-{l-[2-amino-9H-purin-6- ylamino]ethyl}-3-(3-methoxyphenyl)-5-methyl-3H-quinazolin-4-one; 2-{l-[2-amino- 9H-purin-6-ylamino]ethyl}-3-(3-cyclopropylmethoxy-phenyl)-5-methyl-3H- quinazolin-4-one; 2-{l-[2-amino-9H-purin-6-ylamino]ethyl}-5-methyl-3-(3-prop-2- ynyloxy-phenyl)-3H-quinazolin-4-one; 3-(3-ethynyl-phenyl)-5-methyl-2-[l-(9H- purin-6-ylamino)-ethyl] -3H-quinazolin-4-one; 3 - { 5 -methyl-4-oxo-2-[ 1 -(9H-purin-6- ylamino)-ethyl]-4H^quinazolin-3-yl}-benzonitrile; 3-{5-methyl-4-oxo-2-{l-[9H- purin-6-ylamino)-ethyl]-4H-quinazolin-3-yl}-benzamide; 3-(3-acetyl-phenyl)-5- methyl-2- { 1 - [9H-purin-6-ylamino] -ethyl} -3H-quinazolin-4-one; 2-(3 -(5 -methyl-4- oxo-2-{l-[9H-purin-6-ylamino]-ethyl},-4H-quinazolin-3-yl-phenoxy acetamide; 5- methyl-2-{l-[9H-purin-6-ylamino]-ethyl}-3-[3-(tetrahydropuran-4-yloxy)-phenyl]- 3H-quinazolin-4-one; 3-[3-(2-methoxy-ethoxy)-phenyl]-5-methyl-2-[l-(9H-purin-6- ylamino)-ethyl]-3H-quinazolin-4-one; 6-fluoro-2-[l-(9H-purin-6-ylamino)ethyl]-3-[3- (tetrahydro-pyran-4-yloxy)-phenyl]-3H-quinazolin-4-one; 3-[3-(3-dimethylamino- propoxy)-phenyl]-5-methyl-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-(3-ethynyl-phenyl)-5-methyl-3H- quinazolin-4-one; 3-{2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5-methyl-4-oxo-4H- quinazolin-3-yl} -benzonitrile; 3-{2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-5- methyl-4-oxo-4H-quinazolin-3-yl}-benzamide; 3-{2-[l-(2-amino-9H-purin-6- ylamino)-ethyl]-5-methyl-4-oxo-4H-quinazolin-3-yl}-benzamide; 5-methyl-3-(3- morpholin-4-yl-phenyl)-2-[l-(9Hr-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 2-[l- (2-amino-9JJ-purin-6-ylamino)-ethyl] -5 -methyl-3 -(3 -moφholin-4-yl-phenyl)-3H~ quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-ethyl]-3-[3-(2-methoxy- ethoxy)-phenyl]-5-methyl-3H-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)- ethyl]-3-[3-(2-dimethylamino-ethoxy)-phenylJ-5-methyl-3H-quinazolin-4-one; 2-[l- (2-amino-9Η-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3Η-quinazolin-4-one; 2-[l-(2-amino-9H-purin-6-ylamino)-but-3-ynyl]-5-methyl-3-phenyl-3H-quinazolin-4- one; 5-chloro-3-(3,5-difluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-ethyl]-3H- quinazolin-4-one; 2-[l-(2-amino-9H-ρurin-6-ylamino)-propyl]-5-chloro-3-(3,5- difluoro-phenyl)-3H-quinazolin-4-oήe; 2-[ 1 -(2-amino-9H-purin-6-ylamino)-ethyl]-5- chloro-3-(3,5-difluoro-phenyl)-3H-quinazolin-4-one; 3-(3,5-difluoro-phenyl)-6- fluoro-2-[l-(9H-purin-6-ylamino)-ethyl]-3H-quinazolin-4-one; 5-chloro-3-(2,6- difluoro-phenyl)-2-[l-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one; 2-[l-(2- amino-9H-purin-6-ylamino)-propyl]-5-chloro-3-(2,6-difluoro-phenyl)-3H-quinazolin- 4-one; 5-methyl-3-phenyl-2-[l-(9H-purin-6-yloxy)-ethyl]-3H-quinazolin-4-one and, pharmaceutically acceptable salts and solvates thereof.
32. An article of manufacture comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) and a label indicating a method of use according to any one of claims 1-31.
33. Use of a composition comprising at least one selective inhibitor of PI3Kδ in the manufacture of a medicament for treating or preventing a disorder involving undesirable mast cell activity.
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