WO1997010004A1 - Methods for treatment of interstitial cystitis - Google Patents

Abstract

Methods of treatment of interstitial cystitis with IgE antagonists, including anti-IgE antibodies, IgE variants, peptide antagonists, peptidomimetics and other small molecules, are described.

Description

METHODS FOR TREATMENT OF INTERSTITIAL CYSTITIS

FIELD OF THE INVENTION This invention relates to methods of treatment of interstitial cystitis with IgE antagonists, including anti- IgE antibodies. BACKGROUND OF THE INVENTION

Interstitial cystitis (IC) is a condition ofthe bladder characterized by urinary frequency, urgency, and suprapubic pain and pressure. Typically, the disease is diagnosed on the basis of cytoscopic appearance or pathological findings.

Immunological responses are associated with numerous urological diseases such as IC, bladder cancer, and bladder infection (Van de Merwe et al. J. Rheumatol. 20: 962, 1993). These responses include an increased number of macrophages, activated lymphocytes, and vascular endothelial cells expressing HLA class II molecules widiin the submucosa (Christmas et al. Clin. EXP. Immunol. 87:450, 1992), increased expression of

HLA-DR in urothelial cells (Liebert et al. J. Urol. 149:470. 1993), and alteration of lymphocyte sub-populations

(CD4:CD8 ratio) in the bladder tissue of IC patients (MacDermott J. Urol. 145:274, 1991). Some investigators have suggested that the immune responses are likely to be secondary phenomena associated with inflammatory damage to the bladder (Anderson et al. Br. J. Urol. 63:58, 1989). However, autoimmune or allergic disorders have been suggested among the potential causes of non-infectious cystitis, including IC (Hand et al. J. Urol.

61:291, 1949; Messing et al. Campbell's Urology. 6th ed.. P.C. Walsh et al. eds., Philadelphia, W.B. Saunders

Co. pp 982-1005, 1991: Holm-Bentzen et al. J. Urol. 138: 500, 1987). Experimentally-induced autoimmune cystitis has many features similar to those observed in clinical IC (Bullock et al. J. Urol. 148:1951, 1992).

Furthermore, reports on IC indicate that at least 50% of IC patients have some form of allergy (Koziol. Urol.

Clin. of N. Amer. 21:7. 1994). Thus, an immunological etiology involving mast cells appears to occur in at least a subset of IC patients.

Bjorling et al. ⁽J. Urol. 152:1603, 1994) describe an experimental model for non-infectious cystitis in which bladder tissue from humans, guinea pigs, or Rhesus monkeys is passively sensitized in vitro by incubation with serum containing antigen-specific immunoglobulin. In this model, subsequent antigen challenge stimulates contraction and histamine release in the sensitized tissue.

Haak-Frenscho etal. ("Human FCeRI-IgG and Humanized anti-IgE monoclonal Antibody Mae 1 1 Block Passive Sensitization of Human and Rhesus Monkey Bladder", presented at 1995 Interstitial Cystitis Symposium, January 9-11), which is not prior art to this invention, disclose both FCeRI-IgG immunoadhesin and humanized anti-IgE antibody abolished IgE-induced contraction and histamine release in an in vitro IC model system.

The concept of using anti-IgE antibodies as a treatment for allergy has been widely disclosed in the scientific literature. A few representative examples are as follows. Baniyash and Eshhar (European Journal of Immunolog^y 14:799-807 (1984)) demonstrated that an anti-IgE monoclonal antibody could specifically block passive cutaneous anaphylaxis reaction when injected intradermally before challenging with the antigen; U.S. 4,714,759 discloses a product and process for treating allergy, using an antibody specific for IgE; and Rup and Kahn (International Archives Allergy and Applied Immunology. 89:387-393 (1989) discuss the prevention of the development of allergic responses with monoclonal antibodies which block mast cell-IgE sensitization. Anti-IgE antibodies which block die binding of IgE to its receptor on basophils and which fail to bind to IgE bound to the receptor, thereby avoiding histamine release are disclosed, for example, by Rup and Kahn (supra), by Baniyash et al. (Molecular Immunology 25:705-711, 1988), and by Hook et al. (Federation of American Societies for Experimental Biology. 71st Annual Meeting, Abstract #6008, 1987). Antagonists of IgE in the form of receptors, anti-IgE antibodies, binding factors, or fragments thereof have been disclosed in the art. For example, U.S. 4,962,035 discloses DNA encoding the alpha-subunit of he mast cell IgE receptor or an IgE binding fragment thereof. Hook et al. (Federation Proceedings Vol. 40, No. 3, Abstract #4177) disclose monoclonal antibodies, of which one type is anti-idiotypic, a second type binds to common IgE determinants, and a third type is directed towards determinants hidden when IgE is on the basophil surface.

U.S. 4,940,782 discloses monoclonal antibodies which react with free IgE and thereby inhibit IgE binding to mast cells, and react wim IgE when it is bound to the B-cell FcE receptor, but do not bind with IgE when it is bound to the mast ceil FcE receptor, nor block the binding of IgE to the B-cell receptor.

U.S.4,946,788 discloses a purified IgE binding factor and fragments thereof, and monoclonal antibodies which react with IgE binding factor and lymphocyte cellular receptors for IgE, and derivatives thereof.

U.S. 5,091,313 discloses antigenic epitopes associated wid the extracellular segment ofthe domain which anchors immunoglobulins to the B cell membrane. The epitopes recognized are present on IgE-bearing B cells but not basophils or in the secreted, soluble form of IgE. U.S. 5,252,467 discloses a method for producing antibodies specific for such antigenic epitopes. U.S. 5,231,026 discloses DNA encoding murine- human antibodies specific for such antigenic epitopes.

U.S. 4,714,759 discloses an imrnunotoxin in the form of an antibody or an antibody fragment coupled to a toxin to treat allergy.

Presta et al. (J. Immunol. 151 :2623-2632 ( 1993)) disclose a humanized anti-IgE antibody that prevents the binding of free IgE to FceRI but does not bind to FceRI-bound IgE. Copending WO93/04173 discloses polypeptides which bind differentially to die high- and low-affinity IgE receptors. Copending WO93/04173 discloses IgE antagonists comprising one or more ofthe FceRI receptor-binding determinant sites of human IgE.

U.S. 5,428,133 discloses anti-IgE antibodies as a therapy for allergy, especially antibodies which bind to IgE on B cells, but not IgE on basophils. U.S. 5,422,258 discloses a method for making such antibodies.

Haak-Frenscho et al. (J. Immunol. 151:351-358, 1993) disclose an FCeRI-IgG immunoadhesin which is a fusion ofthe extracellular portion ofthe human α-chain of FCeRI, which contains d e high affinity binding site for IgE, with a truncated human IgGl heavy chain constant region.

SUMMARY OF THE INVENTION One embodiment ofthe invention is a method of treatment of interstitial cystitis in a patient comprising administering a therapeutic dose of an IgE antagonist to the patient. Another embodiment of die invention is a method of reducing histamine release from mast cells in the bladder tissue of a patient widi interstitial cystitis comprising administering a therapeutic dose of an IgE antagonist to the patient. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph depicting representative concentration-dependent contraction of human bladder tissue segments sensitized by incubation widi serum from a ragweed allergic patient and induced by cumulative addition of ragweed antigen (antigen E or AgE). The change in the symbols represents cumulative addition of increasing concentrations of AgE (0.01, 0.1, 1.0, and 3.0 μg/ml) or KCI (200 mM). The maximal effect was obtained with 1.0 μg/ml AgE. The highest concentration tested, 3.0 μg/ml, reduced contraction in the tissue. The graph represents tissue contractability and viability results obtained widi eight different bladders.

Figure 2 is a graph depicting concentration-dependent contraction of sensitized human bladder tissue segments induced by addition of a single concentration of AgE ((0.01, 0.1, 1.0, and 3.0 μg/ml). Responses are represented as a percentage ofthe maximum obtained in response to KCI (200 mM) added at the end of die experiment. The graph represents results obtained widi eight different bladders.

Figure 3 (A-D) represents polygraph tracings representative of tissues isolated from eight different bladders. Adjacent segments of each bladder were passively sensitized, suspended in vitro, and challenged with AgE (1 μg/ml) at 10 min (first arrow). Contractions are expressed as grams of tension. Segment A was a non- sensitive negative control; segment B was a sensitized positive control incubated widi control IgG; segment C was incubated widi anti-IgE monoclonal antibody E25 (1 :1 concentration of E25 to IgE) during sensitization; segment D was incubated wid E25 (5: 1 concentration of E25 to IgE) during sensitization. KCI (200 mM) was added at the end ofthe experiment (second arrow).

Figure 4 (A and B) represents IgE antagonist blocking of histamine release (A) and tissue contraction (B) in a concentration-dependent manner in sensitized human bladder tissue. Human bladder strips were incubated widi a 1 : 10 dilution of human ragweed serum for 20 hr. in the presence of equimolar, 5-fold, or 10-fold excess concentrations of E25 or a 10-fold concentration of control IgG in excess ofthe serum IgE concentration.

IgG had no detectable effect on either histamine release (A) or tissue contraction (B). In contrast, E25 blocked both histamine release and tissue contraction in response to IgE challenge in a concentration-dependent manner. Data are presented as the mean ± SEM of eight separate experiments. Peak histamine release occurred at 3 min. and correlated with the onset of contraction. In all experiments, adjacent bladder strips incubated with physiologic salt solution (PSS) only (nonsensitized) for 20 hr did not respond to AgE challenge. Asterisks indicate time points at which a significant difference was observed (p<0.05) compared to control IgG responses.

Figure 5 (A and B) represents IgE antagonist blocking of histamine release (A) and tissue contraction (B) in a concentration-dependent manner in sensitized Rhesus monkey bladder tissue. Rhesus monkey bladder strips were incubated widi a 1 : 10 dilution of human ragweed serum for 20 hr in the presence of equimolar, 5-fold, or 10-fold excess concentrations of E25 or a 10-fold concentration of control IgG in excess ofthe serum IgE concentration. IgG had no detectable effect on either histamine release (A) or tissue contraction (B). In contrast,

E25 blocked both histamine release and tissue contraction in response to AgE challenge in a concentration- dependent manner. Data are presented as the mean ± SEM of 14 separate experiments. Peak histamine release occurred at 3 min and correlated with the onset of contraction. In all experiments, adjacent bladder strips incubated with PSS only (nonsensitized) for 20 hr did not respond to AgE challenge. Asterisks indicate time points at which a significant difference was observed (p<0.05) compared to control IgG responses. Figure 6 is a graph depicting degranulation and tissue contraction in passively sensitized human (full bar) or monkey (empty bar) bladder tissues. Bladder segments were passively sensitized with ragweed serum, washed with PSS and challenged with 1 μg/ml E25. Positive control bladders challenged with AgE exhibited vigorous bladder contraction (percent of KCI maximum and histamine release (ng/g of tissue). In contrast, challenge with E25 did not result in any detectable histamine release or tissue contraction. Non-sensitized bladders segments (negative control) challenged widi AgE did not respond. Results are expressed as die mean responses of tissue from five human bladders and 10 monkey bladders. Asterisks and SEM are not shown for sake of clarity. A significant difference (p<0.05) was found between AgE and E25 challenge of sensitized tissues. DETAILED DESCRIPTION OF THE INVENTION

A. DEFINITIO S

The term "interstitial cystitis' as used herein is intended to refer to a bladder condition characterized by urinary frequency, urgency, and suprapubic pain and pressure.

The term "IgE antagonist" as used herein refers to a substance which inhibits the biological activity of IgE. Such antagonists include but are not limited to anti-IgE antibodies, immunoadhesins, IgE receptors, anti- IgE receptor antibodies, variants of IgE antibodies, ligands for die IgE receptors, and fragments thereof. Antibody antagonists can be ofthe IgA, IgD, IgG, IgE, or IgM class. Bispecific antibodies can also be used. Variant IgE antibodies typically have amino acid substitutions or deletions at one or more amino acid residues. Ligands for IgE receptors include but are not limited to IgE and anti-receptor antibodies, and fragments thereof capable of binding to die receptors, including amino acid substitution and deletion variants, and cyclized variants.

In general, in some embodiments ofthe invention, IgE antagonists act by blocking the binding of IgE to its receptors on B cells, mast cells, or basophils, either by blocking die binding site on die IgE molecule or blocking its receptors. Additionally, in some embodiments of die invention, IgE antagonists act by binding soluble IgE and thereby removing it from circulation. The IgE antagonists of the invention can also act by binding to IgE on B cells, thereby eliminating clonal populations of B cells. The IgE antagonists ofthe instant invention can also act by inhibiting IgE production. Preferably, die IgE antagonists ofthe instant invention do not result in histamine release from mast cells or basophils.

The term "therapeutic amount" as used herein denotes an amount at prevents or ameliorates symptoms of a disorder or responsive patiiologic physiological condition. "Polypeptide" as used herein refers generally to peptides and proteins having at least about two amino acids.

The term "free IgE" as used herein refers to IgE not complexed to a binding partner, such an anti-IgE antibody. The term "total IgE" as used herein refers to the measurement of free IgE and IgE complexed to a binding partner, such as an anti-IgE antibody. The term "baseline IgE" as used herein refers to the level of free IgE in a patient's serum before treatment with an IgE antagonist.

The term "polyol" as used herein denotes a hydrocarbon including at least two hydroxyls bonded to carbon atoms, such as polyethers (e.g. polyediylene glycol), trehalose, and sugar alcohols (such as mannitol). The term "polyether" as used herein denotes a hydrocarbon containing at least three ether bonds. Polyethers can include other functional groups. Polyethers useful for practicing die invention include polyediylene glycol (PEG). B. GENERAL METHODS Polyclonal antibodies to IgE generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of IgE and an adjuvant. It can be useful to conjugate IgE or a fragment containing the target amino acid sequence from die Fc region of IgE to a protein diat is immunogenic in die species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine tiiyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (dirough lysine residues), glutaraldehyde, succinic anhydride, SOCI₂, or R'N = C = NR, where R and R¹ are different alkyl groups.

Animals ordinarily are immunized against the cells or immunogenic conjugates or derivatives by combining 1 mg or 1 μg of IgE with Freund's complete adjuvant and injecting die solution intradermally at multiple sites. One month later the animals are boosted widi 1/5 to 1/10 die original amount of conjugate in Freund's incomplete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, animals are bled and die serum is assayed for anti-IgE titer. Animals are boosted until die titer plateaus. Preferably, die animal is boosted widi a conjugate of the same IgE, but conjugated to a different protein and/or dirough a different cross-linking agent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum can be used to enhance die immune response. Monoclonal antibodies are prepared by recovering spleen cells from immunized animals and immortalizing the cells in conventional fashion, e.g. by fusion widi myeloma cells or by Epstein-Barr (EB)-virus transformation and screening for clones expressing die desired antibody. The hybridoma technique described originally by Koehler and Milstein, Eur. J. Immunol.. £: 51 1 (1976) and also described by Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas. Elsevier, N.Y., pp. 563-681 (1981) has been widely applied to produce hybrid cell lines diat secrete high levels of monoclonal antibodies against many specific antigens.

The hybrid cell lines can be maintained in vitro in cell culture media. The cell lines producing die antibodies can be selected and/or maintained in a composition comprising the continuous cell line in hypoxandiine-aminopterin thymidine (HAT) medium. In fact once the hybridoma cell line is established, it can be maintained on a variety of nutritionally adequate media. Moreover, the hybrid cell lines can be stored and preserved in any number of conventional ways, including freezing and storage under liquid nitrogen. Frozen cell lines can be revived and cultured indefinitely widi resumed syndiesis and secretion of monoclonal antibody.

The secreted antibody is recovered from tissue culture supernatant by conventional methods such as precipitation, ion-exchange chromatography, affinity chromatography, or die like. The antibodies described herein are also recovered from hybridoma cell cultures by conventional metiiods for purification of IgG or IgM, as die case may be, that heretofore have been used to purify tiiese immunoglobulins from pooled plasma, e.g., ethanol or polyethylene glycol precipitation procedures. The purified antibodies are sterile-filtered.

While routinely mouse monoclonal antibodies are used, die invention is not so limited; in fact, human antibodies can be used. Such antibodies can be obtained, for example, by using human hybridomas (Cote et al., Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, p. 77 (1985)). In fact, according to die invention, techniques developed for die production of chimeric antibodies (Cabilly et al., U.S. 4,816,567, Morrison et al., Proc. Natl. Acad. Sci. 81: 6851 (1984); Boulianne et al., jjyjs 312: 643-646 (1984); Neuberger et al., Nature. 312: 604 (1984); Neuberger et al., Nature 314: 268-270 (1985); Takeda et al., Nature 314: 452 (1985); EP 184,187; EP 171,496; EP 173,494; PCT WO 86/01533; Shaw et al., J. Nat. Cane. Inst. 80: 1553-1559 (1988); Morrison, Science 229: 1202-1207 (1985); Oi et al., BioTechniques. 4: 214 (1986)) by coupling an animal antigen-binding variable domain to a human constant domain can be used; such antibodies are within die scope of diis invention. The term "chimeric" antibody is used herein to describe a polypeptide comprising at least the antigen binding portion of an antibody molecule linked to at least part of another protein (typically an immunoglobulin constant domain). In one embodiment, such chimeric antibodies contain about one diird rodent (or odier non-human species) sequence and thus are capable of eliciting a significant anti-globulin response in humans. For example, in die case of die murine anti-CD3 antibody OKT3, much of die resulting anti-globulin response is directed against die variable region rather than die constant region (Jaffers et al., Transplantation 41 : 572-578 (1986)).

Humanized antibodies are used to reduce or eliminate any anti-globulin immune response in humans. In practice, humanized antibodies are typically human antibodies in which some amino acid residues from the complementarity determining regions (CDRs), the hypervariable regions in die variable domains which are directly involved widi formation of die antigen-binding site, and possibly some amino acids from the framework regions (FRs), die regions of sequence diat are somewhat conserved widiin die variable domains, are substituted by residues from analogous sites in rodent antibodies. The construction of humanized antibodies is described in Riechmann et al., MaQjre.332: 323-327 (1988), Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989), Co et al.. Proc. Natl. Acad. Sci. USA 88: 2869-2873 (1991), Gorman et al.. Proc. Natl. Acad. Sci. 88: 4181-4185 (1991), Daugherty et L. Nucleic Acids Res. 19: 2471-2476 (1991), Brown et aL, Proc. Natl. Acad. Sci. USA 88: 2663-2667 (1991), Junghans et aL. Cancer Res. 50: 1495-1502 (1990), Fendly et al., Cancer Res. 50: 1550-1558 (1990) and in PCT applications WO 89/06692 and WO 92 22653. In some cases, substituting CDRs from rodent antibodies for the human CDRs in human frameworks is sufficient to transfer high antigen binding affinity (Jones et aL, NJJJHS 321 : 522-525 (1986); Verhoeyen et al., Science 239: 1534-1536 (1988)) whereas in other cases it is necessary to additionally replace one (Riechmann et al., supra) or several (Queen et al., supra) FR residues. See also Co et al., supra.

The invention also encompasses die use of human antibodies produced in transgenic animals. In this system, DNA encoding the antibody of interest is isolated and stably incorporated into die germ line of an animal host. The antibody is produced by die animal and harvested from die animal's blood or odier body fluid.

Alternatively, a cell line that expresses the desired antibody can be isolated from die animal host and used to produce die antibody in vitro, and the antibody can be harvested from die cell culture by standard mediods.

Anti-IgE antibody fragments can also be used in die methods of die invention. Any fragment of an anti- IgE antibody capable of blocking or disrupting IgE interaction widi its receptor is suitable for use herein.

Suitable anti-IgE antibody fragments can be obtained by screening combinatorial variable domain libraries for DNA capable of expressing the desired antibody fragments. These techniques for creating recombinant DNA versions ofthe antigen-binding regions of antibody molecules which bypass the generation of monoclonal antibodies, are encompassed widiin die practice of diis invention. One typically extracts antibody- specific messenger RNA molecules from immune system cells taken from an immunized animal, transcribes these into complementary DNA (cDNA), and clones die cDNA into a bacterial expression system. "Phage display" libraries are an example of such techniques. One can rapidly generate and screen great numbers of candidates for those diat bind die antigen of interest Such IgE-bindiπg molecules are specifically encompassed widiin die term "antibody" as it is defined, discussed, and claimed herein.

In a further embodiment of die invention, soluble IgE receptor can be used as die IgE antagonist.

Soluble receptors suitable for use herein include, for example, molecules comprising die IgE binding site in die extracellular domain (exodomain) ofthe FceRI a chain. The α chain of FceRI can be genetically modified such diat die exodomain is secreted as a soluble protein in a recombinant expression system according to the mediod of Blank et al., J. Biol. Chem..26£: 2639-2646 (1991) or Qu et al., J. Exp. Med.. 167: 1195.

The invention also encompasses die use of IgE-binding peptides in addition to anti-IgE antibodies and soluble receptor. Any IgE-binding peptide capable of disrupting or blocking die interaction between IgE and its receptors is suitable for use herein.

In addition to IgE antagonists which interfere widi IgE/receptor interaction by binding to IgE, such as anti-IgE antibodies, fragments thereof, soluble IgE receptor and other IgE-binding peptides described above, die invention encompasses die use of IgE antagonists which disrupt IgE/receptor interaction by competing with IgE for binding to its receptor, thereby lowering die available IgE receptor.

IgE variants are an example of a receptor-binding competitor that is suitable for use in the methods of die invention. IgE variants are forms of IgE possessing an alteration, such as an amino acid substitution or substitutions and/or an amino acid deletion or deletions, wherein die altered IgE molecule is capable of competing with IgE for binding to its receptors.

Fragments of IgE variants are also suitable for use herein. Any fragment of an IgE variant capable of competing witii IgE for binding to its receptors can be used in die mediods ofthe invention.

The invention also encompasses die use of IgE receptor-binding peptides in addition to IgE variants and fragments thereof. Any IgE receptor-binding peptide capable of disrupting or blocking the interaction between

IgE and its receptors is suitable for use herein.

The amount of IgE antagonist delivered to the patient to be used in therapy will be formulated and dosages established in a fashion consistent with good medical practice taking into account the disorder to be treated, die condition of die individual patient the site of delivery, the method of administration and odier factors known to practitioners. Similarly, die dose of die IgE antagonist administered will be dependent upon die properties of die IgE antagonist employed, e.g. its binding activity and in vivo plasma half-life, die concentration of die IgE antagonist in die formulation, die administration route, die site and rate of dosage, die clinical tolerance ofthe patient involved, die pathological condition afflicting die patient and the like, as is well widiin die skill of die physician. Typically IgE antagonists are administered by intramuscular, intravenous, intrabronchial, intraperitoneal, intravesical, subcutaneous or other suitable routes. The antagonists can be administered before and/or after the onset of symptoms. In general, a "loading" dose of an IgE antagonist is useful to obtain a rapid and sustained decrease in free IgE. A loading dose is typically a first dose of IgE antagonist that is greater dian a subsequent or "maintenance" dose of IgE antagonist. However, patients can be loaded in other ways. For example, patients can be loaded by administering a dose of antagonist diat is greater than or equal to the same mg/kg amount as die maintenance dose, but increasing the frequency of administration in a "loading regimen". Thus, for example, if the maintenance dose is 1 mg/kg biweekly, the patient can be loaded by administering 1 mg/kg weekly for two or more weeks in a row, then administering die maintenance dose of 1 mg/kg biweekly. Furthermore, patients can be loaded during a course of treatment widi a maintenance dose of IgE antagonist by administering larger or more frequent doses than die maintenance dose. The term "loading dose" is intended as used herein to include such single loading doses, multiple loading doses, loading regimens, and combinations diereof.

A sustained decrease in free IgE can be obtained by administration of a maintenance dose of die antagonist Maintenance doses are delivered widi a frequency of about every day to about every 90 days, more preferably weekly to biweekly, depending on die severity ofthe patient's symptoms, die concentration and in vivo properties of antagonist delivered, and the formulation of die antagonist. For example, slow release formulations can allow less frequent administration. Maintenance doses can be adjusted upwards or downwards over time, depending on the response of die patient. Thus, for example, in one embodiment of die invention, die dose of IgE antagonist is sufficient to reduce free IgE in the patient's serum to less an about 40 ng/ml.

In a further dosing strategy, about 0.05 to 10 mg/kg, more preferably about 0.1 to 1 mg/kg, most preferably about 0.5 mg/kg IgE antagonist can be administered on a weekly basis to a patient having about 40- 200 IU/ml baseline IgE. In another dosing strategy for individuals widi higher baseline IgE, patients are preferably "loaded" with about 1 to about 10 mg/kg, more preferably about 1 to about 5 mg/kg, most preferably about 2 mg/kg, IgE antagonist followed by weekly or biweekly administration of about 0.1 to about 10 mg/kg, most preferably about 1 mg/kg.

In a further dosing strategy, a maintenance dose of IgE antagonist averaging about 0.0005 to 0.05 mg/kg/week for every IU/ml baseline IgE, more preferably 0.001 to about 0.01 mg/kg/week for every IU/ml baseline IgE is used. This maintenance regimen can follow an initial loading dose of about 1 to 10 mg/kg, more preferably about 1 to 5 mg kg IgE antagonist.

In a further embodiment of die invention, sufficient IgE antagonist is provided dirough die maintenance dose, and, optionally, die loading dose, to achieve about a 1 to 20 fold, preferably about 3 to 5 fold, most preferably about a 5 fold greater serum concentration than total serum IgE concentration in die patient. IgE levels are typically assayed by standard ELISA techniques well known in the art. Total serum IgE can be measured by commercially available assays, such as Abbott Laboratories' Total IgE assay. Free IgE, e.g., IgE not bound to antibody can be measured by a capture type assay in which, for example, IgE receptor is bound to a solid support. IgE complexed to an anti-IgE antibody which binds at or near the site on IgE which binds to die receptor will be blocked from binding die receptor, and tiius only free or unbound IgE can react with die receptor bound to die solid support in diis assay. An anti-IgE antibody which recognizes IgE even when die IgE is bound to its receptor can be used to detect die IgE captured by die receptor on the solid support. This anti-IgE antibody can be labeled widi any of a variety of reporter systems, such as alkaline phosphatase, etc.

It is envisioned diat injections (intravenous, intramuscular or subcutaneous) will be die primary route for therapeutic administration of die IgE antagonist of this invention, although delivery through catheter or other surgical tubing is also used. Alternative routes include suspensions, tablets, capsules and die like for oral administration, commercially available nebulizers for liquid formulations, and inhalation of lyophilized or aerosolized microcapsules, and suppositories for rectal or vaginal administration. Liquid formulations can be utilized after reconstitution from powder formulations. Additional pharmaceutical memods may be employed to control the duration of action of die antagonists of this invention. The antagonists also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(metiιylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences. 16th edition, Osol, A., ed., 1980).

In general, the formulations of the subject invention can contain other components in amounts not detracting from die preparation of stable forms and in amounts suitable for effective, safe pharmaceutical administration. For example, odier pharmaceutically acceptable excipients well known to those skilled in die art can form a part of the subject compositions. These include, for example, salts, various bulking agents, additional buffering agents, chelating agents, antioxidants, cosolvents and die like; specific examples of tiiese include tris-(hydroxymetiιyl)aminometiιane salts ("Tris buffer"), and disodium edetate.

In one embodiment of die invention, IgE antagonist formulations comprise a buffer, a salt optionally, a polyol, and optionally, a preservative. One exemplary formulation of die invention is a liquid formulation of about 1 - 100 mg/ml IgE antagonist in 10 mM acetate buffer, pH 5.0-6.5, 100-200 mM sodium chloride, and about 0.01% polysorbate 20, more preferably about 5 mg/ml IgE antagonist in 10 mM acetate buffer, pH 5.2, 142 mM sodium chloride, and 0.01% polysorbate 20. In other embodiments of die invention, the formulation may be freeze-dried and reconstituted for administration. For example, anti-IgE antibody can be formulated at about 25 mg/ml in 5 M histidine, pH 6.0, and 88 M sucrose, freeze-dried, and reconstituted in water to 100 mg/ml antibody for administration. Mixed sugars can also be used, such as a combination of sucrose and mannitol, etc.

In general, unless otherwise specified, die abbreviations used for the designation of amino acids and die protective groups used tiierefor are based on recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature (Biochemistry. 11 : 1726-1732 (1972). The nomenclature used to define compounds ofthe invention is diat specified by die IUPAC, published in European Journal of Biochemistry 138:9-37 (1984).

Therapy of interstitial cystitis can be combined widi odier known therapies for allergy and or interstitial cystitis, including corticosteroids, immunosuppressive drugs, anti-inflammatory drugs, antihistamiπes, pentosanpolyphosphate, heparin, arnitriptyline. dimetiiyl sulfoxide, oxychlorosene sodium, silver nitrate, disodium chromoglycate, etc. Further details ofthe invention can be found in die following examples, which further define die scope of the invention. All references cited herein are expressly incorporated by reference in their entireties.

EXPERIMENTAL RESULTS In this study a model of passively sensitized human and monkey bladder tissues was used to test die therapeutic potential of an IgE antagonist for the treatment of interstitial cystitis. Human bladder segments were obtained from cystectomies performed to treat bladder carcinoma. Only macroscopicaliy healthy portions of die bladder were used. However, all patients were receiving medical therapy to treat the carcinoma at the time tissues were obtained. In contrast monkey bladder tissue was obtained from healthy animals euthanized for research purposes. Bladder tissues were passively sensitized using the mediod of Bjorling et al. (J. Urol. 152: 1603, 1994).

Basically, bladder tissue was cleansed and prepared as entire (neck to dome) full diickness strips (5 X 18 mm). Tissue segments were placed in physiologic salt solutions (PSS) ofthe following composition: 119 mM NaCl/ 4.7 mM KCI/ 1.0 mM NaH₂P0₄/ 0.5 mM MgCl₂/ 2.5 mM CaC 25 mM N21HCO3/ 1 1 mM glucose at pH 7.4, gassed wim 95% O and 5% C0₂, and maintained at 37° C. Bladder segments were washed four times with 50 ml PSS, placed in 50 ml of PSS, and passively sensitized by incubation for 15 to 20 hr at 25°C widi a 1:10 dilution of serum from a ragweed-allergic patient. The total IgE content of the serum was 1250 ng/ml, as determined by ELISA.

To test die ability of an IgE antagonist to block sensitization, tissues were sensitized in the presence of anti-IgE antibody E25 (Presta et al., supra) at concentrations 1, 5, and 10 times die amount of serum IgE or with control IgG at 10 times the concentration of serum IgE. Tissues incubated widi PSS only were used as negative controls, and those incubated with ragweed serum oniy were preserved as positive controls.

After sensitization, each tissue was suspended in an air-filled tissue chamber and superfused with PSS. All solutions, as well as die tissue chamber, were maintained at 37° C. PSS was pumped from a reservoir through Tygon tubing to a water-jacketed coiled glass tube heat exchanger using a Gilson Minipulse II peristaltic pump. To remove excess sera, tissues were allowed to equilibrate for 90 minutes while perfused with PSS ( 1 ml min) and maintained at a tension of 1.5 g. Changes in tension were recorded via force displacement transducers (FT- 03, Grass Instruments, Quincy, MA) on a polygraph (Model 7D, Grass Instruments, Quincy, MA). Following equilibration, tissues were superfused (1 ml/min) with ragweed antigen (AgE) (0.01 to 3 μg/ml) diluted in PSS. Superfusate samples were collected at 60 second intervals, beginning one minute before and continuing 16 minutes during challenge with IgE. Contractile responses were calculated as a percent of maximal contraction induced by KCI (200 M) added at the end of each experiment. After collection, superfusate samples were placed on crushed ice for subsequent analysis of histamine content. The remaining tissue histamine was extracted widi 0.4 N perchloric acid.

To test die safety ofthe anti-IgE monoclonal antibody E25, adjacent segments of human bladder were passively sensitized widi human ragweed serum. Following sensitization, bladder segments were suspended in tissue batiis containing 10 ml of PSS (37° C) and maintained at a tension of 1.5 g for 1 hr of equilibration during which the PSS was changed every 15 minutes. Changes in tension were recorded via force displacement transducers (FT-03) on a Grass polygraph (Model 7D). After the equilibration period, tissues were challenged for 30 min widi E25 (10 μg/ml) or AgE (1 μg ml), die latter used as a positive control. Histamine content ofthe superfusate and tissues was analyzed by an automated fluorometric method widi a sensitivity of 1.5 ng/ml. The net concentration of histamine (release minus spontaneous) in each superfusate sample was expressed as a per cent of total histamine in each tissue prior to collection.

The mean and SEM were calculated for all data. Means were compared by analysis of variance or Student's t test. A value of p< 0.05 was considered indicative of significant difference. When passively sensitized segments of human bladder were challenged in vitro with different AgE concentrations added in a cumulative fashion to die tissue bath, a concentration-dependent contraction was observed (Figure 1). Cumulative responses to AgE were maximal at 1 μg/ml and decreased when tissues were exposed to 3 μg/ml. To verify if die decreased response to high concentration of antigen was due to a desensitization phenomena, individual tissue segments were incubated with single concentrations of antigen and responses were observed over time (Figure 2). The responses were maximal for 1 μg/ml antigen. Indeed, contraction in response to 3 μg ml AgE was lower an concentration induced by 1 μg/ml. The responses of monkey bladder were similar (data not shown). Therefore, a concentration of 1 μg/ml AgE was used in all subsequent experiments as described below. Figure 3 illustrates representative contractile responses typically observed widi antigen challenge of tissues obtained from eight different human bladders utilized in this experiment. The tissue segment in Figure 3A was not sensitized and served as a negative control. Adjacent segments of die same human bladder (3B, C, and D) were passively sensitized in vitro. AgE (1 μg/ml) challenge failed to induce contraction ofthe non-sensitized bladder segment (Figure A), which exhibited only spontaneous motility. The contractile responses to AgE (1 μg/ml) were evident when a bladder tissue segment was used as a positive control for sensitization. Tissue segments treated witii equimolar E25 (1:1 IgE to E25 concentrations) during sensitization had botii a reduced and delayed response to AgE (Figure 3C). Tissue segments treated witii E25 (5-fold excess E25) during sensitization exhibited no measurable response to AgE (Figure 3D). All tissue segments contracted in response to KCI (200 mM) added at the end of experiments, confirming viability. Figure 4 summarizes results of histamine release obtained widi human urinary bladder segments passively sensitized in die presence of E25. E25 blocked AgE-induced histamine release and tissue contraction in a concentration-dependent manner. Equimolar concentrations of serum IgE and E25 resulted in significant inhibition of botii histamine release and tissue contraction. Five-fold excess concentration of E25 completely blocked the response to AgE challenge. In contrast, tissues preincubated widi ragweed serum and a 10-fold excess concentration of control IgG had histamine release and tissue contraction indistinguishable from die positive control tissues. Similar results were observed witii the Rhesus monkey bladder tissue (Figure 5 A-B). There was a dose-dependent inhibition of sensitization and a five-fold molar excess of E25 was need to completely block response to IgE challenge.

Challenge of eitiier passively sensitized human or monkey bladder tissue with AgE at 1 μg/ml induced histamine release and tissue contraction (Figure 6). However, in striking contrast no histamine release or tissue contraction was observed in response to challenge wim up to 10 μg/ml E25. These results indicate mat E25 does not bind human IgE that is already bound to high affinity receptors on human and monkey bladder mast cells. Thus, in these experiments, an IgE antagonist effectively blocked mast cell degranulation and histamine release in a model for non-infectious cystitis. These results indicate that an IgE antagonist can be used therapeutically in the treatment of interstitial cystitis.

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Claims

WE CLAIM:

1. A mediod of treatment of interstitial cystitis in a patient comprising administering a therapeutic dose of an IgE antagonist to the patient.

2. The method of claim 1, wherein die therapeutic dose comprises a maintenance dose of an IgE antagonist and, optionally, a loading dose of die IgE antagonist.

3. The method of claim 2, wherein the maintenance dose is repeated at intervals of about 1 to about 90 days.

4. The method of claim 2, wherein the maintenance dose is repeated weekly.

5. The method of claim 2, wherein the maintenance dose is repeated biweekly.

6. The method of claim 1 , wherein the IgE antagonist is an anti-IgE antibody.

7. The mediod of claim 6, wherein the antibody is chimeric.

8. The method of claim 7, wherein the antibody is humanized.

9. The metiiod of claim 6. wherein the antibody is a human antibody.

10. The mediod of claim 2, wherein the antagonist binds to soluble IgE and blocks the binding of IgE to die IgE receptor on basophils.

11. The method of claim 6, wherein the antibody binds to soluble IgE and blocks die binding of IgE to tiie IgE receptor on basophils.

12. The method of claim 2, wherein the loading dose is greater an die maintenance dose.

13. The method of claim 1, wherein the therapeutic dose reduces die concentration of free IgE in the patient's serum to less than about 40 ng/ml.

14. The method of claim 1, wherein die therapeutic dose of antagonist is about 0.001 to 0.01 mg/kg/week baseline IgE IU/ml.

15. The metiiod of claim 1, wherein the therapeutic dose results in a total serum concentration of antagonist of about 1 to 10 times greater an die patient's total serum IgE concentration. 1 . The method of claim 1 , wherein the therapeutic dose of IgE antagonist averages about 0.001 to

0.01 mg/kg/week IgE antagonist for every IU/ml baseline IgE in the patient's serum.

17. A method of reducing histamine release from mast cells in the bladder tissue of a patient with interstitial cystitis comprising administering a therapeutic dose of an IgE antagonist to the patient.