WO2007047489A2

WO2007047489A2 - Compositions and methods for use in cancer therapy

Info

Publication number: WO2007047489A2
Application number: PCT/US2006/040183
Authority: WO
Inventors: Mark Brann; Hans Schiffer
Original assignee: Acadia Pharmaceuticals Inc.
Priority date: 2005-10-18
Filing date: 2006-10-16
Publication date: 2007-04-26
Also published as: WO2007047489A3

Abstract

Disclosed herein are compositions for co-administering an RTK inhibitor and an RTK ligand. Also disclosed are methods of ameliorating an adverse effect due to the administration of an RTK inhibitor by co-administering an RTK inhibitor and RTK ligand. Also disclosed are methods of informing a subject that co-administration of an RTK inhibitor and an RTK ligand ameliorates an adverse effect due to the administration of an RTK inhibitor.

Description

COMPOSITIONS AND METHODS FOR USE IN CANCER THERAPY

FIELD OF THE INVENTION

[0001] The present invention relates to the field of medicine. In particular, it relates to pharmaceutical compositions and methods for ameliorating one or more adverse effects associated with administering a chemotherapeutic agent.

BACKGROUND

[0002] Overexpression and activation of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), plays an important role in the etiology and progression of cancers, such as advanced non-small-cell lung cancer (NSCLC). Consequently, EGFR is recognized as a key target for the development of anticancer therapies. Two drug development strategies focusing on EGFR inhibition are currently pursued: (I) the identification of reversible or irreversible small molecule drugs that inhibit the intracellular tyrosine kinase activity of EGFR by competitively binding to the ATP- binding site of the kinase domain, and (II) the identification of humanized monoclonal antibodies (Mab) that interact with extra-cellular EGFR domains interfering with ligand binding (e.g. epidermal growth factor, EGF) or EGFR dimerization. The reversible quinazoline EGFR inhibitors gefitinib (Irresa, ZD1839) and erlotinib (Tarceva, OSI-774) and the antibody drug cetuximab (Erbitux, MC-C225) have already been marketed in the U.S. for NSCLC. ^' '

[0003] The presence of certain mutations in EGFRs in the lung tissue of NSCLC patients is significantly associated with a clinical response to treatment with gefitinib or erlotinib. These somatic EGFR mutations include point mutations that change single amino acids and small in frame deletions with or without insertions of amino acids. The majority of mutations is clustered in the activating loop of the EGFR kinase domain and has been more frequently found in females, Asians, never-smokers and adenocarcinomas. Importantly, cancer cell lines, which endogenously express high levels of the mutated EGFRs, are significantly more sensitive to growth inhibition when treated with gefitinib or erlotinib. However, some NSCLC patients who respond to treatment with gefitinib or erlotinib lack such somatic mutations, indicating that additional mechanism(s) exist that promote sensitivity to these drugs.

[0004] The currently marketed quinazoline EGFR inhibitors represent a convenient treatment option for patients that show a response to these compounds because they can be orally, self administered according to a once-daily treatment regimen. However, the amount of quinazoline EGFR inhibitor that can be administered, and thus the inhibitory effect, is limited by the high incidence of adverse side effects. In particular, both gefitinib and erlotinib produce adverse gastrointestinal effects and skin disorders in a large portion of the patient population receiving either of these treatments. Accordingly, there is a need to ameliorate the adverse gastrointestinal effects and skin disorders associated with the oral administration of quinazoline therapeutics. More generally, there is a need to reduce adverse effects associated with administering any RTK inhibitor to patients in need of RTK inhibitor therapy.

SUMMARY OF THE INVENTION

[0005] Aspects of the present invention relate to pharmaceutical compositions for increasing receptor tyrosine kinase (RTK) function in noncancerous tissues of patients that are treated with RTK inhibitors. In some embodiments, pharmaceutical compositions comprise a first dosage form comprising an RTK inhibitor and a second dosage form comprising an RTK ligand. In certain embodiments, the RTK ligand interacts with an RTK that is inhibited by the supplied RTK inhibitor. Pharmaceutical compositions described herein can be for any type of administration including, but not limited to, oral, parenteral and topical administration.

[0006] Further aspects of the present invention relate to methods for increasing RTK function in noncancerous tissues of patients that are treated with RTK inhibitors. In some embodiments, the method comprises co-administering an RTK inhibitor and an RTK ligand. In certain embodiments, the RTK ligand interacts with an RTK that is inhibited by the supplied RTK inhibitor. RTK inhibitors and RTK ligands can be co-administered by various routes of administrations including, but not limited to, oral, parenteral and topical administration.

[0007] Still other aspects of the present invention relate to methods of using an RTK ligand to ameliorate adverse effects associated with administration of an RTK inhibitor in a human. The method comprises informing the human that co-administering RTK ligand with an RTK inhibitor ameliorates at least one adverse effect associated with the administration of the RTK inhibitor. In some embodiments, the RTK ligand interacts with an RTK that is inhibited by the supplied RTK inhibitor.

[0008] Aspects of the invention also relate to methods of manufacturing a pharmaceutical composition, wherein the method comprises obtaining a first oral dosage form comprising an RTK inhibitor, obtaining a second oral dosage form comprising an RTK ligand, and packaging together the first oral dosage form and the second oral dosage form. In some embodiments, the RTK ligand interacts with an RTK that is inhibited by the supplied RTK inhibitor.

[0009] Further aspects of the present invention relate to a pharmaceutical composition produced by the above-described manufacturing methods.

[0010] One aspect of the present invention relates to a pharmaceutical composition comprising a first dosage form, which comprises epidermal growth factor receptor (EGRF) inhibitor and a second dosage form, which comprises an EGFR ligand. In some embodiments, the first and second dosage forms are combined into a single dosage form.

[0011] Another aspect of the present invention relates to methods of ameliorating adverse effects associated with administration of an EGFR inhibitor. The methods comprise co-administering to a patient an EGFR inhibitor and an EGFR ligand.

[0012] A further aspect of the present invention relates to methods of using an EGFR ligand to ameliorate adverse effects associated with administration of an EGFR inhibitor in a human. The method comprises informing the human that co-administering an EGFR ligand with an EGFR inhibitor ameliorates at least one adverse effect associated with the administration of an EGFR inhibitor. [0013] Another aspect of the present invention relates to methods of manufacturing a pharmaceutical composition, wherein the method comprises obtaining a first oral dosage form comprising an EGFR inhibitor, obtaining a second oral dosage form comprising an EGFR ligand, and packaging together the first oral dosage form and the second oral dosage form. In some embodiments, the EGFR ligand comprises EGF. In other embodiments, the EGFR inhibitor comprises gefϊtinib and/or erlotinib.

[0014] Still other aspects of the present invention relate to a pharmaceutical composition produced by the above-described manufacturing methods.

[0015] Another aspect of the invention involves promoting the use of an

EGFR ligand to ameliorate the adverse effects associated with administration of an EGFR inhibitor in humans.

[0016] In certain jurisdictions, there may not be any generally accepted definition of the terms "comprising" and/or "consisting essentially of." As used herein, the term "comprising" is intended to represent "open" language which permits the inclusion of any additional elements. As used herein, the transitional phrase "consisting essentially of includes any elements listed after the phrase and other elements that do not materially alter the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. Additional embodiments are described below.

[0017] One embodiment disclosed herein relates to a pharmaceutical composition that includes a first dosage form which can include an epidermal growth factor receptor (EGFR) inhibitor and a second dosage form which can include an EGFR ligand. In one aspect of this embodiment, the pharmaceutical composition can include a first dosage form and a second dosage form each having an oral dosage form. In another aspect of this embodiment, the EGFR inhibitor can be selected from the group consisting of reversible inhibitors and irreversible inhibitors. In another aspect of this embodiment, the EGFR inhibitor can be a small molecule. In one embodiment when the EGFR inhibitor is a small molecule, the small molecule can include a quinazoline compound. In one embodiment when the molecule is a quinazoline compound, the quinazoline compound can be selected from the group consisting of erlotinib, gefitinib and 4-(4-benzyloxyanilino)-6,7- dimethoxyquinazoline. In another aspect of this embodiment, the quinazoline compound can be present in an amount from about 50 mg/dose to about 50 g/dose. In another aspect of this embodiment, the quinazoline compound can be present in an amount from about 500 mg/dose to about 20 g/dose. hi another aspect of this embodiment, the quinazoline compound can be present in an amount of about 10 g/dose. In one embodiment where the EGFR inhibitor is a small molecule, the small molecule can include a carbohydrate or carbohydrate analog. In one embodiment, the carbohydrate or carbohydrate analog can be selected from the group consisting of lacto-N-neotetraose, 3'-sialyllactose and 6'- sialyllactose. hi another embodiment, the EGFR ligand can include a proteinaceous EGFR ligand. hi this embodiment, the proteinaceous EGFR ligand can be selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs) and mucin 4 (MUC4). hi another aspect of this embodiment, the EGF can be selected from the group consisting of the 53 amino acid form, the 52 amino acid form, the 51 amino acid form, the 48 amino acid form and homologs having at least 30% amino acid identity with any one of the aforementioned EGF forms. In another aspect of this embodiment, the proteinaceous EGFR ligand can be present in an amount from about 1 mg/dose to about 50 g/dose. In another aspect of this embodiment, the proteinaceous EGFR ligand can be present in an amount from about 10 mg/dose to about 10 g/dose. hi another aspect of this embodiment, the proteinaceous EGFR ligand can be present in an amount of about 1 g/dose. In another aspect of this embodiment, the first dosage form and the second dosage form can further include a pharmaceutically acceptable carrier, hi another aspect of this embodiment, the first dosage form and the second dosage form can be selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel. hi another aspect of this embodiment, the first dosage form and the second dosage form can be merged, thereby forming a combined dosage form, hi one embodiment when the dosages are merged, the combined dosage form can further include a pharmaceutically acceptable carrier, hi another aspect of this embodiment, the combined dosage form can be selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel. In another aspect of this embodiment, the EGFR inhibitor can be gefitinib and said EGFR ligand can be EGF. In another aspect of this embodiment, the EGFR inhibitor can be erlotinib and said EGFR ligand can be EGF. In another embodiment, the EGFR inhibitor can be Panitumumab and said EGFR ligand can be EGF. In another aspect of this embodiment, the EGFR ligand can be capable of activating EGFR.

[0018] Another embodiment disclosed herein relates to a method of ameliorating adverse effects associated with the administration of an EGFR inhibitor, the method can include co-administering to a patient an EGFR inhibitor and a therapeutically effective amount of an EGFR ligand. In one aspect of this embodiment, the EGFR inhibitor and the EGFR ligand can be orally co-administered to the patient. In another aspect of this embodiment, co-administering the EGFR inhibitor and the EGFR ligand can include administering the EGFR ligand at least about 1 hour prior to the administration of the EGFR inhibitor. In another aspect of this embodiment, co-administering the EGFR inhibitor and the EGFR ligand can include administering the EGFR ligand at least about 1 hour subsequent to the administration of the EGFR inhibitor. In another aspect of this embodiment, coadministering the EGFR inhibitor and the EGFR ligand can include administering the EGFR ligand at about the same time as administering the EGFR inhibitor. In another aspect of this embodiment, the EGFR ligand and the EGFR inhibitor can be administered at the same time. In another aspect of this embodiment, the EGFR ligand and the EGFR inhibitor can be administered together in a single dosage form. In another aspect of this embodiment, the EGFR inhibitor can be selected from the group consisting of reversible inhibitors and irreversible inhibitors. In another aspect of this embodiment, the EGFR inhibitor can be a small molecule. In one embodiment when the EGFR is a small molecule, the small molecule can include a quinazoline compound. In another aspect of this embodiment, the quinazoline compound can be selected from the group consisting of erlotinib, gefitinib and 4-(4- benzyloxyanilino)-6,7-dimethoxyquinazoline. In another aspect of this embodiment, the quinazoline compound can be administered in a range from about 1 mg/kg/day to about 1 g/kg/day. In another aspect of this embodiment, the quinazoline compound can be administered in a range from about 10 mg/kg/day to about 400 mg/kg/day. In another aspect of this embodiment, the quinazoline compound can be administered at about 200 mg/kg/day. In another aspect of this embodiment, the small molecule can include a carbohydrate or carbohydrate analog, hi one aspect of this embodiment, the carbohydrate or carbohydrate analog can be selected from the group consisting of lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose. In another embodiment, the EGFR ligand can be a proteinaceous EGFR ligand. In one aspect of this embodiment, the proteinaceous EGFR ligand may not be substantially absorbed into the bloodstream, hi another aspect of this embodiment, the proteinaceous EGFR ligand may not substantially alter the activity of EGFRs outside of the gut. hi another aspect of this embodiment, the proteinaceous EGFR ligand may not substantially alter the activity of EGFRs in a cancerous tissue, hi another aspect of this embodiment, the cancerous tissue can be lung tissue, hi another embodiment, the proteinaceous EGFR ligand can be selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs) and mucin 4 (MUC4). In another embodiment, the EGF can be selected from the group consisting of the 53 amino acid form, the 52 amino acid form, the 51 amino acid form, the 48 amino acid form and homologs having at least 30% amino acid identity with any one of the aforementioned EGF forms. In another embodiment, the proteinaceous EGFR ligand can be administered in a range from about 20 μg/kg/day to about 1 g/kg/day. In another embodiment the proteinaceous EGFR ligand can be administered in a range from about 200 μg/kg/day to about 200 mg/kg/day. hi another embodiment, the proteinaceous EGFR ligand can be administered at about 20 mg/kg/day. hi another embodiment, the adverse effects can include adverse gastrointestinal effects. In one aspect of this embodiment, the adverse gastrointestinal effects can be selected from the group consisting of diarrhea, nausea, vomiting, anorexia and weight loss, hi another aspect of this embodiment, the patient can be identified as suffering adverse gastrointestinal effects or at risk of suffering adverse gastrointestinal effects due to the administration of the EGFR inhibitor, hi another aspect of this embodiment, the patient can have condition associated with a mutant EGFR which can be substantially unresponsive to EGF. hi another embodiment, the adverse effects can include adverse skin effects. In one aspect of this embodiment, the adverse skin effects can be selected from the group consisting of rash, acne, dry skin, pruritus, vesiculobullous rash and mouth ulcerations. In another aspect of this embodiment, the patient can be identified as suffering adverse skin effects or at risk of suffering adverse skin effects due to the administration of the EGFR inhibitor. In another aspect of this embodiment, the patient can have a condition associated with a mutant EGFR that is substantially unresponsive to EGF. In another embodiment, the EGFR inhibitor can be gefitinib and the EGFR ligand can be EGF. In another embodiment, the EGFR inhibitor can be erlotinib and the EGFR ligand can be EGF. In another embodiment, the EGFR ligand can be capable of activating EGFR.

[0019] Another embodiment disclosed herein relates to a method of using an EGFR ligand to ameliorate adverse effects associated with the administration of an EGFR inhibitor in a human subject, the method can include informing the human subject that coadministering the EGFR ligand with the EGFR inhibitor ameliorates at least one adverse effect associated with the administration of the EGFR inhibitor. In one embodiment, the EGFR ligand can be capable of activating EGFR. In another embodiment, the administration of the EGFR inhibitor can be oral administration. In another embodiment, at least one adverse effect can be an adverse gastrointestinal effect. In another embodiment, the adverse gastrointestinal effect can be selected from the group consisting of diarrhea, nausea, vomiting, anorexia and weight loss. In another embodiment, at least one adverse effect can be an adverse skin effect. In another embodiment, the adverse gastrointestinal effect can be selected from the group consisting of rash, acne, dry skin, pruritus, vesiculobullous rash and mouth ulcerations. In another embodiment, the method of informing the human subject can include providing printed matter that advises that co-administering the EGFR ligand with the EGFR inhibitor ameliorates at least one adverse effect associated with the administration of the EGFR inhibitor. In one aspect of this embodiment, the printed matter can be a label.

[0020] Another embodiment disclosed herein relates to a method of manufacturing a pharmaceutical composition, the method can include obtaining a first dosage form comprising an EGFR inhibitor, obtaining a second dosage form comprising an EGFR ligand, and packaging together the first dosage form and the second dosage form. In one embodiment, the first dosage form and the second dosage form can each include an oral dosage form. In another embodiment, the EGFR ligand can be capable of activating EGFR. In another embodiment, the EGFR ligand can be EGF. In another embodiment, the EGFR inhibitor can be gefitinib. In another embodiment, the EGFR ligand can be EGF. In another embodiment, the EGFR inhibitor can be erlotinib. In another embodiment, the EGFR ligand can be EGF. In another embodiment, the first dosage form and the second dosage form can be merged together, thereby forming a combined dosage form. Another embodiment disclosed herein relates to a pharmaceutical composition made by the methods disclosed herein.

[0021] Another embodiment disclosed herein relates to a pharmaceutical composition comprising a first dosage form which can include a receptor tyrosine kinase (RTK) inhibitor and a second dosage form which can include an RTK ligand. In one embodiment, the first dosage form and the second dosage form can each include an oral dosage form. In another embodiment, the RTK inhibitor can be selected from the group consisting of reversible inhibitors and irreversible inhibitors. In another embodiment, the RTK inhibitor can include a small molecule. In another embodiment, the small molecule can be selected from the group consisting of erlotinib, gefitinib, 4-(4-benzyloxyanilino)-6,7- dimethoxyquinazoline, imatinib, PKC412, MLN518, CEP-701, SU5402, SU5416, PD0173074 and SMS-354825. In another embodiment, the small molecule can be present in an amount from about 50 mg/dose to about 50 g/dose. hi another embodiment, the small molecule can be present in an amount from about 500 mg/dose to about 20 g/dose. In another embodiment, the small molecule can be present in an amount of about 10 g/dose. hi another embodiment, the RTK ligand can include a proteinaceous RTK ligand. In another embodiment, the proteinaceous RTK ligand can be selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs), mucin 4 (MUC4), fibroblast growth factor- 1 (FGFl), fibroblast growth factor 2 (FGF2), fins- related tyrosine kinase 3 ligand (FMS-TK3), colony stimulating factor-1 (CSF-I), platelet- derived growth factor (PDGF), growth hormone (GH), prolactin (PL), erythropoietin (EP), leptin (LP), stem cell factor (SFC), nerve growth factor (NGF), neutrophin 3 (NTF3) and vegetative growth factor (VEGF). In another embodiment, the proteinaceous RTK ligand can be present in an amount from about 1 mg/dose to about 50 g/dose. In another embodiment, the proteinaceous RTK ligand can be present in an amount from about 10 mg/dose to about 10 g/dose. In another embodiment, the proteinaceous RTK ligand can be present in an amount of about 1 g/dose. In another embodiment, the first dosage form and the second dosage form can further include a pharmaceutically acceptable carrier. In another embodiment, the first dosage form and the second dosage form can be selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel. In another embodiment, the first dosage form and the second dosage form can be merged, thereby forming a combined dosage form. In another embodiment, the combined dosage form can further include a pharmaceutically acceptable carrier. In another embodiment, the combined dosage form can be selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel. In another embodiment, the RTK ligand can interact with an RTK that is inhibited by the RTK inhibitor. In another embodiment, the RTK ligand can be capable of activating an RTK.

[0022] Another embodiment disclosed herein relates to a method of ameliorating adverse effects associated with the administration of an RTK inhibitor, the method can include co-administering to a patient an RTK inhibitor and a therapeutically effective amount of an RTK ligand. In one embodiment, the RTK inhibitor and the RTK ligand can be orally co-administered to the patient, hi another embodiment, co-administering the RTK inhibitor and the RTK ligand can include administering the RTK ligand at least about 1 hour prior to the administration of the RTK inhibitor. In another embodiment, co-administering the RTK inhibitor and the RTK ligand can include administering the RTK ligand at least about 1 hour subsequent to the administration of the RTK inhibitor. In another embodiment, coadministering the RTK inhibitor and the RTK ligand can include administering the RTK ligand at about the same time as administering the RTK inhibitor. In another embodiment, the RTK ligand and the RTK inhibitor can be administered at the same time, hi another embodiment, the RTK ligand and the RTK inhibitor can be administered together in a single dosage form, hi another embodiment, the RTK inhibitor can be selected from the group consisting of reversible inhibitors and irreversible inhibitors. In another embodiment, the RTK inhibitor can be a small molecule, hi another embodiment, the small molecule can be selected from the group consisting of erlotinib, gefitinib, 4-(4-benzyloxyanilino)-6,7- dimethoxyquinazoline, imatinib, PKC412, MLN518, CEP-701, SU5402, SU5416, PD0173074 and SMS-354825. In another embodiment, the small molecule can be administered in a range from about 1 mg/kg/day to about 1 g/kg/day. In another embodiment, the small molecule can be administered in a range from about 10 mg/kg/day to about 400 mg/kg/day. In another embodiment, the small molecule can be administered at about 200 mg/kg/day. In another embodiment, the RTK ligand can include a proteinaceous RTK ligand. In another embodiment, the proteinaceous RTK ligand may not be substantially absorbed into the bloodstream. In another embodiment, the proteinaceous RTK ligand may not substantially alter the activity of RTKs outside of the gut. In another embodiment, the proteinaceous RTK ligand may not substantially alter the activity of RTKs in a cancerous tissue. In another embodiment, the proteinaceous RTK ligand can be selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs), mucin 4 (MUC4), fibroblast growth factor-1 (FGFl), fibroblast growth factor 2 (FGF2), fins-related tyrosine kinase 3 ligand (FMS-TK3), colony stimulating factor-1 (CSF-I), platelet-derived growth factor (PDGF), growth hormone (GH), prolactin (PL), erythropoietin (EP), leptin (LP), stem cell factor (SFC), nerve growth factor (NGF), neutrophin 3 (NTF3) and vegetative growth factor (VEGF). In another embodiment, the proteinaceous RTK ligand can be administered in a range from about 20 μg/kg/day to about 1 g/kg/day. In another embodiment, the proteinaceous RTK ligand can be administered in a range from about 200 μg/kg/day to about 200 mg/kg/day. In another embodiment, the proteinaceous RTK ligand can be administered at about 20 mg/kg/day. In another embodiment, the patient can be identified as suffering adverse effects or at risk of suffering adverse effects due to the administration of the RTK inhibitor. In another embodiment, the patient can have a condition associated with a mutant RTK which is substantially unresponsive to an RTK ligand. In another embodiment, the adverse effects can include adverse gastrointestinal effects. In another embodiment, the adverse effects can include adverse skin effects. In another embodiment, the RTK ligand can interact with an RTK that is inhibited by the RTK inhibitor. In another embodiment, the RTK ligand can be capable of activating an RTK. [0023] Another embodiment disclosed herein relates to a method of using an RTK ligand to ameliorate adverse effects associated with the administration of an RTK inhibitor in a human subject, the method can include informing the human subject that co-administering the RTK ligand with the RTK inhibitor ameliorates at least one adverse effect associated with the administration of the RTK inhibitor. In one embodiment, the administration of the RTK inhibitor can include oral administration. In another embodiment, the RTK ligand can be capable of activating an RTK. In another embodiment, the method of informing the human subject can include providing printed matter that advises that co-administering the RTK ligand with the RTK inhibitor ameliorates at least one adverse effect associated with the administration of the RTK inhibitor. In another embodiment, the printed matter can be a label. In another embodiment, the RTK ligand can interact with an RTK that is inhibited by the RTK inhibitor. t

[0024] Another embodiment disclosed herein relates to a method of manufacturing a pharmaceutical composition, the method can include obtaining a first dosage form comprising an RTK inhibitor, obtaining a second dosage form comprising an RTK ligand, and packaging together the first dosage form and the second dosage form, hi one embodiment, the first dosage form and the second dosage form can each include an oral dosage form, hi another embodiment, the first dosage form and the second dosage form can be merged¹ together, thereby forming a combined dosage form, hi another embodiment, the RTK ligand can interact with an RTK that is inhibited by the RTK inhibitor, hi another embodiment, the RTK ligand can be capable of activating an RTK. Another embodiment disclosed herein relates to a pharmaceutical composition made by the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figs. IA-E are line graphs displaying BRET-2 signal as a function of ligand concentration. Dose-responses for the epidermal growth factor (EGF) agonist (filled symbols) response are compared with dose responses for the inhibition of constitutive receptor activity with the small molecule inhibitor gefitinib (open symbols). Cells were starved for 24 hours in 0.1% FBS before testing (A) EGFR wild type (WT), (B) EGFR G719C, (C) EGFR L858R, (D) EGFR Δ752-759 and (E) EGFR Δ747-749 A750P. The BRET-2 signal is calculated as the ratio between the Renilla luciferase emission and the GFP2 emission corrected by the background emissions of non-transfected cells.

[0026] Figs. 2A-E are bar charts showing the differential effects of somatic EGFR mutations on constitutive EGFR signaling and EGF-induced signaling. The indicated wild type or mutant EGFR isoforms were co-expressed with (A) GFP2-Grb2, (B) Stat5A- GFP2, (C and E) GFP2-p85 or (D) GFP2-PLCγl, in HEK293T cells and analyzed by BRET- 2. The BRET-2 signal of the mutant EGFR isoforms was expressed as percent of wild type EGFR responses and derived from ratios between the Renilla luciferase emission and the GFP2 emission corrected by the background emissions of non-transfected cells.

[0027] Figs. 3A-C are line graphs displaying BRET-2 signal as a function of ligand concentration. Dose responses for the inhibition of constitutive receptor activity with the small molecule inhibitor gefitinib and erlotinib are compared. Cells were starved for 24 hours in 0.1% FBS before testing (A) EGFR T790M, (B) EGFR L858R T790M and (C) EGFR Δ747-749 A750P. The BRET-2 signal was calculated as the ratio between the Renilla luciferase emission and the GFP2 emission corrected by the background emissions of non- transfected cells.

DETAILED DESCRIPTION

[0028] It is known that various mutations in receptor tyrosine kinases (RTKs) cause constitutive or other abnormal activation of these receptors, in the absence of the endogenous activator ligand. RTKs having such mutations are often associated with abnormal cell proliferation diseases, such as cancers. The present disclosure involves the discovery that the activation of certain mutant RTKs does not substantially increase in the presence of their activator ligand. As such, an appropriate RTK ligand can be supplied to patients suffering from RTK-associated cancers without exacerbating the already heightened activity of the mutant receptors. Administration of the RTK ligand to a patient suffering from an RTK-associated cancer is beneficial in cases where the patient is being administered one or more RTK inhibitors in their chemotherapeutic regimen. RTK inhibitors are capable of inhibiting the activity of certain mutant RTKs, thereby reducing, and in some cases, even eliminating the aberrant proliferation response in cancerous tissues. However, RTK inhibitors also affect the RTKs in noncancerous cells, which can lead to unpleasant and even severe adverse effects. Because the frequency, severity, and/or duration of the adverse effects are often directly correlated with the dose of the inhibitor, the maximum tolerable dose of RTK inhibitor is often well below the dose necessary to bring the blood concentration of the inhibitor to a level high enough to eradicate the cells having the mutant RTKs.

[0029] Embodiments of the present invention relate to pharmaceutical compositions and methods for co-administering an RTK ligand and an RTK inhibitor. In some embodiments, the RTK ligand interacts with and activates non-mutant RTKs so as to reduce, and in some cases even, reverse inhibitory effects due to the RTK inhibitor. Because the mutant RTKs are not further substantially activated by RTK ligand, these receptors will be inhibited by the RTK inhibitor. In some embodiments, the dose of the RTK inhibitor can be substantially increased in the presence of an RTK ligand, for example, from about 2-fold to about 1000-fold above its highest normally-prescribed, therapeutic dose. In some embodiments, co-administration of and RTK ligand and an RTK inhibitor results in the reduction of one or more of the adverse effects associated with the administration of the RTK inhibitor.

[0030] In some embodiments of the present invention, an appropriate RTK ligand is administered to a patient suffering from a cell proliferation disorder associated with a mutant RTK. In some embodiments, the cell proliferation disorder is cancer. Cancers resulting from the constitutive or otherwise abnormal activation of RTKs are well known and have been described in Krause et al. (2005). N. Engl. J. Med. 353:172-87, the disclosure of which is incorporated herein by reference in its entirety. RTKs having mutations resulting in constitutive or otherwise abnormal activation include, but are not limited to, EGFR, FGFRl, FGFR3, FLT3, c-FMS, ΝTRK3, PDGFα, PDGFβ and VEGF. Mutations in EGFR include, among other things, deletions and specific point mutations which result in receptor activation, and which are associated with, but not limited to, breast cancer, glioblastoma and lung cancer, including non-small cell lung cancer (NSCLC). For example, some mutant EGFRs have a deletion of amino acids 747 to 750. Others carry point mutations, such as G719C/S (GIy to Cys or Ser at position 719), L858R (Leu to Arg at position 858) and/or L861Q (Leu to GIn at position 861). Mutations in FGFRl, which are typically associated with myeloproliferative syndrome and atypical chronic myeloid leukemia, predominantly include translocations, which result in activation. Such translocations include, for example, translocations between chromosomes 8 and 13 (t8;13), 6 and 8 (t6;8), 8 and 9 (t8;9), 8 and 19 (t8;19) as well as 8 and 22 (t8;22). Mutations in FGFR3, which are associated with multiple myeloma, include point mutations, such as K650E (Lys to Ser at position 650) and translocations, such as between chromosomes 4 and 12 (t4;12). The FLT3 mutants, which are associated with acute myeloid leukemia, can include, for example, internal tandem duplications and point mutations, such as D835X (Asp to any amino acid at position 835). The RTK, c-FMS, is activated by the point mutations L301F/S (Leu to Phe or Ser at position 301) and Y969C (Tyr to Cys at position 969). Such activation is typically associated with myelodysplastic syndrome and acute myeloid leukemia. Translocations in NTRK3 result in activation, which can lead to acute myeloid leukemia. For example, NTRK3 is activated by a translocation between chromosomes 12 and 15 (tl2;15). Activating mutations in PDGFα can include interstitial deletions as well as translocations, for example, the joining of chromosomes 4 and 22 (t4;22). Such activation is typically associated with hypereosinophilic syndrome, systemic mastocytosis, atypical chronic myeloid leukemia. Activating mutations in PDGFβ primarily include translocations, such as between chromosomes 5 and 12 (t5;12), 5 and 7 (t5;7), 5 and 17 (t5;17), 5 and 10 (t5;10) as well as 5 and 14 (t5;14). Such activation is typically associated with chronic myelomonocytic leukemia and acute myeloid leukemia. Mutations in JAK2 that cause activation include, among others things, point mutations and translocations. For example, JAK2 can be activated by the point mutation V617F (VaI to Phe at position 617) and translocations, such as the joining of chromosomes 9 and 12 (t9;12) or 9 and 22 (t9;22). Such mutations are associated with diseases, such as polycythemia vera, essential thrombocythemia openia, idiopathic myelofibrosis, acute myeloid leukemia, acute lymphoblastic leukemia and atypical chronic myeloid leukemia. Mutations in c-KIT that cause activation primarily include point mutations, such as D419X (Asp to any amino acid at position 419), V560X (VaI to any amino acid at position 560) and D816X (Asp to any amino acid at position 816). Such mutations are associated with acute myeloid leukemia and systemic mastocytosis. It will be appreciated that the above description of activating mutations for RTKs is an exemplary listing of such mutations. Patients having RTKs that include activating mutations other than those appearing in the above description can be also be treated using the pharmaceutical compositions and methods described herein.

[0031] A number of RTK inhibitors can be used in the treatment of cell proliferation disorders that are associated with increased activation of RTKs. Some inhibitors include specifically developed monoclonal antibodies targeted to specific RTKs. Other inhibitors include small molecules, such as erlotinib, gefϊtinib, 4-(4-benzyloxyanilino)- 6,7-dimethoxyquinazoline, imatinib, PKC412, MLN518, CEP-701, SU5402, SU5416, PD0173074 and SMS-354825. Some small molecule RTK inhibitors can act to inhibit a wide range of RTKs (broad spectrum inhibitors), whereas others specifically inhibit only one or a few RTKs. Given the wide ranging inhibitory effects of some RTK inhibitors, certain embodiments of the present invention comprise pharmaceutical compositions and coadministration methods that include a plurality of different RTK ligands.

[0032] Many RTKs have more than one known ligand. Natural ligands for RTKs are commonly polypeptides. Many of these polypeptides are endogenously generated by cleavage of a precursor protein to produce the active form, hi addition to polypeptide ligands, RTKs can bind to and/or be activated by other macromolecules as well as small molecules. In some embodiments, RTK ligands can include molecules that are previously unknown to bind to and/or activate an RTK. Detailed methods for the identification of such ligands are described below in connection with EGFR ligands.

[0033] Endogenous polypeptide ligands for RTKs include growth factors and other polypeptides that typically bind to the extracellular ligand binding domain of an RTK, thereby causing activation. However, in some embodiments of the present invention, RTK ligands activate RTKs through mechanisms other than binding at the extracellular ligand binding domain, for example, anti-RTK antibodies that facilitate receptor dimerization. hi still other embodiments, RTK ligands bind to the receptor activation domain but do not activate the RTK.

[0034] RTK ligands for various receptors include, but are not limited to, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs), mucin 4 (MUC4), fibroblast growth factor- 1 (FGFl)₅ fibroblast growth factor 2 (FGF2), fins- related tyrosine kinase 3 ligand (FMS-TK3), colony stimulating factor- 1 (CSF-I), platelet- derived growth factor (PDGF), growth hormone (GH), prolactin (PL), erythropoietin (EP), leptin (LP), stem cell factor (SFC), nerve growth factor (NGF), neutrophin 3 (NTF3) and vegetative growth factor (VEGF). The ligands for EGFR, which include EGF, TFG-α, AR, BTC, HB-EGF, EPR, NRGs and MUC4, are discussed in further detail in connection with material related to EGFR.

[0035] As used herein, "fibroblast growth factor-1 (FGFl)" or "acidic FGF (aFGF)" means any full-length or truncated FGFl protein or FGFl homolog that can act as a ligand for an FGFR. In some, but not all embodiments of the present invention, the FGFl will function so as to activate wild type FGFRs. The term "FGFl" includes, without limitation, wild type and other truncated forms that continue to act as a ligand for an FGFR. Additionally, the term "FGFl" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of an FGFR. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type FGFl. In some embodiments of the present invention, the FGFl is selected from any of SEQ ID NOs: 1-5, homologs thereof, active fragments of SEQ ID NOs: 1-5 or active fragments of homologs of SEQ ID NOs: 1-5.

[0036] As used herein, "fibroblast growth factor-2 (FGF2)" or "basic FGF (bFGF)" means any full-length or truncated FGF2 protein or FGF2 homolog that can act as a ligand for an FGFR. In some, but not all embodiments of the present invention, the FGF2 will function so as to activate wild type FGFRs. The term "FGF2" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for an FGFR. Additionally, the term "FGF2" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of an FGFR. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type FGF2. In some embodiments of the present invention, the FGF2 is selected from any of SEQ ED NOs: 6-10, homologs thereof, active fragments of SEQ ID NOs: 6-10 or active fragments of homologs of SEQ ED NOs: 6-10.

[0037] As used herein, "fins-related tyrosine kinase 3 ligand (FMS-TK3)" means any full-length or truncated FMS-TK3 protein or FMS-TK3 homolog that can act as a ligand for a FLT3. In some, but not all embodiments of the present invention, the FMS-TK3 will function so as to activate wild type FLT3. The term "FMS-TK3" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a FLT3. Additionally, the term "FMS-TK3" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of a FLT3. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type FMS-TK3. In some embodiments of the present invention, the FMS-TK3 is selected from any of SEQ ID NOs: 11-18, homologs thereof, active fragments of SEQ IDNOs: 11-18 or active fragments of homologs of SEQ ED NOs: 11-18.

[0038] As used herein, "colony stimulating factor-1 (CSF-I)" or "macrophage colony stimulating factor (M-CSF)" means any full-length or truncated CSF-I protein or CSF-I homolog that can act as a ligand for a c-FMS. In some, but not all embodiments of the present invention, the CSF-I will function so as to activate wild type c-FMS. The term "CSF-I" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a c-FMS. Additionally, the term "CSF-I" includes homologs of the full- length and truncated forms that have the ability to function as a ligand of a c-FMS. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type CSF-I. hi some embodiments of the present invention, the CSF-I is selected from any of SEQ ED NOs: 19- 23, homologs thereof, active fragments of SEQ ID NOs: 19-23 or active fragments of homologs of SEQ ID NOs: 19-23. [0039] As used herein, "platelet derived growth factor (PDGF)" means any full- length or truncated PDGFα or PDGFβ protein or PDGFα or PDGFβ homolog that can act as a ligand for a PDGFRα and/or PDGFRβ. In some, but not all embodiments of the present invention, the PDGF will function so as to activate wild type PDGFRα and/or PDGFRβ. The term "PDGF" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a PDGFRα and/or PDGFRβ. Additionally, the term "PDGF" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of a PDGFRα and/or PDGFRβ. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type PDGF. In some embodiments of the present invention, the PDGF is selected from any of SEQ ID NOs: 24-34, homologs thereof, active fragments of SEQ ID NOs: 24-34 or active fragments of homologs of SEQ IDNOs: 24-34.

[0040] As used herein, "growth hormone (GH)" means any full-length or truncated GH protein or GH homolog that can act as a ligand for a JAK2. In some, but not all embodiments of the present invention, the GH will function so as to activate wild type JAK2. The term "GH" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a JAK2. Additionally, the term "GH" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of a JAK2. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type GH. hi some embodiments of the present invention, the GH is selected from any of SEQ ID NOs; 35-42, homologs thereof, active fragments of SEQ ID NOs: 35-42 or active fragments of homologs of SEQ ID NOs: 35-42.

[0041] As used herein, "prolactin (PL)" means any full-length or truncated PL protein or PL homolog that can act as a ligand for a JAK2. hi some, but not all embodiments of the present invention, the PL will function so as to activate wild type JAK2. The term "GH" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a JAK2. Additionally, the term "PL" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of a JAK2. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type PL. In some embodiments of the present invention, the PL is selected from any of SEQ E) NOs: 43-47, homologs thereof, active fragments of SEQ ID NOs: 43-47 or active fragments of homologs of SEQ ID NOs: 43-47.

[0042] As used herein, "erythropoietin (EP)" means any full-length or truncated EP protein or EP homolog that can act as a ligand for a JAK2. In some, but not all embodiments of the present invention, the EP will function so as to activate wild type JAK2. The term "EP" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a JAK2. Additionally, the term "EP" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of a JAK2. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type EP. In some embodiments of the present invention, the EP is selected from any of SEQ DD NOs: 48-52, homologs thereof, active fragments of SEQ ID NOs: 48-52 or active fragments of homologs of SEQ ID NOs: 48-52.

[0043] As used herein, "leptin (LP)" means any full-length or truncated LP protein or LP homolog that can act as a ligand for a JAK2. In some, but not all embodiments of the present invention, the LP will function so as to activate wild type JAK2. The term "LP" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a JAK2. Additionally, the term "LP" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of a JAK2. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type LP. In some embodiments of the present invention, the LP is selected from any of SEQ ED NOs: 53-56, homologs thereof, active fragments of SEQ ID NOs: 53-56 or active fragments of homologs of SEQ ID NOs: 53-56.

[0044] As used herein, "stem cell factor (SCF)" or "steel factor (SLF)" or "mast cell growth factor (MCGF)" or "kit ligand (KL)" means any full-length or truncated SCF protein or SCF homolog that can act as a ligand for a c-KIT. In some, but not all embodiments of the present invention, the SCF will function so as to activate wild type c- KIT. The term "SCF" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for a c-KIT. Additionally, the term "SCF" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of a c-KIT. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type SCF. hi some embodiments of the present invention, the SCF is selected from any of SEQ ID NOs: 57-63, homologs thereof, active fragments of SEQ DD NOs: 57-63 or active fragments of homologs of SEQ DD NOs: 57-63.

[0045] As used herein, "nerve growth factor (NGF)" means any full-length or truncated NGF protein or NGF homolog that can act as a ligand for an NTRKl. In some, but not all embodiments of the present invention, the NGF will function so as to activate wild type NTRKl. The term "NGF" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for an NTRKl. Additionally, the term "NGF" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of an NTRKl. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type NGF. In some embodiments of the present invention, the NGF is₍ selected from any of SEQ DD NOs: 64-68, homologs thereof, active fragments of SEQ ID NOs: 64-68 or active fragments of homologs of SEQ ID NOs: 64-68.

[0046] As used herein, "neutrophin 3 (NTF3)" means any full-length or truncated NTF3 protein or NTF3 homolog that can act as a ligand for an NTRK3. In some, but not all embodiments of the present invention, the NTF3 will function so as to activate wild type NTRK3. The term "NTF3" includes, but is not limited to, wild type and other truncated forms that continue to act as a ligand for an NTRK3. Additionally, the term "NTF3" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of an NTRK3. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type NTF3. In some embodiments of the present invention, the NTF3 is selected from any of SEQ ID NOs: 69-73, homologs thereof, active fragments of SEQ ID NOs: 69-73 or active fragments of homologs of SEQ ID NOs: 69-73.

[0047] As used herein, "vegetative growth factors (VEGFs)" refer to VEGFR ligands which include, but are not limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PIGF and homologs of any of these growth factors that can act as a ligand for a VEGFR. Additionally, the term "VEGFs" include, but is not limited to, truncated VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PIGF or any of their homologs that can act as a ligand for a VEGFR. Homologs of the full-length and truncated VEGFs that have the ability to function as a ligand of a VEGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type VEGF. hi some embodiments of the present invention, the VEGF is selected from any of SEQ ID NOs: 74-91, homologs thereof, active fragments of SEQ ID NOs: 74-91 or active fragments of homologs of SEQ ID NOs: 74-91. As used herein "VEGFR" includes, but is not limited to VEGFRl (also known as FLTl), VEGFR2 (also known as FLK-1/KDR) and VEGGR3 (also known as FLT-4). [0048] Embodiments of the present invention relate to pharmaceutical compositions for the co-administration of an RTK inhibitor and an RTK ligand. Some embodiments relate to a pharmaceutical composition comprising a first dosage form, which comprises an RTK inhibitor, and a second dosage form, which comprises an RTK ligand. In some embodiments, the first dosage form and/or the second dosage form further comprises a pharmaceutically acceptable carrier. In certain embodiments of the present invention, the first dosage form and the second dosage form are separate. In other embodiments, the first dosage form and second the dosage form are merged together to form a single combined dosage form.

[0049] In certain embodiments of the present invention, the RTK ligand is responsible for activating a receptor that has been inhibited by, or that is subject to inhibition by, an RTK inhibitor. In such embodiments, the RTK ligand binds to or otherwise interacts with an RTK that is inhibited by, or that is subject to inhibition by, the RTK inhibitor, m certain embodiments where a broad spectrum RTK inhibitor, such as imatinib, is present in the first dosage form, a plurality of RTK ligands can be present in the second dosage form. Additionally, in some embodiments, the plurality RTK ligands are provided in a plurality of separate dosage forms each comprising one to a few RTK ligands.

[0050] Pharmaceutical compositions of the present invention are used to ameliorate adverse effects due to the administration of the RTK inhibitor. By reducing such adverse effects, the pharmaceutical compositions of the present invention permit the RTK inhibitors to be administered at a dose up to 1000-fold above the currently maximum prescribed dose. Some embodiments of the pharmaceutical compositions described herein comprise an RTK inhibitor in an amount ranging from about 50 mg/dose to about 50 g/dose. In preferred embodiments, the pharmaceutical compositions described herein comprise an RTK inhibitor in an amount ranging from about 500 mg/dose to about 20 g/dose. In still other preferred embodiments, the pharmaceutical compositions described herein comprise an RTK inhibitor in an amount of about 5 g/dose.

[0051] As described above, RTK ligands for use in the pharmaceutical compositions described herein can be selected from small molecule ligands or macromolecule ligands. Such ligands can include, but are not limited to, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs), mucin 4 (MUC4), fibroblast growth factor- 1 (FGFl), fibroblast growth factor 2 (FGF2), fms-related tyrosine kinase 3 ligand (FMS-TK3), colony stimulating factor- 1 (CSF-I), platelet-derived growth factor (PDGF), growth hormone (GH), prolactin (PL), erythropoietin (EP), leptin (LP), stem cell factor (SFC), nerve growth factor (NGF), neutrophin 3 (NTF3) and vegetative growth factor (VEGF) and anti-RTK antibodies. The effective amount of RTK ligand that is present in the pharmaceutical compositions described herein will depend on, among other things, the affinity of the ligand for the RTK. In some embodiments, RTK ligand is present in the pharmaceutical compositions described herein in an amount ranging from about 1 mg/dose to about 50 g/dose. In other embodiments, the RTK ligand is present in an amount ranging from about 10 mg/dose to about 10 g/dose. In still other embodiments, the RTK ligand is present at about 1 g/dose. It will be appreciated that an appropriate amount of RTK ligand for inclusion in the pharmaceutical compositions described herein can be determined using methods well known to those or ordinary skill in the art.

[0052] Typical dosage forms comprising RTK inhibitors and/or RTK ligands include, but are not limited to, conventional tablets, capsules (softgel or hard gel), caplets, gelcaps, pills, liquids (e.g., solutions, suspensions or elixirs), powders, lozenges, micronized particles or osmotic delivery systems and any other oral dosage forms known in the pharmaceutical arts. Dosage forms for parenteral administration are also contemplated. Such dosage forms are well within the ordinary skill in the art and include, but are not limited to intravenous and/or subcutaneous injectables, suppositories and aerosols. Also contemplated are dosage forms for topical administration, such as creams, salves ointments and gels.

[0053] Embodiments of the present invention relate to methods of ameliorating adverse effects associated with the administration of an RTK inhibitor in a patient in need of RTK inhibitor administration. Examples of patients in need of RTK inhibitor administration are individuals who suffer from one or more cell proliferation disorders that are amenable to treatment with an RTK inhibitor, such as cancers and cell proliferative syndromes. Administration of the RTK inhibitor causes dose-dependent adverse effects in the majority of the patient population receiving such treatment. With inhibitors, such as gefϊtinib and erlotinib, adverse skin effects and adverse gastrointestinal effects are the most common. Such adverse skin, gastrointestinal and/or other effects typically increase in frequency, severity and/or duration as the dose of the RTK inhibitor increases. Because these adverse effects increase with dose, there is a limit on the amount of RTK inhibitor that can be administered. As a result, certain disorders may not be treatable at the maximum tolerable RTK inhibitor dose. Embodiments of the present invention, which relate to the coadministration of an RTK ligand with the RTK inhibitor, permit an increased dosing of RTK inhibitor, thereby expanding the therapeutic efficacy of such compounds. In some embodiments, the RTK inhibitor dose is increase to levels that would be lethal in the absence of RTK ligand.

[0054] In some embodiments of the co-administration methods described herein, an RTK inhibitor can be administered in a range from about 1 mg/kg/day to about 1 g/kg/day. In a preferred embodiment, the RTK inhibitor is administered in a range from about 10 mg/kg/day to about 100 mg/kg/day. In another preferred embodiment, the RTK inhibitor is administered at about 50 mg/kg/day. In yet another preferred embodiments, the RTK inhibitor is administered at about 20 mg/kg/day.

[0055] As described previously, RTK ligands for use in methods of ameliorating adverse effects associated with the administration of an RTK inhibitor can be selected from small molecule ligands or macromolecule ligands. hi some embodiments, the RTK ligands are administered parenterally, whereas in other embodiments, the RTK ligands are administered orally, m certain embodiments, wherein administration is oral, RTK ligands are not substantially absorbed from the gut into the bloodstream. In such embodiments, RTK ligands do not substantially come into contact with RTKs outside the gut, and thus, do not cause activation of RTKs outside of the gut. In other embodiments, RTK ligands are, at least in part, absorbed from the gut into the bloodstream; however, such ligands do not substantially activate RTKs outside of the gut. In preferred embodiments, RTK ligands do not substantially activate RTKs associated with cancerous tissue or other abnormal tissues.

[0056] In some embodiments of the present invention, the RTK ligands that are co-administered with the RTK inhibitors include, but are not limited to, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs), mucin 4 (MUC4), fibroblast growth factor- 1 (FGFl), fibroblast growth factor 2 (FGF2), fms-related tyrosine kinase 3 ligand (FMS-TK3), colony stimulating factor-1 (CSF-I), platelet-derived growth factor (PDGF), growth hormone (GH), prolactin (PL), erythropoietin (EP), leptin (LP), stem cell factor (SFC), nerve growth factor (NGF), neutrophin 3 (NTF3) and vegetative growth factor (VEGF) and anti-RTK antibodies. The effective dose of RTK ligand that is used for co-administration will depend on, among other things, the route of administration and the affinity of the ligand for the RTK. In preferred embodiments, the RTK ligand is orally coadministered with the RTK inhibitors. In some embodiments of the present invention, the RTK ligand is administered in a range from about 20 μg/kg/day to about 1 g/kg/day. In other embodiments, the RTK ligand is administered in a range from about 200 μg/kg/day to about 200 mg/kg/day. In still other embodiments, the RTK ligand is administered at about 20 mg/kg/day. In yet other embodiments, the RTK ligand is administered at about 2 mg/kg/day. It will be appreciated that an appropriate dose of RTK ligand for co-administration can be determined in view of the dose of RTK inhibitor to be administered, using methods well known to those or ordinary skill in the art.

[0057] In some embodiments of the present invention a plurality of different RTK ligands are co-administered with the RTK inhibitor. Such embodiments are particularly preferred when the administered RTK inhibitor is a broad spectrum RTK inhibitor, such as imatinib, or when a plurality of RTK inhibitors are administered. In some embodiments, the RTK ligand interacts with one or more different RTKs that are inhibited by the RTK inhibitor.

[0058] Additional embodiments of the present invention relate to the timing of the administration of the RTK ligand and RTK inhibitor. In some embodiments of the present invention, the RTK ligand is administered prior to the administration of the RTK inhibitor. In such embodiments, administration of the RTK ligand occurs about 1 hour, about 2 hours, about 3 hours or about 4 hours prior to the administration of the RTK inhibitor. In other embodiments, the RTK ligand in administered after the administration of the RTK inhibitor. In such embodiments, administration of the RTK ligand occurs about 1 hour, about 2 hours, about 3 hours or about 4 hours after the administration of the RTK inhibitor. In still other embodiments, administration of the RTK ligand occurs at about the same time as the administration of the RTK inhibitor. In such embodiments, the RTK ligand and RTK inhibitor can be administered in separate dosage forms or together in a single combined dosage form, hi other embodiments, the RTK ligand is administered at anytime that allows it to reduce or eliminate one or more adverse effects of the RTK inhibitor.

[0059] Embodiments of the present invention relate to co-administering an RTK ligand and an RTK inhibitor to ameliorate adverse effects due to administration of the RTK inhibitor. All of the above-described embodiments are based on the discovery that mutant receptors involved in certain cell proliferative disorders are not substantially activated by the binding of ligand. In such embodiments, the RTK ligand can be administered by a parenteral route, such as intravenously, rectally, subcutaneously, sublingually, or intranasally. In addition, the RTK ligand can be administered either orally or topically.

[0060] Additional embodiments of the present invention include methods of using an RTK ligand to ameliorate adverse effects associated with the administration of an RTK inhibitor in a human subject. Such methods comprise informing a human subject that coadministering an RTK ligand with an RTK inhibitor ameliorates at least one adverse effect associated with the administration of the RTK inhibitor. In some embodiments, the subject is a patient in need of administration of an RTK inhibitor. In such embodiments, the patient may be suffering from one or more adverse effects associated with the administration of the RTK inhibitor or the patient may one who is not suffering from an adverse effect associated with the administration of an RTK inhibitor but who is at risk of suffering from one or more adverse effects if the amount of RTK inhibitor that is administered is increased. In such embodiments, the methods comprise informing the subject that orally co-administering an RTK ligand and an RTK inhibitor ameliorates at least one adverse effect associated with the administration of the RTK inhibitor.

[0061] Pharmaceutical compositions and co-administration methods utilizing RTK ligands and RTK inhibitors have been generally described. Provided below is a detailed description of such pharmaceutical compositions and co-administration methods as relates' to EGFR inhibitors and EGFR ligands. It will be appreciated that the methods of making pharmaceutical compositions and methods of co-administration, as related to EGFR inhibitors and EGFR ligands, which are described below, can be applied with any RTK inhibitors and RTK ligands, including those previously described. For example, a skilled artisan will appreciate that RTK inhibitors and RTK ligand combinations can be used to ameliorate side effects associated with the administration of the RTK inhibitor as well as to increase the maximum tolerable dose of RTK inhibitor.

Embodiments related to EGFR

[0062] It is known that certain mutations in the epidermal growth factor receptor (EGFR) are often present in the lung tissue of patients suffering from non-small-cell lung cancer (NSCLC). Such mutations result in the activation of the tyrosine kinase activities of these receptors independent of endogenous activator ligand, thereby increasing the survival of cells expressing such mutant receptors. Now, as described herein, it has surprisingly been found that the activation of certain mutant EGFRs does not substantially increase in the presence of the activator ligand, epidermal growth factor (EGF). As such, EGF as well as other EGFR ligands can be supplied to patients suffering from NSCLC without exacerbating the already heightened activity of the mutant receptors.

[0063] The heightened activity of mutant EGFRs in NSCLC patients can often be inhibited by quinazoline drugs, such as gefitinib and erlotinib. However, administration of these inhibitors causes an extremely high incidence of adverse effects, such as adverse effects to the skin, eyes, respiratory system and gastrointestinal system. Such adverse effects increase as the dose of the inhibitor is increased. Accordingly, the dose of quinazoline inhibitor that can be administered to a patient suffering from NSCLC is limited by such adverse effects. It is thought that these adverse effects are due to the inhibition of the tyrosine kinase activity of non-mutant EGFRs that are present in affected areas of the body. For example, it is thought that adverse gastrointestinal effects are due to the inhibition of tyrosine kinase activity of non-mutant EGFRs that are present throughout the gastrointestinal system.

[0064] One embodiment of the present invention provides a method for ameliorating adverse effects due to the administration of an EGFR inhibitor by coadministering to a patient the EGFR inhibitor and a therapeutically effective amount of an EGFR ligand. In some embodiments, the patient is one who is identified as suffering adverse effects due to the administration of an EGFR inhibitor or one who is identified as at risk of suffering adverse effects if their normal dose of EGFR inhibitor is increased. Other embodiments relate to co-administering an EGFR ligand in an amount sufficient to counteract the potentially toxic effects of the administration of a substantial overdose of EGFR inhibitor.

[0065] A preferred embodiment of the present invention provides a method for ameliorating adverse effects due to the oral administration of an EGFR inhibitor by orally coadministering to a patient the EGFR inhibitor and a therapeutically effective amount of an EGFR ligand. In some embodiments, the patient is one who is identified as suffering adverse gastrointestinal effects due to the administration of an EGFR inhibitor or one who is identified as at risk of suffering adverse gastrointestinal effects if their normal dose of EGFR inhibitor is increased.

[0066] Without being bound by theory, there are at least two mechanisms by which administration of an EGFR ligand may act to ameliorate the adverse effects resulting from the administration of an EGFR inhibitor. First, it is thought that oral administration of an EGFR ligand will sufficiently activate non-mutant EGFR receptors present in the gastrointestinal tract so as to ameliorate adverse gastrointestinal effects that are due to the EGFR inhibitor-mediated inhibition of these receptors. In some embodiments of the present invention, much of the orally administered EGFR activator ligand is not absorbed from the gastrointestinal environment in an active form, and thus, it is not significantly available to EGFRs at the site of the tumor. In such embodiments, any active EGFR ligand that becomes localized to the site of the tumor does not substantially activate the mutant receptors because these receptors are not substantially responsive to the EGFR activator ligand.

[0067] In cases where the EGFR inhibitor acts at the extracellular EGFR ligand- binding domain (as opposed to the intracellular tyrosine kinase domain), the EGFR ligand may act to ameliorate adverse gastrointestinal effects by competing with an EGFR inhibitor for binding to the ligand-binding domain of EGFR receptors in the gut. As described above, in some embodiments, much of the orally administered EGFR activator ligand is not absorbed from the gastrointestinal environment in an active form, and thus, it is not be available to EGFRs at the site of the tumor. However, in embodiments where EGFR ligand becomes localized to the site of the tumor, the EGFR does not substantially activate the mutant receptors.

Epidermal growth factor receptor (EGFRI

[0068] Subclass I of the receptor tyrosine kinase (RTK) superfamily consists epidermal growth factor receptors (EGFR), which are also known as the ERBB receptors. This group of receptors comprises four members: EGFR/ERBBl, ERBB2, ERBB3 and ERBB4. All members have an extracellular ligand-binding region, a single membrane- spanning region and a cytoplasmic tyrosine-kinase-containing domain. The ERBB receptors are expressed in various tissues of epithelial, mesenchymal and neuronal origin. Under normal physiological conditions, activation of the ERBB receptors is controlled by the spatial and temporal expression of their ligands, which are members of the EGF family of growth factors. Ligand binding to ERBB receptors induces the formation of receptor homodimers and heterodimers and activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for a range of proteins, the recruitment of which leads to the activation of intracellular signaling pathways.

[0069] As used in this disclosure, "epidermal growth factor receptor (EGFR)" means any of the receptors which fall into the EGFR family of receptors including, but not limited to, EGFR, ERBB2, ERBB3 and ERBB4. The term "EGFR" also refers to mutant forms of EGFR, ERBB2, ERBB3 or ERBB4, including those mutant forms which have altered activities as compared to their wild type counterparts, such as mutant EGFR, ERBB2, ERBB3 or ERBB4 forms that are involved in cancer. For examples of EGFR roles in cancer, see Hynes, et al. (2005). Nature Reviews Cancer 5: 341-54, the disclosure of which is incorporated herein by reference in its entirety.

EGFR inhibitors

[0070] Embodiments of the present invention relate to methods and pharmaceutical compositions for the co-administration of an EGFR ligand with one or more molecules that inhibit the tyrosine kinase activity of an EGFR (EGFR inhibitor) so as to ameliorate side effects resulting from the administration of the EGFR inhibitor. A wide variety of EGFR inhibitors can be utilized with such methods and compositions. The inhibitors can be reversible or irreversible and can mediate their inhibitory effects through a variety of mechanisms. The following non-limiting examples describe mechanisms by which EGFR inhibitors may act. For example, an EGRF inhibitor may inhibit EGFR tyrosine kinase activity by binding to the intracellular tyrosine kinase domain or may act as a receptor antagonist by binding at the extracellular ligand binding domain. Alternatively, the EGFR inhibitor may prevent dimerization of EGFRs.

[0071] EGFR inhibitors that are contemplated for use with the present invention can be selected from any administrable inhibitor molecules. In a preferred embodiment, the EGFR inhibitors are orally administrable. A common class of EGFR inhibitors comprises •quinazoline drugs and derivatives thereof (hereinafter "quinazoline compounds"). Methods of synthesizing quinazoline compounds are well known in the art. Such methods are described in, for example, United States Patent Nos. 5,814,630; 5,814,631; 5,866,572; 6,291,455; 6,849,625; 6,897,214 and 6,939,866, the disclosures of which are incorporated herein by reference in their entireties. Preferred quinazoline inhibitors include gefitinib (Astrazenica, Wilmington, DE), erlotinib (OSI Pharmaceuticals, Melville, NY) and 4- 4(benzyloxyanilino)-6,7-dimethoxyquinazoline (Calbiochem, San Diego, CA).

[0072] In addition to the quinazoline EGFR inhibitors, certain carbohydrate and carbohydrate analogs are known to be inhibitors of EGFR activity. Exemplary compounds include, but are not limited to, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose. Methods for making and using such compounds in the treatment of cancer are described in United States Patent No. 6,281,202, the disclosure of which is incorporated herein by reference in its entirety.

[0073] One of ordinary skill in the art will recognize that in addition to the EGFR inhibitors described-above, other EGFR inhibitors that are can be utilized with the methods and compositions described herein. In preferred embodiments, such inhibitors are orally administrable inhibitors. EGFR ligands

[0074] A number EGFR ligands can be used in connection with the methods and pharmaceutical compositions described herein. EGFR ligands can include any molecules that bind to the extracellular, ligand-binding domain of an EGFR or molecules which function to activate EGFR tyrosine kinase activity. An EGFR ligand can act as an agonist of the EGFR or may have no effect on the activity of the EGFR. hi preferred embodiments of the present invention, EGFR ligands bind to and activate the tyrosine kinase activity of EGFRs present in the gut. Activation can occur by binding of the EGFR ligand at the extracellular binding domain of the EGFR, or alternatively, activation can occur by binding of the EGFR ligand at a domain other than the extracellular ligand binding domain. However, in some embodiments, wherein the co-administered EGFR inhibitor mediates its inhibitory effect through binding at the extracellular EGFR ligand binding domain, rather than directly at the tyrosine kinase domain, the EGFR ligand need not function to activate EGFR tyrosine kinase activity (competitive mechanism). Furthermore, an EGFR ligand can bind either reversibly or irreversibly to the EGFR. Another aspect of the invention is the use of agents that result in stimulation of a naturally occurring RTK ligand. Examples of such agents include, without limitation, idebenone, and propentofylline.

[0075] Some embodiments of the present invention relate to methods and/or compositions in which the EGFR ligand is provided either as a small molecule or as a macromolecule. As used herein, the term "small molecule" refers to a chemical compound that has a molecular weight of less than about 10,000 amu. In some embodiments, the small molecule is a previously unknown ligand for an EGFR. Previously unknown small molecule ligands of EGFR can be identified from combinatorial libraries of small molecules by screening such compounds in bioluminescence resonance energy transfer assays (BRET) assays using an EGFR-luminesceήt protein fusion in conjunction with a protein fusion comprising a luminescent protein fused to a signaling protein that interacts with the EGFR. Exemplary signaling proteins that interact with EGFRs include, but are not limited to, PLCγ, CBL, GRB2, SHC, SHPl, CRKH, DOK-R, p85 and GRB7. Exemplary methods of conducting BRET assays using the above-described constructs are provided in the Examples below. [0076] In some embodiments of the present invention, a small molecule EGFR inhibitor is formulated so that it is not substantially absorbed from the gut into the bloodstream. The degree to which a small molecule is absorbed through the gut into the bloodstream can be controlled using methods established in the art. For example, small molecule absorbance in the gut can be delayed or even inhibited by providing the small molecule in a controlled release formulation, providing the small molecule with and appropriate carrier or by attaching the small molecule to a non-absorbing macromolecule. Any other known methods of delaying and/or inhibiting the absorbance of small molecule from the gut into the bloodstream are also contemplated herein.

[0077] Some embodiments of the present invention relate to methods and/or compositions in which the EGFR ligand is provided as a macromolecule. hi some embodiments, the EGFR ligand is a proteinaceous macromolecule. By "proteinaceous" is meant comprised, at least in part, of protein. Proteinaceous material can consist entirely of protein or primarily of protein. Proteinaceous material also includes material that is primarily of a substance other than protein but which also includes a protein component. Examples of proteinaceous EGFR ligands include, but are not limited to, EGF-family ligands and homologs thereof that have the ability to bind to the extracellular binding domain of an EGFR. Other proteinaceous EGFR ligands include members of the mucin-family and homologs thereof that have the ability to activate EGFR tyrosine kinase activity. Additionally, proteinaceous EGFR ligands can include anti-EGFR antibodies and fragments thereof that bind to the extracellular binding domain of an EGFR or which otherwise activate the tyrosine kinase activity of an EGFR.

[0078] EGF-family ligands are typically short polypeptides that bind to an EGFR at the extracellular ligand binding domain, thereby activating the EGFR tyrosine kinase activity. Examples of EGF-family ligands include, but are not limited to, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR) and neuregulins (NRGs).

[0079] As used herein, "epidermal growth factor (EGF)" means any full-length or truncated EGF protein or EGF homolog that can act as a ligand for an EGFR. hi some, but not all embodiments of the present invention, the EGF will function so as to activate wild type EGFRs. The term "EGF" includes, but is not limited to, wild type 53 amino acid form, the 52 amino acid form, the 51 amino acid form (EGF-2), the 48 amino acid form (EGF-5) and any other truncated forms that continue to act as a ligand for an EGFR. Additionally, the term "EGF" includes homologs of the full-length and truncated forms that have the ability to function as a ligand of an EGFR. Homologs may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type EGF. hi some embodiments of the present invention, the EGF is selected from any of SEQ ID NOs: 92-96, homologs thereof, active fragments of SEQ ID NOs: 92-96 or active fragments of homologs of SEQ ED NOs: 92-96.

[0080] As used herein, the term "transforming growth factor-α (TGF-α)" refers to EGFR ligands which include, but are not limited to, wild type TGF-α and homologs thereof as well as truncated TGF-α and homologs thereof, wherein these molecule retain the ability to act as a ligand for an EGFR. Homologs of the full-length and truncated TGF-α that have the ability to function as a ligand of an EGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type TGF-α. In some embodiments of the present invention, the TGFα is selected from any of SEQ ID NOs: 97-103, homologs thereof, active fragments of SEQ ID NOs: 97-103 or active fragments of homologs of SEQ ID NOs: 97-103.

[0081] As used herein, the term "amphiregulin (AR)" refers to EGFR ligands which include, but are not limited to, wild type AR and homologs thereof as well as truncated AR and homologs thereof, wherein these molecule retain the ability to act as a ligand for an EGFR. Homologs of the full-length and truncated AR that have the ability to function as a ligand of an EGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type AR. hi some embodiments of the present invention, the AR is selected from any of SEQ ID NOs: 104-107, homologs thereof, active fragments of SEQ ID NOs: 104-107 or active fragments of homologs of SEQ ID NOs: 104-107.

[0082J As used herein, the term "betacellulin (BTC)" refers to EGFR ligands which include, but are not limited to, wild type BTC and homologs thereof as well as truncated BTC and homologs thereof, wherein these molecule retain the ability to act as a ligand for an EGFR. Homologs of the full-length and truncated BTC that have the ability to function as a ligand of an EGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type iBJTC. In some embodiments of the present invention, the BTC is selected from any of SEQ ID NOs: 108-111, homologs thereof, active fragments of SEQ ID NOs: 108-111 or active fragments of homologs of SEQ ID NOs: 108-111.

[0083] As used herein, the term "heparin-binding EGF (HB-EGF)" refers to EGFR ligands which include, but are not limited to, wild type HB-EGF and homologs thereof as well as truncated HB-RGF and homologs thereof, wherein these molecule retain the ability to act as a ligand for an EGFR. Homologs of the full-length and truncated HB-EGF that have the ability to function as a ligand of an EGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type HB-EGF. hi some embodiments of the present invention, the HB-EGF is selected from any of SEQ ED NOs: 112-117, homologs thereof, active fragments of SEQ ID NOs: 112-117 or active fragments ofhomologs of SEQ ID NOs: 112-117.

[0084] As used herein, the term "epiregulin (EPR)" refers to EGFR ligands which include, but are not limited to, wild type EPR and homologs thereof as well as truncated EPR and homologs thereof, wherein these molecule retain the ability to act as a ligand for an EGFR. Homologs of the full-length and truncated EPR that have the ability to function as a ligand of an EGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type EPR. In some embodiments of the present invention, the EPR is selected from any of SEQ ID NOs: 118-120, homologs thereof, active fragments of SEQ ID NOs: 118-120 or active fragments of homologs ofSEQ ID NOs: 118-120.

[0085] As used herein, "neuregulins (NRGs)" refer to EGFR ligands which include, but are not limited to, NRGl, NRG2, NRG3, NRG4 and homologs of any of these neuregulins that can act as a ligand for an EGFR. Additionally, the term "NRGs" include, but is not limited to, truncated NRGl, NRG2, NRG3, NRG4 or any of their homologs that can act as a ligand for an EGFR. Homologs of the full-length and truncated NRGs that have the ability to function as a ligand of an EGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type NRG. In some embodiments of the present invention, the NGR is selected from any of SEQ ID NOs: 121-124, homologs thereof, active fragments of SEQ ID NOs: 121-124 or active fragments of homologs of SEQ ID NOs: 121-124.

[0086] In addition to the above-described EGF-family ligands, in some embodiments of the present invention, the protein mucin 4 (MUC4) can be supplied so as to activate the tyrosine kinase activity of EGFRs in the presence of an EGFR inhibitor. As used herein, the term "mucin 4 (MUC4)" refers to EGFR ligands which include, but are not limited to, wild type MUC4 and homologs thereof as well as truncated MUC4 and homologs thereof, wherein these molecule retain the ability to activate the tyrosine kinase activity of an EGFR. Homologs of the full-length and truncated MUC4 that have the ability to activate the tyrosine kinase activity of an EGFR may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater than at least 99% amino acid identity with a full-length or truncated wild type MUC4. In some embodiments of the present invention, the MUC4 is selected from any of SEQ ED NOs: 125-134, homologs thereof, active fragments of SEQ ED NOs: 125-134 or active fragments of homologs of SEQ ID NOs: 125-134.

[0087] In some embodiments of the present invention, an anti-EGFR antibody can be supplied as an EGFR ligand. In certain embodiments, the anti-EGFR antibody binds to or facilitates dimerization of EGFRs so as to activate the receptor tyrosine kinase activity. In preferred embodiments, the anti-EGFR antibody binds to or facilitates dimerization of EGFRs present in the gut, thereby activating the receptor tyrosine kinase activity. In other embodiments, wherein the EGFR inhibitor mediates inhibitory effects by binding the extracellular binding domain of the EGFR, the anti-EGFR antibody competes with EGFR inhibitor binding. Methods for producing antibodies that bind to EGFRs are well known in the art. In particular, methods of producing humanized and/or chimeric monoclonal antibodies that recognize EGFRs are described in United States Patent No. 5,558,864 and 6,506,883, the disclosures of which are incorporated herein by reference in their entireties. Methods for producing single chain antibodies that recognize EGFRs are described in United States Patent No. 6,129,915, the disclosure of which is incorporated herein by reference in its entirety.

[0088] A preferred embodiment of the present invention relates to the coadministration of an EGFR inhibitor and EGF. In such embodiments, commercially available preparations of recombinant EGF can be used. For example, recombinantly produced EGF can be obtained from Chiron Corporation (Emeryville, CA), Austral Biological (San Ramon, CA) or other manufactures and/or distributors. Alternatively, recombinant EGF and homologs thereof can be produced using methodology that is known in the art. Exemplary methods are described in United States Patent No. 5,004,686, the disclosure of which is incorporated herein by reference in its entirety. Methods for large scale production of recombinantly-expressed EGF and homologs thereof are also known in the art. For example, United States Patent No. 5,102,789, the disclosure of which is incorporated by reference in its entirety, describes the large-scale production of EGF in the yeast Pichia pastoris. Recombinant EGF can be stored in solution or as a crystalline solid. Methods of producing stabile, crystalline EGF by forming a complex between EGF and a pharmaceutically acceptable metal ion, such as zinc, are described in United States Patent No. 5,130,298, the disclosure of which is incorporated by reference in its entirety.

[0089] It will be appreciated that the above-described methods for obtaining, producing and/or stabilizing EGF can be also be applied to other proteinaceous EGFR ligands, such as TGF-α, AR, BTC, HB-EGF, EPR, NRGs, MUC4 and anti-EGFR antibodies.

Methods of co-administering EGFR ligands and EGFR inhibitors

[0090] Embodiments of the present invention relate to methods of ameliorating adverse effects associated with the administration of an EGFR inhibitor. Preferred embodiments relate to methods of ameliorating adverse gastrointestinal effects associated with the oral administration of an EGFR inhibitor. In some embodiments of the methods described herein, an EGFR inhibitor is orally administered to a patient in need of EGFR inhibitor therapy. Examples of patients in need of EGFR inhibitor therapy are individuals who suffer from one or more cancers that are amenable to treatment with an EGFR inhibitor, such as non-small-cell lung cancer (NSCLC). Administration of the EGFR inhibitor causes dose-dependent adverse effects in the majority of the patient population receiving such treatment. Adverse skin effects and adverse gastrointestinal effects are the most common. Adverse skin effects can include, but are not limited to, rash, acne, dry skin, pruritus, vesiculobullous rash and mouth ulcerations. Adverse gastrointestinal effects can include, but are not limited to, diarrhea, nausea, vomiting, weight loss and anorexia. Other adverse effects can include, but are not limited to, asthenia, peripheral edema, amblyopia, conjunctivitis and dyspnea. Such adverse skin, gastrointestinal and/or other effects typically increase in frequency, severity and/or duration as the dose of the EGFR inhibitor increases. Because these adverse effects increase with dose, there is a limit on the amount of EGFR inhibitor that can be administered. As a result, certain tumors may not be treatable at the maximum tolerable EGFR inhibitor dose. Embodiments of the present invention, which relate to the co-administration of an EGFR ligand with the EGFR inhibitor, permit an increased dosing of EGFR inhibitor, thereby expanding the therapeutic efficacy of such compounds. In some embodiments, the EGFR inhibitor dose is increase to levels that would be lethal in the absence of EGFR ligand. [0091] Some preferred embodiments of the methods described herein relate to orally co-administering an EGFR inhibitor and an EGFR ligand to a patient who is suffering from adverse gastrointestinal effects due to the administration of an EGFR inhibitor or who is at risk of suffering adverse gastrointestinal effects due to the administration of an increased dose of an EGFR inhibitor. As used herein, to "co-administer" means to provide two or more substances to a subject within a time frame that allows therapeutically effective amounts of each substance to be present in the subject at the same time. In some embodiments, to "coadminister" refers to administering two or more substances within about 4 hours of each other. For example, consider the co-administration of substance A and substance B. If substance A is administered at 4:00 PM, then substance B can be administered as early as about 12:00 PM or as late as about 8:00 PM. As used herein, "therapeutically effective amount" means an amount of EGFR ligand that is sufficient to ameliorate the frequency, severity and/or duration of adverse gastrointestinal effects that result from the oral administration of an EGFR inhibitor.

[0092] As described previously, an EGFR inhibitor that can be utilized in connection with the methods described herein can be any molecule for oral administration that is capable of inhibiting the tyrosine kinase activity of an EGFR. Such inhibitors can be reversible or irreversible. In some embodiments, the methods described herein contemplate the administration of an EGFR inhibitor comprising a small molecule. In preferred embodiments, the small molecule EGFR inhibitor is a quinazoline compound. Especially preferred quinazoline inhibitors are selected from the group consisting of gefitinib, erlotinib and 4-(4-benzyloxyanilino)-6,7-dimethoxyquinazoline.

[0093] In the absence of EGFR ligand, quinazoline compounds are typically orally administered in a range from about 3.5 mg/kg/day to about 7 mg/kg/day. In some embodiments of the co-administration methods described herein, a quinazoline inhibitor can be administered in a range from about 1 mg/kg/day to about 1 g/kg/day. In a preferred embodiment, the quinazoline inhibitor is administered in a range from about 10 mg/kg/day to about 100 mg/kg/day. In another preferred embodiment, the quinazoline inhibitor is administered at about 50 mg/kg/day. In yet another preferred embodiments, the quinazoline inhibitor is administered at about 20 mg/kg/day. In preferred embodiments, the quinazoline inhibitor is orally administered.

[0094] In other embodiments of the present invention, the EGFR inhibitor that is administered is a carbohydrate or carbohydrate analog. Such carbohydrate or carbohydrate analogs include, but are not limited to, lacto-N-neotetraose, 3'-sialyllactose and 6'- sialyllactose.

[0095] As described previously, EGFR ligands for use in methods of ameliorating adverse effects associated with the oral administration of an EGFR inhibitor can be selected from small molecule ligands or macromolecule ligands. In some embodiments, the EGFR ligands are administered parenterally, whereas in other embodiments, the EGFR ligands are administered orally. In certain embodiments, wherein administration is oral, EGFR ligands are not substantially absorbed from the gut into the bloodstream. In such embodiments, EGFR ligands do not substantially come into contact with EGFRs outside the gut, and thus, do not cause activation of EGFRs outside of the gut. In other embodiments, EGFR ligands are, at least in part, absorbed from the gut into the bloodstream; however, as described in the Examples below, such ligands do not substantially activate EGFRs outside of the gut. In preferred embodiments, EGFR ligands do not substantially activate EGFRs associated with cancerous tissue, such as EGFRs associated with NSCLC.

[0096] In some embodiments of the present invention, the EGFR ligands that are co-administered with the EGFR inhibitors comprise proteinaceous ligands. Such proteinaceous EGFR ligands include, but are not limited to, EGF, TGF-α, AR, BTC, HB- EGF, EPR, NRGs, MUC4 and anti-EGFR antibodies. The effective dose of EGFR ligand that is used for co-administration will depend on, among other things, the route of administration and the affinity of the ligand for the EGFR. In preferred embodiments, the proteinaceous EGFR ligand is orally co-administered with the EGFR inhibitors. In some embodiments of the present invention, the proteinaceous EGFR ligand is administered in a range from about 20 μg/kg/day to about 1 g/kg/day. In other embodiments, the proteinaceous EGFR ligand is administered in a range from about 200 μg/kg/day to about 200 mg/kg/day. In still other embodiments, the proteinaceous EGFR ligand is administered at about 20 mg/kg/day. In yet other embodiments, the proteinaceous EGFR ligand is administered at about 2 mg/kg/day. It will be appreciated that an appropriate dose of EGFR ligand for coadministration can be determined in view of the dose of EGFR inhibitor to be administered, using methods well known to those or ordinary skill in the art.

[0097] Preferred embodiments of the present invention relate to the oral coadministration of an EGFR inhibitor and an EGF-family ligand, such as EGF. Li some preferred embodiments, the EGF that is administered is selected from the group consisting of the 53 amino acid form, the 52 amino acid form, the 51 amino acid form, the 48 amino acid form and homologs having at least 30% amino acid identity with any one of the aforementioned EGF forms.

[0098] Additional embodiments of the present invention relate to the timing of the administration of the EGFR ligand and EGFR inhibitor. Ih some embodiments of the present invention, the EGFR ligand in administered prior to the administration of the EGFR inhibitor. In such embodiments, administration of the EGFR ligand occurs about 1 hour, about 2 hours, about 3 hours or about 4 hours prior to the administration of the EGFR inhibitor, hi other embodiments, the EGFR ligand in administered after the administration of the EGFR inhibitor. In such embodiments, administration of the EGFR ligand occurs about 1 hour, about 2 hours, about 3 hours or about 4 hours after the administration of the EGFR inhibitor. In still other embodiments, administration of the EGFR ligand occurs at about the same time as the administration of the EGFR inhibitor. In such embodiments, the EGFR ligand and EGFR inhibitor can be administered in separate dosage forms or together in a single combined dosage form. In preferred embodiments, the EGFR ligand and EGFR inhibitor can be administered in separate oral dosage forms or together in a single combined oral dosage form.

[0099] In addition to methods relating to orally co-administering an EGFR ligand and an EGFR inhibitor to ameliorate adverse gastrointestinal effects, such as diarrhea, nausea, vomiting, weight loss and anorexia, it will be appreciated that other adverse effects due to administration of EGFR inhibitors can be ameliorated by co-administering an EGF ligand with the EGF inhibitor, wherein the EGF ligand is not administered orally. Such adverse effects include, but are not limited to, rash, acne, dry skin, pruritus, vesiculobullous rash, mouth ulcerations, asthenia, peripheral edema, amblyopia, conjunctivitis and dyspnea. Such methods are based on the discovery that mutant receptors involved in NSCLC are not substantially activated by the binding of EGF. In such embodiments, the EGFR ligand can be administered by a parenteral route, such as intravenously, rectally, subcutaneously, sublingually, or intranasally. In addition, the EGFR ligand can be administered topically.

Pharmaceutical compositions for co-administering EGFR ligands and EGFR inhibitors

[0100] Embodiments of the present invention relate to pharmaceutical compositions for the co-administration of an EGFR inhibitor and an EGFR ligand. In a preferred embodiment, the pharmaceutical composition comprises a first oral dosage form which comprises an EGFR inhibitor and a second oral dosage form which comprises an EGFR ligand. hi some embodiments, the first oral dosage form and/or the second oral dosage form further comprises a pharmaceutically acceptable carrier. In some embodiments, the first oral dosage form and the second oral dosage form are separate. In other embodiments, the first oral dosage form and second the oral dosage form are merged together to form a single combined oral dosage form. In some embodiments, the combined oral dosage form further comprises a pharmaceutically acceptable carrier.

[0101] Typical oral dosage forms comprising EGFR inhibitors and/or EGFR ligands include, but are not limited to, conventional tablets, capsules (softgel or hard gel), caplets, gelcaps, pills, liquids (e.g., solutions, suspensions or elixirs), powders, lozenges, micronized particles or osmotic delivery systems and any other oral dosage forms known in the pharmaceutical arts. Each dosage form includes an EGFR inhibitor and/or an effective amount of an EGFR ligand along with pharmaceutically inert ingredients, e.g., conventional excipients, vehicles, fillers, binders, disentegrants, solvents, solubilizing agents, sweeteners, coloring agents and any other inactive ingredients which are regularly included in pharmaceutical dosage forms for oral administration. Many such dosage forms and oral vehicles immediately after listings of inactive ingredients therefore are set forth in Remington's Pharmaceutical Sciences, 17th edition (1985). Oral dosage forms comprising EGFR inhibitors and/or EGFR ligands can be made using methods known to those of ordinary skill in the art, such as direct compression tableting, wet granulation followed by tableting and extrusion followed by marumerization. [0102] As described in connection with the previously-disclosed coadministration methods, the EGFR inhibitor used in the pharmaceutical compositions described herein can be any molecule for oral administration that is capable of inhibiting the tyrosine kinase activity of an EGFR. Such inhibitors can be reversible or irreversible. In some embodiments, the pharmaceutical compositions described herein comprise a small molecule EGFR inhibitor. In preferred embodiments, the small molecule EGFR inhibitor is a quinazoline compound. Especially preferred quinazoline inhibitors are selected from the group consisting of gefitinib, erlotinib and 4-(4-benzyloxyanilino)-6,7-dimethoxyquinazoline.

[0103] Currently, oral dosage forms containing either 250 mg or 500 mg of quinazoline inhibitor are approved for the treatment of NSCLC. Some embodiments of the pharmaceutical compositions described herein comprise a quinazoline compound in an amount ranging from about 50 mg/dose to about 50 g/dose. In preferred embodiments, the pharmaceutical compositions described herein comprise a quinazoline compound in an amount ranging from about 500 mg/dose to about 20 g/dose. In still other preferred embodiments, the pharmaceutical compositions described herein comprise a quinazoline compound in an amount of about 10 g/dose.

[0104] In other embodiments of the present invention, the EGFR inhibitor present in the pharmaceutical compositions described herein is a carbohydrate or carbohydrate analog. Such carbohydrate or carbohydrate analogs include, but are not limited to, lacto-N- neotetraose, 3'-sialyllactose and 6'-sialyllactose.

[0105] As described previously, EGFR ligands for use in the pharmaceutical compositions described herein can be selected from small molecule ligands or macromolecule ligands. In some embodiments of such pharmaceutical compositions, the EGFR ligands comprise proteinaceous ligands. Such proteinaceous EGFR ligands include, but are not limited to, EGF, TGF-α, AR, BTC, HB-EGF, EPR, NRGs, MUC4 and anti-EGFR antibodies. The effective amount of EGFR ligand that is present in the pharmaceutical compositions described herein will depend on, among other things, the affinity of the ligand for the EGFR. In some embodiments, the proteinaceous EGFR ligand is present in the pharmaceutical compositions described herein in an amount ranging from about 1 mg/dose to about 50 g/dose. In other embodiments, the proteinaceous EGFR ligand is present in an amount ranging from about 10 mg/dose to about 10 g/dose. In still other embodiments, proteinaceous EGFR ligand is present at about 1 g/dose. It will be appreciated that an appropriate amount of EGFR ligand for inclusion in the pharmaceutical compositions described herein can be determined using methods well known to those or ordinary skill in the art.

[0106] ' Preferred embodiments of the present invention relate to pharmaceutical compositions comprising a first oral dosage form that comprise an EGFR inhibitor and a second oral dosage form that comprises an EGF-family ligand, such as EGF. In some preferred embodiments, the EGF present in the second oral dosage form is selected from the group consisting of the 53 amino acid form, the 52 amino acid form, the 51 amino acid form, the 48 amino acid form and homologs having at least 30% amino acid identity with any one of the aforementioned EGF forms. In some embodiments, the first oral dosage form is separate from the second oral dosage form. In certain preferred embodiments, the first oral dosage form is merged with the second oral dosage form, thereby forming a combined oral dosage form comprising EGF and an EGFR inhibitor. In especially preferred embodiments, the EGF inhibitor is selected from the group consisting of gefitinib and erlotinib.

[0107] It will be appreciated that, while oral dosage forms are preferred, other pharmaceutical dosage forms for the co-administration of an EGFR inhibitor and an EGFR ligand can be made. Such dosage forms are well within the ordinary skill in the art and include, but are not limited to, dosage forms for parenteral administration, such as intravenous and/or subcutaneous injectables, suppositories and aerosols, and dosage forms for topical administration, such as creams, salves ointments and gels.

[0108] The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, oral, intramuscular, intraocular, intranasal, intravenous, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid and the like. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0109] The term "physiologically acceptable" defines a carrier or diluent that does not abrogate the biological activity and properties of the compound.

[0110] The pharmaceutical compositions described herein can be administered to a human patient per se,_. or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990, which is hereby incorporated by reference in its entirety.

[0111] Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, intraocular injections or as an aerosol inhalant.

[0112] Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into the area of pain or inflammation, often in a depot or sustained release formulation. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.

[0113] The pharmaceutical compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.

[0114] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients maybe used as suitable and as understood in the art; e.g., as disclosed in Remington's Pharmaceutical Sciences, cited above.

[0115] For injection, the agents disclosed herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0116] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds disclosed herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with pharmaceutical combination disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0117] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0118] Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. AU formulations for oral administration should be in dosages suitable for such administration.

[0119] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0120] For administration by inhalation, the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0121] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0122] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions. [0123] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0124] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0125] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0126] An exemplary pharmaceutical carrier for the hydrophobic compounds disclosed herein is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. A common co-solvent system used is the VPD co-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; and other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0127] Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free acids or base forms.

[0128] Pharmaceutical compositions suitable for use in the methods disclosed herein include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0129] The exact formulation, route of administration and dosage for the pharmaceutical compositions disclosed herein can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al. 1975, in "The Pharmacological Basis of Therapeutics", Chapter 1, which is hereby incorporated by reference in its entirety). Typically, the dose range of the composition administered to the patient can be from about 0.5 to 1000 mg/kg of the patient's body weight, or 1 to 500 mg/kg, or 10 to 500 mg/kg, or 50 to 100 mg/kg of the patient's body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. Where no human dosage is established, a suitable human dosage can be inferred from ED₅₀ or ID₅₀ values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals. '

[0130] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. The daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.1 mg and 500 mg of each ingredient, preferably between 1 mg and 250 mg, e.g. 5 to 200 mg or an intravenous, subcutaneous, or intramuscular dose of each ingredient between 0.01 mg and 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg of each ingredient of the pharmaceutical compositions disclosed herein or a pharmaceutically acceptable salt thereof calculated as the free base, the composition being administered 1 to 4 times per day. Alternatively the compositions disclosed herein may be administered by continuous intravenous infusion, preferably at a dose of each ingredient up to 400 mg per day. Thus, the total daily dosage by oral administration of each ingredient will typically be in the range 1 to 2000 mg and the total daily dosage by parenteral administration will typically be in the range 0.1 to 400 mg. In some embodiments, the compounds will be administered for a period of continuous therapy, for example for a week or more, or for months or years.

[0131] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety, which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0132] Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.

[0133] In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0134] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

[0135] The compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound disclosed herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Providing information regarding the benefit of co-administering EGFR ligands and inhibitors [0136] Additional embodiments of the present invention include methods of using an EGFR ligand to ameliorate adverse effects associated with the administration of an EGFR inhibitor in a human subject. Such methods comprise informing a human subject that coadministering an EGFR ligand with an EGFR inhibitor ameliorates at least one adverse effect associated with the administration of the EGFR inhibitor. In some embodiments, the subject is a patient in need of administration of an EGFR inhibitor. In such embodiments, the patient may be suffering from one or more adverse effects associated with the administration of the EGFR inhibitor or the patient may one who is not suffering from an adverse effect associated with the administration of an EGFR inhibitor but who is at risk of suffering from one or more adverse effects if the amount of EGFR inhibitor that is administered is increased. In some embodiments, the adverse effects are adverse gastrointestinal effects due to oral administration of the EGFR inhibitor. In such embodiments, the methods comprise informing the subject that orally co-administering an EGFR ligand and an EGFR inhibitor ameliorates at least one adverse gastrointestinal effect associated with the administration of the EGFR inhibitor.

[0137] As used herein, "informing" refers to providing information relating to the pharmacodynamic activities of an EGFR ligand co-administered with an EGFR inhibitor. The act of informing can be performed, for example, by providing a verbal description or by providing printed matter. In instances where printed matter is used, the printed matter may provide, for example, information relating to effects of co-administering an EGFR ligand and an EGFR inhibitor. The printed matter may further provide information relating to the amelioration of specific adverse effects as a result of this co-administration. As used herein, "informing" does not require any more than the mere act of providing the information. It is not required that intended recipients of the information accept, acknowledge receipt of or understand the information.

[0138] Some embodiment of the present invention relate to a method of using an EGFR ligand to ameliorate adverse effects in a human patient who is suffering from adverse effects associated with the administration of an EGFR inhibitor or who is at risk of suffering from adverse effects associated with increasing the amount of administered EGFR inhibitor. The method comprises informing the human patient that co-administering the EGFR ligand and the EGFR inhibitor ameliorates the frequency, severity and/or duration of at least one adverse effect associated with the administration of the EGFR inhibitor. In some embodiments, the adverse effect is selected from the group consisting of rash, acne, dry skin, pruritus, vesiculobullous rash, mouth ulcerations, asthenia, peripheral edema, amblyopia, conjunctivitis, dyspnea, diarrhea, nausea, vomiting, weight loss and anorexia.

[0139] A preferred embodiment of the present invention relates to a method of using an EGFR ligand to ameliorate adverse gastrointestinal effects in a human patient who is suffering from adverse gastrointestinal effects associated with the oral administration of an EGFR inhibitor or who is at risk of suffering from adverse gastrointestinal effects associated with increasing the amount of administered EGFR inhibitor. The method comprises informing the human patient that orally co-administering the EGFR ligand and the EGFR inhibitor ameliorates the frequency, severity and/or duration of at least one adverse gastrointestinal effect associated with the oral administration of the EGFR inhibitor. In some embodiments, the adverse gastrointestinal effect is selected from the group consisting of diarrhea, nausea, vomiting, weight loss and anorexia.

[0140] In some embodiments of the above-described methods, the informing step comprises providing printed matter that advises that co-administering said EGFR ligand with said EGFR inhibitor ameliorates at least one adverse effect associated with the oral administration of said EGFR inhibitor. In certain preferred embodiments, the printed matter comprises a label. As used herein, "label" refers to printed matter that is associated with a container for holding a pharmaceutical composition. By way of non-limiting example, the label and container can be placed together in a box or shrink wrap. Alternatively, the label can be attached directly to the container. In other embodiments, the label need not be physically associated with or in physical proximity with the container, however, the label should be provided at the same time or at a time reasonably near to the time of providing the container.

EXAMPLES

[0141] Having been generally described above, the following examples set out, in detail, certain aspects of the present invention. These examples in no way limit the scope of the invention as described herein.

Signaling pathway specific EGFR bioluminescence energy transfer (BRET-2) assays

[0142] The rapid ligand-stimulated autophosphorylation of specific tyrosine residues in the intracellular carboxy-terminus of receptor tyrosine kinases (RTKs) is an obligatory event in how RTKs transduce growth factor signals across the cell membrane. The phosphorylated tyrosine residues serve as docking sites for a diverse set of proteins, which are involved in building, shaping and directing the specific RTK downstream signaling pathways. Traditionally, RTK pharmacology and signaling have been quantitatively studied using methods that detect RTK phosphorylation or downstream effects on proliferation. Western blotting, immunoprecipitation or ELISA have been the most frequently applied methods to quantitate RTK autophosphorylation. However, these methods are expensive, laborious, time consuming and show limitations in accuracy and resolution. A novel functional RTK assay that utilizes the bioluminescence resonance energy transfer (BRET-2) technology was designed and overcomes most of these limitations. RTKs are good candidates for setting up a quantitative proximity assay due to the ligand-stimulated autophosphorylation of tyrosine residues and subsequent the recruitment of specific signaling proteins to these residues. No additional proteins are required in this initial step in the RTK signaling cascades, reducing the likelihood of interference through other proteins or signaling pathways. The BRET technology was applied to quantitatively monitor in living cells the recruitment of various EGFR signaling proteins, which directly or indirectly interacted with EGFR to link the receptor to the four-major RTK signaling pathways. The adapter proteins Grb2 and She were used as signaling molecules of the MAP-kinase (MAPK) proliferation pathway. STAT5A as a signaling protein of the STAT pathway, phospholipase Cγl as a key protein in the phospholipase Cγl - calcium pathway, and p85, the regulatory subunit of phosphatidyl-inositol-3-kinase (PBK) as signaling protein from the PI3K-Akt survival pathway. The human EGFR protein was in frame carboxy-terminal tagged with Renilla luciferase, which neither affected the expression levels nor the downstream signaling properties. The amino-termini of Grb2, PLCyI and p85 and the carboxy-terminus of STA5A with Green Fluorescence Protein isoform 2 (GFP2) was tagged in frame. Transient co- transfection of EGFR-luciferase (EGFR-Luc) and the GFP2-tagged signaling proteins in HEK293T cells, which endogenously expressed EGFR at very low levels, allowed the quantitative measurement of EGF stimulated recruitment of the GFP2-tagged signaling molecules to EGFR-Luc, and this directly correlated with the activation of EGFR. The EGF stimulated recruitment of the GFP2-tagged signaling proteins to EGFR-Luc was effectively inhibited through the application of various commercially available EGFR inhibitors (IC₅₀ AG1478 5 nM, IC5₀ PD 168393 6.3 nM). This EGFR BRET assay was used as new tool to study the pharmacology and signaling properties of somatic EGFR mutations in lung cancer and in particular compared the activities of gefitinib and erlotinib.

[0143] The assays conducted in the following Examples utilized cell cultures having normal or mutant EGFRs. Methods of constructing the appropriate bioluminescently- tagged mutant EGFRs and downstream signaling proteins as well as methods for transfecting cell lines with such constructs are described as follows:

[0144] Human cDNA's encoding EGFR, Grb2, p85, PLCyI, STAT5A were obtained by standard RT-PCR on poly- A-RNA isolated from various human tissues or tumor cell lines. Identities of all cDNA's were confirmed by completely sequencing the open reading frames. EGFR isoforms containing somatic mutations were generated by standard mutagenesis methods. EGFR and isoforms were in-frame subcloned into the vector pRluc-N (Perkin-Elmer, USA) to generate a chimeric cDNA expressing the EGFR-(i?e«z7/α)-luciferase fusion protein (EGFR-Luc). The cDNA's encoding the EGFR signaling molecules (GRB2, STAT5A, PLCyI and p85) were subcloned into the vector pGFP2-N or pGFP2-C (Perkin- Elmer, USA) to generate chimeric cDNA's expressing the corresponding fusion proteins: GFP2-Grb2, GFP2-p85, GFP2-PLCyl, STAT5A-GFP2.

[0145] HEK293T cells were cultured in DMEM (with 4500 mg/I D-glucose and glutamine, with out sodium pyruvate) (Invitrogen - GIBCO, Carlsbad, CA, USA), 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) supplemented with penicillin-streptomycin- glutamine solution (Invitrogen - GIBCO, Carlsbad, CA, USA). Two days before transfection, 2 million cells were plated in 10 cm cell culture dishes. The cells reached 70-80% confluency at the day of transfection. Plasmid-DNA's were transient transfected using the lipid based Polyfect transfection reagent (Qiagen, Valencia, CA, USA) as instructed by the manufacturer. Transfection efficiencies reached 50-75%, verified by control transfection with beta- galactosidase. One day after transfection cells were serum starved for 24 hours in DMEM with 0.1% FBS and supplemented with penicillin-streptomycin-glutamine solution. Experiments were performed two days after transfection. Cells were cultured at 37⁰C in a humidified 5% CO₂ incubator.

[0146] Small molecule EGFR inhibitors used in bioluminescence energy transfer assays were obtained as by direct synthesis (gefitinib and erlotinib) or purchased from Calbiochem, USA (CL-387,785).

[0147] EGFR bioluminescence resonance energy transfer assays (BRET-2) were performed according to the following protocol. HEK293T cells cultured in 10 cm plates were transiently transfected with plasmid DNAs expressing a bioluminescence donor (1 μg plasmid DNA expressing EGFR-Luc isoform) and a fluorescence acceptor (40 μg plasmid DNA expressing GFP2 tagged RTK signaling molecule). Transfection was performed with Polyfect (Qiagen) as described by manufacturer. One day after transfection cells were serum starved for 24 hours in DMEM, 0.1 % FBS supplemented with penicillin-streptomycin- glutamine solution. Two days after transfection, cells were harvested and resuspended in phosphate-buffered-saline pH 7.5 (PBS) with glucose and sodium pyruvate to a concentration of 2X10⁶ — 4X10⁶ cells/ml dependent on transfection efficiency. Drug dilutions were prepared in Costar 3912, non-treated, white polystyrene, 96-well plates. For agonist assays, 50 μl of any drug concentration tested was incubated with 50 μl of the cell suspension for the time indicated to establish the ligand induced recruitment of GFP2 tagged EGFR signaling proteins to the intracellular carboxy-terminus of EGFR-luciferase. For antagonist assays, 25 μl of any antagonist concentration tested was incubated with 50 μl of the cell suspension for the time indicated followed by additional incubation time as indicated after addition of 25 μl of the used agonist. For both types of assay, 50 μl of Renilla luciferase substrate coelenterazine 400A (DeepBlueC obtained from Biotium INC. (Hayward, CA, USA)) was added at a 5 μM final concentration to activate the luciferase. Luciferase and GFP2 emissions (due to bioluminescence resonance energy transfer, BRET) were measured after DBC addition and a shaking step for one second each. The time after addition of coelenterazine 400A was sufficient to reach equilibrium with luciferase activity. Injection of DBC and recording of luminescence kinetics was automatically performed by the multiplate reader Mithras 940LB (Berthold, Germany). The plate reader was equipped with filters to detect GFP2 emission (505-525 nm) and Renilla luciferase emission (375-445 nM). The BRET signal was calculated as the ratio between the Renilla luciferase emission and the GFP2 emission corrected by the background emissions of non-transfected cells. Dose response curves and non-linear regression analysis were performed with the software PRISM (GraphPad software INC, USA) to obtain IC₅₀ and EC_5O values.

EXAMPLE 1 Somatic mutations in EGFR cause constitutive activity and affect responsiveness to EGF

[0148] Somatic EGFR mutations have been identified in NSCLC, which activate EGFR signaling. Four somatic EGFR mutations were studied in the EGFR BRET-2 assay described herein: L858R, the most frequent in NSCLC identified point mutation (exon 21) was localized in the activation loop of the EGFR TK domain; G719C (exon 18); localized in the nucleotide phosphate binding loop (P-loop), and the two deletion mutations Δ752-759 and Δ747-749 A750P (exon 19), localized close to the ATP binding region. EGFR-Luc isoforms carrying these mutations were co-transfected with GFP2-Grb2 to evaluate the effects of these somatic mutations on the MAP-kinase pathway signaling. In the absence of exogenously added EGF, significant constitutive activity was observed for all 4 mutations tested (no ligand in Figs. IA-E), which was reflected by the higher BRET-2 signal of the mutants compared to the wild type EGFR. While wild type EGFR exhibited a BRET-2 signal of 0.21 in the absence of EGF, the L858R mutant receptor showed the highest constitutive activity with a BRET-2 signal of 0.33. All constitutive activities were fully inhibited by both gefitinib (Figs.lA-E) and erlotinib (Table 1). The G719C mutation (exon 18) had the lowest constitutive activity of the four mutations tested, which coincided with a location in the P- loop, while the three other mutants localized in two mutation hotspots in exons 19 and 21 with the highest constitutive activity. The results demonstrated that gefitinib and erlotinib were able to effectively inhibit the constitutive activity of EGFR receptors that carry somatic mutations, probably by competing with ATP binding at the intracellular catalytic tyrosine kinase domain. These results further provided evidence that the somatic mutations tested caused an increase in constitutive signaling through the MAP-kinase pathway. Similar results were obtained in the EGFR/Shc BRET-2 assay, which monitored the recruitment of the adapter protein Shc42 to EGFR-Luc.

[0149] Importantly, treating the various mutant EGFR isoforms with EGF revealed dramatic differences in their respective EGF responsiveness. EGF was a very potent agonist for wild type EGFR (Table 1, EC₅₀ about 0.1 nM) in the EGFR/Grb2 BRET-2 assay, demonstrating the sensitivity of the EGFR BRET assay. All mutant EGFR isoforms were only slightly less potent in responding to EGF, but showed more dramatic differences in efficacy (Figs. IA-E and Table 1). For the wild type receptor, the BRET-2 signal increased to 0.55 in the presence of EGF (Fig. IA). However, for the EGFR point mutants, the signal with EGF increased to only 0.50 for G719C and 0.45 for L858R (Figs. IB and 1C, respectively). The EGF signal was further impaired in the deletion mutants, which showed only a slight ligand induced increase in the BRET-2 signal to 0.35 (Figs. ID and IE), indicating a strong impairment in transducing EGF signals into the MAP-kinase pathway signaling. Therefore, none of the tested constitutively active EGFR mutants reached wild type EGFR activity level after EGF stimulation. This reduced EGFR response to EGF has previously not been recognized.

Table 1: Signaling pathway specific and quantitative comparison of pharmacology of gefitinib and erlotinib on constitutive active EGFR isoforms EC50 ICSO O n n ICS -Log [EGF) M - Log [Ircssa] M - Log [Tarceva] M n EGFR signaling pathway

EGFRWT 10.14 +/- 0.01 32 6.59 +/- 0.08 15 6.83 +/- 0.08 15 EGFR L858R 9.63 +/- 0.03* 8 7.59 +/- 0.05* 4 8.04 +/- 0.04* 4

MAP kinase EGFR Δ752-759 9.71 +/- 0.06* 8 7.88 +/- 0.06* 4 8.23 +/- 0.05* 4 (Grb2)

EGFR Δ747-749A750P 9.69 +/- 0.08* 7 7.64 +/- 0.03* 3 8.00 +/- 0.06* 4 EGFR G719C 9.78 +/- 0.04* 8 7.41 +/- 0.10* 4 8.05 +/- 0.06* 4

EGFR WT 9,97 +/- 0.09 6 n.d 3 n.d 3 EGFR L8S8R 9.91 +/- 0.05 3 8.46 +/- 0.18 3 8.88 +/- 0.05 3 STAT

(STAT5A) EGFR Δ752-759 9.79 +/- 0.17 3 8.45 +/- 0.02 3 8.74 +/- 0.33 3

EGFR WT 10.22 +/- 0.02 6 8.00 +/- 0.15 6 8.03 +/- 0.03 7

PI3K/Akt EGFR L858R 9.36 +/- 0.21* 3 8.33 +/- 0.11 3 8.79 +/- 0.06* 3 (p8S)

EGFR Δ752-759 9.25 +/- 0.18* 3 8.56 +/- 0.09^& 3 8.60 +/- 0.09* 3

EGFRWT 10.15 +/- 0.03 9 n.d. 4 n.d. 4 EGFR L858R 9.89 +/- 0.02* 5 8.04 +/- 0.30 4 8.50 +/- 0.22 PLCyI -calcium 4 (PLCyI)

EGFRΔ7S2-759 9.93 +/- 0.07 5 8.32 +/- 0.21 5 8.42 +/- 0.30 5

EXAMPLE 2

Gefitinib and erlotinib effectively inhibit constitutive activity of EGFR isoforms [0150] The constitutive activity displayed by the wild type and mutant EGFR isoforms in the EGFR/Grb2 BRET-2 assay was effectively inhibited by gefitinib (Figs. IA-E) and erlotinib (Table 1). The BRET-2 signals were reduced to around 0.19 for wild type and all mutants at the highest concentrations of these EGFR inhibitors, which was likely to be the baseline BRET-2 signal for the EGFR/Grb2 BRET-2 assay. Quantification of the pharmacological dose-responses obtained in the EGFR/Grb2 BRET-2 assays determined a log IC₅₀ for gefitinib and erlotinib acting at the wild type EGFR as -6.59 +/- 0.32 (257 nM) and -6.86 +/- 0.3 (138 nM), respectively. The wild type EGFR also showed a low level of constitutive activity in the MAP -kinase pathway, indicated through the small inhibition with gefitinib or erlotinib in the EGFR/Grb2 BRET-2 assay (Fig. IA and Table 1). The constitutive activity of the wild type EGFR could not be neutralized by anti-human EGF antibodies in contrast to EGF stimulated EGFR activity. The constitutive active mutant EGFR receptors were 5-10 fold more sensitive to inhibition by gefitinib or erlotinib, compared to wild type EGFR (Table 1). No significant differences were observed between gefitinib and erlotinib acting at the various EGFR mutants, except that erlotinib appeared slightly more potent in inhibiting the wild type and mutant EGFR isoforms (ΔIC₅₀ = 0.41 +/- 0.14). EXAMPLE 3

Constitutive activity of EGFR isoforms is predominantly transduced through the PI3K/Akt survival pathway

[0151] Based on the results from the EGFR/Grb2 BRET-2 assay, the effect of the L858R and the Δ752-759 mutations on other EGFR signaling pathways was studied. EGFR/p85 BRET-2 assays to monitor the PI3K/Akt pathway; EGFR/Stat5a BRET-2 assays to monitor the STAT pathway and EGFR/PLCγl BRET-2 assays to monitor the PLCγl- calcium pathway were performed. Additionally, the pharmacology of gefitinib and erlotinib in all experiments was compared. The results of all experiments are summarized in Table 1 and presented in Figs. 2A-E. For each signaling pathway specific assay, the BRET-2 signals obtained for the wild type EGFR in the presence of EGF plus the constitutive activity determined by gefitinib inhibition was normalized to 100% and the signals obtained for the other receptor isoforms were compared to the activated wild type EGFR responses. The results showed that both mutant EGFR variants were constitutively active (in the absence of EGF) in all pathways tested (Figs. 2A-E open bars), but with quantitative differences. Importantly, all EGFR mutants tested predominantly signaled through the PI3K/Akt survival pathway. For the L858R mutant, the constitutive activity (Fig. 2C, L858R open bar) was about 70% of the total wild type response (Fig. 2C, WT filled bar). The corresponding constitutive signaling activity of the L858R mutant through the MAP-kinase, STAT and PLCγl-calcium signaling pathways ranged only between 28% and 40% of the total wild type responses (Figs. 2A, 2B and 2D, L858R open bars). The deletion mutant EGFR Δ752-759 showed a similar profile for constitutive activity and coupling to the different signaling pathways, with 54% activity in the PI3K/Akt pathway (Figs. 2C, Δ752-759, open bar) and lower levels of activity 30-35% in the other pathways (Figs. 2A, 2B, and 2D, Δ752-759 open bar).

EXAMPLE 4

EGF responsiveness differs between the somatic EGFR L858R and Δ752-759 mutant EGFR isoforms [0152] When the L858R and Δ752-759 EGFR isoforms were treated with EGF in BRET assays monitoring STAT, PBK/Akt or PLCγl -calcium signaling, a reduced EGF responsiveness for both mutants compared to EGFR wild type (Figs. 2B-D, filled bars), consistent with the MAP-kinase pathway EGF response (Figs. IA-E, filled symbols and Figs. 2A filled bars) was detected. The L858R mutant showed a response to EGF for all signaling pathways (Figs. 2A-D, compare open bars with filled bars). However, the Δ752-759 mutant isoform showed a dramatic quantitative difference in signaling between the different pathways. EGF stimulated signaling for this mutant was induced for the STAT, PBK/Akt and PLCγl pathways (Figs. 2B-D, compare open and filled bars) but not for the Grb2/MAP- kinase pathway (Fig. 2A). This finding may be due to the difference in the pattern of autophosphorylated tyrosine residues required for Grb2 or Stat5a recruitment to EGFR.

EXAMPLE 5 Signaling responses of somatic EGFR mutants upon gefitinib and erlotinib treatment

[0153] Table 1 shows that with respect to each other, the different mutants did not display significant quantitative differences in the increase in drug sensitivity when comparing results obtained for gefitinib with erlotinib for different mutants or different signaling pathways. However, in general the EGFR isoforms were more sensitive to gefitinib and erlotinib treatment compared to wild type EGFR. This was consistent with the increase in drug sensitivity seen in cancer cell lines that harbor these mutations. Constitutive activity was not detected in the wild type EGFR with the EGFR/Stat5a or EGFR/PLCyl BRET-2 assays, which prevented us from quantitating the increase of drug sensitivity.

EXAMPLE 6 Impact of T790M mutation on inhibition of constitutive EGFR activity by erlotinib and gefitinib

[0154] It is currently unclear why the EGFR inhibitors gefitinib and erlotinib, which are believed to share the same mechanism of action, show differences in the clinical efficacy affecting overall survival in NSCLC. The results revealed that erlotinib is overall slightly more potent than gefitinib in inhibiting EGFR signaling pathways (Table 1). The effective steady state plasma concentrations for gefitinib (0.4 - 1.4 μM) and erlotinib (3 μM) are significantly higher than required to inhibit EGFR signaling in vitro. Based on these data, it is assumed that both drugs saturate EGFRs during treatment. However, skin rash and gastrointestinal side effects appear more prominent with erlotinib than with gefitinib. The development of skin rash is dose-dependent and seems to be correlated to the clinical response and survival, making rash a potential surrogate marker of activity. Therefore, it might be possible that clinical doses of gefitinib do not saturate EGFR in the skin and maybe in the tumor tissue explaining the difference in clinical efficacy. Many lung cancer patients will relapse and acquire drug resistance to both drugs during their treatment regiment. Acquisition of drug resistance is a complex process involving multiple poorly characterized pathways, one involving the occurrence of resistance mutations. Recently, three independent studies reported the identification of an acquired secondary resistance mutation (T790M) in the EGFR kinase domain of patients, who were treated and initially responded to gefitinib and erlotinib. The T790M mutation has only been found in the presence of an activating EGFR mutation in tumor samples, although only in a small fraction of the total tumor cells number. The mutation has also been found in patients that did not undergo treatment with gefitinib and erlotinib. Blencke, et al introduced T790M into the EGFR receptor and found in an in- vitro kinase assay that the mutated receptor had a 100 fold reduced sensitivity to inhibition by the 4-anilino-quinazoline inhibitor PD153035. Structural models of the EGFR kinase domain bearing the T790M mutation predict a steric hindrance for erlotinib (erlotinib) or gefitinib (gefitinib) binding to the ATP binding site due to a chloride substitute at the 3- position of the aniline ring structure, which would structurally clash with the bulkier methionine structure. The pharmacology and signaling properties of these double mutants EGFR isoforms have not been studied in detail.

[0155] A mutant EGFR variant was analyzed bearing the T790M mutation alone and mutant EGFRs that carry T790M in combination with the mutation L858R or Δ747-749 A750P in the BRET/p85 BRET-2 assay, which monitored signaling through the PDK/Akt survival pathway. The results showed that the T790M mutation alone generates a highly constititutively active EGFR receptor (Fig. 2E open bar and Fig. 3A). In agreement with predictions from structural models, erlotinib was not effective in inhibiting the constitutive activity of the EGFR T790M isoform (Fig. 3A, circles). Gefitinib, which has a similar structure as erlotinib, was expected to behave similarly to erlotinib; however, despite similarities with erlotinib in structure and mode of action, gefitinib inhibited the constitutive activity of the T790M isoform (85% inhibition with 33 μM high dose), but with a lower potency compared to the other somatic mutants (pICso - 5.3 +/- 0.033) (Fig. 3A triangles). Additionally, the double mutant EGFRs L858R T790M and Δ747-749 A750P T790M, reported to occur in patients that developed drug resistance in NSCLC was analyzed. Both double mutants showed the highest levels of constitutive activity of all mutants tested in this study. The constitutive activity of the EGFR L858R T790M receptor reached the same activity level as the EGF triggered response at wild type EGFR (Fig. 2E open bars). The constitutive activity level of EGFR Δ747-749 A750P T790M reached 80% of the maximal EGF response at the EGFR wild type. The EGF responsiveness of both double mutants was nearly completely impaired (Fig. 2E filled bars). The results strongly indicated that the development of drug resistant cells carrying the T790M mutation was accompanied by a dramatic increase in constitutive activation of the PI3K/Akt pathway (Fig. 2E and Figs. 3A- C). Similar strong constitutive activation of the MAP-kinase pathway by the double mutant receptors was found. Drug resistant tumor cells that carried the T790M mutation appeared more aggressive and less dependent on growth factor EGF. This may provide these cancer cells with a strong advantage to dominate over the non-resistant cells leading to enrichment and more resistant tumor formation during drug treatment, hi contrast to the prediction from the T790M mutation alone significant but partial inhibition (50-75%) of the double mutants by gefitinib or erlotinib (Figs. 3B and 3C) was observed. These results were in good agreement with experiments in which the proliferation of the cancer cell line NCI-H1975, which harbors the L858R and T790M mutations, was significantly suppressed by gefitinib treatment. Interestingly, erlotinib was slightly more potent in inhibiting EGFR L858R T790M activity than gefitinib in the BRET/p85 BRET-2 assay (erlotinib log IC₅₀ = -5.89 +/- 0.06 vs. gefitinib log IC₅₀ ⁼ -5.25 +/- 0.07). A similar potency difference was observed for the deletion mutant EGFR Δ747-749 A750P T790M (erlotinib log IC₅₀ = - 6.33 vs. gefitinib log IC₅₀ = -5.80). It is important to note, that the IC₅₀ of inhibiting the double mutant EGFRs in the in vitro cell based BRET assay was consistent with the effective concentration of erlotinib and gefitinib reported in the human plasma of treated NSCLC patients. Therefore, small differences in the plasma concentrations of gefitinib or erlotinib would likely cause significant changes in their efficacy to inhibit the EGFR PI3K/Akt survival pathway in the double mutants. The lack of complete inhibition might explain the acquisition of drug resistant cells.

[0156] Recent studies by others demonstrate that irreversible EGFR inhibitors are more effective in inhibiting the constitutive activity of these EGFR double mutants. The irreversible inhibitor CL-387,785 in the EGFR/p85 BRET-2 assay at EGFR L858 T790M and EGFR Δ747-749 A750P T790M was tested and observed complete inhibition of constitutive activity with an log IC₅₀ °f -6-86 +/-0.14 and -6.81 +/- 0.12, respectively (and data not shown). Wild type EGFR showed a higher sensitivity for inhibition with an log IC₅₀ of -9.4 +/- 1.1. Similar results were obtained in the EGFR/Grb2 assay. Accordingly, irreversible inhibitors may be useful in treating relapsed NSCLC patients after gefitinib or erlotinib treatment.

EXAMPLE 7

Amelioration of EGFR inhibitor-mediated adverse gastrointestinal effects by oral coadministration of EGF and gefitinib or EGF and erlotinib

[0157] To determine the efficacy of orally co-administering EGF and gefitinib or EGF and erlotinib for ameliorating adverse gastrointestinal effects due to the administration of an EGFR inhibitor, patients having NSCLC are randomly divided into four groups. A tumor biopsy is obtained from each patient and used for EGFR genotyping analysis. The first group is given 500 mg of gefitinib and 50 mg of placebo in a single, oral administration daily for 14 days. The second group is given 500 mg of gefitinib and 50 mg of EGF in a single, oral administration daily for 14 days. The third and fourth groups are identical to the first and second groups except that gefitinib is replaced with erlotinib.

[0158] Throughout the 14 day dosing regimen, patients are evaluated for frequency and severity of adverse gastrointestinal effects. In particular, patients are asked to record any instance of an adverse gastrointestinal event including a description of the event and the duration. Additionally, patients are asked to rank the severity of the adverse event on a scale of 1-5.

[0159] Appropriate statistical methods, such as a two-way analysis of variance, are used to determine whether co-administration of the quinazoline compound and EGF resulted in a statistically significant difference in the incidence, severity or duration of any of the recorded classes of adverse gastrointestinal effects. A statistically significant difference is observed between patients who received the quinazoline compound and EGF and patients who received EGF alone.

EXAMPLE 8

Somatic mutations in RTKs cause constitutive activity and affect responsiveness to activator ligands

[0160] This Example shows that mutations associated with the constitutive activation of various RTKs also diminish the responsiveness of these receptors to activator ligand. As described above for EGFR, RTK-luc isoforms carrying point, insertion, deletion or translocation mutations, as described previously herein, are co-transfected with an appropriate GFP2-signaling protein fusion to evaluate the effects of these somatic mutations on the downstream pathway signaling. In particular, the mutant isoforms of FGFl, FGF3, FLT-3, c-FMS, PDGFα, PDGFβ, JAK2, c-KTT, NTRKl, NTRK3 and VEGFR that have been previously described are tested. In the absence of exogenously added activator ligand, significant constitutive activity for all the mutations is observed. This is reflected by the higher BRET-2 signal of the mutants as compared to the corresponding wild type RTKs. Wild type RTKs exhibit a low level BRET-2 signal in the absence of activator ligand, whereas the corresponding mutant receptor show the high BRET-2 signal, which indicates strong constitutive activity. For RTKs having known inhibitors, addition of the inhibitor causes a reduction in BRET-2 signal.

[0161] The various mutant RTK isoforms are then treated with an appropriate RTK ligand. In particular, FGFl and FGF2 are agonists for wild type FGFRl and FGFR3 the BRET-2 assay, thus demonstrating the sensitivity of the FGFR BRET assay. Additionally, FMS-TK3, CSF-I, PDGF, GH, SFC, NGF, NTF3 and VEGF are agonists for wild type FLT- 3, c-FMS, PDGFα, PDGFβ, JAK2, c-KIT, NTRKl, NTRK3 and VEGFR, respectively. All mutant RTK isoforms are less potent in responding to their "activator" ligands and show a reduction in efficacy, which is shown by a lack of increase in BRET-2 signal in the presence of the appropriate activator ligand.

EXAMPLE 9

Amelioration of RTK inhibitor-mediated adverse effects by oral co-administration of RTK ligand and RTK inhibitor

[0162] In this Example, the efficacy of orally co-administering an RTK ligand and an RTK inhibitor for ameliorating adverse gastrointestinal effects due to the administration of an RTK inhibitor is demonstrated. First, a tumor biopsy is obtained from each patient and used for RTK genotyping analysis. Next, patients having a cell proliferative disorder due to a specific mutation in an RTK receptor are grouped together. The patients in each "receptor- type group" are randomly divided into two groups. The first group is given 500 mg of an appropriate inhibitor and 50 mg of placebo in a single administration daily for 14 days. The second group is given 500 mg of appropriate inhibitor and 50 mg of RTK "activator" ligand corresponding to the mutant RTK in a single administration daily for 14 days.

[0163] Throughout the 14 day dosing regimen, patients are evaluated for frequency and severity of adverse effects. In particular, patients are asked to record any instance of an adverse event including a description of the event and the duration. Additionally, patients are asked to rank the severity of the adverse event on a scale of 1-5.

[0164] Appropriate statistical methods, such as a two-way analysis of variance, are used to determine whether co-administration of the RTK inhibitor and RTK ligand resulted in a statistically significant difference in the incidence, severity or duration of any of the recorded classes of adverse effects. A statistically significant difference is observed between patients who received the RTK inhibitor and the RTK ligand and those who received RTK inhibitor alone.

[0165] The methods, compositions, and devices described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the disclosure. Accordingly, it will be apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

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Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a first dosage form which comprises an epidermal growth factor receptor (EGFR) inhibitor and a second dosage form which comprises an EGFR ligand.

2. The pharmaceutical composition of claim 1, wherein said first dosage form and said second dosage form each comprise an oral dosage form.

3. The pharmaceutical composition of claim 1, wherein said EGFR inhibitor is selected from the group consisting of reversible inhibitors and irreversible inhibitors.

4. The pharmaceutical composition of claim 1, wherein said EGFR inhibitor comprises a small molecule.

5. The pharmaceutical composition of claim 4, wherein said small molecule comprises a quinazoline compound.

6. The pharmaceutical composition of claim 5, wherein said quinazoline compound is selected from the group consisting of erlotinib, gefitinib and 4-(4- benzyloxyanilino)-6,7-dimethoxyquinazoline.

7. The pharmaceutical composition of claim 5, wherein said quinazoline compound is present in an amount from about 50 mg/dose to about 50 g/dose.

8. The pharmaceutical composition of claim 7, wherein said quinazoline compound is present in an amount from about 500 mg/dose to about 20 g/dose.

9. The pharmaceutical composition of claim 8, wherein said quinazoline compound is present in an amount of about 10 g/dose.

10. The pharmaceutical composition of claim 4, wherein said small molecule comprises a carbohydrate or carbohydrate analog.

11. The pharmaceutical composition of claim 10, wherein said carbohydrate or carbohydrate analog is selected from the group consisting of lacto-N-neotetraose, 3'- sialyllactose and 6'-sialyllactose.

12. The pharmaceutical composition of claim 1, wherein said EGFR ligand comprises a proteinaceous EGFR ligand.

13. The pharmaceutical composition of claim 12, wherein said proteinaceous EGFR ligand is selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin- binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs) and mucin 4 (MUC4).

14. The pharmaceutical composition of claim 13, wherein EGF is selected from the group consisting of the 53 amino acid form, the 52 amino acid form, the 51 amino acid form, the 48 amino acid form and homologs having at least 30% amino acid identity with any one of the aforementioned EGF forms.

15. The pharmaceutical composition of claim 12, wherein said proteinaceous EGFR ligand is present in an amount from about 1 mg/dose to about 50 g/dose.

16. The pharmaceutical composition of claim 15, wherein said proteinaceous EGFR ligand is present in an amount from about 10 mg/dose to about 10 g/dose.

17. The pharmaceutical composition of claim 16, wherein said proteinaceous EGFR ligand is present in an amount of about 1 g/dose.

18. The pharmaceutical composition of claim 1, wherein said first dosage form and said second dosage form further comprise a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 1, wherein said first dosage form and said second dosage form are selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel.

20. The pharmaceutical composition of claim 1, wherein said first dosage form and said second dosage form are merged, thereby forming a combined dosage form.

21. The pharmaceutical composition of claim 20, wherein said combined dosage form further comprises a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, wherein said combined dosage form is selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel.

23. The pharmaceutical composition of claim 1, wherein said EGFR inhibitor comprises gefitinib and said EGFR ligand comprises EGF.

24. The pharmaceutical composition of claim 1, wherein said EGFR inhibitor comprises erlotinib and said EGFR ligand comprises EGF.

25. The pharmaceutical composition of claim 1, wherein said EGFR ligand is capable of activating EGFR.

26. A method of ameliorating adverse effects associated with the administration of an EGFR inhibitor, said method comprising co-administering to a patient an EGFR inhibitor and a therapeutically effective amount of an EGFR ligand.

27. The method of claim 26, wherein said EGFR inhibitor and said EGFR ligand are orally co-administered to said patient.

28. The method of claim 26, wherein co-administering said EGFR inhibitor and said EGFR ligand comprises administering said EGFR ligand at least about 1 hour prior to the administration of said EGFR inhibitor.

29. The method of claim 26, wherein co-administering said EGFR inhibitor and said EGFR ligand comprises administering said EGFR ligand at least about 1 hour subsequent to the administration of said EGFR inhibitor.

30. The method of claim 26, wherein co-administering said EGFR inhibitor and said EGFR ligand comprises administering said EGFR ligand at about the same time as administering said EGFR inhibitor.

31. The method of claim 30, wherein said EGFR ligand and said EGFR inhibitor are administered at the same time.

32. The method of claim 31, wherein said EGFR ligand and said EGFR inhibitor are administered together in a single dosage form.

33. The method of claim 26, wherein said EGFR inhibitor is selected from the group consisting of reversible inhibitors and irreversible inhibitors.

34. The method of claim 26, wherein said EGFR inhibitor comprises a small molecule.

35. The method of claim 34, wherein said small molecule comprises a quinazoline compound.

36. The method of claim 35, wherein said quinazoline compound is selected from the group consisting of erlotinib, gefitinib and 4-(4-benzyloxyanilino)-6,7- dimethoxyquinazoline .

37. The method of claim 35, wherein said quinazoline compound is administered in a range from about 1 mg/kg/day to about 1 g/kg/day.

38. The method of claim 37, wherein said quinazoline compound is administered in a range from about 10 mg/kg/day to about 400 mg/kg/day.

39. The method of claim 38, wherein said quinazoline compound is administered at about 200 mg/kg/day.

40. The method of claim 34, wherein said small molecule comprises a carbohydrate or carbohydrate analog.

41. The method of claim 40, wherein said carbohydrate or carbohydrate analog is selected from the group consisting of lacto-N-neotetraose, 3'-sialyllactose and 6'- sialyllactose.

42. The method of claim 26, wherein said EGFR ligand comprises a proteinaceous EGFR ligand.

43. The method of claim 42, wherein said proteinaceous EGFR ligand is not substantially absorbed into the bloodstream.

44. The method of claim 42, wherein said proteinaceous EGFR ligand does not substantially alter the activity of EGFRs outside of the gut.

45. The method of claim 42, wherein said proteinaceous EGFR ligand does not substantially alter the activity of EGFRs in a cancerous tissue.

46. The method of claim 45, wherein said cancerous tissue comprises lung tissue.

47. The method of claim 42, wherein said proteinaceous EGFR ligand is selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR)₅ betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs) and mucin 4 (MUC4).

48. The method of claim 47, wherein said EGF is selected from the group consisting of the 53 amino acid form, the 52 amino acid form, the 51 amino acid form, the 48 amino acid form and homologs having at least 30% amino acid identity with any one of the aforementioned EGF forms.

49. The method of claim 42, wherein said proteinaceous EGFR ligand is administered in a range from about 20 μg/kg/day to about 1 g/kg/day.

50. The method of claim 49, wherein said proteinaceous EGFR ligand is administered in a range from about 200 μg/kg/day to about 200 mg/kg/day.

51. The method of claim 50, wherein said proteinaceous EGFR Iigand is administered at about 20 mg/kg/day.

52. The method of claim 26, wherein said adverse effects comprise adverse gastrointestinal effects.

53. The method of claim 52, wherein said adverse gastrointestinal effects are selected from the group consisting of diarrhea, nausea, vomiting, anorexia and weight loss.

54. The method of claim 52, wherein said patient is identified as suffering adverse gastrointestinal effects or at risk of suffering adverse gastrointestinal effects due to the administration of said EGFR inhibitor.

55. The method of claim 54, wherein said patient has a condition associated with a mutant EGFR which is substantially unresponsive to EGF.

56. The method of claim 26, wherein said adverse effects comprise adverse skin effects.

57. The method of claim 56, wherein said adverse skin effects are selected from the group consisting of rash, acne, dry skin, pruritus, vesiculobullous rash and mouth ulcerations.

58. The method of claim 56, wherein said patient is identified as suffering adverse skin effects or at risk of suffering adverse skin effects due to the administration of said EGFR inhibitor.

59. The method of claim 58, wherein said patient has a condition associated with a mutant EGFR which is substantially unresponsive to EGF.

60. The method of claim 26, wherein said EGFR inhibitor comprises gefitinib and said EGFR Iigand comprises EGF.

61. The method of claim 26, wherein said EGFR inhibitor comprises erlotinib and said EGFR Iigand comprises EGF.

62. The method of claim 26, wherein said EGFR Iigand is capable of activating EGFR.

63. A method of using an EGFR Iigand to ameliorate adverse effects associated with the administration of an EGFR inhibitor in a human subject, said method comprising informing said human subject that co-administering said EGFR Iigand with said EGFR inhibitor ameliorates at least one adverse effect associated with the administration of said EGFR inhibitor.

64. The method of claim 63, wherein said EGFR ligand is capable of activating EGFR.

65. The method of claim 63, wherein the administration of the EGFR inhibitor comprises oral administration.

66. The method of claim 63, wherein said at least one adverse effect is an adverse gastrointestinal effect.

67. The method of claim 66, wherein said adverse gastrointestinal effect is selected from the group consisting of diarrhea, nausea, vomiting, anorexia and weight loss.

68. The method of claim 63, wherein said at least one adverse effect is an adverse skin effect.

69. The method of claim 66, wherein said adverse gastrointestinal effect is selected from the group consisting of rash, acne, dry skin, pruritus, vesiculobullous rash and mouth ulcerations.

70. The method of claim 63, wherein informing said human subject comprises providing printed matter that advises that co-administering said EGFR ligand with said EGFR inhibitor ameliorates at least one adverse effect associated with the administration of said EGFR inhibitor.

71. The method of claim 70, wherein said printed matter is a label.

72. A method of manufacturing a pharmaceutical composition, said method comprising: obtaining a first dosage form comprising an EGFR inhibitor; obtaining a second dosage form comprising an EGFR ligand; and packaging together said first dosage form and said second dosage form.

73. The method of claim 72, wherein said first dosage form and said second dosage form each comprise an oral dosage form.

74. The method of claim 72, wherein said EGFR ligand is capable of activating EGFR.

75. The method of claim 72, wherein said EGFR ligand comprises EGF.

76. The method of claim 72, wherein said EGFR inhibitor comprises gefitinib.

77. The method of claim 72, wherein said EGFR ligand comprises EGF.

78. The method of claim 72, wherein said EGFR inhibitor comprises erlotinib.

79. The method of claim 72, wherein said EGFR ligand comprises EGF.

80. The method of claim 72, wherein said first dosage form and said second dosage form are merged together, thereby forming a combined dosage form.

81. A pharmaceutical composition made by the method of claim 72.

82. A pharmaceutical composition comprising a first dosage form which comprises a receptor tyrosine kinase (RTK) inhibitor and a second dosage form which comprises an RTK ligand.

83. The pharmaceutical composition of claim 82, wherein said first dosage form and said second dosage form each comprise an oral dosage form.

84. The pharmaceutical composition of claim 82, wherein said RTK inhibitor is selected from the group consisting of reversible inhibitors and irreversible inhibitors.

85. The pharmaceutical composition of claim 82, wherein said RTK inhibitor comprises a small molecule.

86. The pharmaceutical composition of claim 85, wherein said small molecule is selected from the group consisting of erlotinib, gefitinib, 4-(4-benzyloxyanilino)-6,7- dimethoxyquinazoline, imatinib, PKC412, MLN518, CEP-701, SU5402, SU5416, PDO 173074 and SMS-354825.

87. The pharmaceutical composition of claim 85, wherein said small molecule is present in an amount from about 50 mg/dose to about 50 g/dose.-

88. The pharmaceutical composition of claim 87, wherein said small molecule is present in an amount from about 500 mg/dose to about 20 g/dose.

89. The pharmaceutical composition of claim 88, wherein said small molecule is present in an amount of about 10 g/dose.

90. The pharmaceutical composition of claim 82, wherein said RTK ligand comprises a proteinaceous RTK ligand.

91. The pharmaceutical composition of claim 90, wherein said proteinaceous RTK ligand is selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB- EGF), epiregulin (EPR), neuregulins (NRGs), mucin 4 (MUC4), fibroblast growth factor- 1 (FGFl), fibroblast growth factor 2 (FGF2), fins-related tyrosine kinase 3 ligand (FMS-TK3), colony stimulating factor- 1 (CSF-I), platelet-derived growth factor (PDGF), growth hormone (GH), prolactin (PL), erythropoietin (EP), leptin (LP), stem cell factor (SFC), nerve growth factor (NGF), neutrophin 3 (NTF3) and vegetative growth factor (VEGF).

92. The pharmaceutical composition of claim 90, wherein said proteinaceous RTK ligand is present in an amount from about 1 mg/dose to about 50 g/dose.

93. The pharmaceutical composition of claim 92, wherein said proteinaceous RTK ligand is present in an amount from about 10 mg/dose to about 10 g/dose.

94. The pharmaceutical composition of claim 93, wherein said proteinaceous RTK ligand is present in an amount of about 1 g/dose.

95. The pharmaceutical composition of claim 82, wherein said first dosage form and said second dosage form further comprise a pharmaceutically acceptable carrier.

96. The pharmaceutical composition of claim 82, wherein said first dosage form and said second dosage form are selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel.

97. The pharmaceutical composition of claim 82, wherein said first dosage form and said second dosage form are merged, thereby forming a combined dosage form.

98. The pharmaceutical composition of claim 97, wherein said combined dosage form further comprises a pharmaceutically acceptable carrier.

99. The pharmaceutical composition of claim 97, wherein said combined dosage form is selected from the group consisting of a tablet, a capsule, a solution, a suspension, a cream, an ointment and a gel.

100. The pharmaceutical composition of claim 82, wherein said RTK ligand interacts with an RTK that is inhibited by said RTK inhibitor.

101. The pharmaceutical composition of claim 82, wherein said RTK ligand is capable of activating an RTK.

102. A method of ameliorating adverse effects associated with the administration of an RTK inhibitor, said method comprising co-administering to a patient an RTK inhibitor and a therapeutically effective amount of an RTK ligand.

103. The method of claim 102, wherein said RTK inhibitor and said RTK ligand are orally co-administered to said patient.

104. The method of claim 102, wherein co-administering said RTK inhibitor and said RTK ligand comprises administering said RTK ligand at least about 1 hour prior to the administration of said RTK inhibitor.

105. The method of claim 102, wherein co-administering said RTK inhibitor and said RTK ligand comprises administering said RTK ligand at least about 1 hour subsequent to the administration of said RTK inhibitor.

106. The method of claim 102, wherein co-administering said RTK inhibitor and said RTK ligand comprises administering said RTK ligand at about the same time as administering said RTK inhibitor.

107. The method of claim 106, wherein said RTK ligand and said RTK inhibitor are administered at the same time.

108. The method of claim 107, wherein said RTK ligand and said RTK inhibitor are administered together in a single dosage form.

109. The method of claim 102, wherein said RTK inhibitor is selected from the group consisting of reversible inhibitors and irreversible inhibitors.

110. The method of claim 102, wherein said RTK inhibitor comprises a small molecule.

111. The method of claim 110, wherein said small molecule is selected from the group consisting of erlotinib, gefitinib, 4-(4-benzyloxyanilino)-6,7-dimethoxyquinazoline, imatinib, PKC412, MLN518, CEP-701, SU5402, SU5416, PD0173074 and SMS-354825.

112. The method of claim 111, wherein said small molecule is administered in a range from about 1 mg/kg/day to about 1 g/kg/day.

113. The method of claim 112, wherein said small molecule is administered in a range from about 10 mg/kg/day to about 400 mg/kg/day.

114. The method of claim 113, wherein said small molecule is administered at about 200 mg/kg/day.

115. The method of claim 102, wherein said RTK ligand comprises a proteinaceous RTK ligand.

116. The method of claim 115, wherein said proteinaceous RTK ligand is not substantially absorbed into the bloodstream.

117. The method of claim 115, wherein said proteinaceous RTK ligand does not substantially alter the activity of RTKs outside of the gut.

118. The method of claim 115, wherein said proteinaceous RTK ligand does not substantially alter the activity of RTKs in a cancerous tissue.

119. The method of claim 115, wherein said proteinaceous RTK ligand is selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF), epiregulin (EPR), neuregulins (NRGs), mucin 4 (MUC4), fibroblast growth factor- 1 (FGFl), fibroblast growth factor 2 (FGF2), fms-related tyrosine kinase 3 ligand (FMS-TK3), colony stimulating factor- 1 (CSF-I), platelet-derived growth factor (PDGF), growth hormone (GH), prolactin (PL), erythropoietin (EP), leptin (LP), stem cell factor (SFC), nerve growth factor (NGF), neutrophin 3 (NTF3) and vegetative growth factor (VEGF).

120. The method of claim 115, wherein said proteinaceous RTK ligand is administered in a range from about 20 μg/kg/day to about 1 g/kg/day.

121. The method of claim 120, wherein said proteinaceous RTK ligand is administered in a range from about 200 μg/kg/day to about^' 200 mg/kg/day.

122. The method of claim 121, wherein said proteinaceous RTK ligand is administered at about 20 mg/kg/day.

123. The method of claim 102, wherein said patient is identified as suffering adverse effects or at risk of suffering adverse effects due to the administration of said RTK inhibitor.

124. The method of claim 102, wherein said patient has a condition associated with a mutant RTK which is substantially unresponsive to an RTK ligand.

125. The method of claim 102, wherein said adverse effects comprise adverse gastrointestinal effects.

126. The method of claim 102, wherein said adverse effects comprise adverse skin effects.

127. The method of claim 102, wherein said RTK ligand interacts with an RTK that is inhibited by said RTK inhibitor.

128. The method of claim 102, wherein said RTK ligand is capable of activating an RTK.

129. A method of using an RTK ligand to ameliorate adverse effects associated with the administration of an RTK inhibitor in a human subject, said method comprising informing said human subject that co-administering said RTK ligand with said RTK inhibitor ameliorates at least one adverse effect associated with the administration of said RTK inhibitor.

130. The method of claim 129, wherein the administration of the RTK inhibitor comprises oral administration.

131. The method of claim 129, wherein said RTK ligand is capable of activating an RTK.

132. The method of claim 129, wherein informing said human subject comprises providing printed matter that advises that co-administering said RTK ligand with said RTK inhibitor ameliorates at least one adverse effect associated with the administration of said RTK inhibitor.

133. The method of claim 132, wherein said printed matter is a label.

134. The method of claim 129, wherein said RTK ligand interacts with an RTK that is inhibited by said RTK inhibitor.

135. A method of manufacturing a pharmaceutical composition, said method comprising: obtaining a first dosage form comprising an RTK inhibitor; obtaining a second dosage form comprising an RTK ligand; and packaging together said first dosage form and said second dosage form.

136. The method of claim 135, wherein said first dosage form and said second dosage form each comprise an oral dosage form.

137. The method of claim 135, wherein said first dosage form and said second dosage form are merged together, thereby forming a combined dosage form.

138. The method of claim 135, wherein said RTK ligand interacts with an RTK that is inhibited by said RTK inhibitor.

139. The method of claim 135, wherein said RTK ligand is capable of activating an RTK.

140. A pharmaceutical composition made by the method of claim 135.