US 6,339,100 B1Grant

Methods for inhibiting mastocytosis

Issue Date:2002-01-15

•20 Claims

•2 Drawing Sheets

Abstract

A method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject.

Metadata

Assignee

The Trustees of Columbia University in the City of New York

Inventor

B. Jack Longley

Application Information

Application Number:US 09/474,474

Filing Date:1999-12-29

Priority Date:1999-12-29

Art Unit:7

Classifications

IPC:

A61K 3140A61K 31495

Field of Search:

514412514255

Patent Drawings (2 sheets)

Description

[0003] The invention described herein was made with Government support under grant numbers RO1 AR43356 and P30 AR44535 from the National Institutes of Health. Accordingly, the United States Government has certain rights in this invention.

[0004] Throughout this application, various publications are referenced within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found immediately preceding the claims.

BACKGROUND OF THE INVENTION

[0005] Mastocytosis is a neoplastic disease caused at least in part by somatic mutations of the c-KIT proto-oncogene resulting in constitutive activation of its protein product, KIT, the receptor tyrosine kinase for stem cell factor. KIT stimulates mast cell proliferation and prevents apoptosis of neoplastic mast cells. To develop potential therapies for mastocytosis we used indolinones, small molecules which inhibit tyrosine kinases. An indolinone derivative with structure VI (see below) or structure VII inhibited wild-type KIT, but variably inhibited constitutively activated KIT mutants. The compounds with structure VI or structure VII were effective against KIT with juxtamembrane activating mutations. Furthermore, the compounds with structure VI or structure VII killed neoplastic mast cells expressing a juxtamembrane-mutated KIT, whereas the compound with structure VII killed neoplastic mast cells expressing KIT bearing a kinase domain mutation. These data show a direct correlation between inhibition of constitutively activated KIT and the death of neoplastic mast cells, and point to specific tyrosine kinase inhibitors as a novel therapy aimed directly at a cause of mastocytosis.

SUMMARY OF THE INVENTION

[0006] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0007] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein

[0008] R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0009] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0010] R₈, R₉, and R₁₀are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0011] n is 0-3; x is Br, Cl, F or I; each of R and R′ is independently H, alkyl or aryl.

[0012] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0013] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0014] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0015] R₈, R₉, R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0016] Ra and Rb are each independently hydrogen, alkyl and C(O)R, or NRaRb taken together may be a heterocyclic ring of 3 to 8 atoms optionally substituted at one or more positions with hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0017] n is 0-3; x is Br, Cl, F or I; and each of R and R′ is independently H, alkyl or aryl.

[0018] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0019] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0020] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0021] A is a five membered heteroaryl ring thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, or tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO2, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0022] n is 0-3; x is Br, Cl, F or I; and each of R and R′ is independently H, alkyl or aryl.

[0023] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0024] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0025] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0026] R₈, R₉and R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0027] n is 0-3; x is Br, Cl, F or I; each of R and R′ is independetly H, alkyl or aryl.

[0028] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0029] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein RI is HI or alkyl; R₂is O or S; R₃is hydrogen;

[0030] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR ′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH2)_nCO2R, or CONRR′;

[0031] R₈, R₉, R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0032] n is 0-3; Z is Br, Cl, F, I, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl; and each of R and R′ is independently H, alkyl or aryl.

[0033] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0034] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject.

[0035] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0036] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject.

[0037] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0038] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject.

BRIEF DESCRIPTION OF THE FIGURES

[0039] FIG. 1: Effects of indolinone derivatives on phosphorylation of wild-type and mutant KITs. (a) Anti-phosphotyrosine (pTyr) blots of immunoprecipitated wild-type (WT) KIT expressed in COS cells and of mutant KITs expressed in C2 and P815 cells stimulated (+), or not (−), with stem cell factor (SCF, 100 ng/ml, 10 min) after incubation (+), or not (−), with indolinone derivatives (5 μM for COS and C2 cells, 40 μM for P815 cells, 30 min) show that the compound with structure VII or structure VI inhibit wild-type KIT phosphorylation but variably inhibit C2 and P815 KIT phosphorylation. The compound with the structure VII or structure VI are effective against KIT containing an activating juxtamembrane domain mutation in C2 cells. (b) Re-probing the anti-pTyr blots (after stripping) with anti-KIT antibody shows that comparable amounts of receptor are present in different lanes.

[0040] FIG. 2: Effects of indolinone derivatives on neoplastic mast cell growth. Cell proliferation assay of C2 and P815 cells treated daily with 1 μM and 10 μM, respectively, of indolinone derivatives shows that the compound with structure VII or structure VI kill C2 cells, and the compound with structure VII kill P815 cells. Results (mean±SEM) represent averages of triplicate cultures.

DETAILED DESCRIPTION OF THE INVENTION

[0041] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0042] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein

[0043] R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0044] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0045] R₈, R₉, and R₁₀are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂,NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0046] n is 0-3; x is Br, Cl, F or I; each of R and R′ is independently H, alkyl or aryl.

[0047] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0048] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0049] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0050] R₈, R, R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0051] Ra and Rb are each independently hydrogen, alkyl and C(O)R, or NRaRb taken together may be a heterocyclic ring of 3 to 8 atoms optionally substituted at one or more positions with hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R. SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R. NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0052] n is 0-3; x is Br, Cl, F or I; and each of R and R′ is independently H, alkyl or aryl.

[0053] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0054] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0055] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0056] A is a five membered heteroaryl ring thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, or tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0057] n is 0-3; x is Br, Cl, F or I; and each of R and R′ is independently H, alkyl or aryl.

[0058] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0059] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

[0060] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂) CO2R, or CONRR′;

[0061] R₈, R₉and R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0062] n is 0-3; x is Br, Cl, F or I; each of R and R′ is independently H, alkyl or aryl.

[0063] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0064] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R3 is hydrogen;

[0065] R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0066] R₈, R₉, R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

[0067] n is 0-3; Z is Br, Cl, F, I, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl; and each of R and R′ is independently H, alkyl or aryl.

[0068] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0069] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject.

[0070] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0071] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject.

[0072] This invention provides a method of preventing or treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

[0073] or a pharmaceutically acceptable salt thereof so as to thereby prevent or treat mastocytosis in the subject.

[0074] In one embodiment, the above method comprises inhibiting the kinase enzymatic reaction of kit protein. In one embodiment, the method comprises inhibiting chymase, elastase or other SCF cleaving enzymes. In one embodiment, the method comprises inhibiting ligand binding with an antibody, peptide, or nonpeptide chemical. In one embodiment, the method comprises inhibiting kit dimerization with an antibody, peptide, or nonpeptide chemical. In one embodiment of the above method, downstream signaling of the kit activation pathway is inhibited by blocking substrate association with kit kinase domain. In another embodiment of the above method, downstream signaling of the kit activation pathway is inhibited by blocking enzymatic function in the downstream signaling pathway. In another embodiment of the above method, the downstream signaling of the kit activation pathway is inhibited by blocking binding of molecules in the downstream signaling pathway.

[0075] The subject of the above methods includes but is not limited to a mammal. The subject may be a mammal or non-mammal. The subject may be a human, a primate, an equine subject, an opine subject, an avian subject, a bovine subject, a porcine, a canine, a feline or a murine subject. In another embodiment, the subject is a vertebrate. In a preferred embodiment, the mammal is a human being.

[0076] In another embodiment of the above methods, the compound is a small molecule or peptide. In one embodiment, the compound has the following structure or a pharmaceutically acceptable salt thereof:

[0077] In one embodiment, the compound has the following structure or is a pharmaceutically acceptable salt thereof:

[0078] The subject of this method includes but is not limited to a canine or a human being.

[0079] In one embodiment of the above methods, the compound is admixed with a carrier.

[0080] In one embodiment of the above methods, the administration is intralesional, intraperitoneal, intramuscular, subcutaneous, intravenous, liposome mediated delivery, transmucosal, intestinal, topical, nasal, oral, anal, ocular or otic delivery.

[0081] In one embodiment of the above methods, the therapeutically effective amount is from about 1 mg/kg to about 1000 mg/kg. In one embodiment of the above methods, the therapeutically effective amount is from about 50 mg/kg to about 200 mg/kg.

[0082] In one embodiment of the above methods, the pharmaceutically acceptable salt is a hydrochloride salt, a mesylate salt, an ethylsulfonate salt, or a sulfate salt.

[0083] This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

[0084] The proto-oncogene c-KIT encodes KIT (Yarden et al, 1987; Qiu et al, 1988), the receptor tyrosine kinase for stem cell factor (Martin et al, 1990), also known as mast cell growth factor. Somatic c-KIT mutations causing ligand-independent activation of KIT and cell transformation (Furitsu et al, 1993; Kitayama et al, 1995; Tsujimura et al, 1996; Hirota et al, 1998; Ma et al, 1999a) appear causal in certain types of mastocytosis (Nagata et al, 1995; Longley et al, 1996, 1999; Ma et al, 1999a).

[0085] Documented activating c-KIT mutations fall into two groups. One group consists of mutations in codon 816 of human c-KIT, or its equivalent positions in other species, resulting in single residue substitution for Asp816 in the activation loop of the receptor kinase domain (Ma et al, 1999a). The other group of activating mutations includes single residue substitutions and in-frame insertions or deletions in the receptor intracellular juxtamembrane region, which disrupt intramolecular inhibition of the kinase by a putative juxtamembrane α-helix (Ma et al, 1999b). All sporadic adult-onset mastocytosis patients examined to date, and a subset of pediatric cases with atypical clinical presentations, have activating codon 816 mutations (Longley et al, 1999), whereas activating juxtamembrane mutations are common in canine mastocytomas (Ma et al, 1999a) and in human gastrointestinal stromal tumors (Hirota et al, 1998).

[0086] It is shown that inhibition of KIT by indolinone derivatives is sufficient to kill neoplastic mast cells. These results support a causal role for activating c-KIT mutations in the pathogenesis of mastocytosis and identify tyrosine kinase inhibitors as a novel therapy targeting a cause of mastocytosis.

Materials and Methods

[0087] Compounds Indolinones are polycyclic compounds which bind the ATP binding pocket of receptor tyrosine kinases, inhibiting their activities (Mohammadi et al, 1997; Sun et al, 1998). Several indolinone derivatives were used in the present study:

[0088] (a) the compound with following structure:

[0089] (b) the compound with the following structure:

[0090] (c) the compound with the following structure:

[0091] The compound with structure VII has a relatively broad spectrum of inhibition, being effective against platelet-derived growth factor receptor, fibroblast growth factor receptor, and insulin receptor, while the compound with structure VIII effectively inhibits the fibroblast growth factor receptor (Mohammadi et al, 1997; Sun et al, 1998). The compound with structure VI suppresses vascular endothelial growth factor receptor and the platelet-derived growth factor receptor. (Sun L, Tran N, Tang F, Schreck R, Fong TAT, McMahon G, Tang C: Synthesis and biological evaluation of novel 3-(substituted pyrrol-2-yl)indolin-2-ones as potent and selective inhibitors of the Flk-1/KDR receptor tyrosine kinase. 215th ACS National Meeting, Dallas, MEDI-169, 1998 (Abstr.); Sun L, Tran N, Liang C, Tang F, Rice A, Schreck R, Waltz K, Shawver L, McMahon G, Tang C: Design, synthesis, and evaluations of substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methylindenyl]indolin-2-ones as inhibitors of VEGF, PDGF, and FGF receptor tyrosine kinases. The potential activities of these compounds on KIT have not been previously reported.

Assays

[0092] KIT activity was determined by in vitro phosphorylation, as previously described (Ma et al, 1999a). The neoplastic mast cell line C2 and P815 have been previously described (Dunn et al, 1957; Lazarus et al, 1986; DeVinney and Gold, 1990), and their activating c-KIT mutations characterized (Tsujimura et al, 1994; Ma et al, 1999a). To determine cell proliferation and viability, cells from triplicate cultures were stained with trypan blue and counted in a hemocytometer. All experiments were repeated at least three times.

Results and Discussion

[0093] The effects of indolinone derivatives on the activity of wild-type KIT were first examined. A compound with structure VII or structure VI could substantially reduce tyrosine phosphorylation of the wild-type receptor at 5 μM concentration, while the compound of structure VIII could only slightly decrease the receptor phosphorylation at this concentration (FIG. 1).

[0094] The effects of these compounds on constitutive activation of KIT mutants expressed by neoplastic mast cells were then tested. For activating juxtamembrane mutations, the C2 dog mastocytoma cell line was studied (Lazarus et al, 1986; DeVinney and Gold, 1990), in which KIT is constitutively active due to a juxtamembrane insertion mutation (Ma et al, 1999a). Treatment of the cells with 5 μM of the compound with structure VII or structure VI resulted in approximately 50% reduction of the constitutive C2 KIT phosphorylation (FIG. 1). In contrast, 5 μM of the compound of structure VIII could barely repress the receptor phosphorylation. The effects of these compounds on constitutive phosphorylation of KIT bearing in the juxtamembrane region either deletion of Trp556-Lys557 or substitution of Pro for Leu575, mutations previously identified in dog mastocytomas (Ma et al, 1999a) were also examined. Similar inhibitory effects were observed with the additional mutant receptors (data not shown).

[0095] For activating mutations in the kinase domain, the P815 murine mast cell tumor line (Dunn et al, 1957) was examined. P815 c-KIT contains a point mutation resulting in substitution of Tyr for Asp814, the equivalent position of human Asp816, which causes constitutive receptor activation (Tsujimura et al, 1994).

[0096] To determine whether these compounds might be useful therapeutically for mast cell tumors, the effects on C2 and P815 cell proliferation was examined. As shown in FIG. 11, C2 cells were killed by treating them daily with 1 μM of the compound of structure VII or structure VI. By comparison, the compound of structure VIII only retarded the C2 cell proliferation at this concentration. These results were consistent with the effects of the five compounds on the phosphorylation of KIT expressed by C2 cells (FIG. 1).

[0097] The proliferation of P815 cells was not affected by treating the cells daily with the compounds at 1 μM concentration. However, the compound with structure VII killed the P815 cells at 10 μM concentration, whereas the compound with structure VIII or structure VI showed only inhibition of the cell proliferation at this concentration (FIG. 2). The requirement of an order of magnitude or more of the compound of structure VII to kill P815 cells in comparison with their effects on C2 cells might be related to the difference in the in vivo behavior of these two cell lines. P815 cells are able to rapidly form metastasizing tumors in syngenic mice and kill the animals, whereas tumors formed by C2 cells in nude mice do not metastasize and do not kill the host.

[0098] Although the compound with structure VII was able to kill the P815 cells, it may exert its effects through a different mechanism. It has been reported that the mutation found in P815 cells alters KIT's substrate specificity (Piao et al, 1996). In this case, the relatively general inhibitor with the structure VII, although not effectively inhibiting phophorylation of the KIT mutant, might still effectively repress other activating effector(s) in the KIT signaling pathway(s), resulting in the death of P815 cells. The fact that a compound which does not inhibit constitutively activated KIT can kill neoplastic mast cells does not detract from the theory that activating c-KIT mutations may be causal events in mastocytosis. Conversely, effective suppression of constitutively activated KIT is directly associated with the ability to kill neoplastic mast cells, supporting this hypothesis.

[0099] While the mechanism whereby the compound with the structure VII killed neoplastic P815 cells remains to be elucidated, the present study identifies indolinone-derived tyrosine kinase inhibitors as the first potential therapy aimed at a cause of mastocytosis.

References

[0100] Devinney R, Gold W V: Establishment of two dog mastocytoma cell lines in continuous culture. Am J Respir Cell Mol Biol3: 413-420, 1990

[0101] Dunn T B, Potter M: A transplantable mast-cell neoplasm in the mouse. J Natl Cancer Inst18: 587-601, 1957

[0102] Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, et al: Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest92: 1736-1744, 1993

[0103] Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, et al: Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science279: 577-580, 1998

[0104] Kitayama H, Kanakura Y, Furitsu T, Tsujimura T, Oritani K, et al: Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. Blood85: 790-798, 1995

[0105] Lazarus S C, DeVinney R, McCabe L J, Finkbeiner W E, Elias D J, Gold W M: Isolated canine mastocytoma cells: propagation and characterization of two cell lines. Am J Physiol251: C935-944, 1986

[0106] Longley B J, Tyrrell L, Lu S, Ma Y, Langley K, et al: Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nature Genet12: 312-314, 1996

[0107] Longley B J, Metcalfe D D, Tharp M, Wang X, Tyrrell L, et al: Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci USA96: 1609-1614, 1999

[0108] Ma Y, Longley B J, Wang X, Blount J L, Langley K, Caughey G H: Clustering of activating mutations in c-KIT's juxtamembrane coding region in canine mast cell neoplasms. J Invest Dermatol112: 165-170, 1999a

[0109] Ma Y, Cunningham M E, Wang X, Ghosh I, Regan L, Longley B J: Inhibition of spontaneous receptor phosphorylation by residues in a putative α-helix in the KIT intracellular juxtamembrane region. J Biol Chem274: 13399-13402, 1999b

[0110] Martin F H, Suggs S V, Langley K E, Lu H S, Ting J, et al: Primary structure and functional expression of rat and human stem cell factor DNAs. Cell63: 203-211, 1990

[0111] Mohammadi M, McMahon G, Sun L, Tang C, Hirth P, et al: Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science276: 955-960, 1997

[0112] Nagata H, Worobec A S, Oh C K, Chowdhury B A, Tannenbaum S, et al: Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Natl Acad Sci USA92: 10560-10564, 1995

[0113] Piao X, Paulson R, Van Der Geer P, Pawson T, Berstein A: Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1. Proc Natl Acad Sci USA93: 14665-14669, 1996

[0114] Qiu F, Ray P, Brown K, Barker P E, Jhanwar S, et al: Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO J7: 1003-1011, 1988

[0115] Sun L, Tran N, Tang F, App H, Hirth P, McMahon G, Tang C: Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine kinases. J Med Chem41: 2588-2603, 1998

[0116] Tsujimura T, Furitsu T, Morimoto M, Koji K, Nomura S, et al: Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation. Blood83: 2619-2626, 1994

[0117] Tsujimura T, Morimoto M, Hashimoto K, Moriyama Y, Kitayama H, et al: Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain. Blood87: 273-283, 1996

[0118] Yarden Y, Kuang W-J, Yang-Feng T, Coussens L, Munemitsu S, et al: Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J6: 3341-3351, 1987

Claims

What is claimed:

1. A method of treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby treat mastocytosis in the subject, wherein

R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

R₄, R₅, R₆, and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

R₈, R₉, and R₁₀are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

n is 0-3; x is Br, Cl, F or I; each of R and R′ is independently H, alkyl or aryl.

2. A method of treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

R₄, R₅, R₆and R₇are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

R₈, R₉, R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

Ra and Rb are each independently hydrogen, alkyl and C(O)R, or NRaRb taken together may be a heterocyclic ring of 3 to 8 atoms optionally substituted at one or more positions with hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

n is 0-3; x is Br, Cl, F or I; and each of R and R′ is independently H, alkyl or aryl.

3. A method of treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

A is a five membered heteroaryl ring thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, or tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO2, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

n is 0-3; x is Br, Cl, F or I; and each of R and R′ is independently H, alkyl or aryl.

4. A method of treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

R₈, R₉and R₁₀and R₁₁are each independently hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO₂NRR′, SO₃R, SR, NO₂, NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH₂)_nCO2R, or CONRR′;

n is 0-3; x is Br, Cl, F or I; each of R and R′ is independently H, alkyl or aryl.

5. A method of treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby treat mastocytosis in the subject, wherein R₁is H or alkyl; R₂is O or S; R₃is hydrogen;

n is 0-3; Z is Br, Cl, F, I, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl; and each of R and R′ is independently H, alkyl or aryl.

6. A method of treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby treat mastocytosis in the subject.

7. A method of treating in a subject mastocytosis which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof so as to thereby treat mastocytosis in the subject.

8. The method of any one of claims 1-6 and 7, which comprises inhibiting the kinase enzymatic reaction of kit protein.

9. The method of any one of claims 1-6 and 7, wherein downstream signaling of the kit activation pathway is inhibited by blocking substrate association with kit kinase domain.

10. The method of any one of claims 1-6 and 7, wherein downstream signaling of the kit activation pathway is inhibited by blocking enzymatic function in the downstream signaling pathway.

11. The method of any one of claims 1-6 and 7, wherein downstream signaling of the kit activation pathway is inhibited by blocking binding of molecules in the downstream signaling pathway.

12. The method of any one of claims 1-6 and 7, wherein the subject is a mammal.

13. The method of claim 12, wherein the mammal is a human being.

14. The method of claim 9, wherein the compound is a small molecule or peptide.

15. The method of claim 9, wherein the compound has the structure:

or is a pharmacuetically acceptable salt thereof.

16. The method of claim 15, wherein the subject is a canine or a human being.

17. The method of any one of claims 1-6 and 7, wherein the compound is admixed with a carrier.

18. The method of any one of claims 1-6 and 7, wherein the administration is intralesional, intraperitoneal, intramuscular, subcutaneous, intravenous, liposome mediated delivery, transmucosal, intestinal, topical, nasal, oral, anal, ocular or by otic delivery.

19. The method of any one of claims 1-6 and 7, wherein the therapeutically effective amount is from about 1 mg/kg to about 1000 mg/kg.

20. The method of any one of claims 1-6 and 7, wherein the pharmaceutically acceptable salt is a hydrochloride salt, a mesylate salt, an ethylsulfonate salt, or a sulfate salt.

Patent Citations (4)

Patent	Date	Inventor	Cited By
US5883116(A)	1999-03-01	Tang et al.	Applicant
US5905149(A)	1999-05-01	Battistini et al.	Applicant
WO9910325	1999-03-01		Applicant
WO9961422	1999-12-01		Applicant

Non-Patent Literature (35)

Anderson, D.M., et al. (1990) “Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms” Cell, 63:235-243 (Exhibit 1).Applicant
Bradl, M., et al. (1991) “Clonal coat color variation due to a transforming gene expressed in melanocytes of transgenic mice” Proc. Nat. Acad. Sci. USA 88:6447-6451; (Exhibit 2).Applicant
Costa, J. J. et al. (1996) “Recombinant human stem cell factor (KIT ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo” J. Exp. Med 183: 2681-2686; (Exhibit 3).Applicant
Funasaka, Y., et al. (1992) “C-kit-kinasse induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in respnse to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas” Mol. Biol. Cell, 3:197-209; (Exhibit 4).Applicant
Furitsu, T., et al. (1993) Identification of mutations in the coding sequence of the proto-oncogene c-kit in human mast cell leuemia cell line causing ligand independent activation of c-KIT product J. Clin. Invest., 92:1736-1744; (Exhibit 5).Applicant
Grichnik, J. M., et al. (1995) “Human recombinant stem-cell factor induces melanocytic hyperplasia in susceptible patients” J. Am. Acad. Dermol., 33: 577-583; (Exhibit 6).Applicant
Hamann, K., et al. (1995) “Expression of stem cell factor in cutaneous mastocytosis” Br. J. Dermatol., 133: 203-208; (Exhibit 7).Applicant
Harrist, T.J., et al. (1995) Recombinant human stem cell factor in (SCF) (c-kit ligand promotes melanocytes hyperplasia and activation in vivo Lab. Invest., 72:48A; (Exhibit 8).Applicant
Hirobe, T. (1984) “Histochemical survey of the distribution of the epidermal melanoblasts and melanocytes in the mouse during fetal and postnatal periods” Anat. Rec., 208:589-594; (Exhibit 9).Applicant
Longley, B. J. et al. (1993) “Altered metabolism of mast-cell growth factor (c-kit ligand) in cutaneous mastocytosis” N. Engl. J. Med. . 328:1302-1307; (Exhibit 10).Applicant
Longley, B. J. et al. (1995) “The mast cell and mast cell disease” J. Am. Acad. Dermatol., 32:545-561; (Exhibit 11).Applicant
Longley, B. J., et al. (1996) “Somastic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm” Nature Genetics, 12:312-314; (Exhibit 12).Applicant
Lu, H. S., et al. (1991) “Amino acid sequence and post-translational modification of stem cell factor isolated from buffalo rat liver cell-conditioned medium” J. Biol. Chem., 266:8102-8107; (Exhibit 13).Applicant
Nishikawa, S., et al. (1991) “In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development” EMBO J. 10:2111-2118; (Exhibit 14).Applicant
Okura, M., et al. (1995) “Effects of monoclonal anti-c-kit antibody (AKC2) on melanocytes in newborn mice” J. Invest. Dermatol., 105:322-328; (Exhibit 15).Applicant
Tsai, M., et al. (1991) “The rat c-kit ligand, stem cell factor, induces the development of connective tissue-type and mucosal mast cells in vivo: analysis by anatomical distribution, hisochemistry, and protease phenotype” J. Exp. Med., 174:125-131; (Exhibit 16).Applicant
Vassar, R., et al. (1989) “Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice” Proc. Natl. Acad. Sci. USA, 86:1563-1567; (Exhibit 17).Applicant
Weiss, R. R. et al. (1995) “Human dermal endothelial cells express membrane-associated mast cell growth factor” J. Invest. Dermatol. 104:101-106; (Exhibit 18).Applicant
Williams, D.E., et al. (1990) “Identification of a ligand for the c-kit proto-oncogene” Cell, 1990; 63:167-174; (Exhibit 19).Applicant
Yarden, Y., et al. (1987) “Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand” EMBO J. 6:3341-3351; (Exhibit 20).Applicant
Yoshida, H., et al. (1996) “Distinct stages of melanocyte differentiation revealed by analysis of nonuniform pigmentation patterns” Development, 122:1207-1214; (Exhibit 21).Applicant
Yohida, H. et al. (1996) “Neural and skin cell- specific expression pattern conferred by Steel factor regulatory sequence in transgenic mice” Development Dynamic, 207:222-232; (Exhibit 22).Applicant
Zsebo, K. M., et al. (1990) “Stem cell factor is encoded at the S1 locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor” Cell, 63:213-224; (Exhibit 23).Applicant
Devinney R, Gold WV: Establishment of two dog mastocytoma cell lines in continuous culture. Am J Respir Cell Mol Biol 3: 413-420, 1990 (Exhibit 24).Applicant
Dunn TB, Potter M: A transplantable mast-cell neoplasm in the mouse. J Natl Cancer Inst 18: 587-601, 1957 (Exhibit 25).Applicant
Lazarus SC, DeVinney R, McCabe LJ, Finkbeiner WE, Elias DJ, Gold WM: Isolated canine , 1986 mastocytoma cells: propagation and characterization of two cell lines. Am J Physiol 251: C935-944 (Exhibit 26).Applicant
Martin FH, Suggs SV, Langley KE, Lu HS, Ting J, et al: Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63: 203-211, 1990 (Exhibit 27).Applicant
Mohammadi M, McMahon G, Sun L, Tang C, Hirth P, et al: Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276: 955-960, 1997 (Exhibit 28).Applicant
Piao X, Paulson R, Van Der Geer P, Pawson T, Berstein A: Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1. Proc Natl Acad Sci USA 93: 14665-14669, 1996 (Exhibit 29).Applicant
Qiu F, Ray P, Brown K, Barker PE, Jhanwar S, et al: Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family-oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO J 7: 1003-1011, 1988 (Exhibit 30).Applicant
Sun L, Tran N, Tang F, App H, Hirth P, McMahon G, Tang C: Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine kinases. J Med Chem 41: 2588-2603, 1998 (Exhibit 31).Applicant
Tsujimura T, Furitsu T, Morimoto M, Koji K, Nomura S, et al: Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation. Blood 83: 2619-2626, 1994 (Exhibit 32).Applicant
Schrader, John W. and Thomas, Wayne R. “Delayed Hypersensitivity in Mast-Cell-Deficient Mice”, The Journal of Immunology. (1983), vol. 130, No. 6, pp. 2565-2567 (Exhibit 33).Applicant
Galli, Stephen J. and Mekori, Yoseph A., “Undiminished Immunologic Tolerance to Contact Sensitivity in Mast Cell-Deficient W/Wv and S1/S1d Mice1”, The Journal of Immunology. (1995), vol. 135, No. 2, pp. 879-885 (Exhibit 34).Applicant
Askenase, Philip W., Loveren, Henk Van, Kraeuter-Kops, Sandra, Ron, Yacov, Meade, Robin, Theoharides, Theoharis C., Nordlund, James J., Scovern, Henry, Gerhson, Michael D., and Ptak, Wlodzimierz., “Defective Elicitation of Delayed-Type Hypersensitivity in W/Wv and SI/SId Mast Cell-Deficient Mice1”, The Journal of Immunology. (1983), vol. 131, No. 6, pp. 2687-2693 (Exhibit 35).Applicant