MXPA00001592A - Combination of tyrosine kinase inhibitor and chemical castration to treat prostate cancer - Google Patents

Abstract

A method of treating prostate cancer by coadministering a tyrosine kinase inhibitor such as an indolocarbazole and a chemical castration agent is disclosed. A composition containing a tyrosine kinase inhibitor and a chemical castration agent is also disclosed.

Description

COMBINATION OF TIROSINE KINASE INHIBITOR AND CHEMICAL CASTRATION TO TREAT PROSTATE CANCER BACKGROUND OF THE INVENTION The invention relates to oncology, endocrinology, andrology and pharmacology. Prostate cancer is the most frequently diagnosed cancer and is responsible for approximately 41,000 deaths in the United States annually (Parker, S.L. et al., (1996) CA Cancer J. Clin., 65: 5-27). Early-stage prostate cancer, confined to an organ, is usually managed with surgery or radiation therapy until the patient dies of unrelated causes. Carcinomas such as breast cancer, colon cancer and adenocarcinoma are characterized by rapid cell division.

Consequently, these cancers are treatable with chemotherapeutic agents that inhibit the rapid division of cells. In contrast, prostate cancer is not characterized by rapid cell division. Therefore, conventional chemotherapeutic agents generally exhibit low efficacy against prostatic carcinomas. Protative carcinomas are usually sensitive to hormonal manipulation. The currently approved treatment for prostate cancer includes surgical castration, chemical castration, or a combination of surgical castration and chemical castration. The removal of the testes, the primary organ of testosterone production, reduces the level of androgens in circulation, to less than 5% of normal levels. This reduction in androgen levels inhibits tumor growth in the prostate. Although the anti-tumor effects of surgical castration are direct, the anti-tumor effects may be temporary. Surgical castration usually leads to clonal selection of tumor cells in androgen-independent prostate. This results in the further development of the prostate tumor in a form that proliferates without testosterone or stimulation of DHT (Isaacs et al. (1981) Cancer Res.41: 5070-5075; Crawford et al. (1989) N. Eng. J. Med. 321: 419-424). Chemical castration (also known as medical castration) is usually replaced by surgical castration, as an initial treatment. Chemical castration can be achieved through the administration of estrogens, for example, diethylstilbestrol (DES); LHRH analogs, for example, leuprolide acetate (LUPRON®) or goserelin acetate (ZOLADEX®); steroidal anti-androgens, for example cyproterone acetate (CPA); non-steroidal anti-androgens, for example, flutamide, nilutamide or CASODEX®; or a combination of said agents. Tyrosine kinases linked to the receptor are transmembrane proteins that contain an extracellular ligand binding domain, a transmembrane sequence, and a cytoplasmic tyrosine kinase domain. Tyrosine kinases work in cell signal transduction. Proliferation, differentiation, migration, metabolism and programmed cell death are examples of cellular responses mediated by tyrosine kinase. Tyrosine kinases have been implicated in prostate epithelial cell transformation and tumor progression. The tyrosine kinases involved include fibroblast growth factor receptors (FGF), epidermal growth factor receptors (EGF) and platelet-derived growth factor (PDGF) receptors. Also involved are nerve growth factor receptors (NGF), brain derived neurotrophic factor (BDNF) receptors, and neurotrophin-3 (NT-3) receptors and neurotrophin-4 receptors (NT-4). The patents of E.U.A. Nos. 5,516,771; 5,654,427 and 5,65,407 discuss inhibitors of indolocarbazole tyrosine kinase and prostate cancer. The patents of E.U.A. Nos. 5,475,110; 5,591,855; and 5,594,009; and WO 96/11933 discuss inhibitors of tyrosine kinase of the pyrrolocarbazole type and prostate cancer.

COMPENDIUM OF THE INVENTION It has been unexpectedly discovered that tyrosine kinase inhibitors exert their anti-tumor effects against mammalian prostate cancer through a hormone-independent mechanism. It has further been discovered that the combination of tyrosine kinase inhibitor therapy and anti-hormone therapy can be synergistic. Based on the discoveries, the invention relates to a method for treating prostate cancer in a mammal, for example a human being. The method includes administering a therapeutic amount of a tyrosine kinase inhibitor to the mammal, and co-administering a chemical castration agent to the mammal. The tyrosine kinase inhibitor and the chemical castration agent can synergistically inhibit the progression of the prostate tumor. Inhibitors of tyrosine kinase include indolocarbazoles. Preferred indolocarbazoles include the following compounds: Compound 11-12; Compound 11-4; Y Compound II-4-LAE.

The compound II-4-LAE is the lysyl-β-alaninate ester of Compound 11-4, or a pharmaceutically acceptable salt of the ester, for example, the dihydrochloride salt. Compound 11-12 is described in the patent of E.U.A. No. 4,923,986 ("Compound 20"). Compound II-4 is described in the patent of E.U.A. No. 5,461,146. Compound II-4-LAE is described in the patent of E.U.A. No. 5,650,407 (Example 14). The tyrosine kinase inhibitor can also be a pyrrolcarbazole. The tyrosine kinase inhibitor can be administered through any suitable route, for example, orally or parenterally. Chemical castration agents useful in the invention include estrogens; LHRH agonists, for example leuprolide acetate (LUPRON®) and goserelin acetate (ZOLADEX®); LHRH antagonists, for example ANTIDE® (Ares-Serono) and GANIRELIX® (Akzo Nobel); and anti-androgens, for example flutamide and nilutamide. The tyrosine kinase inhibitor and the chemical castration agent can be administered in separate formulations.

Alternatively, they can be formulated together and administered in a single composition. The invention also relates to a composition comprising a tyrosine kinase inhibitor and a chemical castration agent. Preferably, the tyrosine kinase inhibitor in the composition is a trkA inhibitor, a trkB inhibitor, or a trkC inhibitor. Preferably, the tyrosine kinase inhibitor in the composition is an indolocarbazole. Preferred indolocarbazoles are: (Compound 11-12); (Compound II-4); Y (Compound II-4-LAE).

Alternatively, the tyrosine kinase inhibitor in the composition can be a fused pyrrolocarbazole. The composition can be formulated for oral administration or parenteral administration. The chemical castration agent can be an estrogen, an LHRH analog, or an anti-androgen, or a combination of two or more of these compounds. A preferred LHRH analog to be included in the composition is leuprolide acetate. A preferred anti-androgen to be included in the composition is flutamide. The chemical castration agent included in the composition can be a combination of an LHRH analogue and an anti-androgen, for example, leuprolide acetate and flutamide. As used herein, "chemical castration agent" means a compound that: (1) inhibits the production of androgens in serum, (2) blocks the binding of androgens in serum to androgen receptors, or (3) inhibits the conversion of testosterone to DHT, or a combination of two or more of said compounds. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In the case of conflict, the present application, including the definitions, will control. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference. Although methods and materials similar or equivalent to those described herein can be used, in the practice or testing of the present invention, preferred methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other aspects and advantages of the invention will be apparent from the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of the relative tumor volume (change of times) versus time (days) in a rat prostate tumor model system in vivo. Circles, vehicle control; right triangles, monotherapy control of Compound 11-4; cadres, castration controls; inverted triangles, combination treatment of Compound ll-4 / castration. The dose of Compound 11-4 was 10 mg / kg, by subcutaneous injection.The administration of Compound II-4 is indicated by rectangles containing plus signs on the X axis. Figure 2 is a graph of the relative tumor volume (change of times in tumors of Dunning H) against time (days) in castrated rats. Circles, vehicle control; picture, Compound II-4. The administration time of Compound II-4 is indicated by rectangles on the X axis. Figure 3 is a graph of the relative tumor volume. (change of times) against time (days) in rats previously treated with Compound II-4. The circles, Compound II-4; frames, treatment with Compound ll-4 / castration. The administration time of Compound 11-4 is indicated by rectangles on the X axis. Figure 4 is a graph of the relative tumor volume (change of times) against time (days) in a rat prostate tumor model system in vivo Circles, vehicle control; straight triangles, monotherapy control of Compound 11-12; frames, chemically castrated controls that receive leuprolide acetate (LUPRON®); inverted triangles, combination treatment of Compound ll-12 / leuprolide acetate. The dose of compound II-4 was 10 mg / kg of IDB, per os. The administration of Compound 11-12 is indicated by rectangles on the X axis.

DETAILED DESCRIPTION OF THE INVENTION The co-administration of a tyrosine kinase inhibitor and a chemical castration agent can be through the concurrent administration of separate formulations, i.e., a tyrosine kinase formulation and a chemical castration agent. The administration of separate formulations in "concurrent" if the time of their administration is such that the pharmacological activities of the tyrosine kinase inhibitor and the chemical castration agent occur simultaneously in the treatment that the mammal experiences. In some embodiments of the invention, the co-administration of a tyrosine kinase inhibitor and a chemical castration agent is achieved by formulating them into a single composition. Preferably, the dose of the tyrosine kinase inhibitor is from 1 μg / kg to 1 g / kg of body weight per day. Most preferably, the dose of the tyrosine kinase inhibitor is from 0.01 mg / kg to 100 mg / kg of body weight per day. The optimal dose of the tyrosine kinase inhibitor will vary depending on factors such as the type and degree of progression of the prostate cancer, the overall health status of the patient, the potency of the tyrosine kinase inhibitor and the route of administration. Optimization of tyrosine kinase dose is within ordinary skill in the art. Various tyrosine kinase inhibitors suitable for use in this invention are known in the art. Preferably, the tyrosine kinase inhibitor used in this invention is a trkA inhibitor, a trkB inhibitor, or a trk C inhibitor. Suitable indolocarbazole tyrosine kinase inhibitors can be obtained using information found in documents such as Dionne and others, US patent No. 5,516,771, Dionea et al., Patent of E.U.A. No. 5,654,427, Lewis et al., U.S. No. 5,461,146 and Mallamo et al., Patent of E.U.A. No. 5,650,407. In some embodiments of the invention, the compound II-4-LAE was prepared and administered to a human patient according to the following procedure. The compound II-4-LAE (dihydrochloride) and an osmotically adequate amount of mannitol were dissolved in distilled water, and the pH was adjusted to approximately 3.5. This solution was lyophilized to produce a powder. For storage and convenient use, aliquots of the pooled Mof powder were prepared containing 27.5 mg of Compound II-4-LAE and 55 mg of mannitol. At the time of use, an aliquot of the lyophilized powder was redissolved in sterile water for injection, USP, to yield 1.1 ml containing 50 mg / ml mannitol and 25 mg / ml Compound II-4-LAE (dihydrochloride). This reconstituted solution was then diluted with an appropriate volume of 5% dextrose injection, USP, for the administration of the desired dose of compound II-4-LAE through intravenous infusion over a period of about one hour. The dose of Compound II-4-LAE in this procedure can be conveniently started at 1 mg / meter / day and gradually increased, for example, to 64 mg / meter2 / day, or 501 mg / meter2 / day, as The progress of the patient is verified. Several chemical castration agents are known. The known chemical castration agents useful in this invention are sometimes categorized as follows: oestrogens, leutinizing hormone releasing hormone (LHRH) agonists, LHRH antagonists and anti-androgens. The anti-androgens can also be categorized as steroidal or non-steroidal. Estrogens, for example, diethylstilbestrol (DES), raise levels of sex hormone-binding globulin in plasma and prolactin levels. This reduces the secretion of LH and the synthesis of testicular testosterone through a negative feedback response. The dose of DES is usually 1 mg / day to 5 mg / day. Preferably, higher doses of DES are avoided due to possible complications in relation to cardiovascular risk. A preferred LHRH agonist for use in this invention is leuprolide acetate, commercially available as LUPRON® (Takeda Abbott Pharmaceuticals, Inc.). The chemical name of leuprolide acetate is 5-oxo-L-prolyl-L-histidyl-L-tryptopyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-N-ethyl acetate. L-prolinamide (salt). Leuprolide acetate, an LHRH agonist, is a potent inhibitor of gonadotropin secretion when administered continuously and in therapeutic doses. This effect is reversible after discontinuation of the administration of leuprolide acetate. It is believed that leuprolide acetate, for example, LUPRON DEPOT®, acts through a negative feedback mechanism. In humans, subcutaneous administration of individual daily doses of leuprolide acetate results in an initial increase in serum leutinizing hormone (LH) levels. In males or males, in the two to four weeks after the start of the administration of leuprolide acetate, the testosterone level in the serum drops to castration levels. When used in this invention, leuprolide acetate is administered subcutaneously, intramuscularly or intravenously. Leuprolide acetate can be administered, for example, by subcutaneous injection of 1 mg per day. In some embodiments of the invention, leuprolide acetate is administered in a container formulation. A container formulation conveniently provides a sustained release of the drug for an extended period, for example from 1 to 4 months. An illustrative container formulation includes a suspension of microspheres containing leuprolide acetate, purified gelatin, copolymer of DL-lactic and glycolic acids, and D-mannitol. The microspheres can be suspended in a vehicle containing sodium carboxymethylcellulose, D-mannitol and water. Said container formulation is commercially available as LUPRON DEPOT ™ (Takeda Abbott Pharmaceuticals) and is suitable for intramuscular injection. Another LHRH agonist useful in this invention is goserelin acetate, commercially available as ZOLADEX® (Zeneca). The chemical structure of goserelin acetate is pyro-Glu-His-Trp-Ser-Tyr-D-SeryBu ^ -Leu-Arg-Pro-Azgly-NHa acetate. ZOLADEX® is supplied as a formulation designed for subcutaneous injection with continuous release over a period of 28 days. An example of a LHRH antagonist useful in this invention is ANTIDE® (Ares-Serono), whose chemical name is D-alaninamide N-acetyl-3- (2-naphthalenyl) -D-alanyl-4-chloro-D-phenylalanyl -3- (3-pyridinyl) -D-alanyl-L-seryl-N6- (3-pyridinylcarbonyl) -L-lysyl-N6- (3-pyridinylcarbonyl) -D-lysyl-L-leucyl-N6- (1- methylethyl) -L-lysol-prolyl. Another example of a useful LHRH antagonist is GANIRELIX® (Roche / Akzo Nobel), whose chemical name is N-Ac-D-Nal, D-pCI-Phe, D-Pal, D-hArg (Et) 2, hArg ( Et) 2, D-Ala. Examples of steroidal anti-androgens are cyproterone acetate (CPA) and megestrol acetate, commercially available as MEGACE® (Bristol-Myers Oncology). Steroidal anti-androgens can block the prostatic androgen receptors. They can also inhibit the release of LH. The CPA is preferably administered to human patients at doses of 100 mg / day to 250 mg / day. Non-steroidal anti-androgens block androgen receptors. They can also cause an increase in serum LH levels and serum testosterone levels. A preferred non-spheroidal anti-androgen is flutamide (2-methyl-N- [4-nitro-3- (trifluoromethyl) phenyl] propanamide), commercially available as EULEXIN® (Schering Corp.). Flutamide exerts an anti-adrenergic action by inhibiting androgen consumption, inhibiting the androgen binding in target tissues, or both. Flutamide is typically administered orally, for example, in the form of a capsule. One dose of illustrative flutamide is 250 mg, three times a day, ie 750 mg per day. Another non-spheroidal anti-androgen is nilutamide, whose chemical name is 5,5-dimethyl-3- [4-nitro-3- (trifluoromethyl-4'-nitrophenyl) -4,4-dimethylimidazolidine-dione. When used in this invention, an illustrative dose of nilutamide is 300 mg daily, followed by a reduced dose of 150 mg / day.

In some embodiments of the invention, the chemical castration agent is a combination of an LHRH agonist such as leuprolide acetate and an anti-androgen such as flutamide or nilutamide. For example, leuprolide acetate can be administered by subcutaneous, intramuscular or intravenous injection, and concurrently flutamide can be administered orally. The tyrosine kinase inhibitor can be administered separately in a third formulation, or it can be formulated together with the LHRH agonist or the anti-androgen. Another non-steroidal anti-androgen useful in this invention CASODEX®. An illustrative dose of CASODEX® is from 5 mg to 500 mg per day, preferably around 50 mg per day. An illustrative combined formulation according to this invention includes 1-20 mg of Compound 11-12 and 100-1000 mg of flutamide in a capsule for oral administration to a human patient, once, twice or three times per day. In a preferred embodiment, the formulation includes a vehicle containing polysorbate 80 and polyethylene glycol in a ratio of 1: 1 (v / v), to improve the bioavailability of Compound 11-12. In some embodiments, this oral formulation of Compound 11-12 / flutamide is supplemented through an intramuscular injection of a leuprolide acetate container injection, for example, LUPRON DEPOT®. Another tyrosine kinase such as Compound II-4 or Compound II-4-LAE can be substituted for Compound 11-12 in this formulation. Other illustrative combined formulations according to this invention include Compound 11-12, Compound M-4 or Compound II-4-LAE, and a chemical castration agent, in a single dose suitable for intravenous infusion. Tyrosine kinase inhibitors and chemical castration agents can be formulated, individually or in combination, in pharmaceutical compositions by mixing with non-toxic pharmaceutically acceptable excipients and carriers or carriers. Said compositions can be prepared for use in parenteral administration, in particular in the form of liquid solutions or suspensions; for oral administration, particularly in the form of liquid, tablets or capsules; or intranasally, in particular in the form of powders, nasal drops or aerosols. The composition can be conveniently administered in unit dosage form and can be prepared by any of the methods known in the art. Such methods are described in, for example, Remignton's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980). Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilization agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. In addition, of the inert diluents, the oral compositions may also include auxiliaries such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and perfumers. Injectable container forms are made by forming microcapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug to polymer ratio and the nature of the particular polymer employed, the rate of release of the drug can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable container formulations are also prepared by trapping the drug in liposomes or microemulsions, which are compatible with body tissues. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In said solid dosage forms, the active compound is mixed with at least one pharmaceutically acceptable inert excipient or carrier, such as sodium citrate or dicalcium phosphate and / or a) fillers or spreading agents such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures of the same. In the case of capsules, tablets and pills, the dosage form may also comprise pH regulating agents. Solid compositions of a similar type can also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and breastplates such as enteric coatings and other coatings well known in the field of pharmaceutical formulation. Optionally they may contain opacifying agents and may also be of a composition that they release the active ingredient (s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed form. Examples of imbibition compositions that can be used include polymeric substances and waxes.

Solid compositions of similar type can also be used as fillers in soft and hard gelatin capsules, using excipients such as lactose and milk sugar as well as high molecular weight polyethylene glycols and the like. The active compounds may also be in microencapsulated form with one or more excipients as noted above. In solid dose forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose or starch. Said dosage forms may also comprise, as is normal in practice, additional substances other than inert diluents, for example, tabletting lubricants and other tabletting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms can also comprise pH regulating agents. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient (s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of imbibition compositions that can be used include polymeric substances and waxes. The invention is further illustrated by the following examples. The examples are provided for purposes of illustration only, and are not construed as limiting the scope or content of the invention in any way.

EXAMPLES Example 1: Compound II-4 Combined with Surgical Castration Data from animal experiments involving the administration of tyrosine kinase in combination with surgical castration were considered relevant for the administration of tyrosine kinase in combination with chemical castration. Dunning's tumor R-3327 H (derived from a spontaneous prostate tumor in an old Copenhagen rat) was used in these experiments because of its sensitivity to androgen and its slow growth rate (Isaacs, 1989, Cancer Res. 49: 6290-6294). A consideration in the experimental design was that surgical castration of rats harboring Dunning H tumors leads to an almost immediate cessation of tumor growth followed by regression of the androgen-insensitive tumor in approximately 5-6 weeks after castration (Isaacs et al. (1981) Cancer Res. 41: 5070-5075). The Dunning H tumor regression induced by Compound II-4 was not due to an effect on androgen levels, since the experiments were performed on rats with testosterone releasing silastic capsule implants. The implanted capsules were designed to keep testosterone circulating at physiological levels. Serum testosterone levels measured at the end of the experiment confirmed that testosterone was > _ 1-2 ng / ml. The following experiment had three objectives: 1) to determine whether the combination of Compound II-4 with surgical castration can provide greater anti-tumor efficacy than Compound II-4 or surgical castration alone; 2) determining whether Compound II-4 can cause regression of Dunning H tumors that have been insensitivity to selected hormone through anterior castration of the host animals; and 3) determining whether tumors treated with Compound 11-4 over several dosing cycles remain sensitive to surgical castration. Compound 11-4 was synthesized in Cephalon, Inc., and formulated (10 mg / ml) in a vehicle containing 40% polyethylene glycol (PEG 1000, Spectrum, Los Angeles, CA), 10% polyvinylpyrrolidone (C30, ISP Boundbrook, NJ), 2% benzyl alcohol, (Spectrum, Los Angeles, CA) in water (48%). Inbred, male, adult Copenhagen rats (200-240 g) obtained from Harlan Sprague Dawley (Indianapolis, IN) were kept in groups of three rats / cage and were given a commercial diet (Purina Formulab 5001) and water ad libitum. The animals were housed under conditions of controlled humidity and controlled temperature, with a light / dark cycle of 12-hour intervals. The rats were isolated for a week before experimental manipulation. Dunning R-3327 tumors of prostate cancer were transplanted to the rat using trocar pieces.

A Copenhagen male adult rat carrying a Dunning H tumor was sacrificed and the tumor isolated. The tumor was cut, and the small pieces were inoculated subcutaneously in adult male Copenhagen rats. The experiments were carried out under the guidelines of the Johns Hopkins University Animal Care (Animal Care of the Johns Hopkins University) and Use Committee Protocol No. RA91M517 (Protocol of Use Committee No. RA91M517) and Cephalon Institutional Animal Care (Institutional Animal Care of Cephalon) and Use Committee Protocol No. 03 -008 (Protocol of Use Committee No. 03-008). For surgical castration, rats carrying established Dunning H tumors were anesthetized through intramuscular injection of KETAMINE ™ (4.1 mg / 100 g of body weight) and XYLAZINE ™ (0.85 mg / 100 g of body weight) . Each rat was placed on its back. A small incision (0.5-1 cm) was made through the skin at the posterior tip of the scrotum. Another incision was made to break the connective membrane that surrounds the testicles. The epididymis, testes, vas deferens, spermatic blood vessels, and fat were removed and cut. Any remaining tissue was returned to the sac, and the incision was closed with self-fasteners. The self-fasteners were removed 5-7 days after the surgery. Thirty-six rats carrying the Dunning H tumor (size 0.9-18 cm3) were divided into four groups of nine animals each. Group 1 served as a vehicle control. Group 2 was neutered on day 1. Group 3 received injections of Compound II-4 (10 mg / kg, se) as described below. Group 4 was castrated on day 1 and injected with Compound II-4, as described below. Groups 1 and 3 were implanted subcutaneously (flank) with a sealed silastic tube with a length of 2 cm filled with testosterone, on day 1. A silastic implant of this size was adequate to maintain testosterone in the serum in the physiological scale of 1-3 ng / ml chronically for six months. Compound 11-4 was administered subcutaneously (10 ng / ml / day) to Groups 3 and 4 in a 5-day dosing cycle, periodically with approximately 10 days between cycles. The drug was administered on days 1-5, 14-18, 29-33 and 42-46. The drug vehicle was administered to Groups 1 and 2 in the same program as the groups treated with Compound II-4. Eight rats of Group 2 were divided into two groups of four rats each on day 60. One group was treated with Compound II-4, 10 mg / kg, (subcutaneous) for 5 days, followed by withdrawal of the drug during 9 days, followed by a second 5-day dosing regimen of 10 mg / kg / day (subcutaneous). The second group received the vehicle with the same dosing program. Seven rats derived from Group 3 of the preceding experiment were divided into two groups on day 60. One group (N = 3) of rats was castrated. Both groups were treated with Compound II-4, 10 mg / kg (subcutaneous) for 5 days, followed by withdrawal of the drug for 9 days, followed by a second five-day dosing regimen, as before. Tumors were measured in the anesthetized animals (isoflurane vapor for approximately 1-2 minutes) at the indicated intervals using a vernier caliper. The tumor volume was calculated using the formula: V (cm3) = .5236 x length (cm) x width (cm) [(length (cm) + width (cm)) / 2]. The Dunnett test, the Mann-Whitney Rank Sum test, the t test in Pairs, or the Significant Signed Rank test was applied for statistical analysis using the SigmaStat program. The growth of the Dunning H tumors in rats treated with the vehicle, intact, was linear, and an increase of approximately 3.5 times in the volume of the tumor was observed during 60 days (Figure 1). Surgical castration caused a rapid regression of the tumors, that is, 25% on day 5. Additional tumor growth in the castrated rats was observed on day 12, and complete recovery of the returned tumor volume was achieved in the day 38. Compound II-4 only (10 mg / kg,; independent cycles of treatment with Compound II-4: 5 days of drug treatment followed by withdrawal of the drug for approximately 10 days) caused complete inhibition of tumor growth or induced tumor regression. The average tumor volume in the animals treated with the drug was significa smaller (p <0.01) than the control animals treated with the vehicle after each cycle of administration of Compound II-4 (days 5, 19, 34 and 47; data not shown). In addition, each cycle of administration of Compound II-4 caused the regression in relation to the volume of the tumor at the start of each dosage cycle (data not shown). The combination of Compound II-4 with surgical castration caused complete inhibition of tumor growth or induced tumor regression (Figure 1). In summary, the combination of the administration of Compound 11-4 and surgical castration was significa more effective than surgical castration alone. A further additional growth of the tumor was observed, in vivo, after withdrawal of Compound 11-4 administration, both in castrated and non-castrated animals. However, the additional growth was again minimal in castrated animals (p <0.01, Figure 1). These results demonstrated that Compound II-4 can be used in combination with surgical castration to maximize the extent and / or duration of tumor regression in an accepted in vivo model of prostate cancer. Other experiments were conducted to determine whether treatment with Compound II-4 causes regression of selected tumors as hormone-insensitive via anterior androgen ablation. Eight rats from the group of castrated rats from the previous experiment (without previous treatment with Compound II-4) were divided into two groups of four rats each on day 60. One group was treated with Compound 11-4, 10 mg / kg, se, for 5 days followed by withdrawal of the drug for 9 days, followed by a second dosage regimen of 5 days at 10 mg / kg / day, se. The second group received the vehicle in the same dosing program. Treatment with Compound II-4 caused significant regression of androgen-insensitive Dunning H tumors developed in castrated rats on day 3 (p <0.05, Figure 2). A maximum regression was observed on day 6 (p <0.05). the withdrawal of the drug allowed the tumors to grow back. However, the volume of the tumor in the rats treated with Compound 11-4 was significa lower (p <0.05) than the animals treated with the vehicle, even 10 days after the end of the first cycle with Compound 11-4. Dunning H tumors selected as hormone-insensitive through anterior androgen ablation remained sensitive to the anti-tumor action of Compound II-4. Experiments were also conducted to determine whether treatment with the above Compound 11-4 of rats bearing Dunning H tumors could cause the selection of tumors insensitive to the subsequent androgen ablation via surgical castration. Seven rats from the group treated with Compound 11-4 of the preceding experiment were divided into two groups on day 60. One group (N = 3) was castrated as described above. Both groups were treated with Compound 11-4, 10 mg / kg, se, for 5 days followed by withdrawal of the drug for 9 days, followed by a second five-day dosing regimen as before. Surgical castration caused a non-statistically significant regression of tumors that experienced four previous cycles, and two concurrent cycles, of the treatment with Compound M-4 (Figure 3). In summary, the data indicated that repeated exposure to Compound II-4 did not select a population of androgen-insensitive Dunning H tumors. Castration, treatment with Compound II-4, and the combination of Compound II-4 with surgical castration were well tolerated. Limited mortality was observed in the groups of control and castrated animals treated with the vehicle or Compound II-4. In each case, mortality occurred at the time of tumor measurement, presumably due to an overdose of anesthesia.

Example 2: Combination of Compound 11-12 and Chemical Castration Compound II-4 was synthesized in Cephalon, Inc. The dihydrochloride of Compound 11-12 was formulated (10 mg / ml) in a vehicle containing 3: 2 (v / v) ) from Gelucire (Gattefosse, Saint-Priest, France) in propylene glycol (Spectrum, Gardenia, CA). In this experiment, Copenhagen, innate, male, adult rats (200-240 g) obtained from Harlan Sprague Dawley (Indianapolis, IN) were used. The maintenance and handling of the rats, and Dunning H R-3327 tumor transplantation, was as described in Example 1 (above). Forty-six rats (weight: 380 +8 g) carrying Dunning H tumors (size 1 / 6-33.2 cm3) were divided into four groups. Group 1 (N = 12) served as vehicle control (1 ml / kg, po BID, days 0-20 and 31-45). Group 2 (N = 10) was treated with leuprolide acetate (LUPRON DEPOT®) (5.2 mg / kg, on days 0 and 21). Group 3 (N = 12) received Compound 11-12 (10 mg / kg, po BID on days 0-20 and 31-45). Group 4 (N = 12) received a combination of leuprolide acetate (5.2 mg / kg, on days 0 and 21) and Compound 11-12 (10 mg / kg, po BID on days 0-20 and 31-45). The animals of Groups 1 and 3 were implanted, se, a 2 cm long silastic capsule filled with testosterone. Tumor measurements and statistical analyzes were performed as described in Example 1 (above). The in vivo growth of Dunning H tumors was consistent in vehicle treated animals. On day 53 an increase of approximately 2.5 times in tumor volume was observed. The start of each treatment cycle with Compound II-12 (10 mg / kg BID, po for 21 days with a drug-free period, interim, of 10 days) inhibited the growth of the Dunning H tumor and caused a marked regression of the tumor. The inhibition of tumor growth observed in the group with the treatment with Compound 11-12 was statistically significant, as compared to the vehicle control group. This difference was observed from the earliest tumor measurement, ie, day 4, until the end of the experiment). Treatment with leuprolide acetate alone also resulted in tumor regression. A slow additional growth of the tumor was observed approximately 32 days after the start of the leuprolide treatments (Figure 4). As compared only to leuprolide, or Compound 11-12 only, the combination of Compound 11-12 / leuolide was significantly more effective (p <0.05) to maintain a reduced rate of tumor growth than any treatment alone (Figure 4). ). The additional growth of the tumor was observed in the group with the treatment with Compound 11-12 and the group with the leuprolide treatment, approximately 30 days after the start of the treatments. In contrast, the treatment group with the combination exhibited a longer duration of reduced tumor growth and was statistically different from the group with Compound 11-12 and the group with leuprolide from days 35 and 39, respectively, until the end of the experiment on day 54 (p <0.05; Figure 4). These experimental results demonstrated that the combination of Compound 11-12 with chemical castration was synergistic in anti-tumor efficacy (Figures 1 and 4). Other embodiments are within the following claims.

Claims (28)

1. - A method for inhibiting the progression of prostate tumor in a mammal, comprising administering a therapeutically effective amount of a tyrosine kinase inhibitor to the mammal, and co-administering to the mammal a therapeutically effective amount of a group chemical castration agent which consists of an estrogen, an LHRH agonist, an LHRH antagonist and an anti-androgen.

2. The method according to claim 1, wherein the mammal is a human being.

3. The method according to claim 1, wherein the therapeutically effective amount of a tyrosine kinase inhibitor and the therapeutically effective amount of the chemical castration agent synergistically inhibit tumor progression.

4. The method according to claim 1, wherein the tyrosine kinase inhibitor is a trkA inhibitor, a trkB inhibitor, or a trkC inhibitor.

5. The method according to claim 1, wherein the tyrosine kinase inhibitor is an indolocarbazole.

6. The method according to claim 5, wherein the indolocarbazole has the following structure: (Compound 11-12).

7. - The method according to claim 5, wherein the indolocarbazole has the following structure:

(Compound II-4). 8. The method according to claim 5, wherein the indolocarbazole has the following structure: Compound II-4-LAE.

9. - The method according to claim 1, wherein the tyrosine kinase inhibitor is a fused pyrrolocarbazole.

10. The method according to claim 1, wherein the tyrosine kinase inhibitor is administered orally.

11. The method according to claim 1, wherein the tyrosine kinase inhibitor is administered parenterally.

12. The method according to claim 1, wherein the LHRH agonist is leuprolide acetate.

13. The method according to claim 1, wherein the anti-androgen is flutamide.

14. The method according to claim 1, wherein the chemical castration agent is a combination of an LHRH agonist and an anti-androgen.

15. The method according to claim 14, wherein the LHRH agonist is leuprolide acetate, and the anti-androgen is flutamide.

16. The method according to claim 1, wherein the tyrosine kinase inhibitor and the chemical castration agent are administered in separate formulations.

17. The method according to claim 1, wherein the tyrosine kinase inhibitor and the chemical castration agent are formulated together in a single composition.

18. A composition comprising a tyrosine kinase inhibitor and a chemical castration agent selected from the group consisting of an estrogen, an LHRH agonist, an LHRH antagonist and an anti-androgen.

19. The composition according to claim 18, wherein the tyrosine kinase inhibitor is a trkA inhibitor, a trkB inhibitor or a trkC inhibitor.

20. The composition according to claim 18, wherein the tyrosine kinase inhibitor is an indolocarbazole.

21. The composition according to claim 20, wherein the indolocarbazole is selected from the group consisting of an indolocarbazole whose structure is: (Compound 11-12); an indolocarbazole whose structure is: (Compound II-4); and and an indolocarbazole whose structure is: CH2) 4NH2. HCl. HCl Compound II-4-LAE.

22. The composition according to claim 18, wherein the tyrosine kinase inhibitor is a fused pyrrolocarbazole.

23. The composition according to claim 18, wherein the composition is formulated for oral administration.

24. The composition according to claim 18, wherein the composition is formulated for parenteral administration.

25. The composition according to claim 24, wherein the LHRH agonist is leuprolide acetate.

26. The composition according to claim 24, wherein the anti-androgen is flutamide.

27. The composition according to claim 18, wherein the chemical castration agent is a combination of an LHRH agonist and an anti-androgen.

28. The composition according to claim 19, wherein the LHRH agonist is leuprolide acetate and the anti-androgen is flutamide.