MXPA98009051A - Use of protein cinase c inhibitors to increase the clinical efficacy of oncolithic agents and radiac therapy - Google Patents

Abstract

A method for treating neoplasms is described, particularly using the inhibitor of selective PKC of isozyme B, (S) -3,4- (N; N'-1,1 '- ((2"-ethoxy) -3"'(0) -4"' - (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)) - 1 (H) -pyrrole-2,5-dione or one of its salts, such inhibitors of PKC increase the clinical efficacy of oncolytic agents and radiation therapy

Description

USE OF PROTEIN INHIBITORS CINASE C FOR INCREASE THE CLINICAL EFFICACY OF THE AGENTS ONCOLITICS AND RADIATION THERAPY . This application claims the priority benefits of United States Provisional Application Serial No. 60 / 016,658 filed May 1, 1996.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is broadly directed to a method for increasing the antineoplasma effects of chemotherapies and radiation therapies with PKC inhibitors. The present invention is particularly directed to the use of Protein Kinase C (PKC) inhibitors, especially a particular class of PKC inhibitors selective for the isozyme in combination with an oncolytic agent or? -irradiation to increase their anti-HIV effects. -neoplasma in the treatment of neoplasms.

Description of Related Art Over the years, therapeutic treatments have been developed to treat neoplasms. There are two main proposals to treat neoplasms: 1) chemotherapy using agents "oncolytic, and 2) radiation therapy, eg, -radiation.Oncolytic agents and the? -radiation cause cytotoxic effects, preferably tumor cells, and cause cell death.Studies have shown that? -radiation and certain groups of oncolytic agents affirm their cytotoxic effects through the activation of programmed cell death or apoptosis. ün equilibrium between the activities of the apoptotic and antiapoptotic intracellular signal transduction pathways is important with respect to the decision of the cell to undergo apoptosis in response to the chemotherapy mentioned above as well as radiation therapy, PKC inhibitors have been proposed • for cancer therapy, for example see U.S. 5,552,391 and the activities of the PKC have been indicated to exert antiapoptotic effects, especially in response to radiation therapies, for example,? -radiation. In particular, studies have shown that activation of PKC inhibits apoptosis induced by antineoplasma agents such as Ara-c, 2-chloro-2-deoxyadenosine, 9-β-D-arabinosyl-2-f luoroadenine, and therapy tfe f ~ irradiation. Regarding this, there have also been indications that the regulation of PKC activities in tumor cells increases apoptosis stimulated by oncolytic agents. The activation of PKC has been shown to attenuate cell death induced by? -irradiation. There is a need in the art to develop therapeutic agents that increase the apoptotic signal transduction pathways in cells and therefore increase the clinical efficacy of oncolytic agents and radiation therapy.

BRIEF DESCRIPTION OF THE INVENTION It is an object of the invention to provide methods for treating a neoplasm.

It is another object of the invention to provide methods for increasing an anti-neoplasm effect of an oncolytic agent. It is yet another effect of the invention to provide methods for increasing the effects of anti- neoplasm of radiation therapy. These and other objects of the invention are provided by one or more of the embodiments described in the following. In one embodiment of the invention there is provided a method for treating a neoplasm comprising administering to a mammal in need of such treatment an oncolytic agent or? -irradiation in combination with a protein kinase C inhibitor. In yet another embodiment of the invention. invention provides a method for enhancing an anti-neoplasm effect of chemotherapy and radiation therapy comprising administering a protein kinase C inhibitor in combination with the oncolytic agent or radiation therapy. The present invention provides the technique with a method to increase apoptotic effects in cells and thus be effective in increase the effects of anti- neoplasm of chemotherapies and radiation therapies.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the dosage effect of briostatin 1 on the isoforms of PKC. Figure 2 demonstrates the effect of incubation time of briostatin 1 on the isoforms of PKC. Figure 3 demonstrates that the regulation of PKC-β increases the efficacy of? -irradiation. Figure 4 shows that increased expression of PKC-β demonstrates resistance to cell death stimulated by radiation.

DETAILED DESCRIPTION OF THE INVENTION It is a discovery of the present invention that the use of PKC inhibitors, especially a particular class of protein kinase C inhibitors, reduces or inhibits the anti-apoptotic effects in a cell. Accordingly, such compounds can be used to increase the effects of anti-neoplasm of chemotherapies and radiation therapies. The method of this invention can employ any PKC inhibitor known in the art that includes non-specific PKC inhibitors and PKC inhibitors specific to different isoforms. : Information about PKC inhibitors and methods for their preparation are readily available in the art. For example,. Different types of PKC inhibitors and their preparation are described in USPs 5621101, 5621098, 5616577, 5578590, 5545636, 5491242, 5488167, 5481003, 5461146, 5270310, 5216014, 5204370, 5141957, 4990519 and 4937232, all of which are incorporated herein by reference. Preferably, the present invention uses those inhibitors of the protein kinase C that effectively inhibit the β-isozyme. A suitable group of compounds is generally described in the prior art as bis-indolylmaleimides or macrocyclic bis-indolylmalemides. The bis-indolylmaleimides well recognized in the prior art include those compounds described in US Pat. Nos. 5621098, 5552396, 5545636, 5481003, 5491242 and 5057614, all incorporated by reference herein. The macrocyclic bis-indolylmaleimides are represented in particular by the compounds of the formula I. These compounds and method for their preparation have been described in U.S. Patent 5,552,396, which is incorporated herein by reference. In accordance with the present invention, these compounds are administered in combination with other anti-neoplasm therapies to a mammal in need of such treatment. In particular, these compounds can be used to increase the anti-neoplasm effects of chemotherapies and radiation therapies. A preferred class of compounds for use in the method of the invention has the formula: U wherein: W is -O-, -S-, -SO-, -S02-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aryl-, -aryl (CH2) m0'-, -heterocycle-, -heterocycle- (CH2) mO-, -bicylic- fused-, -bicylic- (CH2) m0-, -NR3-, -ÑOR3-, • -CONH-, or -NHCO-; X and Y are independently C1-C4 alkylene, substituted alkylene, or together X, Y and W combine to form - (CH2) n-AA-; the R1's are hydrogen or up to four optional substituents independently selected from halo, C? -C 'hydroxy alkyl, C? -C4 alkoxy, haloalkyl, nitro, NR4R5 or -NHCO (alkyl) C1-C4); R2 is hydrogen, CH3C0-, NH2, or hydroxy; R3 is hydrogen, (CH2) maryl, alkyl. of C1-C4, -COO (C? -C4 alkyl), -C0NR4R5, - (C = NH) NH2, -S0 (C? -C4 alkyl), -S02 (NR4R5), or -S02 (alkyl) C? -C4); R 4 and R 5 are independently hydrogen, C 1 -C 4 alkyl, phenyl, benzyl, or combine to the nitrogen to which they are attached to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently 0, 1, 2 or 3; and n is independently 2, 3, 4 or 5, or a "pharmaceutically acceptable salt, prodrug or ester thereof.

A more preferred class of compounds for use in this invention is represented by formula I, wherein the -X-W-Y-portions contain from 4 to 8 atoms, which may be substituted or unsubstituted. Most preferably, the -X-W-Y- portions contain 6 atoms. Other preferred compounds for use in the method of this invention are those compounds of formula 1, wherein R 1 and R 2 are hydrogen; and is a substituted alkylene, -O-, -S-, -CONH-, -NHCO- or -NR3-. Particularly preferred compounds are the compounds of the formula la: wherein Z is - (CH2) P- or - (CH2) p-0- (CH2) p-; R 4 is hydroxy, -SH, C 1 -C 4 alkyl, (CH 2) maryl, -NH (aryl), -N (CH 3) (CF 3), -NH (CF 3), or -NR 5 R 6; R5 is hydrogen or C? -C4 alkyl; R6 is hydrogen, C? -C4 alkyl or benzyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof. The most preferred compounds of the formula la are those wherein Z is CH2; and R4 is -NH2, -NH (CF3) ,. O -N (CH3) 2. Other preferred compounds for use in the method; of the present invention are the compounds wherein in the formula I is -O-, Y is a substituted alkylene, and X is an alkylene. These preferred compounds are represented by formula I.b: wherein Z is - (CH2) P-; R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or C1-C4 alkyl; p is 0, 1 or 2; m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof. The most preferred compounds of formula Ib are those wherein p is 1; and R5 and R6 are methyl.

Because they contain a basic portion, the compounds of the formulas I, la and Ib may also exist as pharmaceutically acceptable acid addition salts. Acids commonly employed to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acids, as well as organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulphonic, carbonic, succinic, citric acid, Benzoic, acetic and related inorganic and organic acids. Such pharmaceutically acceptable salts of this form include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monoacid phosphate, diacid phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate. , heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleuate, 2-butyn-l, 4-dioate, 3-hexin-2,5-dioate, benzoate, chlorobenzoate, hydroxybenzoate, methoxybenzoate, fatalate, xylene sulfonate , phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propansulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and Similar. Particularly the c-hydroxy salts and mesylate are used. In addition, for pharmaceutically acceptable salts, other salts may also exist. They can serve as intermediaries in the purification of compounds, in. the preparation of other salts, or in the identification and characterization of compounds or intermediates. Pharmaceutically acceptable salts of the compounds of the formulas I, la and Ib may also exist in various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the crystallization solvent, inherent in the preparation or crystallization solvent, or adventitious to such a solvent. It is recognized that various stereoisomeric forms of the compounds of formulas I, la and Ib may exist; for example, W may contain a chiral carbon atom in the substituted alkylene portion.

The compounds are normally prepared as racemate and can conveniently be used as such.

Alternatively, both individual enantiomers can be isolated or synthesized by conventional techniques if so desired. Such racemates and individual enantiomers and mixtures thereof are part of the compounds used in the methods of the present invention. The compounds used in this invention also encompass the pharmaceutically acceptable prodrugs of the compounds "of the formulas I, Ia and Ib. A prodrug is a drug that has been chemically modified and may be biologically inactive at its site of action, but the which can be degraded or modified by one or more enzymatic processes or other invi ve for the mother bioactive forms.This prodrug may probably have a pharmacokinetic profile different from the mother, allowing easier absorption through the mucosal epithelium, better formation or salt solubility, and / or improved systemic stability (an increase in the plasma half-life, for example) Typically, such chemical modifications include the following: 1) the ester or amide derivatives which can be divided by esterases or lipases, 2) peptides that can be recognized by specific or non-specific proteases, or 3) derivatives that accumulate at the site of action through the selection of the membrane of a pro-drug form or a modified prodrug form; or any combination of 1 'to 3, supra. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in H. Bundgaard, Design of Prodrugs, (1985). The synthesis of the various bis-indole-N-maleimide derivatives are described in U.S. Patent 5 / 057,614 to Davis et al., And. the synthesis of preferred compounds suitable for use in this invention are described in U.S. Patent 5,552,396 previously identified and in EP 0 657 411 Al de Faul et al., all of which are incorporated herein by reference . A particularly preferred protein kinase C inhibitor for use in the method of this invention is the compound described in Example 5g (Hydrochloride salt of (S) -3, - [N, N '-l, l' - (( 2"-ethoxy) -3" '(O) -4"' - (N, N-dimethylamino) -butane) -bis (3,3 '-indol'yl)] -1 (H) -pyrrole-2 , 5-dione) of the aforementioned U.S. Patent 5,552,396 This compound is a potent inhibitor of protein kinase C. It is selective for protein kinase C over other kinases and is highly selective isozyme, that is, it is selective for the beta-1 and beta-2 isozymes. Other salts of this compound could also be favored, especially the mesylate salts. A preferred mesylate salt can be prepared by reacting a compound of the formula II with methanesulfonic acid in a non-reactive organic solvent, preferably an organic / water mixture, and more preferably water-acetone. Other solvents such as methanol, acetone, ethyl acetate and mixtures thereof are operable. The ratio of the solvent to water is not critical and is generally determined by the solubility of the reactants. The preferred solvent for water ratios is generally from 0.1: 1 to 100: 1 solvent to water by volume. Preferably, the ratio is from 1: 1 to 20: 1 and more preferably from 5: 1 to 10: 1. The optimum ratio depends on the selected solvent and preferably is acetone in a solvent of 9: 1 for the water ratio. The reaction usually involves approximately equimolar amounts of the two reactants, although other ratios, especially those in which methanesulphonic acid is in excess, are operative. The addition ratio of methanesulfonic acid is not critical to the reaction and can be added quickly (<5 minutes) or slowly for 6 or more hours. The reaction is carried out at temperatures ranging from 0 ° C to reflux. The reaction mixture is stirred until the formation of the salt is complete, as determined by X-ray powder diffraction and can take from 5 minutes to 12 hours. The salts of the present invention are preferably and readily prepared as a crystalline form. The trihydrate form of the salt can be easily converted to the monohydrate by drying or exposure to 20-60% relative humidity. The salt is substantially crystalline demonstrating a defined melting point, bi-refringency and a pattern of x-ray diffraction. In general, crystals have less than 10% amorphous solid and preferably less than 5% and more preferably less than 1% amorphous solid. The mesylate salt is isolated by filtration or other separation techniques appreciated in the art directly from the reaction mixture in yields ranging from 50% to 100%. Recrystallization and other purification techniques known in the art can be used to further purify 1-a salt if desired. PKC inhibitors, which include the compounds described in the foregoing, are used in combination with conventional anti-neoplasm therapies to treat mammals, especially humans with neoplasia. Methods for conventional anti-neoplasm therapies, including chemotherapies, are known, for example, using oncolytic agents and radiation therapies, for example,? -radiation, readily available, and routinely practiced in the art, for example, see Harrison's PRINCIPLES OF INTERNAL MEDICINE llava .edición, McGraw-Hill Boo Company. The neoplasm is characterized by normal cell growth which often results in the invasion of normal tissues, for example, primary tumors or dilation for distant organs, for example, metastasis. The treatment of any neoplasm by conventional anti-neoplasm therapies can be increased by the present invention. Such neoplastic growth includes but is not limited to primary tumors, primary tumors that are incompletely removed by surgical techniques, primary tumors that have been adequately treated but which have a high risk of developing a subsequent metastatic disease, and a disease established metastatic. Specifically, the inhibitors of PKC described above can increase the anti-neoplasm effects of an oncolytic agent. The wide variety of oncolytic agents available is contemplated by the combination therapy according to the present invention. In a preferred embodiment, oncolytic agents that affirm their cytotoxic effects by the activation of programmed cell death or apoptosis are used in combination with the described PKC inhibitors. These include but are not limited to 1-ß-D-arabinofuranosilcitosina or Ara-c, etoposida or VP-16, cis-diaminadicloroplat ino (11) or cis-platino, doxorubicin or adriamycin, 2-chloro-deoxyadenosine, 9-ß-D-arabinosi 1-2-f luoroadenine and glucocorticoids. All neoplastic conditions treatable with such oncolytic agents can be treated according to the present invention using a combination of a PKC inhibitor with one or more oncolytic agents. The oncolytic agents affirm the effects of cytotoxicity or anti-neoplasm in a variety of specific neoplastic conditions. For example, Ara-C is commonly used for the treatment of acute lymphoid leukemia (ALL) absent in childhood, • ALL thymic, ALL of B cell, acute myeloid leukemia, acute granulocytic leukemia and its variants, non-Hodgkins lymphoma, myelomonocytoid leukemia, - acute megakaryocytoid leukemia and Burkitt's lymphoma, adult ALL B, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and T cell leukemia VP-16 is normally used for the treatment of testicular carcinoma, small and large scale non-small cell lung carcinoma, Hodgkin's lymphoma. non-Hodgkin's lymphoma, choriocarcinoma, Ewing's sarcoma, and acute granulocytic leukemia. Cis-platinum can be used for the treatment of carcinoma testicular, germ cell tumors, ovarian carcinomas, prostate cancer, lung cancer, sarcomas, cervical cancer, endomermetrial cancer, gastric cancer, breast cancer and cancer of the head and neck. 2-chloro-2-deoxyadenosine and 9-β-D-arabinosyl-2-fluoroaderiin s.e can be used to treat chronic lymphoid leukemia, lymphomas and hairy cell leukemia. Doxorubicin can be used to treat acute granulocytic leukemia and its variants, ALL, breast cancer, bladder cancer, ovarian cancer, thyroid cancer, cancer of the lung, Hodgkin's lymphoma. , non-Hodgkin's lymphoma, sarcomas, gastric carcinoma, prostate cancer, endometrial cancer, tumor and Wilm's neuroblastoma. The clinical effects of oncolytic agents in all neoplastic conditions treatable with oncolytic agents include the only ones discussed in the foregoing that can be potentiated by the use of combination therapy with the PKC inhibitors identified in accordance with this invention. The inhibitors of PKC identified in the present invention can also increase the effects of anti-neoplasm of a therapy of radiation. Usually, the? -irradiation is used to treat the site of a solid tumor directly. The experimental results provided in the present invention demonstrate that complete down-regulation or loss of protein kinase C-β is associated with synergistic intensification of oncolytic induced apoptosis in human leukemic cells (Figure 1). Similarly, the significant down-regulation of C-β protein kinase in human U937 leukemic cells increases cell death simulated by radiation (Figure 2). Human U937 leukemic cells overexpressing C-β protein kinase demonstrate resistance to cell death stimulated by radiation (Figure 3). These data provide a strong indication that PKC inhibitors, especially selective inhibitors of the β-isozyme, preferably used according to the present invention, can increase tumor elimination or anti-neoplasm effects of chemotherapies and radiation therapies and improve clinical responses for these therapeutic modalities currently used. The PKC inhibitors of the present invention are administered in combination with other anti-neoplasm therapies that include oncolytic agents and radiation therapy. The phrase "in combination with other therapies" means that the compounds can be administered shortly before, shortly after, or together with such other antineoplasma therapies. The compounds can be administered in combination with more than one anti-neoplasm therapy. In a preferred embodiment, the compounds are administered from 2 weeks to 1 day before any chemotherapy, or 2 weeks to 1 day before any radiation therapy. Alternatively, inhibitors of PKC can be administered during chemotherapies and radiation therapies. If they are administered after chemotherapy or 'therapy radiation, PKC inhibitors should be given within 1 to 14 days after primary treatments. One skilled in the art will recognize that the amount of the PKC inhibitor. to be administered according to the present invention in combination with other anti-neoplasm agents or therapies is that amount sufficient to increase the anti-neoplasm effects of oncolytic agents or radiation therapies or that - > enough to induce apoptosis or death cell phone. Such amount may vary inter alia, depending on the size and type of the neoplasm, the concentration of the compound in the therapeutic formulation, the specific anti-neoplasm agents used, the timing of administration of the PKC inhibitors relative to the other therapies, and the age, size and condition of the patient. Tests can be used both in vi and in vi t to assess the amount of compounds needed to induce apoptosis. For example, human leukemic cells could be exposed to various concentrations of oncolytic agents, for example, Ara-c, or to radiation in the presence or absence of the PKC inhibitor compounds used in the present invention. The appropriate neoplastic cell types can be selected for the different oncolytic agents. Other selective inhibitors of protein kinase C can also be used for comparison. At various times, cells should be examined for viability by conventional methods or by any means available in the art. Apoptosis or cell death can be measured by any means known in the art. Cell death can be determined and quantified by d.e the exclusion of trypan blue, and reduced clonogenicity in soft agar. As will be understood by those experts in the technology, apoptosis is a specific mode of cell death recognized by a characteristic pattern of morphological, biochemical and molecular changes that include but are not limited to, endonucleolysis (DNA scale), abnormal DNA breaks and condensation of chromatin and cytoplasm (condensed nuclei and marked with points). These changes can be easily detected by any means known in the art, for example, microscopy; flow cytometric methods based on the increased sensitivity of DNA for denaturation and altered light scattering properties; DNA fragmentation as assessed by agarose gel electrophoresis; terminal DNA transferase assay, (TdT assay), and cutting translation assay (NT assay). Viral studies can be carried out using tumor xenografts inoculated in immunocompromised or siginic animals. After inoculation and growth of the primary implant, the animals should be treated with the compounds of the present invention before exposure to the animal. desired oncolytic or radiation treatment. The size of the tumor implant before and after each treatment in the presence and absence of the compounds in the present invention can be used as an indication of the therapeutic efficacy of the treatment. In general, an amount of the protein kinase C inhibitor to be administered in combination with other anti-neoplasm therapies is decided on a case-by-case basis by the attending physician. As a guide, the extent of the neoplasm, the body weight and the age of the patient will be considered, among other factors, when • an appropriate dose is established. Typically, the PKC inhibitors of the present invention are expected to potentiate the effects of anti-neoplasm of oncolytic agents and radiation therapy from about 2 times to about 10 times. In general, a suitable dose is one that results in a concentration of the protein kinase C inhibitor at the site of the tumor cells in the range of 0.5 nM to 200 μM, and more usually from 20 nM to 80 nM. It is expected that serum concentrations of 40 nM to 150 nM should be sufficient in most circumstances.

To obtain these treatment concentrations, a patient in need of treatment will probably be administered between approximately 0.1 mg per day per kg of body weight and 1.5 mg per day per kg. Usually, no more than about 1.0 mg per day per kg body weight of the protein kinase C inhibitor should be necessary. As noted in the above, the above amounts may vary on a case-by-case basis. The compounds of the formula I and the preferred compounds of the formula la and Ib are preferably formulated before administration. Suitable pharmaceutical formulations are prepared by known procedures using the well-known and readily available ingredients. The preparation of compositions suitable for use in the method of the present invention, the active ingredient will usually be mixed with a carrier, or diluted by a carrier, or included within a carrier which may be in the form of a carrier. capsule, sachet, paper or other container. When the carrier serves as a diluent, it can be a solid, semi-solid or liquid material that acts as a vehicle, excipient or medium for the active ingredient. In this way, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosol (as a solid or in a liquid medium), soft gelatin capsules and hard, suppositories, sterile injectable solutions and sterile packaged powders for oral or topical application. Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone ,. cellulose, aqueous syrup, methylcellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preservatives, sweetening agents or flavoring agents. The compositions of the invention can be formulated in such a way as to provide rapid, sustained or delayed release of the active ingredient after administration to the patient. The compositions preferably are they formulate in a dosage unit form, each dose containing from about 0.05 mg to about 3 g, more usually about 64 mg of the active ingredient. However, it will be understood that the therapeutic dose administered will be determined by the physician in light of the relevant circumstances which include the severity of the condition to be treated, the choice of the compound to be administered and the choice of the route of administration. Therefore, the above dose ranges are not intended to limit the scope of the invention in any way. The term "dose unit form" refers to physically discrete units suitable as unit doses for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier. In addition to the above formulations, most of which can be administered orally, the compounds used in the method of the present invention can also be administered topically. Topical formulations - > - *; They include ointments, creams and gels.

Ointments are generally prepared already (1) an oil base, that is, one consisting of fixed oils or hydrocarbons, such as white petrolatum or mineral oil, or (2) an absorbent base, that is, one consisting of of an anhydrous substance or substances that can absorb water, for example anhydrous lanolin. Usually, after the formation of the base, either oil or absorbent, the active ingredient (compound) is added to an amount that provides the desired concentration. The creams are emulsions in oil / water. They consist of an oil phase (internal phase), comprising typically fixed oils, hydrocarbons, and the like, such as waxes, petrolatum, mineral oil, and the like, and an aqueous phase (continuous phase), which comprises water and any soluble substances. in water, such as added salts. The two phases are stabilized by the use of an emulsifying agent, for example, an active surface agent, such as sodium lauryl sulfate; hydrophilic colloids, such as acacia colloidal clays, veegum, and the like. In the formation of the emulsion, the active ingredient (compound) it is usually added in an amount to achieve the desired concentration. ' The gels comprise a base selected from an oleaginous base, water, or an emulsion-suspension base. To the base is added a gelling agent that forms a matrix in the base, which increases its viscosity. Examples of gelling agents are hydroxypropylcellulose, acrylic acid polymers, and the like. Usually, the active ingredient (compounds) is added to the formulation at the desired concentration at a point preceding the addition of the gelling agent. The amount of the compound incorporated in a topical formulation is not critical; the concentration should be within a sufficient range to allow easy application of the formulation to the area of the affected tissue in an amount that will release the desired amount of the compound to the desired treatment site. The usual amount of a topical formulation to be applied to an affected tissue will depend on the size of tissue affected and the concentration of the compound in the formulation. In general, the formulation will be applied to the affected tissue in an amount that reaches approximately 1 to about 500 μg of the compound per cm2 of an affected tissue. Preferably, the applied amount of the compound will vary from about 30 to about 300 μg / cm 2, more preferably from about 50 to about 250 μg / cm 2, and, most preferably, from about 60 to about 100 μg / cm 2. The following formulation examples are illustrative only and are not intended to be limiting. the scope of the invention in any form.

Formulation 1 Hard gelatine capsules are prepared using the following ingredients: Amount (mg / capsule) Active agent 250 dry starch 200 magnesium stearate 10 Total 460 mg The above ingredients are mixed and filled into hard gelatin capsules in amounts of 460 mg.

Formulation 2 A tablet is prepared using the following ingredients: Amount (mg / capsule) Active agent 250 microcrystalline cellulose 400 - ~ '- vaporized silicon dioxide 10 stearic acid 5 Total 665 mg The components are mixed and compressed to form tablets, each weighing 665 mg.

Formulation 3 Each of the tablets containing 60 mg of the active ingredient are made as follows: Amount (mg / tablet) Active agent 60 mg starch 45 mg microcrystalline cellulose ina 35 mg polyvinylpyrrolidone (as a 10% solution in water) 4 mg sodium carboxymethyl starch 4.5 mg magnesium stearate 0.5 mg talc 1 mg Total 150 mg The active ingredient, starch and cellulose are passed through a U.S. mesh screen. No. 45 and they are completely mixed. The solution of polyvinylpyrrolidone is mixed with the resulting powders which are then passed through a U.S. mesh screen. No. 14. The granules so produced are suspended at 50 ° C and are passed through a U.S. mesh screen. No. 18. Sodium carboxymethyl starch, magnesium stearate and talc, which were previously passed through a U.S. mesh screen. No. 60, are then added to the granules which, after mixing, are compressed in a tabletting machine to produce each of the tablets weighing 150 mg.

EXAMPLES Example 1, Effects of Briostatin for the PKC isoforms This experiment demonstrates the dose and time effects of briostatin for the isoforms of PKC. U937 cells of human leukemia in the amount of 0.5 x. 106 are treated with various amounts of briostatin 1 for 24 hours. Accordingly, the cells are solubilized for Preparation of protein samples according to a routine procedure. Protein samples from the cells treated with briostatin are then used in the Western blot analysis with a specific antiserum of the C-β protein kinase previously described in Ways et al., Cell Growth & Di f ferentiation 1994, 5: 1195-1203. As shown in Figures 1 and 2, treatment with briostatin triggered the activity of PKC-β to decrease within a certain amount of time, ie 10 nM briostatin affects PKC-β within 2 hours, or 1 nM briostatin affects PKC-β within 24 hours. In a repeated experiment, similar results are obtained.

Example 2. The increased efficiency of the? -irradiation caused by down-regulation of PKC-β This experiment demonstrates that down-regulation of PKC-β increases the efficacy of? -irradiation. The U937 cells of human leukemia are treated for 24 hours with either briostatin 1 of 3 nM or the control solution, ie the vehicle for briostatin 1. The cells are irradiated then with either 500 or 1000 rads of? -irradiation. Seventy-two hours after irradiation, cell viability is examined using the exclusion of propidium iodide and quantified by FACS analysis as previously described in Ways et al., Cell Growth & Di f ferent iat ion 1994, 5: 1195-1203. The tests * '- were carried out in triplicate. As shown in Figure 3, apoptosis induced by? -irradiation was increased under the condition when PKC-ß was downregulated significantly using briostatin 1. Similar results are obtained in several repeated experiments.

Example 4. Cells Overexpressing PKC-β Show Resistance to Cell Death Stimulated by Radiation Parental U937 cells and cells overexpressing PKC-? U937 (PKC-? Cells) are treated with 0, 500 or 1000 rads of? -irradiation. It is known that PKC-? exhibit increased ni.vel of PKC-β (Ways et al., Cell Growth & Differentiation 1994, 5: 1195-1203). Seventy-two hours after irradiation, cell viability was examined using the exclusion of propidium iodide and quantified by FACS analysis as previously described in Ways et al., Cell Growth & Different iat ion 1995, 6: 371-382. Feasibility tests were carried out in triplicate. As shown in Figure 4, cells that have an increased level of PKC-β showed resistance to cell death stimulated by radiation. Similar results are obtained in various repeated experiments. The principles, preferred embodiments and modes of operation of the present invention have been described in the above specification. The invention that is intended to be protected by the present, however is not to be construed as limited to the particular forms described, as they are considered as. illustrative rather than restrictive. Variations and changes can be made by those skilled in the art without departing from the spirit of the invention.

Claims (15)

1. A method for treating a neoplasm comprising administering to a mammal in need of such treatment, an oncolytic agent having an anti-neoplastic effect in combination with a protein kinase C inhibitor, characterized in that the inhibitor of protein kinase C increases the anti-neoplastic effect of the oncolytic agent.

2. The method according to claim 1, characterized in that the protein kinase C inhibitor is an inhibitor of the protein isozyme β of protein kinase C and is a bis-indolylmaleimide or a bis-indolylmaleimide macrocyclic.

3. The method according to claim 1, characterized in that the inhibitor of protein kinase C is a selective isozyme and wherein the selectivity of the isozyme is selected from the group consisting of beta-1 and beta-2 isozymes.

4. The method according to claim 3, characterized in that the inhibitor of protein kinase C has the following formula: wherein: W is -0-, -S-, -SO-, -S02-, -CO-, C2-C6 alkylene, • substituted alkylene, C2-C6 alkenylene, -aryl-, -aryl (CH2) m0-, -heterocycle-, -heterocycle- (CH2) m0-, -bicylic-fused-, -bicylic- (CH2) m0-, -NR3-, -ÑOR3-, -CONH-, or -NHC0-; X and Y are independently C al-C alkylene, substituted alkylene, or together X, Y and W combine to form - (CH 2) n-AA-; the R1 are hydrogen or up to four optional substituents independently selected from halo, C1-C4 alkyl, hydroxy, C? -C4 alkoxy, haloalkyl, nitro, NR4R5 or -NHC0 (alkyl) C? -C4); R2 is hydrogen, CH3C0-, NH2, or hydroxy; R3 is hydrogen, (CH2) maryl, C? -C4 alkyl, -C00 (C1-C4 alkyl), -CONR4R5, - (C = NH) NH2, -SO (C1-C4 alkyl), -S02 (NR4R5), or -S02 (C1-C4 alkyl); R 4 and R 5 are independently hydrogen, C 1 -C 4 alkyl, phenyl, benzyl, or combine to the nitrogen to which they are attached to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently 0, 1, 2 or 3; and n is independently 2, 3, 4 or 5, or a pharmaceutically acceptable salt, prodrug or ester thereof.

5. The method according to claim 4, characterized in that the inhibitor of protein kinase C has the following formula: gives) wherein Z is (CH2) (CH2) p-0- (CH2) p- R < is hydroxy, -SH, C1-C4 alkyl, (CH2) maryl, -NH (aryl), -N (CH3) (CF3), -NH (CF3) v or -NR5R6; R5 is hydrogen or C1-C4 alkyl; R6 is hydrogen, C? -C4 alkyl or benzyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

6. The method according to claim 4, characterized in that the inhibitor of protein kinase C has the following formula: wherein Z is - (CH) P-; R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or C? -C4 alkyl; p is 0, 1 or 2; m is independently 2- or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

7. The method according to claim 4, characterized in that the inhibitor of protein kinase C comprises (S) -3, 4- [N, N '- 1, 1' - ((2"-ethoxy) -3" ' (O) -4"'- (N, N-dimethylamino) -5-butane) -bis (3,3'-indolyl)] -1 (H) -pyrrole-2, 5-dione or its pharmaceutically acceptable acid salt .

8. The method according to claim 1, characterized in that the agent Oncolytic is selected from the group consisting of Ara-c, etoposide or VP-16, cis-platinum, adriamycin, 2-chloro-deoxyadenosine, 9-β-D-arabinosyl-2-fluoro-adenine and glucocorticoids.

9. A method for treating a neoplasm comprising administering to a mammal in need of such treatment, -radiation having an anti-neoplastic effect in combination with a protein kinase C inhibitor, characterized in that the Protein kinase C inhibitor increases the anti-neoplastic effect of the? -irradiation.

10. The method according to claim 9, characterized in that the inhibitor - > of protein kinase C is an isozyme inhibitor ß of protein kinase C and is a bis-indolylmaleimide or a macrocyclic indolylmaleimide bis.

11. The method according to claim 9, characterized in that the inhibitor • of protein kinase C is a selective isozyme and wherein the selectivity of the isozyme is selected from the group consisting of beta-1 and beta-2 isozymes.

12. The method according to claim 11, characterized in that the inhibitor of protein kinase C has the following formula: wherein: W is -0-, -S-, -SO-, -S02-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aryl-, -aryl (CH2) m0 -, -heterocycle-, -heterocycle- (CH2) mO-, -bicylic. fused-, -bicylic- (CH2) m0 fused-, -NR3-, -ÑOR3-, -CONH-, or -NHCO-; X and Y are independently C1-C4 alkylene, substituted alkylene, or together X, Y and W combine to form - (CH2) n-AA-; R 1 are hydrogen or up to four optional substituents - independently selected from halo, C 1 -C 4 alkyl, hydroxy, C 1 -C 4 alkoxy, haloalkyl, nitro, NR 4 R 5 or -NHCO (C 1 -C 4 alkyl); R2 is hydrogen, CH3CO-, NH2, or hydroxy; R3 is hydrogen, (CH2) maryl, C1-C4 alkyl, -C00 (C 1 -C 4 alkyl), -CONR 4 R 5, - (C = NH) NH 2, -SO (C 1 -C 4 alkyl), - S02 (NR4R5), or -S02 (C1-C4 alkyl); R 4 and R 5 are independently hydrogen, C 1 -C 4 alkyl, phenyl, benzyl, or combine to the nitrogen to which they are attached to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently 0, 1, 2 or 3; and n is independently 2, 3, 4 or 5, or a pharmaceutically acceptable salt, prodrug or ester thereof.

13. The method according to claim 12, characterized in that the inhibitor of protein kinase C has the following formula: wherein Z is - (CH2) p- or - (CH2) p-0- (CH2) p-; R 4 is hydroxy, -SH, C 1 -C 4 alkyl, (CH 2) maryl, -NH (aryl), -N (CH 3) (CF 3), -NH (CF 3), or -NR 5 R 6; R5 is hydrogen or C? -C4 alkyl; R 6 is hydrogen, C 1 -C 4 alkyl or benzyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

14. The method according to claim 12, characterized in that the inhibitor of protein kinase C has the following formula: wherein Z is - (CH2) p-; R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or CJ.sub.C alkyl; p is 0, 1 or 2; m is independently 2 or 3, or a pharmaceutically acceptable salt, p-r-drug or ester thereof.

15. The method according to claim 12, characterized in that the inhibitor of protein kinase C comprises (S) -3, 4- [N, N '-l, l' - ((2"-ethoxy) -3" ' (0) -4"'- (N, N-dimethylamino) -b-tano) -bis (3,3'-indolyl)] -1 (H) -pyrrole-2, 5-dione or its pharmaceutically acceptable salt acceptable.