Formula I In another embodiment of the invention there is provided a method for inhibiting tumor growth comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of Formula I.

Another embodiment of the invention provides for the use of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of cancer. Still another embodiment of the invention provides for the use of a compound of

Formula I or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for inhibiting tumor growth.

Still another embodiment of the invention provides for the use of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of cancer.

Preferred embodiments of the invention provide for such methods and uses wherein the cancer being treated is lung, bladder, breast, colon, brain, cervical, stomach or kidney cancer.

Other preferred embodiments of the invention provide for such methods and uses for inhibiting the growth of lung, bladder, breast, colon, brain, cervical, stomach or kidney tumors.

Other preferred embodiments of the invention provide for such methods and uses for inhibiting the growth of human SW2 small lung carcinoma, human T98G glioblastoma multiforme, human Calu-6 non-small lung carcinoma, human SKOV-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human H5747T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma tumors. Special embodiments of the invention provide for such methods and uses wherein the cancer being treated is human SW2 small lung carcinoma, human T98G glioblastoma multiforme, human Calu-6 non-small lung carcinoma, human SKON-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human H5747T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma.

The present invention provides the art with a method for increasing apoptotic effects in malignant cells and inhibiting the signal transduction of angiogenic factors in tumors, and is thus effective in treating cancer and inhibiting tumor growth.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that use of a compound of Formula I, reduces or inhibits anti-apoptotic effects in a malignant cell and inhibits the intracellular signal transduction of angiogenic factors. Consequently, this compound can be used to treat cancer and inhibit tumor growth.

This compound may be prepared by methods known in the art (see, for example, U.S. Patent No. 5,668,152).

Because it contains a basic moiety, the compound of Formula I can 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 acid, as well as organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic, acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, mono- hydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, 2-butyne-l,4-dioate, 3-hexyne-2, 5-dioate, benzoate, chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, β- hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1- sulfonate, naphthalene-2-sulfonate, mandelate and the like. Particularly the hydrochloric and mesylate salts are used.

In addition to pharmaceutically acceptable salts, other salts also can exist. They may serve as intermediates in the purification of the compound of Formula I, in the preparation of other salts, or in the identification and characterization of the compound or intermediates.

The pharmaceutically acceptable salts of the compound of Formula I can also exist as 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 solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

Neoplasia is characterized by abnormal growth of cells which often results in the invasion of normal tissues, e.g., primary tumors or the spread to distant organs, e.g., metastasis. Any neoplastic growth may be treated by the methods of the present invention. Such neoplastic growth includes but not limited to primary tumors, primary tumors that are incompletely removed by surgical techniques, primary tumors which have been adequately treated but which are at high risk to develop a metastatic disease subsequently, and an established metastatic disease.

Specifically, the PKC inhibitor of Formula I above can be used to treat cancer and to inhibit tumor growth. The following examples are provided merely to further illustrate the present invention. The scope of the present invention is not to be construed as merely consisting of the following examples, hi each of the following examples, the compound of Formula I is administered as the dihydrochloride salt (the "Compound"), and the amounts administered are given in terms amounts of the dihydrochoride salt. The primary pharmacologic activity of the Compound is the inhibition of vascular endothelial growth factor (VEGF)-induced angiogenesis and basic fibroblast growth factor-induced angiogenesis associated with neoplastic disease. Three assay systems demonstrate this action of the molecule: 1.) the Compound is a potent inhibitor of VEGF- stimulated proliferation of HUNEC cells in culture with an IC₅₀ of 150 nM upon 72 hrs. exposure to the compound; 2.) the Compound is a highly effective inhibitor of VEGF- induced neoangiogenesis in the rat corneal micropocket when administered orally to the animals for 10 days; 3.) the Compound decreases to one-half to one-third the number of intratumoral blood vessels in the human SW2 small cell lung carcinoma grown as a xenograft in nude mice when administered orally for two weeks.

The treatment of neoplastic disease requires eradication of the primary and metastatic disease. The Compound demonstrated activity in each of ten tumors tested. The murine Lewis lung carcinoma or human T98G glioblastoma mutliforme, human SW2 small cell lung carcinoma, human Calu-6 non-small cell lung carcinoma, human SKON-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human HS746T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma was implanted in male C57B1 mice or male or female nude mice and the tumor-bearing animals were treated with either the Compound alone or with standard anticancer chemotherapy (paclitaxel, carboplatin, gemcitabine, BCΝU, 5-fluorouracil, irinotecan or cisplatin) or with fractionated radiation therapy.

Specifically, for most experiments, tumor cells prepared from a brie of donor tumors (5 x 10⁶ cells) were implanted subcutaneously in a hind-leg of mice (Charles River). Intracranial tumors were implanted by injecting 10⁴ T98G cells in 1.5 microliters. Intraperitoneal tumors were implanted by injecting 5 x 10⁶ SKON-3 tumor cells intraperitoneally into female nude mice. Treatment with the Compound (3, 10 or 30 mg/kg) administered orally twice per day for schedules lasting 2 weeks or more. Each standard chemotherapeutic agent was administered in a full murine dose regimen for that agent. Each treatment group as well as a group of untreated control animals consisted of 5 animals per group in each experiment. Subcutaneous tumor response was monitored by tumor volume measurement performed twice per week over the course of 60-120 days. For the survival experiments, long-term survivors (cures) were determined at 120 days post tumor implantation. Body weights were measured as a general measure of toxicity. The subcutaneous tumor data were analyzed by determining the mean tumor volume for each treatment group over the course of the experiment and calculating the tumor growth delay as the difference in days for the treatment versus the control tumors to reach a volume of 500 and 1000 mm³.

Growth Inhibition of Endothelial and Malignant Cells by the Compound

Human Umbilical Vein Endothelial Cell Culture Assay. HUVEC cells in basal medium were stimulated to proliferate by exposure to human VEGF (20 ng/ml). Human tumor cells were grown in media with 2% fetal bovine serum. When the Compound was added to the cultures for 72 hours, the proliferation of the VEGF-stimulated HUVEC cells was profoundly inhibited by 0.6 μM of the compound. The IC₅₀ for the HUVEC assay was 150 nM of the Compound (as shown in Table 1). The Compound was growth inhibitory at concentrations of 3 to >85 μM in eight human tumor cell lines exposed to the compound for the same period of time assuming (Concentration times Time) (as shown in Table 1).

Rat Corneal Micropocket Assay

The cornea is normally an avascular tissue. Surgical implantation of a small filter disc impregnated with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) into the cornea will result in robust neoangiogenesis that is quantifiable in 7 to 10 days. Administration of the Compound orally twice per day on days 1 through 10 post surgical implant of VEGF or bFGF impregnated filters resulted in markedly decreased vascular growth in the cornea. A dose of 10 mg/kg of the Compound decreased vascular growth to about one-half of the VEGF stimulated controls; while a dose of 30 mg/kg of the Compound decreased vascular growth to the level of the un- stimulated surgical control in the VEGF-stimulated corneas and to a level of one-third of control in the bFGF-stimulated corneas. Results for VEGF are shown in Table 2, and for bFGF in Table 3.

Intratumoral Vessel Count Assay.

Nude mice bearing human SW2 small cell lung carcinoma, human Calu-6 non- small cell lung carcinoma, human SKOV-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human HS746T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma growing as a subcutaneous xenograft on the thigh of the animals were treated with the Compound twice daily on days 14 through 30 post tumor cell implantation. On day 31 tumors were collected, preserved in 10% phosphate buffered formalin and 5 mm thick sections were immunohistochemically stained for expression of endothelial specific markers, either Factor VIII, CD31 or CD 105. The number of intratumoral vessels in the samples was quantified by counting stained regions in 10 high power microscope fields (200x). The results are shown in Tables 4 to 11.

Tumor Growth Delay in Human Xeno grafts

The Compound, administered orally twice per day for 2 weeks, has demonstrated activity in a variety of human tumor xenografts grown in nude mice (Tables 12 to 19).

In nine tumor lines grown in vivo in mice, the tumor growth delay produced by the Compound (30 mg/kg) administered twice per day orally for 2 weeks compared very favorably with the tumor growth delay produced by standard anticancer agents administered on a normal full murine dose-schedule (Table 20). In Table 5, RADS is 3 Gray x 10.

TABLE 1

GROWTH INHIBITION OF ENDOTHELIAL AND MALIGNANT CELLS BY

THE COMPOUND

TABLE 2

RAT CORNEAL MICROPOCKET VEGF INDUCED ANGIOGENESIS: RESPONSE TO THE COMPOUND, po, 2x dl-10

TABLE 3 RAT CORNEAL MICROPOCKET bFGF INDUCED ANGIOGENESIS:

RESPONSE TO THE COMPOUND, po, 2x dl-10

TABLE 4

HUMAN CALU-6 NON-SMALL CELL LUNG CA

TABLE 5

HUMAN CaKil RENAL CELL CARCINOMA

TABLE 6

HUMAN HT-29 COLON CARCINOMA

TABLE 7

HUMAN HS746T GASTRIC CARCINOMA

TABLE 8

HUMAN Hep3B HEPATOCELLULAR CARCINOMA

TABLE 9

HUMAN MX-1 BREAST CARCINOMA

TABLE 10 HUMAN SKOV-3 OVARIAN CARCINOMA

TABLE 11 HUMAN SW2 SMALL CELL LUNG CANCER

TABLE 12 RESPONSE OF Calu-6 TO THE COMPOUND AS A SINGLE AGENT

TABLE 13 RESPONSE OF SW2 TO THE COMPOUND AS A SINGLE AGENT

TABLE 14 RESPONSE OF Caki-1 TO THE COMPOUND AS A SINGLE AGENT

TABLE 15

REPONSE OF HS746T TO THE COMPOUND AS A SINGLE AGENT

TABLE 16

RESPONSE OF Hep3B TO THE COMPOUND AS A SINGLE AGENT

TABLE 17

RESPONSE OF T98G TO THE COMPOUND AS A SINGLE AGENT

TABLE 18

RESPONSE OF HT-29 TO THE COMPOUND AS A SINGLE AGENT

TABLE 19

RESPONSE OF MX-1 TO THE COMPOUND AS A SINGLE AGENT

TABLE 20

TUMOR GROWTH DELAY BY THE COMPOUND COMPARED WITH

STANDARD AGENTS

In addition, when administered to animals bearing intracranial human T98G glioblastoma multiforme, the mean survival time of the animals treated with the Compound (30 mg/kg, po) was 72 days. The mean survival time for animals treated with BCNU alone in this study was 42 days and the mean survival time for the untreated control animals was 39 days.

Survival was also used as an endpoint for studies with animals bearing intraperitoneally implanted human SKOV-3 ovarian carcinoma. The mean survival time time for animals treated with the Compound (30 mg/kg, po) was 73 days. The mean survival times for animals in these studies treated with paclitaxel was 84 days and for animals treated with carboplatin was 45 days. In theses studies the untreated control animals survived 40 days.

The PKC inhibitor may be administered chronically or semi-chronically, over a period of from about 2 weeks to about 5 years. One skilled in the art will recognize that the amount of PKC inhibitor to be administered in accordance with the present invention, that is the therapeutically effective amount, is that amount sufficient to produce an anti-neoplastic effect, to induce apoptosis or cell death, and/or to maintain an antiangiogenic effect. Such amount may vary, among other factors, depending upon the size and the type of neoplasia, the concentration of the compound in the therapeutic formulation, the timing of the ad inistration of the PKC inhibitor relative to the other therapies, and the age, size and condition of the patient.

Generally, an amount of protein kinase C inhibitor to be administered is decided on a case by case basis by the attending physician. As a guideline, the extent of the neoplasia, the body weight, and the age of the patient will be considered, among other factors, when setting an appropriate dose.

Generally, a suitable dose is one that results in a concentration of the protein kinase C inhibitor at the site of tumor cells in the range of 50 nM to 50 μM, and more usually from 100 nM to 10 μM. It is expected that serum concentrations of 250 nM to 250 μM should be sufficient in most circumstances.

To obtain these treatment concentrations, a patient in need of treatment will be administered between about 0.5 mg per day per kg of body weight and about 10 mg per day per kg. Usually, not more than about 3 mg per day per kg of body weight of protein kinase C inhibitor should be needed. As noted above, the above amounts may vary on a case-by-case basis.

The compound of the present invention is preferably formulated prior to administration. Suitable pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients, hi making the 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 enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semisolid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosol (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders for either oral or topical application.

Some examples of suitable carriers, excipient, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphates, algiriate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient. The compositions are preferably formulated in a unit dosage form, each dosage 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 dosage administered will be determined by the physician in the light of the relevant circumstances including the severity of the condition to be treated, the choice of compound to be administered and the chosen route of administration.

Therefore, the above dosage ranges are not intended to limit the scope of the invention in any way. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages 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. hi addition to the above formulations, most of which may be administered orally, the compound used in the method of the present invention also may be administered topically. Topical formulations include ointments, creams and gels.

Ointments generally are prepared using either (1) an oleaginous base, i.e., one consisting of fixed oils or hydrocarbons, such as white petrolatum or mineral oil, or (2) an absorbent base, i.e., one consisting of an anhydrous substance or substances which can absorb water, for example anhydrous lanolin. Customarily, following formation of the base, whether oleaginous or absorbent, the active ingredient (compound) is added to an amount affording the desired concentration. Creams are oil/water emulsions. 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), comprising water and any water-soluble substances, such as added salts. The two phases are stabilized by use of an emulsifying agent, for example, a surface active agent, such as sodium lauryl sulfate; hydrophilic colloids, such as acacia colloidal clays, veegum, and the like. Upon formation of the emulsion, the active ingredient (compound) customarily is added in an amount to achieve the desired concentration.

Gels comprise a base selected from an oleaginous base, water, or an emulsion- suspension base. To the base is added a gelling agent which forms a matrix in the base, increasing its viscosity. Examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers, and the like. Customarily, the active ingredient (compounds) is added to the formulation at the desired concentration at a point preceding addition of the gelling agent.

The amount of compound incorporated into a topical formulation is not critical; the concentration should be within a range sufficient to permit ready application of the formulation to the affected tissue area in an amount which will deliver the desired amount of compound to the desired treatment site.

The customary amount of a topical formulation to be applied to an affected tissue will depend upon an affected tissue size and concentration of compound in the formulation. Generally, the formulation will be applied to the effected tissue in an amount affording from about 1 to about 500 μg compound per cm² of an affected tissue.

Preferably, the applied amount of compound will range from about 30 to about 300 μg/cm², more preferably, from about 50 to about 200 μg/cm², and, most preferably, from about .60 to about 100 μg/cm². The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way.

Formulation 1 Hard gelatin capsules are prepared using the following ingredients:

Quantity (mg/capsule) Active agent 250 starch, dried 200 magnesium stearate 10

Total 460 mg The above ingredients are mixed and filled into hard gelatin capsules in 460 mg quantities.

Formulation 2 A tablet is prepared using the ingredients below:

Quantity (mg/capsule)

Active agent 250 cellulose, microcrystalline 400 silicon dioxide, fumed 10 stearic acid 5

Total 665 mg

The components are blended and compressed to form tablets each weighing 665 mg.

Formulation 3 Tablets each containing 60 mg of active ingredient are made as follows:

Quantity (mg/tablet)

Active agent 60 mg starch 45 mg microcrystalline cellulose 35 mg polyvinylpyrrolidone

(as 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 No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50°C and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg.

The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since they are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.

Claims

CLAIMS:

1. A method for treating cancer comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of the formula

or a pharmaceutically acceptable salt or solvate thereof.

2. A method for inhibiting tumor growth comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of the formula

or a pharmaceutically acceptable salt or solvate thereof.

3. The use of a compound of the formula

or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of cancer.

4. The use of a compound of the formula

or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for inhibiting tumor growth.

5. A method according to claim 1 wherein the cancer being treated is lung, bladder, breast, colon, brain, cervical, stomach or kidney cancer.

6. A method according to claim 2 for inhibiting the growth of a lung, bladder, breast, colon, brain, cervical, stomach or kidney tumor.

7. A use according to claim 3 wherein the cancer being treated is lung, bladder, breast, colon, brain, cervical, stomach or kidney cancer.

8. A use according to claim 4 for inhibiting the growth of a lung, bladder, breast, colon, brain, cervical, stomach or kidney tumor.

9. A method according to claim 5 wherein the cancer being treated is human SW2 small lung carcinoma, human T98G glioblastoma multiforme, human Calu-6 non-small lung carcinoma, human SKOV-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human H5747T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma.

10. A method according to claim 6 for inhibiting the growth of a human SW2 small lung carcinoma, human T98G glioblastoma multiforme, human Calu-6 non-small lung carcinoma, human SKOV-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human H5747T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma tumor.

11. A use according to claim 7 wherein the cancer being treated is human SW2 small lung carcinoma, human T98G glioblastoma multiforme, human Calu-6 non-small lung carcinoma, human SKOV-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human H5747T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma.

12. A use according to claim 8 for inhibiting the growth of a human SW2 small lung carcinoma, human T98G glioblastoma multiforme, human Calu-6 non-small lung carcinoma, human SKOV-3 ovarian carcinoma, human HT-29 colon carcinoma, human CaKil renal cell carcinoma, human H5747T gastric cancer, human Hep3B hepatocellular carcinoma or human MX-1 breast carcinoma tumor.