WO1989007441A1

WO1989007441A1 - 6-amino-1,2-benzopyrone antitumorigenic agents and method

Info

Publication number: WO1989007441A1
Application number: PCT/US1989/000516
Authority: WO
Inventors: Alexander Tseng, Jr.; Jerome Mclick; Hakam Alaeddin
Original assignee: The Regents Of The University Of California
Priority date: 1988-02-10
Filing date: 1989-02-09
Publication date: 1989-08-24

Abstract

6-amino-1,2-benzopyrone is administered as a chemopreventative, or antitumorigenic agent, to inhibit tumor-igenicity in a mammalian host. Preferably, the administering is by a daily oral dose and provides about 100 mg per day.

Description

6-AMINO-1,2-BENZOPYRONE ANTITUMORIGENIC AGENTS AND METHOD

Inventors: ALEXANDER TSENG, JR., JEROME McLICK and ALAEDDIN HAKAM

Field of the Invention

The present invention generally relates to anti- tumorigenic agents, and more particularly to use of 6-amino-1,2-benzopyrone, or 6-aminocoumarin, and salts thereof in inhibiting tumor growth in a mammalian host.

Background of the Invention

Some populations are at high risk of developing tumors. For example, immunosuppressed patients, such as post-cardiac or renal transplant patients, can experience a high tumor incidence. Similarly, AIDS (Acquired Immune Deficiency Syndrome) patients or persons with the ARC (AIDS Related Complex) syndrome are another patient group in which tumors are prone to develop.

One approach in attempts to inhibit tumor growth is to administer a phenylalkyamine or the like as a calcium channel blocker compound, as described by U.S. Patent 4,690,935, inventors Taylor et al., issued September 1, 1987. Another approach is described by U.S. Patent 4,714,683, inventor Shoyab, issued December 22, 1987 in whicn administration of a certain polypeptide is said to retard the growth of neoplastic cells. U.S. Patent 4,684,627, inventor LeVeen, issued August 4, 1987, describes administering compounds, such as lonidamine and phlorizin, to humans with lung cancer, which compounds are said to inhibit glucose transport in cancer cells.

Poly(ADF-ribose) polymerase has been implicated as an enzyme system involved in neoplastic transformation. 1,2-benzopyrone (frequently referred to as coumarin) has recently been reported to inhibit poly(ADP-ribose) polymerase at the DNA binding site of the enzyme. Although poly(ADP-ribose) polymerase was inhibited by 1,2-benzopyrone to the same extent in both non-transformed and neoplastic (transformed) cells, the arrest of cell proliferation occurred only in transformed cells expressing oncogenes. Tseng, Jr. et al., "Prevention of Tumorigenesis of Oncogene-Transformed Rat Fibroblasts with DNA Site Inhibitors of Poly(ADP Ribose) Polymerase," Proc. Natl. Acad. Sci. USA, Volume 84, pp. 1107-1111 (February 1987).

1,2-benzopyrone has the structure illustrated by Formula I, below.

Summary of the Invention

In one aspect of the present invention, a method for inhibiting tumorigenicity in a patient comprises administering to the patient an effective amount of a compound having the formula

or a therapeutically acceptable salt, such as chloride or sulfate, thereof. Said compound, designated 6-amino1 ,2-benzopyrone, or 6-aminocoumarin, is surprisingly more efficacious than the unsubsti tuted, 1,2-benzopyrone compound in inhibiting growth of the human renal cancer cell line CRL1611. The compound (like 1,2-benzopyrone) is readily administered, such as by oral doses, and is non-toxic. The compound is believed particularly useful in mammalian hosts, or patients, who are at risk due to exposure to mutagenic agents or are particularly susceptible to neoplastic formation. Thus, use of the 6-amino1,2-benzopyrone compound and its pharmaceutically acceptable salts is particularly preferred as a chemopreventative agent for populations at high risk of developing tumors. By chemoprevention is meant the use of chemical agents to inhibit, block or reverse the growth and/or progression of cancer.

Brief Description of the Drawings:

Figure 1 is a schema ti c represen ta tion of the 4.8 Kb EJ-ras oncogene fragment and pCV108 cloning vector ; and , Figure 2 is a graph showing the effect of 6-amino1,2-benzopyrone on the initial velocity of auto-mono-ADP-ribosylation of the enzyme as a function of varying concentrations of octameric DNA.

Detailed Description of the Preferred Embodiments

The compound 1,2-benzopyrone has been found clinically effective in several metastatic human tumors, i.e., metastatic melanoma and metastatic renal cell cancer, when used in combination with cimetidine.

Practice of the present invention includes use of 6-amino-1,2-benzopyrone or its salts as a chemopreventative, or anti tumorigenic, agent. The compound may be synthesized in good yield and converted to a therapeutically acceptable salt, preferably such as sulfate and chloride, most preferably as the chloride salt. These salts improve the solubility of the free base and are shelf-stable (e.g., are non-hygroscopic and thermally stable at ambient temperatures). The structure of the free base 6-amino-1,2-benzopyrone is illustrated by Formula IIA and that of two preferred salts by Formulas IIB and IIC. The compound is obtained as the free base and can be readily converted to salts with inorganic acids/ but does not form salts with weaker organic acids such as acetic acid.

A number of analogs to the Formula IIA compound are known in the literature which may have similar pharmacological properties. These analogs include 8-amino1,2-benzopyrone, 3-amino-1,2-benzopyrone, 6-amino-3-hydroxy-1,2-benzopyrone, and 8-amino-3-hydroxy-1,2-benzopyrone.

A preferred method for synthesizing the Formula IIA, IIB and IIC anti tumorigenic agents will now be described.

Preferred Synthesi s Method

6-amino-1,2-benzopyrone: To a stirred suspension of 0.50 gm of 10% palladium on activated carbon in 30 ml of degassed water was added a solution of potassium borohydride (2.70 gm, 0.050 mol ) in water (35 ml). A solution of 6-nitro-1,2-benzopyrone (3.82 cm, 0.020 mol; available from Pflatz & Bauer) in methanol (1000 ml) was then slowly added (10 minutes) to the stirred mixture at ambient temperature. After stirring for an additional 15 minutes, the mixture was filtered through a bed of Celite to remove the catalyst, and the filtrate was then stripped of methanol by rotary evaporation. The damp residue was suspended in cold water (100 ml) and collected by suction filtration. The crude yellow product was washed on the filter with additional cold water and then dried and weighed (2.83 gm). The material was finally recrystallized from ethanol to afford 2.32 gm (72% yield), m.p. 166-169°C (lit 166-167°C, see Kokotos et al., J. Heterocyclic Chem., 23, p. 87 (1986). MASS: 161 (M⁺), 133, 104, 78, 52.

6-amino-1,2-benzopyrone hydrochloride : To a chilled slurry of 6-amino-1,2-benzopyrone (1.50 gm, 0.009 mol) in 20 ml of water was added 2 M HCl dropwise in sufficient quantity with stirring to dissolve the base, yielding a yellow solution. The solution was filtered to remove a small amount of cloudiness and then stripped of water by rotary evaporation. The residue was crystallized from 1/1 (v/v) methanol/e thanol to give 1.59 gm (86% yield) of pale yellow prisms, m.p. 280-285°C with decomposition (the material darkens above 230°C).

Elemental analysis for C₉H₈ClNO₂ : Calculated: C,

54.70; H, 4.08; Cl , 17.94; N, 7.09. Found: C, 54.80; H,

4.09; Cl, 18.06; N, 6.98. MASS: 161 (M-HCl)⁺, 133, 104,

78, 52, 51.

6-amino-1,2-benzopyrone sulfate: To a chilled, stirred slurry of 6-amino-1,2-benzopyrone (0.48 gm, 0.003 mol) in 6 ml of water was added 2M H₂ SO₄ dropwise in sufficient quantity to dissolve the base. The solution was filtered to remove cloudiness and then stripped of water by rotary evaporation. The residue, which contained a small amount of excess H₂ SO₄ , was dissolved in hot ethanol, which upon cooling gave tan-colored platelet crystals. The material was collected, washed with cold solvent, and crystallized again from ethane! to afford 0.38 gm (60% yield) of the salt, m.p. 214-215°C with decomposition. Elemental analysis for ( C₉H₈NO₂ )₂SO₄ ; Calculated:

C, 51.43; H, 3.84; N, 6.66; S,7.63. Found : C, 51.40; H, 3.38; N, 6.43; S, 7.66.

The antitumorigenic properties of the 6-amino-1,2-benzopyrone compound, including its growth inhibitory effects (expressed as IC₅₀) and non-toxic concentrations (expressed as IC₀ ) are illustrated by a variety of in vitro and in vivo assays. Thus, Example I describes measurement of the growth inhibitory effects in vitro with a rat fibroblast cell line designated 14C, and Example VI describes the in vivo tumor growth inhibitory effects of the Formula II compound in salt form.

The 14C cell line appears morphologically similar to the parent cell line (Rat-1 fibroblasts) in the absence of dexamethasone in the culture medium. The cells appear flat and are non-refractive to light. After a 24 hour exposure to 0.1 μM dexamethasone, the cells round up and develop cellular projections, heap up into refractile foci, and lose their contact growth inhibition.

The 14C cell line spontaneously forms few and only very small colonies in soft agar, but exposure to 0.1 uM dexamethasone (Dex) greatly increases both the number and size of colonies. Dexamethasone is a potent synthetic corticosteroid. The 14C cell line contains a stercidinducible EJ-ras oncogene isolated from a human bladder carcinoma. The pMTV-EJras plasmid was made by placing the EJ-ras coding domain under the transcriptional control of the mouse mammary tumor virus (MTV; steroidinducible) promoter/enhancer, and it also contains a neomycin-resistance gene (see Figure 1). EJ-ras is a mutant c-Ha-rasl gene isolated from the EJ/T24 bladder carcinoma cell lines. The steroid-responsive portion of the MTV long terminal repeat ( Cla I-BamHI fragment) from plasmid p484 was subcloned between the above restriction sites in the vector pCV108, which carries the neomycinresistance gene, generating pMTV108. A fragment of the pEJ6.6 plasmid [the 4.8-kilobase (kb) fragment from a Sma I site located approximately 20 base pairs (bp) upstream from the initiation ATG to the Bgl II site near the 3' end of the clone] was converted to Bgl II ends with linkers and inserted into the BamHI site of pMTV108, generating pMTV-EJras. Rat-1 fibroblasts were transfected with this plasmid by the calcium phosphate technique and selected in the presence of 400 ug/ml of G418 (GIBCO). Drug-resistant colonies were isolated with the aid of a cloning cylinder.

Because the 14C cell line contains the inducible EJ-ras oncogene, the effect of compounds described in this invention can be determined before and after overt transformation and avoid biochemical complications introduced by the toxicity of chemical carcinogens and irradiation.

EXAMPLE I Growth Assay in 14C Cells

Cell cultures (or the 14C cell line previously discussed) were initiated at a cell density of 20,000 cells per T25 culture flasks, and grown in Dulbecco's modified Eagles 's medium (DMEM) supplemented with 10% fetal calf serum, 0.5% penicillin/streptomycin, and 2mM glutamine. The flasks were incubated at 37°C for 24 hours to allow the cells to attach to the plastic.

The 6-amino-1,2-benzopyrone in free base and in chloride salt form was then added and cell counts were performed after 24, 48 and 72 hours of further incubation.

The cells were trypsinized (0.25% STV trypsin) and cell counts were determined on a Coulter Counter (ZBI model).

Table I shows the growth inhibitory effects of 6-amino-1,2-benzopyrone and its salts on 14C cells exposed to the drug for 72 hours.

TABLE I Growth Inhibition Assay of 14C Cells

IC₅₀ Maximal IC₍₀₎

6-aminocoumarin 1mM 100μM 6-aminocoumarin ·HCl 2mM 100uM

This growth inhibition assay shows that an inhibitory concentration of drug can be defined, as well as a maximal, non-inhibiting concentration. When the maximal, non-inhibitory concentration of 6-aminocoumarin (that is, 6-amino-1,2-benzopyrone) is used in the soft agarose and tumorigenic assays, the drug proves to be effective in preventing growth. Thus, a non-toxic drug concentration for preventing the tumorigenic phenotype can be defined and is set out in Table III. EXAMPLE II Growth Assay in Human Cells

The growth inhibitory effects of the anti tumorigenic agent in accordance with the invention was also measured in human renal carcinoma ce]l lines (CRL1611 and KEC-1) and a human bladder carcinoma cell line (253J). The 253J and KEC-1 cell lines were as described by Williams, Invest. Urol., 17, pp. 359-363 (1980). The CRL1611 cell line was obtained from the ATCC. The results were similar to those described in Example I, and are set forth in Table II.

TABLE II

Growth Inhibition at 72 Hours

253J (Human Bladder Cancer Cell Line)

6-aminocoumarin · HCl IC₅₀=4.5×10^-4M 6-aminocoumarin IC₅₀=10^-6M

KEC-1 (Human Renal Cancer Cell Line)

6-ammocoumarin IC₈₀=10^-4M

CRL1611 (Human Renal Cancer Cell Line)

6-aminocoumarin IC₈₅=10^-4M EXAMPLE III

Soft Agarose Assay with 14C Cells

Assays were conducted to measure anchorage-independent colony formation of cells when plated into soft agarose. 25,000 14C cells were initiated into T25 culture flasks and exposed for 48 hours to either 0.1 μM dexamethasone (Dex) or to sterile water. The 14C cells were then exposed to a four log concentration of 6-amino- 1,2-benzopyrone for an additional 72 hours. Cells (1×10³) were suspended in 8 ml of 0.35% agarose (Calbiochem, type A) in DMEM containing 10% fetal calf serum and overlayed above 5 ml of 0.7% agarose in DMEM containing

10% fetal calf serum in 60 mm petri dishes. Following incubation at 37°C for 10 days, the cells were stained with 1 ml of MTT dye (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte trazolium bromide, Sigma) overnight and counted. The number of colonies measuring over 0.3 mm in diameter (greater than 100 cells) was counted under low power phase microscopy.

The effects of 6-amino-1,2-benzopyrone on colony formation (and of 1,2-benzopyrone as comparison) are illustrated by the data of Table III.

TABLE III

Treatment No. of Colonies IC₅₀

Rat-1 Fibroblasts (Control)

(-) Dex 10 ± 4 --

14C Cells (Control)

(-) Dex 42 ± 48 --

(+) Dex 308 ± 69 --

1,2-benzopyrone at 0.1 mM

(-) Dex 56 ± 53 0.5 mM (+) Dex 74 ± 18 0.5 mM

6-amino-1,2-benzopyrone at 0.1 mM

(-) Dex 34 ± 12 1 mM

(+) Dex 33 ± 7 1 mM

6-amino-1,2-benzopyrone· HCl at 0.1 mM (-) Dex 43 ± 33 2 mM

(+) Dex 49 ± 33 2 mM

6-amino-1,2-benzopyrone· HCl at 1 mM

(-) Dex 46 ± 12 2 mM

(+) Dex 29 ± 6 2 mM

As may be seen from the data of Tab]e III, the control cells were transformed by being induced with dexamethasone, but treatments with 6-amino-1,2-benzopyrone or treatments with 6-amino-1,2-benzopyrone ·HCl inhibited such dexamethasone induced crowch at concentra tions as small as 0.1 mM. As may also be seen, the related compound 1,2-benzopyrone also had an inhibitory effect, but one tending to be less efficacious.

EXAMPLE IV Soft Agarose Assays with Human Cancer Cells

Soft agarose assays were performed as described in Example III, but with CRL1611 human renal carcinoma cells; however, there was no exposure to dexamethasone (since the cells are already transformed) and 10⁵ cells were used. The assays were conducted with varying concentrations of 1,2-benzopyrone and 6-amino-1,2-benzopyrone, and the results are set out in Table IV.

TABLE

Number of Colonies

Control 382 + 85

10^-4M 1,2-benzopyrone 244 ± 85 10^-5M 1,2-benzopyrone 315 ± 43 10^-6M 1,2-benzopyrone 402 ± 21

10^-4M 6-amino-1,2-benzopyrone 175 ± 35 10^-5M 6-amino-1,2-benzopyrone 170 ± 8

10^-6M 6-amino-1,2-benzopyrone 170 ± 37

As may be seen from the data of Table IV, the 6-amino-1,2-benzopyrone treatment at only 1 μM (10^-6 M) was strikingly rrore effective in inhibiting colonies of the human renal cell cancer line than was the 1,2-benzopyrone compound at 100 times the concentration. EXAMPLE V Cell Cycle Assay

Early passage nonsynchronized 14C cells were pretreated with 0.1 μM dexamethasone for 72 hours to induce transformation. This was followed by either 24 hour treatment with 100 uM 6-amino-1,2-benzopyrone or phosphate buffer saline. Non-dexamethasone transformed cells were similarly treated with 100 uM 6-amino-1,2-benzopyrone or with phosphate buffer saline only. Media and drugs were changed daily. When cells reached 70% confluency in T75 flasks, they were pulse labeled with 15 uM 5-bromodeoxyridine (BrdUrd) for 30 minutes, washed with PBS, trypsinized, centrifuged at 4ºC and fixed in 70% cold ethanol. The fixed cells were pelleted, resuspendeά and incubated for 15 minutes in 2.5 M HCl/0.7% TX-100. The cells were then washed in PBS and 0.5% Tween 20. Mouse anti-3rdUrd monoclonal antibody IU-4 diluted 1:4000 in PBS/0.5% Tween 20/0.5% Carnation Milk powder was then incubated with cells for 30 minutes at room temperature. Cells were washed and treated with FITC-con jugated rabbit anti-mouse IgG diluted 1:150 in PBS/Tween 20/milk power and incubated for 20 minutes at room temperature. Cells were washed and stained with 10 αg/ml of propidium iodide for 30 minutes. Cells were filtered through a 27 uM Nitrex mesh and 20,000 cells were analyzed on a FACS II flow cytometer (Becton-Dickenson, Mountain View, CA ). Cells were excited at 488 nm. Red fluorescence from propidium iodide was collected through a 600 nm long pass filter and recorded on linear scale as a measure of DNA content. Green fluorescence from fluorescein was collected through a 514 nm band pass filter and recorded after logarithmic amplification as a measure of the amount of incorporated BrdUrd. List mode data was collected with Electric Desk software and displayed as dual parameter correlated data. The percentage of cells in G₀/G₁, G₂ /M and S phases of the cell cycle was calculated within a 5% error by appropriate gating around those easily defined populations.

The cell cycle effects of the 6-amino-1,2-benzopyrone treatment and the control are illustrated by the data of Table V.

TABLE V

G₀/G₁

Control 67% 21%

0.1 μM Dex 55% 31%

100 μM 6-amino- 1,2-benzopryone 65% 18%

0.1 μM Dex and 100 μM

6-amino-1,2-benzopyrone 61% 19%

As may be seen from the data in Table V, dexamethasone increases the percentage of cells in the S phase with a concomitant decrease in the percentage of cells in the G₀/G₁ phase of the cell cycle. This represents a recruitment of cells from the G₀/G₁ into the S chase.

Treatment with 6-amino-1,2-benzopyrone of 14C cells that have not been transformed by dexamethasone has no significant effect on the percentage of cells in these phases compared to control. However, treatment of dexamethasone transformed 14C cells with 6-amino-1,2-benzo- pyrone reverses the effect of dexamethasone.

As may also be seen from the data of Table V, the 6-amino-1,2-benzopyrone treatment seems to have specificitv in causing a cell cycle block in the G₀/G₁ ohase of transformed cells.

EXAMPLE VI

Tumorigenesis Inhibition in Vivo

An in vivo tumorigenesis assay using 14C cells was performed. Subcutaneous injections of 10 cells in Fischer 344 rats (male, 150-g body weight) led to the development of lethal fibrosarcomas. Groups of at least ten animals were tested per group. Tumor incidence and weight were compared between four different treatment groups, as shown in Table VI. Two groups of animals were injected with 14C cells that were pretreated with 100 uM 6-amino-1,2-benzopyrone ·HCl in vitro, versus two groups that were injected with 14C cells that had not been pretreated. Two groups of animals received 6-amino-1,2-benzopyrone HCl orally as administered in their drinking water, versus two groups that received sterile water only. In the drug-treated groups, each bottle contained 200 cc of 200μM 6-amino-1,2-benzcpyrone ·HCl dissolved in sterile water. The drinking bottles were refilled every three days and monitored for the volume of water consumed. The animals were weighed and survivors were recorded. Animals were sacrificed two weeks after innoculation of 14C cells and tumor weights are measured. The data of Table VI summarizes the results.

TABLE VI

100 UM 6-1 ,2-amlno 200 μM 6-amino- benzopyrone Pre- 1,2 -benzopyrone No. of Injectione No. Tumors/ Average Tumor

Group treatment in Vitro in Water Supply (No. of Rata) In j. (>1gm) we ight

1. (-) (-) 75 (38) 50/75 (66%) 3.5 ± 2.5 gm

2. (+) (-) 20 (10) 2/20 (10%) 0.34 ± 0.78 gm

3. (-) (+) 20 (10) 3/20 (15%) 0.66 ± 1.68 gm

4. (+) (+) 22 (11) 1/22 ( 5%) 0.11 ± 0.49 gm

As may be seen from the data of Table VI, the pretreatment of the transformable 14C cells with 6-amino-1,2-benzopyrone was effective in inhibiting both the number of tumors and tumor weight. Similarly, the dietary administration of 6-amino-1,2-benzopyrone was also effective in reducing the number of tumors and the average tumor weight. Best results were obtained with both pretreatment and oral administration of 6-amino-1,2-benzopyrone in reducing the number of tumors and reducing tumor weight.

It was calculated that a 200μM drug concentration in the drinking water for seven days could maintain a cellular concentration of unme tabolized drug between 50-88 μM, that is, in the same order of magnitude as the competitive Ki obtained with 6-amino-1,2-benzopyrone with purified poly(ADP-ribose) polymerase enzyme. EXAMPLE VII Inhibition of Poly (ADP-ribose) Polymerase

The 6-amino-1,2-benzopyrone compound competitively inhibits the DNA binding site of poly( ADP-ribose) polymerase (also known as adenosine diphosphoribosyl-transferase, EC 2.4.30), which is a nuclear enzyme that catalyzes the cleavage of oxidized NAD with the concomitant covalent attachment of ADP-ribose to acceptor nuclear proteins. As described below, the Ki (inhibitory constant) is 28 uM for 6-amino-1,2-benzopyrone. By comparison, the Ki for 1,2-benzopyrone is 47 uM.

The 6-amino-1,2-benzopyrone compound is a potent inhibitor of the enzyme adenosine diphosphoribosyl-transferase. Specifically, the inhibitory effect of 6-amino-1,2-benzopyrone on the initial velocity of mono-ADP-ribosylation of the enzyme as a function of the concentration of octameric coenzymic DNA was studied. Figure 2 shows a double-reciprocal plot of the data obtained. The graphical results demonstrate that 6-amino-1,2-benzopyrone competitively inhibits at the coenzymic DNA binding site with an apparent K_i of 28μM. (Without the inhibitor, at a fixed NAD concentration a Michaelis-Menten relationship exists between the initial velocity and the concentration of coenzymic DNA (lowest curve in the figure) with an apparent binding constant of 1 μM. This value was obtained by varying the concentration of the octameric DNA over a 10-fold range.) The results establish that a site exists which is structurally unrelated to MAD at which the inhibitor binds. The octameric DNA used in this study was 5'-A-G-A-T- C-A-G-T-3' in the duplex form with its complement. The initial velocity rates were determined using the following enzyme incubation buffer: 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 20 mM MgCl₂, 2.5 mM dithiothreitol, and

7 mM NaCl. NAD concentration was 25 nM and the final volume of each assay was 100 μL. The reaction was started by addition of 1μg of enzyme, and incubation was carried out at 24ºC for 2 minutes and then stopped by addition of 20% aqueous trichloroace tic acid. The amount of product ADP-ribose formed was determined by collection on Whatman glass microfiber filters.

The implications of the tumorigenesis inhibition illustration of Example VI are that administration of 6-amino-1,2-benzopyrone or its salts will be useful with adjuvent therapy in treating different types of cancers. For example, among the present treatments following breast cancer surgery are adjuvant uses of me thotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, vincristine, and prednisone. Patients with advanced Kaposi's sarcoma are treated with vinblastine or tenoposide, but early staged Kaposi's sarcoma is being treated with adjuvant azothymidine (AZT). It is contemplated that 6-amino-1,2-benzopyrone or its salts may be formulated with such other standard cytotoxic cancer therapies, as well as with various excipients or carriers, where such other ingredients are pharmaceutically acceptable. For example, an illustrative tablet formation for the oral administration of 6-amino-1,2-benzopyrone or its salts may include microcrystalline cellulose, dicalcium phosphate (dihydrate), stearic acid, magnesium εtearate, silica, flavorings and the like tableting components known to the art.

The dosage level administered in vivo to a human patient will generally range from about 1 to about 100 mg orally per day, preferably about 100 mg orally per day. This dosage is effective in inhibiting tumors, but is believed to be substantially nontoxic to the patient. A two-year baboon study of the 1,2-benzopyrone compound revealed no liver damage when fed at 67.5 mg per kg baboon body weight per day.

The dosage of compositions useful for the present invention may take a variety of forms, such as a solid tablet, or may be formulated with appropriate physiologically acceptable additives as a powder, dispersion or solution. Particularly if used as a solution, the antitumorigenic agent is preferably in salt form to increase solubility, and an agent such as cimetidine (which inhibits cytochrome P-450) may be included to reduce the rate by which the 6-amino-1,2-benzopyrone or salt thereof is metabolized by the liver in the body.

Although the present invention has been described with reference to specific examples, it should be understood that various modifications and variations can be easily made by those skilled in the art without departing from the spirit of the invention. Accordingly, the foregoing disclosure should be interpreted as illustrative only and not to be interpreted in a limiting sense. The present invention is limited only by the scope of the following claims.

Claims

IT IS CLAIMED:

1. A method for inhibiting tumorigenicity in a mammalian host comprising: administering to the host an effective amount of a compound having the formula

or a therapeutically acceptable salt thereof.

2. The method as in claim 1 wherein the administering is by an oral dose.

3. The method as in claim 2 wherein the oral dose includes the compound or salt thereof in sufficient amount to provide about 100 mg of the compound per day.

4. The method as in claim 1 wherein the effective amount is substantially non-toxic to the host.

5. An antitumorigenic agent having the formula

6. The antitumorigenic agent as in claim 5 combined with a pharmaceutically acceptable carrier or excipient.