Feb. 25, 1997

Methods and compositions for inhibiting 5 alpha -reductase activity

INVENTOR: Liao, Shutsung, Chicago, Illinois Liang, Tehming, Centerville, Ohio

ASSIGNEE-AT-ISSUE: Arch Development Corp., Chicago, Illinois (02)

APPL-N0: 442,055

FILED: May 16, 1995

REL-US-DATA: Continuation-in-part of Ser. No. 904,443, Jul. 1, 1992 now patented 5,422,371 Which is a continuation-in-part of Ser. No. 889,589, May 27, 1992 now abandoned

INT-CL: [6] A61K 31#35; C07D 311#04

US-CL: 514#456; 514#544; 549#406; 560#70;

CL: 514;549;560;

SEARCH-FLD: 514#456, 544; 560#70; 549#406

REF-CITED:

U.S. PATENT DOCUMENTS 4,191,759 3/1980 * Johnston et al. 424#242 4,220,775 9/1980 * Rasmusson et al. 221#18 4,268,517 5/1981 * Niebes et al. 514#456 4,394,389 7/1983 * Van't Riet et al. 514#544 4,840,966 6/1989 * Hara et al. 514#456 5,032,514 7/1991 * Anderson et al. 5,126,129 6/1992 * Wiltrout et al. 514#456 5,318,986 6/1994 * Hara et al. 514#456

FOREIGN PATENT DOCUMENTS 0004949 12/1979 * European Patent Office (EPO)

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Liao, et al., Endocrinology 94:1205 (1974). Liao, et al., J. Biol. Chem. 248:6154 (1973). Liao, Int. Rev. Cytology 41:87 (1975). Liao, et al., J. Steroid Biochem. 34:41-51 (1989). Mock, et al., J. Pediatrics 106:762 (1985). Moguilewsky and Bouton, J. Steroid Biochem. 31:699 (1988). Mooradian et al., Endocrine Rev. 8:1 (1987). Morello, et al., Invest. Derm. 66:319 (1976). Morse, et al., Brit. J. Dermatol. (1989) 121:75-90. Munnich, et al., Lancet 2:1080 (1980). Nalboone, et al., Lipids 25:301 (1990). Needleman, et al., Ann. Rev. Biochem. 55:69 (1986). Newman, Proc. Nat'l Acad. Sci. 87:5543-5547 (1990). O'Malley, Mol. Endocrinol. 4:363 (1990). Pattison and Buchanan, Biochem. J. 92:100 (1964). Phillipson, et al., Eng. J. Med. 312-1210 (1985). Pochi, Ann. Rev. Med. 41:187 (1990). Rasmusson, et al., J. Med. Chem. 29:2298 (1986). Rittmaster, et al., J. Androl. 10:259 (1989). Rittmaster, et al., J. Clin. Endocr. Metab. 65:188-193 (1987). Rose and Connolly, The Prostate 18:243-254 (1991). Sansone and Reisner, J. Invest. Dermatol. 56:366 (1971). Schafer and Kragballe, Lipids 26:557-560 (1991). Schweikert and Wilson, Clin. Endocrinol. Metab. 38:811 (1974). Serafini and Lobo, Fert. Steril. 43:74 (1985). Siiteri and Wilson, J. Clinical Invest. 49:1737 (1970). Strauss and Yesalis, Ann. Rev. Med. 42:499 (1991). Strong, et al., Brit. J. Clin. Prac. Nov/Dec:444-445 (1985). Synder, Ann. Rev. MEd. 35:207 (1984). Szepesi, et al., J. Nutr. 119:161 (1989). Tesoriere et al., J. Neurochem. 51:704 (1988). Tosaki and Hearse, Basic Res. Cardiol. 83:158 (1988). Vallette, et al., J. Steroid Biochem. 263:3639 (1988). Vermeulen et al., The Prostate 14:45 (1989). Voigt, et al., J. Biol. Chem. 248:4248 (1973). Wenderoth and George, Endocrinology, 113:569 (1983). Wilson, Am. J. Med. 68:745 (1980). Wright, Prostaglandins, Leukotrienes and Essential Fatty Acids 38:229 (1989). Ziboh and Miller, Ann. Rev. Nutr. 10:433 (1990). Ziboh, ARch Dermatol. 125:241-245 (1989). Zuniga, et al., J. Nutr. 119:152 (1989). Begin, et al., NJCI, 5:1053-1061 (1986). Anderson, et al., Prostaglandins, Leukotrienes and Essential Fatty Acids, 40:137-141 (1990). Liang, et al., J. Biol. Chem. 260:4890-4895 (1985). International Search Report, PCT/US93/04090. Andersson and Russell, "Structural and biochemical properties of cloned and expressed human and rat steroid 5 alpha -reductases," Proc. Natl. Acad. Sci. USA, 87:3640-3644, May, 1990. Andersson et al., "Deletion of steroid 5 alpha -reductase 2 gene in male pseudohermaphroditism," Nature, 354:159-161, Nov. 1991. Andersson et al., "Expression Cloning and Regulation of Steroid 5 alpha -Reductase, an Enzyme Essential for Male Sexual Differentiation, " J. Biol. Chem., 264(27):16249-16255, Sep. 1989. Berman and Rusell, "Cell-type-specific expression of rat steroid 5 alpha PAGE 4 Pat. No. 5605929, *

-reductase isozymes," Proc. Natl. Acad. Sci. USA, 90:9359-9363, Oct. 1993. Faller et al., "Finasteride: A Slow-Binding 5 alpha -Reductase Inhibitor," Biochemistry, 32:5705-5710, 1993. Giovannucci, "Epidemiologic Characteristics of Prostate Cancer," Cancer (Suppl.), 75(7):1766-1777, Apr. 1995. Harris et al., "Identification and selective inhibition of an isozyme of steroid 5 alpha -reductase in human scalp," Proc. Natl. Acad. Sci. USA, 89:10787-10791, Nov. 1992. Hilpakka and Liao, "Androgen Receptors and Action," Endocrinology, 3rd ed., (DeGroot, L. I., ed.) W. B. Saunders Co., Philadelphia, 2336-2351, 1995. Hirsch et al., "LY191704: A selective, nonsteroidal inhibitor of human steroid 5 alpha -reductase type 1," Proc. Natl. Acad. USA, 90:5277-5281, 1993. Honda et al., "Inhibition of Saccharide Digestive Enzymes by Tea Polyphenols," In: Food Phytochemicals for Cancer Prevention II, ACS Symp. Ser. 547:84-89, American Chemical Society, Washington, D.C., 1994. Liang and Liao, "Inhibition of steroid 5 alpha -reductase by specific aliphatic unsaturated fatty acids," Biochem. J., 285:557-562, 1992. Liang et al., "Species Differences in Prostatic Steroid 5 alpha -Reductases of Rat, Dog and Human," Endocrinology, 117(2):571-579, 1985. McConnell et al., "Finasteride, an Inhibitor of 5 alpha -Reductase, Suppresses Prostatic Dihydrotestosterone in Men with Benign Prostatic Hyperplasia," Journal of Clinical Endocrinology and Metabolism, 74(3):505-508, 1992. Moore and Pizza, "Observations on the inhibition of HIV-1 reverse transcriptase by catechins," Biochem. J., 288:717-719, 1992. Rittmaster, "Finasteride," The New England Journal of Medicine, 330(2):120-125, Jan. 1994. Russell and Wilson, "Steroid 5 alpha -Reductase: Two Genes/Two Enzymes," Annu. Rev. Biochem., 63:25-61, 1994. Wynder et al., "Nutrition and Prostate Cancer: A Proposal for Dietary Intervention," Nutrition and Cancer, 22(1):1-10, 1994. Yang and Wang, "Tea and Cancer," Journal of the National Cancer Institute, 85(13):1038-1049, Jul. 1993.

PRIM-EXMR: Shah, Mukund J.

ASST-EXMR: Sripada, Pavanaram K.

ABST: Disclosed are a novel class of antiandrogenic compounds including saturated and unsaturated fatty acids, catechin gallates, their derivatives, and synthetic analogs, their method of synthesis, and their use in treating disorders associated with androgenic activities. Also disclosed is the use of known compounds not previously known for their antiandrogenic activity in treating disorders related to androgenic activities and cancers.

NO-OF-CLAIMS: 8

EXMPL-CLAIM: <=11> 1

NO-OF-FIGURES: 42

NO-DRWNG-PP: 37

GOVT-INT: The U.S. government owns certain rights in the present invention pursuant to grant DK41670 from the National Institutes of Health. PAGE 5 Pat. No. 5605929, *

PARCASE: The present invention is a continuation-in-part of U.S. Ser. No. 07/904,443, filed Jul. 1, 1992, now U.S. Pat. No. 5,422,371, which is a continuation-in-part application of U.S. Ser. No. 07/889,589 filed May 27, 1992, now abandoned; the entire text and figures of which disclosures are specifically incorporated herein by reference without disclaimer.

SUM: BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compounds, compositions and methods regulating the actions of androgens and other steroid hormones by modulating the activity of 5 alpha -reductase. More particularly, the present invention relates to the use of these compounds to treat disorders that are caused by abnormal androgen action in cells or organs. This invention also deals with the use of natural and synthetic fatty acids and catechins, especially polyunsaturated fatty acids and their derivatives and epigallocatechin gallates, as 5 alpha -reductase inhibitors and as therapeutic agents.

2. Description of the Related Art

Uses of androgens known to the medical arts include, for example, treatment of hypogonadism and anemia (Synder, 1984; Mooradian et al., 1987). The abuse of androgen among athletes to enhance performance is well known (Strauss and Yesalis, 1991). Androgens are also known to promote the development of benign prostatic hyperplasia (BPH) (Wilson, 1980), prostate cancer (Huggins and Hodges, 1940), baldness (Hamilton, 1942), acne (Pochi, 1990), hirsutism, and seborrhea (Hammerstein et al., 1983; Moguilewslcy and Bouton, 1988). Approximately 70% of males in the U.S. over the age of 50 have pathological evidence of BPH (Carter and Coffey, 1990). Prostate cancer is the second leading cause of cancer death in males in the U.S. (Silverberg and Lubera, 1990; Gittes, 1991). Male-patterned baldness can start as early as the teens in genetically susceptible males, and it has been estimated to be present in 30% of Caucasian males at age 30, 40% of Caucasian males at age 40, and 50% of Caucasian males at age 50. Acne is the most common skin disorder treated by physicians (Pochi, 1990) and affects at least 85% of teenagers. In women, hirsutism is one of the hallmarks of excessive androgen action (Ehrmann and Rosenfield, 1990). The ovaries and the adrenals are the major sources of androgen in women.

1. Differential Actions of Testosterone and 5 alpha -Dihydrotestosterone (5 alpha -DHT)

In men, the major androgen circulating in the blood is testosterone. About 98% of the testosterone in blood is bound to serum proteins (high affinity binding to sex-steroid binding globulin and low affinity binding to albumin), with only 1-2% in free form (Liao and Fang, 1969). The albumin-bound testosterone, the binding of which is readily reversible, and the free form are considered to be bioavailable, and account for about 50% of total testosterone. Testosterone enters target cells apparently by diffusion. In the prostate, seminal vesicles, skin, and some other target organs it is converted by a NADPH-dependent 5 alpha -reductase to a more active metabolite, 5 alpha -DHT. 5 alpha -DHT then binds to androgen receptor (AR) in target organs (Anderson and Liao, 1968; Bruchovsky and Wilson, 1968; Liao, 1975). The 5 alpha PAGE 6 Pat. No. 5605929, *

-DHT-receptor complexes interact with specific portions of the genome to regulate gene activities (Liao et al., 1989). Testosterone appears to bind to the same AR, but it has a lower affinity than 5 alpha -DHT. In tissues such as muscle and testes, where 5 alpha -reductase activity is low, testosterone may be the more active androgen.

The difference between testosterone and 5 alpha -DHT activity in different androgen-responsive tissues is further suggested by findings in patients with 5 alpha -reductase deficiency. Males with 5 alpha -reductase deficiency are born with female-like external genitalia. When they reach puberty, their plasma levels of testosterone are normal or slightly elevated. Their muscle growth accelerates, the penis enlarges, voice deepens, and libido toward females develops. However, their prostates remain non-palpable, they have reduced body hair, and they do not develop acne or baldness. Females with 5 alpha -reductase deficiency do not have clinical symptoms (Imperato-McGinley, 1986).

The findings in 5 alpha -reductase deficient patients suggest that inhibitors of 5 alpha -reductase would be useful for the treatment of prostatic cancer, BPH, acne, baldness, and female hirsutism. Clinical observations and animal experiments have indicated that spermatogenesis, maintenance of libido, sexual behavior, and feed-back inhibition of gonadotropin secretion do not require the conversion of testosterone to 5 alpha -DHT (Brooks et al., 1982; Blohm et al., 1986; George et al., 1989). This is in contrast to other hormonal therapies which abolish the actions of both testosterone and 5 alpha -DHT.

Treatments of androgen-dependent skin and prostatic diseases by 5 alpha -reductase inhibitors would be expected to produce fewer side effects than the presently available hormonal therapies. These include castration, estrogen therapy, high doses of superactive gonadotropin-releasing hormone such as Luprolide, and the use of competitive antiandrogens which inhibit AR binding of testosterone and 5 alpha -DHT, such as flutamide, cyproterone acetate and spironolactone. The long term efficacy of 'competitive antiandrogens' is also compromised by their block of the androgenic feedback inhibition of gonadotropin secretion. This results in elevated gonadotropin secretion, which in turn increases testicular secretion of testosterone. The higher level of testosterone eventually overcomes the action of the antiandrogen.

2. Biological Importance of 5 alpha -Reductase

Excessive 5 alpha -DHT is implicated in certain androgen-dependent pathological conditions including BPH, acne, male-pattern baldness, and female idiopathic hirsutism. It has been shown that 5 alpha -reductase activity and the 5 alpha -DHT level are higher in the presence of BPH prostates than that of the patients with normal prostates (Isaacs, 1983; Siiteri and Wilson, 1970). 5 alpha -reductase activity is reported to be higher in hair follicles from the scalp of balding men than that of nonbalding men (Schweikert and Wilson, 1974).

3. Steroidal 5 alpha -Reductase Inhibitors

The most potent inhibitors of 5 alpha -reductase developed so far are steroids or their derivatives. Among these the 4-azasteroidal compounds (Merck Co.) are the most extensively studied (Liang et al., 1983; Rasmusson et al., 1986). These inhibitors are 3-oxo-4-aza-5 alpha -steroids with a bulky functional group at the 17 beta -position, and act by reversibly competing with testosterone for the binding site on the enzyme. PAGE 7 Pat. No. 5605929, *

The A-ring conformation of these compounds is thought to be similar to the presumed 3-enol transition state of the 5 alpha -reduction of 3-oxo- DELTA <4> -steroids. A prototype for 5 alpha -reductase inhibitors is 17 beta -N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha -androstan-3-one (4-MA), which behaves as an inhibitor of 5 alpha -reductase in vivo, decreasing the prostatic concentration of 5 alpha -DHT in intact male rats or in castrated male rats given testosterone propionate. 4-MA attenuated the growth of the prostate of castrated rats induced by testosterone, but had much less of an effect in rats given 5 alpha -DHT (Brooks et al., 1981).

When dogs are treated with 4-MA, the prostate size decreases (Brooks et al., 1982; Wenderoth and George, 1983). Topical applications of 4-MA to the scalp of the stamptail macaque, a primate model of human male pattern baldness, also prevented the baldness which normally occurs at puberty in these monkeys (Rittmaster et al., 1987). These results also suggest that the growth of the prostate in rats and dogs, and baldness in the stamptail macaque depend on 5 alpha -DHT. On the other hand, studies in rat pituitary cultures showed that complete inhibition of testosterone conversion to 5 alpha -DHT by 4-MA did not affect testosterone inhibition of LH release, indicating direct action of testosterone in this system (Liang et al., 1984).

Another potent inhibitor is Proscar TM (Merck Co.) (Finasteride, MK-906, or 17 beta -N-t-butylcarbamoyl-4-aza-5 alpha -androst-1-en-3-one). The inhibitor has no significant affinity for the rat prostate AR. In clinical trials, Proscar TM decreases the plasma level of 5 alpha -DHT and the size of the prostate and also improves urinary flow in patients with benign prostatic hyperplasia (Vermeulen et at., 1989; Rittmaster et al., 1989; Gormley et al., 1990; Imperato-McGinley et al., 1990). In stamptail macaque monkeys, Proscar TM administered orally at 0.5 mg/day, alone or in combination with topical 2% Minoxidil TM , reduced serum 5 alpha -DHT level, and reversed the balding process by enhancing hair regrowth by topical Minoxidil TM (Diani et al., 1992). The effects of Minoxidil TM and Proscar TM were additive.

Among other steroidal compounds shown to inhibit 5 alpha -reductase are 4-androstane-3-one-17 beta -carboxylic acid (Voigt et al., 1985), 4-diazo-21-hydroxymethyl-pregnane-3-one (Blohm et al., 1989), and 3-carboxy A-ring aryl steroids (Brandt et at., 1990).

4. Biological and Biochemical Effects of Fatty Acids and Lipids

Since treatments of androgen-dependent skin and prostatic diseases by 5 alpha -reductase inhibitors can produce fewer side effects than the hormonal therapies which indiscriminately inhibit all androgen actions, it is desirable to provide different types of 5 alpha -reductase inhibitors.

Several membrane-associated enzymes (e.g., 5'-nucleotidase, acetyl CoA carboxylase) have been shown to be affected by the polyunsaturated fatty acid content of dietary fat, and to alter the physicochemical properties of cellular membranes (Zuniga et al., 1989; Szepsesi et al., 1989). Various types of phospholipases in rat ventricular myocytes are modulated differentially by different unsaturated fatty acids in the culture media (Nalboone et al., 1990). In addition, treatment of cerebral cortical slices (Baba et al., 1984) or intact retina (Tesoriere et al., 1988) with unsaturated fatty acids can enhance adenyl cyclase activities. PAGE 8 Pat. No. 5605929, *

Few studies have been directed to the elucidation of the mode of action of free fatty acids on enzymes in cell-free systems. Certain cis-unsaturated fatty acids, at 50 mu M, were shown to stimulate protein kinase C activity (Dell and Severson, 1989; Khan et al., 1991) and to inhibit steroid binding to receptors for androgens, estrogens, glucocorticoids, and progestins (Vallette et al., 1988; Kato, 1989). It has not been shown that unsaturated fatty acids can affect steroid receptor binding of steroid hormones in vivo in an animal or human.

SUMMARY OF THE INVENTION

The present invention relates generally to the utilization of certain long chain fatty acids and catechins including derivatives of these classes of compounds for the control of androgen activity in target organs and cells through the modulation of 5 alpha -reductase activity. In certain aspects, particular fatty acids and catechin compounds are employed to repress androgenic activity by inhibiting the formation and availability of active androgen in target cells. Consequently, the invention is useful for the treatment of a wide variety of conditions including, but not limited to, the treatment of prostatic hyperplasia, prostatic cancer, hirsutism, acne, male pattern baldness, seborrhea, and other diseases related to androgen hyperactivity. Several of these compounds have been shown to effectively decrease body weight and, in some cases, to decrease the weight of an androgen-dependent body organ, such as the prostate and other organs. The effectiveness of these compounds may be dependent also on their action on other mechanisms involved in angiogenesis, cell-cell interaction, and on their interaction with various components of organs or cells.

Compounds useful in the practice of the present invention include various isomers of saturated and unsaturated fatty acids, natural and synthetic analogues, and derivatives from which these fatty acids can be generated as well as the metabolite and oxidation products of these fatty acids. The use of these and other fatty acids and their derivatives is also contemplated. Also useful are catechin compounds, particularly, catechins that are structurally similar to epicatechin gallate (ECG) and epigallocatechin gallate (EGCG). EGCG has an additional hydroxyl group on the epicatechin gallate molecule which has been found to be surprisingly active in modulating several 5 alpha -reductase mediated processes. EGCG derivatives having such an additional OH group on the ECG molecule were shown to be active in inducing body weight loss and particularly in reducing the size of androgen sensitive organs such as preputial glands, ventral prostate, dorsolateral prostate, coagulating glands, seminal vesicles, human prostate tumors, and breast tumors in nude mice.

The inventors have discovered the importance of certain structural features of some catechin compounds which appear to contribute to activity toward 5 alpha -reductase. The presence of an additional hydroxyl group in gallocatechin gallate as compared with catechin gallate has a significant effect on activity as reflected in the ability to reduce body and organ weight and tumor growth in animals. The structural requirements for activity therefore are EGCG which has one extra -OH group on the ECG molecule was considerably more active than ECG in inducing body weight loss, and in reducing the sizes of preputial gland, ventral prostate, dorsolateral prostate, coagulating glands, seminal vesicles, and tumors of the prostate and breast. PAGE 9 Pat. No. 5605929, *

The general formula for 5 alpha -reductase inhibitors is as shown: [See Original Patent for Chemical Structure Diagram] or [See Original Patent for Chemical Structure Diagram]

m, n, and p can be 0 or 1. R1, R2, and R5, can have 0 to 6 atom chain consisting of C, N, S or O.

Each of the atoms in the chain can have a substitution of -H, -OH, -CH3, -OCH3, -OC2H5, -CF3, -CHF2, -SH, -NH2, halogen,=O, -CH(CH3)2 or -C(CH3)3.

Atoms in R5 is connected to atoms in R1 and R2. R3 or R4 can be: -H, -OH, -CH3, -OCH3, -OC2H5, -CF3, -CHF2, -SH, -NH2, halogen,=O, -CH(CH3)2, or -C(CH3)3, or the following groups: [See Original Patent for Chemical Structure Diagram]

R6 to R10 can be: -H, -OH, -CH3, -OC2H5, -CF3, -CHF2, -SH, -NH2, halogen,=O, -CH(CH3)2, -C(CH3)3, galloyl, or gallolyl groups.

Carbon-carbon linkages in R1 to R11 may be saturated or have double bonds.

For example, the following compound has been found to be potent inhibitor of 5 alpha -reductase: [See Original Patent for Chemical Structure Diagram]

The active compounds may include ester linkages that may be hydrolyzed to the active unsaturated fatty acids, catechins, or the structure shown. In addition, R1 and R2 need not be individual substituents, but may represent together aromatic or heterocyclic moieties and containing halogen or alkyl substituents. Alternatively, R1/R2 may represent alicyclic moieties with one or more isolated double bonds. Combining all of the information obtained, the structures shown above comprise a group of novel 5 alpha -reductase inhibitors.

For catechin gallates and their derivatives, the following general structure is noted: [See Original Patent for Chemical Structure Diagram]

The fatty acid and catechin compounds are believed to effect the transformation of androgens by inhibition of 5 alpha -reductase and, as a result, (a) limit the supply of dihydroxytesterone (5 alpha -DHT) to target organs and suppress the 5 alpha -DHT dependent androgen actions, and/or (b) prevent the metabolic loss of testosterone or other androgenic precursors of 5 alpha -DHT and promote or maintain hormone actions that are dependent on testosterone or other 5 alpha -DHT precursors. These compounds may act by controlling organogenesis, angiogenesis and/or cellular interaction with other chemical agents.

Steroids other than testosterone or dihydroxytestosterone are also substrates of 5 alpha -reductase. It is expected, therefore, that the fatty acid and catechin compounds disclosed herein will also regulate the transformation and PAGE 10 Pat. No. 5605929, *

activation of other 3-oxodelta<4> -steroids and therefore control the biological functions of other steroid hormones through the same mechanism. An advantage to the use of fatty acid and catechin compounds of the present invention, and particularly some of their derivatives, is their relative stability in vivo and in vitro. In general, one may prepare derivatives that are not easily metabolized, degraded or incorporated into lipid structures or other derivatives. Stability may, for example, be increased by alkylation, cyclization, fluorination, etc. One will of course not wish to prepare derivatives that interfere with the functional aspects of the fatty acid or catechin. Certain of the fatty acid and catechin compounds are particularly effective in exhibiting organ specificity without significant side effects and in such cases one would choose to prepare derivatives that would not significantly increase side effects.

As discussed, there are certain structural features of the catechins that contribute to their utility and effectiveness in particular uses, such as inhibition of sebum production. This appears also to be true for the selection of fatty acids that are active inhibitors of 5 alpha -reductase. As shown in Tables 1 and 2, a relatively large number of polyunsaturated fatty acids inhibit 5 alpha -reductase activity. While the glycerides, esters, nitriles and chlorides showed little activity in the cell binding assays, some of these compounds are likely to be hydrolyzed outside the cells, or hydrolyzed after entering cells, to form the free fatty acid. In comparison with free fatty acids, it may be desirable to administer free fatty acids as glycerides or other derivatives that are relatively more stable to oxidation and/or are less readily metabolized than the free acids. Such derivatives are, of course, considered to be therapeutically active compounds.

In general, the inventors have observed that where fatty acid compounds are employed for inhibition of 5 alpha -reductase activity, the length of the fatty acid carbon chain, as well as the position and number of double bonds in the molecules, appear to relate to activity. The highest activities are observed with 14 or more carbon atoms and at least one, preferable two or more, double bonds. The effectiveness of the unsaturated fatty acids is dependent on the positions of double bonds in the carbon chains.

In addition to certain fatty acids, it has been shown that gallates of catechins and gallocatechins are effective 5 alpha -reductase inhibitors. This class of inhibitors includes a relatively large group of related compounds, some of which have been isolated and identified. These compounds are found in several types of plant bark and leaves, particularly tea and, most particularly, in green tea. Catechins with galloyl substitution showed surprising activity as inhibitors of 5 alpha -reductase. These compounds include catechin gallate (CG), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), the optical isomers, and conjugated substances such as theaflavins and theaflavin mono- (or di-) gallates. The latter compounds are components of fermented teas including black tea.

The inventors determined that active catechin gallates have three distinct groups in their molecules: (a) a 3-flavonol substituent; (b) a 3',4',5'-trihydroxybenzen (gallolyl) group attached to the 2-position of the flavonol; and (c) a gallic acid that forms an ester linkage (galloyl) with the 3-OH of the flavonol. The three groups may independently contribute to the inhibitory action, but the effect on 5 alpha -reductase appears to be synergistic. Certain synthetic gallate derivatives (such as methyl gallate and PAGE 11 Pat. No. 5605929, *

n-propyl gallate, 3,4,5-trihydroxybenzamide, gallic acid and pyrogallol) were not as active as catechin gallate, indicating that the gallolyl or galloyl structure alone was not sufficient for high inhibitory activity. A low inhibitory activity was found within octyl gallate indicating that for the inhibitory activity, the flavonol group of catechin gallates may be replaced by other groups having similar geometric structures. Based on the lower activities of catechin or epicatechin compared with their gallate derivatives, it appears that the essential structural feature required for high 5 alpha -reductase inhibition is an acyl (galloyl) or a trihydroxybenzen group that forms an ester or ether linkage with the flavonol.

By analogy with the fatty acid compounds, the inventors expect that certain active catechin gallates may not enter target cells easily. However, esterification of hydroxyl groups on the inhibitory compounds should enhance the ability of these compounds to enter the target cells. Once inside the cells, esters would be readily hydrolyzed by esterases to alcohols (e.g., epigallocatechin gallate) that can inhibit 5 alpha -reductases (Williams, 1985).

In another aspect of the invention, gamma -linolenic acid was found to be a particularly potent 5 alpha -reductase inhibitor. The ability of gamma -linolenic acid to inhibit 5 alpha -reductase in solubilized microsomes indicates that the gamma -linolenic acid inhibition may not be rigidly dependent on the native source of endoplasmic reticulum membranes. The fatty acid inhibitor may act by interacting with the reductase and/or other components that are vital for reductase activity. The inhibitory fatty acids may also interact with and potentiate other endogenous inhibitors or interfere with lipids which may potentiate the reductase. A proposed mechanism (Brandt et al., 1990) for the 5 alpha -reductase (E) reaction includes the following steps: [See Original Patent for Chemical Structure Diagram]

It was surprising that two trans isomers of fatty acids, i.e., elaidic acid and linolelaidic acid, had little inhibitory activity in the [<3> H]4-MA binding assay, yet were as potent as their cis-isomers, oleic and linoleic acid, in the enzymatic assay. The cis-unsaturated fatty acids may inhibit the formation of [NADPH-E-T] (step a); whereas the trans isomers act at points after the formation of the ternary complex (step b).

In certain embodiments the disclosed methods are useful for reducing weight in an androgen dependent organ. The inventors have demonstrated that certain fatty acids and catechins are effective in reducing the weight of androgen dependent organs, including the preputial gland, ventral prostate, dorsolateral prostate, seminal vesicles, coagulating gland, and at high doses, also the testes. This effect was observed with several fatty acids. The most effective correlated with those that showed the most inhibitory activity toward 5 alpha -reductase. In a preferred embodiment, gamma -linolenic acid was shown to be particularly effective in reducing the weight of androgen dependent organs; in particular, the ventral, prostate and preputial organ. It is evident that a relatively broad range of long chain polyunsaturated fatty acids will have the desired effect in reducing the weight of androgen dependent organs. One will select such fatty acids based on, for example, in vivo stability, ease of administration, and release in active form. Certain ester or ether derivatives are expected to be hydrolyzed by cell esterases to an active form; for example, glycerides. A particularly preferred long chain polyunsaturated fatty acid is PAGE 12 Pat. No. 5605929, *

gamma -linolenic acid. This fatty acid, as well as related derivatives and compounds, are particularly effective. Contemplated derivatives are esters, particularly hydrolyzable esters.

The invention also includes the inhibition of 5 alpha -reductase in cells by contacting the cells with a composition comprising at least one catechin compound. Several catechins including ( - )epicatechin gallate (ECG) and ( - )epigallocatechin gallate (EGCG) reduced the weight of the androgen dependent organs, ventral prostate and preputial organ; however, EGCG also reduced body weight by as much as 35% in some case, suggesting a potential use of this compound and related species as weight loss agents. EGCG would be ideal for weight loss programs because of its lack of toxicity or apparent side effects. EGCG and related catechins occur naturally in several types of plants, including tea, and thus have a long history of safety as a component of a food item.

EGCG, EGC and gamma -linolenic acid are particular examples of catechins and fatty acids that reduce weight of androgen-sensitive organs. The inventors believe that these compounds reduce lipid or sebum production in male hormone sensitive organs, for example, in ventral and dorsal lateral prostate glands, coagulating glands, and seminal vesicles. EGCG and ECG are structurally similar in that EGCG has eight hydroxyl groups compared with the seven hydroxyl groups in EGC, yet EGCGC is significantly more effective than ECG in promoting weight reduction. The effect of EGCG on lipid production or organ weights may be dependent on a specific EGCG interaction with a macromolecule that is specific for EGCG on the modulation of cell-cell or protein-protein interactions, or regulation of enzyme activity or gene expression. Regulation or modulation of the interaction or the function of the EGCG receptor or protein complex by natural or synthetic compounds would be expected to offer a means to control the lipid synthesis or the growth and function of androgen-sensitive organs.

In more particular aspects of the invention, the inventors have discovered that certain catechins, particularly EGCG, can be administered to promote body weight loss that differentially affects overall body weight and prostate weight loss. In particular examples, it was shown that for a certain percentage of overall body weight loss, prostate weight loss was percentage-wise more than three times as much. The loss in body weight and the organ weight are likely due to EGCG interference of a common step in the pathway controlling body and organ weight gain. EGCG and related compounds may interact and interfere with a receptor macromolecule (probably containing a protein) that modulates specific lipid synthesis or accumulation. Lipids can modulate gene expression, cell development and differentiation, and organ growth. Specific interference of lipid metabolism in the cells and organs may control the growth of the organs, in particular, prostate sebaceus organs, preputial organs and other secretory organs. In certain applications, it is expected that benign or abnormal growth or cancer of these organs may be treated or even prevented by administration of catechin related compounds.

It has been demonstrated that catechin compounds will arrest or reduce human prostate and breast cancer cell growth. The effectiveness of catechin compounds was shown by the inventors to be dependent on the methods these compounds were administered to the experimental animals. The inventors found that intraperitoneal application was much more effective than oral route. It is expected that direct application to the prostate having tumor will be very effective. The inventors demonstrated that EGCG was surprisingly effective in suppressing and even reducing the size of human prostate and breast tumors in PAGE 13 Pat. No. 5605929, *

animal models. The effect was illustrated with EGCG; however, structurally similar catechin compounds should also be effective, particularly those that are structurally similar to EGCG in having at least one additional hydroxyl group as compared with EGC. Thus, the EGCG species that contains eight hydroxyl groups is significantly more effective in reducing body weight than is EGC, which contains seven hydroxyl groups. Compounds of this general structure are expected to be particularly effective in chemoprevention and chemotherapy of human prostate cancer. Compounds having a part of structure similar to a part of structure of EGCG are also expected to be effective also.

A useful animal model for skin is the rat model. In rat sebaceous glands, as in human, sebum lipids are synthesized in the intermediate cells by the smooth endoplasmic reticulum (SER). The volume density of SER, as seen under electron microscopic examination, depends on androgen (Moguilewsky and Bouton, 1988). Since repression of androgen action can cause reduction of this density, the effectiveness of test compounds, systemically or topically administered to rats, can be evaluated by measuring their ability to reduce the volume density of SER.

Polyunsaturated fatty acids can be used as antiandrogenic agents through topical or systemic application. A preparation for this purpose can include a carrier, a protectant, an antioxidant (such as vitamin C or E and various catechins and polyphenols), and other pharmaceutical and pharmacological agents. It is also expected that such fatty acids can be used in a delivery system involving molecular recognition through which the said fatty acids are delivered to target sites. Such a delivery system may involve, among other methods, liposome techniques or immunological devices.

Natural or synthetic chemicals that can modulate the production or cellular action of receptors and macromolecules may be useful in the treatment of abnormalities such as obesity, BPH, prostate cancer, skin diseases, baldness, breast tumors, and hirsutism, which are related to lipid synthesis, body weight, and/or androgen function.

The inventors contemplate that animal models may be used to demonstrate the effectiveness of EGCG and related compounds on a variety of cancers. For example, Shionogi tumor and other tumor induced tumors may be studied in male rats. Human breast and prostate cancer cell growth may be studied in nude mice. Alternatively, rodent breast tumors induced by carcinogens and other cancers induced in transgenic mice or Dunning tumors in rats may be similarly analyzed for their chemotherapy by EGCG and related compounds.

Other aspects of the invention include methods for screening inhibitors of sebum production. While other animal models may be used, the inventors have found it convenient to use humans for screening. The method basically involves applying a compound suspected of inhibiting sebum production to some portion of the human body on the skin area that has sebaceus glands. These areas include the human forehead, as well as other areas of faces and hands. Ideally, the applications will cover two bilaterally similar areas, with one area designated a control area and the other a test area. One will then measure sebum production in each of these areas. Several ways of measuring sebum production may be employed; however, a convenient means is to use a clear tape over each area for a specified length of time. This length of time is conveniently 30-40 minutes, but could be shorter or longer; e.g. 10 minutes or 2-3 or more hours. Longer periods of time, however, will result in generally more sebum production and would be employed only in cases where sebum production is low or difficult to PAGE 14 Pat. No. 5605929, *

obtain. The use of a clear tape is particularly convenient because each tape may then be removed from the subject and the amount of sebum deposited on the tape measured or determined by such means as light scattering, decrease in light transmission, etc.

The inventors have found that regardless of the measurement means employed, it is rapid and convenient to assign a relative and arbitrary value for sebum production to each measurement. Use of arbitrary values avoids the necessity of absolute measurements and outside control samples because the control area tape may be used as a relative control. It has been found that when the ratio of the value for the test area to that of control is lower than the ratio before the application of the test compound to the control area, the test compound is a suitable candidate for use in sebum suppression. When identified by this method of screening, compounds that exhibit a lower ratio will be useful as topical agents.

The use of the fatty acid and catechin compounds disclosed in the present invention, in therapeutically effective amounts of pharmaceutical compositions containing one or more of the compounds of the invention, in some cases in combination with other therapeutic agents and carriers, or in natural or synthetic products, is appropriate in the treatment of various disorders. These disorders include, but not necessarily limited to, those conditions wherein excessive androgenic activities have been implicated, for example, male pattern baldness, female hirsutism, acne, BPH, and cancers of prostate, breast, skin and other organs.

These pharmaceutical compositions, comprising certain fatty acids, catechins or compounds of the invention, can be administered by topical or internal routes, including oral, injection, or other means, such as topical creams, lotions, hair tonics, scalp care products, or transdermic patch applications, alone or in combination with other compounds of the invention and or with other drugs, drug additives, or pharmaceutical compounds. Combination of unsaturated fatty acids and catechins will be beneficial for clinical or cosmetic treatments because they individually may selectively control the activities of different enzymes or isozymes, and they may act to stabilize each other or protect active compound from degradation or alteration by chemical, biological or environmental condition during the preparation, application or storage of the compounds or products. It has been demonstrated that some of these compounds appear to regulate steroid metabolism, and may thereby affect the function of normal or mutated hormone receptors. Therefore, these compositions are useful in the treatment of androgen and other hormone-sensitive or insensitive disorders or tumors. The compounds of the invention are also important in the studies of the mechanism of action of hormones and anti-hormones.

As used herein the terms "contact", "contacted", and "contacting", are used to describe the process by which an effective amount of a pharmacological agent, e.g. , an inhibitor of 5 alpha -reductase, comes in direct juxtaposition with the target cell. As used herein the term "cell" refers to cells capable of fatty acid synthesis. What is meant by an effective amount is the amount of drug necessary to give the therapeutically desired level of 5 alpha -reductase inhibition.

Although the present invention has been described primarily in terms of its clinical usefulness, as indicated by the art accepted model of inhibition of sebum production used in the practice of the present invention, the methods PAGE 15 Pat. No. 5605929, *

and compositions herein will also be useful in methods for screening a candidate substance for 5 alpha -reductase stimulatory properties in combination with compositions of the present invention. Such a method would comprise preparation of different isozyme of 5 alpha reductase including isozymes genetically engineered and expressed in cells; obtaining a candidate substance; contacting a culture of sebaceous cells with said candidate substance; simultaneously contacting said culture with a composition of the present invention having 5 alpha -reductase inhibitory activity; and determining the extent of 5 alpha -reductase inhibition. 5 alpha -reductase inhibition using compositions of the present invention may also be utilized in such methods to provide a baseline control for determining the efficacy of a candidate substance, as well as to test such a candidate substance for synergistically enhancing the 5 alpha -reductase inhibitory activity of the compositions disclosed herein. As used herein, a "candidate substance" is defined as any substance or compound, either naturally occurring or synthetic that is suspected to affect 5 alpha -reductase activity.

DRWDESC: BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Schematic representation of the synthesis of compounds 2 to 6 of Example 1.

FIG. 2. Schematic representation of the synthesis of compounds 4 to 9 of Example 1.

FIG. 3. Schematic representation of the synthesis of compounds 12 to 15 of Example 1.

FIG. 4. Schematic representation of the synthesis of compounds 17, and 19 to 21 of Example 1.

FIG. 5. Schematic representation of the synthesis of compounds 23 to 27 of Example 1.

FIG. 6. Schematic representation of the synthesis of compounds 28 and 29 of Example 1.

FIG. 7. Schematic representation of the synthesis of compounds 32 and 34 to 36 of Example 1.

FIG. 8. Schematic representation of the synthesis of compounds 38 and 40 to 42 of Example 1.

FIG. 9. Schematic representation of the synthesis of compounds 44 and 45 of Example 1 from compound 43 of Example 1.

FIG. 10. Schematic representation of the synthesis of compound 47 of Example 1 from compound 46 of Example 1. PAGE 16 Pat. No. 5605929, *

FIG. 11. Structures for compounds 48 to 50 of Example 1 which can be made using the scheme shown in FIG. 8.

FIG. 12. Fractionation of 5 alpha -reductase inhibitors in the microsomal extract of rat liver by Sephadex Registered TM G-50 column chromatography. 5 alpha -reductase was assayed by the [<3> H]4-MA-binding assay (closed circles). The absorbance at 280 nm for each fraction is also shown (open circles). Most of the inhibitory activity was associated with the fractions No. 19 to No. 29.

FIG. 13A. Effects of oleic acid (C18:1, cis-9) (c-9), linoleic acid (C18:2, cis-9,12) (c-9,12), elaidic acid (C18:1, trans-9) (t-9), and linolelaidic acid (C18:2, trans-9,12) (t-9,12) on rat liver microsomal 5 alpha -reductase activity as determined by [<3> H]4-MA binding assay. The abbreviations in parentheses indicate: the number of carbon atoms in the carbon chain, the number and the position of cis or trans double bonds and abbreviation shown in the figures. The amount of rat liver microsomes was 10 mu g protein. In the absence of lipid, the control value for the [<3> H]4-MA binding assay was 30618 +/- 975 dpm. This value was taken as 100% activity.

FIG. 13B. Effects of oleic acid (C18:1, cis-9) (c-9), linoleic acid (C18:2, cis-9,12) (c-9,12), elaidic acid (C18:1, trans-9) (t-9), and linolelaidic acid (C18:2, trans-9,12) (t-9,12) on rat liver microsomal 5 alpha -reductase activity as determined by enzymatic assay. The abbreviations in parentheses are defined in FIG. 13A. The amount of rat liver microsomes was 2 mu g protein. In the absence of lipid, the control value was 9.0 +/- 0.9 nmol 5 alpha -DHT formed/15 min using 0.5 mu M testosterone as substrate. This value was taken as 100% activity.

FIG. 14. Time course of gamma -linolenic acid inhibition of [<3> H]4-MA-binding to rat liver microsomes (5 mu g protein). The concentration of gamma -linolenic acid was 5 mu M.

FIG. 15A. Inhibition of [<3> H]4-MA binding to 5 alpha -reductase in intact rat liver microsomes by gamma -linolenic acid. The [<3> H]4-MA-binding assay was carried out in the absence (control) and presence of 10 mu M of gamma -linolenic acid and varying amounts of microsomal protein.

FIG. 15B. Inhibition of [<3> H]4-MA binding to 5 alpha -reductase in detergent-solubilized rat liver microsomes by gamma -linolenic acid. The [<3> H]4-MA-binding assay was carried out in the absence (control) and presence of 10 mu M of gamma -linolenic acid and varying amounts of microsomal protein.

FIG. 16A. Inhibition of 5 alpha -reductase activity by gamma -linolenic acid at varying concentrations of NADPH. The concentrations of gamma -linolenic acid are shown.

FIG. 16B. Inhibition of 5 alpha -reductase activity by gamma -linolenic acid at varying concentrations of testosterone. The concentrations of gamma -linolenic acid are shown.

FIG. 17. General formula for compounds that are part of the present disclosure. R1, R2, R3, R4, R5 ,R6, R7, R8, or R9 may be a hydrogen, a fluorine or other halogen, or a methyl, ethyl, propyl, other alkyl or aryl group; one or two fluorine or other halogen atom(s) may replace hydrogen attached to any carbon atom(s) and 'l', 'm', 'n', 'p', 'q', 'r', and 't' are each PAGE 17 Pat. No. 5605929, *

independently 0 or an integer from 1 to about 50 and, preferably from 1 to about 30. The alkyl or aryl group and fluorine or other halogens attached to the molecules may protect them from degradation by oxidation of the unsaturated double bonds and alpha , beta or omega oxidation. Oxidation products and metabolites of these fatty acids are also included since they are also expected to regulate 5 alpha -reductase activity. Also -CH and the -OH groups can be in a substituted form (-CR and/or -OR) wherein -R represents an alkyl or an aryl group. Also included are acylates and esters that, upon hydrolysis, can form the carboxylic acid shown. 'X' can be a carbon, a sulfur, an oxygen, or a -NH-. This X-linkage is not limited to link carbon 2 and the carbon at the end of the chain; the link can be between any two carbons in the carbon chain. For protection of a fatty acid from oxidative degradation, it may be useful to incorporate one or two sulfur atoms into the backbone carbon chains. The total carbon chain length can be 6 to 28.

FIG. 18. Fatty acids which can be used to regulate 5 alpha -reductase activity.

FIG. 19. Examples of fluorinated and cyclic derivatives of fatty acids that are part of the present disclosure.

FIG. 20A. General structure of catechin derivatives.

FIG. 20B. Structure of galloyl moiety.

FIG. 21. Structure of important catechins.

FIG. 22. Structure of important catechin gallates.

FIG. 23. Novel classes of 5 alpha -reductase inhibitors. R1 and R2 are alkyl, allyl, or groups having general structures of VII, VIII, IX, X, XI, XII. R3 and R4 are groups having general structures of XI or XII. R5 and R6 are hydrogen or halogen atoms. R7, R8, R9, R10, R11, R12, R13, R14, R15, and R16 are hydrogen, halogen, hydroxyl, methyl, ethyl, methoxyl, acetyl, or acetoxyl group. R is oxygen, nitrogen, or sulfur atom.

FIG. 24. gamma -Linolenic acid inhibition of forehead sebum production in a human male.

FIG. 25. Catechin inhibition of forehead sebum production in a human male.

FIG. 26. Stimulation of hamster flank organ by topical application of testosterone (T) and dihydrotestosterone (5 alpha -DHT). The right flank organs of immature castrated male hamsters (5 each group) were treated topically with 5 mu l/day of ethanol solution alone (C), or ethanol containing 0.5 mu g T or 5 alpha -DHT for 17 days. One representative animal from each group is shown.

FIG. 27. Effect of testosterone (T) stimulation on the application site (right flank organ) versus the contralateral site (left flank organ). The right flank organ of immature castrated male hamsters was treated with T (0.5 mu g/day) for 17 days. The left flank organ was not treated.

FIG. 28. Inhibition of testosterone-stimulated growth of the pigmented macule of the hamster flank organ by gamma -linolenic acid, but not by stearic acid. Male hamsters (4 weeks old) were castrated and treatment was started 2 weeks PAGE 18 Pat. No. 5605929, *

later for 18 days. The animals were treated with 5 mu l of ethanol (C), ethanol containing testosterone (T, 0.5 mu g), T (0.5 mu g) + gamma -linolenic acid (LA, 1 mg), T (0.5 mu g) + stearic acid (SA, 1 mg), or T (0.5 mu g) + SA (2 mg). Only the right flank organ was treated and shown here. The data collected from these animals are shown in Table 7.

FIG. 29. gamma -Linolenic acid applied to the right flank organ of intact male hamster produced a localized inhibition of the growth of the pigmented macule stimulated by endogenous androgens. The right flank organs of intact male hamsters, 4 weeks old, were treated for 156 days as described in Table 7. The treatment consisted of topical solutions of vehicle (ethanol) alone (C) or gamma -LA (1 mg/flank organ/day). The fight flank organs of 2 representative hamsters are shown.

FIG. 30. gamma -Linolenic acid topically applied to the right flank organ of intact hamsters inhibited only the application site and not the contralateral (left) flank organ. A representative intact hamster treated with 1 mg gamma -LA from group 3 of the study described in Table 7 is shown.

FIG. 31. gamma -Linolenic acid reduced the pigmentation and the length of the hair in the flank organ. The hamsters shown here are the same animals shown in Table 7. The picture represents the hair growth on the flank organ during the last two days. The hair of the flank organ of the group treated with 1 mg gamma -LA/5 mu l ethanol/day (A) was markedly lighter in color and shorter in length than the vehicle (C) treated hamsters.

FIG. 32. The effect of gamma -linolenic acid treatment on the growth rate of the pigmented macules from intact male hamsters. The right flank organs of intact prepubertal male hamsters, 4 weeks old, were treated topically with vehicle alone (control), gamma -LA 1 mg or 2 mg/5 mu l ethanol/flank organ/day. Them were 10 animals per treatment group. The index of the area of the pigmented macules was determined at the beginning (Day 0) and after various days of treatment. The left flank organs received vehicle only. The growth rates of the pigmented macules of the left flank organs of all 3 groups were similar to that of the right flank organ of the control group.

FIG. 33. Tumor size suppression by EGCG in nude mice.

FIG. 34. Reduction of tumor size in nude mice following EGCG therapy.

FIG. 35. Effects of ECG and EGCG on hamster ventral prostate size.

FIG. 36. Effects of ECG and EGCG on hamster preputial gland size.

FIG. 37. Effects of EC, ECG, EGC and EGCG on body weight gain in rats.

FIG. 38. Restoration of normal body weight gain following cessation of EGC and EGCG treatment in rats.

DETDESC: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Steroid Hormones and 5 alpha -Reductase Activity PAGE 19 Pat. No. 5605929, *

1. Androgens

Androgens are one of the six major classes of steroid hormones. Steroid hormones form complexes with specific receptor proteins in selective cells of target organs (Jensen et at., 1968; Liao, 1975; Gorski, et al., 1976). Steroid receptors are members of a superfamily of transcription factors that can regulate gene expression, and this function is dependent on the binding of a specific hormonal ligand to an appropriate receptor (Evans, 1989; Beato, 1989; O'Malley, 1990).

Studies of the specificity and affinity of steroid hormones for their receptors have contributed greatly to the understanding of the relationships among steroid and receptor structures and biological activity, target organ specificity, and the mechanism of action of many antihormones, including "competitive antiandrogens". "Competitive antiandrogens" are defined herein as those antiandrogens that interact with receptors and competitively prevent receptor binding of active androgens (Fang and Liao, 1969; Liao et al., 1973; Liao et al., 1974; Chang and Liao, 1987; Liao et al. 1989), although it should be noted that some compounds with an antiandrogenic activity may act by a different mechanism.

Androgens, produced in the testis, stimulate the differentiation of the male reproductive organs, including the penis, scrotum, prostate, seminal vesicles, epididymis, and vas deferens. With the onset of puberty, an increase in the production of androgen promotes the growth of these tissues. Androgen is required for spermatogenesis and accelerates skeletal muscular growth and bone formation. In the central nervous system, it stimulates libido and produces feedback inhibition of gonadotropin secretion. In skin, androgen increases the size of sebaceous glands and apocrine glands and converts villus hairs in the axillae, pubic region, and the beard to form coarser and longer terminal hairs. Androgen causes thickened vocal cords and lowers the pitch of the voice. Androgen also stimulates hematopoiesis.

Androgen action in many organs, such as prostate is dependent on the conversion of testosterone by a NADPH-dependent 5 alpha -reductase to 5 alpha -dihydrotestosterone (5 alpha -DHT), which then binds to AR to exert its biological function (Liao et al., 1989). The inhibition of 5 alpha -reductase limits the availability of 5 alpha -DHT but not testosterone, therefore, 5 alpha -reductase inhibitors are useful in selective treatment of 5 alpha -DHT-dependent abnormalities, such as benign prostate hyperplasia, prostate cancer, hirsutism, male pattern alopecia and acne, without affecting testosterone-dependent testicular function, sexual behavior, and muscle growth (Russell and Wilson, 1994; Hipakka and Liao, 1995). Most 5 alpha -reductase inhibitors are steroids or compounds with steroid-like structures. The present invention, however, also has identified specific fatty acids and catechins, including gamma -linolenic acid and epigallocatechin-3-gallate, which are potent 5 alpha -reductase inhibitors.

It is known that polyunsaturated fatty acids can correct the effects of fatty acid deficiencies that manifest as dermatitis, kidney necrosis, infertility, and cardiovascular diseases (Herold and Kinsella, 1986; Phillipson et al., 1985; Ziboh and Miller, 1990) and also can exhibit anti-tumor activities (Begin, 1990; Karmali et al., 1984). Many unsaturated fatty acids are essential components of mammalian membranes, typically in the acylated form of triglycerides and phospholipids (Lands, 1965). PAGE 20 Pat. No. 5605929, *

Arachidonic acid serves as a specific precursor in the biosynthesis of prostaglandins and leukotrienes (Needleman et al., 1986). These metabolites of unsaturated fatty acids are mediators of inflammation. Unsaturated essential fatty acids have been implicated as dietary factors that influence acne. However, no firm support for this view has developed, and no successful treatment based on this idea has appeared (Downing et al., 1986). Synthetic retinoids and AR binding competitive antiandrogens have been used to obtain therapeutic improvement of acne in some individuals. These anti-acne agents increase the proportion of linoleic acid in sebum in parallel with clinical improvement (Wright, 1989).

2. 5 alpha -reductase

Selective inhibitors of the different types of 5 alpha -reductase, therefore, are desirable for studies of androgen action and for therapy for androgen-dependent tumors and other abnormalities.

Two 5 alpha -reductase isozymes have been demonstrated in rats and humans. In the human, type 1 and 2 isozymes have only 50% amino acid sequence homology (Anderson et al., 1991). Type 1 isozyme has a neutral to basic pH optimum and is rather insensitive to the 5 alpha -reductase inhibitor finasteride. Type 2 isozyme has an acidic pH optimum and is 30 times more sensitive to finasteride inhibition than type 1 isozyme. In the prostate, type 2 isozyme is the major form (Anderson et al., 1991), whereas, in the scalp, type 1 isozyme predominates (Harris et al., 1992). In the rat, it has been shown that the liver contains mainly type 1 isozyme (Berman and Russell, 1993); however, the prostate contains both type 1 (60%) and type 2 (40%) isozymes (Normington and Russell, 1992). gamma -LA was found to inhibit 5 alpha -reductase activity in both the liver and prostate (Liang and Liao, 1992). gamma -LA therefore, is an inhibitor of both type 1 and type 2 isozymes. 5 alpha -reductase isozymes in the hamster flank organ have not been characterized at the molecular level. However, 5 alpha -reductase activity in hamster flank has an optimum of pH 8 (Takayasu and Adachi, 1972), indicating that the major 5 alpha -reductase isozyme in the flank organ may be type 1, rather than the type 2 isozyme.

In a given individual, 5 alpha -reductase activity is found to be higher in balding skin than from hairy skin (Bingham and Shaw, 1973). Some idiopathic hirsute women have a normal circulating level of testosterone, but their affected skin has a higher 5 alpha -reductase activity than that of nonhirsute women (Serafini and Lobo, 1985). An increased 5 alpha -reductase activity has also been reported for skin with acne (Sansone and Reisner, 1971).

Genetic evidence also supports the suggestion that 5 alpha -DHT plays an important role in the development of BPH and the above skin conditions. In males with hereditary 5 alpha -reductase deficiency, their prostates remain small or nonpalpable after puberty. They do not develop acne, temporal hairline recession, or baldness. Compared to their fathers and brothers, they have scanty beards and reduced body hair.

B. FATTY ACID METABOLISM

Fatty acids fluorinated at alpha , beta , and omega positions (Gershan and Parmegiani, 1967; Pattison and Buchanan, 1964; Gent and Ho, 1978) and omega -oleic acids (Tosaki and Hearse, 1988) have been identified in plants and microorganisms, and have been chemically synthesized. Many of these PAGE 21 Pat. No. 5605929, *

fluorinated acids are toxic. Degradation of some fluorinated fatty acids can yield fluoro-acetic acid, which can be incorporated into fluorocitrate and can then block aconitase action. This can cause inhibition of the citric acid cycle and cellular energy production (Hall, 1972). Fluorinated fatty acids are often useful in the studies of fatty acid degradation, metabolism and transport in biological systems (Stoll et al., 1991), and biophysical studies of protein-lipid interaction and membranes functions (Gent et al., 1981).

Biotin is a cofactor of major carboxylases which are necessary for orderly production and metabolism of fatty acids. Alopecia caused by biotin-deficiency can be completely treated by biotin administration to patients. Oral administration and cutaneous application of unsaturated fatty acids can also improve biotin-dependent dermatological conditions including scalp hair growth (Munnich et al., 1980; Mock et al., 1985). The fatty acid effect is apparently due to supplementation of the deficient fatty acids and not related to regulation of androgen action involved in male pattern-alopecia.

C. PHARMACEUTICAL COMPOSITIONS

Aqueous compositions of the present invention comprise an effective amount of the 5 alpha -reductase inhibitory agent dissolved or dispersed in a pharmaceutically acceptable aqueous medium. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.

The preparation of an aqueous composition that contains such an inhibitory compound as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

The pharmaceutical compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be formulated for control release, such as transdermic and osmotic pressure devices, injectable devices and implantable devices, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the compositions and preparations may, of course, be varied and may conveniently be 100% (application of pure compounds). The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or PAGE 22 Pat. No. 5605929, *

both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus ny additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. PAGE 23 Pat. No. 5605929, *

For oral administration the composition may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The composition can be formulated in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

In other embodiments, one may desire a topical application of compositions disclosed herein. Such compositions may be formulated in creams, lotions, solutions, or in solid form depending upon the particular application. The formulation of pharmaceutically acceptable vehicles for topical administration is well known of skill in the art (see i.e., "Remington's Pharmaceuticals Sciences" 15th edition). Variation of the dosage of the compositions disclosed herein, will necessarily depend upon the particular subject, and the nature of the condition(s) being treated.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

D. ASSAYS FOR CANDIDATE SUBSTANCES

In still further embodiments, the present invention concerns a method for identifying new agents that act to inhibit the activity of 5 alpha -reductase. Those new agents may be termed as "candidate substances." Different types of 5 PAGE 24 Pat. No. 5605929, *

alpha -reductase isozymes have been found to be present in different combinations in different cells of various organs (Russel and Wilson, 1994). Therefore, it is desirable to have isozyme-selective inhibitors for therapeutic purposes. For the sources of type 1 and type 2 5 alpha -reductase Rat 1A cells were genetically engineered to certain only type 1 or type 2 isozyme. Rat 1A cells or microsomes were used for the screening of isozyme-selective inhibitors. It is contemplated that this screening technique will prove useful in the general identification of any compound that will serve the purpose of inhibiting the activity of 5 alpha -reductase or specific types of 5 alpha -reductase. It is further contemplated that useful compounds in this regard will in no way be limited to the specific compositions disclosed herein, but any analogs, derivatives, synthetic modifications, or substitutions of constituents of those compositions which can effectively inhibit this activity either in vitro or in vivo.

Accordingly, in screening assays to identify pharmaceutical agents which inhibit 5 alpha -reductase activity, it is proposed that compounds isolated from natural sources such as plants, animals or even sources such as marine, forest or soil samples, may be assayed for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived from chemical compositions or man-made compounds.

The active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.

E. METHODS OF INHIBITING 5 alpha -REDUCTASE ACTIVITY

In still further embodiments, the present invention is concerned with a method of inhibiting 5 alpha -reductase which includes subjecting a cell to an effective concentration of a 5 alpha -reductase inhibitor such as one of the family of fatty acid or ECGC compounds disclosed herein, or with a candidate substance identified in accordance with the candidate screening assay embodiments. This is, of course, an important aspect of the invention in that it is believed that by inhibiting the activity of 5 alpha -reductase, one will be enabled to treat various aspects of disease and cancers, such as prostate-related cancers and diseases caused by abnormal androgen actions. It is believed that the use of such inhibitors to block abnormal androgen action will serve to treat cancers and diseases and may be useful by themselves or in conjunction with other anti-cancer therapies, including chemotherapy, resection, radiation therapy, and the like. The compounds of this invention, besides acting as 5 alpha -reductase inhibitor, may have other effects that can lead to antitumor activity or to suppress abnormal growth of prostate or other organs.

The following examples illustrate the rationale and practice of the invention. Although many of the examples are based on the actions of androgens and ARs, they may also apply to the function of other steroid hormones which is dependent on or regulated by 5 alpha -reductase or their isozymes. They are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can PAGE 25 Pat. No. 5605929, *

be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Synthesis of Fatty Acid Analogs and Related Molecules

1. Synthesis of beta -Fluoro Fatty Acids

The synthesis of beta -fluoro acid analogs of linolenic acid is a relatively straightforward process. Starting with the appropriate 16-carbon acid 1 (FIG. 1), aldehyde 2 can be obtained through reduction using isopentyl boron hydride beta -Hydroxy acetate 3 can be made from 2 using zinc and ethyl bromoacetate in a Reformatski-type reaction. The beta -keto ester 4 can be made from 3 using pyridinium dichromate in dichloromethane at room temperature. The difluoro ester 5 can be made from 4 using diethylaminosulfur trifluoride (DAST) in methylene chloride at room temperature. DAST is a fluorination reagent which is very selective for aldehydes, ketones and alcohols at room temperature. The free acid 6 is obtained via base hydrolysis of the ester group.

Alternatively, one could produce the acid chloride 7 from 1 by reaction with thionyl chloride (FIG. 2). Compound 8 can be made by reaction of 7 with Meldrum's acid, which is the product from the reaction of malonic acid with acetone. Compound 8 undergoes ring opening and decarboxylation to form beta -keto acid 9. From the beta -keto acid 9 the sequence continues to produce compounds 4, 5, and the target compound 6 as previously described above.

2. Synthesis of 6-Membered Ring Linolenic Acid Mimetics

Since the two key groups present in linolenic acid are 1) three cis double bonds and 2) the carboxylic acid group, a molecule can be designed that possess the same key groups and retains the activity of linolenic acid. Examples of such compounds are 15 and 21, the synthetic routes to which are shown in FIG. 3 and FIG. 4.

Starting from 1,3-hexanedione 10 one can produce compound 12 by forming the enolate anion of 10 followed by reaction with ethyl 6-bromocaproate 11. Reduction using sodium borohydride then gives compound 13. Reaction of 13 with trifluoromethylsulfonic anhydride gives the ditrifluoromethylsufonate (ditriflate) 14. Compound 14 can be trans/brined into the target linolenic acid mimic 15 by double elimination of triflate using 1,5-diazabicyclo[5.4.0]undec-5-ene (DBU). The free acid can be obtained or it can be formed in vivo by enzymatic hydrolysis of the ester group.

Another linolenic acid mimetic is compound 21, the synthesis of which is shown in FIG. 4. Starting from cyclohexanone 16, one can obtain the dibromoketone 17 through reaction with pyridinium bromide perbromide. Reaction of 17 with the Grignard reagent 18 gives the dibromohydroxyl ester 19. Fluorination of 19 at room temperature using DAST gives compound 20, which when stirred with DBU at room temperature undergoes double elimination of HBr to give the target linolenic acid mimetic 21. Again the free acid can be obtained by chemically removing the ester function or by enzymatic hydrolysis in vivo. PAGE 26 Pat. No. 5605929, *

3. Synthesis of C17-Ring Linolenic Acid Analogs

Cyclic analogs of linolenic acid should be more stable in vivo than linolenic acid itself, due to greater resistance to beta -oxidative cleavage. An example of one class of cyclic compounds is compound 27, which is a 17-membered hydrocarbon ring possessing a double bond and the required carboxylic acid group. The synthetic route to compound 27 is depicted in FIG. 5. Starting from civetone 22, the silated cyanohydrin 23 can be formed by reaction with trimethylsilyl cyanide. The cyanohydrin can be converted to the alpha -hydroxy acid 24 via reduction with stannous chloride and hydrochloric acid. Esterification of 24 gives compound 25, which can be converted to the alpha -fluoro ester 26 through reaction with DAST. Cleavage of the ester group then gives the free acid 27.

Fluoro ester 26 can also undergo allelic bromination to give compound 28 (FIG. 6), which undergoes double elimination by reaction with DBU to give the tri-ene 29, most likely as a mixture of cis and trans isomers. Fluoro ester 26 can also be catalytically hydrogenated to give the C17-saturated ring compound as well.

4. Synthesis of C16-Ring and 16-Membered Hetero Atom-Substituted Ring Linolenic Acid Analogs

The synthesis of this class of compounds begins with 3-hexenedial 30 (FIG. 7). Reaction of 30 with compound 31 (prepared in six steps from butyrolactone) and triphenylphosphine gives the Wittig product 32. Reaction of 32 with aldehyde 33 (prepared in four steps from cyclohexene) gives compound 34, which leads to compound 35 after removal of the ketal protecting groups. Compound 36 is obtained from the carbonyl coupling of 35 via titanium (III) chloride and lithium in dry dimethyoxyethane.

Insertion of heteroatoms into the ring system is accomplished by the procedure described in FIG. 7 using compounds with appropriate modification of compound 31. For example, compound 37 can be made from precursors to 31. Compound 37, after reacting with compound 30, yields compound 38 which, after several steps, yields compound 41. Compound 41, stirred in the presence of DBU, can undergo cyclization. After hydrolysis of the ester group, the acid 42 is formed (FIG. 8).

The sulfur and oxygen-substituted ring compounds 45 and 47 can be made in an analogous fashion (FIG. 9 and FIG. 10).

5. Synthesis of 17-Membered Hetero Atom-Substituted Ring Linolenic Acid Analogs

Compounds 48, 49, and 50 (FIG. 11) can be made using the chemistry outlined in FIG. 8 by merely replacing the 5-carbon aldehyde 39 with its 6-carbon homologue 33.

6. Synthesis of Polyunsaturated Fatty Acids With CF2 Group(s) in Between cis Double Bonds

A general methodology for the synthesis of unsaturated fatty acids, in which one of the methylene group between cis double bonds is replaced by a CF2 group is available (Kwok et al., 1987). This is exemplified by the preparation of PAGE 27 Pat. No. 5605929, *

10, 10-difluoroarachidonic acids (compound 51) and 11, 11-difluoro- gamma -linoleic acid (compound 52 in FIG. 11).

7. Chemical Names of Compounds in FIG. 1 to FIG. 11

1. Any acid.

2. The corresponding aldehyde.

3. Ethyl 3-hydroxyacid ester.

4. Ethyl 3-ketoacid ester.

5. Ethyl 3,3-difluoroacid ester.

6. 3,3-Difluoro acid.

7. Any acid chloride.

8. Meldrum's acid adduct.

9. 3-Keto acid.

10. 1,3-Cyclohexanedione.

11. Ethyl 6-bromohexanoate.

12. Ethyl 6-(2,6-cyclohexanedion-yl)hexanoate.

13. Ethyl 6-(2,6-dihydroxycyclo-hexanyl)hexanoate.

14. Ethyl 6-[2,6-bis(trifluoromethane sulfonyl)cyclohexanyl]heaxanoate.

15. Ethyl 6-(cyclohex-2,5-dienyl)hexanoate.

16. Cyclohexanone.

17. 2,6-Dibromocyclohexanone.

18. Ethyl 6-magnesiumbromo-hexanoate.

19. Ethyl 6-(1-hydroxy-2,6-dibromo-cyclohexyl)hexanoate.

20. Ethyl 6-(1-fluoro-2,6-dibromo-cyclohexyl)hexanoate.

21. Ethyl 6-(1-fluoro-cyclohex-2,5-dienyl)hexanoate.

22. Civetone.

23. Civetone trimethylsilyl-cyanohydrin.

24. 1-Hydroxycyclohept-9-ene-1-carboxylic acid.

25. Ethyl 1-hydroxycyclohept-9-ene-1-carboxylate. PAGE 28 Pat. No. 5605929, *

26. Ethyl 1-fluorocyclohept-9-ene-1-carboxylate.

27. Ethyl 1-fluorocyclohept-9-ene-1-carboxylic acid.

28. Ethyl 1-fluoro-8,11-dibromocyclohept-9-ene-1-carboxylate.

29. Ethyl 1-fluorocyclohept-7,9,11-triene-1-carboxylic acid.

30. 3-Hexenedial.

31. Tert-butyl 6-bromo-3-ketohex-anoate resorcinol ketal.

32. Tert-butyl 3-keto-dodec-6,9-diene-12-carboxaldehydoate resorcinol ketal.

33. 6,6- Dimethoxyhexanal.

34. Tert-butyl 3-keto-18,18-dimethoxyoctadec-6,9,12-trienoate resorcinol ketal.

35. Tert-butyl 3-ketooctadeca-6,9,12-triene-18-carbox-aldehydoate.

36. 2-(1-Cyclohexadec-1,7,10,13-tetraenyl)acetic acid.

37. Tert-butyl 6-bromo-3-N-phthal-amidohexanoate.

38. Tert-butyl 3-N-phthalamido-dodec-6,9-diene-12-carbox-aldehydoate.

39. 5,5-Dimethoxypentanal.

40. Tert-butyl 3-N-phthalamido-18,18-dimethoxyoctadec-6,9,12-trienoate.

41. Tert-butyl 3-keto-18-bromo-octadeca-6,9,12-trienoate.

42. 2-(2-Azacyclohexadec-7,10,13-trienyl)acetic acid.

43. Tert-butyl 3-thio-18-bromo-octadeca-6,9,12-trienoate.

44. Tert-butyl 3-sulfhydryl-18-bromooctadeca-6,9,12-trienoate.

45. 2-(2-Thiacyclohexadec-7,10,13-trienyl)acetic acid.

46. Tert-butyl 3-hydroxy-18-bromo-octadeca-6,9,12-trienoate.

47. 2-(2-Oxacyclohexadec-7,10,13-trienyl)acetic acid.

48. 2-(2-Oxacycloheptadec-8,11,14-trienyl)acetic acid.

49. 2-(2-Azacycloheptadec-8,11,14-trienyl)acetic acid.

50. 2-(2-Thiacycloheptadec-8,11,14-trienyl)acetic acid.

51. 10,10-Difluoro-arachidonic acid.

52. 11,11-Difluoro- gamma -linolenic acid. PAGE 29 Pat. No. 5605929, *

EXAMPLE 2

Inhibition of 5 alpha -Reductase Activity

A. ASSAYS

In mammalian cells, 5 alpha -reductase is very tightly associated with intracellular membranes, including the membrane of the endoplasmic reticulum and contiguous nuclear membranes. Attempts to solubilize and purify active 5 alpha -reductase have not been very successful. The assay of 5 alpha -reductase activity, therefore, has been performed by measuring the rate of conversion of testosterone to 5 alpha -DHT by whole cells or by microsomal and nuclear preparations in the presence of NADPH (enzymatic assay). Alternatively, the 5 alpha -reductase activity can be reliably assayed by following NADPH-dependent noncovalent binding of a potent radioactive inhibitor, such as [<3> H]4-MA ([<3> H]4-MA-binding assay), which strongly competes with testosterone for binding to the reductase. The results of the two assays correlate very well when microsomal preparations from different organs or animals are used for comparison (Liang et at., 1983).

1. [<3> H]4-MA Binding Assay for 5 alpha -Reductase

The procedure was described in detail previously (Liang et al., 1983, 1990). Briefly, the binding assay solution, in a final volume of 0.15 ml, contained microsomes (2-20 mu g of protein), 0.08 mu Ci of [<3> H]4-MA, 0.1 mM-NADPH, 1 mM-dithiothreitol and 50 mM-potassium phosphate, pH 7.0, with or without the indicated amount of a lipid or an inhibitor preparation. Lipids were dissolved in ethanol and added in 1-5 mu l volumes. Control tubes received the same amount of ethanol. After incubation at 0o C. for 1 h, the [<3> H]4-MA bound to microsomes was determined by collecting microsomes on a Whatman GF/1F glass fibre filter and washing with 10 ml of 20 mM-potassium phosphate, pH 7.0, containing 0.01% CHAPS to remove unbound [<3> H]4-MA.

2. Assay of the Enzymatic Activity of Microsomal 5 alpha -Reductase

The standard reaction mixture, in a final volume of 0.15 ml, contained microsomes (1 mu Ci of [<3> H]testosterone, 0.5-3.0 mu M non-radioactive testosterone, 0.1 mM-NADPH, 1 mM-dithiothreitol and 50 mM-potassium phosphate, pH 7.0, with or without the indicated amount of a lipid or an inhibitor preparation. The reaction was started by the addition of microsomes and the incubation was carried out at 37o C. for 15 min. Steroids were extracted and separated by t.l.c. as described previously (liang & Heiss, 1981; Liang et al., 1984a, 1985a). Radioactive steroids were located by fluorography and the amount of radioactivity present was determined by scintillation counting. The 5 alpha -reductase activity was measured by analyzing the extent of the conversion of [<3> H]testosterone to [<3> H]5 alpha -DHT.

B. SOURCES OF 5 alpha -REDUCTASE ACTIVITY

Microsomes were prepared at 4o C. from a buffered 0.32M-sucrose homogenate of human liver and from the livers of adult Sprague-Dawley female rats by differential centrifugation as described previously (Liang et al., 1990), and were used in the assay of 5 alpha -reductase activity. In some experiments, PAGE 30 Pat. No. 5605929, *

microsomes were solubilized with 0.1% polyoxyethylene ether W-1 as described previously (Liang et al., 1990), except for the substitution of polyoxyethylene ether W-1 for Lubrol-WX.

Cells genetically engineered to express specific types of 5 alpha -reductase isozymes may also be used as sources of 5 alpha -reductase activity. Intact cells containing 5 alpha -reductase, their microsomes, or nuclear preparation may also be used to screen 5 alpha -reductase inhibitors.

C. INHIBITORS OF 5 alpha -REDUCTASE ACTIVITY

Animal and plant sources were tested for the presence of compounds affecting 5 alpha -reductase activity. Inhibitory activities were found in extracts of rat and beef liver microsomes, beef kidney, human placenta, rat and human prostate as well as in yeast and vegetable plant oils, e.g., corn, peanut and olive oils, indicating the presence of 5 alpha -reductase inhibitors in a wide range of sources including animal, plant and microorganisms.

1. Rat Liver Microsomes

When the microsomal fraction of rat liver was solubilized with acetic acid and then mixed with methanol, more than 80% of microsomal proteins were removed as precipitates. This procedure inactivated the 5 alpha -reductase activity completely. The soluble fraction, but not the precipitated fraction, contained compounds that inhibited 5 alpha -reductase activity (determined by the enzymatic assay or [<3> H]4-MA-binding assay) of rat liver microsomes. As shown in FIG. 12, Sephadex Registered TM G-50 column chromatography of the methanol soluble fraction showed separation of the inhibitory activity from the majority of the protein peak which eluted in the void volume. The inhibitory activity was also found in methylene chloride extracts of rat liver microsomes, suggesting that some of the inhibitors were lipids.

2. Plants and Fungi

Preparations were also obtained and specific compounds were isolated from various plant materials. Some of these were able to regulate both the type 1 and type 2 5 alpha -reductase isozymes of rat and human. While some of these agents were inhibitory, other agents stimulated 5 alpha -reductase activities.

Each plant material (1 to 2 g) was extracted by 2 to 10 ml of water, ethanol, isopropyl alcohol, ether, chloroform, or ethyl acetate. Organic solvents can contain 0-90% of water and the extraction can be carried out at 0o-100o C. for 30 minutes to 20 hours.

When 1 g of the plant material was extracted with 4 ml of ethanol or water, and 3 mu l of the extract was tested in the liver microsomal 5 alpha -reductase assay system (the final volume of the reaction mixture was 0.15 ml), a significant inhibitory activity (over 20% inhibition) was observed with extracts from various brands of green tea, Yunnan tea, special gunpowder tea, oolong tea, black tea, chlorella, black shiitake mushroom, basil leaves, parsley leaves, and Chinese herbs, including Angelica sinensis, Anisi stellati fructus, Codonopsis pilosula, Ligustici rhizoma, Salvia mitiorrhiza, and Golden Lilly flower, seeds of borage, evening primose, black current, sesame, pumpkin, sunflower, and wheat germ. PAGE 31 Pat. No. 5605929, *

The inhibitory substances in basil, oolong tea, green tea, and Angelica sinensis, could be separated from other inactive substances by one or two dimensional silica gel thin-layer chromatography or by Sephadex TM gel column chromatography. The chemical structures of some purified compounds were determined by comparing their chemical properties with that of standard compounds, including chromatographic mobility, melting point, ultra-violet and visible-light spectra and NMR. Commercially-available standard compounds were also used in 5 alpha -reductase assays to show that some of them were indeed 5 alpha -reductase inhibitors that inhibit the formation of 5 alpha -DHT.

3. Fatty Acids

Certain long chain fatty acids were found to inhibit 5 alpha -reductase activity. The assay procedures and active compounds are described in Example 3. In general, it was found that long chain polyunsaturated fatty acids were most effective, particularly those with at least two double bonds and with a chain length of at least 12.

4. Catechins and Epicatechin Gallates

The major inhibitory substances in various brands of tea preparations, especially in green tea, were found to be catechin derivatives (FIG. 20A). Catechins without a galloyl (FIG. 20B) substitution (FIG. 21 ) were much less active than catechin gallate, epicatechin gallate, epigallocatechin gallate, and their optical isomers (FIG. 22) or their conjugated substances such as theaflavins and theaflavin mono- (or di-) gallates. These gallates showed significant inhibitory activities (30 to 90% inhibition) at concentrations of 0.5 to 40 mu M in the assay systems containing (a) rat liver microsomal preparations or (b) cells infected with retrovirus containing genes for type 1 or type 2 5 alpha -reductases and expressing specific type of the reductases. Catechin and epicatechin (FIG. 21) were much less active (less than 25% inhibition at 40 mu M).

Although these inhibitory polyphenolic substances are antioxidants, they did not significantly oxidize NADPH under the assay conditions (in the presence of liver microsomal preparation and in the absence of testosterone or 4-MA, indicating that the inhibitory activity was due to the inhibition of 5 alpha -reductases and not due to a nonspecific oxidation of NADPH by these polyphenols.

Various synthetic gallate derivatives (methyl gallate, n-propyl gallate, 3,4,5-trihydroxybenzamide), gallic acid, and pyrogallol were not as active as catechin gallate. This indicated that the gallol or galloyl structure alone was not sufficient for the high inhibitory activity. A low inhibitory activity was found with n-octyl gallate, indicating that, for the inhibitory activity, the flavonol group of catechin gallates may be replaced by other alcoholic group having similar geometric structures.

The results indicated that the gallate moiety incorporating an acyl (galloyl) or alcoholic (trihydroxybenzyl) group may be required for inhibition of 5 alpha -reductase. These groups may form an ester or ether linkage with the flavonol (FIG. 23).

EXAMPLE 3 PAGE 32 Pat. No. 5605929, *

Fatty Acid Inhibition of 5 alpha -Reductase Activity

Identification of compounds that inhibited 5 alpha -reductase utilized two types of assays; an enzymatic assay and a binding assay as described in Example 2. Both assays identified similar activities for the active fatty acids.

When various lipids were tested for their ability to affect binding of [<3> H]4-MA to rat liver microsomes, only certain unsaturated fatty acids were inhibitory, as shown in Tables 1 and 2. Among the lipids tested, the highly inhibitory fatty acids have 14 to 22 carbon chains and one to six double bonds. The presence of a double bond was required for higher inhibitory activity; saturated fatty acids were generally not as active as corresponding unsaturated fatty acids. With the [<3> H]4-MA binding assay, only compounds with double bonds in the cis configuration were active at low concentrations ( < 10 mu M), whereas the trans isomers were inactive even at high concentrations ( > 0.2 mM). However, as is shown in Example 4, the trans isomers were active inhibitors when the reductase activity was analyzed using the enzyme assay. The difference in the effect of cis and trans isomers of fatty acids in the [<3> H]4-MA binding assay is obvious when the following sets of fatty acids are compared: oleic acid (C18:1, cis-9) vs. elaidic acid (C18:1, trans-9) and linoleic acid (C18:2, cis-9,12) vs. linolelaidic acid (C18:2, trans-9,12). The results presented in Tables 1 and 2 also demonstrate that the number and the position of the double bonds also affected the potency. When the [<3> H]4-MA binding assay was used, the inhibitory potency for the C18 fatty acids were, in decreasing order: gamma -linolenic acid (cis-6,9,12) > cis-6,9,12,15-octadecatetraenoic acid > alpha -linolenic acid (cis-9,12,15) > linoleic acid (cis-9,12) > oleic acid (cis-9) > petroselinic acid (cis-6). Erucidic acid (C22:1, cis-13) was inactive; whereas cis-4,7,10,13,16,19-docosahexaenoic acid was a potent inhibitor. Undecylenic acid (C11:1,10) and nervonic acid (C24:1, cis-15) were also inactive.

A free carboxyl group is important since the methyl ester and alcohol analogs of these inhibitory unsaturated fatty acids were either inactive or only slightly active. Prostaglandin E2, F2a and 12 were not active; whereas the prostaglandin A1, A2, B1, B2, D2, E1, and F1a were somewhat active at 0.2 mM. Carotenes, retinals, and retinoic acid were also inactive. Phosphatidylcholine, phosphatidyl ethanolamine, 3-diolein, retinol, 13-cis-retinoic acid, and 13-cis-retinol were slightly stimulatory.

When the inhibitory effects of fatty acids were tested by the enzymatic assay, the relative potency of saturated and cis-unsaturated fatty acids were in agreement with that obtained by the [<3> H]4-MA-binding assay (Tables 1 and 2), regardless of whether rat liver microsomes or prostate microsomes were used as the source of the enzyme. The trans isomers, elaidic acid (C18:1, trans-9) and linolelaidic acid (C18:2, trans-9,12) were much less inhibitory than their cis isomers, oleic acid (C18:1, cis-9) and linoleic acid (C18:2, cis 9,12), in the [<3> H]4-MA binding assay (Tables 1 and 2, and FIG. 13A); however, they were as potent as their cis isomers in the enzymatic assay using either prostate microsomes or liver microsomes (FIG. 13B). The results suggested that the trans isomers inhibited 5 alpha -reductase through a different mechanism. TABLE 1 Inhibition of [<3> H]4-MA Binding to 5 alpha -Reductase of Rat Liver Microsomes by Lipids PAGE 33 Pat. No. 5605929, * % Inhibition of [<3> H]4-MA binding* Concentration of test compounds

Numeric * * Test compounds symbol # 10 mu M 40 mu M 200 mu M Control * * * (no addition) Undecylenic acid C11:1 (10) * NA 13 +/- 2 Myristoleic acid C14:1 (cis-9) NA 25 +/- 4 43 +/- 1 Palmitic acid C16:0 * * NA Palmitoleic acid C16:1 (cis-9) NA 16 +/- 5 73 +/- 7 Palmitoleic acid * * NA NA methyl ester Palmitoleyl alcohol * * NA 16 +/- 4 Stearic acid C18:0 NA NA NA Petroselinic acid C18:1 (cis-6) * NA 52 +/- 9 Oleic acid C18:1 (cis-9) NA 16 +/- 6 63 +/- 12 Elaidic acid C18:1 NA NA NA (trans-9) Oleic acid methyl * * NA NA ester Oleyl alcohol * * NA NA Linoleic acid C18:2 (cis- NA 12 +/- 3 86 +/- 4 9,12) Linolelaidic acid C18:2 (trans- * NA 19 +/- 5 9,12) Linoleic acid methyl * * NA NA ester Linoleyl alcohol * NA NA 25 +/- 5 alpha -Linolenic acid C18:3 (cis- 19 +/- 3 27 +/- 7 84 +/- 6 9,12,15) alpha -Linolenic acid * NA NA NA methyl ester alpha -Linolenyl alcohol * NA NA 24 +/- 1 gamma -Linolenic acid C18:3 (cis- 50 +/- 2 83 +/- 12 96 +/- 2 6,9,12) Octadecatetraenoic C18:4 (cis- NA 40 +/- 6 88 +/- 2 acid 6,9,12,15) Arachidonic acid C20:4 (cis- NA 30 +/- 10 88 +/- 5 5,8,11,14) Docosahexaenoic C22:6 (cis-4,7, NA 27 +/- 1 87 +/- 6 acid 10,13,16,19) Erucic acid C22:1 (cis-13) * NA NA Nervonic acid C24:1 (cis-15) * NA NA

nLipids were tested at concentrations ranged from 0.01 to 0.2 mM. Each study was carried out in duplicates and several experiments were performed to assure that the results shown are representative. Compounds that showed less than 10% inhibition were considered not active (NA). At 200 mu M, no significant effect was observed with - PAGE 34 Pat. No. 5605929, *

n(a) saturated aliphatic fatty acids including caproic acid, heptanoic acid, caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, behenic acid, tricosanoic acid, and lignoceric acid, -

n(b) fatty acyl esters and alcohols including stearic acid methyl ester, S-stearoyl CoA, palmitic acid methyl ester, S-palmitoyl CoA, cis-9-tetradecenol, and arachidonyl alcohol, and -

n(c) vitamin A related compounds including alpha - and beta -carotenes, retinoic acid, 9-cis-retinal, retinal, and 13-cis-retinal. At this high concentration, some aliphatic lipids showed inhibitory activities that were significantly lower than the corresponding unsaturated fatty acids (percent inhibition in the parentheses): myristoleic acid methyl ester (27%), gamma -linolenic acid methyl ester (32%), and cis-4,7,10,13,,16,19- docosahexenol (51%). Retinal, 13-cis retinoic acid, and 13-cis-retinol showed 58% stimulation at 200 mu M but no stimulation or inhibition at 40 mu M. IC50 (the concentrations needed to show 50% inhibition) for potent fatty acids were: gamma -linolenic acid (10 mu M, octadecatetraenoic acid (57 mu M), gamma -linolenic acid (60 mu M), arachidonic acid (65 mu M), palmitoleic acid (108 mu M), linoleic acid (117 mu M), and oleic acid (128 mu M). -

n# The numeric symbol indicates the number of carbon atoms and double bondS in the molecule. The numbers in parentheses indicate the position of double bonds (numbered from the carboxyl end) in cis- or trans-forms. -

In addition to the compounds shown in Table 1, the fatty acids, their methyl esters and glycerides shown in Table 2 were tested. The carbon chain length of these fatty acids ranged from 11 to 24 carbons with one to 6 double bonds. Some of the inhibitory compounds and the concentrations required for 50% inhibition (shown in parenthesis; NA indicates not inhibitory at 200 mu M or lower concentrations) are: 10-pentadecenoic acid (100 mu M), 10-heptadecenoic acid (28 mu M), 10-trans-heptadecenoic acid (NA), methyl 10-heptadecenoate (NA), 13-octadecenoic acid (93 mu M), 12-octadecenoic acid (NA), 11-octadecenoic acid (26 mu M), monogamma linolenin (86 mu M), gamma -linolenyl alcohol (NA), gamma -linolenyl acetate (NA), methyl gamma -linolenate (NA), cholesteryl gamma -linolenate (NA), di- gamma -linolenin (NA), gamma -linolenoyl chloride (NA), tri- gamma -linolenin (NA), 6,9,12,15-octadecatetraenoic acid (74 mu M), nonadecane nitrile (NA), 12-nonadecenoic acid (90 mu M), 10-nonadecenoic acid (130 mu M), 10-trans nonadecenoic acid (NA), 10,13-nonadecadienoic acid (86 mu M), linoleyl cyanide nitrile (NA), linolelaidyl cyanide nitrile (NA), 11 eicosenoic acid (146 mu M), 8-eicosenoic acid (48 mu M), 5-eicosenoic acid (NA), 11,14 eicosadienoic acid (131 mu M), trans 11,14-eicosadienoic acid (NA), methyl 11,14 eicosadienoate (NA), 11,14-eicosadienoyl chloride (NA), 11,14,17-eicosatrienoic acid (29 mu M), 11,14,17-eicosatrienoyl chloride (NA), 8,11,14-eicosatrienoic acid (15 mu M), homo- gamma -linolenoyl chloride (NA), methyl homo- gamma -linolenate (NA), 5,8,11-eicosatrienoic acid (50 mu M), archidoyl chloride (NA), heneicosenoic acid (154 mu M), heneicosene nitrile (NA), erucic acid (NA), 13,16-docosadienoic acid (118 mu M), 13,16,19-docosatrienoic acid (163 mu M), methyl 13,16,19-docosatrienoate (NA), 7,10,13,16-docosatetraenoic acid (46 mu M), methyl docosatetraenoate (NA), 4,7,10,13,16,19-docosahexaenoic acid (47 mu M), 14 tricosenoic acid (NA), PAGE 35 Pat. No. 5605929, *

15-tetracosanoic acid (NA). TABLE 2 Inhibition of [<3> H]4-MA Binding to 5 alpha -Reductase of Rat Liver Microsomes by Lipids

% Inhibition of [<3> H]4- MA binding* Concentration of test compounds

Numeric 5 10 40 200 Test compounds symbol # mu M mu M mu M mu M Control (no * * * * addition) Undecylenic Acid C11:1 (cis-10) * NA NA 13 Myristoleic Acid C14:1 (cis-9) NA NA 25 43 10-Pentadecenoic C15:1 (cis-10) NA NA NA 71 Acid Palmitic Acid C16:0 * * * NA Palmitoleic Acid C16:1 (cis-9) * NA 16 73 Palmitelaidic Acid C16:1 (trans-9) * * * NA 10-cis-Hepta- C17:1 (cis-10) NA NA 83 84 decenoic Acid 10-trans-Hepta- C17:1 (trans-10) NA NA NA 40 decenoic Acid Stearic Acid C18:0 * NA NA NA 11-Octadecenoic C18:1 (cis-11) NA 14 55 81 Acid 12-Octadecenoic C18:1 (cis-12) NA NA NA NA Acid 13-Octadecenoic C18:1 (cis-13) NA NA NA 57 Acid trans-Vaccenic Acid C18:1 (trans-11) * * * 39 Oleic Acid C18:1 (cis-9) NA NA 16 63 Elaidic Acid C18:1 (trans-9) * NA NA NA Petroselinic Acid C18:1 (cis-6) * * NA 52 Linoleic Acid C18:2 (cis-9,12) * NA 12 86 Linolelaidic Acid C18:2 (trans-9,12) * NA NA 19 alpha -Linolenic Acid C18:3 (cis-9,12,15) NA 19 27 84 Linolenoyl Chloride chloride * * * NA gamma -Linolenic Acid C18:3 (cis-6,9,12) 30 50 83 96 Mono gamma -Linolenin monoglyceride NA NA 35 87 gamma -Linolenyl Alcohol alcohol NA NA NA 41 gamma -Linolenyl Acetate acetate NA NA NA 27 Di- gamma -Linolenin Diglyceride * NA NA NA gamma -Linolenoyl chloride * NA NA NA Chloride Tri- gamma -Linolenin triglyceride NA NA NA NA 6,9,12,15-Octadera- C18:4 (cis-6, * NA 40 88 tetraenoic Acid 9,12,15) 10-cis-Nonadecenoic C19:1 (cis-10) NA NA 13 79 Acid 10-trans-Non- C19:1 (trans-10) NA NA NA 32 PAGE 36 Pat. No. 5605929, * adecenoic Acid 12-cis-Nonadecenoic C19:1 (cis-12) NA NA 32 91 Acid 10,13-Non- C19:2 (cis-10,13) NA NA 37 83 adecadienoic Acid 5-Eicosenoic Acid C20:1 (cis-5) NA NA NA NA 8-Eicosenoic Acid C20:1 (cis-8) 14 41 52 81 11-Eicosenoic Acid C20:1 (cis-11) NA NA 15 76 11,14-cis- C20:2 (cis-11,14) NA NA NA 89 Eicosadienoic Acid 11,14-trans- C20:2 (trans- NA NA NA NA Eicosadienoic Acid 11,14) 11,14-cis- methyl ester NA NA NA NA Eicosadienoate 11,14-cis- chloride NA NA NA NA Eicosaidienoyl Chloride 11,14,17-cis- C20:3 (cis- NA 1 78 94 Eicosatrienoic Acid 11,14,17) 11,14,17-cis- chloride NA NA NA NA Eicosatrienoyl Chloride 8,11,14-cis- C20:3 (cis-8,11,14) NA 42 92 82 Eicosatrienoic Acid Arachidonic Acid C20:4 (cis- * NA 30 88 5,8,11,14) Arachidoyl chloride * NA NA NA Chloride Heneicosenoic Acid C21:1 (cis-12) NA 15 25 60 Erucic Acid C22:1 (cis-13) * NA NA NA 13,16-Docosadienoic C22:2 (cis-13,16) NA NA 21 93 Acid 13,16,19-Docosa- C22:3 (cis- NA NA NA 65 trienoic Acid 13,16,19) 7,10,13,16-Docosa- C22:4 (cis-7, NA NA 41 79 tetraenoic Acid 10,13,16) 4,7,10,13,16,19- C22:6 (cis-4,7, NA 18 49 86 Docosahexenoic 10,13,16,19) Acid 14-Tricosenoic Acid C23:1 (cis-14) NA NA NA 36 15-Tetracosenoic C24:1 (cis-15) * * NA NA Acid

EXAMPLE 4

gamma -Linolenic Acid Inhibition of 5 alpha -Reductase

gamma -LA appeared to be one of the more potent inhibitors of 5 alpha -reductase and was therefore further examined with respect to its 5 alpha -reductase binding characteristics. PAGE 37 Pat. No. 5605929, *

1. 5 alpha -reductase Inhibition

With either the enzymatic assay or with the [<3> H]4-MA binding assay (FIG. 14), inhibition was observed within a minute after gamma -LA was mixed with the microsomal enzyme preparation and was observed with both intact (FIG. 15A) and detergent (polyoxyethylene ether) solubilized (FIG. 15B) rat liver microsomes. As the concentration of protein increased from 2 to 20 mu g, the extent of inhibition by 10 mu M gamma -LA decreased from 93% to 52% for intact microsomes and from 96% to 88% for solubilized microsomes.

When [<3> H]4-MA was allowed to bind to microsomes in the presence of NADPH, followed by addition of gamma -LA to a final concentration of 10 mu M, about 60% of the microsome-bound [<3> H]4-MA dissociated from the microsomes within 2 min. The remaining microsome-bound [<3> H]4-MA dissociated at a much slower rate over the next 60 min. To determine whether gamma -LA inhibition is reversible, microsomes were incubated with gamma -LA and then reisolated to remove free gamma -LA. The results showed that the inhibition was only partially reversed (reduced from 78% to 63% inhibition). It is possible that gamma -LA was bound tightly to microsomes and/or irreversibly inactivated components which were essential for the reductase activity.

By either the enzymatic or the [<3> H]4-MA binding assay, the inhibition could not be overcome by increasing the level of NADPH (FIG. 16A) or testosterone (FIG. 16B). gamma -LA did not appear to compete with testosterone or NADPH for their binding to the microsomal reductase. Double reciprocal plots of the data showed that 5 mu M of gamma -LA increased the apparent K m value for NADPH (from 2.0 to 3.1 mu M) and testosterone (from 2.4 to 4.5 mu M), and decreased the V max from 7.5 to 2.8 pmol 5 alpha -DHT formed/mg protein/15 min. gamma -LA at 5 and 10 mu M increased the apparent K i values for [<3> H]4-MA from 13 to 20 and 40 mu M, respectively, and decreased the maximal binding from 0.56 to 0.45 and 0.40 pmol/10 mu g protein, respectively.

2. NADH:Menadione Reductase and UDP-Glucuronic Acid:5 alpha -DHT Glucuronyl Transferase Inhibition

The effect of gamma -LA on the activities of another microsomal reductase and a microsomal enzyme that uses asteroid as a substrate was tested to determine the specificity of the effect of gamma -LA. Results showed that gamma -LA at 10 to 40 mu M did not affect the activities of NADH:menadione reductase or UDP-glucuronic acid:5 alpha -DHT glucuronosyl transferase.

Mammalian 5 alpha -reductase is a cellular membrane-bound enzyme. Perturbation of the lipid matrix of the membranes may affect reductase activity nonspecifically. The fact that only unsaturated fatty acids with specific configurations were potent inhibitors of 5 alpha -reductase in a specific assay and that two other microsomal enzymes examined were not affected suggests that the inhibition was selective.

3. Effect of gamma -LA on Human Microsomes and Prostate Cancer Cells

gamma -LA inhibited NADPH-dependent [<3> H]4-MA binding to human liver microsomes to the same degree as in experiments with rat liver microsomes. The 5 alpha -reduction of [<3> H]testosterone by human prostate cancer cells in culture was also selectively affected by gamma -LA. Table 3 shows that gamma -LA, at 5 to 50 mu M, inhibited 5 alpha -reductase reduction of [<3> PAGE 38 Pat. No. 5605929, *

H]testosterone in both the androgen-sensitive LNCaP cells (Horszewicz et al., 1983) and the androgen insensitive PC-3 cells (Kaighn et al., 1979). gamma -LA, however, did not affect the metabolism of testosterone to 4-androstenedione, suggesting that 17 beta -steroid dehydrogenase was not sensitive to the unsaturated fatty acid. Stearic acid (5 to 20 mu M) did not affect the 5 alpha -reductase reduction or 17 beta -steroid dehydrogenase of PC-3 cells in culture. The specific 5 alpha -reductase inhibition observed with intact prostate cells in culture indicated that externally added fatty acids were able to enter cells and exert an inhibitory action on the endoplasmic reticulum or nuclear membrane-bound 5 alpha -reductase in situ. TABLE 3 Inhibition of the Formation of Radioactive 4-Androstenedione and 5 alpha -DHT from [<3> H]Testosterone by Human Prestatic Cancer Cells by gamma -LA

Prostate Fatty acid Metabolites formed*

cell added 4-Androstendione 5 alpha -DHT line ( mu M) (% of control) (% of control) PC-3 None (control) 100 100 gamma -LA 1 102 +/- 6 98 +/- 6 5 110 +/- 1 50 +/- 3 20 99 +/- 2 2 +/- 2 Stearic acid 5 103 +/- 2 123 +/- 2 20 106 +/- 5 121 +/- 5 LNCaP None (control) ND 100 gamma -LA 50 ND 27 +/- 0 100 ND 9 +/- 4

n*The control values for the formation of 4-androstenedione and 5 alpha -DHT by PC-3 cells were 400,851 +/- 9,507 dpm and 12,183 +/- 74 dpm, respectively. The control value for the formation of 5 alpha -DHT by LNCaP was 4,569 +/- 505 dpm. -

nNo 4-androstenedione formation was detected when LNCaP was used. gamma -LA and stearic acid, at the concentrations tested, did not produce any visible change in cell morphology during the 2 hour incubation. IC50 values (four studies) for gamma -LA with the prostate cancer cells were 10 +/- 5 mu M. -

EXAMPLE 5

Effects of Polyunsaturated Fatty Acids and Other Compounds on Androgen Action in the Hamster Flank Organ Model PAGE 39 Pat. No. 5605929, *

The inventors sought an inhibitor of 5 alpha -reductase that would be active topically and inactive systemically, as such an agent would be ideal for treatment of androgen-dependent dermatological disorders. Of the aliphatic unsaturated fatty acids tested for inhibition of 5 alpha -reductase activity in liver and prostate from rats and humans, gamma -LA was found to be the most potent fatty acid inhibitor when topically applied to hamster flank organs.

In this study, inhibition of androgen action by topical administration of gamma -LA in hamster flank organs is investigated. Especially useful in the evaluation of the effects of these compounds on skin cells or sebaceous glands is the hamster flank organ (Frost and Gomez, 1972). The paired flank organs, one on each side of the costovertebral angle, are highly sensitive to androgen stimulation. The androgen sensitive structures in the flank organ include dermal melanocytes, sebaceous glands, and hair follicles (Hamilton and Montagna, 1950). This animal model has been widely used for testing androgenic (Hamilton and Montagna, 1950; Frost et al., 1973) and antiandrogenic compounds (Voigt and Hsia, 1973; Weissmann et al., 1985; Chakrabarty et at., 1980). The unique advantage of this animal model is that a testing compound can be applied topically to only one of the flank organs and the effect observed on both organs. If the test compound has only a local effect then only the treated flank organ is affected. However, if the effect is systemic then both flank organs are affected. Results indicate that gamma -LA applied topically inhibits androgen action locally without a systemic effect.

A. MATERIALS AND METHODS

1. Chemicals