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
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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