DEPRIVATION THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/095,567, filed December 22, 2014, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods for androgen deprivation therapy. Specifically, the present invention relates to the use of the selective estrogen receptor modulator cis-clomiphene or an analogue or salt thereof to reduce the level of testosterone in a human in order to treat or prevent a disease mediated by testosterone such as prostate cancer or benign prostatic hypertrophy.

BACKGROUND

[0002] Prostate cancer is one of the most common types of cancer in men and is the second leading cause of cancer death in men in the United States. In 2014, an estimated 233,000 men in the United States will be diagnosed with prostate cancer. Prostate cancer is usually slow growing and confined to the prostate and nearby organs. The prognosis is quite good if the cancer has not metastasized.

[0003] Benign prostatic hypertrophy (BPH), characterized by an enlarged prostate gland resulting from elevated level of male hormones such as dihydrotestosterone (DHT) in prostate tissue, is a common condition of the aging male. Left untreated, BPH can result in blocked urine flow and urinary tract and/or kidney problems.

[0004] Androgen deprivation therapy (ADT) has been used as a method for treating certain types of cancer, especially prostate cancer and can also be an effective treatment for BPH. According to ADT, the level of androgen hormone (mainly testosterone) is chemically or surgically reduced. Because androgens stimulate the growth of prostate cancer cells, this approach inhibits the growth of prostate cancer cells and can make prostate cancers shrink or grow more slowly.

[0005] Surgical castration (orchiectomy) consists of removing the testicles of men with prostate cancer. It is an effective but irreversible method of decreasing androgens. [0006] Chemical castration methods include LHRH agonists and antagonists which work by inhibiting the formation of LH in the pituitary gland. These drugs are injected into the skin or placed as small implants under the skin and achieve the same result as surgical castration. Anti-androgens (or androgen antagonists), which prevent androgens from expressing their biological effects on responsive tissues, are also used to induce chemical castration. Most commonly, anti-androgens are androgen receptor antagonists that competitively inhibit the binding of androgen to their receptor.

[0007] ADT alone does not cure prostate cancer and for this reason is generally employed as an adjuvant to radiation therapy.

[0008] Side effects of ADT include reduced or absent libido, impotence, hot flashes, breast tenderness, osteoporosis, anemia, decreased mental sharpness, loss of muscle mass, weight gain, fatigue, increased cholesterol and depression.

SUMMARY

[0009] In several embodiments, the present invention provides compositions and methods for reducing androgen (testosterone) levels in a mammal. In particular, the present invention provides a method for treating an androgen-responsive disease or to ameliorate one or more symptoms thereof, by administering to a subject in need thereof an effective amount of cis- clomiphene or an analogue or salt thereof.

[0010] In preferred embodiments, a therapeutically effective amount of cis-clomiphene or an analogue thereof, essentially free of trans-clomiphene, is administered to a subject in order to treat benign prostate hypertrophy (BPH) or prostate cancer.

[0011] Preferred cis-clomiphene analogs for use according to the invention are the hydroxylated, de-ethylated and di-de-ethylated metabolites of cis-clomiphene and their pharmaceutically acceptable salts including, without limitation, 4-OH-cis-clomiphene, N- desethyl-cis-clomiphene and N,N-di-desethyl-cis-clomiphene.

[0012] In other related embodiments, the present invention is related to methods for inhibiting the proliferation of prostate cancer cells in a subject by administering to the subject a therapeutically and/or prophylactically effective amount of a composition comprising cis- clomiphene, an analogue thereof or a pharmaceutically acceptable salt thereof, the composition preferably being essentially free of trans-clomiphene. The subject may be a human male diagnosed with prostate cancer or at risk for developing prostate cancer.

[0013] In some embodiments, the present invention is related to the use of a composition comprising cis-clomiphene, an analogue thereof or a pharmaceutically acceptable salt thereof, the composition preferably being essentially free of trans-clomiphene, as an adjuvant therapy to treat prostate cancer.

[0014] Preferred dosages of cis-clomiphene or an analogue or salt thereof include 1 mg to 200 mg, 1 mg to 100 mg, 2 mg to 100 mg, 2 mg to 80 mg, 5 mg to 200 mg, 5 mg to 100 mg, 5 mg to 80 mg, 5 mg to 50 mg, 5 mg to 20 mg, 10 mg to 200 mg, 10 mg to 100 mg, 10 mg to 50 mg, 12.5 mg to 200 mg, 12.5 mg to 100 mg, 12.5 mg to 50 mg, 12.5 mg to 25 mg, 25 mg to 200 mg, 25 mg to 100 mg, 50 mg to 200 mg, 50 mg to 100 mg cis-clomiphene or an analogue or salt thereof. Preferably cis-clomiphene or an analogue or salt thereof is administered daily or every other day. In a particularly preferred embodiment, a relatively high dose of cis-clomiphene from 50-200 mg or any range there between such as 50-100, 60- 100, 70-100, or 80-100 mg of cis-clomiphene is administered to the subject, preferably as a daily dosage. In related embodiments, a daily dosage of 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg is administered to the subject.

[0015] Cis-clomiphene or an analogue or salt thereof according to the methods described herein is administered for a period of time sufficient to achieve the desired result, e.g.

treatment of BPH or prostate cancer. In one aspect, cis-clomiphene or an analogue or salt thereof is administered for treatment period of at least 3 months, at least 6 months, at least 12 months or even at least 2, 3, 4, or 5 years.

BRIEF DESCRIPTION OF THE DRAWING

[0016] FIGS. 1 A-D. Tissue weights. Collected tissues expressed as percent body weight. Group I: Solid white bar-Placebo; Group II: Solid black bar-40 milligrams per kilogram (MPK) Enclomiphene citrate; Group III: Solid grey bar-4 MPK Enclomiphene citrate; Group IV: Vehicle hatch marks-40 MPK Zuclomiphene citrate; Group V: Horizontal hatch marks-4 MPK Zuclomiphene citrate.

[0017] FIG. 2 is a testicular histology micrograph. [0018] FIG. 3. Differences in body weight after daily administration of Enclomiphene citrate and Zuclomiphene citrate for 91 days. Group 1 : Solid line with diamonds- Placebo; Group II: Solid line with squares-40 MPK Enclomiphene citrate; Group III: Solid line with triangles-4 MPK Enclomiphene citrate; Group IV: Solid lines with circles-40 MPK Zuclomiphene citrate; Group V: Dashed lines with diamonds-4 MPK Zuclomiphene citrate.

DETAILED DESCRIPTION

[0019] The present invention is based on the discovery that cis-clomiphene, administered at relatively high dosage, decreases testosterone by causing major deleterious effects on the Leydig cells of the testes and also leads to a failure to gain weight. The castrating effect, of cis-clomiphene - reducing both testosterone and Leydig cells - renders the compound particularly useful as an androgen deprivation therapy. Thus the compositions can be administered to human men treat or prevent androgen responsive diseases such as BPH or prostate cancer.

[0020] In some embodiments, cis-clomiphene or a salt thereof, essentially free of trans- clomiphene, is administered therapeutically or prophylactically as a monotherapy to a human to treat or prevent prostate cancer. Preferably cis-clomiphene is administered as a monotherapy to subjects with prostate cancer that is metastatic or is recurring.

[0021] In other embodiments, cis-clomiphene or a salt thereof, essentially free of trans- clomiphene, is administered therapeutically or prophylactically as an adjuvant to a human to treat or prevent prostate cancer. In some embodiments, cis-clomiphene is administered to a subject prior to, concurrently with, or subsequently to surgery and/or radiation therapy. Preferably, the subject is a human male with early-stage prostate cancer or a human male identified as having a high risk of recurrence.

[0022] In related embodiments, cis-clomiphene or a salt thereof, essentially free of trans- clomiphene, is administered therapeutically or prophylactically as an adjuvant to a subject after surgery and/or radiation therapy.

[0023] In other related embodiments, cis-clomiphene or a salt thereof, essentially free of trans-clomiphene, is administered therapeutically or prophylactically as an adjuvant to a subject prior to surgery and/or radiation therapy [0024] In other embodiments, a metabolite of cis-clomiphene or salt thereof, essentially free of cis-clomiphene, trans-clomiphene and any other metabolite of clomiphene, is administered therapeutically or prophylactically to a subject to treat or prevent an androgen responsive disease such as BPH or prostate cancer. Preferred cis-clomiphene analogs for use according to the invention include the hydroxylated, de-ethylated and di-de-ethylated metabolites of cis- clomiphene and their pharmaceutically acceptable salts including, without limitation, 4-OH- cis-clomiphene, N-desethyl-cis-clomiphene and N,N-di-desethyl-cis-clomiphene.

[0025] "Monotherapy" refers to administering a single active agent for treating or preventing a condition, such as prostate cancer. Thus, if cis-clomiphene is administered as a

monotherapy for treating or preventing prostate cancer, no other active agent is administered sequentially or concurrently for this purpose. .

[0026] As used herein, a "subject" means a human or animal.

[0027] It is to be understood that the methods described herein may be used prophylactically, meaning to protect, in whole or in part, against a an androgen-responsive disease. The methods may also be used therapeutically to ameliorate, in whole or in part, an androgen- responsive disease, or to protect, in whole or in part, against further progression of an androgen-responsive disease.

[0028] "Effective amount" refers to the amount of active agent required to treat a patient with an androgen-responsive disease in a clinically relevant manner or prevent an androgen- responsive disease in a subject at risk for developing such a disease.

[0029] In a preferred embodiment, compositions of the invention comprise cz ^' s-clomiphene or a salt thereof such as cis-clomiphene citrate at a dose which may range from 1 to 200 mg or from 5 to 100 mg. The dosage of cz ^' s-clomiphene may also be from 5 to 10 mg, from 5 to 12.5 mg, from 5 to 15 mg, from 5 to 20 mg, from 10 to 15 mg, from 10 to 20 mg, from 12.5 to 25 mg, from 12.5 to 50 mg, or from 25 mg to 50 mg The dosage of cw-clomiphene may also be 12.5 mg , 25 mg or 50 mg.

[0030] In one embodiment, compositions of the invention comprise one or more

pharmaceutically acceptable salts of cis-clomiphene or an analogue thereof. Depending on the process conditions the salt compound obtained may be either in neutral or salt form. Salt forms include hydrates and other solvates and also crystalline polymorphs. Both the free base and the salts of these end products may be used in accordance with the invention.

[0031] Acid addition salts may be transformed into the free base using basic agents such as alkali or by ion exchange. The free base obtained may also form salts with organic or inorganic acids.

[0032] In the preparation of acid addition salts, preferably such acids are used which form suitably pharmaceutically acceptable salts. Examples of such acids are hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, aliphatic acid, alicyclic carboxylic or sulfonic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, fumaric acid, maleic acid, hydroxymaleic acid, pyruvic acid, aspartic acid, glutamic acid, p-hydroxybenzoic acid, embonic acid, ethanesulfonic acid, hydroxyethanesulfonic acid, phenylacetic acid, mandelic acid, alogenbensenesulfonic acid, toluenesulfonic acid, galactaric acid, galacturonic acid or naphthalenesulfonic acid. All crystalline form polymorphs may be used in accordance with the invention. A preferred salt is the citrate salt.

[0033] Base addition salts may also be used in accordance with the invention and may be prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkali earth metals or organic amines. Examples of metals used as cations are sodium, potassium, calcium, magnesium and the like. Examples of suitable amines are amino acids such as lysine, choline, diethanolamine, ethylenediamine, N-methylglucamine and the like.

[0034] Compositions of the instant invention can be prepared in the form of a dose unit or dose units suitable for oral, parenteral, transdermal, rectal, transmucosal, or topical administration. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular. Preferably, compositions of the instant invention are prepared in a form suitable for oral administration. [0035] The terms "oral administration" or "orally deliverable" herein include any form of delivery of a therapeutic agent or a composition thereof to a subject wherein the agent or composition is placed in the mouth of the subject, whether or not the agent or composition is swallowed. Thus, "oral administration" includes buccal and sublingual as well as esophageal (e.g. inhalation) administration.

[0036] Compositions of the present invention may also be formulated for inhalation, for transdermal delivery, for parenteral administration including, but not limited to, by injection or continuous infusion, as a depot preparation, which may be administered by implantation or by intramuscular injection, or as a liposome preparation.

[0037] A composition of the invention can be in the form of solid dosage units such as tablets, (e.g. suspension tablets, bite suspension tablets, rapid dispersion tablets, chewable tablets, effervescent tablets, bilayer tablets, etc.), caplets, capsules (e.g., a soft or a hard gelatin capsule), powder (e.g. a packaged powder, a dispensable powder or an effervescent powder), lozenges, sachets, cachets, troches, pellets, granules, microgranules, encapsulated microgranules, powder aerosol formulations, or any other solid dosage form reasonably adapted for administration. A preferable dosage form is a soft or hard gelatin capsule.

Another preferable dosage form is a tablet.

[0038] Tablets can be prepared according to any of the many relevant, well known pharmacy techniques. In one embodiment, tablets or other solid dosage forms can be prepared by processes that employ one or a combination of methods including, without limitation, (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion.

[0039] Suitable liquid dosage forms of a composition of the invention include solutions, aqueous or oily suspensions, elixirs, syrups, emulsions, liquid aerosol formulations, gels, creams, ointments, etc. Such compositions may also be formulated as a dry product for constitution with water or other suitable vehicle before use.

[0040] Compositions of the invention can, if desired, include one or more pharmaceutically acceptable excipients. The term "excipient" herein means any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition. Excipients include, by way of illustration and not limitation, diluents, disintegrants, binding agents, adhesives, wetting agents, lubricants, glidants, surface modifying agents or surfactants, fragrances, suspending agents, emulsifying agents, nonaqueous vehicles, preservatives, antioxidants, adhesives, agents to adjust pH and osmolarity (e.g. buffering agents), preservatives, thickening agents, sweetening agents, flavoring agents, taste masking agents, colorants or dyes, penetration enhancers and substances added to improve appearance of the composition.

[0041] Compositions of the present invention may be administered in any manner including, but not limited to, orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, intraarticular, intracisternal and intraventricular.

[0042] A therapeutically effective amount of the composition required for use in therapy varies with the length of time that activity is desired, and the age and the condition of the patient to be treated, among other factors, and is ultimately determined by the attendant physician. In general, however, doses employed for human treatment typically are in the range of about 0.001 mg/kg to about 500 mg/kg per day, for example about 1 μg/kg to about 1 mg/kg per day or about 1 μg/kg to about 100 μg kg per day. For most large mammals, the total daily dosage is from about 1 to 100 mg, preferably from about 2 to 80 mg. The dosage regimen may be adjusted to provide the optimal therapeutic response. The desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more subdoses per day.

[0043] Illustratively, a composition of the invention may be administered to a subject to provide the subject with cis-clomiphene or an analog thereof in an amount of about 1 μg/kg to about 1 mg/kg body weight, for example about 1 μg kg, about 25 μg kg, about 50 μg kg, about 75 μg/kg, about 100 μg/kg, about 125 μg/kg, about 150 μg/kg, about 175 μg/kg, about 200 μg/kg, about 225 μg/kg, about 250 μg kg, about 275 μg/kg, about 300 μg kg, about 325 μg/kg, about 350 μg/kg, about 375 μg/kg, about 400 μg/kg, about 425 μg/kg, about 450 μg kg, about 475 μg/kg, about 500 μg/kg, about 525 μg/kg, about 550 μg kg, about 575 μg kg, about 600 μg/kg, about 625 μg/kg, about 650 μg/kg, about 675 μg kg, about 700 μg kg, about 725 μg/kg, about 750 μg/kg, about 775 μg/kg, about 800 μg/kg, about 825 μ§/1¾ about 850 μg/kg, about 875 μ /kg, about 900 μπ/kg, about 925 μg/kg, about 950 μg/kg, about 975 μg/kg or about 1 mg/kg body weight..

[0044] In a preferred embodiment, compositions according to the present invention comprise cw-clomiphene at a dosage between one mg to about 200 mg (although the determination of optimal dosages is with the level of ordinary skill in the art). The composition may comprise cw-clomiphene at a dosage of about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 12 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 1 10 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg or there between. The composition is preferably substantially free of /rara-clomiphene and may comprise about 0% w/w trans-cloimphene. Analogs of the cw-isomer of clomiphene are also useful in the practice of the present invention.

[0045] Compositions of the present invention may also be administered long-term. In this regard, the compositions may be administered for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more months. The compositions may also be administered for an administration period of at least 1, 2, 3, 4, 5, 6, 7 8, 9, 10 or more years. Preferably the composition is administered for a period of between 3 months and 5 years. During the administration period, the composition may be administered daily or periodically such as every other day and the like.

[0046] Compositions of the present invention may also be administered intermittently. For example, the compositions may be administered for an administration period of 1, 2, 3, 4, 5, or more weeks, followed by a period of discontinuance, followed by an administration period of 1 , 2, 3, 4, 5 or more weeks, and so on.

[0047] All of the references referred to herein are incorporated by reference in their entirety.

[0048] The following Examples are meant to be illustrative of the invention and are not intended to limit the scope of the invention as set out is the appended claims EXAMPLE 1

Hormonal Effect of Cis-Clomiphene on Mice

[0049] A study was conducted to assess the acute oral toxicity potential of the test substances Enclomiphene citrate (trans-clomiphene) and Zuclomiphene citrate (cis-clomiphene) when administered to male mice by oral gavage. Seventy-five mice were divided into five treatment groups of fifteen mice each. Mice in Group I received placebo (1 %

carboxymethylcellulose (CMC) in PBS) and served as a control group. Mice in Group II received the high dose (40 milligrams per kilogram (MPK)) of Enclomiphene citrate and mice in Group III received the low dose (4 MPK) of Enclomiphene citrate. Mice in Group IV received the high dose (40 MPK) of Zuclomiphene citrate and mice in Group V received the low dose (4 MPK) of Zuclomiphene citrate. Body weights were measured weekly and cage side observations were taken at least twice a day for morbidity, mortality, injury and availability of food and water. Food consumption was monitored weekly and at the end of the study serum samples were obtained from each animal and hormones were analyzed.

[0050] Animals

[0051] Male albino mice were acclimated for at least 5 days prior to testing. Normal weight gain, appearance and behavior were factors used to select healthy animals for testing. Only naive animals were selected. A weight-stratified randomization procedure was employed. Mice were housed 5 per group per box and fed PMI Feeds, Inc. Formulab #5008, or equivalent, available ad libitum prior to fasting and after dosing; analyzed by manufacturer for nutritional content. Tap water was available ad libitum. There were no known contaminants in the feed or water available to laboratory animals that would be expected to interfere with the study. The room animals were housed had a target temperature of 22°C ± 3° and relative humidity of 30-70% with a 12-hour light/dark cycle (regulated automatically).

[0052] Histology

[0053] At the end of the study, a detailed necropsy was performed on all mice and the following tissues were weighed preserved in formalin: adrenal gland, epididymis, heart, kidney, liver, seminal vesicle, skeletal muscle (biceps femoris), spleen, testis, thymus and thyroid (with parathyroid). Histopathologic analysis was performed on the preserved tissues. [0054] Statistical Analysis

[0055] Statistical analysis was conducted on the mean body weights and absolute organ weights by an unpaired t-test and the p value was determined for each individual treatment group when compared to the placebo control. ANOVA was used for analysis of groups as appropriate. Hormonal data that was skewed, kurtotic, or abnormally distributed was analyzed by non-parametric methods Mann- Whitney (Wilcoxon) or Kruskal Wallis.

Categorical distributions among groups were analyzed by Fisher's exact test.

[0056] RESULTS

[0057] Organ Weights

[0058] Reproductive tissues and kidneys were affected by the administration of test compounds (Figure 1 A-D). Testes weight (Figure 1A) was significantly lower with 40 MPK Zuclomiphene citrate treatment compared to placebo, Enclomiphene citrate or 4 MPK Zuclomiphene citrate treatment groups. Examination of the testes showed testicular degeneration and an absence of sperm in the seminiferous tubules. Leydig cells surrounding the tubules were reduced in size and numbers and appeared to be degenerating.

Histopathology of the testes in the 40 MPK Zuclomiphene citrate treatment group defined lesions of moderately severe to severe testicular degeneration in 13 of the 15 animals evaluated, compared to light (3/14) and moderate (1/13) animals in the 40 or 4 MPK Enclomiphene citrate treatment groups (Table 1).

Table 1. Summary of testicular degeneration.

[0059] Testes were described to have very few spermatozoa observed in the seminiferous tubules. The tubules were described as containing germinative cells within the lumina, reduced layers of germinative epithelium and numerous mitotic figures in the spermatogonia levels. No mature spermatozoa were observed. Widespread tubules filled with large, germinal epithelial cells and reduced numbers of spermatozoa were noted. There was widespread loss of tubules with prominent interstitial cells. Scattered tubules were filled with mineralized debris. Reduced numbers of tubules were noted. Examples of micrographs of each group are seen in Figure 2.

[0060] Analysis of other organs of reproductive significance showed disruptions. The epididymis was reduced in weight in both of the Zuclomiphene citrate treatment groups (Figure IB). Microscopic examination of the Group IV (40 MPK Zuclomiphene citrate) showed an absence of sperm in the epididymis. This observation correlates with the testicular degeneration and lack of sperm in the testes seen in this treatment group. Seminal vesicles were reduced in weight compared to controls (Figure 1C). Histopathology confirmed no significant findings other that a reduction in size. Kidneys decreased in overall weight with Zuclomiphene citrate treatment (Figure 1 D). Kidney weights in both doses of Zuclomiphene citrate were significantly reduced. Histopathology indicated that there were no significant findings for the kidneys in any treatment group. Organs examined that had no change in weight after 12 weeks of drug administration compared to control animals were spleen, thymus, femoral muscle, liver, heart, adrenal gland and thyroid/parathyroid (with trachea).

[0061] Animal Weight Changes

[0062] Overall the weight of Zuclomiphene citrate (4 and 40 MPK) treated animals significantly decreased by the end of the 91 day treatment period (Figure 3). Overall the health of the animals in these treatment groups was described as "agitated" manifesting as shredding of food. Due to excessive shredding of food data dealing with food consumption could not be calculated. Animals dosed with Enclomiphene citrate (4 and 40 MPK) showed no changes in body weight the duration of the study.

[0063] Hormone Analysis

[0064] Hormone levels were determined in treated animals after 91 days of treatment (Table 2). Table 2. Hormone profile.

*Δ from coniiols

t Δ from Group IV

[0065] At the highest dose given, Zuclomiphene citrate reduced the level of LH and testosterone versus the control group (p = 0.0025 and p = 0.0006, Mann- Whitney

(Wilcoxon)). The levels of serum testosterone were variable among the groups but the use of 40 MPK Zuclomiphene citrate resulted in levels of testosterone lowered than any other group. In fact, the very lowest values measured (<50 ng/dL) were more concentrated in Group IV (7/8) than all the other groups combined (5/45), highly suggestive of an effect (p < 0.001, Fisher's exact test). The effects were dose-dependent since the 40 MPK group (Group IV) was lower and different form the 4 MPK group (Group V) which did not differ from controls (p = 0.23, Mann- Whitney (Wilcoxon)). Although the high dose Enclomiphene citrate group tended to have lower serum T, it could not be shown to be statistically significant versus controls (p = 0. 10, Mann-Whitney (Wilcoxon)) nor were Groups I, II, III and V shown to have significant differences among them (p = 0.06, Kruskal-Wallis). Groups I, II and V were not different in terms of serum T (p = 0.34, Kruskal-Wallis) and the mean was 585 ± 588 ng/dL suggesting the normal level of serum T in these mice and its variability. The effects of 40 MPK were similar in the case of serum LH. The use of 40 MPK of Zuclomiphene citrate resulted in low levels of serum LH (p = 0.0025, Mann- Whitney (Wilcoxon)) compared to controls. The 40 MPK group of Zuclomiphene citrate was lower in LH than any other group considered individually or in total. Luteinizing Hormone was lower in Group III (40 MPK of Zuclomiphene citrate) than in Group V (4 MPK of Zuclomiphene citrate) again showing a dose-dependence. Using a value of 2 mlU/ml as a lower cut-off value, Group IV demonstrated the lowest proportion of low values (9/14) than all the other groups combined (7/55), highly suggestive distribution (p < 0.0002, Fisher's exact test). Groups I, II, III and V were the same level taken as a whole (p = 0.97, ANOVA) and would appear to represent the mean LH value for these mice at 6.24 ± 7.13 mlU/ml. Comparing serum T values to serum LH values showed at, in general, as LH was higher, so was T. A linear correlation showed a relatively weak association mathematically (r = 0.14, p = 0.35, ANOVA) and noted was a number of outliner or 'spikes" of LH that seemed to work against a better correlation.

[0066] The effects on serum FSH were not analogous to the effects on T and LH. The highest serum FSH values were found in the control group. The best determination of serum FSH for these mice would be that in the control group (71 ± 20 mlU/ml), a value with a smaller range of variation than serum T or LH. High dose Zuclomiphene citrate was associated with a low FSH compared to control (p = 0.00003, Mann- Whitney (Wilcoxon)) and the low dose Zuclomiphene citrate group was different from both the high dose group (p = 0.0002, Mann-Whitney (Wilcoxon)) and the control (p = 0.031, Mann- Whitney

(Wilcoxon)). The low dose of Enclomiphene citrate was associated with lower serum FSH than the high dose of Enclomiphene citrate and the control. There was a relatively weak relationship between serum T and FSH when compared in a linear correlation (r = 0.25, p = 0.088, ANOVA). Serum LH and FSH did not show a linear correlation that suggested a strong relationship (r = -0.02, p = 0.89, ANOVA).

[0067] DISCUSSION

[0068] In the present study, the effects of administration of Enclomiphene citrate and Zuclomiphene citrate were assessed in mice. Differences in the different treatment groups were observed. Overall weight was decreased in animals treated with Zuclomiphene citrate, compare to both controls and Enclomiphene citrate treated animals. Normal spermatogenesis was present in all groups, with the exception of the high Group IV high dose (40 MPK) of Zuclomiphene citrate. Testicular degeneration in this group was evident and no sperm production was seen. Spermatogonia and primary spermatocytes are present, but spermatogenesis was arrested. Leydig cells exterior to the seminiferous tubules appear to be degenerating. Conclusion of the Pathology summary was that Zuclomiphene citrate (40 MPK) treatment by oral gavage daily for 91 days appeared to produce consistent testicular degeneration. Tissue weight was decreased in the 4 MPK group, but normal spermatogenesis was seen. In addition to testicular degeneration other reproductive tissues were adversely affected by Zuclomiphene citrate administration Epididymis size in the Zuclomiphene citrate treated group (4 MPK and 40 MPK) and weight was significantly decreased with the epididymal sperm content being absent in the 40 MPK group confirming the lack of testicular sperm production in that group. Also, seminal vesicles were significantly decreased in size and weight in the 4 and 40 MPK Zuclomiphene citrate treated groups.

[0069] There were strong effects on serum T, LH and FSH at 91 days of dosing with Zuclomiphene citrate at the highest dose. There was much less of an effect at the lower dose and a clear dose-dependency. The effects of Enclomiphene citrate were not the same as Zuclomiphene citrate, suggesting the clear dichotomy for these two compounds. At high dose, Zuclomiphene citrate appeared to have estrogen agonist activity in lowering LH and FSH. Serum T tended to be lower with Enclomiphene citrate but the effects were not proven here. The clear effects of enclomiphene citrate in men in clinical trials may have been due to the fact that those men had low serum T before dosing whereas, these mice were normal. The effects on LH were not the same as on FSH and mice seemed more sensitive to treatments with regard to FSH.

[0070] The decreases in weight and function of reproductive tissues could be due to the estrogenic effects of zuclomiphene acting on the hypothalamic-pituitary-testes (HPT) axis. We cannot rule out that a direct effect of zuclomiphene on the Leydig cells resulting in poor T production as a proximal cause. These effects seen in the 91 day study would be predicted to be amplified in longer studies due to the long half-life of zuclomiphene.

[0071] CONCLUSIONS

[0072] In conclusion, this study demonstrates differences in the mouse when comparing administration of the clomiphene isomers, enclomiphene and zuclomiphene. Dramatic effects on weight, sexual organs and hormones were seen in the mouse and such results raise the question of the kinds of effects that might be seen in men taking Zuclomiphene citrate alone, a situation not usually encountered since clomid is provided as the mixture of isomers. The observation that mice treated with Zuclomiphene citrate displayed an agitated behavior manifesting as shredding of food that may be indicative of a behavioral effect that has not been described previously, to our knowledge. Clomid raises serum T and Enclomiphene citrate does as well. It is tempting to speculate that enclomiphene effectively counters zuclomiphene given the superior binding affinity of enclomiphene. At the same time, the longer half-life of zuclomiphene might suggest that pernicious effects may occur over time. Perhaps this may account for whatever failures may be attributed to Clomid. The effects of high dose zuclomiphene described in the mouse model herein are consistent with our findings that zuclomiphene depresses testosterone levels in primates and indicates the use of zuclomiphene as an agent for androgen deprivation therapy in human men.