The Isomers of Clomiphene Citrate have Dissimilar Dispositions Once Ingested: Results of a Mouse ADME Study

Fontenot GK*, Wiehle RD, Hsu K and Podolski J

Repros Therapeutics, The Woodlands, Texas, USA

*Corresponding Author:
Gregory K Fontenot, PhD
Repros Therapeutics 2408 Timberloch Place B-7
The Woodlands, Texas 77380, USA
Tel: 281 719 3455
Fax: 281 719 3446
Email: [email protected]

Received date: January 17, 2017; Accepted date: January 18, 2017; Published date: January 25, 2017

Citation: Fontenot GK, Wiehle RD, Hsu K, et al. The Isomers of Clomiphene Citrate have Dissimilar Dispositions Once Ingested: Results of a Mouse ADME Study. Adv Tech Clin Microbiol. 2017, 1:1.

Copyright: © 2017 Fontenot GK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 
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Abstract

Background: Clomiphene Citrate is composed of two structural isomers: cis- or Zuclomiphene citrate (Zu) and trans- or Enclomiphene citrate (En). Clomiphene, as well as isolate isomers, has recently been shown to have effects on Ebola virus infections. It has been shown that the enclomiphene molecule is an estrogen antagonist and that zuclomiphene is inactive. We demonstrate here that the two isomers have different fates once ingested and the tissues that absorb each are distinct from each other to lead to different biologic effects. Methods and findings: We studied these molecules by employing 14C-labelled versions of each in C-57 black mice. Mice were given the same oral dose, but sacrificed at different time periods. Each isomer could be followed separately. Enclomiphene was rapidly lost such that the majority was found in low amounts after 24 h. Zuclomiphene was distributed to more organs and remained associated with discrete tissues for longer periods of time. Remarkable exceptions were the pigmented organs of the eye, which retained both compounds. Notable was the specific absorption in individual tissues and the lack of clearance in certain cases. The ratio of zuclomiphene to enclomiphene (Zu/En) demonstrated the promiscuous nature of the zuclomiphene and the specific absorption. The tissue/plasma ratios demonstrated those tissues that were accrued or failed to clear compounds. Important differences were found in the lack of clearance of isomers in the eye, gall blabber/bile, brain, lung, fat, adrenals, kidneys and reproductive tissues. Conclusion: The adverse effects of Clomiphene citrate in the eye and male reproductive organs may be rationalized by lack of clearance of higher levels of zuclomiphene as well as effects of Ebola virus infection. An additional element of this study was to determine levels that would not be expected to represent a significant radiation exposure risk to human male volunteers in future ADME studies.

Keywords

Clomiphene; Enclomiphene; Zuclomiphene; Blood-brain barrier; Metabolism; Uveal tract.

Introduction

Clomiphene citrate has been approved for use in women to induce ovulation [1-3] and has been used off-label in men to raise serum testosterone [4-9]. The latter use was rationalized early [10] by the recognition that a bolus dose of Clomiphene citrate could be used as a diagnostic tool to determine the functionality of the hypothalamic-pituitary axis by the stimulation of release of Luteinizing Hormone (LH) in a subject with low gonadotropins. Clomiphenes and its isolated isomes, have also been shown to have an effect on progression of disease after Ebola virus infection in cell culture and mouse model systems [11, 12].

Clomiphene citrate is considered to be a SERM, i.e., a compound possessing estrogen agonist or antagonist properties depending on the tissue. Clomiphene citrate was described as a mixture of two isoforms, enclomiphene (trans-isomer) and zuclomiphene (cis-isomer), with estrogen agonist or antagonist properties [13-16]. The structure of the isomers described in the initial papers is shown in Figure 1. Clomiphene citrate is available commercially as a mixture of the two isomers. Clomiphene citrate is approximately 62% enclomiphene and 38% zuclomiphene.

advanced-techniques-clinical-microbiology-Structure-isomers

Figure 1: Structure of the isomers.

Enclomiphene citrate has anti-estrogenic properties [17] and appears to block the negative feedback inhibitory effects of estradiol on the hypothalamic-pituitary axis resulting in increased levels in both LH and follicle stimulating hormone (FSH) which stimulate endogenous intragonadal testosterone production and spermatogenesis in men [8, 18].

Clomiphene citrate presented no adverse sequelae due to zuclomiphene which is, at worst, an inactive congener. That view was not blunted despite the known much longer half-life of zuclomiphene [19, 20] and the absence of estrogen antagonism [17, 21], the key feature of the activity of the mixture. Early ADME studies of Clomiphene citrate describe loss through the feces and urine after a single dose [13, 22]. Previous descriptions of The ADME profiles of Clomiphene citrate are thus inadequate since the longer-lived zuclomiphene would be in excess over the more active enclomiphene.

We have determined that one of the two isomers of Clomiphene citrate, Zuclomiphene citrate, a component of a drug believed to be safe, has deleterious effects on the male reproductive organs of mice [23]. There are known effects of clomiphene on the eyes [24-26] as has been recognized in the product label [27]. These effects might be a result of an accumulation of the zuclomiphene isomer over time.

Materials and Methods

The 14C-labelling of Enclomiphene and Zuclomiphene was done at Ricerca Concord, OH. The labeled compounds as citrate salts were done after synthesis. The positon of the 14C-label was distributed among the carbons of one of the rings (Figure 1). The in-life dosing to C-57 black mice was performed at Covance Laboratories Inc. Madison, WI under the direction of Randall Press. All procedures performed in animals were under the guidance of the “Guide for the Care and Use of Laboratory Animals”. Tissue distribution of 14CEnclomiphene related radioactivity was assessed following a single oral administration of 14CEnclomiphene citrate to male and female pigmented (C57 black) mice (Group 1). Tissue distribution of 14CZuclomiphene related radioactivity was also investigated following a single oral administration of 14CZuclomiphene citrate to male pigmented (C57 Black) mice (Group 2). Dose formulations were prepared as solutions on the day of dose administration by combining appropriate amounts of radiolabeled and nonradiolabeled Enclomiphene citrate or radiolabeled and non-radiolabeled Zuclomiphene citrate in 0.5% methylcellulose and 0.2% Tween 80 in reverse osmosis water at a nominal concentration of 2.0 mg/mL and a target radioactivity dose of 100 μCi/20 mg/kg. In Group 1, two animals/sex/time points were sacrificed at 0.25, 1, 4, 24, 72, 168, 240, 336, 504 and 840 h. For Group 2, two animals/time points were sacrificed at 1, 4, 24, 72, 168, 240, 336, 504, 840 and 1008 h post dose. Blood was collected and plasma was harvested at specified time points and carcasses were prepared for QWBA. Sections were collected from one carcass per time point and exposed to phosphor imaging screens. The images were processed for determination of the radioactivity concentrations in selected tissues. Blood and plasma were analyzed for concentrations of radioactivity using liquid scintillation counting (LSC). The radioactivity concentrations were expressed as ng equivalents 14CEnclomiphene/g or ng equivalents 14CZuclomiphene/g of sample, as applicable. The pharmacokinetic analysis of 14CEnclomiphene citratederived and 14CZuclomiphene citratederived radioactivity were conducted for blood, plasma and tissues and radiation dosimetry parameters were calculated. There was no attempt to separate and thus determine the identity of the 14C-derived products, i.e., to determine the metabolites of each drug. A description of the kinds of metabolites derived from each of the drugs in women has already been shown [28, 29]. We have also determined metabolites of each in liver hepatocytes of four species and characterized the metabolites detected in dogs after both acute and chronic dosing using non-labelled materials (internally generated data not shown).

Results

Test article and dose formulation analysis

Radiopurity and stability

The HPLC analyses performed by Covance showed the radiopurities of 14C-Enclomiphene and 14C-Zuclomiphene to be 98.2 and 96.6%, respectively, prior to dose preparation. The mean radiopurity values from HPLC analysis for 14CEnclomiphene of predose and postdose aliquots were 98.0 and 97.9%, respectively. The mean radiopurity values from HPLC analysis for 14C-Zuclomiphene of pre-dose and post-dose aliquots were 97.2 and 97.3%, respectively. These values confirmed stability of the test articles under conditions of the study.

Concentrations of radioactivity in blood and plasma

Group 1, 14C-Enclomiphene

After a single 20 mg/kg oral dose of 14C-Enclomiphene to male and female mice, the maximum average plasma concentrations of radioactivity were 1370 and 2140 ng equivalents 14C-Enclomiphene/g, respectively, observed at 1 and 4 h post-dose, respectively, concentrations declined to BLQ, by 240 h post-dose. The mean concentration versus time profile is presented graphically in Figure 2A. All reported concentrations are in ng equivalents of free base/g of matrix.

Group 2, 14C-Zuclomiphene

After a single 20 mg/kg oral dose of 14C-Zuclomiphene to male mice, the maximum average plasma concentration of radioactivity was 697 ng equivalents 14C-Zuclomiphene/g, observed at 4 h post-dose, concentrations declined to BLQ by 336 h post-dose. The mean concentration versus time profile is presented graphically in Figure 2B.

advanced-techniques-clinical-microbiology-Mean-concentration

Figure 2:Mean concentration versus time profile.

Tissue distribution of radioactivity by QWBA

Group 1, 14C-Enclomiphene

The concentrations of radioactivity in tissues are presented in Table 1 (for males) and Table 2 (for females). All reported concentrations are in ng equivalents of free base/g of matrix.

Tissue 4 h Tissue 24 h
Bile 728000 Eye uveal tract 6680
Gall bladder 583000 Liver 4180
Liver 29800 Gall bladder 3960
Pancreas 14200 Bile 2420
Kidney cortex 12700 Harderian gland 2210
Kidney medulla 12700 Eye(s) 1560
Kidneyls) 12700 Pancreas 1380
Harderian gland 11600 Intra-orbital lacrimal gland 1280
Eye uveal tract 10300 Kidney cortex 1220
Lung(s) 9840 Preputial gl 1170
Urine 9710 Kidneyls) 1130
Small intestine 9340 Kidney medulla 1040
Pituitary gland 6160 Cecum 977
Urinary bladder 6010 Pituitary gland 970
Esophagus 5690 Prostate gland 961
Exorbital lacrimal gland 5550 Small intestine 689
Intra-orbital lacrimal gland 5020 Exorbital lacrimal gland 676
Salivary(s) 4260 Large intestine 635
Spleen 4180 Salivary(s) 469
Thyroid 4120 Epididymis 456
Bone marrow 3850 Thyroid 444
Lymph node(s) 3760 Testisles) 442
Diaphragm 3010 Diaphragm 440
Adrenals 2880 Thymus 436
Cecum 2830 Lymph node(s) 431
Fat (brown) 2810 Lung(s) 384
Stomach 2770 Esophagus 383
Thymus 2760 Spleen 349
Eye Is) 2450 Urinary bladder 302
Large intestine 2360 Fat (brown) 258
Myocardium 1840 Skin (pigmented) 233
Preputial gland 1660 Bone marrow 222
Prostate gland 1390 Adrenals 221
Skin (pigmented) 1260 Stomach 197
Fat (abdominal) 1210 Fat (abdominal) BLQ
Muscle 919 Muscle BLQ
Brain cerebrum 855 Brain cerebrum BLQ
Seminal vesicle(s) 823 Seminal vesicle(s) BLQ
Blood 815 Blood BLQ
Brain cerebellum 786 Brain cerebellum BLQ
Spinal cord 716 Spinal cord BLQ
Brain medulla 678 Brain medulla BLQ
Bone 672 Bone BLQ
Testisles) 669 Myocardium BLQ
Brain olfactory lobe 595 Brain olfactory lobe BLQ
Nasal turbinates 417 Nasal turbinates BLQ
Epididymis 382 Urine BLQ
Eye lens BLQ Eye lens BLQ

Table 1: ng equivalents [14C]-enclomiphene/g in Group 1 males.

Tissue 4 h Tissue 24 h
Bile 383000 Gall bladder 19400
Gall bladder 256000 Bile 16400
Liver 28500 Liver 7650
Kidney medulla 12100 Cecum 4310
Pancreas 11700 Eye uveal tract 3720
Harderian gland 11400 Small intestine 2890
Small intestine 10500 Kidney cortex 1970
Lung(si_ 10400 Kidney(s) 1940
Cecum 9340 Kidney medulla 1870
Kidney(s) 8830 Pancreas 1510
Eye uveal tract 7350 Harderian gland 1470
Kidney cortex 5800 Large intestine 1170
Large intestine 5200 Intra-orbital lacrimal gland 1140
Adrenals 4310 Stomach 1090
Exorbital lacrimal gland 4130 Adrenals 997
Pituitary gland 3810 Thyroid 916
Esophagus 3340 Diaphragm 901
Spleen 3310 Esophagus 778
Salivary(s) 3240 Eye(s) 773
Lymph node(s) 3160 Exorbital lacrimal gland 730
0vary(ies) 3000 lung(s) 713
Thyroid 2920 Ovary(ies) 628
Bone marrow 2880 Spleen 577
Fat (brown) 2860 Pituitary gland 542
Intra-orbital lacrimal gland 2720 Fat (brown) 458
Diaphragm 2710 Uterus 458
Urine 2700 Salivary(s) 448
Stomach 2610 Urinary bladder 423
Thymus 1990 Urine 423
Fat (abdominal) 1780 Lymph node(s) 377
Eye(s) 1520 Thymus 304
Uterus 1480 Blood 236
Myocardium 1440 Myocardium 198
Urinary bladder 1290 Fat (abdominal) 197
Blood 1060 Bone marrow BLQ
Skin (pigmented) 999 Skin (pigmented) BLQ
Muscle 687 Muscle BLQ
Brain medulla 594 Brain medulla BLQ
Spinal cord 558 Spinal cord BLQ
Brain cerebrum 549 Brain cerebrum BLQ
Brain cerebellum 454 Brain cerebellum BLQ
Brain olfactory lobe 427 Brain olfactory lobe BLQ
Nasal turbinates 397 Nasal turbinates BLQ
Bone 292 Bone BLQ
Eye lens BLQ Eye lens BLQ

Table 2 ng equivalents [14C]-enclomiphene/g in Group 1 females.

The single 20 mg/kg oral dose of 14C-Enclomiphene to male and female pigmented mice was well absorbed and widely distributed to tissues by 1 h post-dose. Peak tissue concentrations were reached at 1 or 4 h post-dose and the concentrations of radioactivity in tissues declined over time in both genders. For both genders, the highest concentrations of drug-derived radioactivity were in the contents of the gastrointestinal (GI) tract and bile. The radioactivity concentrations in the contents of the GI tract were based on qualitative visual assessment of the autoradiographic data. In male and female mice, bile had a maximum concentration of 728000 and 383000 ng equivalents 14C-Enclomiphene/g, respectively, observed at 4 h post-dose. Hepato-biliary excretion was an important route of elimination.

Tissue 4 h Tissue 24 h
Bile 105000 Harderian gland 43900
Gall bladder 90200 Bile 39800
lung(s) 72400 Gall bladder 33600
Kidneycortex 53500 lung(s) 33600
Kidneyls) 43700 Eye uveal tract 30000
Pituitary gland 34200 Kidneycortex 29400
Kidney medulla 32400 Kidney(s) 24600
Spleen 31600 Liver 18200
Harderian gland 29300 Kidney medulla 17800
Liver 29100 Exorbital lacrimal gland 15000
Adrenals 26100 Adrenals 14800
Pancreas 24900 Intra-orbital lacrimal gland 14000
Small intestine 21900 Spleen 13500
Cecum 21000 Pancreas 12700
Salivary(s) 20300 Salivary(s) 12700
Exorbital lacrimal gland 20000 Pituitary gland 12600
Fat (brown) 19800 Thymus 11700
Bone marrow 19500 Fat (brown) 11400
Intra-orbital lacrimal gland 18700 Lymph node(s) 10600
Eye uveal tract 18400 Epididymis 10500
Thyroid 17400 Small intestine 10000
Diaphragm 15200 Bone marrow 9930
Stomach 14900 Urinary bladder 9630
Myocardium 14700 Cecum 9260
Fat (abdominal) 14000 Diaphragm 8190
Thymus 12500 Thyroid 7410
Lymph node(s) 11900 Brain cerebrum 7110
Esophagus 10700 Testisles) 6810
Urinary bladder 9140 Myocardium 6790
Large intestine 7820 Large intestine 6070
Preputial gland 7630 Eye(s> 5760
Brain cerebrum 7070 Fat (abdominal) 5530
Muscle 5860 Prostate gland 4760
Brain cerebellum 5310 Spinal cord 4460
Spinal cord 5000 Stomach 4170
Seminal vesicle(s) 4630 Nasal turbinates 4020
Brain medulla 4620 Brain cerebellum 3920
Prostate gland 4460 Brain medulla 3800
Brain olfactory lobe 4390 Esophagus 3770
Epididymis 4390 Seminal vesicle(s) 3490
Nasal turbinates 4290 Brain olfactory lobe 3300
Blood 4250 Skin (pigmented) 3220
Bone 3060 Preputial gland 3140
Eye Is) 2840 Muscle 2910
Testis(es) 2830 Urine 2530
Skin (pigmented) 2680 Blood 1670
Urine 2350 Bone 1440
Eye lens BLQ Eye lens 302

Table 3: ng equivalents [14C]-zuclomiphene in Group 2 males.

The highest peak tissue radioactivity concentrations in males were in gall bladder, liver, kidney cortex, kidney and pancreas with values of 583000, 46800, 16800, 15600 and 14200 ng equivalents 14C-Enclomiphene/g, respectively. All other analyzed tissues had peak concentration values less than 13000 ng equivalents 14C-Enclomiphene/g. For females, the highest peak tissue radioactivity concentrations were gall bladder, liver, kidney medulla, pancreas and harderian gland with values of 256000, 51700, 12100, 11700 and 11400 ng equivalents 14CEnclomiphene/ g, respectively. All other analyzed tissues had peak concentration values less than 11000 ng equivalents 14CEnclomiphene/ g. The lowest peak concentrations were detected in nasal turbinates (448) of males and bone (292) of females.

Group 2, 14C-Zuclomiphene

The concentrations of radioactivity in tissues are presented in Table 3 (males only). All reported concentrations are in ng equivalents of free base/g of matrix.

14C-Zuclomiphene was well absorbed and widely distributed to tissues by 1 h post-dose. Peak tissue concentrations were reached at 1 or 4 h post-dose for most tissues, with some tissues having peak concentrations at 24 and 72 h post-dose. The highest concentrations of drug-derived radioactivity were in the contents of the GI tract and bile. The radioactivity concentrations in the contents of the GI tract were based on qualitative visual assessment of the autoradiographic data. Bile had a maximum concentration of 105000 ng equivalents 14CZuclomiphene/ g observed at 4 hours post-dose. Hepato-biliary excretion was an important route of elimination.

Dosimetry

Group 1, 14C-Enclomiphene

Dosimetry calculations from pigmented male mice data predict that uveal tract, eye, liver, pancreas and renal cortex will be exposed to the highest doses of radiation in humans following a single oral dose of 14C-Enclomiphene. In man, these matrices are estimated to be exposed to 215, 53.1, 11.4, 8.45 and 6.02 mRad or mrem, respectively (2.15, 0.531, 0.114, 0.0845 and 0.0602 mGy, respectively).

Dosimetry calculations from pigmented female mice data predict that uveal tract, eye, liver, small intestine and pancreas will be exposed to the highest doses of radiation in humans following a single oral dose of 14C-Enclomiphene. In women, these matrices are estimated to be exposed to 186, 49.7, 16.7, 9.93 and 8.01 mRad or mrem, respectively (1.86, 0.497, 0.167, 0.0993 and 0.0801 mGy, respectively).

Group 2, 14C-Zuclomiphene

Dosimetry calculations from pigmented male mice data predict that uveal tract, epididymis, eye, testes and renal cortex will be exposed to the highest doses of radiation in humans following a single oral dose of 14C-Zuclomiphene. In man, these matrices are estimated to be exposed to 365, 185, 91.8, 88.9 and 62.0 mRad or mrem, respectively (3.65, 1.85, 0.918, 0.889 and 0.620 mGy, respectively).

Discussion

There were no apparent gender related differences in the tissue concentration data for male and female mice. Tissue to plasma concentration ratios were greater than one for most tissues through 24 h post-dose and were all above one where measurable through 168 h post-dose. Radioactivity concentrations in central nervous system tissues protected by the blood:brain barrier (cerebellum, cerebrum, medulla, olfactory lobe and spinal cord) were low and dropped to nonmeasurable levels by 24 h post-dose. Low levels of 14CEnclomiphene- derived radioactivity distributed to the reproductive tissues of the male and female mice, but cleared by 72 h post-dose. Elimination was nearly complete for most tissues by 72 h post-dose. By the final sampling time of 840 h post-dose, no tissues, other than the eye and uveal tract of the eye, contained measurable concentrations of radioactivity.

The accrual in 47 male tissues individually assessed are summed and given for both zuclomiphene and enclomiphene in Figure 3. We chose to use “accrual” to indicate amounts found in tissue in distinction to “accumulation” which might indicate an active process of tissue uptake or lack of clearance over that found in biological tissues. Peak tissue concentrations were reached at 1 or 4 h post-dose for most tissues, with some tissues having peak concentrations at 24 and 72 h post dose. There were major differences in the uptake and disposition of zuclomiphene and enclomiphene. Enclomiphene has been shown to have an approximate half-life of 7 h compared to the 14 day half-life of zuclomiphene. Every tissue examined demonstrated high lack of clearance of 14C-zuclomiphene over plasma at 1, 4, 24 and 72 h post dose (Figure 4). These differences were an effect of the differing half-lives of the two isomers. We propose there is not a differential accumulation of the two isomers, rather a difference of half-life and of each of the isomers resulting in a differing elimination of each isomer in a specific tissue.

advanced-techniques-clinical-microbiology-zuclomiphene-enclomiphene

Figure 3:Accrual in 47 male tissues for both zuclomiphene and enclomiphene.

advanced-techniques-clinical-microbiology-post-dose

Figure 4:14C-zuclomiphene over plasma at 1, 4, 24 and 72 h post dose.

The highest concentrations of radioactivity in eye uveal tract (10300 and 7350 ng equivalents 14C-Enclomiphene/g in male and female pigmented mice, respectively) were observed at 4 h post-dose. Radioactivity concentrations in eye uveal tract declined, but remained measurable through 840 h post-dose (1410 and 1150 ng equivalents 14C-Enclomiphene/g in male and female pigmented mice, respectively). The radioactivity concentrations in eye uveal tract at 840 h post-dose in male and female mice represented approximately 7- and 6-fold decreases in radioactivity concentrations, respectively, from the observed peak concentrations. The highest concentrations in pigmented skin were 1260 ng equivalents 14C-Enclomiphene/g at 4 h postdose in males and 999 ng equivalents 14C-Enclomiphene/g at 4 h post-dose in females. The radioactivity concentrations in pigmented skin declined over time and dropped to BLQ by 72 and 24 h post-dose in male and female mice, respectively. These data suggested that 14C-Enclomiphene-related radioactivity was selectively associated with melanin-containing tissues of the eye.

For pigmented male mice, the 14C-Enclomiphene-derived radioactivity declined with half-life (T1/2) values ranging from 2.47 h (myocardium) to 1012 h (eye), with most matrices showing T1/2 values less than 20 h. The area under the concentration-time curve from 0 to infinity (AUC0-∞) ranged from 11064 ng equivalents 14C-Enclomipheneh/g in spinal cord to 7567172 ng equivalents 14C-Enclomipheneh/g in gall bladder. In pigmented female mice, the 14C-Enclomiphene-derived radioactivity declined with half-life values ranging from 5.17 hours (lung) to 707 h (eye), with most matrices showing T1/2 values less than 20 h. The AUC0-∞ ranged from 5115 ng equivalents 14C-Enclomipheneh/g in the olfactory lobe to 3973695 ng equivalents 14C-Enclomipheneh/g in the gall bladder.

The highest peak tissue radioactivity concentrations in male mice were in gall bladder, lung, kidney cortex, liver and harderian gland with values of 90200, 72400, 53500, 45900 and 43900 ng equivalents 14C-Zuclomiphene/g, respectively. All other analyzed tissues had peak concentration values less than 43800 ng equivalents 14C-Zuclomiphene/g. The lowest peak concentrations were detected in plasma (644) and lens of eye (302). Elimination was nearly complete for most tissues by 504 h post-dose. By the final sampling time of 1008 hours post-dose, no tissues, other than the eye and uveal tract of the eye, contained measurable concentrations of radioactivity. Unlike 14C-Enclomiphene, tissue to plasma concentration ratios following single oral dosing with 14C-Zuclomiphene were greater than one for all tissues with measurable concentrations of radioactivity over the time course examined, except the lens of the eye. Radioactivity concentrations in central nervous system tissues protected by the blood:brain barrier (cerebellum, cerebrum, medulla, olfactory lobe and spinal cord) were low and dropped to non-measurable levels by 168 h post-dose. Unlike 14C-Enclomiphene, 14C-Zuclomiphene-derived radioactivity concentrations remained measurable in the noncircumventricular CNS tissues through 72 h post-dose. Low levels of 14C-Zuclomiphene-derived radioactivity distributed to the testis, but cleared by 240 h post-dose.

The highest concentration of radioactivity in eye uveal tract (30000 ng equivalents 14C-Zuclomiphene/g) was observed at 24 h post-dose. Radioactivity concentrations in eye uveal tract declined, but remained measurable through 1008 hours postdose (1020 ng equivalents 14C-Zuclomiphene/g). The radioactivity concentrations in eye uveal tract at 1008 h postdose represented approximately a 29-fold decrease in radioactivity concentration from the observed peak concentration. The highest concentration in pigmented skin was 3220 ng equivalents 14C-Zuclomiphene/g at 24 h post-dose. The radioactivity concentration in pigmented skin declined over time and dropped to BLQ by 168 h post-dose.

The 14C-Zuclomiphene-derived radioactivity declined with half-life (T1/2) values ranging from 18.7 h (urinary bladder) to 411 h (uveal tract of the eye), with most matrices showing T1/2 values less than 40 h. The AUC0-∞ ranged from 60644 ng equivalents 14C-Zuclomiphene h/g in plasma to 6204197 ng equivalents 14C-Zuclomipheneh/g in uveal tract of the eye.

Conclusion

In conclusion, 14C-Enclomiphene and 14C-Zuclomiphenerelated radioactivity were widely distributed in tissues and organs of mice and were selectively associated with melanincontaining tissues. For 14C-Enclomiphene, there were no apparent gender related differences in the tissue concentration data for male and female mice. In general, 14C-Zuclomiphenerelated radioactivity had a longer biological half-life in most tissues as compared to 14C-Enclomiphene. Unlike 14CEnclomiphene, 14C-Zuclomiphene-related radioactivity was preferentially distributed to the cellular components of blood and tissue to plasma concentration ratios were greater than one for all tissues with measurable concentrations of radioactivity over the time course examined, except the lens of the eye. Based on the pharmacokinetic and dosimetry data, administration of a single 100 μCi (3.7 MBq) oral dose of 14CEnclomiphene would not be expected to represent a significant radiation exposure risk to human male or female volunteers. Administration of an oral dose of 14C-Zuclomiphene at the same level would not be expected to represent a significant radiation exposure risk to human male volunteers.

Acknowledgement

Repros would like to acknowledge and thank Randy Press and Erin Ballard at Covance for their work in performing this study.

Authorship Contributions

Participated in research design: Fontenot, Wiehle, Podolski.

Conducted experiments: Covance Laboratories Inc. Madison, WI (Under direction of Randall Press).

Contributed new reagents: Hsu

Performed data analysis: Fontenot, Wiehle

Wrote or Contributed to writing of the manuscript: Fontenot, Wiehle, Podolski.

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