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The 2014 outbreak of Ebola virus (EBOV) in Western Africa highlighted the need for anti-EBOV therapeutics. Clomiphene is a U.S. Food and Drug Administration (FDA)-approved drug that blocks EBOV entry and infection in cells and significantly protects EBOV-challenged mice. As provided, clomiphene is, approximately, a 60:40 mixture of two stereoisomers, enclomiphene and zuclomiphene. The pharmacokinetic properties of the two isomers vary, but both accumulate in the eye and male reproductive tract, tissues in which EBOV can persist. Here we compared the ability of clomiphene and its isomers to inhibit EBOV using viral-like particle (VLP) entry and transcription/replication-competent VLP (trVLP) assays. Clomiphene and its isomers inhibited the entry and infection of VLPs and trVLPs with similar potencies. This was demonstrated with VLPs bearing the glycoproteins from three filoviruses (EBOV Mayinga, EBOV Makona, and Marburg virus) and in two cell lines (293T/17 and Vero E6). Visual problems have been noted in EBOV survivors, and viral RNA has been isolated from semen up to nine months post-infection. Since the clomiphene isomers accumulate in these affected tissues, clomiphene or one of its isomers warrants consideration as an anti-EBOV agent, for example, to potentially help ameliorate symptoms in EBOV survivors.

Objectives: To determine, in a chronic dosing study, the oral toxicity potential of the test substances, enclomiphene citrate (ENC) and zuclomiphene citrate (ZUC), when administered to male mice by oral gavage., Materials and Methods: Mice were divided into five treatment groups. Group I, placebo; Group II, 40 mg/kg body weight/day ENC; Group III, 4 mg/kg/day ENC; Group IV, 40 mg/kg/day ZUC; Group V, 4 mg/kg/day ZUC. Serum samples and tissues were obtained from each mouse for analysis and body weights were measured., Results: In this chronic dosing study in mice, profound effects on Leydig cells, epididymis, seminal vesicles, and kidneys were seen, as well as effects on serum testosterone, follicle-stimulating hormone and luteinising hormone levels that were associated with ZUC treatment only. Treatment with the isolated enclomiphene isomer had positive effects on testosterone production and no effects on testicular histology., Conclusions: The present study suggests that an unopposed high dose of zuclomiphene can have pernicious effects on male mammalian reproductive organs. The deleterious effects seen when administering ZUC in male mice, justifies the case for a monoisomeric preparation and the development of ENC for clinical use in human males to increase serum levels of testosterone and maintain sperm counts., (© 2015 The Authors BJU International © 2015 BJU International Published by John Wiley & Sons Ltd.)

Objective: To determine the effect of enclomiphene citrate in men with secondary hypogonadism., Design: Phase II clinical trial., Setting: Community dwelling men making visits to physician offices., Patient(s): Men with secondary hypogonadism., Intervention(s): Oral administration of enclomiphene citrate or 1% topical T gel., Main Outcome Measure(s): Luteinizing hormone, FSH, T, and semen analysis., Result(s): Treatment with enclomiphene citrate resulted in increased morning serum T, E2, and LH levels similar to those obtained with a topical T gel in men with secondary hypogonadism. Follicle-stimulating hormone and LH were increased with enclomiphene, and sperm counts were conserved., Conclusion(s): Enclomiphene citrate reverses the two hallmarks of secondary hypogonadism, namely, low serum total T and low or inappropriately normal LH while preserving sperm production., Clinical Trial Registration Number: NCT01270841 (ClinicalTrials.gov Identifier NCT01270841)., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objectives: To determine the pharmacodynamic (PD) profile of serum total testosterone levels (TT) and luteinizing hormone (LH) in men with secondary hypogonadism following initial and chronic daily oral doses of enclomiphene citrate in comparison to transdermal testosterone. To determine the effects of daily oral doses of enclomiphene citrate (Androxal®) in comparison to transdermal testosterone on other hormones and markers in men with secondary hypogonadism., Patients and Methods: This was a randomized, single blind, two-center phase II study to evaluate three different doses of enclomiphene citrate (6.25mg, 12.5mg and 25 mg Androxal®), versus AndroGel®, a transdermal testosterone, on 24-hour LH and TT in otherwise normal healthy men with secondary hypogonadism. Forty-eight men were enrolled in the trial (ITT Population), but 4 men had T levels >350 ng/dL at baseline. Forty-four men completed the study per protocol (PP population). All subjects enrolled in this trial had serum TT in the low range (<350 ng/dL) and had low to normal LH (<12 IU/L) on at least two occasions. TT and LH levels were assessed each hour for 24 hours to examine the effects at each of three treatment doses of enclomiphene versus a standard dose (5 grams) of transdermal testosterone (AndroGel). In the initial profile TT and LH were determined in a naïve population following a single initial oral or transdermal treatment (Day 1). This was contrasted to that seen after six weeks of continuous daily oral or transdermal treatment (Day 42). The pharmacokinetics of enclomiphene was performed in a select subpopulation. Serum samples were obtained over the course of the study to determine levels of various hormones and lipids., Results: After six weeks of continuous use, the mean ± SD concentration of TT at Day 42 C0hrTT, was 604 ± 160 ng/dL for men taking the highest of dose of enclomiphene citrate (enclomiphene, 25 mg daily) and 500 ± 278 ng in those men treated with transdermal testosterone. These values were higher than Day 1 values but not different from each other (p = 0.23, T-test). All three doses of enclomiphene increased C0hrTT, CavgTT, CmaxTT, CminTT and CrangeTT. Transdermal testosterone also raised TT, albeit with more variability, and with suppressed LH levels. The patterns of TT over 24 hour period following six weeks of dosing could be fit to a non-linear function with morning elevations, mid-day troughs, and rising night-time levels. Enclomiphene and transdermal testosterone increased levels of TT within two weeks, but they had opposite effects on FSH and LH Treatment with enclomiphene did not significantly affect levels of TSH, ACTH, cortisol, lipids, or bone markers. Both transdermal testosterone and enclomiphene citrate decreased IGF-1 levels (p<0.05) but suppression was greater in the enclomiphene citrate groups., Conclusions: Enclomiphene citrate increased serum LH and TT; however, there was not a temporal association between the peak drug levels and the Cmax levels LH or TT. Enclomiphene citrate consistently increased serum TT into the normal range and increased LH and FSH above the normal range. The effects on LH and TT persisted for at least one week after stopping treatment., (This article is protected by copyright. All rights reserved.)

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.

Insulin resistance occurs in 20%-25% of the human population, and the condition is a chief component of type 2 diabetes mellitus and a risk factor for cardiovascular disease and certain forms of cancer. Herein, we demonstrate that the sphingolipid ceramide is a common molecular intermediate linking several different pathological metabolic stresses (i.e., glucocorticoids and saturated fats, but not unsaturated fats) to the induction of insulin resistance. Moreover, inhibition of ceramide synthesis markedly improves glucose tolerance and prevents the onset of frank diabetes in obese rodents. Collectively, these data have two important implications. First, they indicate that different fatty acids induce insulin resistance by distinct mechanisms discerned by their reliance on sphingolipid synthesis. Second, they identify enzymes required for ceramide synthesis as therapeutic targets for combating insulin resistance caused by nutrient excess or glucocorticoid therapy.

Recombinant human zona pellucida (rhZP) proteins (minus the N-terminal leader and the C-terminal transmembrane-like domain) were expressed in four different expression systems: bacteria, yeast, insect cells, and Chinese Hamster Ovary (CHO) cells. The recombinant proteins in each system were engineered with a C-terminal six histidine (His6) segment that was used to purify the proteins by metal affinity [either nickel (Ni) or cobalt (Co)] column chromatography. Each of the rhZP proteins was a candidate antigen as an immunocontraceptive vaccine. However, the rhZP proteins produced in bacteria, yeast and insect cell culture could only be purified after being solubilized by strong denaturants. After purification the final products of each of these expression systems required 6 M urea to maintain solubility. However, the rhZP proteins expressed by CHO cells were secreted into the media, and the soluble proteins could be purified to near homogeneity. In this report the expression and purification procedures used to produce and isolate these secreted proteins are described., (Copyright 1999 Academic Press.)

The zona pellucida (ZP) is an extracellular matrix composed of multiple glycoproteins that surrounds all mammalian oocytes. Analysis of the genes encoding the ZP proteins indicates that there are three classes ZPA (largest), ZPB (intermediate) and ZPC (smallest). Polyclonal antibodies developed against recombinant proteins of human and pig ZP have been used to identify immunohistochemically the components of the ZP of cynomolgus monkeys during the course of follicular development. Digoxigenin-labelled cDNA probes specific for the mRNA encoding ZPA, ZPB or ZPC were used to probe sections of cynomolgus monkey ovaries by in situ hybridization. mRNA encoding ZPA was identified in growing follicles at all stages and was seen in the granulosa cells of mature preovulatory follicles. ZPB protein and mRNA encoding ZPB were present in oocytes in secondary follicles and to a lesser extent in tertiary follicles, but were not found in primordial, primary or antral follicles or granulosa cells. ZPC protein and mRNA encoding ZPC were present in oocytes at all stages of folliculogenesis. These results suggest that each component of the ZP is produced and secreted at specific stages of folliculogenesis and that granulosa cells may be contributing to the synthesis of ZPA and ZPC.

Full length zona pellucida cDNAs from cat, dog and pig that are homologous to the ZP2/rc75 genes from mouse, human and rabbit, a full length zona pellucida cDNA from cat and a gene and full length cDNA from human that are homologous to the rc55/ZP3 alpha genes from rabbit and pig, and full length zona pellucida cDNAs from cat, cow, dog, pig and rabbit that are homologous to the ZP3 genes from mouse, hamster, human and marmoset have been cloned and characterized. The members of these gene families are herein referred to as ZPA, ZPB and ZPC genes to avoid the confusion that currently exists in the zona pellucida of nomenclature. This report is the first to describe the presence all three major zona pellucida genes within individual mammalian species. Within the ZPA, ZPB and ZPC gene families, the DNA and deduced amino acid sequences are highly homologous to each other, and are most homologous between members of the same order within the class mammalia. These results imply that all or most mammalian species express the ZPA, ZPB and ZPC proteins, which form the zona pellucida layer surrounding the oocyte.

We isolated a cDNA encoding the pig oocyte zona pellucida protein ZP3 alpha from a pig ovary lambda gt11 cDNA library. The 1699 bp cDNA contains a short 3' untranslated region characteristic of cDNAs encoding zona proteins. The deduced amino acid sequence for ZP3 alpha consists of 536 amino acid residues and shares 66% overall identity with a 55 kDa rabbit zona protein. Important features of the ZP3 alpha polypeptide include a predicted N-terminal signal sequence, twenty-two cysteine residues, an O-glycosylated domain and potential attachment sites for five N-linked sugar chains. A multibasic tetrapeptide occurs upstream of a predicted C-terminal transmembrane sequence; this suggests proteolytic processing of an integral membrane precursor within the constitutive secretory pathway.