Clomiphene Isomers Research Articles

Inclusion Complex of Clomiphene Citrate with Hydroxypropyl-β-Cyclodextrin for Intravenous Injection: Formulation and Stability Studies.

Clomiphene citrate is the first-line treatment for women with abnormal or failed ovulation. Currently, it is available as oral tablets, and the parenteral formulation does not exist. In this study, we prepared clomiphene citrate-hydroxypropyl-β-cyclodextrin inclusion complex for its use in intravenous injection. The inclusion complex was characterized in the liquid state (phase solubility) and solid state by differential scanning calorimetry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy analyses. The sterile intravenous injection containing 0.5% clomiphene citrate was prepared and characterized for its physical properties, assay, pH, and osmolality. A stability-indicating high-performance liquid chromatography (HPLC) method for the injection was developed. The HPLC method was validated for the assay, linearity, precision and repeatability, benchtop stability, and forced degradation to elute clomiphene isomers from the degradation products. The injection was packed in sterile 10-ml glass vials with butyl rubber stoppers and stored at 40°C, room temperature, and 4°C. The samples at 0, 0.5, 1, 2, 3, and 6months were analyzed for clarity, pH, osmolality, and drug assay. The HPLC method was linear (R2 = 0.9999), precise (0.86% relative standard deviation), and stability indicating. The stability data at the accelerated (40°C) storage condition for 6months showed satisfactory results: the drug assay in the injection was between 90 and 105%, the injection remained clear, pH was between 4.0 and 4.4, and osmolality was between 270 and 350mOsm. The stability data suggests that the product is stable and meets the given analytical specifications.

A phase II, dose finding, placebo-controlled, study of zuclomiphene citrate to amerliorate the frequency and severity of hot flashes caused by androgen deprivation in men with advanced prostate cancer.

TPS338 Background: Androgen deprivation therapy (ADT) is the mainstay of treatment for advanced prostate cancer. ADT not only lowers testosterone, but also decreases estrogen levels which can cause significant side effects including hot flashes, loss of bone and bone fractures, and decreases in libido. Up to 80% of the men on ADT report hot flashes and 30-40% of men have moderate to severe hot flashes. Concern over hot flashes make patients less likely to begin ADT and can lead to early discontinuation of ADT. While the off-label use of potent steroidal estrogens has demonstrated efficacy, the appropriate dose as well as dosing route or schedule of these potent estrogens, has not been established. Furthermore, the potential for safety issues with potent steroidal estrogens remains a significant limitation to their clinical utility. Zuclomiphene citrate, is novel weak nonsteroidal estrogenic agent that should ameliorate hot flashes caused by ADT, and as one of the isomers of clomiphene, has a 50 year safety history of being well tolerated in men. Methods: The Phase 2, placebo controlled, dose finding clinical trial (V72203) evaluating zuclomiphene citrate (VERU-944) capsules, oral daily dosing, for the treatment of moderate to severe hot flashes in men with prostate cancer on ADT is in progress. Men are randomized to daily doses of placebo or zuclomiphene 10mg, 50mg or 100mg. V72203 is enrolling approximately 36 men per arm in 10 sites in the United States. The primary efficacy endpoint is the mean change in frequency of moderate and/or severe hot flashes from baseline to week 4 and maintained until week 12. Secondary endpoints include changes from baseline in bone turnover markers, free and total testosterone, SHBG, PSA, and safety. Hot flashes are being measured in real time utilizing an electronic data capture device (ePRO) provided to each subject. We anticipate completion of enrollment by the end of 2018 with study results by second quarter of 2019. Clinical trial information: NCT03646162.

Clomiphene and Its Isomers Block Ebola Virus Particle Entry and Infection with Similar Potency: Potential Therapeutic Implications.

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.

Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry

Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 μm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene.

Determination by liquid chromatography–mass spectrometry of clomiphene isomers in the plasma of patients undergoing treatment for the induction of ovulation

A rapid, sensitive and selective LC–MS method is described for the simultaneous determination of zuclomiphene and enclomiphene in plasma from patients undergoing treatment with clomiphene citrate for the induction of ovulation. Samples spiked with N-didesmethyltamoxifen, the internal standard, were extracted into methyl tertiary butyl ether. The compounds were separated on a Luna C 18 analytical column, and a mobile phase of methanol–water (70:30 v/v) containing 0.05% trifluoroacetic acid at a flow rate of 1 ml/min. The limits of determination were 35 pg/ml and 7 pg/ml for zu- and enclomiphene, respectively. Within-day coefficients of variation ranged from 2.1% to 7.2%.

Multivariate analysis of capillary electrophoresis separation conditions for Z- E isomers of clomiphene

Plackett-Burman (P-B) experimental design has been used to optimize the factors affecting separation of Z and E isomers of clomiphene (zuclomiphene and enclomiphene respectively) using capillary electrophoresis. The P-B design was used to simultaneously investigate the following five factors: buffer ionic strength, buffer pH, heptakis (2,3,6-tri- o-methyl) β-cyclodextrin (TMCD) concentration, methanol concentration and injection time, each at three levels. In addition to these, a dummy variable was added to estimate the variability of the system. Effects on resolution and analysis time were calculated. Based on the information gained from the P-B design, the following set of conditions was chosen: 100 mM phosphate buffer pH 2.3, 5 mM TMCD, 5% methanol, and 1.7 s hydrodynamic injection time. These conditions gave well-resolved peaks for zuclomiphene and enclomiphene.

Simple and rapid determination of clomiphene cis and trans isomers in human plasma by high-performance liquid chromatography using on-line post-column photochemical derivatization and fluorescence detection

A validated, sensitive and rapid high-performance high-performance liquid chromatographic method has been developed for the analysis of both cis and trans isomers of clomiphene in human plasma. The method involves a new off-line pre-column solid-phase extraction for sample preparation of clomiphene from human plasma in the presence of an internal standard. Analysis was performed by isocratic elution on a LiChrospher 100 RP-18 5-μm column using on-line post-column photochemical derivatization, with fluorescence detection at 247 nm (excitation) and 378 nm (emission). The limit of quantitation was 0.75 ng/ml for the cis isomer and 1.25 ng/ml for the trans isomer. The precision and accuracy of method were between good laboratory practice (GLP) required limits.

Inhibition of decidual induction in rats by clomiphene and tamoxifen.

The objective of this study was to characterize the estrogen action that confers endometrial sensitization to nontraumatic deciduogenic stimuli by use of antiestrogens. Tamoxifen, ethamoxytriphetol, and clomiphene and its two component enantiomers inhibited decidual induction in pseudopregnant rats when administered 17 h before pyrathiazine. Unexpectedly, clomiphene (250 micrograms/rat) and tamoxifen (25 micrograms/rat) proved inhibitory at all times up to and including the time of induction. Clomiphene, administered in the hours preceding decidual induction, inhibited the increase of ornithine decarboxylase activity, which normally marks the end of the induction phase. Clomiphene had no inhibitory effect on the availability or receptor binding of progesterone. Clomiphene also inhibited implantation of blastocysts when administered at the time of their adherence to the uterus. The inhibition by antiestrogens of decidual induction could not be explained on the basis of the current understanding of mechanisms of estrogen action. The discrepancies were that no latent period between the time of antiestrogen administration and decidual induction was observed and no difference was observed in the inhibitory activities of the isomers of clomiphene.

Short communication

We compared the gonadotrophin and oestradiol responses to clomiphene citrate (CC) in women aged 18-25 versus 35-40 years. Despite similar baseline hormone concentrations and the attainment of similar concentrations of the E and Z isomers of clomiphene, higher follicle stimulating hormone (FSH) concentrations were observed in the older women, possibly indicative of their ageing ovaries. Furthermore, diminished oestradiol responses occurred in a subgroup of women showing FSH hyper-responsiveness to CC. These preliminary results support the concept that the hormonal response to CC may be an indicator of approaching perimenopause.

Effect of clomiphene isomers on oestradiol synthesis in cultured human granulosa cells

The effects of en-clomiphene and zu-clomiphene (10(-7) - 10(-5) M) on oestradiol synthesis in human granulosa cells were studied. Granulosa cells were obtained from stimulated cycles in women undergoing oocyte retrieval, in the course of in-vitro fertilization/embryo transfer due to mechanical infertility. En-clomiphene and zu-clomiphene (10(-5) M) significantly reduced basal and LH-stimulated oestradiol synthesis after 2-4 days of culture. The LH-stimulated synthesis was also significantly and reversibly reduced by zu-clomiphene at lower concentrations (10(-6) - 10(-7) M) and by a mixture of en- and zu-clomiphene (10(-6) M). It is concluded that both the mixture of en- and zu-clomiphene and the isomers alone have direct inhibitory effects on oestradiol synthesis in human granulosa cells in vitro. Furthermore, it cannot be excluded that the LH-stimulated oestradiol synthesis is inhibited by zu-clomiphene at concentrations found in follicular fluid after administration of clomiphene citrate.

Pharmacokinetics of intravenous clomiphene isomers.

British Journal of Clinical PharmacologyVolume 27, Issue 5 p. 639-640 Free Access Pharmacokinetics of intravenous clomiphene isomers. M Szutu, M Szutu Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorDJ Morgan, DJ Morgan Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorM McLeish, M McLeish Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorG Phillipou, G Phillipou Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorGL Blackman, GL Blackman Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorLW Cox, LW Cox Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorW Dollman, W Dollman Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this author M Szutu, M Szutu Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorDJ Morgan, DJ Morgan Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorM McLeish, M McLeish Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorG Phillipou, G Phillipou Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorGL Blackman, GL Blackman Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorLW Cox, LW Cox Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this authorW Dollman, W Dollman Victorian College of Pharmacy, Melbourne, Australia.Search for more papers by this author First published: May 1989 https://doi.org/10.1111/j.1365-2125.1989.tb03430.xCitations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume27, Issue5May 1989Pages 639-640 ReferencesRelatedInformation

Effect of clomiphene isomers on progestin synthesis in cultured human granulosa cells.

The effect of en-clomiphene and zu-clomiphene (10(-9)-10(-5) M) on progestin synthesis in cultured human granulosa cells was studied under basal conditions and in the presence of LH (100 ng/ml). Granulosa cells were obtained from either pre-ovulatory follicles of clomiphene-HMG-stimulated cycles or from large follicles of mid-to-late follicular phase of spontaneous cycles. The basal and LH-stimulated progesterone accumulation was dose-dependently reduced by en- and zu-clomiphene (10(-6)-10(-5) M) in cells from both groups studied, an effect similar to that of oestradiol. In contrast to oestradiol, both en- and zu-clomiphene (10(-6)-10(-5) M) reduced the basal and LH-stimulated pregnenolone accumulation in cells of stimulated cycles. The effect of clomiphene on progesterone, and pregnenolone accumulation was more pronounced in LH-stimulated cells than under basal conditions. There were no qualitative differences between the two isomers and the results were principally the same in cells from both groups of follicles studied. It is concluded that en-clomiphene and zu-clomiphene have similar inhibitory effects on progestin synthesis in human granulosa cells in vitro.

Analysis of clomiphene isomers in human plasma and detection of metabolites using reversed-phase chromatography and fluorescence detection

A method is described for the quantitative clinical analysis of plasma concentrations of the E and Z isomers of clomiphene, which is used in the induction of ovulation. The isomers of clomiphene, in addition to metabolites, are extracted from plasma with tert-butyl methyl ether (MTB). The MTB layer is dried, reconstituted and an aliquot subjected to chromatography. The drug and metabolites are separated by reversed-phase high-performance liquid chromatography. The eluent is fed into a knitted or braided, cylindrical reaction coil made of Teflon, into which is inserted a low-energy mercury lamp. This results in a photoinduced stilbene-to-phenanthrene oxidation yielding highly fluorescent analytes; this provides excellent sensitivity for the quantitation of the intact drug isomers and the detection of presently uncharacterized metabolites. Use of the reversed-phase chromatographic mode results in elution of the polar metabolites prior to the intact drug isomers. A combination of reversed-phase chromatography and an in-line post-column reaction coil results in a sensitive method that is more reliable and rapid than those previously reported and is applicable to the routine analysis of clinical samples. The method has been applied to individual isomers of clomiphene in plasma at concentrations of 0.06–600 ng/ml.

The inhibition of the estrogen receptor's positive cooperative [3H]estradiol binding by the antagonist, clomiphene.

The calf uterine estrogen receptor showed positive cooperativity of [3H]estradiol equilibrium binding; the Scatchard plot was convex and the Hill coefficient was 1.69 +/- 0.021 (n = 14). The effects of the estrogenic antagonists, zuclomiphene (cis-2-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate) and enclomiphene (trans-23-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate), on the positive cooperativity of [3H]estradiol binding were measured by titrating the receptor with a variable concentration of [3H]estradiol and antagonist while maintaining a constant excess in a specific ratio of the antagonist to the [3H]estradiol. With a 45- to 55-fold molar excess of zuclomiphene or an 820- to 900-fold molar excess of enclomiphene above the [3H]estradiol concentration, the receptor's positive cooperative [3H]estradiol binding was inhibited. A transition from a convex to a linear Scatchard plot and a decrease in the Hill coefficient from 1.69 to 1.10 +/- 0.02 (n = 6) were induced. The specifically bound [3H]estradiol was inhibited 43 to 50% by the zuclomiphene and enclomiphene. The addition of unlabeled estradiol in a 1- or 2.3-fold molar excess above that of the [3H]estradiol concentration produced a 50 to 75% competitive displacement of the specifically bound [3H]estradiol; nevertheless, the Scatchard plot remained convex and the Hill coefficient was 1.74 and 1.80, respectively. Thus, inhibition of the positive cooperativity of [3H]estradiol binding by the clomiphene isomers was not due to dilution of the specifically bound [3H]estradiol by the antagonist. These data demonstrate that there are two molecular mechanisms by which an estrogen antagonist interferes with the function of the receptor: as a competitor, thus blocking the estrogen receptor's binding site to an agonist, and second by inducing conformational changes that inhibit site:site interactions and receptor activation.

Inhibition of rabbit liver microsomal oxidative metabolism and substrate binding by tamoxifen and the geometric isomers of clomiphene

The inhibitory effects of the triarylethylene antiestrogens tamoxifen (TAM) and enclomiphene (EC), and of the estrogenic geometric isomer of the latter compound (zuclomiphene, ZC) on the binding and metabolism of standard substrates in the presence of rabbit liver cytochrome P-450 have been studied. In the presence of microsomes at pH 7.4, the triarylethylenes interfered with the binding of β-diethylaminoethyl diphenylpropylacetate (SKF 525-A) and with the N-demethylation of ethylmorphine. Their inhibitory effects were less pronounced on the binding and metabolism (hydroxylation) of aniline. Kinetic studies showed that EC and TAM interfered with SKF 525-A binding by a mixed mechanism. These results suggest that the triarylethylenes affect preferentially the binding of ‘Type I’ substrates with cytochrome P-450.

Stereochemistry of geometric isomers of clomiphene: A Correction of the Literature and a Reexamination of Structure‐Activity Relationships

A correction of the literature and a reexamination of structure-activity relationships are reported with special emphasis on stereochemistry of geometric isomers of clomiphene. The establishment of the stereochemistry of isomers of 1 has corrected an error in the literature permitted a consistent explanation of the biological and physical data available on isomers of 1 and 4 and provided insight into the molecular mechanisms of action and direction for additional studies.

Female endocrine control mechanisms during the neonatal period. 3. The effect of clomiphene citrate.

ABSTRACT The purpose of this paper is to report on the in vivo effect of different doses of cis- and trans-clomiphene on the RNA and protein synthesis in the ovary, liver, adrenal gland and uterus of newborn guinea pigs. After the sc injection of either isomer the biochemical parameters were determined at different time intervals. Both isomers induced an increase of the RNA- and protein-synthesizing activity in the uterus, but failed to do so in the ovary, liver and adrenal gland. According to data reported here and also by other investigators it may be assumed that the lack of ovarian response to the treatment with cis- or trans-clomiphene is primarily due to the functional immaturity of the female hypothalamus and/or pituitary during the neonatal period. Both isomers failed to modify the pattern of gonadotrophin release, as shown by the constancy of the ovarian RNA and protein synthesis after the administration of either of the isomers. In the uterus, both cis- and trans-clomiphene strikingly imitated the biochemical stimulation produced by natural oestrogens. In contrast to oestradiol-17β, both clomiphene isomers showed a later onset and longer duration of the oestrogenic activity. The first increase of the amino acid incorporation into uterine proteins was noted after 5 hours in the cis-clomiphene treated group and after 8 hours in the trans-clomiphene treated animals as compared to 1 hour when oestradiol-17β was used. This difference seems to be due to a delayed transfer of the cytoplasmic oestradiol receptor to the nucleus after the injection of cis- or trans-clomiphene. As compared to oestradiol-treated animals, the clomiphene injection provoked a prolonged elevation of the uterine protein-synthesizing activity, most likely reflecting an effect of the enterohepatic recirculation of both isomers.

Role of estrogens in spontaneous ovulation: evidence for the positive feedback in hamsters.

Existence of the positive feedback of estrogen for release of ovulating hormone has been explored in cyclic hamsters by using antiestrogens. Oral administration of either ICI 46,474 (10 mg/kg) or clomiphene (20 mg/kg) at 17:00 hr on Day 3 (day of external vaginal discharge Day 1) of the cycle interfered with ovulation, vaginal discharge and mating behavior. Of the two isomers of clomiphene used only the isomer with known antiestrogenic properties (clomiphene B) blocked ovulation (10 mg/kg). Simultaneous administration of estradiol benzoate (.025 to 0.5 mg/kg, sc) but not of progesterone (4 mg/kg, sc) prevented the inhibitory effects of both antiestrogens and fully restored ovulation and external vaginal discharge. Intravenous injection of pituitary gonadotropins on Day 4 also restored ovulation to a varying degree but did not induce vaginal discharge in the treated hamsters. A similar administration of LH-RF (10 ME) proved ineffective. These results demonstrate the existence of the positive feedback of estrogen for the release of ovulating hormone in the hamster similar to that reported earlier in the 4-day cyclic rat. (Endocrinology90: 941, 1972)

Effect of some triphenylethylenes on oestradiol binding in vitro to macromolecules from uterus and anterior pituitary.

SUMMARY The effects of a series of triphenylethylenes (I.C.I. 46,474, I.C.I. 47,699 and clomiphene isomers A and B) on oestradiol binding to receptors from rat, rabbit and mouse uteri and rat anterior pituitary were studied. Binding of oestradiol to receptors from uteri was inhibited competitively by I.C.I. 46,474. Receptors from uteri of the three species and from rat anterior pituitary have very similar affinities for oestradiol and the four triphenylethylenes. The order of activity of the triphenylethylenes as inhibitors of oestradiol binding is: clomiphene B > I.C.I. 46,474 > I.C.I. 47,699 > clomiphene A. Some correlations are made with the activity of the compounds in vivo.

Effect of cis and trans isomers of clomiphene on reproductive performance in ewes.

Trans-clomiphene injected intramuscularly daily for 16 days in doses ranging from 0.17 mg to 4.5 mg/day did not result in synchronized estrus cycles following withdrawal and had no effect on ovulation rate or fertility. On the other hand, 1.5 mg of cis-clomiphene injected daily for 16 days resulted in 4 of 5 ewes being in estrus within a 48-hr, period at the end of the treatment period. This dose of cis-clomiphene completely blocked ovulation and caused a significant reduction in the number of follicles greater than 4 mm in diameter (P<.01) and a significant increase in the number of services required for conception (P<.01). Doses of .012, .06 and 0.3 mg of cis-clomiphene per day had no effect on the occurrence of estrus, ovulation rate or fertility, except that the dose of .06 mg caused a significant increase in the number of functional corpora lutea (P<.05). Neither isomer of clomiphene stimulated body weight gains or wool growth. However, cis-clomiphene could prove useful if used prior to gonadotropin injections in synchronization techniques designed for inseminating animals at a predetermined date without regard to the occurrence of estrus.