Depletion of clomiphene residues in eggs and muscle after oral administration to laying hens.

The selective oestrogen receptor modulator (SERM) clomiphene is therapeutically used to induce ovulation. While prohibited as a doping agent in sports, it is frequently detected in sports drug testing urine samples. Few reports exist on clomiphene's (illicit) use in the farming industry to increase the egg production rate of laying hens, which creates a risk that eggs as well as edible tissue of these hens contain residues of clomiphene. To investigate the potential transfer of clomiphene into eggs and muscle tissue, laying hens were orally administered with clomiphene citrate at 10 mg/day for 28 days. To determine clomiphene residues in eggs, chicken breast and chicken thigh, the target analyte was extracted from homogenised material with acetonitrile and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The test method reached a limit of quantification (LOQ) of 1 µg/kg and was characterised concerning specificity, precision, trueness and linearity. Analyses were performed on whole egg, egg white and yolk separately, and chicken muscle from breast and thigh. Clomiphene was detectable in eggs two days after the beginning of the drug administration period. The drug concentrations increased to 10-20 µg per egg within one week, and after withdrawal of clomiphene, residues decreased after 4 days, but traces of clomiphene were still detectable until the end of the study (14 days after the last administration). In the chicken's muscle tissue, clomiphene levels up to 150 µg/kg (thigh) and 36 µg/kg (breast) were found. Six days after the last dose, tissue clomiphene concentrations fell below the LOQ. Overall, these results underline the concerns that clomiphene may be transferred into animal-derived food and future research will therefore need to focus on assessing and minimising the risk of unintentional adverse analytical findings in doping controls.

Flavin-Containing Monooxygenase 3 Polymorphic Variants Significantly Affect Clearance of Tamoxifen and Clomiphene.

Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolising enzyme that oxygenates many drugs and xenobiotics in the liver. This enzyme is also known to exhibit single nucleotide polymorphisms (SNPs) that can alter the rates of monooxygenation of therapeutic agents. The purpose of this study was to investigate the effect of the three common polymorphic variants of hFMO3 (V257M, E158K and E308G) on the metabolism and clearance of three structurally similar compounds: tamoxifen (breast cancer medication), clomiphene (infertility medication) and GSK5182 (antidiabetic lead molecule). For GSK5182, none of the three variants showed any significant differences in its metabolism when compared to the wild-type enzyme. In the case of clomiphene, two of the variants, V257M and E308G, exhibited a significant increase in all the kinetic parameters measured with nearly two times faster clearance. Finally, for tamoxifen, a mixed behaviour was observed; E158K variant showed a significantly higher clearance compared to the wild type, whereas V257M mutation had the opposite effect. Overall, the data obtained demonstrate that there is no direct correlation between the SNPs and the metabolism of these three hFMO3 substrates. The metabolic capacity is both variant-dependent and substrate-dependent and therefore when testing new drugs or administering already approved therapies, these differences should be taken into consideration.

Effect of the CYP2D6*10 allele on the pharmacokinetics of clomiphene and its active metabolites.

Clomiphene citrate, a selective estrogen receptor modulator, is metabolized into its 4-hydroxylated active metabolites, primarily by CYP2D6. In this study, we investigated the effects of the most common CYP2D6 variant allele in Asians, CYP2D6*10, on the pharmacokinetics of clomiphene and its two active metabolites (4-OH-CLO and 4-OH-DE-CLO) in healthy Korean subjects. A single 50-mg oral dose of clomiphene citrate was given to 22 Korean subjects divided into three genotype groups according to CYP2D6 genotypes, CYP2D6*wt/*wt (n = 8; *wt = *1 or *2), CYP2D6*wt/*10 (n = 8) and CYP2D6*10/*10 (n = 6). Concentrations of clomiphene and its metabolites were determined using a validated HPLC-MS/MS analytical method in plasma samples collected up to 168 h after the drug intake. There was a significant difference only in the Cmax of clomiphene between three CYP2D6 genotype groups (p < 0.05). Paradoxically, the elimination half-life (t1/2) and AUC of both active metabolites were all significantly increased in the CYP2D6*10 homozygous carriers, compared with other genotype groups (all p < 0.001). The AUCinf of corrected clomiphene active moiety in CYP2D6*10/*10 subjects was 2.95- and 2.05-fold higher than that of CYP2D6*wt/*wt and *wt/*10 genotype groups, respectively (both p < 0.001). Along with the partial impacts on the biotransformation of clomiphene and its metabolites by CYP2D6 genetic polymorphism, further studies on the effects of other CYP enzymes in a multiple-dosing condition can provide more definite evidence for the inter-individual variabilities in clomiphene pharmacokinetics and/or drug response.

Potential Teratogenic Effects of Clomiphene Citrate.

Clomiphene citrate (CC) is the oldest drug used to regulate the process of ovulation. Considering the great use of CC over the last 40 years, it is important to understand the possible risks associated with its use. The aim of this review was to evaluate the possible teratogenic effects of CC, analyzing results obtained from animal and human studies. The pharmacokinetics of CC and possible mechanisms involved in teratogenesis are examined. Fetal exposure to CC is possible due to the long half-life of CC and its metabolites. Alarming data have emerged from animal studies, although controversial results come from human studies. There is some evidence regarding a possible association of CC exposure and fetal malformations, mainly neural tube defects and hypospadias, which would require further investigation in order to allow safer use of this useful drug.

(125)I labeling of clomiphene and biodistribution studies for possible use as a model in breast cancer imaging.

Clomiphene has growth-inhibitory effects of breast cancer cells, clomiphene was successfully labeled with (125)I via direct electrophilic substitution reaction with labeling yield 97%. It was obtained at optimum substrate amount of 0.5mg, Chloramine-T was used as an oxidizing agent at optimum amount of 25µg. Labeling reactions was done at pH 5 at ambient temperature. This study showed good in vitro and in vivo stability of the (125)I-clomiphene. The radiolabeled compound showed high ascetic fluid uptake of 18.12±0.27% at 30min post-injection. Solid tumor uptake of (125)I-clomiphene was 12.48±0.32% at 30min post-injection. This data revealed the localization of tracer in tumor tissue with high percent sufficient to use (125)I-clomiphene as a promising tool for the diagnosis of breast cancer.

Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies.

The use of selective oestrogen receptor modulators has been prohibited since 2005 by the World Anti-Doping Agency regulations. As they are extensively cleared by hepatic and intestinal metabolism via oxidative and conjugating enzymes, a complete investigation of their biotransformation pathways and kinetics of excretion is essential for the anti-doping laboratories to select the right marker(s) of misuse. This work was designed to characterize the chemical reactions and the metabolizing enzymes involved in the metabolic routes of clomiphene, tamoxifen and toremifene. To determine the biotransformation pathways of the substrates under investigation, urine samples were collected from six subjects (three females and three males) after oral administration of 50 mg of clomiphene citrate or 40 mg of tamoxifen or 60 mg of toremifene, whereas the metabolizing enzymes were characterized in vitro, using expressed cytochrome P450s and uridine diphosphoglucuronosyltransferases. The separation, identification and determination of the compounds formed in the in vivo and in vitro experiments were carried out by liquid chromatography coupled with mass spectrometry techniques using different acquisition modes. Clomiphene, tamoxifen and toremifene were biotransformed to 22, 23 and 18 metabolites respectively, these phase I reactions being catalyzed mainly by CYP3A4 and CYP2D6 isoforms and, to a lesser degree, by CYP3A5, CYP2B6, CYP2C9, CYP2C19 isoforms. The phase I metabolic reactions include hydroxylation in different positions, N-oxidation, dehalogenation, carboxylation, hydrogenation, methoxylation, N-dealkylation and combinations of them. In turn, most of the phase I metabolites underwent conjugation reaction to form the corresponding glucuro-conjugated mainly by UGT1A1, UGT1A3, UGT1A4, UGT2B7, UGT2B15 and UGT2B17 isoenzymes.

Safety and efficacy of clomiphene citrate and L-carnitine in idiopathic male infertility: a comparative study.

PURPOSE: To compare the effects of L-carnitine with clomiphene citrate in idiopathic infertile men. MATERIALS AND METHODS: Fifty-two men with idiopathic infertility were recruited in this randomized controlled trial. They were randomly assigned into 2 treatment groups, group 1 (n = 20) and group 2 (n = 32), who received L-carnitine 25 mg/day and clomiphene citrate 2 gr/day, respectively, for a period of 3 months. RESULTS: Comparing the effect of L-carnitine and clomiphene on sperm parameters before and after the treatment, both medications had influence on sperm count and motility (P = .01). L-carnitine significantly increased the semen volume (P = .001), while clomiphene citrate was significantly associated with the motility percentage and normal morphology (P = .008). CONCLUSION: It seems that the use of clomiphene citrate and L-carnitine, either individually or in combination, as the first step of idiopathic male infertility treatment is reasonable, safe, and effective.

Clomiphene citrate and enclomiphene for the treatment of hypogonadal androgen deficiency.

Hypogonadism has a number of important clinical consequences related to androgen deficiency and impaired spermatogenesis. The cause of this condition is multifactorial and can result from hypothalamic, pituitary or gonadal dysfunction as well as factors that affect hormonal signaling along the hypothalamic-pituitary-gonadal axis. While testosterone replacement is the most common treatment, it can paradoxically lead to infertility, and may be a less physiologic therapy for patients with secondary hypogonadism due to pituitary dysfunction. Clomiphene citrate, and its derivatives, may allow for restoration of gonadal function by restoring physiologic pituitary function in a subset of patients with hypogonadism.

Inseminación artificial intrauterina en mujeres con síndrome de ovario poliquístico resistentes al citrato de clomifeno / Intrauterine insemination in women with polycystic ovary syndrome and clomiphene citrate resistance

Objetivo: Evaluar los resultados del tratamiento mediante inducción de la ovulación con gonadotropinas e inseminación artificial intrauterina (IAC-IU) realizados en mujeres con síndrome de ovario poliquístico (SOP) resistentes al citrato de clomifeno (CC). Material y métodos: Estudio observacional retrospectivo de 462 ciclos de inducción de la ovulación con IAC-IU en mujeres diagnosticadas de SOP resistentes al tratamiento con CC. Resultados. La tasa de embarazo clínico fue del 11,9% por ciclo iniciado y del 14,74% por inseminación, finalizando en aborto el 45,45% de los embarazos. El porcentaje acumulativo efectivo de embarazo a término por ciclo iniciado fue del 8% al primer ciclo y del 10,23% por inseminación, manteniéndose en el 26,15% a partir del quinto ciclo y en 32,24% a partir de la quinta inseminación. Se canceló el 19,26% de los ciclos. Conclusiones: La IAC-IU es un método útil en mujeres con SOP resistentes al CC, con resultados similares a los obtenidos con otras indicaciones. Estas pacientes presentan altas tasas de aborto del primer trimestre y de cancelación de ciclo. Esta elevada tasa de aborto debe dar lugar al desarrollo de una línea de investigación que logre encontrar la pauta que disminuya la pérdida reproductiva (AU)

Objective: To evaluate the results of ovulation induction cycles with gonadotropins and intrauterine insemination (IUI) carried out in women with polycystic ovary syndrome (POS) resistant to clomiphene citrate. Material and methods: We performed an observational retrospective study of 462 ovulation induction cycles with IUI in women with SOP and clomiphene citrate resistance. Results: The clinical pregnancy rate was 11.9% per cycle initiated and 14.74% per insemination and 45.45% of the pregnancies terminated in abortion. The effective accumulative percentage of term pregnancies per cycle initiated was 8% for the 82 first cycle and 10.23% for insemination, increasing to 26.15% from the fifth cycle and to 32.24% from the fifth insemination. A total of 19.26% of the cycles was cancelled. Conclusions: IUI is a useful method in women with SOP who are resistant to clomiphene citrate, with similar results to those obtained with other indications. These patients have high abortion and cycle cancellation rates. The high abortion rate should prompt research into ways of reducing reproductive loss (AU)

Single-dose pharmacokinetic study of clomiphene citrate isomers in anovular patients with polycystic ovary disease.

The pharmacokinetics of the zuclomiphene (Zu) and enclomiphene (En) isomers of clomiphene citrate following a single oral dose (50 mg) were characterized for the first time in patients receiving the drug (ie, infertile women with polycystic ovary syndrome). Plasma concentrations of Zu and En were measured in 9 patients from the second day of their menstrual cycle (day 1 of dosing) up to 21 days. The mean (+/- coefficient of variation) of C(max), t(max), and AUC of Zu was 15 +/- 41 ng/mL, 7 +/- 87 h, and 1289 +/- 34 ng/mL.h (AUC(0-456 h)), and that of En was 15 +/- 18 ng/mL, 3 +/- 68 h, and 65 +/- 35 ng/ml.h (AUC(0-72h)), respectively. These parameters appeared to be different for Zu from those reported previously in healthy participants, except for t(max). The pharmacokinetic parameters of En in patients with polycystic ovary syndrome were not generally different from the healthy subjects. The effect of obesity on Zu kinetics was stronger than that on En. The conventional model-dependent pharmacokinetics of clomiphene citrate isomers could not be determined due to a very flat terminal half-life and the long-tailed residence time, signifying the lipophilic nature and potentially extensive distribution of the compound.

Clomiphene citrate and ovulation induction.

Clomiphene can be used to treat anovulation due to hypothalamus or pituitary gland dysfunction, and it normalizes the luteal phase in stimulated patients. It can be used to estimate ovarian follicle reserve, and may be predictive of ovulation in women aged >/=35 years or with failed IVF. Contraindications include risk of congenital anomalies, chronic liver disease and visual disorders. Clomiphene may impair fertility through its effects on cervical mucus and in causing various endometrial dysfunctions. However, if clomiphene is administered in 50 mg doses, side-effects are avoided and efficacy is similar to that of a 100 mg dose, although daily dosages of 200 mg/day over 5 days can induce ovulation in approximately 70% of treated patients. Gonadotrophin concentrations increase up to days 5-9 when follicles are selected, and clomiphene is effective in patients with polycystic ovary syndrome (PCOS). Fifty percent of normal patients conceive, a value perhaps biased by the antagonistic effects of clomiphene on cervical mucus in some women. Clomiphene is valuable for IVF, and is used by some clinics in combination with HMG or recombinant FSH. Resistance to clomiphene can develop, and human chorionic gonadotrophin may be needed to induce ovulation in clomiphene cycles. Corticosteroids and human menopausal gonadotrophin (HMG) can be combined with clomiphene for stimulation, its combination with HMG long having been a standard protocol in assisted reproduction. PCOS patients may become insulin resistant, a condition improved by the administration of metformin. Other adverse effects include multiple pregnancies, an increase in the rate of multiple births, ovarian hyperstimulation and unsubstantiated claims of ovarian cancer.

Serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate therapy in anovulatory infertile women.

OBJECTIVE: To determine the serum concentrations of enclomiphene and zuclomiphene across consecutive cycles of clomiphene citrate treatment in anovulatory infertile women. DESIGN: Prospective cohort. SETTING: Tertiary institutional infertility clinic. PATIENT(S): Fourteen consenting anovulatory infertile women receiving standardized, cyclic, incremental treatment with clomiphene citrate for ovulation induction. INTERVENTION(S): Clomiphene citrate treatment (50-150 mg/d, cycle days 5-9), titrated to the minimum effective ovulation-inducing dose, was administered for three to six total cycles. Blood samples were obtained on cycle days 3 and 10 in each treatment cycle. MAIN OUTCOME MEASURE(S): Serum concentrations of enclomiphene and zuclomiphene. RESULT(S): Cycle day 3 zuclomiphene levels were below assay limits in all initial cycles, increased progressively across three consecutive cycles, and thereafter plateaued. Cycle day 3 enclomiphene concentrations were uniformly undetectable. Cycle day 10 enclomiphene levels increased with dose administered, but these observations were not statistically significant. CONCLUSION(S): Clomiphene citrate induction of ovulation results in an isomer-specific systemic accumulation of zuclomiphene across consecutive cycles of treatment. The combined maximum concentration of enclomiphene and zuclomiphene attained in practice approximates 100 nmol/L and is generally well below levels previously demonstrated to have adverse effects in vitro.

Development, pharmacology and clinical experience with clomiphene citrate.

This review describes the development and pharmacology of clomiphene and those specific characteristics of both drug and patients which determine its clinical efficacy. The studies reviewed describe clinical observation of patient characteristics (age, additional infertility diagnosis, semen quality), vaginal ultrasound observations of ovaries (number and size of pre-ovulatory follicles) and endometrial lining (thickness, pattern) in 2841 clomiphene cycles in patients who required intrauterine insemination (IUI) because of poor sperm quality or an unsatisfactory postcoital test. They show that (i) conception in clomiphene cycles is related to the number and size of pre-ovulatory follicles, endometrial thickness, patient age, pelvic adhesions, type of anovulatory disorder and semen quality; (ii) pregnancy rates per clomiphene-IUI cycle are constant through at least six cycles; (iii) multiple births cannot be prevented by withholding human chorionic gonadotrophin or advising against coitus when multiple pre-ovulation follicles are present unless all follicles down to 10-12 mm diameter are counted. We also reviewed pregnancy outcome (number of gestational sacs, babies, preclinical and clinical abortion, ectopic pregnancy and birth sex) in 1744 clomiphene pregnancies from our clinic. We found that (i) preclinical and clinical abortions are increased only slightly by clomiphene use, compared to spontaneous pregnancy; (ii) clinical abortions are decreased in patients with polycystic ovaries and luteal insufficiency who use clomiphene; (iii) conception and preclinical abortions are related to endometrial thickness prior to ovulation; (iv) ectopic pregnancies are not increased by clomiphene and (v) the ratio of male births is not altered by clomiphene, except possibly in timed insemination cycles. These studies repudiate many misconceptions regarding clomiphene. They also show that clinical outcome may be improved by pre-ovulation ultrasound monitoring of ovarian and endometrial response.

Equilibrium binding analysis of estrogen agonists and antagonists: relation to the activation of the estrogen receptor.

The equilibrium binding kinetics of the interaction between the estrogen receptor and natural estrogens (estradiol, estriol and estrone), non-steroidal estrogen agonists (11 beta-chloromethyl-estradiol-17 beta, diethyl-stilbestrol, hexestrol) and non-steroidal antiestrogens (clomiphene, tamoxifen) have been characterized. It is proposed that positive cooperative binding of ligands by the estrogen receptor reflects conformational changes in the DNA binding domain of the receptor dimer which increase its affinity to estrogen responsive elements. Weak estrogens fail to induce maximal cooperativity and are less efficient in activating the receptor complex. Antiestrogens, that inhibit the [3H]estradiol-induced cooperative binding, suppress the activation of the receptor and inhibit its nuclear interactions. Another class of antiestrogens (e.g., 4-hydroxytamoxifen) interacts with the receptor in a manner that is indistinguishable from the cooperative interaction of estradiol, and the resulting complex may also exhibit increased affinity for estrogen responsive elements. However, these complexes cannot activate transcription, presumably due to an aberrant induction of transcription-activating domain in the receptor. We suggest that the positive cooperativity of the estrogen receptor results from conformational changes in the receptor that are transmitted also to the DNA binding domain. On the other hand, conformational changes in the transcription activating domain are not revealed by equilibrium binding kinetics. Thus, compounds that block the positive cooperative binding of [3H]estradiol by the receptor act as antiestrogens. Other compounds that interact cooperatively with the receptor can activate the receptor DNA binding domain, however, they may or may not induce the full array of conformational changes required for transactivation of transcription.

Induction of ovulation in the patient with polycystic ovarian disease.

Induction of ovulation is difficult to achieve in patients with PCOD when they are resistant to therapy with clomiphene citrate; moreover, treatment with human menopausal gonadotropins subjects the PCOD patient to the risk of multifollicular ovulation and hyperstimulation. This article summarizes the hormonal picture and the initial therapeutic approach to the patient with PCOD. The proper administration, usage, and monitoring of conventional ovulation-inducing agents for these patients are discussed. Some of the newer approved agents that can be used in special patients who have not responded to conventional ovulation induction are also described.