Pharmacoinformatics study of Piperolactam A from Piper betle root as new lead for non steroidal anti fertility drug development.

Fertility control is a burning problem all over the world to regulate population overflow and maintain ecological balance. This study is an in-silico approach to explore a non-steroidal lead as contraceptive agent in order to avoid several contraindications generated by steroidal analogues. Piperolactam A, an aristolactam isolated from Piper betle Linn. showed binding affinity towards estrogen and progesterone receptor as -8.9 and -9.0Kcal/mol (inhibition constant Ki=0.294µM and 0.249µM) respectively which is even larger than that of reported antagonists such as Rohitukine and OrgC (binding affinity -8.7 and -8.4Kcal/mol; Ki 0.443µM and 0.685µM respectively). The binding site exploration displayed more hydrogen bonding of Piperolactam A (His 524, Leu 346, Thr 347) than Rohitukine and OrgC (Leu 718) with associated receptors which was further confirmed by molecular dynamics simulations. The drug-likeliness of the compound has been proved from its tally with Lipinsky's Rule of Five and lowered toxicity such as cardiac toxicity, liver toxicity, mutagenicity and ecological toxicity. Endocrine disruptome and later docking guided molecular simulations revealed that Piperolactam A has weaker binding affinity and/or lower probability of binding with nuclear receptors especially hERG and cytochrome P450. The high Caco-2 permeability suggested more bioavailability hence more therapeutic efficacy of the drug.

A critical examination of the mode of action of quinacrine in the reproductive tract in a 2-year rat cancer bioassay and its implications for human clinical use.

A rat carcinogenicity bioassay (CaBio) of quinacrine was reanalyzed to investigate its mode of tumor induction. Quinacrine's effects in the rat uterus when administered as a slurry in methylcellulose were contrasted with the human clinical experience which uses a solid form of the drug, to determine the relevance of the tumors produced in the rat to safe clinical use of quinacrine for permanent contraception (QS). A review was performed of the study report, dose feasibility studies, and clinical evaluations of women who had undergone the QS procedure. The top three doses of quinacrine in the CaBio exceeded the maximum tolerated dose, and produced chronic damage, including inflammation, resulting in reproductive tract tumors. Chronic inflammation was significantly correlated with the tumors; there was no evidence of treatment-related tumors in animals without chronic inflammation or other reproductive system toxicity. Because such permanent uterine damage and chronic toxicity have not been observed in humans under therapeutic conditions, we conclude that this mode of action for tumor production will not occur at clinically relevant doses in women who choose quinacrine for permanent contraception.

The synthetic gestagen levonorgestrel impairs metamorphosis in Xenopus laevis by disruption of the thyroid system.

Synthetic gestagens, including levonorgestrel (LNG), are active compounds in contraceptives, and several studies report their occurrence in surface waters. However, information about endocrine-disrupting effects in nontarget organisms is scarce. The present study investigated effects of LNG exposure on thyroid hormone-dependent metamorphosis of Xenopus laevis. Premetamorphic X. laevis tadpoles at Nieuwkoop and Faber (NF) stage 48 were exposed in a flow-through culture system to four LNG concentrations (10(-11), 10(-10), 10(-9), and 10(-8)M) over the period of metamorphosis. At NF 58 and 66, tadpoles were examined sex specifically. Developmental time and organismal responses were recorded and correlated with molecular and histopathological endpoints. Exposure to 10(-8)M LNG caused an inhibition of metamorphosis resulting in developmental arrest at early climax stages as giant tadpoles or tailed frogs. In brain-pituitary tissue of NF 58 tadpoles, gene expression of thyroid-stimulating hormone (ß-subunit; TSHß), TH receptor ß (TRß), and deiodinase type 3 (D3) was not changed. Instead, prolactin (PRL) messenger RNA (mRNA) was significantly increased by 10(-9)M LNG in females and by 10(-8)M LNG in both sexes. In NF 66 tadpoles, mRNA levels of TSHß mRNA were significantly increased in the 10(-9) and 10(-8)M LNG treatment groups indicating a hypothyroid state. No changes of TRß, D3, and PRL gene expression were detected. Histopathological evaluation of thyroid gland sections revealed no typical sign of hypothyroidism but rather an inactivated appearance of the thyroid. In conclusion, our data demonstrate for the first time a completely new aspect of thyroid system disruption caused by synthetic gestagens in developing amphibians.

Preclinical studies of alkylureas as anti-HIV-1 contraceptive.

The HIV-1 epidemic continues to spread at a rate of over 15, 000 new cases daily. HIV-1 transmission through heterosexual contact became the dominant risk for women globally. About half of the over 40 million HIV-1 infected individuals worldwide are now women. The lack of empowerment of women is the fundamental cause for the rampant spread of HIV-1 in women. Topical microbicides applied intravaginally offer an option for female-initiated HIV-1 prevention. There is an urgent need to develop microbicides with and without contraceptive qualities to also address socio-cultural settings where the woman's status is linked to fertility. A safe and efficacious anti-HIV-1 vaginal formulation is not yet available though a large number of candidates are in preclinical or clinical studies. Presently marketed topical microbicides are by and large toxic and damage the vaginal mucosa with frequent use. The microbicidal system of alkylureas evaluated here lends itself to contraceptive and non-contraceptive anti- HIV-1 formulations. Alkylureas are agents that irreversibly disrupt free and intracellular HIV-1, have a wide margin of safety and are spermicidal above their virucidal concentration without any mucosal toxicity. Butylurea, the lead compound is also effective against other sexually transmitted diseases (STDs) while sparing the normal vaginal flora. Alkylureas with longer alkyl chains still have to be explored and may have a greater selective microbicidality.

Progestogen only contraception and endometrial break through bleeding.

Progestogen only contraceptives (POC) provide a safe and effective method of fertility regulation. Unfortunately, they are commonly associated with the problem of endometrial break through bleeding (BTB), often leading to discontinuation of use. An increase in endometrial vascular fragility has been demonstrated as an important mechanism that contributes to BTB but our understanding of the interaction between exogenous steroid use and endometrial vasculature remains incomplete. This review sets out to describe a number of commonly used POC, their effects on endometrial morphology and possible molecular and cellular mechanisms that may lead to unscheduled bleeding.

Phytochemical screening and pharmacological evaluations for the antifertility effect of the methanolic root extract of Rumex steudelii.

The practice of traditional medicine for the control of fertility in most parts of Ethiopia is based on the uses of plant medicines for many years. The fact that herbal medicines have been employed for such a long time does not guarantee their efficacy and safety. The aim of the present study was, therefore, to carry out phytochemical screening, efficacy and safety studies on one of the traditionally used antifertility plants: Rumex steudelii. The secondary metabolites of the root of this plant were determined. The methanolic extract of the roots of this plant were investigated for their antifertility activity in female rats and oral LD50 was determined in mice. The identification of the secondary metabolites showed that the roots of the plant contained phytosterols and polyphenols. It was found that the extract reduced significantly (p<0.01) the number of litters. It also produced antifertility effect in a dose dependent manner and the contraceptive effect was manifested for a definite period of time. Furthermore, the extract prolonged significantly the estrus cycle (p<0.05) and the diestrous phase (p<0.01) of the rats. The wet weights of the ovaries and uterus were shown to be reduced significantly (p<0.01) and (p<0.05), respectively. The oral LD50 of the extract was found to be 5 g/kg in mice. All these observations suggest that the extract has antifertility effect and is safe at the effective antifertility doses employed in this study.

Pre-clinical safety evaluation of novel nucleoside analogue-based dual-function microbicides (WHI-05 and WHI-07).

Compounds WHI-05 [5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-methoxyphenyl)-methoxyalaninyl phosphate] and WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxyalaninyl phosphate] are aryl phosphate derivatives of zidovudine (ZDV) with anti-HIV and contraceptive activity. WHI-05 and WHI-07 differ fundamentally from currently used surfactant microbicides that are cytotoxic to genital tract epithelial cells at spermicidal concentrations. These drugs were rationally designed to bypass the thymidine kinase dependency of ZDV activation in genital tract secretions, as well as to achieve spermicidal activity. WHI-05 and WHI-07 were formulated via a non-toxic gel-microemulsion for intravaginal use as potential anti-HIV spermicides. Pre-clinical safety studies of intravaginally administered WHI-05 and WHI-07 gel-microemulsions were performed in mice and rabbits to mimic closely the intravaginal application of a microbicidal preparation in women. In addition, systemic toxicity studies were performed in mice and non-human primates. The LD10 doses for WHI-05 and WHI-07 when administered intravenously or intraperitoneally were >500 mg/kg for mice. Female cynomolgus monkeys treated with 20 mg/kg WHI-05 and WHI-07 intravenously developed no grade 2-4 systemic toxicities. Repetitive intravaginal administration of 2% WHI-05 and WHI-07 via a gel-microemulsion to achieve concentrations as high as 6.1 x 10(4) and 5.7 x 10(6) times their respective in vitro anti-HIV IC50 values, and 1200 and 5700 times their spermicidal EC50 values, for up to 13 weeks, was not associated with mucosal, systemic or reproductive toxicity. Furthermore, long-term (2 years) intravaginal administration of 2% WHI-07 gel-microemulsion was not associated with systemic toxicity or increased carcinogenicity in mice. The improved potency, as well as the lack of mucosal, systemic and reproductive toxicity of WHI-05 and WHI-07, means that these compounds have clinical potential as safe, prophylactic contraceptives in addition to their microbicide activity to curb the sexual transmission of HIV.

Toxicology of progestogens of implantable contraceptives for women.

There are currently four progestogens used in implantable contraceptives marketed or tested in clinical trials: levonorgestrel in Norplant and Jadelle, etonogestrel (3-keto-desogestrel) in Implanon, nestorone in Elcometrine, and nomegestrol acetate in Uniplant and Surplant. Each progestogen was evaluated for hormonal activity and for safety in a wide variety of tests in vitro and in animals prior to their use in women. All four progestogens underwent pre-clinical testing that generally followed the format for animal testing of steroidal contraceptives published by the World Health Organization and the US Food and Drug Administration (FDA). Most of the progestogens have been tested for genotoxicity in bacterial and mammalian cultured cells and in rodents. All were tested for toxicity in short- and long-term toxicology studies in rodents and dogs or monkeys, and all were tested for their effects on reproduction and fetal development. In most cases, the progestogens were tested for carcinogenicity in two rodent species, rats and mice. Early clinical trials in small numbers of women provided additional safety data prior to the exposure of large numbers of women in Phase 3 clinical trials. The published data and data submitted to the FDA demonstrate that the implantable progestogens have no significant or unusual toxicities and have a similar safety profile to the progestogens found in the approved oral contraceptives.

The development and regression of deciduosarcomas and other lesions caused by estrogens and progestins in rabbits.

A series of experiments were conducted to study the histopathological effects of a combination of exogenous estrogens and progestins in mature rabbits. Estradiol (14-45 microg/day) and levonorgestrel (30-233 microg/day) were administered by intravaginal or subdermal Silastic devices for various time intervals to study the development of lesions with time and to determine if lesions regressed following withdrawal of the steroids. The origin of splenic decidual tumors (primary or metastasis from the uterus) was determined by administering the same steroid combination to castrated male rabbits. It was determined that uterine decidualization is present after 7 days of steroid treatment and that neoplasms of decidual cells may appear in the uterus after only 30 days of steroid administration. Decidual changes were observed frequently in uterine arteries, often concurrent with infarct-like areas of necrosis of the uterine wall. Withdrawal of contraceptive steroids for 14-120 days after 60 days' administration resulted in atrophy and disappearance of decidual cells and decidual tumors. Decidual neoplasms developed in the spleen of all castrated male rabbits given subdermal steroids, demonstrating that these tumors can arise as primary neoplasms of the spleen. The foregoing lesions appear to be peculiar to the rabbit and, together with previous data, suggest the rabbit to be a poor model for evaluating the effects of contraceptive steroids in other species.

Anticoncepción con implantes subdérmicos en mujeres adolescentes: observaciones preliminares en 174 casos

Se presentan resultados de 174 mujeres adolescentes, usuarias de primera vez del Norplant. Se encontró que la edad promedio fue de 17.7 años; 39.9 por ciento eran nulíparas. La tasa acumulada de emabarazos a 2 años fue de 0 por ciento. El 48 por ciento tuvieron ciclos irregulares( 9 por ciento manchando, 20 por ciento sangrando y 19 por ciento amenorrea). Otros efectos adversos reportados fueron: mareos (6 por ciento), cefalea (4 por ciento) y aumento de peso (4 por ciento). La tasa de retiro fue de 6.8 por ciento. Las restantes usuarias están satisfechas con el método. Los anteriores datos nos permiten sugerir el uso del Norplant como método contraceptivo en este etario. Sin embargo, un estudio prolongado (5 años), es necesario para corroborar estos hallazgos

Animal toxicity studies performed for risk assessment of the once-a-month injectable contraceptive Mesigyna.

Results from toxicity studies performed for risk assessment of the combined injectable hormonal preparation Mesigyna are reviewed. Both components of Mesigyna, i.e., estradiol valerate (E2Val) and norethisterone enanthate (NET-EN), have been thoroughly investigated as individual compounds and some limited toxicity data have been obtained for the combination. Most findings which were gathered in these studies from different animal species occurred in the gonads, accessory genital and endocrine organs and can be related to the known species-specific pharmacological activity of a typical estrogen or progestin, respectively. No additional or unexpected information which might indicate a possible estrogen/progestin interaction was gained from the administration of the combined preparation to animals. Based on the results from toxicity testing, there were no objections to the long-term therapeutic use of Mesigyna for hormonal contraception. The predictive value of the effects (including the tumorigenicity) observed in the common laboratory animals with regard to human safety is critically discussed, taking the vast amount of previous experience with hormonal contraceptives into consideration. The conclusion is drawn that there is no animal model for safety assessment of sex steroids that adequately represents the human situation. Quantitative extrapolations from animal toxicity findings to humans, therefore, are not possible. Especially, the value of long-term studies and of toxicity studies on estrogen/progestin combinations is put into question. Like endocrine pharmacology studies, the toxicity studies with these steroid hormones are useful for the characterization of the possible endocrine pharmacological profile only.

PIP: Considerable research has been conducted on the 2 steroid components of the once-a-month injectable contraceptive, Mesigyna. These steroids are estradiol valerate and norethisterone enanthate. Most findings from the limited toxicity studies of the combined injectable in different animal species were limited to the gonads and accessory genital and endocrine organs. The steroids have a toxicological activity profile in each of the species, which indicate that they act as a typical steroidal estrogen or progestin. They are no different than comparable compounds as used in oral contraceptives. Other than these findings, researchers did not acquire any more or unexpected information that would suggest a possible estrogen/progestin interaction from administering the combined preparation to animals. The findings of the toxicity testing do not suggest problems with the long-term use of Mesigyna for hormonal contraception. Yet, the fundamental species differences in endocrinology, metabolism of compounds, and pharmacokinetics make it virtually impossible to quantitatively extrapolate from findings of animal toxicity studies to human, e.g., those on tumorigenicity. A critical review of in vitro and animal toxicity studies of both compounds individually and combined lead to the conclusion that no animal model for safety assessment of sex steroids exists that correctly replicates the human environment. Toxicologists with Schering AG even question the value of long-term studies and toxicity studies on estrogen/progestin combinations. Animal models can be used, however, to characterize a possible endocrine pharmacological profile of newly developed steroids. Any such animal studies need not last any longer than 6 months.

Mouse sperm-egg interaction in vitro in the presence of neem oil.

In vitro evidence is presented showing toxicity of neem oil on sperm-egg interaction in mouse. Cumulus oophorus-enclosed ova, inseminated with capacitated spermatozoa, were cultured in 1 ml of in vitro fertilization (IVF) medium and overlayered by 1 ml of different concentrations of neem oil (1, 5, 10, 25, 50 and 100%) for IVF duration of 4h. At the end of incubation, ova were allowed to grow in neem oil-free culture medium and assessed for fertilization, first cleavage (2-cell formation) and blastocyst formation in vitro at 4-14h, 24h and 108h post-insemination respectively. The study showed that the presence of neem oil at concentrations of 10, 25 and 50% caused inhibition of IVF in a dose-dependent manner. The toxic effect of exposure of 25 and 50% neem oil was further carried over to the first cleavage of the resulting fertilized ova and the toxic effect of 5, 10, 25 and 50% was carried over to the blastocyst formation from the resulting fertilized ova when grown in neem-oil free culture medium. A total of 94.1% inhibition of 2-cell formation and 100% inhibition of blastocyst formation from the inseminated ova was observed in 50 and 25% neem oil-treated groups respectively. Neem oil at 100% concentration caused 100% degeneration of ova at 1h of sperm-ova coculture. The study showed a direct toxic effect of neem oil on sperm-egg interaction in vitro and encourages research investigations of this herbal product as a pre-coital contraceptive.

Transdermal dual-controlled delivery of contraceptive drugs: formulation development, in vitro and in vivo evaluations, and clinical performance.

Several transdermal contraceptive device (TCD) formulations were developed to provide a dual-controlled transdermal delivery of levonorgestrel (LN), a potent progestin, and 17 beta-estradiol (E2), a natural estrogen. Using a sensitive HPLC method, the in vitro release and skin permeation profiles of LN and E2 from various TCD formulations were simultaneously characterized in the hydrodynamically well-calibrated Valia-Chien skin permeation cells and both were found to follow zero-order kinetics. The rates of drug release and skin permeation were observed to vary significantly depending upon some formulation parameters. Six-month stability studies were performed on seven formulations at room and elevated temperatures (37 and 45 degrees C), and two (Formulations 4 and 5) were found to be acceptable, based on drug recovery, release rate, and skin permeation rate data. Judging from the 6-month accelerated stability studies, it is projected these two formulations will have shelf-life of at least 2 years. As a result of development of an efficient manufacturing process, Formulation 4 was selected for further evaluation. One-week primary skin irritation evaluation in 6 rabbits indicated that Formulation 4 is nonirritating, and it was thus selected for Phase I clinical bioavailability/dose proportionality studies in 12 healthy female volunteers of child-bearing age. Results of pharmacokinetic and pharmacodynamic analyses demonstrated that it is capable of achieving and maintaining a steady-state serum level of LN throughout the 3-week treatment period by weekly applications of one or two TCD patches (10 or 20 cm2). A dose proportionality was obtained in the serum drug levels, daily dose delivered, and contraception efficacy. An excellent correlation was obtained for the rates of transdermal delivery determined by the in vitro studies using human cadaver skin, the in vivo studies in rabbits, and the clinical studies in living subjects.

Malignant neoplasms of decidual origin (deciduosarcomas) induced by estrogen-progestin-releasing intravaginal devices in rabbits.

A combination of estrogen and levonorgestrel was continuously delivered to 23 adult rabbits for up to 2 years via a Silastic ring device sutured into the vagina. Twenty-one control rabbits were given similar rings devoid of drugs. A marked decidual reaction of the endometrium occurred in 16 of 23 test rabbits. In 14 test rabbits (61%) malignant tumors developed of decidual type cells not heretofore described. The deciduosarcomas were composed of anaplastic cells that invaded the uterine walls, uterine lymphatics, and in 4 of 13 (31%) rabbits that survived 2 years of treatment, the tumors metastasized to the lungs. Several deciduosarcomas appeared to arise within the spleen or other abdominal organs. Other drug-related lesions included uterine or vaginal polyps, endometrial atrophy, and focal necrosis and mineralization of the uterine wall. Cells from several deciduosarcomas failed to produce tumors in nude mice or to colonize on soft agar. No decidualization or decidual neoplasms were seen in the controls.

Endometrial morphology after 6 months of continuous treatment with a new gonadotropin-releasing hormone superagonist for contraception.

Light and electron microscopic studies were performed on endometrial curettage specimens from 27 women after 6 months of contraceptive treatment with continuous intranasal gonadotropin hormone-releasing hormone (GnRH) superagonist. The GnRH superagonist nafarelin acetate (D-Nal[2]6-GnRH) was used in single daily doses of 125 or 250 micrograms. Ovulation was inhibited during all but one of the 159 treatment months. No pregnancies occurred. In 6 women with fairly regular bleedings, the endometrium displayed weak to normal proliferation. Twenty women developed oligomenorrhea or amenorrhea, 16 of them had inactive endometrium, 1 had weakly proliferative endometrium, and 3 endometrial biopsies were too sparse for adequate evaluation. One woman reported repeated episodes of heavy uterine bleedings. The endometrial biopsy from this woman showed weak proliferation. No signs of endometrial hyperplasia were observed. Generally, the electron microscopy showed signs of low metabolic activity and weak protein synthesis. Thus, long-term continuous treatment with nafarelin acetate for inhibition of ovulation does not appear to have untoward effects on the endometrium.

Contraceptive steroids and coronary artery atherosclerosis in cynomolgus macaques.

The influence of two types of steroidal contraception on the extent of coronary, aortic, carotid, and iliaco-femoral atherosclerosis was assessed in 57 cynomolgus macaques with moderate diet-induced hyperlipoproteinemia. Thirteen animals were treated with an intravaginal ring that released 17 beta-estradiol and levonorgestrel. Fifteen females were treated with an oral contraceptive (OC) composed of ethinyl estradiol and norgestrel. Fifteen females received placebo vaginal rings, and 14 males were untreated. The contraceptive treatments resulted in similar large reductions in plasma high-density lipoprotein (HDL) cholesterol concentrations. Neither treatment influenced the prevalence of coronary artery atherosclerosis. However, treatment with the contraceptive vaginal ring was associated with increased extent of coronary artery atherosclerosis (plaque size) relative to untreated females, whereas treatment with the OC was not. The contrasting effects of the two treatments could not be explained by differences in total plasma cholesterol, HDL cholesterol, or blood pressure. The results suggest that the greater estrogenic influence associated with the ethinyl estradiol-containing OC resulted in inhibition of coronary artery atherosclerosis despite a pronounced progestin-induced lowering of plasma HDL cholesterol concentration and, further, that hormonal balance may have a marked influence on the relationship between plasma lipids and atherogenesis.

Contraceptive steroid toxicology in the Beagle dog and its relevance to human carcinogenicity.

Problems associated with the use of the Beagle dog in chronic toxicological studies of contraceptive steroids are described. A short review is presented on the occurrence of spontaneous tumours in dogs and in bitches of various breeds. The current status of knowledge of canine reproductive hormones and endocrinology is outlined, together with effects of contraceptive steroids. The pathology and histological classification of spontaneous and induced mammary neoplasia in the dog is discussed and compared with breast cancer in women. A series of recommendations are included for future research in this field which it is hoped may resolve some of the outstanding issues and lead to a more suitable toxicological model for contraceptive steroids.

PIP: Many scientists have criticized the mandatory use of dogs for studies of the chronic toxicity of synthetic steroidal contraceptive hormones. The estimated annual incidence rates for cancer of all sites in dogs is 381.2/100,000 dogs. The estimated relative risk (R) value for the occurrence of tumors in the Beagle breed is 0.9; for malignant tumors, the R value in the Beagle is 0.8. A review of the hormonal potency of various contraceptive steroids in the Beagles indicates that progestogenic compounds generally produce a much lower progestational activity in dogs than in women, and the the predominant hormonal action of norethisterone in dogs is estrogenicity rather than progestogenicity. This weak activity for the canine species may account for some of the toxicological findings for norethisterone and related compounds in the Beagle. It is also possible that there are species differences in the relative affinities of estrogen and progesterone receptors for contraceptive steroids. Studies on long-term administration to female Beagle dogs suggest that the nodules found in the mammary gland are not histologically comparable to mammary tumors found in the human female although there is a superficial morphological resemblance to some forms of human mammary dysplasia. Several authors suggest that the results of testing progestational compounds in Beagles are unlikely to be indicative of a potential hazard to the human female. In testing megestrol acetate, it is suggested that the unique sensitivity of the canine females to megestrol acetate is exemplified by intense mammary development at dose levels 10 times the human oral contraceptive level. In contrast, daily dose levels of 500 mg/day in women as a palliative for endometrial cancer have been used with no serious side effects or mammary enlargement. Also the canine mammary gland produces certain pathological changes following administration of natural or synthetic progesterones in a way not readily seen in other species. Possible alternative models (cat, pig) for contraceptive steroid toxicological studies and recommendations for future research are discussed.