Biocatalytic modifications of ethynodiol diacetate by fungi, anti-proliferative activity, and acetylcholineterase inhibitory of its transformed products.

The fungal transformations of ethynodiol diacetate (1) were investigated for the first-time using Botrytis cinerea, Trichothecium roseum, and R3-2 SP 17. The metabolites obtained are as following: 17α-Ethynyl-17ß-acetoxyestr-4-en-3-one-15ß-ol (2), 19-nor-17a-ethynyltestosterone (3), and 17α-ethynyl-3ß-hydroxy-17ß-acetoxyestr-4-ene (4). The new metabolite, 2 (IC50 = 104.8 µM), which has ketone group at C-3, and the ß-hydroxyl group at C-15, resulted in an almost equipotent strength with the parent compound (IC50 = 103.3 µM) against proliferation of SH-SY5Y cells. The previously reported biotransformed product, 3, showed almost equal strength to 1 against acetylcholinesterase. Molecular modelling studies were carried out to understand the observed experimental activities, and also to obtain more information on the binding mode and the interactions between the biotransformed products, and enzyme.

Novel reversed-phase HPLC method for simultaneous determination of ethynodiol diacetate (EDA)/ethinyl estradiol (EE) in pharmaceutical dosage form.

Ethynodiol diacetate (EDA) and ethinyl estradiol (EE) tablets are indicated to prevent pregnancy in women who use oral contraceptives as a contraception method. EDA and EE were separated by reversed-phase HPLC using Agilent ZORBAX SB-Phenyl column, 4.6 mm × 15 cm, 5 µm, using a gradient mixture of acetonitrile and Milli-Q water as mobile phase. The linearity and recovery were found in the range of 0.025-0.25 mg/mL and 0.05-0.18 mg/mL for EDA and 0.001-0.01 mg/mL and 0.002-0.007 mg/mL for EE, respectively. The method is validated according to the regulatory guidelines concerning system suitability, specificity, repeatability, recovery, linearity, robustness, and stability of the sample solution.

Transient analysis of drug delivery from a toroidal membrane: Applications for medicated vaginal rings.

A mathematical construct is proposed to analyze drug released from matrix-type vaginal rings. This work is intended to support experimental studies and promote the fabrication of these devices. The transport of a dissolved drug through a toroidal membrane was predicted using diffusion equations and their solutions. This dynamic framework led to the estimation of the time elapsed before releasing 98% of the ethynodiol diacetate from the polymer. Closed-form expressions, easily adaptable to spreadsheet implementation, were developed to simulate the controlled delivery of levonorgestrel initially dispersed in a silicone vaginal ring. As the loading increased, a greater amount of medication was delivered. However, the fractional release decreased from 32.6% to 23.1% when the dosage changed from 4.137 g/cm3 to 8.274 mg/cm3. The expressions were further simplified for thin rings.

The Effect of Oral Contraceptive Hormones on Anterior Cruciate Ligament Strength.

BACKGROUND: Women are 2 to 9 times more likely to experience an anterior cruciate ligament (ACL) injury than men. Various hormones including relaxin, progesterone, and estrogen influence ACL strength. Oral contraceptives (OCs) alter these hormone levels; however, studies have yet to comprehensively compare different OCs' effects on the ACL. HYPOTHESIS: OCs with increased progestin-to-estrogen ratios will (1) increase ACL collagen expression, (2) decrease ACL matrix metalloproteinase expression, and (3) increase ACL strength. STUDY DESIGN: Controlled laboratory study. METHODS: Untreated female rats were compared with rats treated with 1 of 5 clinically used OCs: norethindrone (NE) only, NE plus ethinylestradiol (EE), etynodiol diacetate (ED) plus EE, norgestimate (NG) plus EE, and drospirenone (DS) plus EE. Doses were scaled from human doses to account for differences in bioavailability and body weight, and OCs were administered daily via oral gavage for 4 rat estrous cycles (20 days). A total of 36 rats were then sacrificed (6 rats/group). ACLs underwent biomechanical testing to assess ACL strength, stiffness, and maximum load before failure. ACL specimens were also isolated for quantitative real-time polymerase chain reaction analysis to assess collagen, matrix metalloproteinase, and relaxin receptor-1 expression. RESULTS: While the primary structural property of interest (ACL maximum load before failure) was not significantly improved by OC treatment, the main material property of interest (ACL strength) in rats treated with NE only, DS + EE, ED + EE, and NE + EE was significantly increased compared with untreated controls (P = .001, P = .004, P = .004, and P = .04, respectively). The order from strongest to weakest ACLs, which was also the same order as the highest to lowest progestin-to-estrogen ratios, was groups treated with NE only, DS + EE, ED + EE, NE + EE, and lastly NG + EE. Higher ratio formulations also increased the expression of type I collagen (P = .02) and decreased the expression of matrix metalloproteinase-1 (P = .04). CONCLUSION: OC formulations with higher progestin-to-estrogen ratios may be more protective for the ACL than formulations with lower ratios. CLINICAL RELEVANCE: OC formulations with high progestin-to-estrogen ratios may benefit female athletes by reducing their ACL injury risk by decreasing the effects of relaxin on the ACL.

Progestin-only pills for contraception.

BACKGROUND: The introduction of a new progestin-only oral contraceptive in Europe has renewed interest in this class of oral contraceptives. Unlike the more widely used combined oral contraceptives containing an estrogen plus progestin, these pills contain only a progestin (progestogen) and are taken without interruption. How these pills compare to others in their class or to combined oral contraceptives is not clear. OBJECTIVES: This review examined randomized controlled trials of progestin-only pills for differences in efficacy, acceptability, and continuation rates. SEARCH METHODS: Through October 2013, we searched the computerized databases MEDLINE, Cochrane Central Register of Controlled Trials (CENTRAL), POPLINE, and LILACS for studies of progestin-only pills. We also searched for current trials via ClinicalTrials.gov and ICTRP. Previous searches also included EMBASE. SELECTION CRITERIA: We included all randomized controlled trials in any language that included progestin-only pills for contraception.  We incorporated any comparison with a progestin-only pill; this could include different doses, other progestin-only pills, combined oral contraceptives, or other contraceptives. DATA COLLECTION AND ANALYSIS: The first author abstracted the data and entered the information into RevMan 5. Another author performed a second, independent data abstraction to verify the initial data entry.We attempted to extract life-table rates (actuarial or continuous) and used the rate difference as the effect measure. Where life-table rates were not published, we used the incidence rate ratio (ratio of Pearl rates). Where only the crude number of events was published, we calculated the Peto odds ratio with 95% confidence interval (CI) using a fixed-effect model. For continuous variables, the mean difference (MD) was computed with 95% CI. Because of disparate exposures, we were not able to combine studies in meta-analysis. MAIN RESULTS: Six trials met the inclusion criteria. We have not found any new studies since the initial review. In the trial comparing the desogestrel versus levonorgestrel progestin-only pill, desogestrel was not associated with a significantly lower risk of accidental pregnancy; the rate ratio was 0.27 (95% CI 0.06 to 1.19). However, the desogestrel progestin-only pill caused more bleeding problems, although this difference was not statistically significant. The trial comparing low-dose mifepristone versus a levonorgestrel progestin-only pill found similar pregnancy rates. In the trial comparing ethynodiol diacetate versus a combined oral contraceptive, irregular cycles occurred in all women assigned to the progestin-only pill (odds ratio 135.96; 95% CI 7.61 to 2421.02). In a trial comparing two progestin-only and two combined oral contraceptives, the progestin-only pill containing levonorgestrel 30 µg had higher efficacy than did the pill containing norethisterone 350 µg. An early trial found megestrol acetate inferior to other progestin-only pills in terms of efficacy. A study of the timing of pill initiation after birth found no important differences, but high losses to follow up undermined the trial. AUTHORS' CONCLUSIONS: Evidence is insufficient to compare progestin-only pills to each other or to combined oral contraceptives.

Leptin, fat mass, and bone mineral density in healthy pre- and postmenopausal women.

The purpose was to examine relationships between age, fat mass, and bone mineral density (BMD) with resting leptin levels in premenopausal and postmenopausal women. Young (aged 18-30 yr, n=30) and estrogen-deficient postmenopausal (aged 55-75 yr, n=43) women were recruited. Total body and segmental fat mass and bone-free lean body mass (BFLBM) and total body, lumbar spine, and proximal femur BMD were assessed using dual-energy X-ray absorptiometry. Serum-resting, fasted leptin levels were measured by Immunoradiometric Assay (IRMA), and leptin-to-fat mass ratios were calculated. Young and older women had similar amounts of BFLBM, but older women had greater (p<0.05) amounts of fat mass and 35% higher leptin levels. Age differences in leptin concentrations were no longer significant after controlling for fat mass. Older women had significantly (p<0.05) lower hip BMD values. Age was negatively related (r=-0.29, p<0.05) to leptin:trunk fat ratio. Increases in fat mass, not menopause per se, contributes to higher leptin levels in older women. Relationships between leptin and BMD may be age dependent.

Progestin-only pills for contraception.

BACKGROUND: The introduction of a new progestin-only oral contraceptive in Europe has renewed interest in this class of oral contraceptives. Unlike the more widely used combined oral contraceptives containing an estrogen plus progestin, these pills contain only a progestin (progestogen) and are taken without interruption. How these pills compare to others in their class or to combined oral contraceptives is not clear. OBJECTIVES: This review examined randomized controlled trials of progestin-only pills for differences in efficacy, acceptability, and continuation rates. SEARCH STRATEGY: We searched the computerized databases MEDLINE, Cochrane Central Register of Controlled Trials (CENTRAL), POPLINE, LILACS, and EMBASE for studies of progestin-only pills. We also searched for current trials via ClinicalTrials.gov and ICTRP. SELECTION CRITERIA: We included all randomized controlled trials in any language that included progestin-only pills for contraception. We incorporated any comparison with a progestin-only pill; this could include different doses, other progestin-only pills, combined oral contraceptives, or other contraceptives. DATA COLLECTION AND ANALYSIS: The first author abstracted the data and entered the information into RevMan 5. Another author performed a second, independent data abstraction to verify the initial data entry. Because of disparate exposures, we were not able to combine studies in meta-analysis. MAIN RESULTS: Six trials met the inclusion criteria. In the trial comparing the desogestrel versus levonorgestrel progestin-only pill, desogestrel was not associated with a significantly lower risk of accidental pregnancy; the rate ratio was 0.27 (95% CI 0.06 to 1.19). However, the desogestrel progestin-only pill caused more bleeding problems, although this difference was not statistically significant. The trial comparing low-dose mifepristone versus a levonorgestrel progestin-only pill found similar pregnancy rates. In the trial comparing ethynodiol diacetate versus a combined oral contraceptive, irregular cycles occurred in all women assigned to the progestin-only pill (odds ratio 135.96; 95% CI 7.61 to 2421.02). In a trial comparing two progestin-only and two combined oral contraceptives, the progestin-only pill containing levonorgestrel 30 mug had higher efficacy than did the pill containing norethisterone 350 mug. An early trial found megestrol acetate inferior to other progestin-only pills in terms of efficacy. A study of the timing of pill initiation after birth found no important differences, but high losses to follow up undermined the trial. AUTHORS' CONCLUSIONS: Evidence is insufficient to compare progestin-only pills to each other or to combined oral contraceptives.

The use of oral contraceptives as a prevention of recurrent premenstrual psychosis.

Premenstrual psychosis is a rare and not formally recognized disorder (DSM-IVR, ICD-10). The literature mainly consists of clinical cases. There have been preliminary reports of improvement in such cases after administration of oral contraceptives. We present a case of premenstrual psychosis in which hormonal treatment was effective in preventing symptomatic relapses.

Protective role of nordihydroguaiaretic acid (NDGA) against the genotoxic damage induced by ethynodiol diacetate in human lymphocytes in vitro.

Antioxidants and plant products are reported to reduce the genotoxic damage of steroids. In our present study we have tested different dosages of nordihydroguaiaretic acid (NDGA) against the genotoxic damage induced by ethynodiol diacetate in the presence of S9 mix. Treatments with nordihydroguaiaretic acid (NDGA) results in the reduction of the genotoxic damage. A significant decrease was observed at all the tested doses of NDGA in sister chromatic exchanges of number of abnormal cells. The results suggest a protective role of NDGA against the genotoxic damage.

Do AshSplit haemodialysis catheters provide better flow rates in the long term?

BACKGROUND: Recently, interventional radiologists have adopted an increasingly prominent role in the placement and management of hemodialysis catheters, as well as in the research and development of new and better catheters. The purpose of this study was to evaluate the viability and hemodialysis efficiency of the AshSplit catheter and the Permcath catheter. METHODS: 204 consecutive patients requiring radiological insertion of hemodialysis catheters were followed, retrospectively, over a 42-month period. Both hemodialysis catheters were placed using a combination of ultrasonic and fluoroscopic guidance and tunneled appropriately. Information collected included catheter insertion sites, insertion complications, catheter duration, and final outcome. RESULTS: Over the study period of two years, 269 catheters were placed into 204 patients with end stage renal failure. Patients received either an AshSplit (101 patients, 127 catheters) or a Permcath (103 patients, 142 catheters). Vascular access route of choice was the right internal jugular vein (67% AshSplit, 71% Permcath). Insertion complications occurred in 18 patients overall (6.6%), with only 1 requiring further intervention (hemopneumothorax). Flow rates averaged 259 mls/min for AshSplits and 248 mls/min for Permcaths (p < 0.001). Follow-up of catheter viability for 42 months yielded a mean AshSplit catheter duration of 246 days (range 6-932) and 239 days (range 1-1,278) for Permcath (p = 0.46). Reasons for catheter failure and elective catheter removal were similar in both groups; however, Permcaths required significantly more thrombolysis than AshSplits, p < 0.001. CONCLUSION: The AshSplit provides significantly better flow rates and less thrombolysis compared to the Permcath, with similar catheter dwell times.

Oral contraceptives as substrates and inhibitors for human cytosolic SULTs.

Cytosolic sulfotransferases (SULTs) in mammals are involved in the biotransformation and homeostasis of various endogenous and xenobiotic compounds. The current study aimed to examine the sulfation of contraceptive compounds by various human cytosolic SULTs and to investigate the inhibitory effects and mode of action of these compounds on the sulfation of 17beta-estradiol, a major endogenous estrogen. A systematic study using all eleven known human cytosolic SULTs revealed the differential substrate specificity of these enzymes for the eight representative contraceptive compounds and two endogenous estrogens (estrone and 17beta-estradiol) tested as substrates. Activity data showed that SULT1A1 displayed the strongest activity toward 17alpha-ethynylestradiol. Kinetic studies revealed that the V (max) value of the sulfation of 17alpha-ethynylestradiol by SULT1A1 was 1.64 times that of the sulfation of 17beta-estradiol, while the K (m) values were almost equal for the two compounds. The inhibitory effects of three contraceptive compounds on the sulfation of 17beta-estradiol by SULT1A1 were examined. IC(50) values determined were 0.193, 1.84, and 2.98 mM, respectively, for 19-norethindrone acetate, ethynodiol diacetate and mifepristone. Kinetic analyses indicated that the mechanism underlying the inhibition by these contraceptives is of a mixed noncompetitive type. Metabolic labeling experiments confirmed the sulfation of contraceptive compounds and the release of their sulfated derivatives by HepG2 human hepatoma cells. Collectively, the results obtained suggest a role of sulfation in the metabolism of contraceptive compounds in vivo. Moreover, in view of their inhibitory effects on the sulfation of 17beta-estradiol, these compounds may potentially act to disrupt the homeostasis of endogenous estrogens.

Suppressive effect of neonatal treatment with a phytoestrogen, coumestrol, on lordosis and estrous cycle in female rats.

The neural control systems for the ovulatory cycle and lordosis behavior are sexually differentiated by estrogen during the perinatal period in rats. In the present study, the effects of a single neonatal injection with the phytoestrogen, coumestrol, on female reproductive functions were investigated. Female rats were injected subcutaneously with 1 or 3mg coumestrol (CM1, CM3), 1mg genistein (GS1), 1mg estradiol (E2), or oil at day 5 after birth (birth day=day 1) and an estrous cycle check and lordosis behavior test were performed. As a result, vaginal opening was advanced in CM1-, CM3- or E2-treated females. A vaginal smear check indicated that oil- or GS1-treated females showed a constant 4- or 5-day estrous cycle, whereas CM1-, CM3- or E2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovary weights in the CM1-, CM3- or E2-treated groups were lower than those in the oil- and GS1-treated groups and no corpora lutea were found in any rats of these three groups, except for two E2-treated rats. Behavioral tests were carried out after implantation of E2-tubes. All rats in the CM1-, GS1-treated groups showed a high lordosis quotient (LQ), being comparable to that in the oil-treated females. On the other hand, LQs in the CM3, E2 or male groups were lower than that in the control female group. These results suggest that a single neonatal injection of 3 mg coumestrol was effective in suppressing the functions of ovulation-inducing mechanisms and the induction of lordosis, but 1mg coumestrol was effective in only the estrous cycle of female rats.

New observations on endometrial physiology after transcervical injection of methylene blue dye.

We describe the in vivo features of endometrium stained with methylene blue dye and observed via microhysteroscopy, showing the patterns of endometrial glands and superficial cell changes during the midproliferative, periovulatory, and midluteal phases. These preliminary observations have allowed us to identify a series of changes occurring in the different phases of the ovulatory cycle of potential value in reproductive medicine for specific groups of infertile patients.

Chemical and analytical characterization of related organic impurities in drugs.

A system is proposed for the classification of related organic impurities in drugs and drug products including among others (separated and non-separated) intermediates, various kinds of by-products, among them products of different side reactions, epimeric/diastereomeric, enantiomeric impurities, impurities in natural products, and finally degradation products. Examples are taken mainly from the author's own experience and from among the named impurities in the European Pharmacopoeia with focus on impurities in hydrocortisone, prednisolone, enalapril maleate, lisinopril, ethynodiol diacetate, pipecuronium bromide, cimetidine, and ethynylsteroids. The methodological aspects of impurity profiling from the detection to the identification/structure elucidation and quantitative determination of impurities are briefly summarized.

Membrane-initiated steroid signaling (MISS): genomic steroid action starts at the plasma membrane.

UNLABELLED: Plasma membrane (PM) steroid recognition sites are thought to be responsible only for rapid, non-genomic responses without any link to the nuclear receptor-mediated genomic effects of steroids. We focused on a PM "glucocorticoid-importer" (GC-importer) that imports GC into rat liver cells. This site interacts also with particular gestagens (progesterone, P; medroxyprogesterone, MP; ethynodiol, Ethy) and estrogens (ethinylestradiol, EE(2); mestranol), which do not bind to the nuclear GC receptor (GR). To elucidate the role of the GC-importer, we transfected a rat wild-type hepatocyte (CC-1) and a hepatoma cell line, unable to import GC (MH 3924), with a GC<-->GR-responsive luciferase (luc)-reporter gene. Selected steroids were tested for their ability to induce or inhibit luc expression. Corticosterone (B) and dexamethasone (Dex), but also the GC-antagonists cortexolone (Cortex), P and MP, induced luc. Even the PM-impermeable BSA-derivatives of B, Dex and Cortex did so to almost the same extent as the free steroids. MH 3924 cells respond stronger than CC-1 to luc inducing steroids. Luc expression was inhibited by RU 38 486, but also by EE(2) and Ethy. The thiol reactive mesylate-derivatives of B, Dex and Cortex induced to a considerably lesser extent than the free or BSA-steroids. The thiol reagent mersalyl blocks cellular entry of GC and inhibits luc induction in CC-1 cells. Incubation with EE(2) and B of PM-vesicles, isolated from liver cells, resulted in a decrease of the density of two 75 and 52kDa G-proteins reflecting a diminished exchange of GDP by GTP. CONCLUSION: the PM-residing GC-importer, now renamed "Steroid Hormone Recognition and Effector Complex" (SHREC) is an interdependent part of the complete GC signal propagation in which G-proteins are involved. Free SH-groups of SHREC are a prerequisite for genomic GC activity. Specific interactions between SHREC and GC-agonist/-antagonist trigger steroid-dependent signaling. However, import of the ligand into the cell terminates it. Thus, the PM-related non-genomic steroid responses are clearly linked to the GR-related genomic effects.