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Women in developing countries still face enormous challenges when accessing reproductive health care. Access to voluntary family planning empowers women allowing them to complete their education and join the paid workforce. This effectively helps to end poverty, hunger and promotes good health for all. According to the United Nations (UN) organization, in 2022, an estimated 257 million women still lacked access to safe and effective family planning methods globally. One of the main barriers is the associated cost of modern contraceptive methods. Funded by the Bill & Melinda Gates Foundation, Almac Group worked on the development of a novel biocatalytic route to etonogestrel and levonorgestrel, two modern contraceptive APIs, with the goal of substantially decreasing the cost of production and so enabling their use in developing nations. This present work combines the selection and engineering of a carbonyl reductase (CRED) enzyme from Almac's selectAZyme™ panel, with process development, to enable efficient and economically viable bioreduction of ethyl secodione to (13 R ,17 S )-secol, the key chirality introducing intermediate en route to etonogestrel and levonorgestrel API. CRED library screening returned a good hit with an Almac CRED from Bacillus weidmannii , which allowed for highly stereoselective bioreduction at low enzyme loading of less than 1% w/w under screening assay conditions. However, the only co-solvent tolerated was DMSO up to ∼30% v/v, and it was impossible to achieve reaction completion with any enzyme loading at substrate titres of 20 g L -1 and above, due to the insolubility of the secodione. This triggered a rapid enzyme engineering program fully based on computational mutant selection. A small panel of 93 CRED mutants was rationally designed to increase the catalytic activity as well as thermal and solvent stability. The best mutant, Mutant-75, enabled a reaction at 45 °C to go to completion at 90 g L -1 substrate titre in a buffer/DMSO/heptane reaction medium fed over 6 h with substrate DMSO stock solution, with a low enzyme loading of 3.5% w/w wrt substrate. In screening assay conditions, Mutant-75 also showed a 2.2-fold activity increase. Our paper shows which computations and rational decisions enabled this outcome.

Background: Highly reactive oxygen species (ROS) could lead to serious damage in living cells and are associated with many diseases like cancers. Metal cluster with strong fluorescence has great potential in biosensing and many thiolate ligands-protected clusters have been applied in ROS sensing., Results: In this work, we synthesized levonorgestrel protected Au 10 cluster with specific sensing ability for highly ROS via crystal transformation from Au 8 cluster, demonstrating the significance of inner core structure on detecting performance. The detection limit of Au 10 cluster for ClO - could reach as low as 0.1 μM. This fluorescent probe not only achieving detection of exogenous ClO - in living cells and zebrafish, but also successful imaging of endogenous ClO - in HeLa and HepG2 cells., Significance: In comparison to previously reported cluster-based sensors for ROS, this work proposes a different reaction mechanism of metal nanoclusters for ROS detection (breakage of gold-alkynyl bond and oxidation of alkynyl group). This provides new directions for designing specific ROS probes and broadens the applications of metal clusters in disease diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The development of Levonorgestrel Intrauterine Systems (LNG-IUSs) stands as a formidable challenge due to their intricate design and reliance on specialized manufacturing methods. Pharmaceutical manufacturers face a labyrinth of process variables that demand precise identification and comprehension to establish a robust product design to ensure consistent performance. The current manuscript navigates through this complexity, describing a small-scale processing method for LNG-IUSs via addition and condensation curing processes, as well as investigating the influence of key manufacturing variables on LNG-IUS product performance. Different mixing speeds and time exhibited distinct impact on drug content uniformity within the IUS drug-polymer reservoirs. Surprisingly, no variation in drug release rates were observed. Curing temperature and time were the critical processing parameters of IUSs which were dependent on the polymer type (polydimethylsiloxane, PDMS) and drug loading. At lower curing temperatures, crosslinking in PDMS remained relatively unaffected, irrespective of drug loading. By contrast, elevating curing temperatures resulted in a drastic reduction in PDMS crosslinking densities at higher drug loading. This was attributed to increased drug volume fraction within the matrix, impeding optimal prepolymer chain mobility and rearrangement which is crucial for complete crosslinking. Interestingly, rapid curing led to increased PDMS crystallinity, thereby retarding drug release rates while concurrently compromising mechanical properties. PDMS curing chemistry, such as condensation cure (no filler) and addition cure (cured at room temperature), did not affect drug release rates of the LNG-IUSs. In the condensation cure-based LNG-IUS, the formulations prepared without filler had higher drug release rates than those containing silica or diatomaceous earth fillers. Overall, the present study unravels the intricate interplay between PDMS characteristics, processing variables, and product performance, offering fundamental insights into product design and manufacturing of brand and generic LNG-IUS products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The wide array of polydimethylsiloxane (PDMS) variants available on the market, coupled with the intricate combination of additives in silicone polymers, and the incomplete understanding of drug release behavior make formulation development of levonorgestrel intrauterine systems (LNG-IUSs) formidable. Accordingly, the objectives of this work were to investigate the impact of excipients on formulation attributes and in vitro performance of LNG-IUSs, elucidate drug release mechanisms, and thereby improve product understanding. LNG-IUSs with a wide range of additives and fillers were prepared, and in vitro drug release testing was conducted for up to 12 months. Incorporating various additives and/or fillers (silica, silicone resins, silicone oil, PEG, etc.) altered the crystallization kinetics of the crosslinked polymer, the viscosity, and the microstructure. In addition, drug-excipient interactions can occur. Interestingly, additives which increased matrix hydrophobicity and hindered PDMS crystallization facilitated dissolution and permeation of the lipophilic LNG. The influence of additives and lubricants on the mechanical properties of LNG-IUSs were also evaluated. PDMS chemical substitution and molecular weight were deemed to be most critical polymer attributes to the in vitro performance of LNG-IUSs. Drugs with varying physicochemical characteristics were used to prepare IUSs, modeling of the release kinetics was performed, and correlations between release properties and the various physicochemical attributes of the model drugs were established. Strong correlations between first order release rate constants and both drug solubility and Log P underpin the partition and diffusion-based release mechanisms in LNG-IUSs. This is the first comprehensive report to provide a mechanistic understanding of material-property-performance relationships for IUSs. This work offers an evidence-based approach to rational excipient selection and tailoring of drug release to achieve target daily release rates in vivo. The novel insights gained through this research could be helpful for supporting development of brand and generic IUS products as well as their regulatory assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gold nanoclusters exhibit significant potential in antimicrobial applications due to their good stability and desirable biocompatibility in the mammalian cell model. However, most of the previously reported gold nanocluster antimicrobial agents do not have an atomic-precise structure, causing difficulties in understanding the structure-property correlation. In this study, structurally defined gold-levonorgestrel clusters, named Au 8 (C 21 H 27 O 2 ) 8 (Au 8 NCs) and Au 10 (C 21 H 27 O 2 ) 10 (Au 10 NCs), with the same ligand-to-metal ratio but different inner cores were prepared for antibacterial activity investigations, demonstrating that Au 8 NCs exhibited a stronger antibacterial activity owing to the more significant damage it causes on the bacteria wall and membrane, and a stronger inhibition of glutathione reductase activity in bacteria. The leakage of the intracellular components and enzyme inhibition caused an imbalance of the intracellular antioxidant defence system, and consequently killed bacteria. These results indicated that the structure of gold nanoclusters has an important effect on their biological activity, indicating that it as a key factor to consider in the future design of antimicrobial agents.

The cation channel of sperm (CatSper) is the principal entry point for calcium in human spermatozoa and its proper function is essential for successful fertilization. As CatSper is potently activated by progesterone, we evaluated a range of steroids to define the structure-activity relationships for channel activation and found that CatSper is activated by a broad range of steroids with diverse structural modifications. By testing steroids that failed to elicit calcium influx as inhibitors of channel activation, we discovered that medroxyprogesterone acetate, levonorgestrel, and aldosterone inhibited calcium influx produced by progesterone, prostaglandin E 1 , and the fungal natural product l -sirenin, but these steroidal inhibitors failed to prevent calcium influx in response to elevated K + and pH. In contrast to these steroid antagonists, we demonstrated for the first time that the T-type calcium channel blocker ML218 acts similarly to mibefradil, blocking CatSper channels activated by both ligands and alkalinization/depolarization. These T-type calcium channel blockers produced an insurmountable blockade of CatSper, whereas the three steroids produced antagonism that was surmountable by increasing concentrations of each activator, indicating that the steroids selectively antagonize ligand-induced activation of CatSper rather than blocking channel function. Both the channel blockers and the steroid antagonists markedly reduced hyperactivated motility of human sperm assessed by computer-aided sperm analysis, consistent with inhibition of CatSper activation. Unlike the channel blockers mibefradil and ML218, which reduced total and progressive motility, medroxyprogesterone acetate, levonorgestrel, and aldosterone had little effect on these motility parameters, indicating that these steroids are selective inhibitors of hyperactivated sperm motility. SIGNIFICANCE STATEMENT: The steroids medroxyprogesterone acetate, levonorgestrel, and aldosterone selectively antagonize progesterone- and prostaglandin E 1 -induced calcium influx through the CatSper cation channel in human sperm. In contrast to T-type calcium channel blockers that prevent all modes of CatSper activation, these steroid CatSper antagonists preferentially reduce hyperactivated sperm motility, which is required for fertilization. The discovery of competitive antagonists of ligand-induced CatSper activation provides starting points for future discovery of male contraceptive agents acting by this unique mechanism., (Copyright © 2021 by The Author(s).)

Cunninghamella blakesleeana -mediated biotransformation of an oral contraceptive drug, levonorgestrel ( 1 ), yielded a new metabolite, 13 β -ethyl-17 α -ethynyl-10,17 β -dihydroxy-4,6-dien-3-one ( 2 ), and two known metabolites 3 (13 β -ethyl-17 α -ethynyl-10 β ,17 β -dihydroxy-4-en-3-one), and 4 (13 β -ethyl-17 α -ethynyl-6 β ,17 β -dihydroxy-4-en-3-one) at an ambient temperature using aqueous media. Hydroxylation and dehydrogenation of compound 1 was observed during the bio-catalytic transformation. The structure of a new metabolite 2 was determined by 1 H, 13 C, and 2DNMR and HR-EIMS spectroscopic techniques.

Background: To develop and validate a simple and consistent reversed phase high performance liquid chromatography (RP-HPLC) method for the estimation of Levonorgestrel (LNG) drug from silicone based intrauterine device., Methods: Sample solution was prepared using tetrahydrofuran (THF) as solvent for the drug extraction, and RP-HPLC analysis was performed using Luna C18 analytical column (150 × 4.6 mm, 5 μm, 100 Å - Phenomenex), with a mobile phase consisting of a mixture of acetonitrile and water (50:50, v/v) at a flow rate of 1.0 ml/min and injection volume of 20 μl. Detection was carried out at 241 nm in PDA detector, with a total run time of 15 min. The method was validated in accordance with ICH guidelines. Method applicability was tested for optimizing formulation using quality-by-design approach, to check the stability and content uniformity of levonorgestrel-silicone mixture (core blend), and quantifying the amount of LNG from commercially available silicone based formulation., Results: The retention time for LNG drug was obtained at 8.5 min (± 0.3 min). A linear relationship was observed over the concentration range of 2.6-15.6 μg/ml with the correlation coefficient (r) value 0.9999. The method was found to be precise within the acceptable limit (RSD < 2%) and the drug recovery from the intrauterine device was found in the range 99.78-100.0%. Content uniformity for different prototypes developed was observed in the range of 91.6-101.4%, and assay of optimized core blend was in the range of 97.78-106.79% during the 10 days of retention period for stability studies., Conclusion: The validated method is found to be a simple, accurate, precise, reproducible, and hence can be used for the routine analysis of LNG such as in-process, quality control and stability assays of silicone based intrauterine devices by RP-HPLC.

Polydimethylsiloxane (PDMS)-based levonorgestrel intrauterine systems (LNG-IUSs) contain a large amount of potent LNG, and therefore it is important to understand the impact of product design parameters on the in vitro and in vivo performance to ensure safety and efficacy, as well as to avoid serious side effects resulting from dose dumping. LNG-IUS is a complex drug-device combination product, and its formulation design, requires consideration of additional factors such as device configuration and dimensions, in addition to formulation and processing parameters. In this study, ten qualitatively (Q1) and quantitatively (Q2) equivalent LNG-IUSs were manufactured with differences in source (supplier) and dimensions (i.e., thickness) of the outer membrane, drug particle size, dimensions of the drug reservoir (i.e., inner diameter), as well as configuration of the entire IUS. A real-time in vitro release testing method was developed for the LNG-IUSs. In addition, an accelerated release testing method was developed using hydro-alcoholic media in order to reduce the time associated with formulation design. Source variations and thickness of their outer membranes had a great impact on the in vitro drug release from the LNG-IUSs. It was demonstrated that the thicker the outer membrane, the slower the drug release rate. The physicochemical properties of the outer membranes obtained from different sources were characterized to understand their impact on the in vitro drug release of the LNG-IUSs. The composition and mechanical strength may play a role in differences in drug release. The LNG-IUS formulation prepared with the larger drug particle size showed a slightly slower daily release rate. The drug release rates from the compositionally equivalent LNG-IUSs linearly correlated to the surface area of the corresponding drug reservoirs. Another factor that affected the drug release rate was the configuration of the entire IUS. It was shown that the placement of the outer membrane was significant, i.e. whether the ends of the drug reservoir were covered or not. It is important to note that real-time release showed zero-order release kinetics over the test period of approximately 900 days. The current study provides a comprehensive understanding of the impact of product design parameters on the in vitro drug release of LNG-IUSs. In addition, the developed real-time and accelerated release testing methods showed good discriminatory ability for compositionally equivalent LNG-IUSs prepared using different product design parameters., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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/cm 3 to 8.274 mg/cm 3 . The expressions were further simplified for thin rings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

With a dapivirine-releasing vaginal ring having successfully completed late-stage clinical testing for HIV prevention and currently undergoing regulatory review, there is now growing interest in next-generation multipurpose prevention technologies that seek to combine antiretroviral and contraceptive drugs within a single product. Here, we focus on ongoing efforts to develop a silicone elastomer vaginal ring releasing both dapivirine and levonorgestrel. Specifically, we evaluate various strategies aimed at both better understanding and reducing the tendency of levonorgestrel to bind with the elastomer, including: (i) formulation and post-manufacturing strategies aimed at reducing the extent of levonorgestrel reaction with addition-cure silicone elastomers; (ii) evaluation of a simple silicone system to model the complex elastomer; (iii) use of model compounds representing the enone and ethinyl moieties of levonorgestrel to probe the mode of addition of levonorgestrel to addition-cure silicone elastomers; and (iv) solution and solid-state 13 C NMR analysis to probe the structural features of the levonorgestrel-silicone system. The results demonstrate that both the enone and ethinyl groups within levonorgestrel undergo hydrosilylation reactions with the hydrosiloxane groups in the silicone elastomer leading to covalent binding. The results also highlight potential strategies for further optimising the dapivirine + levonorgestrel silicone vaginal ring formulation to ensure that the levonorgestrel is available for release., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gold nanoclusters have become promising radiosensitizers due to their ultrasmall size and robust ability to adsorb, scatter, and re-emit radiation. However, most of the previously reported gold nanocluster radiosensitizers do not have a precise atomic structure, causing difficulties in understanding the structure-activity relationship. In this study, a structurally defined gold-levonorgestrel nanocluster consisting of Au 8 (C 21 H 27 O 2 ) 8 (Au 8 NC) with bright luminescence (58.7% quantum yield) and satisfactory biocompatibility was demonstrated as a nanoradiosensitizer. When the Au 8 NCs were irradiated with X-rays, they produced reactive oxygen species (ROS), resulting in irreversible cell apoptosis. As indicated by in vivo tumor formation experiments, tumorigenicity was significantly suppressed after one radiotherapy treatment with the Au 8 NCs. In addition, compared with tumors treated with X-rays (4 Gy) alone, tumors treated with the nanosensitizer exhibited an inhibition rate of 74.2%. This study contributes to the development of atomically precise gold nanoclusters as efficient radiosensitizers.

The presence of levonorgestrel (LNG) in water bodies via direct discharge and human excretion has been reported worldwide, but its effects on the reproduction of aquatic species and humans are still unknown. Owing to its recalcitrant properties, LNG is not completely removed during wastewater treatment plants, and many species may be exposed to low traces of this compound from discharged effluents. Thus, in this study, a photocatalytic process for removing LNG along with screening of endocrine disruptor effects for risk assessment was applied. Although the removal rate of LNG by ultraviolet C (UV-C) radiation was >90%, reproductive toxicity testing using the BeWo cell line exposed to LNG and its degraded fraction showed the reduced production of basal human chorionic gonadotropin hormone (β-hCG) by more than 73%, from 8.90 mIU mL -1 to <2.39 mIU mL -1 , with both LNG and the degraded fraction. β-hCG hormone has been implicated in the viability of trophoblastic cells during the first trimester of pregnancy; therefore, degraded fractions and waterborne LNG may affect reproduction in some aquatic species and humans with low level of exposure., (Copyright © 2019. Published by Elsevier B.V.)

Levonorgestrel (LNG) and quinestrol (QUN) are typical endocrine disruptors that enter the soil via sewage irrigation and sludge return. However, the fates of both compounds in soil are not well-understood. Laboratory microcosm studies were conducted to fill the gap of understanding of LNG and QUN behavior in soils. High values of goodness-of-fit indices (GFIs) were obtained using the double-first-order in parallel (DFOP) model and the single-first-order (SFO) model to fit the degradation kinetics of LNG and QUN in soils, respectively. The end-points (DT 50 and DT 90 ) of LNG and QUN were positively correlated with soil total organic carbon (TOC). Soil water content and temperature were observed to be critical factors in degradation of LNG and QUN. The degradation rates of LNG and QUN were very slow under sterile and flooded conditions, indicating that the aerobic microbial degradation was dominant in the degradation of LNG and QUN. Moreover, major transformation products were identified, and biodegradation pathways of LNG and QUN were proposed. The present study is expected to provide basic information for ecological risk assessment of LNG and QUN in the soil compartment.

Prazosin (PRZ) and levonorgestrel (LNG) are widely used as an anti-disease drugs due to their biological activity in the human body. The frequent detection of these compounds in water samples requires alternative technologies for the removal of both compounds. After electrochemical degradation of PRZ and LNG, the parent compounds could be completely removed after treatment, but the identification and characterization of by-products are necessary as well. In this study, the effects of NaCl concentration and applied voltage were investigated during the electrochemical degradation process. The results revealed that the increase of NaCl concentration and applied voltage could promote the generation of hypochlorite OCl - and then enhance the degradation of PRZ and LNG. After initial study, 6V and 0.2g NaCl were selected for further experiments (96% and 99% removal of PRZ and LNG after 40min, respectively). Energy consumption was also evaluated and calculated for PRZ and LNG at 3, 6 and 8V. Solid phase extraction (SPE) method plays an important role in enhancing the detection limit of by-products. Furthermore, characterization and identification of chlorinated and non-chlorinated by-products were conducted using an accurate liquid chromatography-time of flight/mass spectrometry LC-TOF/MS instrument. The monitoring of products during the electrochemical degradation process was performed at 6V and 0.2g NaCl in a 50mL solution. The results indicated that two chlorinated products were formed during the electrochemical process. The toxicity of by-products toward E. coli bacteria was investigated at 37°C and 20hr incubation time., (Copyright © 2018. Published by Elsevier B.V.)

Mirena® is long-acting (5 years) contraceptive intrauterine device. It is composed of a hollow cylindrical drug reservoir (containing Levonorgestrel and polydimethylsiloxane), which is covered with a release rate controlling silicone membrane. This structure presents a manufacturing challenge and to date, there have been no literature reports on the manufacturing, product design and quality evaluation of these hollow cylindrical intrauterine devices. It is vital to develop a reproducible and robust manufacturing process for these long-acting intrauterine devices or systems to obtain an understanding the in vitro and in vivo performance of such drug-device combinations. In this study, a twin-syringe method with a customized mold was developed to manufacture hollow cylindrical polydimethylsiloxane (PDMS)-based levonorgestrel intrauterine systems (LNG-IUSs). Different mold materials, curing temperatures and times were screened to fabricate PDMS-drug reservoirs with good quality characteristics (easy demolding, good appearance and appropriate physicochemical characteristics). The prepared PDMS-drug reservoirs were covered with the release rate controlling membrane to fabricate the LNG-IUSs. Physicochemical characterization (drug content and content uniformity, powder X-Ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FTIR) of the PDMS-drug reservoirs with different drug loadings (10%, 25% and 50% w/w) was conducted. Real-time in vitro drug release testing of LNG-IUSs with different drug loading was performed in normal saline (0.9% w/v NaCl) at 37 °C using a water bath shaker rotating at 100 rpm. The prepared PDMS-drug reservoirs demonstrated good and reproducible quality characteristics including appearance (smooth surfaces), targeted drug loading and good drug content uniformity in the PDMS matrix. The PXRD showed that the crystallinity of the API was maintained inside the PDMS matrix. DSC, TGA and FTIR confirmed the structure of the drug and the PDMS, indicating no interaction between the drug and the PDMS matrix in the prepared LNG-IUSs. Real-time in vitro drug release from the LNG-IUSs with different drug loadings showed zero-order release kinetics, and the drug release rate (based on daily release percentage) was inversely proportional to the drug loading., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An effort with the goal of discovering single-dose, long-lasting (>6 months) injectable contraceptives began using levonorgestrel (LNG)-17-β esters linked to a sulfonamide function purposed as human carbonic anhydrase II (hCA 2) ligands. One single analog from this first series showed noticeably superior anti-ovulatory activity in murine models, and a subsequent structure-activity relationship (SAR, the relationship between a compound's molecular structure and its biological activity) study based on this compound identified a LNG-phenoxyacetic acid ester analog exhibiting longer anti-ovulatory properties using the murine model at 2 and 4 mg dose than medroxyprogesterone acetate (MPA). The same ester function linked to etonogestrel (ENG) furnished a compound which inhibited ovulation at 2 mg for 60 days, the longest duration of all compounds tested at these doses. By comparison, MPA at the same dose inhibited ovulation for 32 days., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Levonorgestrel (LNG) is a synthetic progestin that is available in oral contraceptive tablets, a subdermal implant, and an intrauterine system for contraception. LNG pharmacokinetics are a pivotal determinant of contraceptive efficacy and essential in assessing drug-drug interactions influencing LNG exposure following different routes of LNG administration. A highly sensitive LC-MS/MS method was developed and validated to quantify levonorgestrel in human plasma. Liquid-liquid extraction was utilized with a sample volume of 500 μL to extract levonorgestrel from plasma. Chromatographic separation of LNG was achieved with a Fortis™ C18 (3 μm: 100 mm × 2.1 mm) reverse phase analytical column. The mobile phases consisted of de-ionized water plus 0.1% NH 4 OH (100:0.1%, v/v) (A), and methanol plus 0.1% NH 4 OH (100:0.1%, v/v) (B) delivered as a gradient at a flow rate of 400 μL/min. Detection of LNG and internal standard (D-(-)-norgestrel-d7) was achieved using positive polarity mode monitoring at 313.2-245.2 amu and 320.1-251.2 amu, respectively. The assay was linear over the calibration range of 49.6 to 1500 pg/mL. This method was used to quantify plasma LNG released by subdermal implant in support of a drug interaction study among women with HIV receiving efavirenz- or nevirapine-based antiretroviral therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: For decades, progestins have been included in hormone therapies (HT) prescribed to women to offset the risk of unopposed estrogen-induced endometrial hyperplasia. However, the potential effects on cognition of subcategories of clinically used progestins have been largely unexplored., Methods: In two studies, the present investigation evaluated the cognitive effects of norethindrone acetate (NETA), levonorgestrel (LEVO), and medroxyprogesterone acetate (MPA) on the water radial-arm maze (WRAM) and Morris water maze (MM) in middle-aged ovariectomized rats., Results: In Study 1, six-weeks of a high-dose NETA treatment impaired learning and delayed retention on the WRAM, and impaired reference memory on the MM. Low-dose NETA treatment impaired delayed retention on the WRAM. In Study 2, high-dose NETA treatment was reduced to four-weeks and compared to MPA and LEVO. As previously shown, MPA impaired working memory performance during the lattermost portion of testing, at the highest working memory load, impaired delayed retention on the WRAM, and impaired reference memory on the MM. NETA also impaired performance on these WRAM and MM measures. Interestingly, LEVO did not impair performance, but instead enhanced learning on the WRAM., Conclusions: The current study corroborates previous evidence that the most commonly prescribed FDA-approved progestin for HT, MPA, impairs learning and memory in the ovariectomized middle-aged rat. When progestins from two different additional subcategories were investigated, NETA impaired learning and memory similarly to MPA, but LEVO enhanced learning. Future research is warranted to determine LEVO's potential as an ideal progestin for optimal health in women, including for cognition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Despite a long history of incorporating steroids into silicone elastomers for drug delivery applications, little is presently known about the propensity for irreversible drug binding in these systems. In this study, the ability of the contraceptive progestin levonorgestrel to bind chemically with hydrosilane groups in addition-cure silicone elastomers has been thoroughly investigated. Cure time, cure temperature, levonorgestrel particle size, initial levonorgestrel loading and silicone elastomer type were demonstrated to be key parameters impacting the extent of levonorgestrel binding, each through their influence on the solubility of levonorgestrel in the silicone elastomer. Understanding and overcoming this levonorgestrel binding phenomenon is critical for the ongoing development of a number of drug delivery products, including a multi-purpose technology vaginal ring device offering simultaneous release of levonorgestrel and dapivirine - a lead candidate antiretroviral microbicide - for combination HIV prevention and hormonal contraception., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this research, a rapid efficient and automated instrument based on hollow fiber liquid-phase microextraction (HF-LPME) followed by high performance liquid chromatography (HPLC) with UV-vis detection was applied for the preconcentration and determination of two hormonal drugs (megestrol acetate and levonorgestrel) in water and urinary samples. n-Dodecane was used as the supported liquid membrane (SLM) and methanol was used as the acceptor phase in the hollow fiber lumen. The effects of different parameters such as fiber length, extraction time, stirring rate, and ionic strength on the extraction efficiency were investigated using modified simplex and central composite design as the screening and optimization methods, respectively. The composition effect of SLM and type of acceptor phase were optimized separately. For adjustment of the SLM composition, trioctylphosphine oxide (TOPO) was chosen. Under optimized condition, the calibration curves were linear (r(2)>0.997) in the range of 0.5-200 μg L(-1). LOD for both of the drugs were 0.25 μg L(-1). The applicability of this technique was examined by analyzing drugs in water and urine samples. The relative recoveries of the drugs were in the range of 86.2-102.3% that show the capability of the method for the determination of the drugs in various matrices., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The ubiquitous use of pharmaceuticals has resulted in a continuous discharge into wastewater and pharmaceuticals and their metabolites are found in the environment. Due to their design towards specific drug targets, pharmaceuticals may be therapeutically active already at low environmental concentrations. Several human drug targets are evolutionary conserved in aquatic organisms, raising concerns about effects of these pharmaceuticals in non-target organisms. In this study, we hypothesized that the toxicity of a pharmaceutical towards a non-target invertebrate depends on the presence of the human drug target orthologs in this species. This was tested by assessing toxicity of pharmaceuticals with (miconazole and promethazine) and without (levonorgestrel) identified drug target orthologs in the cladoceran Daphnia magna. The toxicity was evaluated using general toxicity endpoints at individual (immobility, reproduction and development), biochemical (RNA and DNA content) and molecular (gene expression) levels. The results provide evidence for higher toxicity of miconazole and promethazine, i.e. the drugs with identified drug target orthologs. At the individual level, miconazole had the lowest effect concentrations for immobility and reproduction (0.3 and 0.022 mg L-1, respectively) followed by promethazine (1.6 and 0.18 mg L-1, respectively). At the biochemical level, individual RNA content was affected by miconazole and promethazine already at 0.0023 and 0.059 mg L-1, respectively. At the molecular level, gene expression for cuticle protein was significantly suppressed by exposure to both miconazole and promethazine; moreover, daphnids exposed to miconazole had significantly lower vitellogenin expression. Levonorgestrel did not have any effects on any endpoints in the concentrations tested. These results highlight the importance of considering drug target conservation in environmental risk assessments of pharmaceuticals.

Fish and other aquatic wildlife, including frogs, turtles, and alligators, have been used as vertebrate sentinels for the effects of endocrine disrupting and other emerging chemicals of concern found in aquatic ecosystems. Research has focused on the effects of estrogenic, androgenic, and thyroidogenic compounds, but there is a growing body of literature on the reproductive health exposure effects of environmental gestagens on aquatic wildlife. Gestagens include native progestogens, such as progesterone, and synthetic progestins, such as gestodene and levonorgestrel, which bind progesterone receptors and have critically important roles in vertebrate physiology, especially reproduction. Roles for progestogen include regulating gamete maturation and orchestrating reproductive behavior, both as circulating hormones and as secreted pheromones. Gestagens enter the aquatic environment through paper mill effluent, wastewater treatment plant effluent, and agricultural runoff. A number of gestagens have been shown to negatively affect reproduction, development, and behavior of exposed fish and other aquatic wildlife at ng/L concentrations, and these compounds have been measured in the environment at single to 375 ng/L. Given the importance of endogenous progestogens in the regulation of gametogenesis, secondary sex characteristics, and reproductive behavior in vertebrates and the documented exposure effects of pharmaceutical progestins and progesterone, environmental gestagens are an emerging class of contaminants that deserve increased attention from researchers and regulators alike. The potential for environmental gestagens to affect the reproductive health of aquatic vertebrates seems evident, but there are a number of important questions for researchers to address in this nascent field. These include identifying biomarkers of gestagen exposure; testing the effects of environmentally relevant mixtures; and determining what other physiological endpoints and taxa might be affected by exposure to environmental gestagens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Electrospun drug-eluting fabrics have enormous potential for the delivery of physicochemically diverse drugs in combination by controlling the underlying material chemistry and fabric microarchitecture. However, the rationale for formulating drugs at high drug loading in the same or separate fibers is unknown but has important implications for product development and clinical applications., Methods: Using a production-scale free-surface electrospinning instrument, we produced electrospun nanofibers with different microscale geometries for the co-delivery of tenofovir (TFV) and levonorgestrel (LNG) - two lead drug candidates for multipurpose prevention of HIV acquisition and unintended pregnancy. We investigated the in vitro drug release of TFV and LNG combinations from composites that deliver the two drugs from the same fiber (combined fibers) or from separate fibers in a stacked or interwoven architecture. For stacked composites, we also examined the role that fabric thickness has on drug-release kinetics. We also measured the cytotoxicity and antiviral activity of the drugs delivered alone and in combination., Results: Herein, we report on the solution and processing parameters for the free-surface electrospinning of medical fabrics with controlled microarchitecture and high drug loading (up to 20 wt%). We observed that in vitro release of the highly water-soluble TFV, but not the water-insoluble LNG, was affected by composite microarchitecture, fabric thickness, and drug content. Finally, we showed that the drug-loaded nanofibers are noncytotoxic and that the antiviral activity of TFV is preserved through the electrospinning process and when combined with LNG., Conclusion: Electrospun fabrics with high drug loading create multicomponent systems that benefit from the independent control of the nanofibrous microarchitecture. Our findings are significant because they will inform the design and production of composite electrospun fabrics for the co-delivery of physicochemically diverse drugs that may be useful for multipurpose prevention.

Purpose: To screen crystallization inhibitors, perform accelerated stability testing and predict saturation solubility of levonorgestrel in drug-in-adhesive patches., Methods: Differential scanning calorimetry (DSC) studies were compared against slide crystallization studies for screening additives. Release studies were performed from crystallized and supersaturated patches. Die cutting was used for accelerated stability testing of patches. Time lag experiments were performed to predict saturation solubility of levonorgestrel in acrylate adhesive, DuroTak-2516., Results: DSC studies indicated poloxamer to be the best additive whereas slide crystallization studies showed polyvinylpyrrolidone to be better. Supersaturated patches showed higher release profiles relative to crystallized patches. Crystals were observed in crystallized patches even after 96 h of release studies. Die-cutting of patches helped in development of crystals in less time as compared to uncut sheets indicating its usefulness in accelerated stability testing. Saturation solubility of levonorgestrel in DuroTak-2516 was predicted to be 0.09% w/w which was in close agreement with value of 0.1% w/w from solubility calculator on vendor's website., Conclusions: Crystallization was shown to have negative impact on drug release and patch performance. Slide crystallization studies, die cutting and time lag experiments can be used as tools to help stabilize the otherwise unstable patches.

The novel steroidal conjugates [M(η(5)-C(5)Me(5))Cl(LEV-ppy)] (M = Rh (1) and Ir (2)) bearing the lipophilic levonorgestrel group 17-α-[2-phenylpyridyl-4-ethynyl]-19-nortestosterone (LEV-ppy), where the chelating ligand is N and C-bound, have been prepared and characterized. Both compounds are more active than cisplatin (about 6-fold) in T47D (breast cancer) at 48 h incubation time. On the other hand, very low resistance factors (RF) of 1 and 2 in A2780cisR (cisplatin-resistant ovarian carcinoma) at 48 h were observed (RF = 0.9 and 1.1, respectively). The iridium steroidal compound 2 is twice as active as the non-steroidal analogue 2', whose promising anticancer activity has recently been reported by Sadler. Theoretical DFT calculations on complexes 1 and 2 at the B3LYP-D/def2-TZVP-ecp level of theory show that the strongest bond to the metal atom is the η(5)-interaction to the Cp* ligand and that both of them feature a rather strong metal-chlorine bond. The new steroidal conjugates 1 and 2 are able to bind to DNA according to Hoechst 33258 displacement experiments and ESI-TOF MS spectrometry studies. Complexes 1 and 2 are also cathepsin B inhibitors, an enzyme implicated in a number of cancer related events.

Levonorgestrel, a synthetic progesterone used as an oral contraceptive or emergency contraceptive pill, has been shown to be an endocrine-disrupting chemical. To assess the environmental risk of levonorgestrel, batch experiments and laboratory microcosm studies were conducted to investigate the adsorption and degradation of levonorgestrel in five contrasting soils of China. Freundlich and Langmuir models were applied to sorption data to examine the affinity of levonorgestrel for soils with varying physical and chemical properties. The K(f) of levonorgestrel in the tested soils ranged from 10.79 to 60.92 mg(1-n) L(n) kg(-1) with N between 0.69 and 1.23, and the Q(m) ranged from 18.18 to 196.08 mg/kg. The multiple regression analysis was conducted between K(f) and soil properties. Results indicate that total organic carbon plays a dominant role in the adsorption process. Gibbs free energy values less than 40 kJ/mol demonstrate that levonorgestrel sorption on soils could be considered as a physical adsorption. The degradation of levonorgestrel in five soils was fitted by the first-order reaction kinetics model. The half-lives of levonorgestrel were between 4.32 and 11.55 days. The initial concentration and sterilization experiments illustrated that the degradation rate of levonorgestrel in soil was concentration-dependent and microbially mediated. The low mobility potential of levonorgestrel in soils was predicted by the groundwater ubiquity score (GUS) and retardation factor (R(f)).

Preparations formulated as coated or film-coated tablets, containing levonorgestrel and the combination ethinylestradiol/levonorgestrel, were evaluated in a comparative study. This study comprised in vitro dissolution, assay and content uniformity. The analytical methods were previously validated according to international guidelines. All examined products complied with the postulated requirements.

The novel steroidal conjugate 17-α-[2-phenylpyridyl-4-ethynyl]-19-nortestosterone (LEV-ppy) (1) and the steroid-C,N-chelate ruthenium(II) conjugate [Ru(η(6)-p-cymene)(LEV-ppy)Cl] (2) have been prepared. At 48 h incubation time, complex 2 is more active than cisplatin (about 8-fold) in T47D (breast cancer) and also shows an improved efficiency when compared to its nonsteroidal analogue [Ru(η(6)-p-cymene)(ppy)Cl] (ppy = phenylpyridine) (3) in the same cell line. The act of conjugating a levonorgestrel group to a ruthenium(II) complex resulted in synergistic effects between the metallic center and the steroidal ligand, creating highly potent ruthenium(II) complexes from the inactive components. The interaction of 2 with DNA was followed by electrophoretic mobility. Theoretical density functional theory calculations on complex 2 show the metal center far away from the lipophilic steroidal moiety and a labile Ru-Cl bond that allows easy replacement of Cl by N-nucleophiles such as 9-EtG, thus forming a stronger Ru-N bond. We also found a minimum energy location for the chloride counteranion (4(+)·Cl(-)) inside the pseudocavity formed by the α side of the steroid moiety, the phenylpyridine chelating subsystem, and the guanine ligand, i.e., a host-guest species with a rich variety of nonbonding interactions that include nonclassical C-H···anion bonds, as supported by electrospray ionization mass spectra.

Cotton-top tamarins (Saguinus oedipus) are a critically endangered species that have been bred successfully in captivity for many years. For two decades, the Cotton-top Tamarin SSP(©) has been challenged with a high rate of reproduction combined with a history of contraceptive failures and nonrecommended births using the current Depo Provera(®) (medroxyprogesterone acetate) injection followed by MGA (melengestrol acetate) implant contraception combination. To address these issues we have developed and tested the use of levonorgestrel (LNG) as an effective contraception option for cotton-top tamarins. LNG was delivered in an injectable, gel matrix consisting of polylactic-co-glycolic acid, triethyl citrate and N-methylpyrrolidone. This gel matrix forms a biodegradable depot at the subcutaneous injection site providing slow release of the active ingredient. Gel matrix composition and LNG concentration were adjusted in four gel formulations to maximize the duration of contraceptive efficacy while minimizing immediate post-injection increases in fecal LNG concentration. LNG treatment (68.44 ± 8.61 mg/kg) successfully eliminated ovarian cycles (fecal pregnanediol-3-glucuronide (PdG) and estrone conjugates (E(1) C)) for 198.8 ± 70.3 days (formulation four; range 19-50 weeks). It was demonstrated that subcutaneous LNG depot injection was an effective, reversible contraceptive option for the management of cotton-top tamarins in captivity., (© 2010 Wiley Periodicals, Inc.)

The characteristics of levonorgestrel (LNG), low solubility and the quick degradation under ultraviolet, limited its study and application in rodent contraception. The inclusion complex of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) with LNG was investigated in present study. The inclusion complex was prepared by solution method and characterized by ultraviolet absorption spectrum and infrared spectrum spectra. And the stability was evaluated by being exposed to ultraviolet. The authors' results showed that the accurate and simple method of quantitative determination for LNG was established by ultraviolet spectrum, the molar ratio of the complex was 1:1 calculated from the phase solubility diagram, the stability constant was 187.3 L x mol(-1) at 25 degrees C, and the formation of the inclusion complex was validated by UV-Vis and Fourier transform infrared spectroscopy. Moreover, the degradation rate of the inclusion complex was less than 5%, which was slower than the LNG monomer. The present study indicated that HP-beta-CD could be formed inclusion complexes with LNG and the solubility, and stability were obviously enhanced.

In the pharmaceutical industry fast and efficient separation techniques play an increasing role among analytical methods because the samples to be investigated grow both in complexity and number, and there is an increasing time pressure to complete the analysis. Reducing the analysis time without decreasing the efficiency is possible using higher pressures, elevated temperatures, smaller particle sizes, or a combination of these approaches. Recently developed chromatographic techniques such as the UHPLC (ultra high performance liquid chromatography) and HTLC (high temperature liquid chromatography) are highly promising in meeting these demands. In this study, high temperature liquid chromatography (HTLC) with a zirconia-based column and temperatures elevated up to 150°C was used. We investigated the chromatographic behaviour of a steroid active pharmaceutical ingredient (levonorgestrel) and its structurally related impurities as model compounds. The effect of the temperature in the range of 50-150°C and the flow-rate in the range of 0.5-3.0 ml/min, and using methanol as an organic modifier, were studied for optimisation of the separation method., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The drug release from medium molecular weight polyisobutene patches containing adsorbates (drug content: 0.2% ethinyl estradiol, 1.0% levonorgestrel; adsorbent content: 20%, w/w) increased in the order of no adsorbent

The morphology and the micro-structural changes of levonorgestrel-releasing intrauterine systems (IUSs) were studied in relation to the duration of their application. The morphology of the removed IUSs was examined without pre-treatment by scanning electron microscopy. The micro-structural changes of the different layers of IUSs were tracked by positron annihilation lifetime spectroscopy. Besides the previously found incrustation formation, the free volume of the hormone containing reservoir was remarkably increased after 3 years of application, thus increasing the real volume of the core of the systems. Although the free volume of the membrane encasing the core was not significantly changed in the course of the application, as a result of the core expansion, microcracks could be formed on the membrane surface. Along these cracks, deposits of different compositions can be formed, causing inflammatory complications and influencing the drug release of IUSs. Stability tests in combination with micro-structural screening of such IUSs could be required during their development phase to avoid the undesired side effects., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

In this study the ability of various additives to inhibit crystallization of two model drugs, captopril and levonorgestrel, in acrylate and silicone adhesives was investigated. Among the various additives tested, PVP was found to be the most effective in inhibiting the crystallization of both drugs. Incorporation of PVP in patches (PVP stabilized patches) allowed incorporation of both drugs in amounts higher than their respective saturation solubility in pure adhesives (saturated patches). Skin permeation profiles of the drugs from the patches across hairless rat skin were obtained using Franz diffusion cells. For the hydrophilic drug captopril the skin flux over the first 24h was the same for the saturated and PVP stabilized patches, but after 24h the PVP stabilized patches produced higher skin flux values. However this may be because the saturated patch was depleted of the drug after 24h. It is not clear if PVP performs as a solubilizer or a crystallization inhibitor for hydrophilic drugs. For the lipophilic drug levonorgestrel, the skin flux profile from the saturated and PVP stabilized patches was the same, suggesting that PVP acts just as a drug solubilizer and does not produce supersaturation., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Today sub-2 microm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 microm shell particles (1.9 microm nonporous core surrounded by a 0.35 microm porous shell, Kinetex, Core-Shell), packed with other shell-type particles (Ascentis Express, Fused-Core), totally porous sub-2 microm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW=270-430) and a high molecular weight one (MW approximately 900) was conducted. This study proves that the Kinetex column packed with 2.6 microm shell particles is worthy of rivaling to sub-2 microm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency., (Copyright 2010 Elsevier B.V. All rights reserved.)

The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min(-1), respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L(-1) was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.

This study investigates the oxidation of pharmaceuticals, endocrine disrupting compounds and pesticides during ozonation applied in drinking water treatment. In the first step, second-order rate constants for the reactions of selected compounds with molecular ozone (k(O3)) were determined in bench-scale experiments at pH 8.10: caffeine (650+/-22M(-1)s(-1)), progesterone (601+/-9M(-1)s(-1)), medroxyprogesterone (558+/-9M(-1)s(-1)), norethindrone (2215+/-76M(-1)s(-1)) and levonorgestrel (1427+/-62M(-1)s(-1)). Compared to phenolic estrogens (estrone, 17beta-estradiol, estriol and 17alpha-ethinylestradiol), the selected progestogen endocrine disruptors reacted far slower with ozone. In the second part of the study, bench-scale experiments were conducted with surface waters spiked with 16 target compounds to assess their oxidative removal using ozone and determine if bench-scale results would accurately predict full-scale removal data. Overall, the data provided evidence that ozone is effective for removing trace organic contaminants from water with ozone doses typically applied in drinking water treatment. Ozonation removed over 80% of caffeine, pharmaceuticals and endocrine disruptors within the CT value of about 2 mg min L(-1). As expected, pesticides were found to be the most recalcitrant compounds to oxidize. Caffeine can be used as an indicator compound to gauge the efficacy of ozone treatment.

Many progestins have been developed for use in contraception, menopausal hormone therapy, and treatment of gynecological diseases. They are derived from either progesterone or testosterone, and they act by binding to the progesterone receptor (PR), a hormone-inducible transcription factor belonging to the nuclear receptor superfamily. Unlike mineralocorticoid, glucocorticoid, and androgen receptors, the steroid-receptor contacts that trigger the switch of the ligand-binding domain from an inactive to an active conformation have not yet been identified for the PR. With this aim, we solved the crystal structure of the ligand-binding domain of the human PR complexed with levonorgestrel, a potent testosterone-derived progestin characterized by a 13-ethyl substituent. Via mutagenesis analysis and functional studies, we identified Met909 of the helix 12 as the key residue for PR activation by both testosterone- and progesterone-derived progestins with a 13-methyl or a 13-ethyl substituent. We also showed that Asn719 contributes to PR activation by testosterone-derived progestins only, and that Met759 and Met909 are responsible for the high potency of 19-norprogestins and of 13-ethyl progestins, respectively. Our findings provide a structural guideline for the rational synthesis of potent PR agonist and antagonist ligands that could have therapeutic uses in women's health.

The aim of this study was to formulate and characterize a microparticulate system of progestin-only contraceptive. Another objective was to evaluate the effect of gamma radio-sterilization on in vitro and in vivo drug release characteristics. Levonorgestrel (LNG) microspheres were fabricated using poly(lactide-co-glycolide) (PLGA) by a novel solvent evaporation technique. The formulation was optimized for drug/polymer ratio, emulsifier concentration, and process variables like speed of agitation and evaporation method. The drug to polymer ratio of 1:5 gave the optimum encapsulation efficiency. Speed of agitation influenced the spherical shape of the microparticles, lower speeds yielding less spherical particles. The speed did not have a significant influence on the drug payloads. A combination of stabilizers viz. methyl cellulose and poly vinyl alcohol with in-water solvent evaporation technique yielded microparticles without any free drug crystals on the surface. This aspect significantly eliminated the in vitro dissolution "burst effect". The residual solvent content was well within the regulatory limits. The microparticles passed the test for sterility and absence of pyrogens. In vitro dissolution conducted on the product before and after gamma radiation sterilization at 2.5 Mrad indicated no significant difference in the drug release patterns. The drug release followed zero-order kinetics in both static and agitation conditions of dissolution testing. The in vivo studies conducted in rabbits exhibited LNG release up to 1 month duration with drug levels maintained within the effective therapeutic window.

A molecularly imprinted polymer (MIP) has been prepared using levonorgestrel (LEV) as template. The polymer was synthesised in a non-covalent approach using methacrylic acid (MAA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linking monomer via a free radical polymerization. An equivalent blank polymer was also synthesised in the absence of the template compound. Batch adsorption experiments were used to evaluate the binding affinity of the imprinted polymer. After packing MIP into a stainless steel column (150 mm x 4.6 mm i.d.), retention and elution of the template and related compounds were evaluated by high-performance liquid chromatography (HPLC). This LEV imprinted polymer was further applied for selective solid phase extraction (SPE) of LEV from human serum. It was confirmed that the binding ability of the prepared MIP for LEV was essentially sufficient in the presence of other compounds coexisting in serum sample. Therefore, as a selective and efficient solid phase material, LEV imprinted polymer has a high potential application in analysis of this steroidal hormone in clinical purposes.

The microbial transformation of levonorgestrel (1) by Cunningham elegans resulted in the formation of five hydroxylated metabolites, 13-ethyl-10beta, 17beta-dihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one(2), 13-ethyl-6beta,17beta-dihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one (3) 13-ethyl 6beta, 10beta, 17beta-trihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one (4) 13-ethyl-15alpha-17beta-dihydroxy-18,19-dinor-17alpha-pregn-4-en-20-yn-3-one (5) and 13-ethyl-11alpha, 17beta-dihydroxy-18,19-dinor-17alpha-pregn-4en-20-yn-3-one. The fermentation of one with Rhizopus stolonifer, Fusarium lini and Curvularia lunata afforded compound 2 as a major metabolise. These metabolites were structurally characterized on the basis of spectroScopic techniques. Metabolite 6 was identified as a new compound. Compounds 2 2 ad 5 displayed inhibitory activity against the acetylcholinesterase ( AChE, EC. 3.1.1.7) with IC50 values of 79.2 and 24.5 microM, respectively. The metabolites 2 and 5 also showed inhibitory activity against the butyryLcholinesterase ( BChE, E.C 3.1.1.8) with IC50 values ranging between 9.4 and 309.8 microM.

Progesterone (P4) is an essential reproductive steroid hormone required for many aspects of female reproductive physiology. Progestins are compounds that demonstrate progesterone-like activity and are used in oral contraception, hormone therapy, and treatment of some reproductive disorders, but differ widely in their chemical structures, potency, and pharmacokinetics. While numerous studies have assessed progestins on specific endpoints, little is known about the activation of global gene expression by progestins. We used Affymetrix GeneChip U133A expression arrays to examine the action of P4 and six clinically relevant synthetic progestins (3-ketodesogestrel, drospirenone, levonorgestrel, medroxyprogesterone acetate, norethindrone acetate, and trimegestone) on the progesterone receptor (PR)-positive T47Dco and the PR-negative T47D-Y breast cancer cell lines. Excluding drospirenone, one or more of the progestins-regulated 329 genes, with 30 genes regulated by at least 2.0-fold by all progestins in the T47Dco cells. The synthetic progestins show a high degree of similarity in their transcriptional responses, and each progestin regulates between 77 and 91% of the genes regulated by P4. Independent quantitative RT-PCR analysis confirmed a similar regulation for S100P, PPL, IL20RA, NET1, ATP1A1, HIG2, and CXCL12 (SDF-1) by all seven progestins. Attempts to find differentially regulated genes by any progestin compared to all other treatments failed, suggesting any differences are quantitative, not qualitative. This analysis demonstrates a high degree of similarity among these progestins on PR-regulated gene expression in T47D cells, suggesting a similar and fairly specific mode of action.

Poly(D,L-lactide-co-glycolide) (PLG) biodegradable microspheres containing a contraceptive drug, levonorgestrel (LNG), were prepared using both the solvent evaporation method and a modified solvent extraction-evaporation method. The microspheres prepared with the solvent evaporation process had porous surfaces with low product yields and poor encapsulation efficiencies. On the other hand, the microspheres prepared using the modified solvent extraction-evaporation method were nonporous with encapsulation efficiencies close to 100%. In vitro drug release showed the nonporous microspheres had a lower initial burst and a slightly prolonged duration of release than those porous microspheres. In vivo release kinetics of the low burst microspheres were determined by measuring LNG plasma levels after a single intramuscular injection to female rats. At a LNG dose of 41.1 mg/kg, average plasma LNG levels were 6-10 ng/ml in the first 24 h and subsequently remained above 1 ng/ml until 126 days. The duration above the minimum effective LNG plasma level of 0.2 ng/ml was 168 days. By comparison, a similar dose of LNG microcrystals used as control produced a much higher plasma level of 15-21 ng/ml in the first day followed by a fast and continuous decline of LNG levels with a duration of only about 35 days.

The analysis of bases is of special interest in pharmaceutical research because numerous active substances contain basic functional groups. Capillary and conventional size LC separations of drug substances spiked with potential impurities were compared. In the case of the nonpolar drug levonorgestrel equal separation efficiency was readily attained by both techniques. The peaks of basic substances, however, showed extensive tailing when separated by capillary LC. The peak deformation was attributable to interactions of the basic substances with the polar inner surface of the fused silica capillaries employed in capillary LC and does not appear with the steel tubing generally used in conventional size LC. This drawback of capillary LC was overcome by use of deactivated fused silica capillaries for column hardware and transfer lines.

The objective of this research is to develop injectable controlled delivery systems for the contraceptive hormone, levonorgestrel (LNG), using phase sensitive and thermosensitive polymers. A combination of poly (lactide) (PLA) and a solvent mixture of benzyl benzoate (BB) and benzyl alcohol (BA) was used in the phase-sensitive polymer delivery systems. The effects of solvent systems and polymer concentrations on the in vitro LNG release were evaluated. In the case of thermosensitive polymer delivery systems, a series of low-molecular-weight poly (lactide-co-glycolide)-poly (ethylene glycol)-poly (lactide-co-glycolide) (PLGA-PEG-PLGA) triblock copolymers with varying ratios of lactide/glycolide (LA/GA, 2.0-3.5) were used. The effects of varying block length of copolymers 1, 2, 3, and 4 on the in vitro LNG release were evaluated. Phosphate buffer saline (pH 7.4) containing 0.5% w/v Tween-80 was used as in vitro release medium. The amount of the released LNG was determined by an high pressure liquid chromatography (HPLC) method. A controlled (zero-order) in vitro release of LNG was observed from both phase-sensitive and thermosensitive-polymer delivery systems. Increasing the concentration of the phase-sensitive polymer from 5% to 30% significantly (p < 0.05) decreased the release rate of LNG from 38.32 microg/day to 31.45 microg/day; and increasing the hydrophilic fraction of the solvents mixture (i.e., BA) significantly (p < 0.05) increased the release rate of LNG. In the case of the thermosensitive polymer delivery system, increasing the hydrophobic PLGA block length of copolymers significantly (p < 0.05) decreased the release rate of LNG (98.65 microg/day to 67.60 microg/day). It is evident from this study that both the phase sensitive and thermosensitive polymers are suitable for developing prolonged-release injectable delivery systems for the contraceptive hormone.

Physico-chemical interactions between drugs and plant extracts can result in the formation of nanoparticles, microparticles and precipitates. The extraction method may have an influence on the components of the plant preparations and the interactions between drugs and plant extracts. In this study the particle formation in different drug-containing preparations of Hypericum perforatum L. was compared. The formation of nanoparticles was investigated by PCS and scanning electron microscopy. Precipitates were separated from nanoparticles by centrifugation or filtration. The particle formation in drug-containing infusions and reconstituted methanolic extracts of Hypericum perforatum L. was different. In reconstituted dry extracts 21.5% 17alpha-ethinylestradiol and 36.6% levonorgestrel was bound to precipitates, in aqueous Hypericum infusions less than 4% of the hormones was bound. In infusions containing desipramine nanoparticles were formed while the formation of nanoparticles in desipramine-containing reconstituted extracts was negligible. Up to 10% of desipramine was bound to precipitates in reconstituted extracts, but only 4% was associated with the precipitate of infusions. The interactions between extracts of Hypericum perforatum L. and drugs depended on the extraction method. Reconstituted methanolic extracts showed a more intensive binding with oral contraceptives than aqueous infusions., (2004 John Wiley & Sons, Ltd.)

Investigations were undertaken to determine the cause of decreases over time in the release rate from levonorgestrel (LNG) implants consisting of silicone elastomer tubing filled with crystalline steroid. Emptying and refilling with the same steroid partially restored release rate. Surprisingly, a further increment in release rates was attained if the tubing was briefly irrigated with methanol before refill. Fractional crystallization showed that release-modulating factors could be concentrated in mother liquors and were initially present as impurities. Boiling LNG in ethanol or methanol produced a number of release-modulating factors of which the most prominent was also found in one production lot of LNG. It was identified as 6beta-hydroxy-levonorgestrel (6beta-OH-LNG). Added to LNG at the 2% level, 6beta-OH-LNG decreased the release rate by 27%., (Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association)

The main objective of this work was to develop a system consisting of polymeric microspheres loaded with steroid drugs. The drugs were encapsulated using biodegradable poly(lactide-co-glycolide) (PLG) and poly(epsilon-caprolactone) (PCL) by double emulsion solvent evaporation method. The lipophilic drugs, levonorgestrel and ethinylestradiol were made soluble by adding ethanol/water mixture. The effects of parameters like polymer concentration and stabilizer concentration were studied on the size, size distribution, surface properties and loading efficiencies of microspheres. The formulated microspheres were smooth, spherical and uniform in shape and size. Fourier transformed infrared spectroscopy and differential scanning calorimetry studies seemed to confirm the absence of chemical interaction between the drugs and the polymers, while the drugs were dispersed in the polymer. The increase in polymer concentrations increased the size as well as the loading efficiency of microspheres. Data obtained in this study demonstrated that the PLG/PCL microspheres may be a suitable polymeric carrier for long acting injectable drug delivery.

A variety of progestins are available for therapeutic use. It is convenient to classify them into those related in chemical structure to progesterone or testosterone. Progestins related to progesterone can be subdivided into pregnanes and 19-norpregnanes, whereas those related to testosterone can be subdivided into those with and without a 17-ethinyl group. 17-Ethinylated progestins consist of the families of norethindrone (estranes) and levonorgestrel (13-ethylgonanes). Progestins administered orally undergo extensive hepatic first pass metabolism primarily by reduction and conjugation, and in most instances, relatively high progestin doses are required for therapeutic use. There are limited reliable data on the pharmacokinetics of most progestins. Some progestins are prodrugs, requiring transformation prior to exhibiting progestational activity. Qualitative and quantitative tests utilizing either human or animal species have been used to establish progestin potency. However, profound differences in progestational activity are often observed between human and animal tissues. Also, there is a misconception about androgenicity of progestins due largely to extrapolation of data from rat studies to the human. Progestins differ widely in their chemical structures, structure-function relationships, metabolism, pharmacokinetics, and potencies; they are not created equal.