Preparation of a sustainable magnetic sorbent for the extraction and preconcentration of progestogens in natural water samples.

A film composed of agarose and graphene (G) and magnetic nanoparticles (G-MNPs) is proposed as a sorbent for the extraction and determination of medroxyprogesterone (MED), levonorgestrel (LEV), norethisterone (NOR) and progesterone (PRO) in natural water samples. Both the preparation of the film and the extraction procedure were optimized. The optimal extraction parameters were as follows: isopropyl alcohol as activation solvent, sample pH value of 3.0, extraction time of 30 min, 1.00 mL of acetonitrile as eluent, elution time of 5 min and sample volume of 100.00 mL. HPLC with photodiode array detector was used for the separation and determination. The method presented a linear range between 2.50 and 75.0 µg L-1 for all analytes, and the LODs were between 1.40 and 1.80 µg L-1. The method was applied to natural water samples, obtaining satisfactory recovery values (75-111 %). In conclusion, for the immobilization of the G-MNPs, agarose was used, which is a non-toxic, renewable and biodegradable material. The G-MNPs-agarose film was reused up to 70 times, without losing its extraction capacity significantly and presenting excellent sorbent properties, which allow the extraction and preconcentration of the progestogens under study.

Characterisation of progestins used in hormonal contraception and progesterone via the progesterone receptor.

Different progestogens are widely used in hormonal therapy and mediate their therapeutic actions via the progesterone receptor (PR). Little published data exist on their relative efficacies and potencies via the PR, while those available may be confounded by off-target receptors, different methodologies and model systems. We performed dose-response analysis to investigate the efficacies and potencies for transcription of progesterone and several progestins widely used in contraception via the B isoform of human PR (PR-B). We compared responses using three different cell lines and two different transient transfection conditions. Results show that in vitro biological responses via PR-B for the select progestogens can vary significantly in biocharacter, rank order and absolute values for efficacies and potencies, depending on the cell line and transfection condition. Progestogen rank orders for published relative binding affinities are mostly different to those for relative efficacies and potencies. These in vitro differences suggest that rank orders and absolute values of the efficacies and potencies of the progestogens are likely to vary in vivo in a cell-specific and progestogen-specific manner, and cannot easily be extrapolated from in vitro data, as is usually the practice. While obtaining such data in vivo is not possible, these in vitro data show proof of concept for likely significant cell- and progestogen-specific PR-B effects.

A progestin isn't a progestin: dienogest for endometriosis as a blueprint for future research - Review as a contribution for discussion.

The different etiopathogenetic mechanisms and the diversity of clinical features of endometriosis has not yet allowed to identify a causal pharmacological monotherapy satisfying the unresolved medical needs in this important female disease. Therefore, despite the search for new therapeutic principles for the indication, the strategy of gradual optimization of established therapeutic principles should not be disregarded.In the case of progestins, the fact that each compound has its own, specific profile may allow to study the therapeutic relevance of the various signal cascades influenced by their receptors.Using the example of the progestin dienogest, the different genomic and non-genomic mechanisms of action are discussed. It is pharmacodynamic profile is unique compared to other progestins.In light of the emerging multitude of pathomechanisms in endometriosis, a monotherapy may not be possible, and then the search for broad spectrum compounds or combination therapies with dual or multiple mode of action in a clinically relevant dose range might be considered. The progestogenic action may greatly benefit from, by way of example, additional anti-inflammatory and/or anti-fibrotic and/or pro-apoptotic activities. Such a strategy could lead to new drug classes.

On the dimorphism and the pressure-temperature state diagram of gestodene, a steroidal progestogen contraceptive.

The pressure-temperature phase diagram of the dimorphism of the contraceptive drug gestodene is constructed using the temperature and enthalpy of fusion of form I (469.5K, 107Jg-1), and those of the endothermic transition from form II to form I (311K, 8.52Jg-1). At ordinary pressure, the sign of the enthalpy of this transition indicates that these polymorphs are enantiotropically related and that form II, whose melting temperature is calculated to be about 452K, is the stable form at room temperature. Considering the inequality in the specific volumes of the two polymorphs, it is shown that the two forms remain enantiotropically related on increasing pressure, because the I-II equilibrium and the melting equilibria I-L and II-L diverge as a consequence of the negative slope dP/dT of the solid-solid equilibrium. In addition, it is demonstrated that the heats of dissolution, inferred from solubility measurements, lead to virtually the same value of the heat of transition from II to I as for the differential scanning calorimetry measurements.

Progestins Related to Progesterone and Testosterone Elicit Divergent Human Endometrial Transcriptomes and Biofunctions.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use.

New Selective Progesterone Receptor Modulators and Their Impact on the RANK/RANKL Complex Activity.

Breast cancer depends on women's age. Its chemotherapy and hormone therapy lead to the loss of bone density and disruption of the skeleton. The proteins RANK and RANKL play a pivotal role in the formation of osteoclasts. It is also well established that the same proteins (RANK and RANKL) are the main molecules that play an important role in mammary stem cell biology. Mammary stem cells guarantee differentiation of the epithelial mammary cells, the growth of which is regulated by the progesterone-induced RANKL signaling pathway. The crosstalk between progesterone receptor, stimulated by progesterone and its analogues results in RANKL to RANK binding and activation of cell proliferation and subsequently unlimited expansion of the breast cancer cells. Therefore downstream regulation of this signaling pathway is desirable. To meet this need, a new class of selective estrogen receptor modulators (SPRMs) with anti- and mesoprogestin function were tested as potential anti-RANK agents. To establish the new feature of SPRMs, the impact of tested SPRMs on RANK-RANKL proteins interaction was tested. Furthermore, the cells proliferation upon RANKL stimulation, as well as NFkB and cyclin D1 expression, induced by tested SPRMs were analyzed. Conducted experiments proved NFkB expression inhibition as well as cyclin D1 expression limitation under asoprisnil and ulipristal treatment. The established paracrine anti-proliferative activity of antiprogestins together with competitive interaction with RANK make this class of compounds attractive for further study in order to deliver more evidence of their anti-RANK activity and potential application in the breast cancer therapy together with its accompanied osteoporosis.

Fecal sex steroids and reproductive behaviors in harpy eagles (Harpia harpyja).

Aiming to improve our reproductive knowledge of large birds of prey, behavioral data and fecal steroids were assessed in captive pairs of Harpy eagles, a keystone species that is monogamous and resides in the Neotropics year-round. Adult individuals exhibited different reproductive outcomes and a breeding season extending beyond summer solstice (5-9 months) suggests that harpy eagles may not be absolutely photorefractory. Comparisons among breeding stages in males revealed that mean androgen levels in courtship were higher than in copulation and incubation, but no differences were detected in fecal progestagens or estrogens. Females had higher mean estrogen concentrations in courtship and copulation, whereas mean progestagen levels peaked during egg laying. Mean androgen concentrations were not significantly different among breeding stages in females. Assessment of six egg-lay cycles from three females demonstrated that fecal estrogens peaked predominantly between 31 and 18 days before oviposition (-31 to -18 days), and then remained low until 45 days after laying the first egg (+45 days). In contrast, fecal progestagens raised mostly between -20 and +1 day, lowering to baseline concentrations by +3 days. To our knowledge, this is the first study to describe in detail endocrine and behavioral data regarding reproduction in tropical eagles, which may serve in the future as a reference to developing breeding programs.

Parallel derivatization strategy coupled with liquid chromatography-mass spectrometry for broad coverage of steroid hormones.

Steroid hormones are a type of crucial substances that mediate numerous vital physiological functions. The comprehensive detection of steroid hormones can help understand the physiopathologic mechanism of steroid hormone-related diseases. It is very difficult to determine steroid hormones in biological samples due to their low endogenous concentrations and poor ionization efficiency. In this study, an efficient and sensitive approach was developed for profiling steroid hormones by combining liquid-liquid extraction and parallel derivatization with liquid chromatography-tandem mass spectrometry. Methoxyamine and dansyl chloride were used to derivatize steroid hormones containing carbonyl and phenolic hydroxyl groups, respectively. Our established method achieved simultaneous analysis of carbonyl and phenolic hydroxyl-containing steroid hormones and could cover estrogens, androgens, corticoids and progestogens. Twenty-nine steroid hormones were detected at pg/mL levels with the sensitivity enhanced by three orders of magnitude after derivatization. The linearity (with linear range of 2-4 orders of magnitude), precision (less than 15%) and recovery (71.1-128.7%) were satisfactory for quantitative analysis of steroid hormones. Finally, the established method was successfully employed to the determination of steroid hormones in serum samples of healthy males and females as well as ovarian cancer patients. The results showed that this approach was suitable and reliable for routine test of steroid hormones containing carbonyl and phenolic hydroxyl groups.

A high resolution LC-MS targeted method for the concomitant analysis of 11 contraceptive progestins and 4 steroids.

In the context of hormonal contraception and hormone replacement therapy (HRT), many women are exposed to exogenous hormones. Current use of hormonal contraception with combined ethinyl estradiol and different progestins bestows a breast cancer relative risk (RR) of 1.2- while combined HRT has a RR of 2. Although these exposures present an important public health issue, little is known about the effects of individual progestins on the breast and other tissues. Increasing availability of large scale biobanks, high throughput analyses and data management tools enable ever expanding, sophisticated population studies. In order to address the impact of distinct progestins on various health indicators, it is desirable to accurately quantify progestins in clinical samples. Here we have developed and validated a high resolution liquid chromatography mass spectrometry (LC-MS) targeted method for the simultaneous quantification of 11 synthetic progestins widely used in oral contraceptives, gestodene, levonorgestrel, etonogestrel, chlormadinone acetate, cyproterone acetate, drospirenone, desacetyl norgestimate, medroxyprogesterone acetate, norethindrone, dienogest, nomegestrol acetate, and 4 endogenous steroid hormones, progesterone, testosterone, androstenedione, and cortisol in blood samples. This highly specific quantitative analysis with high resolution Orbitrap technology detects and quantifies 15 compounds using their internal standard counterparts in a single 12 min LC-MS run. Sensitivity is attained by the use of the instrument in targeted selected ion monitoring mode. Lower limit of quantitation ranges from 2.4 pg/ml for drospirenone to 78.1 pg/ml for chlormadinone acetate. The method provides comprehensive progestin panel measurements with as little as 50 µl of murine or human plasma.

Merits of sponge-like PLGA microspheres as long-acting injectables of hydrophobic drug.

Our study was initiated to challenge the preconception that nonporous PLGA microspheres with compact matrices should be used to develop long-acting depot injectables of hydrophobic drugs. A simple, new oil-in-water emulsion technique was utilized to produce porous PLGA microspheres with a sponge-like skeleton. Then, their applicability to developing sustained-release depots of hydrophobic drugs was explored in this study. As control, nonporous microspheres with a compact matrix were produced following a typical solvent evaporation process. Both microsphere manufacturing processes used non-halogenated isopropyl formate and progesterone as a dispersed solvent and a model hydrophobic drug, respectively. Various attempts were made to evaluate critical quality attributes of the porous microspheres and the nonporous ones. Surprisingly, the former displayed interesting features from the viewpoints of manufacturability and microsphere quality. For example, the spongy microspheres improved drug encapsulation efficiency and particle size uniformity, inhibited drug crystallization during microencapsulation, and minimized the residual solvent content in microspheres. Furthermore, the porous microspheres provided continual drug release kinetics without a lag time and much faster drug release than the non-porous microspheres did. In summary, the porous and sponge-like PLGA microspheres might find lucrative applications in developing sustained release dosage forms of hydrophobic drugs.

Glucocorticoid receptor modulators CpdX and CpdX-D3 exhibit the same in vivo antiinflammatory activities as synthetic glucocorticoids.

We previously reported that the nonsteroidal compound CpdX, which was initially characterized 20 y ago as a possible gestagen and, shortly afterward, as a possible drug for treatments of inflammatory diseases, selectively triggers the NFκB/AP1-mediated tethered indirect transrepression function of the glucocorticoid receptor (GR), and could therefore be a selective glucocorticoid receptor agonistic modulator (SEGRAM). We now demonstrate that, upon administration to the mouse, CpdX and one of its deuterated derivatives, CpdX-D3, repress as efficiently as a synthetic glucocorticoid (e.g., Dexamethasone) an induced skin atopic dermatitis, an induced psoriasis-like inflammation, a house dust mite (HDM)-induced asthma-like allergic lung inflammation, a collagen-induced arthritis, an induced ulcerative colitis, and an ovalbumin-induced allergic conjunctivitis. Interestingly, in the cases of an HDM-induced asthma-like allergic lung inflammation and of a collagen-induced arthritis, the CpdX antiinflammatory activity was selectively exerted by one of the two CpdX enantiomers, namely, CpdX(eA) or CpdX-D3(eA).

Occurrence and removal of progestagens in municipal wastewater treatment plants from different regions in China.

Progestagens discharged from municipal wastewater treatment plants (WWTPs) have increasingly gained attention due to their potential risks to the aquatic organisms. However, limited information is available on the occurrence and removal of various progestagens in WWTPs in different cities of China. This work investigated the occurrence and removal of 11 progestagens in 21 WWTPs from 19 Chinese cities. Results showed that progestagens are widely distributed in the investigated WWTPs, with higher influent concentrations of total progestagens in northern WWTPs. The concentration of progestagens in WWTP influent were closely correlated with influent quality, service population and daily service volume of the WWTPs. Additionally, progesterone (PGT) and dydrogesterone (DDT) were two predominant progestagens in influent, effluent and excess sludge. Up to 5 of 11 progestagens showed high aqueous removal efficiencies (median removal efficiency >90%), whereas megestrol acetate (MTA), chlormadinone acetate (CMA), drospirenone (DSP) and levonorgestrel (LNG) had a removal efficiency of below 50%. Specially, the behaviors of progestagens along the anaerobic-anoxic-oxic of a WWTP were further explored and the aerobic tank is the main contributor to the removal of progestagens. Finally, in the effluent of these 21 WWTPs, daily mass loadings of the total progestagens ranged from 0.51 to 10.4 g d-1. Notably, LNG exhibited high potential risk to the fish base on risk quotient.

Recent Developments in Male Contraception.

Unplanned pregnancies are an ongoing global burden, posing health and economic risks for women, children, and families. Advances in male contraception have been historically stymied by concerning failure rates, problematic side effects, and perceived market limitations. However, increased interest in reliable and reversible options for male contraception have resulted in resurgent efforts to introduce novel contraceptives for men. Hormonal male contraception relies on exogenous androgens and progestogens that suppress gonadotropin production, thereby suppressing testicular testosterone and sperm production. In many men, effective suppression of spermatogenesis can be achieved by androgen-progestin combination therapy. Small-scale contraceptive efficacy studies in couples have demonstrated effectiveness and reversibility with male hormonal methods, but side effects related to mood, sexual desire and cholesterol remain concerning. A number of novel androgens have reached clinical testing as potential contraceptive agents; many of these have both androgenic and progestogenic action in a single, modified steroid, thereby holding promise as single-agent contraceptives. Currently, these novel steroids hold promise as both a "male pill" and long-acting injections. Among non-hormonal methods, studies of reversible vaso-occlusive methods (polymers that block transport of sperm through the vas deferens) are ongoing, but reliable reversibility and long-term safety in men have not been established. Proteins involved in sperm maturation and motility are attractive targets, but to date both specificity and biologic redundancy have been challenges for drug development. In this review, we aim to summarize landmark studies on male contraception, highlight the most recent advances and future development in this important field of public health and medicine.

Characterization of the behavior and reproductive endocrinology of giant river otters (Pteronura brasiliensis) in managed care.

Propagation of giant river otters (GRO) in zoos is inconsistent: some pairs never reproduce while others are prolific in producing young but can be hindered by low cub survival. Developing effective breeding programs requires understanding normal reproductive parameters and behavior. Fecal samples were collected for 6-16 months from five breeding pairs, two individual females, and one female pair at seven zoos, and analyzed for fecal progesterone, estrogen, testosterone, and glucocorticoid (FGM) metabolites via enzyme immunoassay. Enclosure characteristics and management routines were recorded at six facilities where behavior was assessed over 1 week. Median fecal progestogens during pregnancy and pseudopregnancy were ∼2.5-3.8× greater than basal concentrations. Gestation lasted 66.5 ± 3.5 days (62-70 days); pseudopregnancies lasted 58 ± 11.6 days (41-69 days). Elevated progestogens indicate ovulation but cannot distinguish pregnancy from pseudopregnancy. Periodically sustained, elevated progestogens observed in two females housed without a male indicated spontaneous ovulation. Elevations in fecal estrogens were not associated with estrus, and seasonality in male testosterone was not observed. Wavering scream and contact call vocalizations among reproductively successful males and females, respectively, suggested the importance of social communication. Most facilities housing successful pairs had larger enclosures with more water than land area, vegetation, and limited public exposure. Baseline FGM were negatively correlated with enclosure size and percentage of water area (p < 0.05), and lower baseline FGM were associated with reproductive success (p < 0.05). These results suggest that housing GRO in spacious enclosures with open water and some insulation from disturbance might promote appropriate behavior, lower FGM, and reproduction.

Do progestins contribute to (anti-)androgenic activities in aquatic environments?

Unknown compounds with (anti-)androgenic activities enter the aquatic environment via municipal wastewater treatment plants (WWTPs). Progestins are well-known environmental contaminants capable of interfering with androgen receptor (AR) signaling pathway. The aim of the present study was to determine if 15 selected progestins have potential to contribute to (anti-)androgenic activities in municipal wastewaters and the respective recipient surface waters. AR-specific Chemically Activated LUciferase gene eXpression bioassay in agonistic (AR-CALUX) and antagonistic (anti-AR-CALUX) modes and liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) methods were used to assess (anti-)androgenic activity and to detect the target compounds, respectively. The contribution of progestins to (anti-)androgenic activities was evaluated by means of a biologically and chemically derived toxicity equivalent approach. Androgenic (0.08-59 ng/L dihydrotestosterone equivalents - DHT EQs) and anti-androgenic (2.4-26 µg/L flutamide equivalents - FLU EQs) activities and progestins (0.19-75 ng/L) were detected in selected aquatic environments. Progestins displayed androgenic potencies (0.01-0.22 fold of dihydrotestosterone) and strong anti-androgenic potencies (9-62 fold of flutamide). Although they accounted to some extent for androgenic (0.3-29%) and anti-androgenic (4.6-27%) activities in influents, the progestins' contribution to (anti-)androgenic activities was negligible (≤2.1%) in effluents and surface waters. We also tested joint effect of equimolar mixtures of target compounds and the results indicate that compounds interact in an additive manner. Even if progestins possess relatively strong (anti-)androgenic activities, when considering their low concentrations (sub-ng/L to ng/L) it seems unlikely that they would be the drivers of (anti-)androgenic effects in Czech aquatic environments.

Developmental profiles of progesterone receptor transcripts and molecular responses to gestagen exposure during Silurana tropicalis early development.

Environmental gestagens are an emerging class of contaminants that have been recently measured in surface water and can interfere with reproduction in aquatic vertebrates. Gestagens include endogenous progestogens, such as progesterone (P4), which bind P4-receptors and have critically important roles in vertebrate physiology and reproduction. Gestagens also include synthetic progestins, which are components of human and veterinary drugs, such as melengestrol acetate (MGA). Endogenous progestogens are essential in the regulation of reproduction in mammalian species, but the role of P4 in amphibian larval development remains unclear. This project aims to understand the roles and the regulatory mechanisms of P4 in amphibians and to assess the consequences of exposures to environmental gestagens on the P4-receptor signaling pathways in frogs. Here, we established the developmental profiles of the P4 receptors: the intracellular progesterone receptor (ipgr), the membrane progesterone receptor ß (mpgrß), and the progesterone receptor membrane component 1 (pgrmc1) in Western clawed frog (Silurana tropicalis) embryos using real-time qPCR. P4-receptor mRNAs were detected throughout embryogenesis. Transcripts for ipgr and pgrmc1 were detected in embryos at Nieuwkoop and Faber (NF) stage 2 and 7, indicative of maternal transfer of mRNA. We also assessed the effects of P4 and MGA exposure in embryonic and early larval development. Endocrine responses were evaluated through transcript analysis of a suite of gene targets of interest, including: ipgr, mpgrß, pgrmc1, androgen receptor (ar), estrogen receptor α (erα), follicle stimulating hormone ß (fshß), prolactin (prl), and the steroid 5-alpha reductase family (srd5α1, 2, and 3). Acute exposure (NF 12-46) to P4 caused a 2- to 5-fold change increase of ipgr, mpgrß, pgrmc1, and ar mRNA levels at the environmentally relevant concentration of 195 ng/L P4. Acute exposure to MGA induced a 56% decrease of srd5α3 at 1140 ng/L MGA. We conclude that environmental exposure to P4 induced multiple endocrine-related transcript responses in amphibians; however, the differential responses of MGA suggest that the effects of MGA are not mediated through the classical P4 signaling pathway in S. tropicalis.

The development of progesterone-loaded nanofibers using pressurized gyration: A novel approach to vaginal delivery for the prevention of pre-term birth.

Recent evidence has continued to support the applicability of progesterone in preventing preterm birth, hence the development of an appropriate vaginal delivery system for this drug would be of considerable interest. Here, we describe the development of progesterone-loaded bioadhesive nanofibers using pressurized gyration for potential incorporation into a vaginal insert, with a particular view to addressing the challenges of incorporating a poorly water-soluble drug into a hydrophilic nanofiber carrier. Polyethylene oxide and carboxymethyl cellulose were chosen as polymers to develop the carrier systems, based on previous evidence of their yielding mucoadhesive nanofibers using the pressurized gyration technique. The fabrication parameters such as solvent system, initial drug loading and polymer composition were varied to facilitate optimisation of fiber structure and efficiency of drug incorporation. Such studies resulted in the formation of nanofibers with satisfactory surface appearance, diameters in the region of 400 nm and loading of up to 25% progesterone. Thermal and spectroscopic analyses indicated that the drug was incorporated in a nanocrystalline state. Release from the drug-loaded fibers indicated comparable rates of progesterone dissolution to that of Cyclogest, a commercially available progesterone pessary, allowing release over a period of hours. Overall, the study has shown that pressurized gyration may produce bioadhesive progesterone-loaded nanofibers which have satisfactory loading of a poorly water-soluble drug as well as having suitable structural and release properties. The technique is also capable of producing fibers at a yield commensurate with practical applicability, hence we believe that the approach shows considerable promise for the development of progesterone dosage forms for vaginal application.

Characterization of estrous cycles and pregnancy in Somali wild asses (Equus africanus somaliensis) through fecal hormone analyses.

Although reproduction in the domestic horse has been well described, less is known about reproduction in wild equids. This study describes endocrine patterns associated with estrous cycles and pregnancy for Somali wild asses (Equus africanus somaliensis), an endangered African equid. Fecal samples were collected three times per week for more than 2 years from five female Somali wild asses at the Saint Louis Zoo; progestagen and estrogen metabolites were quantified using commercially available immunoassays. Progestagen analysis indicated that cycle lengths were 27.2 ± 1.2 days and females cycled throughout the year. Progestagen levels during early pregnancy were low and not sustained above baseline until approximately 40 weeks prior to partition. Concentrations increased markedly around 16 weeks prior to delivery and peaked 2-3 weeks before birth. Fecal estrogen levels also increased significantly starting 40-45 weeks before parturition and reached their maximal value approximately 20 weeks prior to birth. Neither foal heat nor lactational suppression of estrus was observed, and females cycled within 45 days after delivery. These data are the first to describe the reproductive physiology of Somali wild asses. As the species faces increasing threats in the wild, this information may support conservation efforts by assisting with ex situ breeding programs.

In Vitro-In Vivo Relationship of Amorphous Insoluble API (Progesterone) in PLGA Microspheres.

PURPOSE: The mechanism of PRG release from PLGA microspheres was studied and the correlation of in vitro and in vivo analyses was assessed. METHODS: PRG-loaded microspheres were prepared by the emulsion-evaporate method. The physical state of PRG and microstructure changings during the drug release period were evaluated by powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM) respectively. Pharmacokinetic studies were performed in male Sprague-Dawley rats, and the in vivo-in vitro correlation (IVIVC) was established by linear fitting of the cumulative release (%) in vitro and fraction of absorption (%) in vivo. RESULTS: PXRD results indicated recrystallization of PRG during release. The changes of microstructure of PRG-loaded microspheres during the release period could be observed in SEM micrographs. Pharmacokinetics results performed low burst-release followed a steady-released manner. The IVIVC assessment exhibited a good correlation between vitro and in vivo. CONCLUSIONS: The burst release phase was caused by diffusion of amorphous PRG near the surface, while the second release stage was impacted by PRG-dissolution from crystal depots formed in microspheres. The IVIVC assessment suggests that the in vitro test method used in this study could predict the real situation in vivo and is helpful to study the release mechanism in vivo.