Pharmaceutical and Pharmacological Evaluation of the Effect of Nano-Formulated Spironolactone and Progesterone on Inflammation and Hormonal Levels for Managing Hirsutism Experimentally Induced in Rats.

Hirsutism is a dermatological condition that refers to the excessive growth of hair in androgen-sensitive areas in women. Recently, the enhancement of the visible signs of a hairy female has taken special concern that affected the quality of life. The present study was developed to compare the follicular targeting effect of topical spironolactone (SP) or progesterone (PG)-loaded nanostructured lipid carrier (NLC) on the management of hirsutism. Four NLC formulations were prepared using cold homogenization techniques and pharmaceutically evaluated. SP-NLC and PG-NLC topical hydrogels were prepared to explore their pharmacological effect on letrozole induced polycystic ovarian syndrome (PCOS) in rats. Inflammatory mediators, antioxidant, and hormonal parameters were assayed. Additionally, histopathological examination was carried out to confirm the successful induction of PCOS. Results confirmed that all NLC formulations have a spherical shape with particle size ranged from 225.92 ± 0.41 to 447.80 ± 0.66 nm, entrapment efficiency > 75%, and zeta potential (- 31.4 to - 36.5 mV). F1 and F3 NLCs were considered as selected formulations for SP and PG, respectively. Female Wistar rats treated with F1 formulation for 3 weeks displayed better outcomes as manifested by the measured parameters as compared to the other tested groups. A significant reduction in hair follicle diameter and density was observed after topical application of SP or PG nano-gels. Finally, the outcomes pose a strong argument that the development of topically administered SP-NLC can be explored as a promising carrier over PG-NLC for more effectual improvement in the visible sign of hirsutism.

A Dual Role Reductase from Phytosterols Catabolism Enables the Efficient Production of Valuable Steroid Precursors.

4-Androstenedione (4-AD) and progesterone (PG) are two of the most important precursors for synthesis of steroid drugs, however their current manufacturing processes suffer from low efficiency and severe environmental issues. In this study, we decipher a dual-role reductase (mnOpccR) in the phytosterols catabolism, which engages in two different metabolic branches to produce the key intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a 4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA), respectively. Inactivation or overexpression of mnOpccR in the Mycobacterium neoaurum can achieve exclusive production of either 4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-HBC can be efficiently converted into PG in a scalable manner (100 gram scale). This study deciphers a pivotal biosynthetic mechanism of phytosterol catabolism and provides very efficient production routes of 4-AD and PG.

SAR based in-vitro anticholinesterase and molecular docking studies of nitrogenous progesterone derivatives.

Progesterone is a steroidal hormone that has been described with pathogenic features of brain dysfunction, realized with advanced age-related neurodegenerative diseases such as Alzheimer's disease. In this study, sixteen nitrogenous derivatives of progesterone which we previously synthesized have been used for Alzheimer targets. The progesterone derivatives (1-16) were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potentials in a dose-dependent manner. All the compounds exhibited overwhelming AChE inhibitions, with IC50 values ranging from 14.40 to 40.37 µM. Similarly, the BChE inhibitory potentials of our compounds were also dominant with IC50values between 20.08 and 46.84 µM. In comparison to our compounds, the standard drug galantamine attain IC50 values of 12.03 and 18.20 µM against AChE and BChE respectively. Molecular docking studies suggested that the compounds exerted their inhibitory activity by binding to the active site of the enzyme. The cholinergic system plays an important role in the regulation of learning and memory processes and has been a major target for the design of anti-Alzheimer's drugs. Therefore, these nitrogen-containing progesterone derivatives will be of potential interest to researchers working in AD for developing new drugs or chemical tools to study the disease.

Neurodevelopmental effects of natural and synthetic ligands of estrogen and progesterone receptors in zebrafish eleutheroembryos.

Natural and synthetic estrogens and progestins are widely used in human and veterinary medicine and are detected in waste and surface waters. Our previous studies have clearly shown that a number of these substances targets the brain to induce the estrogen-regulated brain aromatase expression but the consequences on brain development remain virtually unexplored. The aim of the present study was therefore to investigate the effect of estradiol (E2), progesterone (P4) and norethindrone (NOR), a 19-nortestosterone progestin, on zebrafish larval neurogenesis. We first demonstrated using real-time quantitative PCR that nuclear estrogen and progesterone receptor brain expression is impacted by E2, P4 and NOR. We brought evidence that brain proliferative and apoptotic activities were differentially affected depending on the steroidal hormone studied, the concentration of steroids and the region investigated. Our findings demonstrate for the first time that steroid compounds released in aquatic environment have the capacity to disrupt key cellular events involved in brain development in zebrafish embryos further questioning the short- and long-term consequences of this disruption on the physiology and behavior of organisms.

Improvements in corticosteroid 21-acetoxy-17α-hydroxy-16α-methyl-pregna-1,4,9(11)-triene-3,20-dione synthesis and its use as common intermediate in the synthesis of some impurities related to dexamethasone and mometasone.

There are quite substantial number of impurities related to dexamethasone or mometasone which cannot be made from respective Active Pharmaceutical Ingredients but from common intermediate 21-acetoxy-17α-hydroxy-16α-methyl-pregna-1,4,9(11)-triene-3,20-dione (12). As such, a robust and economical synthesis of this key intermediate is important for delivering a resilient and economically viable supply chain for these impurities. Therefore, it is critical to have a robust and economically viable process to synthesize the intermediate 12 in good yield and quality. We report here an improved synthesis of 12 and eight impurities related to dexamethasone and mometasone from this common intermediate.

Synthesis, pharmacological evaluation and docking studies of progesterone and testosterone derivatives as anticancer agents.

Steroidal hormones progesterone and testosterone play a vital role in breast and prostate cancers. In this research, we have synthesized and characterized a total of thirty-one (31) new nitrogenous derivatives of progesterone and testosterone. The synthesized derivatives (1-31) were screened for their anti-cancer potential against MCF-7 and PC-3 cell lines of breast using MTT assay. The compounds 1-31exhibited significant inhibitory potentials against MCF-7 and PC-3 cell lines. In MCF-7 assay, compound 17 displayed IC50 value of 04 ±â€¯0.02 µM while compound 18 was leading in PC-3 assay with IC50 of 03.14 ±â€¯0.4 µM. Tamoxifen was used as positive control which exhibited an IC50of 0.12 ±â€¯0.03 and 0.26 ±â€¯0.01 µM against MCF-7 and PC-3 respectively. The compounds also showed good anti-inflammatory activity according to oxidative burst inhibition by chemiluminescence technique where ibuprofen was used as positive control with 73.2 ±â€¯1.4% ROS inhibition. The compounds showed the percent ROS inhibition between 23.2 ±â€¯0.2 and -3.2 ±â€¯4.1. The results of the compounds were compared with the positive control ibuprofen. Molecular docking correlations suggest that the compounds exerted their inhibitory activity by binding to the active of the enzyme.

Influência da neoplasia mamária na concentração sérica de hormônios e na expressão de receptores de estrógeno e progesterona em cadelas / Influence of mammary neoplasm on the serum concentration of hormones and on the expression of estrogen and progesterone receptors in bitches

O objetivo desse estudo foi avaliar as concentrações séricas de estradiol, progesterona e prolactina, bem como a expressão gênica dos receptores de estrógeno α e β e de progesterona em cadelas com neoplasias mamárias. Foram utilizadas 60 cadelas adultas, sem raça definida que foram distribuídas em dois grupos. O Grupo I constituído por 30 cadelas portadoras de neoplasias mamárias e o Grupo II constituído por 30 cadelas saudáveis, não portadoras de neoplasia. Para os tutores, foram aplicados questionários sobre fatores epidemiológicos da doença. Após avaliação dos exames pré-operatórios, as cadelas com neoplasia mamária foram submetidas à mastectomia, coletaram-se fragmentos das neoplasias e linfonodos regionais, os quais foram processados para análise histopatológica. Para as dosagens hormonais de estradiol, progesterona e prolactina foram colhidas amostras de sangue em tubos sem anticoagulante e os soros foram submetidos à técnica de eletroquimioluminescência. A expressão gênica dos receptores hormonais foi realizada por meio da técnica de Real-time PCR e para isso foram coletados fragmentos das neoplasias mamárias e extraído o RNA para obtenção do cDNA. A expressão do mRNA para os REα, REβ e RP foi avaliada a partir da amplificação desses genes utilizando primers específicos. Verificaram-se maiores níveis séricos de estradiol (média de 38,98±13,68pg/mL) em cadelas portadoras de neoplasias mamárias malignas quando comparadas as cadelas do grupo controle (p<0,05). Já os níveis séricos de prolactina foram maiores (média de 0,231±0,201ng/mL) nas cadelas que não possuíam neoplasias mamárias quando comparadas ao Grupo I (p<0,05). Para os níveis de progesterona não foram observadas diferença entre os diferentes grupos (p>0,05). Tanto os tumores malignos como os benignos expressaram REα, REβ e RP, não havendo diferença (p>0,05) na expressão entre tumores malignos ou benignos ou relacionada aos outros fatores prognósticos investigados (estadiamento clínico, presença de ulceração, vascularização e tempo de evolução do processo). Os níveis séricos de estradiol aumentaram significativamente com o estadiamento clínico da doença (p<0,05). Verificou-se moderada correlação negativa entre os níveis séricos de estradiol e prolactina. Dessa forma, conclui-se que as dosagens séricas de estradiol e PRL foram influenciadas pela malignidade do tumor e pelo estadiamento clínico das neoplasias. Os receptores hormonais foram expressos pelas neoplasias, independentemente do tipo tumoral e não estão associados aos outros fatores prognóstico clássicos, como presença de ulceração, vascularização ou estadiamento clínico.(AU)

The aim of this study was to evaluate the serum concentrations of estradiol, progesterone, prolactin, the gene expression of estrogen α and β and progesterone receptors in bitches with mammary neoplasms. Sixty adult crossbred bitches distributed in two groups were used. Group I consisted of 30 bitches with mammary neoplasms and Group II consisted of 30 healthy bitches without neoplasia. For the tutors, interviews were made about the disease epidemiology. After preoperative examinations, bitches with mammary neoplasia were submitted to mastectomy; fragments of the neoplasms and regional lymph nodes were collected and processed for histopathological analysis. Blood samples were collected in tubes without anticoagulant and the serum was analyzed by electrochemiluminescence to measure estradiol, progesterone and prolactin. The gene expression of the hormonal receptors was performed by means of the Real-time PCR technique, thus fragments of mammary neoplasms were collected and the RNA was extracted to obtain cDNA. Expression of the mRNA for ERα, ERβ and PR was assessed from the amplification of these genes using specific primers. Higher serum levels of estradiol (mean 38.98±13.68pg/mL) were observed in bitches with malignant neoplasms when compared to the control bitches (p<0.05). Serum prolactin levels were higher (mean of 0.231±0.201ng/mL) in bitches that did not have mammary neoplasms when compared to Group I (p<0.05). No difference was observed for related to the progesterone levels between the groups (p>0.05). Both malignant and benign tumors expressed ERα, ERβ and RP with no statistical difference (p>0.05) and there were no difference related to the other prognostic factors investigated (clinical staging, presence of ulceration, vascularization and aging of neoplasms). Serum estradiol levels increased significantly with the clinical staging of the disease (p<0.05). There was a moderate negative correlation between serum levels of estradiol and prolactin. It was concluded that serum levels of estradiol and PRL were influenced by tumor malignancy and clinical staging of neoplasms. Hormonal receptors were expressed by neoplasms, regardless of tumor type and are not associated with other classical prognostic factors, such as ulceration, vascularization or clinical staging.(AU)

The effect of newly synthesized progesterone derivatives on apoptotic and angiogenic pathway in MCF-7 breast cancer cells.

Due to its high potency and selectivity, anticancer agents consisting of combined molecules have gained great interests. The current study introduces newly synthesized progesterone derivatives of promising anticancer effect. Moreover, the pro-apoptotic and anti-angiogenic effects of these compounds were studied extensively. Several thiazole, pyridine, pyrazole, thiazolopyridine and pyrazolopyridine progesterone derivatives were synthesized. The structure of the novel progesterone derivatives was elucidated and confirmed using the analytical and spectral data. This novel derivatives were tested for their cytotoxic effect against human breast cancer cells (MCF-7) using neutral red uptake assay. Tested compounds showed anticancer activity against MCF-7 cancer cell line in the descending order of 7>2>3>8>6>9>4. The expression levels of Bcl-2, survivin, CCND1, CDC2, P53 and P21, VEGF, Hif-1α, MMP-2, MMP-9, Ang-1, Ang-2, and FGF-1 genes were investigated using QRT-PCR (Quantitative real time-polymerase chain reaction). The study clarified that compounds 2, 3, 4, 6, 7, 8 and 9 showed significant pro-apoptotic effect through the down regulation of Bcl-2., besides, survivin and CCND1 expression levels were down regulated by compounds 3, 4, 6, 7, 8, 9. However, Compound 4 may exert this pro-apoptotic effect through the up-regulation of P53 gene expression. On the other hand, the anti-angiogenic effect of these newly synthesized derivatives was due to their down regulation of VEGF, Ang-2, MMP-9 and FGF-1; and the up-regulation of HIF-1α and ang-1. This study recommended promising pro-apoptotic and anti-angiogenic anticancer agents acting through the regulation of key regulators of apoptosis, cell cycle genes, and pro-angiogenic genes.

Selection of Progesterone Derivatives Specific to Membrane Progesterone Receptors.

The search of selective agonists and antagonists of membrane progesterone receptors (mPRs) is a starting point for the study of progesterone signal transduction mechanisms mediated by mPRs, distinct from nuclear receptors. According to preliminary data, the ligand affinity for mPRs differs significantly from that for classical nuclear progesterone receptors (nPRs), which might indicate structural differences in the ligand-binding pocket of these proteins. In the present work, we analyzed the affinity of several progesterone derivatives for mPRs of human pancreatic adenocarcinoma BxPC3 cell line that is characterized by a high level of mPR mRNA expression and by the absence of expression of nPR mRNA. The values were compared with the affinity of these compounds for nPRs. All tested compounds showed almost no affinity for nPRs, whereas their selectivity towards mPRs was different. Derivatives with an additional 19-hydroxyl group and removed 3-keto group had the highest selectivity for mPRs. These results suggest these compounds as the most selective progesterone analogs for studying the mechanisms of progestin action via mPRs.

Optimisation and validation of a new analytical method for the determination of four natural and synthetic hormones using LC-ESI-MS/MS.

A rapid liquid chromatographic-tandem mass spectrometric method was developed for the simultaneous determination of four natural and synthetic hormone residues (progesterone, testosterone, trenbolone acetate and zeranol) in animal tissue samples. Sample preparation was optimised to minimise time and solvent consumption. Meat samples were mechanically homogenised and digested in a procedure that gave similar recoveries to those enzymatically hydrolysed by Helix pomatia. Efficient extraction was achieved using acidified acetonitrile (1% acetic acid). Chromatographic conditions were optimised to minimise matrix effects. Analytes were separated using a C18 column with gradient elution using ammonium formate solution in methanol (MeOH)/water (1:9) and MeOH mobile phases. Finally, residues were qualitatively and quantitatively determined by electrospray ionisation tandem mass spectrometry in multiple reaction monitoring mode. Different parameters for LC-MS/MS (e.g., declustering potential and collision energy) were optimised using API 6500QT; all analytes were measured using positive-mode electrospray ionisation (ESI+) except zeranol which was measured in negative mode (ESI-). Due to LC-MS/MS signal enhancement/suppression, the determination of hormones was based on matrix-matched standard calculations. The method was validated for the four hormones on meat samples at different fortification levels and showed accepted performance criteria according to European Commission Decision 2002/657/EC. Decision limits and detection capabilities were estimated for all analytes.

Synthesis of new steroidal inhibitors of P-glycoprotein-mediated multidrug resistance and biological evaluation on K562/R7 erythroleukemia cells.

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/ß-pregnane-3,20-dione or 5ß-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/ß-pregnane-3,20-dione, among which the 5ß-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 µM versus 1.2 and 10.6 µM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5ß-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 µM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

Synthesis and cytotoxic effect on cancer cell lines and macrophages of novel progesterone derivatives having an ester or a carbamate function at C-3 and C-17.

In this study we report the cytotoxic effect on human cancer cells of two series of novel progesterone derivatives; the first containing an aromatic ester (8a-e) or a carbamate functions both linked to C-3 (9a-e) on the pregn-4,16-diene-6,20-dione skeleton. In the second series, both functional groups (ester and carbamate) are bound to C-17 on the pregn-4,6-diene-3,20-dione scaffold (13a-e and 14a-e). The panel cancer cell lines used in this study were the following: PC-3 (human prostate cancer cell line), MCF-7 (human breast cancer cell line), HCT-15 (human colon cancer cell line) and J774 (noncancerous murine macrophages) for comparison. The results from this study showed that steroid 14a, having a carbamate function at C-17, was the most potent against PC-3 cell line (96.6%) while 8c and 8e showed much higher cytotoxic activity (100%) for MCF-7 cell line. Finally, compounds 8c and 14a displayed selective properties towards tumor cell lines than noncancerous murine macrophages.

Mucor hiemalis mediated 14α-hydroxylation on steroids: in vivo and in vitro investigations of 14α-hydroxylase activity.

Transformation of testosterone and progesterone into synthetically challenging 14α-hydroxy derivatives was achieved by using fungal strain Mucor hiemalis. Prolonged incubation led to the formation of corresponding 6ß/7α,14α-dihydroxy metabolites. The position and stereochemistry of newly introduced hydroxyl group was determined by detailed spectroscopic analyses. The time course experiment indicated that fungal strain initiated transformation by hydroxylation at 14α-position followed by at 6ß- or 7α-positions. Studies using cell-free extracts suggest that the 14α-hydroxylase activity is NADPH dependent and belongs to the cytochrome P450 family.

Synthesis of sterically encumbered 11ß-aminoprogesterone derivatives and evaluation as 11ß-hydroxysteroid dehydrogenase inhibitors and mineralocorticoid receptor antagonists.

11ß-Hydroxyprogesterone is a well-known nonselective inhibitor of 11ß-hydroxysteroid dehydrogenase (11ßHSD) types 1 and 2. It also activates the mineralocorticoid receptor (MR). Modulation of corticosteroid action by inhibition of 11ßHSDs or blocking MR is currently under consideration for treatment of electrolyte disturbances, metabolic diseases and chronic inflammatory disorders. We established conditions to synthesize sterically demanding 11ß-aminoprogesterone, which following subsequent nucleophilic or reductive amination, allowed extension of the amino group to prepare amino acid derivatives. Biological testing revealed that some of the 11ß-aminoprogesterone derivatives selectively inhibit 11ßHSD2. Moreover, two compounds that did not significantly inhibit 11ßHSDs had antagonist properties on MR. The 11ß-aminoprogesterone derivatives form a basis for the further development of improved modulators of corticosteroid action.

Synthesis and cytotoxicity of some novel 21E-benzylidene steroidal derivatives.

A series of novel derivatives of 21E-benzylidene-pregn-1,4-diene-3,20-dione 7a-g and 21E-benzylidene-4-chloro-pregn-1,4-diene-3,20-dione 8a-g was synthesized from the commercially available progesterone. These title compounds were evaluated for their cytotoxic activity against brine shrimp (Artemia salina) and murine Lewis lung carcinoma cells (LLC). It was found that compounds 7a-g exhibited stronger activities than 8a-g against the brine shrimps, and some of the tested compounds possessed weak inhibition of LLC cells.

Synthesis of 6-azaprogesterone and 19-hydroxy-6-azasteroids.

19-Hydroxy-6-azapregnanes were obtained from pregnenolone via a 7-azido-5-oxo-6-nor-5,7-secopregnane intermediate. The 6-azapregnane core was built in good yield in a straightforward way from the secosteroid, by means of a Staudinger (aza-Wittig) reaction. Finally the 19-hydroxy-6-azapregnane was transformed into 19-hydroxy-6-azaprogesterone (that cyclized spontaneously to the 19→3 hemiketal) and 6-azaprogesterone. The 6-azapregnanes lacked agonistic/antagonistic activity on the progesterone receptor.

Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

Preparation and preliminary bioevaluation of 99mTc(CO)3-11ß-progesterone derivative prepared via click chemistry route.

INTRODUCTION: Progesterone receptors (PRs) overexpressed in breast cancers serve as potential targets for developing radiotracers for use in nuclear medicine. Hence, suitably derivatized progesterone can be envisaged as a potential vector for targeting overexpression of receptors in breast cancer. In the present article, we report the preparation of a (99m)Tc(CO)(3)-progesterone triazole using the Cu(I)-catalyzed novel click chemistry route. Preliminary evaluation of the radiolabeled derivative has been carried out in binding studies with MCF 7 cell lines. METHODS: 11-Hydroxyprogesterone has been synthetically derivatized to 11-azidoprogesterone. Subsequently, the cycloaddition reaction between progesterone azide and propargyl glycine was carried out to prepare 1,4-bifunctionalized progesterone triazole analogue. The clicked progesterone triazole derivative was radiolabeled with (99m)Tc and characterized by HPLC. The chemical characterization of (99m)Tc(CO)(3)-progesterone triazole has been carried out by preparing its corresponding rhenium complex using the [NEt(4)](2)[Re(CO)(3)Br(3)] precursor. While in vitro studies were carried out in MCF7 cell lines, in vivo distribution studies were performed in female Swiss mice. RESULTS: The radiolabeled complex could be prepared in >95% radiochemical yield as determined by HPLC. In vitro studies of (99m)Tc(CO)(3)-progesterone complex in MCF7 cell lines overexpressing receptors for breast cancer showed binding up to 30%. In vivo distribution studies in female Swiss mice have shown uterine uptake of 0.41 (0.06) % ID/g at 3 h postinjection (pi) and retention therein till 24 h pi. CONCLUSION: The present study demonstrates a novel and facile route for preparation of (99m)Tc-labeled progesterone complex using click chemistry. This strategy can be further extended towards preparation of radiolabeled complexes of other steroidal derivatives.

Progesterone receptor agonists and antagonists as anticancer agents.

Progesterone is a major female steroid hormone produced by the ovarian corpus luteum and by the placental syncytiotrophoblast during the second trimester. The biological effects of this steroid hormone are mediated by the ubiquitously expressed progesterone receptor. The exact link between progesterone and female reproductive organ cancer is a controversial issue with various cross-talks. The present review summarizes recent trends in the development of some (anti)progestagen in the cure and management of breast and uterine cancers.