Bioanalytical strategies in drug discovery and development.

A reliable, rapid, and effective bioanalytical method is essential for the determination of the pharmacokinetic, pharmacodynamic, and toxicokinetic parameters that inform the safety and efficacy profile of investigational drugs. The overall goal of bioanalytical method development is to elucidate the procedure and operating conditions under which a method can sufficiently extract, qualify, and/or quantify the analyte(s) of interest and/or their metabolites for the intended purpose. Given the difference in the physicochemical properties of small and large molecule drugs, different strategies need to be adopted for the development of an effective and efficient bioanalytical method. Herein, we provide an overview of different sample preparation strategies, analytical platforms, as well as procedures for achieving high throughput for bioanalysis of small and large molecule drugs.

An 'Omics Approach to Unraveling the Paradoxical Effect of Diet on Perfluorooctanesulfonic Acid (PFOS) and Perfluorononanoic Acid (PFNA)-Induced Hepatic Steatosis.

Perfluoroalkyl substances (PFAS) are a family of toxicants universally detected in human serum and known to cause dyslipidemia in animals and humans. Hepatic steatosis, which is defined as lipid deposition in the liver, is known to be a consequence of poor diet. Similarly, PFAS are known to induce hepatic steatosis in animals on a low-fat chow. This study explored diet-PFAS interactions in the liver and their potential to modulate hepatic steatosis. Male C57BL/6J mice were fed with either a low-fat diet (10% kcal from fat, LFD) or a moderately high-fat diet (45% kcal from fat, HFD) with or without perfluorooctanesulfonic acid (3 ppm, PFOS) or perfluorononanoic acid (3 ppm, PFNA) in feed for 12 weeks. Livers were excised for histology and quantification of PFAS and lipids. The PFOS and PFNA coadministration with HFD reduced the hepatic accumulation of lipid and PFAS relative to the LFD treatment groups. Furthermore, transcriptomic analysis revealed that PFAS administration in the presence of an HFD significantly reduces expression of known hepatic PFAS uptake transporters, organic anion transporter proteins. Transcriptomics and proteomics further revealed several pathways related to lipid metabolism, synthesis, transport, and storage that were modulated by PFAS exposure and further impacted by the presence of dietary fat. Both dietary fat content and the chemical functional head group exerted significant influence on hepatic PFAS accumulation and the resulting biochemical signature, suggesting that diet and structure should be considered in the design and interpretation of research on PFAS induced hepatic steatosis.

Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Alter the Blood Lipidome and the Hepatic Proteome in a Murine Model of Diet-Induced Obesity.

Perfluoroalkyl substances (PFAS) represent a family of environmental toxicants that have infiltrated the living world. This study explores diet-PFAS interactions and the impact of perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic (PFHxS) on the hepatic proteome and blood lipidomic profiles. Male C57BL/6J mice were fed with either a low-fat diet (10.5% kcal from fat) or a high fat (58% kcal from fat) high carbohydrate (42 g/l) diet with or without PFOS or PFHxS in feed (0.0003% wt/wt) for 29 weeks. Lipidomic, proteomic, and gene expression profiles were determined to explore lipid outcomes and hepatic mechanistic pathways. With administration of a high-fat high-carbohydrate diet, PFOS and PFHxS increased hepatic expression of targets involved in lipid metabolism and oxidative stress. In the blood, PFOS and PFHxS altered serum phosphatidylcholines, phosphatidylethanolamines, plasmogens, sphingomyelins, and triglycerides. Furthermore, oxidized lipid species were enriched in the blood lipidome of PFOS and PFHxS treated mice. These data support the hypothesis that PFOS and PFHxS increase the risk of metabolic and inflammatory disease induced by diet, possibly by inducing dysregulated lipid metabolism and oxidative stress.

Nonalcoholic Fatty Liver Disease (NAFLD) and Hepatic Cytochrome P450 (CYP) Enzymes.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive fat in the liver. An international consensus panel has recently proposed to rename the disease to metabolic dysfunction associated with fatty liver disease (MAFLD). The disease can range from simple steatosis (fat accumulation) to nonalcoholic steatohepatitis (NASH) which represents a severe form of NAFLD and is accompanied by inflammation, fibrosis, and hepatocyte damage in addition to significant steatosis. This review collates current knowledge of changes in human hepatic cytochrome P450 enzymes in NAFLD. While the expression of these enzymes is well studied in healthy volunteers, our understanding of the alterations of these proteins in NAFLD is limited. Much of the existing knowledge on the subject is derived from preclinical studies, and clinical translation of these findings is poor. Wherever available, the effect of NAFLD on these proteins in humans is debatable and currently lacks a consensus among different reports. Protein expression is an important in vitro physiological parameter controlling the pharmacokinetics of drugs and the last decade has seen a rise in the accurate estimation of these proteins for use with physiologically based pharmacokinetic (PBPK) modeling to predict drug pharmacokinetics in special populations. The application of label-free, mass spectrometry-based quantitative proteomics as a promising tool to study NAFLD-associated changes has also been discussed.

Perfluorooctanesulfonic acid (PFOS) administration shifts the hepatic proteome and augments dietary outcomes related to hepatic steatosis in mice.

Hepatic steatosis increases risk of fatty liver and cardiovascular disease. Perfluorooctanesulfonic acid (PFOS) is a persistent, bio-accumulative pollutant that has been used in industrial and commercial applications. PFOS administration induces hepatic steatosis in rodents and increases lipogenic gene expression signatures in cultured hepatocytes. We hypothesized that PFOS treatment interferes with lipid loss when switching from a high fat diet (HFD) to a standard diet (SD), and augments HFD-induced hepatic steatosis. Male C57BL/6 N mice were fed standard chow diet or 60% kCal high-fat diet (HFD) for 4 weeks to increase body weight. Then, some HFD mice were switched to SD and mice were further divided to diet only or diet containing 0.0003% PFOS, for six treatment groups: SD, HFD to SD (H-SD), HFD, SD + PFOS, H-SD + PFOS, or HFD + PFOS. After 10 weeks on study, blood and livers were collected. HFD for 14 weeks increased body weight and hepatic steatosis, whereas H-SD mice returned to SD measures. PFOS administration reduced body weight in mice fed a SD, but not H-SD or HFD. PFOS administration increased liver weight in H-SD + PFOS and HFD + PFOS mice. PFOS increased hepatic steatosis in H-SD and HFD groups. Hepatic mRNA expression and SWATH-MS proteomic analysis revealed that PFOS induced lipid and xenobiotic transporters, as well as metabolism pathways. Overall, the findings herein suggest that PFOS treatment did interfere with lipid loss associated with switch to a SD and similarly augmented hepatic lipid accumulation in mice established on an HFD.

Development and validation of an assay for a novel ghrelin receptor inverse agonist PF-5190457 and its major hydroxy metabolite (PF-6870961) by LC-MS/MS in human plasma.

PF-5190457 is a selective and potent ghrelin receptor inverse agonist presently undergoing clinical trials to treat alcohol use disorder (AUD). We describe the development and validation of a selective and sensitive liquid chromatography-tandem mass spectrometry-based method for quantification of PF-5190457 and its recently discovered hydroxy metabolite PF-6870961 in human plasma. Analytes were extracted after simple protein precipitation using methanol (2.5 ng mL-1 tacrine as an internal standard). A gradient liquid chromatography method was used to separate the analytes on an Acquity UPLC BEH C18 analytical column. The separation was achieved at a flow rate of 0.25 mL min-1 and the total chromatographic runtime was 11.30 min. Positive electrospray ionization and multiple reaction monitoring mode were used for the quantification of all the analytes. The calibration curves from six validation runs were linear with a correlation coefficient of ≥0.996 for the concentration range of 1-1000 ng mL-1 and 2-250 ng mL-1 for PF-5190457 and PF-6870961, respectively. The retention time for PF-5190457, PF-6870961 and tacrine were 4.4, 3.8, and 4.6 min, respectively. The lower limit of quantification for PF-5190457 and PF-6870961 was 1 and 2 ng mL-1, respectively. The inter-assay precision and accuracy results obtained were within the Food and Drug Administration recommended ±15% limit of nominal values. All the analytes were found to be stable under varied stability conditions. The recovery of PF-5190457 and PF-6870961 ranged from 95 to 103%. Further, the application of the method was demonstrated by measuring the concentration of PF-5190457 and its hydroxy metabolite in patient plasma samples from 100 mg dose.

Role of Molybdenum-Containing Enzymes in the Biotransformation of the Novel Ghrelin Receptor Inverse Agonist PF-5190457: A Reverse Translational Bed-to-Bench Approach.

(R)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)-1-(2-(5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)ethan-1-one (PF-5190457) was identified as a potent and selective inverse agonist of the ghrelin receptor [growth hormone secretagogue receptor 1a (GHS-R1a)]. The present translational bed-to-bench work characterizes the biotransformation of this compound in vivo and then further explores in vitro metabolism in fractions of human liver and primary hepatocytes. Following oral administration of PF-5190457 in a phase 1b clinical study, hydroxyl metabolites of the compound were observed, including one that had not been observed in previously performed human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation was shown to be on the pyrimidine using nuclear magnetic resonance spectroscopy. The aldehyde oxidase (AO) inhibitor raloxifene and the xanthine oxidase inhibitor febuxostat inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. However, greater inhibition was observed with raloxifene, indicating AO is a dominant enzyme in the biotransformation. The intrinsic clearance of the drug in human liver cytosol was estimated to be 0.002 ml/min per milligram protein. This study provides important novel information at three levels: 1) it provides additional new information on the recently developed novel compound PF-5190457, the first GHS-R1a blocker that has moved to development in humans; 2) it provides an example of a reverse translational approach where a discovery in humans was brought back, validated, and further investigated at the bench level; and 3) it demonstrates the importance of considering the molybdenum-containing oxidases during the development of new drug entities. SIGNIFICANCE STATEMENT: PF-5190457 is a novel ghrelin receptor inverse agonist that is currently undergoing clinical development for treatment of alcohol use disorder. PF-6870961, a major hydroxyl metabolite of the compound, was observed in human plasma, but was absent in human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation on the pyrimidine ring was characterized. Inhibitors of aldehyde oxidase and xanthine oxidase inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. This information is important for patient selection in subsequent clinical studies.

Docosahexaenoic Acid Increases the Potency of Soluble Epoxide Hydrolase Inhibitor in Alleviating Streptozotocin-Induced Alzheimer's Disease-Like Complications of Diabetes.

Diabetes is a risk factor for Alzheimer's disease and it is associated with significant memory loss. In the present study, we hypothesized that the soluble epoxide hydrolase (sEH) inhibitor N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl)-urea (also known as TPPU) could alleviate diabetes-aggravated Alzheimer's disease-like symptoms by improving memory and cognition, and reducing the oxidative stress and inflammation associated with this condition. Also, we evaluated the effect of edaravone, an antioxidant on diabetes-induced Alzheimer's-like complications and the additive effect of docosahexaenoic acid (DHA) on the efficacy of TPPU. Diabetes was induced in male Sprague-Dawley rats by intraperitoneally administering streptozotocin (STZ). Six weeks after induction of diabetes, animals were either treated with vehicle, edaravone (3 or 10 mg/kg), TPPU (1 mg/kg) or TPPU (1 mg/kg) + DHA (100 mg/kg) for 2 weeks. The results demonstrate that the treatments increased the memory response of diabetic rats, in comparison to untreated diabetic rats. Indeed, DHA + TPPU were more effective than TPPU alone in reducing the symptoms monitored. All drug treatments reduced oxidative stress and minimized inflammation in the brain of diabetic rats. Expression of the amyloid precursor protein (APP) was increased in the brain of diabetic rats. Treatment with edaravone (10 mg/kg), TPPU or TPPU + DHA minimized the level of APP. The activity of acetylcholinesterase (AChE) which metabolizes acetylcholine was increased in the brain of diabetic rats. All the treatments except edaravone (3 mg/kg) were effective in decreasing the activity of AChE and TPPU + DHA was more efficacious than TPPU alone. Intriguingly, the histological changes in hippocampus after treatment with TPPU + DHA showed significant protection of neurons against STZ-induced neuronal damage. Overall, we found that DHA improved the efficacy of TPPU in increasing neuronal survival and memory, decreasing oxidative stress and inflammation possibly by stabilizing anti-inflammatory and neuroprotective epoxides of DHA. In the future, further evaluating the detailed mechanisms of action of sEH inhibitor and DHA could help to develop a strategy for the management of Alzheimer's-like complications in diabetes.

Bioavailable curcumin formulations: A review of pharmacokinetic studies in healthy volunteers.

Curcumin is a widely studied natural compound which has shown tremendous in vitro therapeutic potential. Despite that, the clinical efficacy of the native curcumin is weak due to its low bioavailability and high metabolism in the gastrointestinal tract. During the last decade, researchers have come up with different formulations with a focus on improving the bioavailability of curcumin. As a result, a significant number of bioavailable curcumin-based formulations were introduced with the varying range of enhanced bioavailability. The purpose of this review is to collate the published clinical studies of curcumin products with improved bioavailability over conventional (unformulated) curcumin. Based on the literature search, 11 curcumin formulations with available human bioavailability and pharmacokinetics data were included in this review. Further, the data on clinical study design, analytical method, pharmacokinetic parameters and other relevant details of each formulation were extracted. Based on a review of these studies, it is evident that better bioavailability of formulated curcumin products is mostly attributed to improved solubility, stability, and possibly low first-pass metabolism. The review hopes to provide a quick reference guide for anyone looking information on these bioavailable curcumin formulations. Based on the published reports, NovaSol® (185), CurcuWin® (136) and LongVida® (100) exhibited over 100-fold higher bioavailability relative to reference unformulated curcumin. Suggested mechanisms accounting for improved bioavailability of the formulations and details on the bioanalysis methods are also discussed.

Nonalcoholic Fatty Liver Disease and Diabetes Are Associated with Decreased CYP3A4 Protein Expression and Activity in Human Liver.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease in the Western population. We investigated the association of nonalcoholic fatty liver disease (NAFLD) and diabetes mellitus on CYP3A4 activity in human liver tissue from brain dead donors ( n = 74). Histopathologically graded livers were grouped into normal ( n = 24), nonalcoholic fatty liver (NAFL, n = 26), and nonalcoholic steatohepatitis (NASH, n = 24) categories. The rate of conversion of midazolam to its 1-hydroxy metabolite was used to assess in vitro CYP3A4 activity in human liver microsomes (HLM). A proteomics approach was utilized to quantify the protein expression of CYP3A4 and related enzymes. Moreover, a physiologically based pharmacokinetic (PBPK) model was developed to allow prediction of midazolam concentration in NAFL and NASH livers. CYP3A4 activity in NAFL and NASH was 1.9- and 3.1-fold ( p < 0.05) lower than normal donors, respectively. Intrinsic clearance (CLint) was 2.7- ( p < 0.05) and 4.1-fold ( p < 0.01) lower in donors with NAFL and NASH, respectively. CYP3A4 protein expression was significantly lower in NAFL and NASH donors ( p < 0.05) and accounted for significant midazolam hydroxylation variability in a multiple linear regression analysis (ß = 0.869, r2 = 0.762, P < 0.01). Diabetes was also associated with decreased CYP3A4 activity and protein expression. Both midazolam CLint and CYP3A4 protein abundance decreased significantly with increase in hepatic fat accumulation. Age and gender did not exhibit any significant association with the observed alterations. Predicted midazolam exposure was 1.7- and 2.3-fold higher for NAFL and NASH, respectively, which may result in a longer period of sedation in these disease-states. Data suggests that NAFLD and diabetes are associated with the decreased hepatic CYP3A4 activity. Thus, further evaluation of clinical consequences of these findings on the efficacy and safety of CYP3A4 substrates is warranted.

Multiplex and Label-Free Relative Quantification Approach for Studying Protein Abundance of Drug Metabolizing Enzymes in Human Liver Microsomes Using SWATH-MS.

We describe a sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) based method for label-free, simultaneous, relative quantification of drug metabolism enzymes in human liver microsomes (HLM; n = 78). In-solution tryptic digestion was aided by a pressure cycling method, which allowed a 90 min incubation time, a significant reduction over classical protocols (12-18 h). Digested peptides were separated on an Acquity UHPLC Peptide BEH C18 column using a 60 min gradient method at a flow rate of 0.100 mL/min. The quadrupole-time-of-flight mass spectrometer (ESI-QTOFMS) was operated in positive electrospray ionization mode, and data were acquired by data-dependent acquisition (DDA) and SWATH-MSALL mode. A pooled HLM sample was used as a quality control to evaluate variability in digestion and quantification among different batches, and inter-batch %CV for various proteins was between 3.1 and 7.8%. Spectral library generated from the DDA data identified 1855 distinct proteins and 25 681 distinct peptides at a 1% global false discovery rate (FDR). SWATH data were queried and analyzed for 10 major cytochrome P450 (CYP) enzymes using Skyline, a targeted data extraction software. Further, correlation analysis was performed between functional activity, protein, and mRNA expression for ten CYP enzymes. Pearson correlation coefficient (r) between protein and activity for CYPs ranged from 0.314 (CYP2C19) to 0.767 (CYP2A6). A strong correlation was found between CYP3A4 and CYP3A5 abundance and activity determined using midazolam and testosterone (r > 0.600, p < 0.001). Protein-to-activity correlation was moderate (r > 0.400-0.600, p < 0.001) for CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP2E1 and significant but poor (r < 0.400, p < 0.05) for CYP2C8, CYP2C19, and CYP2D6. The findings suggest the suitability of SWATH-MS based method as a valuable and relatively fast analytical technique for relative quantification of proteins in complex biological samples. We also show that protein abundance is a better surrogate than mRNA to predict the activity of CYP activity.

Ultra-high performance liquid chromatography tandem mass-spectrometry for simple and simultaneous quantification of cannabinoids.

Cannabis is used widely in the United States, both recreationally and for medical purposes. Current methods for analysis of cannabinoids in human biological specimens rely on complex extraction process and lengthy analysis time. We established a rapid and simple assay for quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy Δ9-tetrahydrocannabinol (11-OH THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannbinol (THCCOOH) in human plasma by U-HPLC-MS/MS usingΔ9-tetrahydrocannabinol-D3 (THC-D3) as the internal standard. Chromatographic separation was achieved on an Acquity BEH C18 column using a gradient comprising of water (0.1% formic acid) and methanol (0.1% formic acid) over a 6 min run-time. Analytes from 200µL plasma were extracted using acetonitrile (containing 1% formic acid and THC-D3). Mass spectrometry was performed in positive ionization mode, and total ion chromatogram was used for quantification of analytes. The assay was validated according to guidelines set forth by Food and Drug Administration of the United States. An eight-point calibration curve was fitted with quadratic regression (r2>0.99) from 1.56 to 100ngmL-1 and a lower limit of quantification (LLOQ) of 1.56ngmL-1 was achieved. Accuracy and precision calculated from six calibration curves was between 85-115% while the mean extraction recovery was >90% for all the analytes. Several plasma phospholipids eluted after the analytes thus did not interfere with the assay. Bench-top, freeze-thaw, auto-sampler and short-term stability ranged from 92.7 to 106.8% of nominal values. Application of the method was evaluated by quantification of analytes in human plasma from six subjects.

Erectogenic and Aphrodisiac Property of Moringa oleifera: Involvement of Soluble Epoxide Hydrolase Enzyme.

Soluble epoxide hydrolase (sEH) inhibitors have been reported to improve penile erection; therefore, sEH could be useful for management of erectile dysfunction. Methanolic and aqueous extracts of 30 Indian medicinal plants were screened for their sEH inhibition potential. Fifteen extracts showed >50% inhibition when screened at 50 µg/mL in sEH inhibition assay. Methanolic extract of Moringa oleifera Lam. (Moringaceae) seeds (MEMO) was most potent with IC50 1.7 ± 0.1 µg/mL and was selected for in vitro studies on isolated rat corpus cavernosum smooth muscle and in vivo sexual behaviour studies on healthy and diabetic rats. Rats were divided into five groups, each containing six animals and treated orally with either water, vehicle (1% Tween-20), MEMO (45 and 90 mg/kg/day for 21 days), and standard drug, sildenafil (5 mg/kg/day for 7 days). An equal number of female rats were used, and the effect of MEMO and sildenafil was compared with that of vehicle. MEMO significantly relaxed isolated rat corpus cavernosum smooth muscle at 0.1-100 µg/mL in vitro and significantly increased (p < 0.05) sexual activity, intracavernous pressure/mean arterial pressure in normal and diabetic rats. The increase in erectile function of rats by MEMO could be because of its sEH inhibitory activity. Copyright © 2016 John Wiley & Sons, Ltd.

Antioxidant Potential and Ability of Phloroglucinol to Decrease Formation of Advanced Glycation End Products Increase Efficacy of Sildenafil in Diabetes-Induced Sexual Dysfunction of Rats.

INTRODUCTION: Diabetes-induced sexual dysfunction is associated with an increase in oxidative stress. Scavengers of reactive oxygen species (ROS) have been shown to reduce oxidative stress and aid in the management of sexual dysfunction in diabetes. AIM: The aim of the study was to test the hypothesis that antioxidant, which scavenge ROS and reduce formation of advanced glycation end products (AGEs), can potentiate efficacy of phosphodiesterase type 5 inhibitors in diabetes-induced sexual dysfunction that is associated with oxidative stress. MATERIALS AND METHODS: Effect of phloroglucinol and sildenafil on serum glucose level, sexual function, penile smooth muscle : collagen ratio, and phenylephrine precontracted corpus cavernosum smooth muscle (CCSM) was studied. The ability of phloroglucinol to reduce the formation of AGEs and its ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) was also evaluated. MAIN OUTCOME MEASURES: Antioxidant potential of phloroglucinol was studied in addition to its effect on diabetes-induced sexual dysfunction in presence and absence of sildenafil. RESULTS: Phloroglucinol (50 mg/kg, p.o.) significantly decreased serum glucose level and increased sexual function in streptozotocin-induced diabetic rats when compared with diabetic control rats. Sildenafil (5 mg/kg, p.o.) had no effect on glycemia but significantly increased sexual function of diabetic rats. Coadministration of phloroglucinol increased the efficacy of sildenafil by improving sexual function. Treatment of diabetic rats with phloroglucinol + sildenafil maintained smooth muscle : collagen levels similar to that of normal rat penile tissue. Phloroglucinol decreased formation of AGEs and significantly scavenged DPPH radical activity in vitro. Sildenafil relaxed isolated CCSM of normal rat and diabetic rat significantly, but phloroglucinol did not show any significant effect. Phloroglucinol also inhibited human CYP3A4 enzyme activity in vitro. CONCLUSION: Phloroglucinol coadministration increases efficacy of sildenafil in diabetes-induced sexual dysfunction. However, further studies are required to ascertain the benefits of phloroglucinol owing to its undesirable CYP3A4 inhibition activity.

Effect of Cinnamomum cassia methanol extract and sildenafil on arginase and sexual function of young male Wistar rats.

INTRODUCTION: Herbs have been used as an aphrodisiac since ages. Cinnamomum cassia is an important ingredient of many Ayurvedic formulations to treat male sexual disorder including erectile dysfunction (ED). AIM: The objective of the present study was to evaluate erectogenic and aphrodisiac activity of methanol extract of C. cassia bark in young male rats. METHODS: Methanol extract of C. cassia was screened in vitro for arginase inhibition potential and IC50 was determined. Effect of the extract was observed in vitro on phenylephrine pre-contracted isolated rat corpus cavernosum smooth muscle (CCSM) at 0.1, 1, 10, and 100 µg/mL. Young male Wistar rats were dosed with extract at 100 mg/kg body weight for 28 days and its effects on sexual behavior and penile smooth muscle : collagen level were observed. MAIN OUTCOME MEASURE: Effect of C. cassia was studied on arginase activity in vitro and sexual behavior of young male rats. RESULTS: C. cassia inhibited arginase activity in vitro with an IC50 of 61.72 ± 2.20 µg/mL. The extract relaxed phenylephrine pre-contracted isolated rat CCSM up to 43% and significantly increased (P < 0.05) sexual function of young male rats. Treatment with the extract also increased smooth muscle level and decreased collagen level in rat penile tissue. CONCLUSION: The study proves usefulness of methanol extract of C. cassia bark for increasing sexual function.

Erectogenic and Aphrodisiac Effects of Butea frondosa Koenig ex Roxb. in Rats: Involvement of Enzyme Inhibition.

Butea frondosa Koenig ex Roxb. (BF) is traditionally used to manage male sexual disorders including erectile dysfunction (ED). Methanol extract of BF (bark) inhibited Rho-kinase 2 (ROCK-II) enzyme activity in vitro with an IC50 of 20.29 ± 1.83 µ g/mL. The relaxant effect of methanol extract of BF (MEBF) was studied on phenylephrine precontracted corpus cavernosum smooth muscle (CCSM) isolated from young rats. The effect of MEBF treatment on sexual behaviour of both young (5 month) and aged (24 month) rats was also studied in addition to the influence on smooth muscle, collagen (collagen-I and -III) level in penis, and sperm characteristics of young and aged rats. MEBF relaxed CCSM up to 21.77 ± 2.57% and increased sexual behavior of young and aged rats. This increase in sexual function could be attributed to ROCK-II inhibition and increase in ratio of smooth muscle to collagen level in rat penile tissue. Increased sperm production and decreased defective sperms in young and aged rats corroborate the usefulness of Butea frondosa in male infertility in addition to ED.

Efficacy of Cinnamomum cassia Blume. in age induced sexual dysfunction of rats.

OBJECTIVE: Cinnamomum cassia has been suggested in Ayurveda for the management of sexual dysfunction. This research work was conducted to shed some light on the mechanism of action of the extract, and evaluate the efficacy of its methanol extract in age induced sexual dysfunction in male Wistar rats. Secondary objective of the project was to study the effect of treatment on sperm parameters and smooth muscle:collagen level in rat penile tissue. METHODS: Young and aged male rats were treated with methanol extract of Cinnamomum cassia at a dose of 100 mg/kg and sexual behavior was observed on 28th day in presence of female rats in estrous phase. Sildenafil was used as standard medicine. Effect of treatment was studied on epididymal sperm parameters, and Massons trichrome staining of rat penile tissues was performed to know the level of smooth muscle:collagen. RESULTS: The treatment significantly increased sexual function in aged rats that had decreased in comparison to young rats, but did not have any significant effect on sperm count, live and defective sperm percentage. However, treatment induced an increase in smooth muscle level and a decrease in collagen level in the aged rat penile tissue in comparison to that of age matched control. CONCLUSION: Based on our studies, we found that Cinnamomum cassia extract was effective in management of sexual dysfunction in aged rats and hence we propose a possible mechanism of action for Cinnamomum cassia which could be responsible for restoring sexual activity in aged rat.

Screening for Rho-kinase 2 inhibitory potential of Indian medicinal plants used in management of erectile dysfunction.

THE AIM OF THE STUDY: Activation of Rho-kinase 2 (ROCK-II) results in contraction of corpus cavernosum smooth muscle and ROCK-II inhibitors relax corpus cavernosum in vitro and in vivo hence, plant extracts capable of inhibiting ROCK-II enzyme may be useful in management of erectile dysfunction (ED). The aim of the study was to screen selected Indian medicinal plants, having similar ethnopharmacological use for ROCK-II inhibition. MATERIALS AND METHODS: Some Indian medicinal plants reported as aphrodisiacs in Ayurveda and modern scientific literature were collected, authenticated and extracted. Direct methanol and successive aqueous extracts of these plants were screened for ROCK-II inhibitory activity using HTRF(®)KinEASE™ STK S2 Kit (Cisbio Bioassays). Relaxant effect of potent extract was recorded on isolated rat corpus cavernosum. RESULTS: Methanolic and successive aqueous extracts of 30 plants were screened for ROCK-II inhibition and 15 herbal extracts showed inhibition ranging between 50 and 88% at 50 µg/mL. While IC(50) of Y-27632, a standard ROCK-II inhibitor, was found to be 163.8 ± 1.2 nM. The Methanolic extract of Terminalia chebula (METC) with IC(50) value of 6.09 ± 0.17 µg/mL was found to be most potent and relaxed isolated rat corpus cavernosum significantly (p<0.01). Chebulagic and chebulinic acid of METC were found to inhibit ROCK-II and might be responsible for the inhibitory potential of the extract. The traditional use of plants like Butea frondosa, Syzygium aromaticum, Butea superba, Chlorophytum borivilianum and Mucuna pruriens, as aphrodisiacs and for male sexual disorder (MSD) might be in part due to the ROCK II inhibitory potential of these plants. CONCLUSION: Some of the Indian medicinal plants have ROCK-II inhibitory potential and those deserve further investigation.