Elucidation of plasma protein binding, blood partitioning, permeability, CYP phenotyping and CYP inhibition studies of Withanone using validated UPLC method: An active constituent of neuroprotective herb Ashwagandha.

ETHNOPHARMACOLOGICAL RELEVANCE: Withanone (WN), an active constituent of Withania somnifera commonly called Ashwagandha has remarkable pharmacological responses along with neurological activities. However, for a better understanding of the pharmacokinetic and pharmacodynamic behavior of WN, a comprehensive in-vitro ADME (absorption, distribution, metabolism, and excretion) studies are necessary. AIM OF THE STUDY: A precise, accurate, and sensitive reverse-phase ultra-performance liquid chromatographic method of WN was developed and validated in rat plasma for the first time. The developed method was successfully applied to the in-vitro ADME investigation of WN. MATERIAL AND METHODS: The passive permeability of WN was assayed using PAMPA plates and the plasma protein binding (PPB) was performed using the equilibrium dialysis method. Pooled liver microsomes of rat (RLM) and human (HLM) were used for the microsomal stability, CYP phenotyping, and inhibition studies. CYP phenotyping was evaluated using the specific inhibitors. CYP inhibition study was performed using specific probe substrates along with WN or specific inhibitors. RESULTS: WN was found to be stable in the simulated gastric and intestinal environment and has a high passive permeability at pH 4.0 and 7.0 in PAMPA assay. The PPB of WN at 5 and 20 µg/mL concentrations were found to be high i.e. 82.01 ± 1.44 and 88.02 ± 1.15%, respectively. The in vitro half-life of WN in RLM and HLM was found to be 59.63 ± 2.50 and 68.42 ± 2.19 min, respectively. CYP phenotyping results showed that WN was extensively metabolized by CYP 3A4 and1A2 enzymes in RLM and HLM. However, the results of CYP Inhibition studies showed that none of the CYP isoenzymes were potentially inhibited by WN in RLM and HLM. CONCLUSION: The in vitro results of pH-dependent stability, plasma stability, permeability, PPB, blood partitioning, microsomal stability, CYP phenotyping, and CYP inhibition studies demonstrated that WN could be a better phytochemical for neurological disorders.

Pyroglutamylated RFamide peptide 43: A putative modulator of testicular steroidogenesis.

BACKGROUND: We have recently shown that QRFP and its receptor are predominantly expressed in germ cells, Sertoli cells and Leydig cells in mice testes. OBJECTIVE: The present study investigated the role of QRFP in testicular steroidogenesis in mice. MATERIALS AND METHODS: Both ex vivo and in vivo experiments were performed. For ex vivo, testicular tissues were cultured with 0, 10, 100 and 1000 nM QRFP, with or without hCG, for 6, 12 and 24 h, and media were used for testosterone assay. The hCG-stimulated testicular tissues were used for immunoblot of SF1, StAR, CYP11A1, 3ß- and 17ß-HSD. For in vivo, mice received bilateral intratesticular injection of saline or 0.3, 1 and 3nmol QRFP and were killed at 6, 12 and 24 h post-injection. Testosterone in serum was measured at above durations, while qRT-PCR of HMG-CoA synthase 1 and SR-B1 and immunoblot of steroidogenesis-related markers were performed at 24 h post-injection. RESULTS: Testosterone production under basal and hCG-stimulated conditions increased in a time-dependent manner, and QRFP supplementation to testicular culture caused an increase and a decrease in hormone production. The effect of QRFP on testosterone production under hCG-stimulated culture or in vivo conditions at 6 and 24h was similar. At 6h, testosterone production increased at 10 and 100 nM and also at 0.3 and 1nmol QRFP, while it decreased at 1000 nM and 3 nmol doses. At 24 h, testosterone level decreased at lower concentrations (10 nM and 0.3 nmol) and thereafter increased at middle (100nM and 1nmol) and higher (1000 nM and 3 nmol) concentrations under both hCG-stimulated culture and in vivo. DISCUSSION AND CONCLUSION: QRFP induced production of testosterone by modulating steroidogenic machinery at optimal doses and durations. Further, findings of in vivo study indicate that QRFP besides directly regulating testicular steroidogenesis may also have modulated other factors which act together in a holistic manner to control steroidogenesis.

Determination of permeability, plasma protein binding, blood partitioning, pharmacokinetics and tissue distribution of Withanolide A in rats: A neuroprotective steroidal lactone.

Preclinical Research & Development Withanolide A (WA), a steroidal lactone is a major bioactive constituent of Withania somnifera (L.) with remarkable neuropharmacological activity. In this study, we investigated the permeability, plasma protein binding (PPB), blood partitioning, intravenous (i.v.), and oral pharmacokinetics as well as i.v. tissue distribution (TD) of pure WA in a rat model. The PPB, RBCs partitioning, and permeability of WA were determined by Ultra Performance Liquid Chromatography (UPLC) method. However, the pharmacokinetics and TD of WA were evaluated by validated and sensitive liquid chromatography coupled mass spectrometry (LC-ESI-MS/MS) method. The PPB and permeability of WA were determined by equilibrium dialysis and parallel artificial membrane permeability assay method, respectively. The results demonstrated that WA has high PPB and passive permeability. Furthermore, WA was found to have fast equilibration between RBCs and plasma. Following i.v. (2 mg/kg) and per-oral (25 mg/kg) administration of WA, the max concentration (Cmax ) in plasma was found as 85.53 ± 6.54 and 48.04 ±5.78 ng/mL, respectively. The TD study results indicated that WA has a rapid and wide TD. The maximum concentration in various tissues was found in following order: Clung > Cliver > Ckidney ≈ Cspleen > Cheart > Cbrain . The preclinical in vitro, as well as pharmacokinetics and TD results, are anticipated to support the future preclinical and clinical application of WA.

Simultaneous quantification of proposed anti-malarial combination comprising of lumefantrine and CDRI 97-78 in rat plasma using the HPLC-ESI-MS/MS method: application to drug interaction study.

BACKGROUND: Lumefantrine is the mainstay of anti-malarial combination therapy in most endemic countries presently. However, it cannot be used alone owing to its long onset time of action. CDRI 97-78 is a promising trioxane-derivative anti-malarial candidate that is currently being investigated as a substitute for artemisinin derivatives owing to their emerging resistance. METHODS: In the present study, a sensitive, simple and rapid high-performance liquid chromatography coupled with positive ion electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the simultaneous determination of lumefantrine and CDRI 97-78's metabolite, 97-63, in rat plasma using halofantrine as an internal standard. Lumefantrine and 97-63 were separated on a Waters Atlantis C18 (4.6×50 mm, 5.0 µm) column under isocratic condition with mobile phase consisting of acetonitrile: methanol (50:50, v/v) and ammonium formate buffer (10 mM, pH 4.5) in the ratio of 95:5 (v/v) at a flow rate of 0.65 mL/min. RESULTS: The method was accurate and precise within the linearity range 3.9-500 ng/mL for both lumefantrine and 97-63 with a correlation coefficient (r2) of ≥0.998. The intra- and inter-day assay precision ranged from 2.24 to 7.14% and 3.97 to 5.90%, and intra- and inter-day assay accuracy was between 94.93 and 109.51% and 96.87 and 108.38%, respectively, for both the analytes. Upon coadministration of 97-78, the relative bioavailability of lumefantrine significantly decreased to 64.41%. CONCLUSIONS: A highly sensitive, specific and reproducible high-throughput LC-ESI-MS/MS assay was developed and validated to quantify lumefantrine and CDRI 97-78. The method was successfully applied to study the effect of oral co-administration of lumefantrine on the pharmacokinetics of 97-78 in male Sprague-Dawley rats and vice versa. Co-administration of 97-78 significantly decreased the systemic exposure of lumefantrine.

Flavone-based novel antidiabetic and antidyslipidemic agents.

The hybrid congeners 62-90 of 6- and 7-hydroxyflavones with aminopropanol have been synthesized and evaluated for their antidiabetic activity in sucrose-challenged low-dosed streptozotocin (STZ)-induced diabetic rats and db/db mice. The optical enantiomers 70a, 70b, 90a, and 90b of two congeners 70 and 90 exhibiting consistent antidiabetic and antidyslipidemic activities were also prepared, and their antidiabetic activity results indicate its association mainly with S isomers. These compounds also lower cholesterol and TG profiles while improving high-density lipoprotein cholesterol to CHOL ratio in db/db mice. The bioavailability of compound 70 and its isomer varies between 27 and 29% whereas that of the more polar compound 90a is poor as determined in rat by oral and intraperitoneal administrations.

Pharmacokinetics, in-situ absorption and protein binding studies of a new neuroleptic agent centbutindole in rats.

The present study reports the absorption kinetics, plasma protein binding and pharmacokinetic profile of the centbutindole (I) after i.v. and oral dosing in rats. In addition, an in-situ absorption study was carried out using a closed-loop technique at pH 2.6 and 7.4. The rate of absorption at pH 2.6 was 5-fold less compared to that observed at pH 7.4. In-vitro and in-vivo protein binding (ultra filtration technique) was independent of substrate concentration over a range of 1.25-10.0 microg/ml. Pharmacokinetic parameters of I were determined in male rats after administering a single 4 mg/kg oral dose and 2 mg/kg intravenous dose. The peak serum concentration of I was found to be 50.1 ng/ml at 30 min after oral administration followed by a secondary Cmax of 43.2 ng/ml at 180 min. For the hydroxy metabolite (II), a Cmax of 6.4 ng/ml was measured at 360 min after oral administration of I. After oral dosing an irregular concentration-time profile with secondary peaks was observed for both I and II. The terminal half-lives for I and II after oral dosing were 163 and 263 min, respectively. After intravenous dosing, the levels of I decreased biexponentially with a distribution (t(1/2) alpha) and elimination (t(1/2) beta) half-lives of 5.7 and 128 min, respectively. Comparison of the AUC after oral and intravenous dosing of I indicates that only about 24% of the oral dose reaches the systemic circulation. The limited bioavailability can either be due to the poor solubility of the compound and/or extensive first pass metabolism in the gastrointestinal (GI) tract. Co-administration of polyethylene glycol (PEG) at oral dosing improves solubilization and increases bioavailability.

Antispermatogenic and antifertility effects of 20,25-diazacholesterol dihydrochloride in mice.

The effect of intraperitoneal administration for 28 days of 10, 20, and 30 mg/kg body weight per day of 20,25-diazacholesterol dihydrochloride (SC 12937), a hypocholesterolemic agent, on the testis of Parkes (P) strain mice was investigated. Histologically, testes in mice treated with 10 or 20 mg/kg body weight of SC 12937 showed non-uniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; the affected tubules showed intraepithelial vacuolation, occurrence of giant cells, exfoliation of germ cells, and marginal condensation of chromatin in round spermatids. In both dosage groups, only 11-12% of the seminiferous tubules were affected, and no significant differences were found in the frequency of affected tubules between the two groups. By contrast, testes in mice treated with 30 mg/kg body weight of the drug exhibited a degenerated appearance of germ cells in all seminiferous tubules. The treatment also had adverse effects on motility, viability, morphology, and number of spermatozoa in the cauda epididymidis, and on fertility. Even 56 days after drug withdrawal, the above parameters remained markedly affected. Our results thus suggest that SC 12937 treatment causes antispermatogenic and antifertility effects in P mice and that the effects are not reversible up to 56 days after drug withdrawal. This compound may prove useful in the control of rodent populations.

Isolation and characterization of metabolites of centpropazine in rat liver, intestine, and red blood cell homogenates.

The potential sites for metabolism of centpropazine (CPZ) (an antidepressant) were evaluated in male Sprague-Dawley rats. The isolation and identification of the major metabolites formed in the presence of rat liver S9 fraction, intestine, and red blood cells under aerobic conditions were performed using high-performance liquid chromatography and electrospray ionization mass spectrometry. CPZ was found to be extensively metabolized to seven possible metabolites by liver S9 fraction in the presence of a nicotinamide adenine dinucleotide phosphate generating system at 37 degrees C. Both intestinal wall and red blood cells were also found to metabolize the compound. This metabolite structure was confirmed by comparison with that of its synthetic standard. The drug was stable in intestinal contents. On the basis of our finding, we propose the in vitro metabolic pathways for CPZ.

Primary antiphospholipid syndrome presenting with cerebral ischemia, thrombocytopenia, anemia and proteinuria successfully treated with warfarin potassium.

A 30-year-old woman with primary antiphospholipid syndrome (PAPS) presented with cerebral ischemia, thrombocytopenia, anemia and proteinuria. Administration of warfarin potassium, without concomitant corticosteroid administration, significantly improved all of these symptoms along with a decrease in the titers of antiCL-beta2-GP-I antibodies and a shortening of prolonged APTT. Therefore, the antiphospholipid antibodies in this patient could have been evoked by vitamin-K-dependent coagulation factors or plasma proteins which are assumed to undergo conformational changes exposing cryptic epitopes. This case report provides clues to the mechanisms underlying the production of antiphospholipid antibodies in patients with PAPS.

Purification and characterization of a paralytic polypeptide from larvae of Myrmeleon bore.

A toxic substance was purified from larvae of the antilion, Myrmeleon bore, by DEAE Sephacel and Phenyl Superose column chromatography. The substance was a large polypeptide with a molecular weight of about 165-167 kDa. Its paralytic activity measured by injection against German cockroaches was about 130 times higher than that of tetrodotoxin on a molar basis.

[A case of pulmonary paraganglioma].

A pulmonary paraganglioma that was discovered incidentally as a solitary coin lesion on abdominal CT was successfully resected. Though this type of paraganglioma is extremely rare, it needs to be differentiated from certain other pulmonary neoplasms.

A case of postnephrectomy arteriovenous fistula.

A rare case of a postnephrectomy arteriovenous fistula involving the major renal vessels is presented herein. This type of postnephrectomy arteriovenous fistula is very unusual; in fact, since first documented in 1934, only 65 cases have been reported in the world literature, including 2 cases from Japan. Even though postoperative arteriovenous fistulae are rare, one should bear this complication in mind when unknown causes of bruit or heart failure develop following surgery.

[Concentrations of cefotaxime in serum and myocardial tissue].

Serum and myocardial concentrations of cefotaxime (CTX) were measured in 20 adult patients undergoing cardiac surgery. To all of these patients 1.0 g of CTX was given intravenously (in the range of 13.7--29.0 mg/kg) at the beginning of operation. The serum concentrations of CTX were determined at 5, 10, 30, 60 minutes and 120 minutes after administration. Myocardial concentrations of CTX were also determined at about 30 minutes (group I), 60 minutes (group II) and 120 minutes (group III). The following results were obtained. Average serum CTX concentrations were 132.8 +/- 34.1 micrograms/ml at 5 minutes, 92.0 +/- 23.1 micrograms/ml at 10 minutes, 44.6 +/- 12.3 micrograms/ml at 30 minutes, 24.5 +/- 7.7 micrograms/ml at 60 minutes and 12.3 +/- 4.9 micrograms/ml at 120 minutes after administration. Average myocardial CTX concentrations were 10.0 +/- 3.7 micrograms/g in group I, 3.6 +/- 2.1 micrograms/g in group II and 2.3 +/- 1.8 micrograms/g in group III. The myocardial/serum concentration ratio was 0.22 +/- 0.14 in group I, 0.15 +/- 0.08 in group II and 0.18 +/- 0.12 in group III, respectively. These results suggested that the serum and myocardial concentrations of CTX were high enough to be prophylactic and therapeutic against not only aerobic but also anaerobic and opportunistic infections during and after cardiac surgery.