Protective effects of Croton hookeri on streptozotocin-induced diabetic nephropathy.

In this study, the protective effects of Croton hookeri (CH) extract on renal injury were investigated in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced by a single injection of STZ (45 mg/kg) to Sprague-Dawley rats. After 5 days, CH extract (200 mg/kg) was administered daily by oral gavage for 2 weeks. Administration of CH extracts significantly reduced blood glucose levels in STZ-induced diabetic rats. STZ-induced changes in total cholesterol, LDL, HDL, ALT, AST, BUN, and serum creatinine levels were significantly restored by treatment with CH extract. Abnormal levels of SOD, catalase, glutathione, and oxidized GSH (GSSG) in STZ-treated rats were also significantly recovered by CH extract treatment. CH extract markedly reduced the expression of collagen-1, fibronectin, and α-SMA in the kidney of STZ-induced diabetic rats. In particular, oxidative DNA damages, MDA, TGF-ß, IL-1ß, and IL-6 levels were significantly reduced in STZ-treated rats following treatment with CH extract, whereas IL-10 showed opposite trend. STZ-induced SIRT1, SIRT3 downregulation and cloudin-1 upregulation in the kidney were dramatically recovered by CH extract treatment. Our data suggest that CH extract protects against diabetic-induced nephropathy by inhibiting oxidative stress and inflammation. Therefore, it has potential as a food supplement to alleviate renal dysfunction caused by diabetes-induced nephropathy.

Application of statistical design to evaluate critical process parameters and optimize formulation technique of polymeric nanoparticles.

In advanced medication, drug-loaded polymeric nanoparticles (NPs) appeared as a novel drug delivery system with lots of advantages over conventional medicines. Despite all the advantages, NPs do not gain popularity for manufacturing hurdles. The study focused on the formulation difficulties and implementation of statistical design to establish an effective model for manufacturing NPs. In this study, physico-chemical properties of the drug and polymer (PLGA) were incorporated to understand the mechanistic insights of nanoformulations. Primarily, the process controlling parameters were screened by Plackett-Burman design and the critical process parameters (Cpp) were further fabricated by Box-Behnken design (BBD). The TLM-PLGA-NPs (telmisartan loaded PLGA NPs) exhibited particle size, encapsulation efficiency and zeta potential of 232.4 nm, 79.21% and -9.92 mV respectively. The NPs represented drug loading of 76.31%. Korsmeyer-Peppas model (R 2 = 0.925) appeared to be the best fitted model for in vitro release kinetics of NPs. The model identified Fickian diffusion of TLM from the polymeric nanoparticles. The ANOVA results of variables indicate that BBD is a suitable model for the development of polymeric NPs. The study successfully identified and evaluated the correlation of significant parameters that were directly or indirectly influencing the formulations which deliberately produce desired nanoparticles with the help of statistical design.

Simultaneous determination of paclitaxel and lansoprazole in rat plasma by LC-MS/MS method and its application to a preclinical pharmacokinetic study of investigational PTX-LAN-PLGA nanoformulation.

In spite of having a remarkable anti tumor activity against a wide variety of cancers, the clinical effectiveness of the major chemotherapeutic drug paclitaxel is often limited by the issues of drug resistance that hampers the therapeutic effectiveness of the drug. The combination of proton pump inhibitor with paclitaxel is an effective approach to overcome therapeutic resistance caused by the acidic microenvironment (Warburg effect) in tumor. In the present study a new simple, precise and selective liquid chromatography tandem mass spectrometry method was developed for quantification of paclitaxel and lansoprazole using esomeprazole as an internal standard and applied for the pharmacokinetic study of investigational paclitaxel - lansoprazole loaded PLGA nanoparticles. The developed method quantifies both the drugs simultaneously irrespective of their dissimilar stability concerns. The detection was exercised with multiple reaction-monitoring mode in positive ionization that yielded highly intense response of parent-product (m/z) transition pair 854.4 → 286.1, 370.1 → 251.9 and 346 → 198 for paclitaxel, lansoprazole and Esomeprazole respectively. The chromatographic separation was achieved using phenomenex Kinetex 5 µ C18 100A 50 × 3.0 mm column and a gradient mobile phase combination of ammonium acetate in deionized water (pH 6.8, 2 mM, w/v) and acetonitrile spiked with formic acid (1:1000, v/v ). This method showed good linearity over a concentration range of 10-320 ng/mL and 100-3200 ng/mL with correlation coefficient (R2) 0.98 and 0.94 for paclitaxel and lansoprazole respectively. Using liquid liquid extraction process both the drugs were extracted from rat plasma. The intra- and inter-day precision and accuracy values were within the variability limits and both the analytes were found to be stable throughout the freeze-thaw, auto-sampler, bench top and long term stability studies. The liquid chromatography tandem mass spectrometry method was successfully validated in accordance with United States Food and Drug administration guidelines and the results were within the acceptable limits. The liquid chromatography tandem mass spectrometry method was successfully utilized for the pharmacokinetic investigation of experimental paclitaxel - lansoprazole loaded PLGA nanoparticles in rat plasma.

LC-MS/MS determination of 4-hydroxynimesulide, an active metabolite of nimesulide and application to bioequivalence study in Indian subjects.

A simple and highly sensitive bioanalytical method was developed and validated for simultaneous quantification of nimesulide (NSD) and its active metabolite 4-hydroxy-nimesulide (M1) in human plasma by liquid chromatography-tandem mass spectrometer (LC-MS/MS) and applied in a bioequivalence study performed on Indian subjects. The bioanalytical method was carried out by LC-MS/MS with celecoxib (CXB) as an internal standard (IS) using liquid-liquid extraction technique. The chromatographic separation was performed on a reversed-phase Agilent eclipse plus C18 (75 mm × 4.6 mm, particle size 3.5 µm) column with a mobile phase of acetronitrile and water containing 5 mM ammonium formate (9:1, v/v). Method validation and clinical sample were analysed as per USFDA and EMA guidelines and results met the acceptance criteria. The lower limit of quantitation of NSD and M1 was found 10 ng/mL with a large linearity range from 10 to 6000 ng/mL for both NSD and M1 using only 100 µL of plasma and reported no matrix effect. The multiple reaction monitoring transitions of m/z 307.20 → 229.20, m/z 323.00 → 245.00 and m/z 380.20 → 316.20 were used to measure NSD, M1 and CXB (IS), respectively. The assay method was successfully applied for the simultaneous quantification of both NSD and M1 in plasma samples after oral administration of nimesulide 100 mg tablet in healthy human subjects.

A chemotherapeutic approach targeting the acidic tumor microenvironment: combination of a proton pump inhibitor and paclitaxel for statistically optimized nanotherapeutics.

Paclitaxel (PTX) is a major chemotherapeutic drug that is effective against a wide variety of cancers, particularly breast, ovarian and lung cancer. For a weakly basic chemotherapeutic drug such as PTX, the development of the acidic tumor microenvironment (Warburg effect) has a remarkable impact on therapeutic resistance. The present approach takes advantage of the acidic tumor microenvironment by incorporating lansoprazole (LAN), a proton pump inhibitor (PPI), with PTX as a potent therapeutic combination that is capable of reversing PTX resistance. To deliver optimal amounts of the drugs to neoplastic cells, a nano drug delivery system was selected. To design the nanoformulation process in a limited framework, typical formulation parameters were optimized and validated by the application of response surface methodology (RSM) using Box-Behnken design (BBD). On the basis of critical quality aspects, the experimental design helped to determine the optimal particle size (243.7 nm), zeta potential (-9.14 mV) and encapsulation efficiencies (88.91% and 80.35% for PTX and LAN respectively). The optimized formulation (PTX-LAN-PLGA-NPs) exhibited sustained in vitro release profiles over 384 hours for both the encapsulated drugs. The Korsmeyer-Peppas model was found to be the best fitted model for the release kinetics, where the release mechanism follows Fickian diffusion. In in vitro anti-tumor efficacy experiments using Michigan Cancer Foundation-7 (MCF-7) breast cancer cells, the PTX-LAN-PLGA-NPs exhibited a steep decrease in cell viability compared to the pure drugs. Taken together, the results strongly support that incorporation of PTX and LAN in nanoparticles (NPs) is a promising approach for cancer chemotherapy.

LC-MS/MS assay for quantitation of enalapril and enalaprilat in plasma for bioequivalence study in Indian subjects.

BACKGROUND: Enalapril (EPL) is an angiotensin-converting enzyme inhibitor for the treatment of hypertension and chronic heart failure. Enalaprilat (EPLT) is an active metabolite that contributes to the overall activity of EPL. AIM: To quantitate EPL along with its metabolite EPLT using LC-MS/MS, a bioanalytical method was developed and validated with tolbutamide in human plasma using a protein precipitation technique. RESULTS: The sensitive and selective method has an LLOQ of 1 ng/ml with a linearity range of 1-500 ng/ml for both EPL and EPLT using 300 µl of plasma without any matrix effect. CONCLUSION: Linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples after oral administration of 20 mg of EPL maleate in healthy volunteers demonstrate applicability to bioavailability/bioequivalence studies.

Development and Validation of an LC-MS/MS-ESI Method for Comparative Pharmacokinetic Study of Ciprofloxacin in Healthy Male Subjects.

A sensitive, specific and reproducible liquid chromatography coupled to tandem mass spectrometric method was developed and validated for the estimation of ciprofloxacin, an extensively used second-generation quinolone antibiotics, in human plasma. A liquid-liquid extraction of ciprofloxacin and the internal standard, ofloxacin, has been approached from the biological matrix using chloroform. Chromatographic separation was achieved in positive ion modes, isocratically on a 3.5 µm C18 analytical column (75 mm×4.6 mm, i.d.) with 0.2% formic acid solution in water: methanol (10:90, v/v) as mobile phase, at a flow rate of 0.5 mL.min-1. The MS/MS ion transitions were monitored as 332.0→231.3 for ciprofloxacin and 362.2→261.0 for IS. The method showed good linearity in the range of 0.01-5.00 µg.mL-1 (r2 >0.99) with a good precision (3.37-12.60%) and accuracy (87.25-114%). At the same time, ciprofloxacin was found to be stable during stability studies viz. bench-top, auto-sampler, freeze-thaw cycle and long-term. The developed and validated method was successfully applied to measure plasma ciprofloxacin concentrations in a single dose bioequivalence study.

A rapid LC-ESI-MS/MS method for the quantitation of choline, an active metabolite of citicoline: Application to in vivo pharmacokinetic and bioequivalence study in Indian healthy male volunteers.

A rapid, simple, and sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-ESI-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of choline (CL), active metabolite of citicoline in human plasma using metformin (MF) as IS. The chromatographic separation was performed on a reversed-phase Phenomenx Gemini C18 column with a mobile phase of methanol:water (containing 10mM ammonium formate) (9:1, v/v). The calibration curves were linear over the range of 0.05-5µg/ml. The validated LC-ESI-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters and bioequivalence study of test and reference control release (CR) tablet preparation of citicoline 1000mg after a single oral administration to all 12 healthy male volunteers.

Putative virulence traits and pathogenicity of Vibrio cholerae Non-O1, Non-O139 isolates from surface waters in Kolkata, India.

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (>/=100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.