Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride.

Besifloxacin is a unique chiral broad-spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R-form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S-isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high-performance liquid chromatography (HPLC) method for determination of R-besifloxacin and S-besifloxacin (BES impurity A) was developed and validated for in-process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD-H (250 x 4.6 mm, 5 µm) column using n-heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S-isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal-to-noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 µg/mL, while the LOQ was 0.90 µg/mL. The calibration curves were linear in the range of 0.9-7.5 µg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628-632, 2016. © 2016 Wiley Periodicals, Inc.

Effect of polydimethylsiloxane and ethylcellulose on in vitro permeation of centchroman from its transdermal patches.

This research aims at the development of controlled release contraceptive transdermal patches of centchroman using ethylcellulose (EC) as film-forming polymer, polydimethylsiloxane (PDMS) as pressure sensitive adhesive with propylene glycol and Di-n-butyl-phthalate for their penetration enhancer and plasticizing properties, respectively. The physicochemical compatibility of the drug and the polymers was performed by differential scanning calorimetry and Fourier transform infrared (FTIR) spectroscopic technique. Effects of EC and PDMS ratios on moisture uptake, moisture content, tensile strength (TS), Young's modulus, adhesive strength, water vapor transmission rate (WVTR) and in vitro permeation of centchroman through Sprague-Dawley rats abdominal skin using Franz's diffusion cell were evaluated. A 3(2) full factorial design was employed to observe the effect of independent variables; concentration of ethyl cellulose and PDMS on drug permeated after 32 h, which was selected as dependent variable. Compatibility studies suggested that there were no significant interaction between the drug and polymers used. It was found that incorporation of only EC resulted in too hard patches and addition of PDMS produced patches with lower TS, increased percentage elongation, WVTR and Young's modulus. Statistical analyses suggested that independent variables have a significant effect on the dependent variable. All formulation follows zero-order release kinetics with r(2) > 0.990. In conclusion, drug in adhesive transdermal patches can be successfully fabricated for non-steroidal contraceptive centchroman to obtain a zero-order release systems.

Neuro-protective potential of a vesicular system of a standardized extract of a new chemotype of Withania somnifera Dunal (NMITLI118RT+) against cerebral stroke in rats.

Withania somnifera Dunal is an Indian medicinal plant with significant pharmacological properties, such as adaptogenic, anti-inflammatory, anti-oxidant, anti-platelet, anti-hypertensive, hypoglycemic and hypolipidemic effects. Several chemotypes of W. somnifera include NMITLI-101, NMITLI-118 and NMITLI-128. The present work elaborates the optimization and development of a liposomal delivery system for efficient delivery of NMITLI118RT+ [a standardized ethanolic extract of a new chemotype of W. somnifera Dunal (NMITLI-118) roots] against cerebral stroke in rats. Liposomal systems were prepared using thin-film hydration method and characterized on the basis of size, zeta potential, physical stability, FT-IR, DSC-TGA analysis and surface morphological studies by TEM. NMITLI118RT+ and its formulations (NMITLI118RT+LF) were evaluated for biological activity utilizing middle cerebral artery occlusion model in rats. The Z average of the developed liposomal formulation was about 142.6 ± 0.09 nm with a zeta potential of -31.20 ± 1.0 mV. Results of TEM revealed spherical particles in the range of 200 nm. The entrapment efficiency was found to be 94.603 ± 2%. The formulation was found to be physically stable over a 3-week period. Results were suggestive of the fact that both NMITLI118RT+ and its delivery system possess significant neuroprotective activity in cerebral ischemia. The liposomal system largely exhibits better performance over NMITLI118RT+ precisely in the post-treatment group. The present studies could elucidate the successful development of a delivery system for NMITLI118RT+ and demonstrate their beneficial neuro-protective potential in overcoming and reversing the consequences of I/R injury following stroke.

Validation of RP-HPLC Method for Simultaneous Quantification of Bicalutamide and Hesperetin in Polycaprolactone-Bicalutamide-Hesperetin-Chitosan Nanoparticles.

Bicalutamide is a non-steroidal anti-androgen drug used for the treatment of androgen-dependent prostate cancer. Hesperetin is a natural bioflavonoid that can be used in combination with bicalutamide to improve efficacy and decrease tolerance. The aim of the present work was to develop and validate a simple, sensitive, rapid reverse phase-high performance liquid chromatographic method for simultaneous estimation of bicalutamide and hesperetin. The validation parameters such as specificity, linearity, precision and accuracy, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. Chromatographic separation was achieved on Lichrocart(®) CN column (250 × 4 mm, 5 µm, MERCK) with isocratic elution. The retention times and detection wavelength, for hesperetin and bicalutamide were 4.28 min, 288 nm and 5.90 min, 270 nm respectively. The intra-day and inter-day assay precision and accuracy were found to be <2% over linearity of 50-2000 ng/mL with R(2) 0.999. LOD and LOQ, of bicalutamide and hesperetin was 14.70, 44.57 ng/mL and 16.11, 48.84 ng/mL, respectively. The method was successfully applied for encapsulation efficiency and drug release studies from bicalutamide and hesperetin loaded nanoparticles.

Arteether nanoemulsion for enhanced efficacy against Plasmodium yoelii nigeriensis malaria: an approach by enhanced bioavailability.

The present work is focused on the preparation of nanoemulsions (NEs) loaded with arteether (ART) for its enhanced efficacy against malaria parasites. ART-NEs have been prepared using high pressure homogenization (HPH) technique with the aim of improving its solubility and thus its bioavailability. ART-NEs were optimized in terms of pressure and number of cycles. Globule size and size distributions were chosen as quality parameters. The maximum drug loading was achieved up to 93 ± 7.4% with globule size 156 ± 10.2 nm and zeta potential of -23.3 ± 3.4 mV. The developed ART-NEs were found to be stable in terms of globule size and size distribution at different pH. The in vitro release profile of the ART-NEs showed 62% drug release within 12h. The percentage cell viability of blank NEs were within acceptable limits. A sensitive assay method for the determination of ART in rat plasma by liquid chromatography-mass spectrometry (LC-MS) was employed after oral administration of ART-NEs. The pharmacokinetic study showed significantly enhanced bioavailability of ART in ART-NE-V. The area under curve (AUC) of ART-NE-V was AUC0-t 1988.411 ± 119.66 h ng/ml which was significantly higher (p<0.05) than ART in ground nut oil (GNO) AUC0-t 671.852 ± 187.05 h ng/ml. The Cmax of ART-NE-V (1506 ± 161.22 ng/ml) was also significantly higher (p<0.05) than ART in GNO (175.2 ± 16.54 ng/ml) and ART given intramuscularly (IM) (278.05 ± 38.59 ng/ml). The ART-NE-V was having significantly high antimalarial efficacy and survival rate of mice giving 80% cure rate at 12.5 mg/kg for 5 days in comparison to 30% cure rate of ART in GNO at the same daily dose and it was also comparable to the 100% cure rate at 12.5 mg/kg for 5 days for ART given intramuscularly. In conclusion ART-NE can be a promising oral delivery system for ART.

Stability indicating studies on NMITLI 118RT+ (standardized extract of withania somnifera dunal).

BACKGROUND: Withania somnifera Dunal (Ashwagandha) is an Indian medicinal plant of great medicinal value; used in many clinically proven conditions. NMITLI-118RT+ is a candidate drug under a Council of Scientific and Industrial Research (CSIR) networking project. It is a chemotype of W. somnifera's root extract, which has been used for the present study. OBJECTIVES: The present investigation aims to develop and validate a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) system for the detection and estimation of Withanolide A (marker compound) and its analytical application for stability indicating studies on NMITLI-118RT+. MATERIAL AND METHODS: A validated RP-HPLC method for Withanolide A was established on a Waters HPLC system and the same was used on NMITLI-118RT+ for quantification and fingerprinting purposes, and for establishing forced degradation, isothermal stress tests, and drug-excipient testing protocols as per International Conference on Harmonization (ICH) guidelines. RESULTS: A validated method was established, which could detect the marker at a retention time of around 6.3 minutes, with a linearity range of 2-100 µg/mL, by varying the amounts of the said marker, which were estimated in four different batches of NMITLI-118RT+. Photostability as per ICH guidelines suggested a slight loss of the active constituent and maximum degradation was afforded with alkali followed by acid, and then peroxide, in the forced degradation studies. In the drug-excipient studies, the maximum amount of active constituent could be detected in the samples with ethyl cellulose and the least with hydroxy propyl cellulose. CONCLUSION: The method developed here was simple and rapid. The various stability indicating studies carried out in the present investigation would be useful for formulation development and were suggestive of deciding the recommended storage conditions for NMITLI-118RT+.

Design and synthesis of γ-butyrolactone derivatives as potential spermicidal agents.

A series of γ-butyrolactone derivatives has been designed and synthesized from commercially available 2-acetyl butyrolactone (3-acetyldihydrofuran-2(3H)-one, 1) by aminoalkylating its active methylene followed by condensation with different aldehydes. Compounds having amino group were further converted to their respective tartrate salts and were evaluated for spermicidal activity against human sperm in vitro. Compounds showing appreciable spermicidal activity at ⩽0.5% [3c, 4d (0.5%); 2c, 3d (0.1%); 2d, 4c (0.05%)] were tested for safety studies against human cervical (HeLa) cell line. These compounds were found safer than, Nonoxynol-9. One of the two most active compounds was also found to be the safest (IC50=961 µg/ml; 4c), while the second compound exhibited lower safety against HeLa (IC50=269 µg/ml; 2d). The compound 4c significantly reduced the number of free thiols on human sperm. All the compounds were inactive against Trichomonas vaginalis.

Pharmacokinetics study of arteether loaded solid lipid nanoparticles: an improved oral bioavailability in rats.

Arteether (ART), an artemisinin derivative, is a life saving drug for multiple drug resistant malaria. It has a deliverance effect in Falciparum malaria and cerebral malaria. We have prepared solid lipid nanoparticles (SLN) by high pressure homogenization (HPH) technique. ART-loaded SLN (ART-SLN) has been produced reproducibly with homogeneous particle size. ART-SLN was characterized for their size measured by Zetasizer Nano-ZS, Malvern, UK and by high resolution transmission electron microscopy (HR-TEM) and which was found to be 100 ± 11.2 nm. The maximum percentage entrapment efficiency (%EE) determined with the high-performance liquid chromatography (HPLC) has been found to be 69 ± 4.2% in ART-SLN-3. The release pattern from ART-SLN revealed that the release of ART is slow but time-dependent manner, which is desirable as it will help to protect the acid degradation of ART in stomach. The percentage cytotoxicity of blank SLN has been found within the acceptable range. The pharmacokinetics results indicated that ART-SLN-3 absorption has been significantly enhanced in comparison to ART in aqueous suspension and ART in ground nut oil (GNO) in rats. The % relative bioavailability (RB%) of ART-SLN to the ART in GNO and ART in aqueous suspension in rats was 169.99% and 7461%, respectively which was found to be significantly high in both the cases. From the results, it can be concluded that ART-SLN offers a new approach to improve the oral bioavailability of ART.

Ameliorative effect of Noni fruit extract on streptozotocin-induced memory impairment in mice.

This study evaluated the effects of a standardized ethyl acetate extract of Morinda citrifolia L. (Noni) fruit on impairment of memory, brain energy metabolism, and cholinergic function in intracerebral streptozotocin (STZ)-treated mice. STZ (0.5 mg/kg) was administered twice at an interval of 48 h. Noni (50 and 100 mg/kg, postoperatively) was administered for 21 days following STZ administration. Memory function was evaluated using Morris Water Maze and passive avoidance tests, and brain levels of cholinergic function, oxidative stress, energy metabolism, and brain-derived neurotrophic factor (BDNF) were estimated. STZ caused memory impairment in Morris Water Maze and passive avoidance tests along with reduced brain levels of ATP, BDNF, and acetylcholine and increased acetylcholinesterase activity and oxidative stress. Treatment with Noni extract (100 mg/kg) prevented the STZ-induced memory impairment in both behavioral tests along with reduced oxidative stress and acetylcholinesterase activity, and increased brain levels of BDNF, acetylcholine, and ATP level. The study shows the beneficial effects of Noni fruit against STZ-induced memory impairment, which may be attributed to improved brain energy metabolism, cholinergic neurotransmission, BDNF, and antioxidative action.

Assay method for quality control and stability studies of a new antimalarial agent (CDRI 99/411).

CDRI compound no. 99/411 is a potent 1,2,4-trioxane antimalarial candidate drug under development at our Institute. An HPLC method for determination of CDRI 99/411 with its starting material and intermediates has been developed and validated for in process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and lower limit of quantification (LLOQ) were determined according to International Conference on Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on a RP-select B Lichrosphere® column (250 mm×4 mm, 5 µm, Merck) using water containing 0.1% glacial acetic acid and acetonitrile as the mobile phase in a gradient elution. The eluents were monitored by a photo diode array detector at 245 and 275 nm. Based on signal to noise ratio of 3 and 10 the LOD of CDRI 99/411 was 0.55 µg/mL, while the LLOQ was 1.05 µg/mL. The calibration curves were linear in the range of 1.05-68 µg/mL. Precision of the method was determined by inter- and intra-assay variations within the acceptable range.

Assay method for quality control and stability studies of a new antimalarial agent (CDRI 99/411)$ / 药物分析学报

CDRI compound no. 99/411 is a potent 1,2,4-trioxane antimalarial candidate drug under development at our Institute. An HPLC method for determination of CDRI 99/411 with its starting material and intermediates has been developed and validated for in process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and lower limit of quantification (LLOQ) were determined according to International Conference on Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on a RP-select B Lichrospheres column (250 mm ? 4 mm, 5μm, Merck) using water containing 0.1%glacial acetic acid and acetonitrile as the mobile phase in a gradient elution. The eluents were monitored by a photo diode array detector at 245 and 275 nm. Based on signal to noise ratio of 3 and 10 the LOD of CDRI 99/411 was 0.55 mg/mL, while the LLOQ was 1.05 mg/mL. The calibration curves were linear in the range of 1.05-68 mg/mL. Precision of the method was determined by inter- and intra-assay variations within the acceptable range.

Exploiting 4-sulphate N-acetyl galactosamine decorated gelatin nanoparticles for effective targeting to professional phagocytes in vitro and in vivo.

The present study was focused on the development of surface modified gelatin nanoparticles (SGNPs) using novel ligand 4-sulfated N-acetyl galactosamine (4-SO(4)GalNAc) for specific targeting to macrophages. The gelatin has been modified with the potential targeting moiety 4-SO(4)GalNAc, which was further used for the preparation of modified nanoparticles. The nanoparticles have been prepared by two step desolvation method. The SGNPs and unmodified gelatin nanoparticles (GNPs) were loaded with doxorubicin (DxR) and its targeting potential was compared. Developed DxR-loaded SGNPs (DxR-SGNPs) were found to have negative zeta potential (-19.8 ± 0.22 mV) whereas DxR-loaded GNPs (DxR-GNPs) have the positive zeta potential of around +12.2 ± 0.36 mV. The mean particle size of DxR-SGNPs and DxR-GNPs was found to be 283 ± 7 and 134 ± 5 nm, respectively. Flow cytometric data confirmed the enhanced uptake of DxR-SGNPs in J774A.1 and PBMC when compared with DxR-GNPs. Intracellular localization studies indicate that the fluorescence intensity of DxR-SGNPs was significantly higher when compared to DxR-GNPs. DxR-SGNPs rendered significantly higher localization of DxR in liver and spleen as compared to DxR-GNPs after i.v. administration. The study stipulates that 4-SO(4)GalNAc assures for targeting resident macrophages.

Protective effect of fruits of Morinda citrifolia L. on scopolamine induced memory impairment in mice: a behavioral, biochemical and cerebral blood flow study.

ETHNOPHARMACOLOGICAL RELEVANCE: Noni (Morinda citrifolia L.) is widely used for different illnesses including CNS disorders. Recently Noni has been reported to prevent amyloid beta induced memory impairment in mice. However, the influence of Noni on cholinergic system has not been explored so far. Therefore, present study was designed to investigate effect of Noni fruit on memory, cerebral blood flow (CBF), oxidative stress and acetylcholinesterase (AChE) activity in scopolamine induced amnesia model. MATERIALS AND METHODS: Mice were orally treated with ethanolic extract of Noni fruit and chloroform, ethyl acetate and butanol fractions of ethanolic extract for three days. Scopolamine was administered 5 min prior to acquisition trial and memory function was evaluated by passive avoidance test. CBF was measured by laser doppler flowmetry. AChE activity and oxidative stress parameters were estimated in mice brain at the end of behavioral studies. Further, effect of ethanolic extract and its fractions (5-400 µg/ml) on AChE activity was measured in vitro. RESULTS: Scopolamine caused memory impairment along with reduced CBF, increased AChE activity and oxidative stress in mice brain. Ethanolic extract of Noni fruits and its chloroform and ethyl acetate fractions significantly improved memory and CBF. However, butanol fraction had no effect. Further, increased oxidative stress and AChE activity following scopolamine was significantly attenuated by ethanolic extract of Noni and its fractions. Moreover ethanolic extract and its fractions showed dose dependent inhibition of AChE activity in vitro. CONCLUSION: These observations suggest that Noni may be useful in memory impairment due to its effect on CBF, AChE and oxidative stress.