Hari Narayan Kushwaha

Photosensitizing mechanism and identification of levofloxacin photoproducts at ambient UV radiation.

Levofloxacin (LVFX) is a broad spectrum third generation fluoroquinolone antibiotic, used in the treatment of severe or life‐threatening bacterial infections. Photosensitizing mechanism of LVFX was investigated under the ambient environmental intensities of UV‐A, UV‐B and sunlight exposure. Phototoxic effects of LVFX were assessed on NIH‐3T3 and HaCaT cell lines. Results identified first time three photoproducts of LVFX at ambient levels of UV‐R by LC‐MS/MS. The generation of reactive oxygen species (ROS) was investigated photochemically as well as intracellularly in HaCaT cell line. ROS were significantly quenched by specific quenchers like DABCO, NaN3, d‐mannitol and NAC. Photosensitized LVFX caused lipid peroxidation at different concentrations. Quenching study with superoxide dismutase confirms the LVFX‐induced lipid photoperoxidation. Further, photocytotoxicity of LVFX showed significant reduction in cell viability by MTT and neutral red uptake assays. LVFX caused cell arrest in G2/M phases as well as induced apoptosis through ROS‐dependent pathway. In addition, photosensitized LVFX also induced upregulation of p21 and Bax/Bcl‐2 genes ratio. India is a tropical country and most of the human activities such as agriculture, commerce, sports, etc. take place in bright sunlight; therefore, photosensitive LVFX may lead to skin/ocular disorders and immune suppression. Information is needed regarding the phototoxicity of LVFX for human safety.

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro.

BackgroundRiboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.ResultsResults indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.ConclusionThus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.

Ambient UVA-Induced Expression of p53 and Apoptosis in Human Skin Melanoma A375 Cell Line by Quinine

This study aimed to analyze the phototoxic mechanism and photostability of quinine in human skin cell line A375 under ambient intensities of UVA (320–400 nm). Photosensitized quinine produced a photoproduct 6‐methoxy‐quinoline‐4‐ylmethyl‐oxonium identified through LC‐MS/MS. Generation of 1O2, O2•−, and •OH was measured and further substantiated through their respective quenchers. Photosensitized Quinine (Q) caused degradation of 2‐deoxyguanosine, the most sensitive nucleotide to UV radiation. The intracellular ROS was increased in a concentration‐dependent manner. Significant reduction in metabolic status measured in terms of cell viability (54%) at 25 μg mL−1 was observed through MTT assay. Results of MTT assay accord NRU assay. Single strand DNA breaks and apoptosis were increased significantly (P < 0.01) as observed through comet assay and EB/AO double staining. Photosensitized quinine caused cells to arrest in G2 phase of cell cycle and induced apoptosis (5.08%) as revealed through FACS. Real‐Time PCR showed upregulation of p21 (4.56 folds) and p53 (2.811 folds) genes expression. Thus, our study suggests that generation of reactive oxygen species by quinine under ambient intensity of UVA may result into deleterious phototoxic effects among human population.

Piperine attenuates UV-R induced cell damage in human keratinocytes via NF-kB, Bax/Bcl-2 pathway: An application for photoprotection

Chronic ultraviolet radiation (UV-R) exposure causes skin disorders like erythema, edema, hyperpigmentation, photoaging and photocarcinogenesis. Recent research trends of researchers have focused more attention on the identification and use of photo stable natural agents with photoprotective properties. Piperine (PIP), as a plant alkaloid, is an important constituent present in black pepper (Piper nigrum), used widely in ayurvedic and other traditional medicines and has broad pharmacological properties. The study was planned to photoprotective efficacy of PIP in human keratinocyte (HaCaT) cell line. We have assessed the UV-R induced activation of transcription factor NF-κB in coordination with cell death modulators (Bax/Bcl-2 and p21). The LC-MS/MS analysis revealed that PIP was photostable under UV-A/UV-B exposure. PIP (10μg/ml) attenuates the UV-R (A and B) induced phototoxicity of keratinocyte cell line through the restoration of cell viability, inhibition of ROS, and malondialdehyde generation. Further, PIP inhibited UV-R mediated DNA damage, prevented micronuclei formation, and reduced sub-G1 phase in cell cycle, which supported against photogenotoxicity. This study revealed that PIP pretreatment strongly suppressed UV-R induced photodamages. Molecular docking studies suggest that PIP binds at the active site of NF-κB, and thus, preventing its translocation to nucleus. In addition, transcriptional and translational analysis advocate the increased expression of NF-κB and concomitant decrease in IkB-α expression under UV-R exposed cells, favouring the apoptosis via Bax/Bcl-2 and p21 pathways. However, PIP induced expression of IkB-α suppress the NF-κB activity which resulted in suppression of apoptotic marker genes and proteins that involved in photoprotection. Therefore, we suggest the applicability of photostable PIP as photoprotective agent for human use.

UFLC method development and validation of a novel triethylamine containing thiophene S006‐830 ‐ an antitubercular molecule and its application to pharmacokinetic and bioavailability studies in SD rats

A sensitive and selective ultra fast liquid chromatography (UFLC) method has been developed and validated for the determination of a potent and novel antitubercular compound S006-830 in Sprague Dawley (SD) rat plasma. Samples were extracted and processed by protein precipitation method using acetonitrile. Chromatographic separation was achieved on a Phenomenex, Luna C-18 column (3μm, 100mm x 2mm i.d.) under isocratic condition. Detection was performed on UFLC-NEXERA system (LC-30AD, Shimadzu, Kyoto, Japan) with a degasser (DGU-20A), auto-injector (SIL-30AC), fixed with a 100-μL loop. Method was found sensitive and reproducible over a linearity range of 15.6-2000 ng/mL. Recovery of S006-830 and internal standard was found >90% for spiked matrix control and standard quality control plasma samples. This validated method was successfully applied to generate pharmacokinetic profile of S006-830 in SD rats. Oral dose proportionality studies were conducted at 100, 50, 25 mg/Kg dose levels, while an IV study was conducted at 25 mg/Kg dose. There was dose dependent increase in AUC and Cmax indicating S006-830 to exhibit linear pharmacokinetics. S006-830 exhibited favorable bioavailability in the range of 45-55%.

In-situ absorption, protein binding and pharmacokinetic studies of S002-853, a novel antidiabetic and antidyslipidaemic flavone derivative in rats.

Objectives The aim of the study was to investigate the in‐situ absorption kinetics, plasma protein binding and pharmacokinetic characteristics of a novel synthetic flavone derivative, S002‐853, which shows pronounced antidiabetic and antidyslipidaemic activity.

Preferential desulfurization of dibenzyl sulfide by an isolated Gordonia sp. IITR100

Several organosulfur compounds are present in the crude oil, and are required to be removed before its processing into transport fuel. For this reason, biodesulfurization of thiophenic compounds has been studied extensively. However, studies on the sulfide compounds are scarce. In this paper, we describe desulfurization of a model sulfidic compound, dibenzyl sulfide (DBS) by an isolated Gordonia sp. IITR100. The reaction was accompanied with the formation of metabolites dibenzyl sulfoxide, dibenzyl sulfone and benzoic acid. Studies with recombinant E. coli revealed that enzyme DszC of this isolate metabolizes DBS into dibenzyl sulfoxide and dibenzyl sulfone, but the reaction downstream to it is mediated by some enzyme other than its DszA. In reactions where DBS and dibenzothiophene (DBT) were present together, both IITR100 and recombinant E. coli exhibited preference for the desulfurization of DBS over DBT. The newly identified capability of IITR100 for desulfurization of both thiophenic and sulfidic compounds suggests its potential use in improved desulfurization of petroleum fractions.

Intersex effect of lamotrigine on the pharmacokinetic parameters of CDRI-97/78, a novel trioxane antimalarial compound, in rats.

Reports regarding drug toxicity and adverse events resulting from coadministration of multiple drugs are increasing at an alarming rate. CDRI-97/78 is an 1,2,4-trioxane antimalarial agent under development which gets metabolized to the in vivo active metabolite 97/63. In order to assess its drug interaction potential, CDRI-97/78 was administered alone and in combination with lamotrigine to male and female rats via the oral route. Quantification of the active metabolite 97/63 in rat plasma was achieved with an LC-MS/MS assay. After oral administration of 97/78, the Tmax and Cmax values of 97/63 in male rats were 1.75±0.77 h and 862±306 ng/mL while female rats showed values for Cmax of 622.75±95.09 ng/mL and for Tmax of 7.5±0.5 h. Coadministration of 97/78 and lamotrigine resulted in decreased Tmax and Cmax values in both male and female rats (Tmax and Cmax of 0.77±0.16 h and 58.58±6.43 ng/mL in male rats; 1.13±0.22 h and 62.95±12.00 ng/mL in female rats, respectively). A statistically significant difference (P<0.05) was observed for the pharmacokinetic parameters of 97/63 after oral administration of 97/78 alone and upon its coadministration with lamotrigine except for the Cmax and Tmax values in male and for the T1/2 value in female rats. Statistically, no significant difference for the pharmacokinetic parameters of 97/63 between male and female rats after oral administration of 97/78 alone or in combination with lamotrigine was determined except for Tmax. The study indicates that coadministration of 97/78, an antimalarial agent, and the antiepileptic lamotrigine may require dose adjustments. Additional clinical drug interaction trials may be required to confirm these findings.

Liquid chromatography tandem mass spectrometry method for determination of antidiabetic chalcones derivative S001-469 in rat plasma, urine and feces: application to pharmacokinetic study.

A sensitive and selective liquid chromatography tandem mass spectrometry assay was developed for quantitation of a novel antidiabetic chalcones derivative S001-469 in rat matrices. Plasma and urine samples were prepared by double liquid-liquid extraction with diethyl ether and feces by protein precipitation using acetonitrile. Chromatographic elution was carried on cyano guard column (30 mm × 4.6 mm i.d., 5 µm) in isocratic mode at a flow rate of 0.75 mL/min using mobile phase comprising of methanol: ammonium acetate buffer (pH 4.6, 10 mM) (90:10, v/v). Run time was 6 min. Detection was achieved by employing positive ionization mode on a triple-quadrupole LC-MS/MS system with an electrospray ionization (ESI) source. The calibration curves were linear over the range of 0.78-400 ng/mL for all 3 matrices. The method was validated and proved reliable through high and consistent intra- and inter- day accuracy and precision (<15%) values. Recoveries was >85% from spiked plasma, urine and feces samples. S001-469 was stable in plasma at room temperature till 8 h and at -60 °C for 30 d and 3 freeze-thaw cycles.

Pharmacokinetic studies of a novel trioxane antimalarial (99/411) in rats and monkeys using LC-MS/MS.

The pharmacokinetic profile of 99/411, a novel anti-malarial drug, was established in rats (12 mg/kg of body weight) and monkeys (20 mg/kg of body weight). Following oral administration, the presence of 99/411 was rapidly determined in rat plasma, tissues, urine, feces and monkey plasma using a validated LC-MS/MS method. The tissue distribution studies in rats indicated that the drug was partially distributed in all major tissues and plasma, and peak concentration levels were achieved within 0.5-4 h. Area under the curve in different rat tissues and plasma was found in order of blood > lung > intestine > heart > muscle > brain > kidney > spleen > liver. The total recoveries (within 86 h) of 99/411 were <0.0017% and <0.08% in urine and feces, respectively. The peak plasma concentration was 3499 ng/mL in rats after ~2 h of oral administration and 697-767 ng/mL in monkeys after ~6 h of oral administration. No plasma accumulation was observed in both male and female monkeys, even after multiple dosing. The preclinical pharmacokinetic profile and tissue distribution data are expected to assist in future clinical explorations of 99/411 as a promising anti-malarial agent.