Utkarsh Doshi - Academia.edu (original) (raw)

Papers by Utkarsh Doshi

Levels of selected carbonyls deposited in PBS-filled wells in the exposure system for the four te... more Levels of selected carbonyls deposited in PBS-filled wells in the exposure system for the four tested products. This dataset was generated in an in vitro systems toxicology study where Human organotypic buccal and small lung airway cultures were exposed at the air-liquid interface to cigarette smoke and aerosols of different formulations of electronic cigarettes, namely "Test Mix" (flavours, nicotine, humectants), "Base" (nicotine, humectants) and "Carrier" (humectants).

This study provides morphological and mechanistic insights into the impact on human lung and oral... more This study provides morphological and mechanistic insights into the impact on human lung and oral epithelium of the exposure to cigarette smoke and aerosols of electronic cigarettes. Human organotypic buccal and small lung airway cultures were exposed at the air-liquid interface to cigarette smoke and aerosols of different formulations of electronic cigarettes, namely "Test Mix" (flavours, nicotine, humectants), "Base" (nicotine, humectants) and "Carrier" (humectants). The acute impact of these test products on cellular morphology, toxicology and gene expression has been assessed through a system toxicology approach that combined the evaluation of functional biological endpoints with multiplex and omics assays. Nicotine and carbonyls levels measurement provided information about the exposure to harmful aerosol substances, while histopathology and ciliary beating frequency assessment estimated the grade of the induced tissue damage, and inflammatory prot...

Recent research findings have provided strong evidence that the metabolic fate of a xenobiotic is... more Recent research findings have provided strong evidence that the metabolic fate of a xenobiotic is a result of the integrated activities of uptake transporters, drug metabolizing enzymes, and efflux transporters. An ideal in vitro hepatic model will be cultured hepatocytes that retain all of these three major functions. Freshly isolated or cryopreserved human hepatocytes in general retain in vivo levels of drug metabolizing enzymes and uptake transporters, but would lack efflux transporter functions. Prolonged (e.g. 5 day) culturing of the hepatocytes as sandwich cultures would allow expression of the efflux transporters, but drug metabolizing enzymes, especially critical P450 isoforms such as CYP3A4 would be decreased to approximately 5 to 10% of the level of freshly isolated hepatocytes. We report here the development of a novel medium (Lis Differentiation Maintenance Medium (LDMM)) that has the potential to maintain all three critical hepatic functions: uptake transport, efflux tr...

Journal of Biomolecular Screening, 2011

The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inh... more The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitaz...

Drug Metabolism Letters, 2011

Time-dependent or mechanism-based CYP3A4 inhibition is an important adverse drug property that sh... more Time-dependent or mechanism-based CYP3A4 inhibition is an important adverse drug property that should be carefully managed during drug development. Evaluation of time-dependent inhibition is traditionally performed using liver microsomes or recombinant P450 isoforms. We report here higher throughput approaches to evaluate time-dependent CYP3A4 inhibition assay using cultured cryopreserved human hepatocytes. The assays were performed in human hepatocytes cultured in 96-well plates, with luciferin-IPA as the CYP3A4 specific substrate. The advantages of the approach are as follows: 1. The use of 96-well plates minimizes the quantity of human hepatocytes and test materials required for the assays. 2. The use of luciferin-IPA allows CYP3A4 activity to be quantified rapidly using a plate reader, thereby avoiding the need for LC/MS that is required for traditional substrates such as testosterone and midazolam. 3. The use of cultured (plated) hepatocytes allows effective removal of treatment medium and washing of the cells without the laborious centrifugation step that is required for hepatocytes in suspension. Two assays were developed: 1. IC(50) shift assay; and 2. enzyme kinetic assay. The IC-50 shift assay is intended for general screening purpose with which a time-dependent CYP3A4 inhibitor would be identified by an increase in inhibitory potency (quantified as a decrease in IC(50)) upon a 30 min. pre-incubation of hepatocytes with the inhibitor at 37 deg. C. Results with model inhibitors showed that the IC50 assay readily distinguished the time-dependent inhibitors (1-aminobenzotriazole, erythromycin) from the non-time-dependent inhibitor (ketoconazole). The enzyme kinetic assay is used for the derivation of the kinetic parameters K(I) and k(inact). With this assay, time and concentration dependent inhibition of CYP3A4 were observed for 1-aminobenzotriazole and erythromycin. With hepatocytes from 4 donors, K(I) and k(inact) values were calculated to be 22.0 to 70.7 uM, and 0.09 to 0.51 min(-1), respectively, for 1-aminobenzotriazole; and 47.3 to 75.1 uM, and 0.26 to 1.48, respectively, for erythromycin. DMSO (tested up to 2% v/v) was found to significantly attenuate the time-dependent inhibitory effects of 1-aminobenzotriazole, and had no apparent effects on erythromycin. Acetonitrile and methanol at 1% v/v had no significant effects. The higher throughput assays describe here can to be used routinely for the evaluation of time-dependent CYP3A4 inhibitory potential of drug candidates during early phases of drug development.

Current Drug Metabolism, 2012

We report here a comprehensive evaluation of the effects of culture duration on the gene expressi... more We report here a comprehensive evaluation of the effects of culture duration on the gene expression of P450 isoforms, uptake transporters and efflux transporters in human hepatocyte cultured in the absence and presence of the prototypical proinflammatory cytokine, interleukin-6 (IL-6). Primary collagen-matrigel sandwich cultures of human hepatocytes were cultured in supplemented William&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s E medium containing 0, 0.1, 0.5 and 5 ng/mL of IL-6 for the time periods of 2, 6, 12, 24 and 48 hrs. Real-time PCR was performed to quantify gene expression of acute phase proteins (suppressor of cytokine signaling 3 (SOCS-3), c-reactive protein (CRP) and lipopolysaccharide (LPS)-binding proteins (LBP)); P450 isoforms (CYPs 1A2, 2B6, 2C8, 2C9, 2D6, 3A4, and 3A5), uptake transporters (SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1) and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2). SOCS-3, CRP, and LBP were extensively induced by IL-6, with maximal induction observed at 2 (SOCS-3) and 12 hrs (CRP; LBP), demonstrating that the cultured human hepatocytes responded to IL-6 treatment. In the untreated group (control), gene expression of P450 isoforms and uptake transporters decreased while efflux transporters remained relatively stable or increased with cultured duration. IL-6 predominantly caused down regulations of the genes studied, with the most significant changes observed at different treatment durations, apparently related to the stability of the basal levels of gene expression. For instance, for genes with unstable expression, which would decrease rapidly in culture (e.g CYP3A4), the most definitive down regulatory effects were observed at a relatively early time point (e.g. 12 hrs). In contrast, a longer treatment duration (e.g. 48 hrs) was required for genes with relatively stable expression levels in culture (e.g. ABCB1). Based on our findings, evaluation of multiple treatment durations rather than single treatment duration is recommended for the evaluation of biotherapeutics in cultured human hepatocytes where down regulation is expected.

Bioorganic & Medicinal Chemistry, 2012

Synthesis and anticonvulsant activity of new N-Mannich bases of 3-(2-fluorophenyl)- and 3-(2-brom... more Synthesis and anticonvulsant activity of new N-Mannich bases of 3-(2-fluorophenyl)- and 3-(2-bromophenyl)-pyrrolidine-2,5-diones have been described. Initial anticonvulsant screening was performed in mice after intraperitoneal administration in the maximal electroshock seizure test (MES) and subcutaneous pentylenetetrazole seizures test (scPTZ). The neurotoxicity was determined applying the rotarod test. The in vivo results in mice showed that majority of compounds were effective in the MES test. Only seven molecules showed protection in the scPTZ test. The quantitative evaluation in the MES seizures after oral administration into rats showed that the most active were 1-[{4-(4-fluorophenyl)-piperazin-1-yl}-methyl]-3-(2-bromophenyl)-pyrrolidine-2,5-dione (14) with ED(50) of 7.4mg/kg and 1-[{4-(3-bromophenyl)-piperazin-1-yl}-methyl]-3-(2-bromophenyl)-pyrrolidine-2,5-dione (16) with ED(50) of 26.4mg/kg. These molecules were more potent and also less neurotoxic than phenytoin which was used as reference antiepileptic drug.

Asthma is a chronic inflammatory disease of the airways characterized by fibrosis of the airways,... more Asthma is a chronic inflammatory disease of the airways characterized by fibrosis of the airways, hyperplasia and hypertrophy of smooth muscle cells and mucous secreting cells due to infiltration of activated eosinophils and activation of resident mast cells and lymphocytes. These chronic inflammatory changes are mediated by secretion of cytokines from inflammatory cells. Cytokines play integral role in the coordination and persistence of inflammatory process in chronic inflammation of the airways. Based on the recent understanding of pathogenesis of asthma, cytokines are classified as a) Lymphokines, b) Proinflammatory cytokines, c) Antiinflammatory cytokines and d) Chemokines. The biology of cytokines seem to be complex and may not confine to inflammatory reactions as some of them do possess antiinflammatory properties. Current therapies for asthma provide only symptomatic relief to patients and do not control inflammation. Modern treatment of asthma is directed towards lessening ...

Applied In Vitro Toxicology

Journal of Applied Toxicology

Internal and Emergency Medicine

In the context of tobacco harm-reduction strategy, the potential reduced impact of electronic cig... more In the context of tobacco harm-reduction strategy, the potential reduced impact of electronic cigarette (EC) exposure should be evaluated relative to the impact of cigarette smoke exposure. We conducted a series of in vitro studies to compare the biological impact of an acute exposure to aerosols of "test mix" (flavors, nicotine, and humectants), "base" (nicotine and humectants), and "carrier" (humectants) formulations using MarkTen ® EC devices with the impact of exposure to smoke of 3R4F reference cigarettes, at a matching puff number, using human organotypic air-liquid interface buccal and small airway cultures. We measured the concentrations of nicotine and carbonyls deposited in the exposure chamber after each exposure experiment. The deposited carbonyl concentrations were used as representative measures to assess the reduced exposure to potentially toxic volatile substances. We followed a systems toxicology approach whereby functional biological endpoints, such as histopathology and ciliary beating frequency, were complemented by multiplex and omics assays to measure secreted inflammatory proteins and whole-genome transcriptomes, respectively. Among the endpoints analyzed, the only parameters that showed a significant response to EC exposure were secretion of proteins and whole-genome transcriptomes. Based on the multiplex and omics analyzes, the cellular responses to EC aerosol exposure were tissue type-specific; however, those alterations were much smaller than those following cigarette smoke exposure, even when the EC aerosol exposure under the testing conditions resulted in a deposited nicotine concentration approximately 200 times that in saliva of EC users.

CPT: pharmacometrics & systems pharmacology, 2018

Plateable human hepatocytes with human plasma were utilized to generate the uptake transporter ki... more Plateable human hepatocytes with human plasma were utilized to generate the uptake transporter kinetic data for pravastatin, an organic anion-transporting polypeptide (OATP) transporter substrate. The active hepatic uptake of pravastatin was determined with a J value of 134.4 pmol/min/million cells and K of 76.77 µM in plateable human hepatocytes with human plasma. The physiologically-based pharmacokinetic (PBPK) model with incorporation of these in vitro kinetic data successfully simulated the i.v. pharmacokinetic profile of pravastatin without applying scaling factor (the mean predicted area under the curve (AUC) is within 1.5-fold of the observed). Furthermore, the PBPK model also adequately described the oral plasma concentration-time profiles of pravastatin at different dose levels. The current investigation demonstrates an approach allowing us to build upon the translation of in vitro OATP uptake transporter data to in vivo, with a hope of utilizing the in vitro data for the p...

Drug metabolism letters, Jan 12, 2017

A recent advancement in isolation and cryopreservation has resulted in commercially available pri... more A recent advancement in isolation and cryopreservation has resulted in commercially available primary human enterocytes that express various drug metabolizing enzymes (DMEs) and transporters. The main objective of this study was to further evaluate the utility of pooled cryopreserved enterocytes, specifically MetMax™ cryopreserved human enterocytes (In Vitro ADMET Laboratories), as an in vitro model for assessing intestinal clearance in comparison to hepatocytes. It was found that, for CYP3A4 substrates such as midazolam, amprenavir and loperamide, in vitro metabolic clearance is generally lower in enterocytes compared to that of hepatocytes, which is consistent with the relative abundance of the enzyme between the intestine and liver. In contrast, raloxifene, a surrogate UGT activity substrate, showed 3-fold greater turnover in enterocytes than hepatocytes, which is likely attributed to the differential expression of individual UGTs in human liver and intestine. For procaine, a kno...

Drug metabolism and disposition: the biological fate of chemicals, Jun 1, 2017

We report in this work successful isolation and cryopreservation of enterocytes from human small ... more We report in this work successful isolation and cryopreservation of enterocytes from human small intestine. The enterocytes were isolated by enzyme digestion of the intestinal lumen, followed by partial purification via differential centrifugation. The enterocytes were cryopreserved directly after isolation without culturing to maximize retention of in vivo drug-metabolizing enzyme activities. Post-thaw viability of the cryopreserved enterocytes was consistently over 80% based on trypan blue exclusion. Cryopreserved enterocytes pooled from eight donors (four male and four female) were evaluated for their metabolism of 14 pathway-selective substrates: CYP1A2 (phenacetin hydroxylation), CYP2A6 (coumarin 7-hydroxylation), CYP2B6 (bupropion hydroxylation), CYP2C8 (paclitaxel 6α-hydroxylation), CYP2C9 (diclofenac 4-hydroxylation), CYP2C19 (S-mephenytoin 4-hydroxylation), CYP2D6 (dextromethorphan hydroxylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4 (midazolam 1'-hydroxylatio...

Toxicological sciences : an official journal of the Society of Toxicology, Dec 9, 2016

Nefazodone, an antagonist for the 5-hydroxytryptanine receptor, has been used for the treatment o... more Nefazodone, an antagonist for the 5-hydroxytryptanine receptor, has been used for the treatment of depression. Acute liver injury has been documented to be associated with the use of nefazodone; however, the mechanisms of nefazodone-induced liver toxicity are not well defined. In this report, using biochemical and molecular analyses, we characterized the molecular mechanisms underlying the hepatotoxicity of nefazodone. We found that nefazodone induced endoplasmic reticulum (ER) stress in HepG2 cells, as the expression of typical ER stress markers, including CHOP, ATF-4, and p-eIF2α, was significantly increased, and splicing of XBP1 was observed. Nefazodone-suppressed protein secretion was evaluated using a Gaussia luciferase reporter assay that measures ER stress. The ER stress inhibitors (4-phenylbutyrate and salubrinal) and knockdown of ATF-4 gene attenuated nefazodone-induced ER stress and cytotoxicity. Nefazodone activated the MAPK signaling pathway, as indicated by increased ph...

Bioorganic Medicinal Chemistry Letters, 2006

Human hepatic organic anion-transporting polypeptides (OATPs), including OATP1B1, 1B3, and 2B1, a... more Human hepatic organic anion-transporting polypeptides (OATPs), including OATP1B1, 1B3, and 2B1, are expressed at the basolateral membrane of hepatocytes and mediate the uptake of a variety of compounds from blood into hepatocytes. The liver-specific OATPs are increasingly recognized as playing important roles in the pharmacokinetic (PK) of many drugs and thus, involved in the clinically significant drug-drug interactions (DDIs). However, the evaluation of the specific roles of individual OATPs in hepatocytes is challenging because of the lack of selective inhibitors and probe substrates for each OATP member. In the present study, the uptake activity of OATP1B3 was examined in human hepatocytes cultured up to 14 days using an in vitro uptake assay. The results showed that OATP-mediated uptake of rosuvastatin, a substrate for OATPs declined substantially in cultured human hepatocytes. In contrast, the uptake of OATP1B3-selective substrate telmisartan was not measureable at earlier culture periods, but became detectable on Day 7 and showed culture duration-dependent changes from Day 7 to 14. Quantitative polymerase chain reaction (qPCR) analyses illustrated that the OATP functional change was not correlated with messenger ribonucleic acid (mRNA) expression alteration in hepatocytes cultured for 3 hours or 7 days. The OATP1B3-mediated telmisartan uptake was also culture medium- and donor-dependent, and only observed in 3 of 5 lots of hepatocytes cultured in 2 of 3 media tested. These results show that using human hepatocytes cultured in certain conditions may provide an excellent addition to transfected cell lines as a way to distinguish OATP1B3 from other hepatic OATP family members, such as OATP1B1, to provide more understanding of OATP-mediated clinical DDI.

Chemico-biological interactions, Jan 12, 2015

We report here the results of a collaborative research program to develop a robust and reliable i... more We report here the results of a collaborative research program to develop a robust and reliable in vitro system to allow an accurate definition of the drug-induced liver injury (DILI) potential of new drug entities during drug development. The in vitro hepatotoxic potential of 152 drugs with known DILI profiles were evaluated in primary cultured human hepatocytes with four mechanistically-relevant endpoints: cellular ATP depletion, reactive oxygen species (ROS), glutathione (GSH) depletion, and caspase activation for apoptosis. The drugs, 80 in the testing set and 72 in the validation set, were classified based on serious clinical/regulatory outcomes as defined by reported acute liver failure, boxed warning, and/or withdrawal. The drugs were further sub-categorized for dominant types of liver injury. Logistic regression models were performed to calculate the area under the receiver operating characteristics curve (AUROC) and to evaluate the prediction potential of the selected endpo...

Drug metabolism letters, Jan 18, 2015

A hepatocyte relay assay has recently been established by Di et al (2012) for the evaluation of c... more A hepatocyte relay assay has recently been established by Di et al (2012) for the evaluation of compounds with low hepatic clearance. In the assay, the compounds are subjected to multiple 4 h incubations (relays) using human hepatocyte suspensions, with each incubation using freshly thawed human hepatocytes, leading to an accumulated incubation duration of 20 h after 5 relays. We report here the Plated Hepatocyte Relay Assay (PHRA) using plated, rather than suspended, human hepatocytes. The use of plated hepatocytes allows extension of each relay incubation to 24 h, leading to total incubation durations of 24 h (initial incubation), 48 h (1st relay), and 72 h (2nd relay). 96 h (3rd relay), and 120 h (4th relay). We report here a proof-of-concept study with two low clearance compounds, diazepam and tolbutamide, and a validation study with 15 compounds with known human in vivo hepatic clearance which included the ultra low clearance compounds (CLnon-renal < 1 mL/min/kg) meloxicam, ...

Obesity is a serious public health problem and a major risk factor for cardiovascular disease, ce... more Obesity is a serious public health problem and a major risk factor for cardiovascular disease, certain types of cancer, and type 2 diabetes. Hence there is a need for relevant cellular models that can be used to test for drugs that may inhibit adipogenesis. Currently, most cellular models utilize cultures of fibroblastic 3T3-L1 preadipocytes that can be induced to undergo adipogenesis. However the effect of other cell types on adipocyte function has not yet been elucidated. Since hepatocytes are the major xenobiotic metabolizing cells of the body, drug testing in the presence of hepatocytes is relevant. In this study we have used a proprietary technology, developed in our laboratory, termed Integrated Discrete Multi Organ Co-culture (IdMOCTM) for the co-culture of 3T3-L1 preadipocytes and hepatocyes. Using quantitative PCR for the amplification of an early marker for adipogenic differentiation namely AP2 we show that hepatocyte presence causes an increase in 3T3-LI adipogenesis. Usi...