Survey of Pharmaceutical Industry's Best Practices around In Vitro Transporter Assessment and Implications for Drug Development: Considerations from the International Consortium for Innovation and Quality for Pharmaceutical Development Transporter Working Group.

The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.

Serological and Molecular Detection of Toxoplasma gondii in Domestic Pigs Intended for Human Consumption and Potential Occupational Hazard to Pig Farmers in India.

Toxoplasma gondii, an important food-borne zoonotic parasite, poses a worldwide public health hazard. Domestic pigs are considered one of the main intermediate hosts in the zoonotic transmission of T. gondii. To date, seroepidemiological information on T. gondii in domestic pigs in India is very scarce, and there are no reports of occupational hazards to pig farmers in this country. Here, we aimed at estimating the occurrence of T. gondii (antibodies and parasite DNA) in slaughtered pigs and pig farmers in Central India. Seroprevalence was determined in 410 serum samples from slaughtered pigs and 103 sera from pig farmers using an in-house prepared antigen-based modified agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and indirect-fluorescent antibody test (IFAT). Anti-T. gondii IgG antibodies were detected in 200 pigs (up to 48.8%, confidence interval [95% CI]: 40.4-52.2) and 44 pig farmers (up to 42.7%, 95% CI: 35.6-47.3) using MAT, ELISA, and IFAT. Inter-rater agreement showed an excellent agreement (kappa κ = 0.9) among the different serological tests suggesting similar detection potential of these tests. Recently acquired infections in all seropositive subjects were determined using IgG avidity testing and polymerase chain reaction (PCR). IgG avidity showed that 20 (10.3%) of slaughtered pigs and 8 (19.5%) pig farmers had a recently acquired infection. PCR for B1 and 529 repeats was performed in the heart tissues of slaughtered pigs and the blood cells of pig farmers. T. gondii DNA was detected in 14 (7.2%) slaughtered pigs and 5 (12.2%) pig farmers. Univariate analysis revealed that adult animals (>1 year), cats and rodents on the farm, and outdoor access are common factors (p ≤ 0.05) associated with T. gondii infection in pigs. Our results indicate that T. gondii is widely distributed in slaughtered pigs and pig farmers at risk of infection, highlighting a potential zoonotic transmission and health risk to consumers.

Drug transporters in drug disposition - the year 2022 in review.

On behalf of all the authors, I am pleased to share our third annual review on drug transporter science with an emphasis on articles published and deemed influential in signifying drug transporters' role in drug disposition in the year 2022. As the drug transporter field is rapidly evolving several key findings were noted including promising endogenous biomarkers, rhythmic activity, IVIVE approaches in transporter-mediated clearance, new modality interaction, and transporter effect on gut microbiome. As identified previously (Chothe et Cal. 2021, 2022) the goal of this review is to highlight key findings without a comprehensive overview of each article and to this end, each coauthor independently selected 1-3 peer-reviewed articles published or available online in the year 2022 (Table 1). Each article is summarized in synopsis and commentary with unbiased viewpoints by each coauthor. We strongly encourage readers to consult original articles for specifics of the study. Finally, I would like to thank all coauthors for their continued support in writing this annual review on drug transporters and invite anyone interested in contributing to future versions of this review.

Mechanistic Static Model based Prediction of Transporter Substrate Drug-Drug Interactions Utilizing Atorvastatin and Rifampicin.

OBJECTIVE: An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair. METHODS: In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations. RESULTS: The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR. CONCLUSIONS: This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.

Serological and molecular detection of Toxoplasma gondii and Neospora caninum in free-ranging rats from Nagpur, India.

Toxoplasma gondii and Neospora caninum are cyst-forming coccidian parasites that infect both wild and domestic non-felids as intermediate hosts, with rodents serving as important reservoir hosts during their life cycles. This study was aimed at investigating T. gondii and N. caninum infections and identifying factors favouring T. gondii infection in free-ranging rats from India. A total of 181 rodents were trap-captured, and blood and brain samples were subsequently collected for serological and molecular examination of T. gondii and N. caninum. Antibodies against T. gondii and N. caninum were detected by MAT/NAT and IFAT in 13.8% (25/181) and 1.65% (3/181) of rodents, respectively. All three N. caninum samples positive by NAT/IFAT were also positive for ELISA, while for T. gondii, 19 of 25 MAT/IFAT positive samples were also positive for ELISA. The antibody titers (MAT/NAT/IFAT) of rodents seropositive for T. gondii ranged from 25 to 400, while those of rats seropositive for N. caninum ranged from 25 to 100. Also, using PCR, DNA from T. gondii (B1 gene) and N. caninum (NC5 gene) was found in 2.76% (5/181) of brain samples and 0.55% (1/181) of brain samples. All PCR positive samples were also seropositive. No mixed infections were observed in the serological and molecular detections. A Chi-square analysis revealed that older rats and rats living in urban areas are significantly associated with T. gondii infection; however, rodent species, gender, location, habitat types, and seasonality were statistically nonsignificant. Overall, this study demonstrated that T. gondii was widely distributed while N. caninum was less prevalent among free-ranging rats in the studied area.

Novel insights in drug transporter sciences: the year 2021 in review.

On behalf of the team I am pleased to present the second annual 'novel insights into drug transporter sciences review' focused on peer-reviewed articles that were published in the year 2021. In compiling the articles for inclusion, preprints available in 2021 but officially published in 2022 were considered to be in scope. To support this review the contributing authors independently selected one or two articles that were thought to be impactful and of interest to the broader research community. A similar approach as published last year was adopted whereby key observations, methods and analysis of each paper is concisely summarized in the synopsis followed by a commentary highlighting the impact of the paper in understanding drug transporters' role in drug disposition. As the goal of this review is not to provide a comprehensive overview of each paper but rather highlight important findings that are well supported by the data, the reader is encouraged to consult the original articles for additional information. Further, and keeping in line with the goals of this review, it should be noted that all authors actively contributed by writing synopsis and commentary for individual papers and no attempt was made to standardize language or writing styles. In this way, the review article is reflective of not only the diversity of the articles but also that of the contributors. I extend my thanks to the authors for their continued support and also welcome Diane Ramsden and Pallabi Mitra as contributing authors for this issue (Table 1).[Table: see text].

Rho Protein: Roles and Mechanisms.

At the end of the multistep transcription process, the elongating RNA polymerase (RNAP) is dislodged from the DNA template either at specific DNA sequences, called the terminators, or by a nascent RNA-dependent helicase, Rho. In Escherichia coli, about half of the transcription events are terminated by the Rho protein. Rho utilizes its RNA-dependent ATPase activities to translocate along the mRNA and eventually dislodges the RNAP via an unknown mechanism. The transcription elongation factor NusG facilitates this termination process by directly interacting with Rho. In this review, we discuss current models describing the mechanism of action of this hexameric transcription terminator, its regulation by different cis and trans factors, and the effects of the termination process on physiological processes in bacterial cells, particularly E. coli and Salmonella enterica Typhimurium.

Seroprevalence, risk factors, and serological cross-reactivity for diagnosis of Toxoplasma gondii and Neospora caninum infections in goats in India.

Toxoplasma gondii and Neospora caninum are genetically related cyst-forming protozoan parasites that cause reproductive failures in ruminants. Given the limited information on the epidemiology of these infections in goats in India, the study aimed to estimate the seroprevalence, assess antibody cross-reactivity for diagnosis, and identify associated risk factors. A total of 695 sera were evaluated for antibodies to T. gondii and N. caninum infections using Modified Agglutination Test (MAT for Toxoplasma)/Neospora agglutination test (NAT), Enzyme-linked immunosorbent assay (ELISA), and Indirect Fluorescent Antibody Test (IFAT for tachyzoite and bradyzoite stages). The seroprevalence rate of T. gondii and N. caninum infections was 56.9% and 10.9%, respectively. Inter-rater agreement (kappa value - κ) was calculated to assess agreements between various diagnostic assays, using the IFAT as the gold standard (for detecting both infections), the agreements for MAT/NAT (κ = 0.97) and the ELISA (κ = 0.95) were excellent. The acute infection among seropositive goats were determined using serological (IgG avidity test - measures the binding strength between IgG antibodies and parasite antigens) and molecular diagnoses (PCR for repetitive DNA sequences - Toxoplasma B1 gene: 131 bp and Neospora NC5 gene: 328 bp). Among seropositive goats ≥80% had high IgG avidity and <10% of animals had low IgG avidity antibodies for both infections. Most low IgG avidity goats were PCR positive for the TgB1 gene (94.4%) or Nc5 gene (85.7%). In the serological assays, we used different dilutions of test serum to rule out the cross-reactivity owing to similar antigenic makeup between these two parasites. When the serological cross-reactivity was analyzed using invasion assay at a serum titer of ≥200, more than 90% T. gondii positive sera showed host cell invasion of N. caninum and vice versa. Largely, the serological results indicate that cut-off serum dilution of ≥1:200 for ELISA and IFAT and ≥1:25 for MAT/NAT avoids serological cross-reactivity between T. gondii and N. caninum. Further, the Univariate and multivariate analyses showed that adult animals (>2 years), reservoir hosts, and extensive rearing systems are common risk factors for these infections. However, the history of abortion was identified as a significant risk factor for T. gondii infection. This study revealed that T. gondii and N. caninum infections are highly prevalent in this region and the use of an appropriate cut-off serum dilution is necessary to avoid cross-reactivity between these closely related parasites.

Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans.

Toxoplasma gondii differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission. Strong humoral immune response has been reported against tachyzoite antigens, however, antibody-mediated response towards bradyzoite antigens is poorly characterized. This work aimed to study the humoral immune response towards bradyzoite and associated cyst wall antigens particularly CST1. The immunoreactivity of 404 goats, 88 sheep and 92 human sera to recombinant (CST1 and SRS9) and native proteins of encysted bradyzoite along with well-established tachyzoite antigens (SAG1 and GRA7) was determined using ELISA, Western blot and immunofluorescence analysis (IFA). ELISA results revealed nearly 50% of sera contain T. gondii specific antibodies. Results were further validated using Western blot and IFA. T. gondii positive sera predominantly recognized the cyst wall besides the known tachyzoite surface antigens. The presence of CST1 antibodies in seropositive samples were in line with the staining patterns which were consistent with CST localization. Notably, T. gondii IgM- IgG+ sera recognize the cyst wall whereas IgM + IgG-sera recognize tachyzoite antigens indicating acute infection consistent with presence of parasite DNA. The study demonstrates a strong humoral response against bradyzoite associated cyst wall antigens across naturally infected animals and humans. CST1 emerged as a key immunomodulatory antigen which may have direct implications for clinical immunodiagnostics.

Prediction and Quantification of Hepatic Transporter-Mediated Uptake of Pitavastatin Utilizing a Combination of the Relative Activity Factor Approach and Mechanistic Modeling.

Quantification of the fraction transported (ft) by a particular transporter will facilitate more robust estimations of transporter interactions. Using pitavastatin as a model uptake transporter substrate, we investigated the utility of the relative activity factor (RAF) approach and mechanistic modeling to estimate ft in hepatocytes. The transporters evaluated were organic anion-transporting polypeptides OATP1B1 and OATP1B3 and sodium-taurocholate cotransporting polypeptide. Transporter-expressing human embryonic kidney 293 cells and human hepatocytes were used for determining RAF values, which were then incorporated into the mechanistic model to simulate hepatocyte uptake of pitavastatin over time. There was excellent agreement between simulated and observed hepatocyte uptake of pitavastatin, indicating the suitability of this approach for translation of uptake from individual transporter-expressing cells to more holistic in vitro models. Subsequently, ft values were determined. The largest contributor to hepatocyte uptake of pitavastatin was OATP1B1, which correlates with what is known about the in vivo disposition of pitavastatin. The ft values were then used for evaluating in vitro-in vivo correlations of hepatic uptake inhibition with OATP inhibitors rifampicin and cyclosporine. Predictions were compared with previously reported plasma exposure changes of pitavastatin with these inhibitors. Although hepatic uptake inhibition of pitavastatin was 2-3-fold underpredicted, incorporation of scaling factors (SFs) into RAF values significantly improved the predictive ability. We propose that calibration of hepatocytes with standard transporter substrates and inhibitors would allow for determination of system-specific SFs, which could subsequently be used for refining predictions of clinical DDI potential for new chemical entities that undergo active hepatic uptake.

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion.

Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5-Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.

Functional dissection of proliferating-cell nuclear antigens (1 and 2) in human malarial parasite Plasmodium falciparum: possible involvement in DNA replication and DNA damage response.

Eukaryotic PCNAs (proliferating-cell nuclear antigens) play diverse roles in nucleic acid metabolism in addition to DNA replication. Plasmodium falciparum, which causes human malaria, harbours two PCNA homologues: PfPCNA1 and PfPCNA2. The functional role of two distinct PCNAs in the parasite still eludes us. In the present study, we show that, whereas both PfPCNAs share structural and biochemical properties, only PfPCNA1 functionally complements the ScPCNA mutant and forms distinct replication foci in the parasite, which PfPCNA2 fails to do. Although PfPCNA1 appears to be the primary replicative PCNA, both PfPCNA1 and PfPCNA2 participate in an active DDR (DNA-damage-response) pathway with significant accumulation in the parasite upon DNA damage induction. Interestingly, PfPCNA genes were found to be regulated not at the transcription level, but presumably at the protein stability level upon DNA damage. Such regulation of PCNA has not been shown in eukaryotes before. Moreover, overexpression of PfPCNA1 and PfPCNA2 in the parasite confers a survival edge on the parasite in a genotoxic environment. This is the first evidence of a PfPCNA-mediated DDR in the parasite and gives new insights and rationale for the presence of two PCNAs as a parasite survival strategy and its probable success.

The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing.

Plasmodium falciparum origin recognition complex 1 (ORC1) protein has been implicated in DNA replication and silencing var gene family. However, the mechanism and the domain structure of ORC1 related to the regulation of var gene family are unknown. Here we show that the unique N-terminus of PfORC1 (PfORC1N(1-238)) is targeted to the nuclear periphery in vivo and this region binds to the telomeric DNA in vitro due to the presence of a leucine heptad repeats. Like PfORC1N(1-238), endogenous full length ORC1, was found to be associated with sub telomeric repeat regions and promoters of various var genes. Additionally, binding and propagation of ORC1 to telomeric and subtelomeric regions was severely compromised in PfSir2 deficient parasites suggesting the dependence of endogenous ORC1 on Sir2 for var gene regulation. This feature is not previously described for Plasmodium ORC1 and contrary to yeast Saccharomyces cerevisiae where ORC function as a landing pad for Sir proteins. Interestingly, the overexpression of ORC1N(1-238) compromises the binding of Sir2 at the subtelomeric loci and var gene promoters consistent with de-repression of some var genes. These results establish role of the N-terminus of PfORC1 in heterochromatin formation and regulation of var gene expression in co-ordination with Sir2 in P. falciparum.

The role of Toxoplasma TFIIS-like protein in the early stages of mRNA transcription.

BACKGROUND: The mRNA transcription is a multistep process involving distinct sets of proteins associated with RNA polymerase II (RNAPII) through various stages. Recent studies have highlighted the role of RNAPII-associated proteins in facilitating the assembly of functional complexes in a crowded nuclear milieu. RNAPII dynamics and gene expression regulation have been primarily studied in model eukaryotes like yeasts and mammals and remain largely unchartered in protozoan parasites like Toxoplasma gondii, where considerable gene expression changes accompany stage differentiations. Here we report a key modulator of RNAPII activity, TFIIS in Toxoplasma gondii (TgTFIIS). METHODS: A Pull-down assay demonstrated that TgTFIIS binds to RNAPII subunit TgRPB1. Truncation mutants of TFIIS help us define the regions critical for its binding to TgRPB1. Co-immunoprecipitation analysis confirmed the interaction between the native TgTFIIS and TgRPB1. Confocal microscopy revealed a predominantly nuclear localization. Native TgTFIIS was able to bind promoter DNA which was consistent with the CHIP results. RESULTS: TgTFIIS complements initiation defects in yeast mutants, and the regions implicated in RNAPII binding appeared essential for this function. Interestingly, the C-terminal zinc finger domain necessary for its potential elongation function is dispensable for TgRPB1 binding. TgTFIIS was found to be associated with the promoter region along with its association with the ORF on an RNAPII transcribed gene. CONCLUSION: The observations were in line with the potential role of TgTFIIS in early events of RNAPII transcription in addition to elongation. GENERAL SIGNIFICANCE: The study elucidates the potential role of RNAPII-associated proteins in multiple steps of transcription.

A functionally divergent transcription elongation factor 1-like protein in Toxoplasma gondii.

Zinc ribbons, one of the largest fold groups among zinc fingers, often include proteins involved in the transcription machinery. Here, we identify and characterize one such zinc ribbon-bearing protein in the apicomplexan parasite Toxoplasma gondii, annotated as putative transcription elongation factor 1 (ELF1), with predicted functions in transcription and chromatin maintenance. We show that this ELF1 homolog, referred to as T. gondii ELF1-like divergent (TgELD), is expressed in both tachyzoite and bradyzoite developmental stages. TgELD associates with the cytoskeleton in the tachyzoites, while it transiently becomes a part of the cyst wall in the early bradyzoites, followed by a cytosolic and peripheral localization in late bradyzoites. TgELD is phosphorylated by a casein kinase 2-like protein, which has potential implications for its localization and function in the parasite.

Seroprevalence and associated risk factors of Toxoplasma gondii and Neospora caninum infections in cattle in Central India.

Toxoplasma gondii and Neospora caninum are closely related cyst-forming parasites identified as important causes of reproductive failures in ruminants. While these parasites have been reported worldwide, seroprevalence and associated risk factors for cattle infections have not been determined in India. A total of 576 serum samples of cattle were analyzed for antibodies to T. gondii and N. caninum using enzyme-linked immunosorbent assay (ELISA), modified/Neospora agglutination test (MAT/NAT), and an indirect fluorescent antibody test (IFAT-tachyzoite and bradyzoite). Additionally, general information about cattle, movement of cats and dogs, the menace of rodents, management, and reproductive disorders were assessed to identify the potential risk factors. Overall, 32.9% (190/576) serum samples reacted positively to T. gondii and 24.8% (143/576) to N. caninum. The performance of the diagnostic tests showed excellent agreement between IFAT and ELISA (kappa [κ] = 0.98) and between MAT/NAT and ELISA (κ = 0.97). Combining both infections on avidity test, 94% sera had high-IgG avidity, and 3% had low-IgG avidity antibodies, indicating chronic infection in the majority of the cases. The identified risk factors (p < 0.05) for exposure to T. gondii were: increasing age (Odds Ratio [OR]: 2.02), movement of cat (OR: 4.8) and rodents (OR: 1.57) in the farm; and for N. caninum: increasing age (OR: 1.6), movement of dogs in the farm (OR: 2.07), drinking pond water (OR: 1.64) and abortion (OR: 1.82). These findings revealed that T. gondii and N. caninum infections are widespread in the study area and suggest conducting nationwide epidemiological studies owing to their economic importance.

Preclinical disposition (in vitro) of novel µ-opioid receptor selective antagonists.

Recently, two novel N-heterocyclic derivatives of naltrexone [designated 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-[(4'-pyridyl)acetamido]morphinan (NAP) and 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl) acetamido]morphinan (NAQ)] have been proposed as µ-opioid receptor (MOR) selective antagonists. The goal of this study was to examine their absorption and metabolism. The bidirectional transport of NAP and NAQ was determined in Caco-2 and MDCKII-MDR1 cells, and the permeability directional ratio (PDR) was estimated (PDR = P(app, B-A)/P(app, A-B), where P(app) is the apparent permeability, A is apical, and B is basolateral). Oxidative metabolism of NAQ (0.5-80 µM) and NAP (0.5-30 µM) was determined in pooled human liver microsomes. The reaction monitored the disappearance of NAQ/NAP. NAP and NAQ were quantitated by high-performance liquid chromatography-UV at 270 or 232 nm, respectively. The permeability of NAQ or NAP was similar to that of naltrexone or paracellular markers, respectively. NAP also exhibited a high PDR and was determined to be a P-glycoprotein (P-gp) substrate. Unbound fractions in human plasma for NAQ and NAP were 0.026 ± 0.019 and 0.85 ± 0.12, respectively. The metabolic oxidative reaction rates, fitted to a Michaelis-Menten model, yielded K(m) and V(max) values of 15.8 ± 5.5 µM and 192 ± 24 pmol/min for NAQ and 1.8 ± 1.5 µM and 8.1 ± 1.4 pmol/min for NAP. Intrinsic hepatic clearance was estimated to be 13 and 5 ml · min(-1) · kg(-1) for NAQ and NAP, respectively. Neither NAQ nor NAP underwent detectable glucuronidation. Thus, NAP was a P-gp substrate with low apparent permeability, whereas NAQ was not a P-gp substrate and showed better permeability. Therefore, in contrast to NAP, NAQ would be more suitable for oral absorption and penetration of the blood-brain barrier, yielding potential pharmacokinetic and pharmacodynamic advantages over naltrexone.

Molecular chaperone function of stress inducible Hsp70 is critical for intracellular multiplication of Toxoplasma gondii.

Intracellular pathogens like Toxoplasma gondii often target proteins and pathways critical for host cell survival and stress response. Molecular chaperones encoded by the evolutionary conserved Heat shock proteins (Hsps) maintain proteostasis and are vital to cell survival following exposure to any form of stress. A key protein of this family is Hsp70, an ATP-driven molecular chaperone, which is stress inducible and often indiscernible in normal cells. Role of this protein with respect to intracellular survival and multiplication of protozoan parasite like T. gondii remains to be examined. We find that T. gondii infection upregulates expression of host Hsp70. Hsp70 selective inhibitor 2-phenylethynesulfonamide (PES) attenuates intracellular T. gondii multiplication. Biotinylated PES confirms selective interaction of this small molecule inhibitor with Hsp70. We show that PES acts by disrupting Hsp70 chaperone function which leads to dysregulation of host autophagy. Silencing of host Hsp70 underscores its importance for intracellular multiplication of T. gondii, however, attenuation achieved using PES is not completely attributable to host Hsp70 indicating the presence of other intracellular targets of PES in infected host cells. We find that PES is also able to target T. gondii Hsp70 homologue which was shown using PES binding assay. Detailed molecular docking analysis substantiates PES targeting of TgHsp70 in addition to host Hsp70. While establishing the importance of protein quality control in infection, this study brings to the fore a unique opportunity of dual targeting of host and parasite Hsp70 demonstrating how structural conservation of these proteins may be exploited for therapeutic design.

Seroprevalence and risk factors of Toxoplasma gondii infection among veterinary personnel and abattoir workers in Central India.

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is an important zoonotic infection. Veterinary personnel and abattoir workers are considered to be at a high risk of T. gondii infection owing to their occupational exposure. However, the association of T. gondii infection with occupational exposure to animals has not been determined in India. Hence, we analysed 139 and 126 blood samples of veterinary personnel and abattoir workers, respectively, for anti-T. gondii antibodies using enzyme-linked immunosorbent assay (ELISA), modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT). The association of seroprevalence with sociodemographic profiles, work activities and dietary habits was determined in the study population. MAT, ELISA and IFAT results demonstrated nearly 46%, 48% and 47% seropositivity, respectively. MAT (kappa = 0.924) and IFAT (kappa = 0.962) results showed good agreement with ELISA results. Of the ELISA positive samples, 46% was copositive for IgG antibody, 1.5% for IgM antibody and 1.5% for both IgG and IgM antibodies. High IgG avidity was observed only in IgG+ IgM- and IgG+ IgM+ samples and not in IgM+ IgG- samples, indicating chronic T. gondii infection in most of the cases. Furthermore, multivariate analysis revealed that T. gondii seropositivity was associated with age > 30 years (odds ration [OR] = 1.992), cat at home (OR = 1.991), not wearing gloves (OR = 1.886), not wearing safety glasses (OR = 1.985) and contact with soil (OR = 1.695). These findings support the presence of a potentially significant association between T. gondii seropositivity and occupational exposure to animals.

A new species and new record of the genus Platybaetis Mller-Liebenau, 1980 (Ephemeroptera: Baetidae) from Eastern Himalaya, India.

A new species of Platybaetis viz., P. selvai sp. nov. is described herein based on larval collections from Tangon stream in the state of Arunachal Pradesh in Eastern region of Indian Himalaya. It can be differentiated by the following combination of characters: (i) posterior margin of abdominal segments IX with rounded 'U shaped spines; (ii) anterolateral margin of gills IVII with minute setae; (iii) claw with 78 denticles; (iv) paracercus composed of 1011 segments; (v) hindwing pads reduced, small. Brief ecological notes are appended.