Chronic and neurotropic: A paradigm-challenging case of dengue virus encephalitis in patient with advanced human immunodeficiency virus infection.

A 32-year-old female with advanced HIV infection presented to an Australian hospital with subacute but worsening symptoms of encephalitis. Metagenomic sequencing and Dengue NS3 antigen staining of brain tissue confirmed active Dengue virus (DENV) encephalitis. The most recent possible DENV exposure was months prior in West Africa, indicating chronicity.

Cluster of invasive Mycobacteria chimaera infections following cardiac surgery demonstrating novel clinical features and risks of aortic valve replacement.

There is a global outbreak of infections due to Mycobacterium chimaera associated with cardiac surgery. The most serious infections involve prosthetic material implantation, and all have followed surgical procedures involving cardiopulmonary bypass. We describe a cluster of four cases following cardiac surgery at a tertiary referral centre in Sydney, Australia. We report novel clinical findings, including haemolysis and kidney rupture possibly related to immune reconstitution inflammatory syndrome. The positive effect of corticosteroids on haemodynamic function in two cases and the failure of currently recommended antimicrobial therapy to sterilise prosthetic valve material in the absence of surgery despite months of treatment are also critically examined. Positron emission tomography was positive in two cases despite normal transoesophageal echocardiograms. The proportion of cases with M. chimaera infection after aortic valve replacement (4/890, 0.45%; 95% confidence interval 0.18-1.15%) was significantly higher than after all other cardiothoracic surgical procedures (0/2433, 0%; 95% confidence interval 0-0.16%).

Fanconi syndrome due to tenofovir disoproxil fumarate reversed by switching to tenofovir alafenamide fumarate in an HIV-infected patient.

A case of tenofovir-induced Fanconi syndrome in a patient receiving antiretroviral therapy for HIV infection, with resolution of the related electrolyte abnormalities upon switch from tenofovir disoproxil fumarate to tenofovir alafenamide fumarate, is reported. Tenofovir alafenamide fumarate, a novel prodrug of tenofovir containing significantly lower doses of tenofovir than tenofovir disoproxil fumarate, has been associated with a favourable renal profile compared to tenofovir disoproxil fumarate. Generally, the rare complication of tenofovir disoproxil fumarate-induced Fanconi syndrome is managed by cessation of tenofovir. There are limited reports of the impact of a switch strategy from tenofovir disoproxil fumarate to tenofovir alafenamide fumarate, which may be necessary in patients unable to discontinue tenofovir.

Species differences in sinusoidal and canalicular efflux transport of mycophenolic acid 7-O-glucuronide in sandwich-cultured hepatocytes.

Metabolism and sinusoidal/canalicular efflux of mycophenolic acid (MPA) was investigated using sandwich-cultured hepatocytes (SCHs). After applying MPA to SCHs from humans, wild-type rats, and multidrug resistance-associated protein (Mrp) 2-deficient rats, the MPA metabolites 7-O-glucuronide (MPAG) and acyl glucuronide (AcMPAG) were detected in the intracellular compartment of the SCHs. Sinusoidal efflux of MPAG was detected in all SCH preparations including Mrp2-deficient rat SCHs, whereas canalicular efflux of MPAG was observed in wild-type rat and human SCHs but not in Mrp2-deficient rat SCHs. The ratio of canalicular efflux to net (canalicular plus sinusoidal) efflux was 37 ± 8% in wild-type rat SCHs, while the ratio in human SCHs was significantly lower (20 ± 2%, P < 0.05), indicating species differences in the direction of hepatic MPAG transport. This 20% ratio in human SCHs corresponds to a high sinusoidal MPAG efflux (80%) that can in part account for the urine-dominated recovery of MPAG in humans. Both sinusoidal and canalicular MPAG efflux in rat SCHs shows a good correspondence to urinary and biliary recovery of MPAG after MPA dosing. The sinusoidal efflux of AcMPAG in human SCHs was detected from one out of three donors, suggesting donor-to-donor variation. In conclusion, this study demonstrates the predictive value of SCHs for elucidating the interplay of metabolism and efflux transport, in addition to demonstrating a species difference between rat and human in sinusoidal and canalicular efflux of MPAG.

Glucuronidation and subsequent biliary excretion of mycophenolic acid in rat sandwich-cultured hepatocytes.

Rat sandwich-cultured hepatocytes (SCH) were used to correlate the in vitro hepatic disposition of mycophenolic acid (MPA) with published in vivo data, as well as mechanistic studies on drug-drug interaction. The major metabolite of MPA in SCH was 7-O-glucuronide (MPAG) followed by acyl-glucuronide (AcMPAG). MPAG and AcMPAG, but not MPA, showed significant in vitro biliary excretion with biliary excretion indexes (BEI) of 40% for MPAG and 45% for AcMPAG. While these BEIs were similar, the biliary excretion amount (BEA) of MPAG (120 pmol/mg protein) was orders of magnitude higher than that of AcMPAG (0.34 pmol/mg protein). Since MPAG is the major metabolite in in vivo bile, we propose that BEA is a better qualifier of biliary excretion. Quercetin inhibited MPAG and AcMPAG production, while chrysin inhibited only MPAG production, showing that chrysin is not a pan-glucuronidation inhibitor. Cyclosporin A (CysA) reduced the BEI of MPAG and increased intracellular MPA accumulation without changing MPAG amounts. These results suggest that CysA causes inhibition of biliary excretion of MPAG, as well as a mixed inhibition of glucuronidation of MPA and sinusoidal efflux of MPA/MPAG. In conclusion, the present study demonstrates a good agreement of hepatic MPA disposition between SCH and in vivo rats.

A quantitative approach to hepatic clearance prediction of metabolism by aldehyde oxidase using custom pooled hepatocytes.

We describe the usability of human pooled hepatocytes for non-CYP metabolism evaluation and an in vivo-in vitro correlation analysis for aldehyde oxidase (AO) substrate compounds using pooled hepatocytes. By comparing intrinsic clearance values of 18 compounds primarily metabolized by AO, UDP-glucuronosyltransferase, carbonyl/aldo-keto reductase, flavin-containing monooxygenase, and monoamineoxidase in individual hepatocytes and pooled hepatocytes from the same individual donors, intrinsic clearance in the pooled hepatocytes was ± 30% of the average clearance value in individuals for 15 of 18 compounds, suggesting that pooled hepatocytes maintained the average activity of the individual hepatocytes. Although the results of an in vivo-in vitro correlation analysis for AO substrate compounds showed a trend toward under-prediction, the underestimation ratios for all AO substrates were nevertheless comparable (7.2- to 14.9-fold), suggesting that hepatic clearance prediction for these compounds can be quantified using empirical scaling. These observations enabled us to obtain specific pooled hepatocytes that showed the expected non-CYP enzyme activities by pre-characterization and to quantify hepatic clearance prediction for AO compounds using an empirical scaling factor.

A practical and direct comparison of intrinsic metabolic clearance of several non-CYP enzyme substrates in freshly isolated and cryopreserved hepatocytes.

Human hepatocytes are a physiologically relevant tool useful in evaluating liver-related pharmacokinetics, including non-cytochrome P-450 (CYP) metabolism, due to their broad spectrum of metabolic enzyme activity. To verify the usefulness of human hepatocytes in evaluating non-CYP metabolism for drug discovery, we compared intrinsic clearance values (CL(int)) in freshly isolated and cryopreserved hepatocytes using 14 compounds primarily metabolized by non-CYP enzymes, including UDP-glucuronosyltransferase, carbonyl/aldo-keto reductase, aldehyde oxidase, flavin-containing monooxygenase, and monoamineoxidase. Cryopreservation resulted in a >20% reduction (maximum: 50%) in CL(int) in 7/14 compounds (statistically significant for 5 compounds) on comparing CL(int) values in freshly isolated and cryopreserved hepatocytes from the same donors (n = 4). However, the number of compounds with >20% CL(int) reduction decreased to 3 on comparing average of CL(int) values including un-matched donors (dolasetron: -27%, naltorexone: -32%, and phthalazine: -48%; statistically significant for phthalazine, n = 6-11). These findings suggest that fresh hepatocytes are useful in evaluating intact non-CYP enzyme activities. However, we must note that the reduction in CL(int) by cryopreservation could be rendered negligible if high-activity lots are selected for assay. We therefore recommend using cryopreserved hepatocytes for large-scale screening for non-CYP metabolism in drug discovery research considering the advantages in usability with cryopreserved hepatocytes.

Comparative pharmacology of human dopamine D(2)-like receptor stable cell lines coupled to calcium flux through Galpha(qo5).

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.

A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors.

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening microARCS) that tests 8640 compounds in the footprint of a standard microtiter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the G alpha(q) protein can readily be assessed by fluorescence-based Ca(2+) release measurements, many GPCRs that are coupled to G alpha(s) or G alpha(i/o) proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D(4.4) receptor, which normally couples through the G alpha(i/o) protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca(2+) release by stably coexpressing this receptor with a chimeric G(alpha qo5) protein in HEK-293 cells. In microARCS format, the cells expressing D(4.4) receptor and G alpha(qo5) protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable microARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists.

Differential activation of dual signaling responses by human H1 and H2 histamine receptors.

Stimulation of human H1 and H2-histamine receptors (HRs) primarily activates signaling pathways to increase intracellular calcium [Ca2+]i and cyclic AMP (cAMP), respectively. Activation of H2-HR in human embryonic kidney (HEK) cells by histamine and dimaprit increases both cAMP formation and [Ca2+]i, as determined by cAMP-scintillation proximity assays and fluorescence imaging plate reader (FLIPR) assays. In HEK cells expressing relatively high levels of H2-HR (Bmax=26 pmol/mg protein), histamine and dimaprit are full agonists in eliciting cAMP responses with pEC50 values of 9.30 and 7.72 that are 1000-fold more potent than their respective pEC50 values of 6.13 and 4.91 for increasing [Ca2+]i. The agonist potencies decrease for both responses at lower H2-HR density (5 pmol/mg protein) and dimaprit exhibits partial agonist behavior for the [Ca2+]i response. The inverse agonists ranitidine and cimetidine more potently inhibit cAMP production in the higher expressing H2-HR line. Histamine also activated both signaling pathways via human H1-HRs highly expressed (Bmax=17 pmol/mg protein) in HEK cells, with a 1000-fold greater potency for [Ca2+]i vs. cAMP responses (pEC50=7.86 and 4.82, respectively). These studies demonstrate a markedly different potency for activation of multiple signaling pathways by H1- and H2-HRs that may contribute to the selectivity of histamine responses in vivo.

Use of a fluorescent imaging plate reader--based calcium assay to assess pharmacological differences between the human and rat vanilloid receptor.

The cloned vanilloid receptor 1 (VR1) is a ligand-gated calcium channel that is believed to be the capsaicin-activated vanilloid receptor found in native tissues, based on similarities regarding molecular mass, tissue distribution, and electrophysiological properties. Using a Fluorescent Imaging Plate Reader (FLIPR), along with Fluo-3 to signal intracellular calcium levels ([Ca(++)](i)), rat VR1 (rVR1) and a human orthologue (hVR1) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVR1 or hVR1, exhibited dose-dependent increases in [Ca(++)](i) when challenged with capsaicin (EC(50)s congruent with 10 nM). Responses to capsaicin were blocked by the VR1 antagonist capsazepine and were dependent on VR1 expression. Potencies for 10 structurally diverse VR1 agonists revealed rVR1 potencies highly correlated to that of hVR1 (R(2) = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVR1 compared to hVR1. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVR1, hVR1 was more sensitive to blockade by both antagonists. For both rVR1 and hVR1, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVR1 but showed rapid responses with rVR1. Thus, FLIPR technology can be used to readily reveal differences between rVR1 and hVR1 pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands.