The simultaneous inhibition of SLC6A19 and BCRP transporters leads to an increase of indoxyl sulfate (a uremic toxin) in plasma and kidney.

SLC6A19 inhibitors are being studied as therapeutic agents for Phenylketonuria. In this work, a potent SLC6A19 inhibitor (RA836) elevated rat kidney uremic toxin indoxyl sulfate (IDS) levels by intensity (arbitrary unit) of 13.7{plus minus}7.7 compared to vehicle 0.3{plus minus}0.1 (P=0.01) as determined by tissue mass spectrometry imaging (tMSI) analysis. We hypothesized that increased plasma and kidney levels of IDS could be caused by the simultaneous inhibition of both Slc6a19 and a kidney IDS transporter responsible for excretion of IDS into urine. To test this, we first confirmed the formation of IDS through tryptophan metabolism by feeding rats a Trp-free diet. Inhibiting Slc6a19 with RA836 led to increased IDS in these rats. Next, RA836 and its key metabolites were evaluated in vitro for inhibiting kidney transporters OAT1, OAT3 and BCRP. RA836 inhibits BCRP with an IC50 of 0.045 µM but shows no significant inhibition of OAT1 or OAT3. Finally, RA836 analogs with either potent or no inhibition of SLC6A19 and/or BCRP were synthesized and administered to rats fed a normal diet. Plasma and kidney samples were collected to quantify IDS using LC-MS. Neither a SLC6A19 inactive but potent BCRP inhibitor nor a SLC6A19 active but weak BCRP inhibitor raised IDS levels, while compounds inhibiting both transporters caused IDS accumulation in rat plasma and kidney, supporting the hypothesis that rat Bcrp contributes to the excretion of IDS. In summary, we identified that inhibiting Slc6a19 increases IDS formation, while simultaneously inhibiting Bcrp results in IDS accumulation in the kidney and plasma. Significance Statement This is the first publication to decipher the mechanism for accumulation of IDS (a uremic toxin) in rats via inhibition of both Slc6a19 and Bcrp. Specifically, inhibition of Slc6a19 in the GI track increases IDS formation and inhibition of Bcrp in the kidney blocks IDS excretion. Therefore, we should avoid inhibiting both SLC6A19 and BCRP simultaneously in humans to prevent accumulation of IDS, a known risk factor for cardiovascular disease, psychic anxiety, and mortality in chronic kidney disease patients.

A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells.

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.

Impedance Measurement in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Impedance-based measurement is a useful tool for assessing the contractility of plated cardiomyocytes in the context of early preclinical cardiosafety assessment. Induced pluripotent stem cell-derived cardiomyocytes (iPSCs) can be used for this purpose as these cells display similar electrochemical properties to primary cardiomyocytes and beat reliably and in synchronicity in culture. Here we describe a method for measuring the contractility of iPSCs using the xCELLigence RTCA impedance measurement system.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4ß,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4ß,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4ß,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4ß-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Sulforaphane is not an effective antagonist of the human pregnane X-receptor in vivo.

Sulforaphane (SFN), is an effective in vitro antagonist of ligand activation of the human pregnane and xenobiotic receptor (PXR). PXR mediated CYP3A4 up-regulation is implicated in adverse drug-drug interactions making identification of small molecule antagonists a desirable therapeutic goal. SFN is not an antagonist to mouse or rat PXR in vitro; thus, normal rodent species are not suitable as in vivo models for human response. To evaluate whether SFN can effectively antagonize ligand activation of human PXR in vivo, a three-armed, randomized, crossover trial was conducted with 24 healthy adults. The potent PXR ligand - rifampicin (300mg/d) was given alone for 7days in arm 1, or in daily combination with 450µmol SFN (Broccoli Sprout extract) in arm 2; SFN was given alone in arm 3. Midazolam as an in vivo phenotype marker of CYP3A was administered before and after each treatment arm. Rifampicin alone decreased midazolam AUC by 70%, indicative of the expected increase in CYP3A4 activity. Co-treatment with SFN did not reduce CYP3A4 induction. Treatment with SFN alone also did not affect CYP3A4 activity in the cohort as a whole, although in the subset with the highest basal CYP3A4 activity there was a statistically significant increase in midazolam AUC (i.e., decrease in CYP3A4 activity). A parallel study in humanized PXR mice yielded similar results. The parallel effects of SFN between humanized PXR mice and human subjects demonstrate the predictive value of humanized mouse models in situations where species differences in ligand-receptor interactions preclude the use of a native mouse model for studying human ligand-receptor pharmacology.

The dietary isothiocyanate sulforaphane is an antagonist of the human steroid and xenobiotic nuclear receptor.

Sulforaphane (SFN) is a biologically active phytochemical found abundantly in broccoli. SFN has been promoted as a putative chemopreventive agent to reduce cancer, and most studies have associated its anti-cancer effects with the induction of phase II xenobiotic metabolism enzymes via activation of the Keap1/Nrf2 antioxidant response pathway. Interestingly, SFN can significantly down-regulate cytochrome P450 3A4 (CYP3A4) expression in human primary hepatocytes. CYP3A4 is responsible for the hepatic and intestinal metabolism of numerous protoxicants, pharmaceutical compounds, and endogenous sterols. Among the most important mediators of CYP3A4 expression is the nuclear hormone receptor, steroid and xenobiotic receptor (SXR; also called "hPXR"). SXR functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Here, we report that SFN is a specific antagonist of human SXR and that it inhibits SXR-mediated induction of drug clearance. SFN can bind directly to SXR, inhibit SXR coactivator recruitment, and efficiently repress SXR activities. Furthermore, SFN inhibited SXR-mediated CYP3A4 expression and CYP3A4-catalyzed midazolam clearance in human primary hepatocytes. Thus, SFN is the first identified naturally occurring antagonist for SXR (hPXR). Because induction of CYP3A4 can result in adverse drug responses (e.g., lack of efficacy), which are a major public health problem, this discovery could lead to the development of important new therapeutic and dietary approaches to reduce the frequency of undesirable inducer-drug interactions.