Efficacy and safety of an inhaled pan-Janus kinase inhibitor, nezulcitinib, in hospitalised patients with COVID-19: results from a phase 2 clinical trial.

BACKGROUND: The inhaled lung-selective pan-Janus kinase inhibitor nezulcitinib had favourable safety and potential efficacy signals in part 1 of a phase 2 trial in patients with severe COVID-19, supporting progression to part 2. METHODS: Part 2 was a randomised, double-blind phase 2 study (NCT04402866). Hospitalised patients aged 18-80 years with confirmed symptomatic COVID-19 requiring supplemental oxygen (excluding baseline invasive mechanical ventilation) were randomised 1:1 to nebulised nezulcitinib 3 mg or placebo for up to 7 days with background standard-of-care therapy (including corticosteroids). Efficacy endpoints included respiratory failure-free (RFF) days through day 28 as the primary endpoint. Secondary endpoints included safety and change from baseline oxygen saturation (SaO2)/fraction of inspired oxygen (FiO2) ratio on day 7, and 28-day mortality rate was a prespecified exploratory endpoint. RESULTS: Between June 2020 and April 2021, 205 patients were treated (nezulcitinib, 103; placebo, 102). There was no statistically significant difference between nezulcitinib versus placebo in the primary endpoint (RFF days; median, 21.0 vs 21.0; p=0.6137) or secondary efficacy endpoints. Nezulcitinib was generally well tolerated with a favourable safety profile. CONCLUSIONS: Although the prespecified primary, secondary and exploratory efficacy endpoints, including RFF through day 28, change from baseline SaO2/FiO2 ratio on day 7, and 28-day mortality rate, were not met, nezulcitinib was generally well tolerated and had a favourable safety profile. Further studies are required to determine if treatment with nezulcitinib confers clinical benefit in specific inflammatory biomarker-defined populations of patients with COVID-19.

A randomized, double-blind, placebo-controlled, phase 2b study of the efficacy and safety of velusetrag in subjects with diabetic or idiopathic gastroparesis.

BACKGROUND: This study assessed the efficacy and safety of velusetrag-a 5-HT4 agonist with pan-gastrointestinal prokinetic activity-for gastroparesis symptom management and gastric emptying (GE). METHODS: In this multicenter, double-blind, randomized, placebo-controlled study, subjects with diabetic or idiopathic gastroparesis received velusetrag 5, 15, or 30 mg or placebo for 12 weeks. The primary efficacy outcome was a 7-day mean Gastroparesis Cardinal Symptom Index 24-h composite score (GCSI-24H) change from baseline at week 4; GE was evaluated using scintigraphy (GES) and breath tests, and safety from adverse events (AEs). KEY RESULTS: 232 subjects (183 females; 113 idiopathic gastroparesis) received treatment from February 2015 through June 2017. Least-squares mean improvement from baseline GCSI-24H (primary endpoint) at week 4 was -1.5 following velusetrag 5 mg vs -1.1 following placebo (treatment difference, -0.4; 95% confidence interval, -0.75 to -0.03; nominal p = 0.0327; Hochberg-adjusted p = 0.0980 [not significant]). Symptom improvement from baseline was achieved only with velusetrag 5 mg, which resulted in greater improvement from baseline vs placebo in all gastroparesis core symptoms, especially in subjects with idiopathic gastroparesis. Improvement from baseline GE by GES was greater in subjects receiving velusetrag (all doses) vs placebo; >70% of subjects receiving velusetrag 30 mg had GE normalization at 4 h. Treatment-emergent AEs were generally mild. CONCLUSIONS AND INFERENCES: Velusetrag treatment was generally well-tolerated and associated with improved GE vs placebo in subjects with diabetic or idiopathic gastroparesis; however, only the lowest dose, velusetrag 5 mg, was associated with short-term improvement in gastroparesis symptoms. CLINICALTRIALS: GOV: NCT02267525.

Phase I study in healthy participants to evaluate safety, tolerability, and pharmacokinetics of inhaled nezulcitinib, a potential treatment for COVID-19.

Nezulcitinib (TD-0903), a lung-selective pan-Janus-associated kinase (JAK) inhibitor designed for inhaled delivery, is under development for treatment of acute lung injury associated with coronavirus disease 2019 (COVID-19). This two-part, double-blind, randomized, placebo-controlled, single ascending dose (part A) and multiple ascending dose (part B) phase I study evaluated the safety, tolerability, and pharmacokinetics (PK) of nezulcitinib in healthy participants. Part A included three cohorts randomized 6:2 to receive a single inhaled dose of nezulcitinib (1, 3, or 10 mg) or matching placebo. Part B included three cohorts randomized 8:2 to receive inhaled nezulcitinib (1, 3, or 10 mg) or matching placebo for 7 days. The primary outcome was nezulcitinib safety and tolerability assessed from treatment-emergent adverse events (TEAEs). The secondary outcome was nezulcitinib PK. All participants completed the study. All TEAEs were mild or moderate in severity, and none led to treatment discontinuation. Overall (area under the plasma concentration-time curve) and peak (maximal plasma concentration) plasma exposures of nezulcitinib were low and increased in a dose-proportional manner from 1 to 10 mg in both parts, with no suggestion of clinically meaningful drug accumulation. Maximal plasma exposures were below levels expected to result in systemic target engagement, consistent with a lung-selective profile. No reductions in natural killer cell counts were observed, consistent with the lack of a systemic pharmacological effect and the observed PK. In summary, single and multiple doses of inhaled nezulcitinib at 1, 3, and 10 mg were well-tolerated in healthy participants, with dose-proportional PK supporting once-daily administration.

Adalimumab long-term safety: infections, vaccination response and pregnancy outcomes in patients with rheumatoid arthritis.

BACKGROUND: Adalimumab has been used in patients with moderately to severely active rheumatoid arthritis (RA) for over 10 years and has a well-established safety profile across multiple indications. OBJECTIVE: To update adverse events (AEs) of special interest from global adalimumab clinical trials in patients with RA. METHODS: This analysis includes 15 132 patients exposed to adalimumab in global RA clinical trials. AEs of interest included overall infections, laboratory abnormalities and AEs associated with influenza vaccination. Pregnancy outcome data were collected from the Adalimumab Pregnancy Registry. RESULTS: Serious infections and tuberculosis occurred at a rate of 4.7 and 0.3 events/100 patient-years, respectively. Two patients experienced hepatitis B reactivation. No significant laboratory abnormalities were reported with adalimumab-plus-methotrexate compared with placebo-plus-methotrexate. Influenza-related AEs occurred in 5% of vaccinated patients compared with 14% of patients not vaccinated during the study. Relative risk of major birth defects and spontaneous abortions in adalimumab-exposed women were similar between that of unexposed women with RA and healthy women. CONCLUSIONS: This analysis confirms and expands the known safety profile of adalimumab and reports no additional safety risk of laboratory abnormalities, hepatitis B reactivation and pregnancy outcomes, including spontaneous abortions and birth defects. The benefits of influenza vaccination are reinforced. TRIAL REGISTRATION NUMBERS: NCT00195663, NCT00195702, NCT00448383, NCT00049751, NCT00234845, NCT00650390, NCT00235859, NCT00647920, NCT00649545, NCT00647491, NCT00649922, NCT00538902, NCT00420927, NCT00870467, NCT00650156, NCT00647270, NCT01185288, NCT01185301.

Species differences in hepatobiliary disposition of taurocholic acid in human and rat sandwich-cultured hepatocytes: implications for drug-induced liver injury.

The bile salt export pump (BSEP) plays an important role in bile acid excretion. Impaired BSEP function may result in liver injury. Bile acids also undergo basolateral efflux, but the relative contributions of biliary (CLBile) versus basolateral efflux (CLBL) clearance to hepatocellular bile acid excretion have not been determined. In the present study, taurocholic acid (TCA; a model bile acid) disposition was characterized in human and rat sandwich-cultured hepatocytes (SCH) combined with pharmacokinetic modeling. In human SCH, biliary excretion of TCA predominated (CLBile = 0.14 ± 0.04 ml/min per g liver; CLBL = 0.042 ± 0.019 ml/min per g liver), whereas CLBile and CLBL contributed approximately equally to TCA hepatocellular excretion in rat SCH (CLBile = 0.34 ± 0.07 ml/min per g liver; CLBL = 0.26 ± 0.07 ml/min per g liver). Troglitazone decreased TCA uptake, CLBile, and CLBL; membrane vesicle assays revealed for the first time that the major metabolite, troglitazone sulfate, was a noncompetitive inhibitor of multidrug resistance-associated protein 4, a basolateral bile acid efflux transporter. Simulations revealed that decreased CLBile led to a greater increase in hepatic TCA exposure in human than in rat SCH. A decrease in both excretory pathways (CLBile and CLBL) exponentially increased hepatic TCA in both species, suggesting that 1) drugs that inhibit both pathways may have a greater risk for hepatotoxicity, and 2) impaired function of an alternate excretory pathway may predispose patients to hepatotoxicity when drugs that inhibit one pathway are administered. Simulations confirmed the protective role of uptake inhibition, suggesting that a drug's inhibitory effects on bile acid uptake also should be considered when evaluating hepatotoxic potential. Overall, the current study precisely characterized basolateral efflux of TCA, revealed species differences in hepatocellular TCA efflux pathways, and provided insights about altered hepatic bile acid exposure when multiple transport pathways are impaired.

An experimental approach to evaluate the impact of impaired transport function on hepatobiliary drug disposition using Mrp2-deficient TR- rat sandwich-cultured hepatocytes in combination with Bcrp knockdown.

Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR(-)) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1-10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR(-) status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR(-) rat SCH decreased by 28.6 (95% CI: 5.8-51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5-9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR(-) rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR(-) rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0-12.8) and 7.5 (3.7-11.3), respectively, relative to noninfected SCH. These findings may be due to off-target effects on hepatic bile acid transporters, even though no changes in protein expression levels of the hepatic bile acid transporters were observed. This study established a strategy for optimization of the knockdown system, and demonstrated the potential use of RNAi in SCH as an in vitro tool to predict altered hepatobiliary drug disposition when canalicular transporters are impaired.

Role of hepatic efflux transporters in regulating systemic and hepatocyte exposure to xenobiotics.

Hepatic efflux transporters include numerous well-known and emerging proteins localized to the canalicular or basolateral membrane of the hepatocyte that are responsible for the excretion of drugs into the bile or blood, respectively. Altered function of hepatic efflux transporters due to drug-drug interactions, genetic variation, and/or disease states may lead to changes in xenobiotic exposure in the hepatocyte and/or systemic circulation. This review focuses on transport proteins involved in the hepatocellular efflux of drugs and metabolites, discusses mechanisms of altered transporter function as well as the interplay between multiple transport pathways, and highlights the importance of considering intracellular unbound concentrations of transporter substrates and/or inhibitors. Methods to evaluate hepatic efflux transport and predict the effects of impaired transporter function on systemic and hepatocyte exposure are discussed, and the sandwich-cultured hepatocyte model to evaluate comprehensively the role of hepatic efflux in the hepatobiliary disposition of xenobiotics is characterized.

Hepatic basolateral efflux contributes significantly to rosuvastatin disposition II: characterization of hepatic elimination by basolateral, biliary, and metabolic clearance pathways in rat isolated perfused liver.

Basolateral efflux clearance (CLBL) contributes significantly to rosuvastatin (RSV) elimination in sandwich-cultured hepatocytes (SCH). The contribution of CLBL to RSV hepatic elimination was determined in single-pass isolated perfused livers (IPLs) from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor, elacridar (GF120918); clearance values were compared with SCH. RSV biliary clearance (CLBile) was ablated almost completely by GF120918 in TR(-) IPLs, confirming that Mrp2 and Bcrp primarily are responsible for RSV CLBile. RSV appearance in outflow perfusate was attributed primarily to CLBL, which was impaired in TR(-) IPLs. CLBL was ≈ 6-fold greater than CLBile in the linear range in WT IPLs in the absence of GF120918. Recovery of unchanged RSV in liver tissue increased in TR(-) compared with WT (≈ 25 versus 6% of the administered dose) due to impaired CLBL and CLBile. RSV pentanoic acid, identified by high-resolution liquid chromatography-tandem mass spectroscopy, comprised ≈ 40% of total liver content and ≈ 16% of the administered dose in TR(-) livers at the end of perfusion, compared with ≈ 30 and 3% in WT livers, consistent with impaired RSV excretion and "shunting" to the metabolic pathway. In vitro-ex vivo extrapolation between WT SCH and IPLs (without GF120918) revealed that uptake clearance and CLBL were 4.2- and 6.4-fold lower, respectively, in rat SCH compared with IPLs; CLBile translated almost directly (1.1-fold). The present IPL data confirmed the significant role of CLBL in RSV hepatic elimination, and demonstrated that both CLBL and CLBile influence RSV hepatic and systemic exposure.

Hepatic basolateral efflux contributes significantly to rosuvastatin disposition I: characterization of basolateral versus biliary clearance using a novel protocol in sandwich-cultured hepatocytes.

Transporters responsible for hepatic uptake and biliary clearance (CLBile) of rosuvastatin (RSV) have been well characterized. However, the contribution of basolateral efflux clearance (CLBL) to hepatic and systemic exposure of RSV is unknown. Additionally, the appropriate design of in vitro hepatocyte efflux experiments to estimate CLBile versus CLBL remains to be established. A novel uptake and efflux protocol was developed in sandwich-cultured hepatocytes (SCH) to achieve desired tight junction modulation while maintaining cell viability. Subsequently, studies were conducted to determine the role of CLBL in the hepatic disposition of RSV using SCH from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor elacridar (GF120918). RSV CLBile was nearly ablated by GF120918 in TR(-) SCH, confirming that Mrp2 and Bcrp are responsible for the majority of RSV CLBile. Pharmacokinetic modeling revealed that CLBL and CLBile represent alternative elimination routes with quantitatively similar contributions to the overall hepatocellular excretion of RSV in rat SCH under baseline conditions (WT SCH in the absence of GF120918) and also in human SCH. Membrane vesicle experiments revealed that RSV is a substrate of MRP4 (Km = 21 ± 7 µM, Vmax = 1140 ± 210 pmol/min per milligram of protein). Alterations in MRP4-mediated RSV CLBL due to drug-drug interactions, genetic polymorphisms, or disease states may lead to changes in hepatic and systemic exposure of RSV, with implications for the safety and efficacy of this commonly used medication.

Determination of intracellular unbound concentrations and subcellular localization of drugs in rat sandwich-cultured hepatocytes compared with liver tissue.

Prediction of clinical efficacy, toxicity, and drug-drug interactions may be improved by accounting for the intracellular unbound drug concentration (C(unbound)) in vitro and in vivo. Furthermore, subcellular drug distribution may aid in predicting efficacy, toxicity, and risk assessment. The present study was designed to quantify the intracellular C(unbound) and subcellular localization of drugs in rat sandwich-cultured hepatocytes (SCH) compared with rat isolated perfused liver (IPL) tissue. Probe drugs with distinct mechanisms of hepatocellular uptake and accumulation were selected for investigation. Following drug treatment, SCH and IPL tissues were homogenized and fractionated by differential centrifugation to enrich for subcellular compartments. Binding in crude lysate and cytosol was determined by equilibrium dialysis; the C(unbound) and intracellular-to-extracellular C(unbound) ratio (K(pu,u)) were used to describe accumulation of unbound drug. Total accumulation (K(pobserved)) in whole tissue was well predicted by the SCH model (within 2- to 3-fold) for the selected drugs. Ritonavir (K(pu,u) ∼1) was evenly distributed among cellular compartments, but highly bound, which explained the observed accumulation within liver tissue. Rosuvastatin was recovered primarily in the cytosolic fraction, but did not exhibit extensive binding, resulting in a K(pu,u) >1 in liver tissue and SCH, consistent with efficient hepatic uptake. Despite extensive binding and sequestration of furamidine within liver tissue, a significant portion of cellular accumulation was attributed to unbound drug (K(pu,u) >16), as expected for a charged, hepatically derived metabolite. Data demonstrate the utility of SCH to predict quantitatively total tissue accumulation and elucidate mechanisms of hepatocellular drug accumulation such as active uptake versus binding/sequestration.

Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues.

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 µg mL(-1) (0.040 µg mg(-1)) to 20 µg mL(-1) (4.0 µg mg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.

Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity.

Sandwich-cultured hepatocytes (SCH) are a powerful in vitro tool that can be utilized to study hepatobiliary drug transport, species differences in drug transport, transport protein regulation, drug-drug interactions, and hepatotoxicity. This review provides an up-to-date summary of the SCH model, including a brief history of, and introduction to, the use of SCH, as well as methodology to evaluate hepatobiliary drug disposition. A summary of the literature that has utilized this model to examine the interplay between drug-metabolizing enzymes and transport proteins, drug-drug interactions at the transport level, and hepatotoxicity as a result of altered hepatic transport also is provided.

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells.

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.