Enhancing the Endocytosis of Phosphatidylserine-Containing Liposomes through Tim4 by Modulation of Membrane Fluidity.

Phosphatidylserine (PS) is a unique lipid that is recognized by the endogenetic receptor, T-cell immunoglobulin mucin protein 4 (Tim4), and PS-containing liposomes have potential use in therapeutic applications. We prepared PS-containing liposomes of various lipid compositions and examined how lipid membrane fluidity affects PS recognition by Tim4 and the resulting endocytosis efficiency into Hela cells. Surface plasmon resonance and laurdan studies showed that increasing lipid membrane fluidity increased the stability of the PS-Tim4 interaction but hampered the entry of liposomes into cells. These results show that endocytosis efficiency is determined by balancing opposing forces induced by membrane fluidity. We found that inclusion of the zwitterionic helper lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, into liposomes ensured efficient cellular internalization because the presence of this lipid provides an ideal balance of lipid fluidity and Tim4 affinity. The results showed that PS recognition by Tim4 and the resulting endocytosis efficiency can be maximized by modulating the membrane fluidity of liposomes by selecting a zwitterionic helper lipid. This study improves our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

Effect of Sample Concentration on Nanoparticle Tracking Analysis of Small Extracellular Vesicles and Liposomes Mimicking the Physicochemical Properties of Exosomes.

For quantitative analysis, data should be obtained at a sample concentration that is within the range of linearity. We examined the effect of sample concentration on nanoparticle tracking analysis (NTA) of small extracellular vesicles (sEVs), including exosomes, by comparing NTA results of sEVs with those obtained for polystyrene nanoparticles (PSN) and liposomes, which mimic lipid composition and physicochemical properties of exosomes. Initially, NTA of PSN at different concentrations was performed and the particle sizes determined were validated by dynamic light scattering. The major peak maxima for PSN mixtures of different sizes at the higher particle numbers were similar, with some fluctuation of the minor peak maxima observed at the lower particle number, which was also observed for sEVs. Sample concentration is critical for obtaining reproducible data for liposomes and exosomes and increasing the sample concentration caused an increase in data variability because of particle interactions. The inter-day repeatability of particles sizes and concentration for sEVs were 7.47 and 4.51%, respectively. Analysis of the linearity range revealed that this was narrower for sEVs when compared with that of liposomes. Owing to the use of liposomes that mimic the lipid composition and physicochemical properties of exosomes and proteinase-treated sEVs, it was demonstrated that these different analytical results could be possibly caused by the protein corona of sEVs. Consideration of the sample concentration and linearity range is important for obtaining reproducible and reliable data of sEVs.

First-in-human study to assess the safety, pharmacokinetics and pharmacodynamics of BMS-962212, a direct, reversible, small molecule factor XIa inhibitor in non-Japanese and Japanese healthy subjects.

AIMS: The aims of the present study were to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of BMS-962212, a first-in-class factor XIa inhibitor, in Japanese and non-Japanese healthy subjects. METHODS: This was a randomized, placebo-controlled, double-blind, sequential, ascending-dose study of 2-h (part A) and 5-day (part B) intravenous (IV) infusions of BMS-962212. Part A used four doses (1.5, 4, 10 and 25 mg h-1 ) of BMS-962212 or placebo in a 6:2 ratio per dose. Part B used four doses (1, 3, 9 and 20 mg h-1 ) enrolling Japanese (n = 4 active, n = 1 placebo) and non-Japanese (n = 4 active, n = 1 placebo) subjects per dose. The PK, PD, safety and tolerability were assessed throughout the study. RESULTS: BMS-962212 was well tolerated; there were no signs of bleeding, and adverse events were mild. In parts A and B, BMS-962212 demonstrated dose proportionality. The mean half-life in parts A and B ranged from 2.04 to 4.94 h and 6.22 to 8.65 h, respectively. Exposure-dependent changes were observed in the PD parameters, activated partial thromboplastin time (aPTT) and factor XI clotting activity (FXI:C). The maximum mean aPTT and FXI:C change from baseline at 20 mg h-1 in part B was 92% and 90%, respectively. No difference was observed in weight-corrected steady-state concentrations, aPTT or FXI:C between Japanese and non-Japanese subjects (P > 0.05). CONCLUSION: BMS-962212 has tolerability, PK and PD properties suitable for investigational use as an acute antithrombotic agent in Japanese or non-Japanese subjects.

Single- and multiple-ascending dose studies to evaluate the safety, tolerability, and pharmacokinetics of daclatasvir and asunaprevir in healthy male Japanese subjects.

OBJECTIVES: Assess the safety, tolerability, and pharmacokinetic (PK) profiles of daclatasvir (DCV) and asunaprevir (ASV) in healthy male Japanese subjects. METHODS: AI444-007 and AI447-005 were phase I, double-blind, placebo-controlled, sequential, single-ascending dose (SAD), and multiple-ascending dose (MAD) studies assessing DCV or ASV, respectively. Eight subjects per panel were randomized to study drug or placebo (3 : 1). In the SAD part of each study, subjects received single oral dose DCV 1/10/50/100/200 mg or ASV 200/400/600/900/1,200 mg. In MAD, subjects received 14-day oral multiple dose DCV 1/10/100 mg once-daily or ASV 200/400/600 mg every 12 hours. Serial PK blood sampling occurred from predose to 72-hours postdose or post-last-dose. Safety and tolerability was assessed throughout. RESULTS: 64 (SAD, n = 40; MAD, n = 24) and 65 (SAD, n = 40; MAD, n = 25) subjects were enrolled in AI444-007 and AI447-005, respectively. DCV and ASV were generally well tolerated, with no serious adverse events or clinicallyrelevant changes in vital signs or ECG parameters. Baseline demographic characteristics were comparable across treatment groups in both studies. DCV was readily absorbed, with median tmax of ~ 1 - 2 hours postdose and concentrations declining in a multi-phasic manner. Exposure generally increased dose-proportionally within dose-range studied. Steady-state was achieved between days 4 and 5 of multiple dosing. ASV was readily absorbed, with median tmax of ~ 2 - 4 hours postdose and concentrations declining in a biphasic manner. Exposure generally increased dose-proportionally within dose-range studied. Steady-state appeared to be achieved between days 3 - 5 of multiple dosing. CONCLUSIONS: Results suggest no clinically significant short-term safety signals with DCV and ASV at single or multiple doses in this population.

A State-of-the-Art Roadmap for Biomarker-Driven Drug Development in the Era of Personalized Therapies.

Advances in biotechnology have enabled us to assay human tissue and cells to a depth and resolution that was never possible before, redefining what we know as the "biomarker", and how we define a "disease". This comes along with the shift of focus from a "one-drug-fits-all" to a "personalized approach", placing the drug development industry in a highly dynamic landscape, having to navigate such disruptive trends. In response to this, innovative clinical trial designs have been key in realizing biomarker-driven drug development. Regulatory approvals of cancer genome sequencing panels and associated targeted therapies has brought personalized medicines to the clinic. Increasing availability of sophisticated biotechnologies such as next-generation sequencing (NGS) has also led to a massive outflux of real-world genomic data. This review summarizes the current state of biomarker-driven drug development and highlights examples showing the utility and importance of the application of real-world data in the process. We also propose that all stakeholders in drug development should (1) be conscious of and efficiently utilize real-world evidence and (2) re-vamp the way the industry approaches drug development in this era of personalized medicines.

Safety, pharmacokinetics, and pharmacodynamics of milvexian in healthy Japanese participants.

This randomized, double-blind, placebo-controlled, multiple ascending-dose study evaluated safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple doses of milvexian, an oral small-molecule FXIa inhibitor, in healthy Japanese participants. Participants received oral milvexian daily under fasted (50 mg and 200 mg) or fed conditions (500 mg) or placebo over 14 days; 24 participants (8/cohort: 6 milvexian; 2 placebo) were planned. Due to an unblinding event, participants in one cohort (200 mg daily) were discontinued, and a second cohort enrolled; 32 participants were included in safety and pharmacodynamic analyses, and 24/32 in pharmacokinetic analyses. Milvexian up to 500 mg daily for 14 days was generally well tolerated, with no deaths, serious adverse events, or discontinuations due to adverse events. Milvexian exposure increased between 50-mg and 200-mg doses. Median Tmax was similar with 50-mg and 200-mg doses (2.5-3.0 h) and delayed under fed conditions (500 mg, 7.0-8.0 h). Median T1/2 was similar across doses (8.9-11.9 h). Multiple oral milvexian administrations resulted in concentration-related prolongation of aPTT and decreased FXI clotting activity. Milvexian was generally safe and well tolerated. The pharmacokinetic and pharmacodynamic profile of milvexian demonstrates suitability for further clinical development in Japanese participants.

Safety Exposure-Response Analysis for Daclatasvir, Asunaprevir, and Beclabuvir Combinations in HCV-Infected Subjects.

The combination regimen of daclatasvir, asunaprevir, and beclabuvir has been developed for the treatment of hepatitis C virus infection. The objectives of this analysis were to characterize the relationship between the exposures of the daclatasvir, asunaprevir, and beclabuvir regimen and liver-related laboratory elevations (Grade 3 or 4 alanine aminotransferase [ALT] and total bilirubin [Tbili]), and to evaluate the impact of selected covariates on the exposure-response relationships. The exposure-response analysis was performed with data from 1 phase 2 and 3 phase 3 studies in hepatitis C virus-infected subjects. The probability of liver-related laboratory elevations were modeled using linear logistic regression. Selected covariates were tested using a forward-addition and backward-elimination approach. The final model for ALT elevation included Asian race, body weight in non-Asian subjects, and asunaprevir exposure. The final model for Tbili elevation included Asian race, fibrosis score (F0-F3 or F4) and asupanprevir exposure. Asian subjects had greater the Grade 3 or 4 ALT and Tbili elevation rates than non-Asians. The Grade 3 or 4 ALT elevation rate increased with decreasing body weight in non-Asian subjects. Subjects with F4 fibrosis score had a higher rate of Grade 3 or 4 Tbili elevation compared to subjects with F0 to F3 fibrosis score. Higher asunaprevir exposure was associated with increases in Grade 3 or 4 ALT and Tbili elevation rates; however, the impact on the ALT elevation was not clinically relevant and the effect on Tbili elevation was smaller than the other significant covariates.

Population Pharmacokinetic Analysis of Daclatasvir, Asunaprevir, and Beclabuvir Combination in HCV-Infected Subjects.

A fixed-dose combination of daclatasvir (pangenotypic NS5A inhibitor), asunaprevir (NS3/4A protease inhibitor), and beclabuvir (nonnucleoside NS5B inhibitor) was approved for hepatitis C virus treatment in Japan. The objectives of the analyses were to develop the daclatasvir, asunaprevir, and beclabuvir population pharmacokinetic models for the combination regimen. First, an original population pharmacokinetic model was developed using the data in non-Japanese hepatitis C virus-infected subjects. The model was subsequently updated after a phase 3 study in Japanese hepatitis C virus-infected subjects was available. A total of 11,382, 11,300, and 10,728 pharmacokinetic records from 1,228 subjects were included for daclatasvir, asunaprevir, and beclabuvir in the updated model, respectively. Daclatasvir and beclabuvir pharmacokinetics (PK) were described by a 1-compartment model with linear elimination and asunaprevir PK was described by 2-compartment model with linear elimination. Cirrhosis, baseline, and time-varying ALT were significant covariates on asunaprevir apparent oral clearance. Asian subjects had greater asunaprevir and beclabuvir exposures than white subjects. The effects of all covariates on daclatasvir PK were modest and not considered clinically significant. With the exception of race on asunaprevir and beclabuvir PK, no other parameters for daclatasvir, asunaprevir and beclabuvir population PK models were meaningfully impacted during the refinement with Japanese subjects.

Exposure-Response (Efficacy) Analysis of Daclatasvir and Asunaprevir in Japanese Patients With Hepatitis C Virus Infection.

The treatment of hepatitis C virus (HCV) infection has been revolutionized by the development of all-oral combination regimens of direct-acting antiviral agents. The current analysis characterized the relationship between exposures of daclatasvir (DCV; tablets) and asunaprevir (ASV; capsules) and sustained virologic response (SVR) in Japanese patients who are HCV genotype (GT) 1b nonresponders to pegylated interferon (IFN) α/ribavirin or IFNß/ribavirin, and IFN-based therapy-ineligible naive/intolerant patients receiving DCV and ASV, and provided insight into patient covariates that were most closely associated with efficacy. The relationship between the probability of achieving SVR at 12 weeks after treatment (SVR12) and average steady-state plasma concentrations estimated from population pharmacokinetic models for DCV and ASV is described using a logistic regression model with data from a phase 2 and a phase 3 study in Japanese patients infected with HCV GT 1b (N=265). The functional form characterization, which describes a relationship between DCV and ASV average steady-state plasma concentrations and SVR12, as well as covariate identification (demographic, laboratory, and prognostic and treatment covariates) were investigated during model development. The presence of the signature nonstructural protein 5A Y93H mutation at baseline was the only significant parameter of SVR12 in the final exposure-response model. Model evaluation plots demonstrate that the final model was able to predict the observed SVR rates. Exposure-response analysis supports the clinical utility of the combination regimen of 60-mg once-daily DCV and 100-mg twice-daily ASV in Japanese patients infected with HCV GT 1b.

Population Pharmacokinetic Analysis for Daclatasvir and Asunaprevir in Japanese Subjects With Chronic Hepatitis C Virus Infection.

Daclatasvir is a nonstructural protein 5A replication complex inhibitor, and asunaprevir is a nonstructural protein 3 protease inhibitor for hepatitis C virus (HCV). In 2014, the combination therapy of daclatasvir and asunaprevir received the first global approval in Japan as the first nonribavirin, all-oral therapy for HCV treatment. The population pharmacokinetics (popPK) of daclatasvir and asunaprevir were characterized by nonlinear mixed-effects modeling using 3801 and 2626 concentration data from 336 and 265 Japanese HCV subjects, respectively. The plasma pharmacokinetic profiles of daclatasvir and asunaprevir were described by a 1-compartment model. Parameter estimates (interindividual variability) of daclatasvir apparent clearance (CL/F) and apparent volume of the central compartment (V/F) were 5.29 L/h (39.4%) and 64.2 L (38.1%). The effects of all statistically significant covariates on daclatasvir PK parameters were within or overlapped the 80% to 125% boundaries, suggesting a lack of clinical relevance. Parameter estimates (interindividual variability) of asunaprevir CL/F and V/F were 52.1 L/h (41.5%) and 75.1 L (93.4%), respectively. Baseline and time-varying aspartate aminotransferase (AST) and cirrhosis on CL/F and formulation (soft-gel capsule or tablet) on F were included as significant covariates in the asunaprevir popPK model. The effects of all covariates exceeded the 80% to 125% boundaries, indicating that the asunaprevir soft-gel capsule had higher bioavailability than the tablet and that asunaprevir exposure increased with cirrhosis and increasing baseline and time-varying AST values. The popPK models adequately described the PK profiles of daclatasvir and asunaprevir in Japanese HCV subjects.

Population Pharmacokinetics of Apixaban in Subjects With Nonvalvular Atrial Fibrillation.

This analysis describes the population pharmacokinetics (PPK) of apixaban in nonvalvular atrial fibrillation (NVAF) subjects, and quantifies the impact of intrinsic and extrinsic factors on exposure. The PPK model was developed using data from phase I-III studies. Apixaban exposure was characterized by a two-compartment PPK model with first-order absorption and elimination. Predictive covariates on apparent clearance included age, sex, Asian race, renal function, and concomitant strong/moderate cytochrome P450 (CYP)3A4/P-glycoprotein (P-gp) inhibitors. Individual covariate effects generally resulted in < 25% change in apixaban exposure vs. the reference NVAF subject (non-Asian, male, aged 65 years, weighing 70 kg without concomitant CYP3A4/P-gp inhibitors), except for severe renal impairment, which resulted in 55% higher exposure than the reference subject. The dose-reduction algorithm resulted in a ~27% lower median exposure, with a large overlap between the 2.5-mg and 5-mg groups. The impact of Asian race on apixaban exposure was < 15% and not considered clinically significant.

Population Pharmacokinetic Analysis of Asunaprevir in Subjects with Hepatitis C Virus Infection.

INTRODUCTION: Asunaprevir (ASV) is a potent, pangenotypic, twice-daily hepatitis C virus (HCV) NS3 inhibitor indicated for the treatment of chronic HCV infection. METHODS: A population pharmacokinetic (PPK) model was developed using pooled ASV concentration data from 1239 HCV-infected subjects who received ASV either as part of the DUAL regimen with daclatasvir or as part of the QUAD regimen with daclatasvir and peg-interferon/ribavirin. RESULTS: A two-compartment model with first-order elimination from the central compartment, an induction effect on clearance, and an absorption model consisted of zero-order release followed by first-order absorption adequately described ASV PK after oral administration. A typical value for ASV clearance (CL/F) was 50.8 L/h, increasing by 43% after 2 days to a CL/F of 72.5 L/h at steady-state, likely due to auto-induction of cytochrome P450 3A4 (CYP3A4). Factors indicative of hepatic function were identified as key influential covariates on ASV exposures. Subjects with cirrhosis had an 84% increase in ASV area under the concentration time curve (AUC) and subjects with baseline aspartate aminotransferase (AST) above 78 IU/L had a 58% increase in area under the concentration time curve (AUC). Asians subjects had a 46% higher steady-state AUC relative to White/Caucasian subjects. Other significant covariates were formulation, age, and gender. CONCLUSION: The current PPK model provided a parsimonious description of ASV concentration data in HCV-infected subjects. Key covariates identified in the model help explain the observed variability in ASV exposures and may guide clinical use of the drug. FUNDING: Bristol-Myers Squibb.

Population Pharmacokinetic Analysis of Daclatasvir in Subjects with Chronic Hepatitis C Virus Infection.

BACKGROUND AND OBJECTIVE: Daclatasvir is a potent, pangenotypic once-daily hepatitis C virus (HCV) NS5A inhibitor that is approved for the treatment of chronic HCV infection. The objective of this analysis was to characterize the pharmacokinetics of daclatasvir in subjects with chronic HCV infection. METHODS: A population pharmacokinetic (PPK) model was developed to evaluate effects of covariates on daclatasvir pharmacokinetics in subjects with chronic HCV infection (n = 2149 from 11 studies). All significant demographic, laboratory, prognostic and treatment covariates (p < 0.05) from univariate screening were included in the full model. The final model was reached by backward elimination (p < 0.001) and simulations were performed to further evaluate the effects of covariates on daclatasvir exposures. The plasma pharmacokinetics of daclatasvir was described by a two-compartment model with linear elimination. Absorption was modeled as a zero-order release followed by a first-order absorption into the central compartment. RESULTS: The typical value of apparent clearance (CL/F) was 5.7 L/h (1.58% relative standard error [RSE]) and of apparent volume of the central compartment (V c/F) was 58.6 L (2.00% RSE). Modest inter-individual variability was estimated for CL/F (35.1%) and V c/F (29.5%). Statistically significant covariates in the final model were sex, race, virus genotype, baseline creatinine clearance, and alanine aminotransferase (ALT) on CL/F and sex, race, and body weight on V c/F. Covariate effects demonstrated a 30% higher area under the plasma concentration-time curve at steady state (AUCss) in female subjects; effects of all other covariates were <16%. CONCLUSIONS: The model adequately described the daclatasvir pharmacokinetics and estimated relatively small covariate effects. Considering the exposure range for the therapeutic dose of daclatasvir 60 mg once daily and the favorable safety profile, the small difference in exposures due to these covariates is not considered clinically relevant.