Alessandro Schipani

Population Pharmacokinetic Modeling of the Association between 63396C→T Pregnane X Receptor Polymorphism and Unboosted Atazanavir Clearance

ABSTRACT Atazanavir (ATV) plasma concentrations are influenced by CYP3A4 and ABCB1, which are regulated by the pregnane X receptor (PXR; NR1I2). PXR expression is correlated with CYP3A4 in liver in the absence of enzyme inducers. The PXR single nucleotide polymorphism (SNP) 63396C→T (rs2472677) alters PXR expression and CYP3A4 activity in vitro, and we previously showed an association of this polymorphism with unboosted ATV plasma concentrations. The aim of this study was to develop a population pharmacokinetic analysis to quantify the impact of 63396C→T and diurnal variation on ATV clearance. A population analysis was performed with 323 plasma samples from 182 randomly selected patients receiving unboosted ATV. Two hundred fifty-nine of the blood samples were collected at random time points, and 11 patients had a full concentration-time profile at steady state. Nonlinear mixed effects modeling was applied to explore the effects of PXR 63396C→T, patient demographics, and diurnal variation. A one-compartment model with first-order absorption and lag time best described the data. Population clearance was 19.7 liters/h with interpatient variability or coefficient of variation (CV) of 21.5%. Homozygosity for the T allele for PXR 63396 was associated with a 17.0% higher clearance that was statistically significant. Evening dosing was associated with 34% higher bioavailability than morning dosing. Patient demographic factors had no effect on ATV clearance. These data show an association of PXR 63396C→T and diurnal variation on unboosted ATV clearance. The association is likely to be mediated through an effect on hepatic PXR expression and therefore expression of its target genes (e.g., CYP3A4, SLCO1B1, and ABCB1), which are known to be involved in ATV clearance.

Integration of population pharmacokinetics and pharmacogenetics: an aid to optimal nevirapine dose selection in HIV-infected individuals

Background Nevirapine is metabolized by CYP2B6 and polymorphisms within the CYP2B6 gene partly explain inter-patient variability in pharmacokinetics. The aim of this study was to model the complex relationship between nevirapine exposure, weight and genetics (based on combined analysis of CYP2B6 516G > T and 983T > C single nucleotide polymorphisms). Methods Non-linear mixed-effects modelling was used to estimate pharmacokinetic parameters from 275 patients. Simulations of the nevirapine concentration profile were performed with dosing regimens of 200 mg twice daily and 400 mg once daily for individuals with body weights of 50, 70 and 90 kg in combination with CYP2B6 genetic variation. Results A one-compartment model with first-order absorption best described the data. Population clearance was 3.5 L/h with inter-patient variability of 24.6%. 516T homozygosity and 983C heterozygosity were associated with 37% and 40% lower clearance, respectively. Body weight was the only significant demographic factor influencing clearance, which increased by 5% for every 10 kg increase. For individuals with higher body weight, once-daily nevirapine was associated with a greater risk of sub-therapeutic drug exposure than a twice-daily regimen. This risk was offset in individuals who were 516T homozygous or 983C heterozygous in which drug exposure was optimal for  > 95% of patients with body weight of ≤70 kg. Conclusions The data suggest that a 400 mg once-daily dose could be implemented in accordance with CYP2B6 polymorphism and body weight. However, the use of nevirapine once daily (immediate release; off-label) in the absence of therapeutic drug monitoring is not recommended due to the risk of inadequate exposure to nevirapine in a high proportion of patients. There are different considerations for the extended-release formulation (nevirapine XR) that demonstrate minimal peak-to-trough fluctuations in plasma nevirapine levels.

Antiretroviral solid drug nanoparticles with enhanced oral bioavailability: production, characterization, and in vitro-in vivo correlation.

Nanomedicine strategies have produced many commercial products. However, no orally dosed HIV nanomedicines are available clinically to patients. Although nanosuspensions of drug particles have demonstrated many benefits, experimentally achieving >25 wt% of drug relative to stabilizers is highly challenging. In this study, the emulsion-templated freeze-drying technique for nanoparticles formation is applied for the first time to optimize a nanodispersion of the leading non-nucleoside reverse transcriptase inhibitor efavirenz, using clinically acceptable polymers and surfactants. Dry monoliths containing solid drug nanoparticles with extremely high drug loading (70 wt% relative to polymer and surfactant stabilizers) are stable for several months and reconstitute in aqueous media to provide nanodispersions with z-average diameters of 300 nm. The solid drug nanoparticles exhibit reduced cytoxicity and increased in vitro transport through model gut epithelium. In vivo studies confirm bioavailability benefits with an approximately four-fold higher pharmacokinetic exposure after oral administration to rodents, and predictive modeling suggests dose reduction with the new formulation may be possible.

Pharmacokinetic and Pharmacodynamic Analysis of Efavirenz Dose Reduction Using an In Vitro–In Vivo Extrapolation Model

The pharmacokinetics (PK) of efavirenz (EFV) is characterized by marked interpatient variability that correlates with its pharmacodynamics (PD). In vitro–in vivo extrapolation (IVIVE) is a “bottom‐up” approach that combines drug data with system information to predict PK and PD. The aim of this study was to simulate EFV PK and PD after dose reductions. At the standard dose, the simulated probability was 80% for viral suppression and 28% for central nervous system (CNS) toxicity. After a dose reduction to 400 mg, the probabilities of viral suppression were reduced to 69, 75, and 82%, and those of CNS toxicity were 21, 24, and 29% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. With reduction of the dose to 200 mg, the probabilities of viral suppression decreased to 54, 62, and 72% and those of CNS toxicity decreased to 13, 18, and 20% for the 516 GG, 516 GT, and 516 TT genotypes, respectively. These findings indicate how dose reductions might be applied in patients with favorable genetic characteristics.

Estimation of the effect of SLCO1B1 polymorphisms on lopinavir plasma concentration in HIV-infected adults.

BACKGROUND The organic anion transporting polypeptides (OATP)/SLCO family represents an important class of hepatic drug uptake transporters that mediate the sodium independent transport of a diverse range of amphipathic organic compounds, including the protease inhibitors. The SLCO1B1 521T>C (rs4149056) single nucleotide polymorphism (SNP) has been consistently associated with reduced transport activity in vivo, and we previously showed an association of this polymorphism with lopinavir plasma concentrations. The aim of this study was to develop a population pharmacokinetic (PK) model to quantify the impact of 521T>C. METHODS A population PK analysis was performed with 594 plasma samples from 375 patients receiving lopinavir/ritonavir. Non-linear mixed effects modelling was applied to explore the effects of SLCO1B1 521T>C and patient demographics. Simulations of the lopinavir concentration profile were performed with different dosing regimens considering the different alleles. RESULTS A one-compartment model with first-order absorption best described the data. Population clearance was 5.67 l/h with inter-patient variability of 37%. Body weight was the only demographic factor influencing clearance, which increased 0.5 l/h for every 10 kg increase. Homozygosity for the C allele was associated with a 37% lower clearance, and 14% for heterozygosity, which were statistically significant. CONCLUSIONS These data show an association between SLCO1B1 521T>C and lopinavir clearance. The association is likely to be mediated through reduced uptake by hepatocytes leading to higher plasma concentrations of lopinavir. Further studies are now required to confirm the association and to assess the influence of other polymorphisms in the SLCO family on lopinavir PK.

CYP3A4*22 (c.522-191 C>T; rs35599367) is associated with lopinavir pharmacokinetics in HIV-positive adults.

The CYP3A4*22 (c.522-191 C>T; rs35599367) single nucleotide polymorphism has been associated with lower CYP3A4 mRNA expression and activity. We investigated the association of CYP3A4*22 with the pharmacokinetics of lopinavir through a population pharmacokinetic approach. The minor allele frequency for CYP3A4*22 was 0.035, and seven of 375 patients had a combination of CYP3A4*22 and SLCO1B1 521T>C alleles. Lack of information on the ethnicity in this cohort should be considered as a limitation. However, in the final model, the population clearance was 5.9 l/h and patients with CYP3A4*22/*22 had 53% (P=0.023) lower clearance compared with noncarriers. In addition, the combined effect of CYP3A4*22 with SLCO1B1 521T>C (previously shown to be associated with lopinavir plasma concentration) was analysed. We observed a 2.3-fold higher lopinavir trough concentration (Ctrough) in individuals with CYP3A4*22/*22, a 1.8-fold higher Ctrough with SLCO1B1 521CC and a 9.7-fold higher Ctrough in individuals homozygous for both single nucleotide polymorphisms, compared with noncarriers. A simulated dose-reduction scenario showed that 200/100 mg lopinavir/ritonavir was adequate to achieve therapeutic concentration in individuals with CYP3A4*22/*22 alone or in combination with SLCO1B1 521CC. These data further our understanding of the genetic basis for variability in the pharmacokinetics of lopinavir.

Observational infant exploratory [14C]‐paracetamol pharmacokinetic microdose/therapeutic dose study with accelerator mass spectrometry bioanalysis

AIMS The aims of the study were to compare [(14)C]-paracetamol ([(14)C]-PARA) paediatric pharmacokinetics (PK) after administration mixed in a therapeutic dose or an isolated microdose and to develop further and validate accelerator mass spectrometry (AMS) bioanalysis in the 0-2 year old age group. METHODS [(14)C]-PARA concentrations in 10-15 µl plasma samples were measured after enteral or i.v. administration of a single [(14)C]-PARA microdose or mixed in with therapeutic dose in infants receiving PARA as part of their therapeutic regimen. RESULTS Thirty-four infants were included in the PARA PK analysis for this study: oral microdose (n = 4), i.v. microdose (n = 6), oral therapeutic (n = 6) and i.v. therapeutic (n = 18). The respective mean clearance (CL) values (SDs in parentheses) for these dosed groups were 1.46 (1.00) l h(-1), 1.76 (1.07) l h(-1), 2.93 (2.08) l h(-1) and 2.72 (3.10) l h(-1), t(1/2) values 2.65 h, 2.55 h, 8.36 h and 7.16 h and dose normalized AUC(0-t) (mg l(-1) h) values were 0.90 (0.43), 0.84 (0.57), 0.7 (0.79) and 0.54 (0.26). CONCLUSIONS All necessary ethical, scientific, clinical and regulatory procedures were put in place to conduct PK studies using enteral and systemic microdosing in two European centres. The pharmacokinetics of a therapeutic dose (mg kg(-1)) and a microdose (ng kg(-1)) in babies between 35 to 127 weeks post-menstrual age. [(14)C]-PARA pharmacokinetic parameters were within a two-fold range after a therapeutic dose or a microdose. Exploratory studies using doses significantly less than therapeutic doses may offer ethical and safety advantages with increased bionalytical sensitivity in selected exploratory paediatric pharmacokinetic studies.

Simultaneous Population Pharmacokinetic Modelling of Atazanavir and Ritonavir in HIV-Infected Adults and Assessment of Different Dose Reduction Strategies

Objectives:The objective of this study was to develop a simultaneous population pharmacokinetic (PK) model to describe atazanavir/ritonavir (ATV/RTV) PK (300/100 mg) and to assess the effect of RTV dose reduction on ATV PK. Simulations of ATV concentration–time profiles were performed at doses of ATV/RTV 300/50 mg, 200/50 mg, and 200/100 mg once daily. Methods:A total of 288 ATV and 312 RTV plasma concentrations from 30 patients were included to build a population PK model using the stochastic approximation expectation maximization algorithm implemented in MONOLIX 3.2 software. Results:A one-compartment model with first-order absorption and lag-time best described the data for both drugs in the final simultaneous model. A maximum-effect model in which RTV inhibited the elimination of ATV was used to describe the relationship between RTV concentrations and ATV clearance (CL/F). An RTV concentration of 0.22 mg/L was associated with 50% maximum inhibition of ATV CL/F. The population prediction of ATV CL/F in the absence of RTV was 16.6 L/h (relative standard error, 7.0%), and the apparent volume of distribution and absorption rate constant were 106 L (relative standard error, 8%) and 0.87 per hour (fixed), respectively. Simulated average ATV trough concentrations at ATV/RTV 300/50 mg, 200/50 mg, and 200/100 mg once daily were 45%, 63%, and 33% lower, respectively, than that of the standard dose. Conclusion:Although simulated median ATV trough concentrations after dose reductions were still more than the ATV minimum effective concentration (2.9-, 1.9-, and 3.6-fold for ATV/RTV 300/50 mg, 200/50 mg, and 200/100 mg, respectively); our modeling predicted a high proportion of individuals with subtherapeutic trough concentrations on the 200/50 mg dose. This suggests that 300/50 mg and 200/100 mg dosing are preferred candidate regimens in future clinical studies.

Investigation of Elimination Rate, Persistent Subpopulation Removal, and Relapse Rates of Mycobacterium tuberculosis by Using Combinations of First-Line Drugs in a Modified Cornell Mouse Model

ABSTRACT Currently, the most effective tuberculosis control method involves case finding and 6 months of chemotherapy. There is a need to improve our understanding about drug interactions, combination activities, and the ability to remove persistent bacteria using the current regimens, particularly in relation to relapse. We aimed to investigate the therapeutic effects of three main components, rifampin (RMP), isoniazid (INH), and pyrazinamide (PZA), in current drug regimens using a modified version of the Cornell mouse model. We evaluated the posttreatment levels of persistent Mycobacterium tuberculosis in the organs of mice using culture filtrate derived from M. tuberculosis strain H37Rv. When RMP was combined with INH, PZA, or INH-PZA, significant additive activities were observed compared to each of the single-drug treatments. However, the combination of INH and PZA showed a less significant additive effect than either of the drugs used on their own. Apparent culture negativity of mouse organs was achieved at 14 weeks of treatment with RMP-INH, RMP-PZA, and RMP-INH-PZA, but not with INH-PZA, when conventional tests, namely, culture on solid agar and in liquid broth, indicated that the organs were negative for bacteria. The relapse rates for RMP-containing regimens were not significantly different from a 100% relapse rate at the numbers of mice examined in this study. In parallel, we examined the organs for the presence of culture filtrate-dependent persistent bacilli after 14 weeks of treatment. Culture filtrate treatment of the organs revealed persistent M. tuberculosis. Modeling of mycobacterial elimination rates and evaluation of culture filtrate-dependent organisms showed promise as surrogate methods for efficient factorial evaluation of drug combinations in tuberculosis in mouse models and should be further evaluated against relapse. The presence of culture filtrate-dependent persistent M. tuberculosis is the likely cause of disease relapse in this modified Cornell mouse model.

Evaluation of universal versus genotype-guided efavirenz dose reduction in pregnant women using population pharmacokinetic modelling

Objectives Lack of data on the pharmacokinetics of efavirenz in pregnant women at the 400 mg reduced dose currently prevents universal roll-out. Population pharmacokinetic modelling was used to explore pharmacokinetic endpoints at 200, 400 and 600 mg daily doses in pregnant women stratified by CYP2B6 metabolic status. Methods The analysis was based on 252 plasma efavirenz concentrations from 77 pregnant women (77 sparse, 175 intensive) who received antiretroviral regimens containing 600 mg of efavirenz. The model was developed using NONMEM®. The effect of genetics was investigated and concentration-time courses at steady-state were simulated for individuals (n = 1000 each) classified as CYP2B6 slow, intermediate and fast metabolizers at 200, 400 and 600 mg daily doses. Results At a 400 mg reduced dose, predicted mean (90% CI) mid-dose efavirenz concentration (C12) was 2.24 μg/mL (0.89-4.18) in pregnant women classified as slow metabolizers, compared with 0.87 μg/mL (0.34-1.64) in intermediate metabolizers and 0.78 μg/mL (0.30-1.47) in fast metabolizers. C12 was below the 0.47 μg/mL threshold determined within the ENCORE 1 trial in 10% at 400 mg, 4.6% at 600 mg and 3.4% with genotype-guided dosing. The 4.0 μg/mL toxicity threshold was exceeded in 4.6% at 400 mg, 13.5% at 600 mg and 5.2% with genotype-guided dosing. Conclusions These data provide context for the ongoing debate about reduction in efavirenz dose to 400 mg during pregnancy and should be interpreted alongside the lower toxicity expected with the lower dose. Additional research is required to investigate genotype-guided dose reduction in pregnant women.