Jan Snoeys

Physiologically based pharmacokinetic modeling in drug discovery and development: A pharmaceutical industry perspective

The application of physiologically based pharmacokinetic (PBPK) modeling has developed rapidly within the pharmaceutical industry and is becoming an integral part of drug discovery and development. In this study, we provide a cross pharmaceutical industry position on “how PBPK modeling can be applied in industry” focusing on the strategies for application of PBPK at different stages, an associated perspective on the confidence and challenges, as well as guidance on interacting with regulatory agencies and internal best practices.

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Human ApoA-I Transfer Attenuates Transplant Arteriosclerosis via Enhanced Incorporation of Bone marrow–derived Endothelial Progenitor Cells

Objective—Transplant arteriosclerosis is the leading cause of graft failure and death in patients with heart transplantation. Endothelial progenitor cells (EPCs) contribute to endothelial regeneration in allografts. We investigated whether increased HDL cholesterol induced by adenoviral human apoA-I (AdA-I) transfer increases number and function of EPCs, promotes incorporation of EPCs in Balb/c allografts transplanted paratopically in C57BL/6 ApoE−/− mice, and attenuates transplant arteriosclerosis. Methods and Results—EPC number in ApoE−/− mice was increased after AdA-I transfer as evidenced by 1.5-fold (P<0.01) higher Flk-1 Sca-1–positive cells and 1.4-fold (P<0.01) higher DiI-acLDL isolectin-positive spleen cells. In addition, HDL enhanced EPC function in vitro. Incorporation of bone marrow–derived EPCs was 5.8-fold (P<0.01) higher at day 21 after transplantation in AdA-I-treated apoE−/− mice compared with control mice. Enhanced endothelial regeneration in AdA-I-treated apoE−/− mice as evidenced by a 2.6-fold (P<0.01) increase of CD31-positive endothelial cells resulted in a 1.4-fold (P=0.059) reduction of neointima and a 3.9-fold (P<0.01) increase of luminal area. Conclusion—Human apoA-I transfer increases the number of circulating EPCs, enhances their incorporation into allografts, promotes endothelial regeneration, and attenuates neointima formation in a murine model of transplant arteriosclerosis.

Species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the size of endothelial fenestrae

Sinusoidal fenestrae may restrict the transport of gene transfer vectors according to their size. Using Vitrobot technology and cryo-electron microscopy, we show that the diameter of human adenoviral serotype 5 vectors is 93u2009nm with protruding fibers of 30u2009nm. Thus, a diameter of fenestrae of 150u2009nm or more is likely to be sufficient for passage of vectors from the sinusoidal lumen to the space of Disse and subsequent uptake of vectors in hepatocytes. The average diameter of fenestrae in New Zealand White rabbits (103±1.3u2009nm) was 1.4-fold (P<0.0001) lower than in C57BL/6 mice (141±5.4u2009nm). The percentage of sinusoidal fenestrae with a diameter larger than 150u2009nm was 10-fold (P<0.01) lower in rabbits (3.2±0.24%) than in C57BL/6 mice (32±5%), and this resulted in 8.8-fold (P=0.01) lower transgene DNA levels in hepatocytes in rabbits after adenoviral transfer. Injection of N-acetylcysteine combined with transient liver ischemia preceding intraportal transfer in rabbits increased the percentage of sinusoidal fenestrae above 150u2009nm 2.0-fold (P<0.001) and increased transgene DNA levels in hepatocytes 6.6-fold (P<0.05). In conclusion, species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the diameter of fenestrae.

Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer

Hepatocytes are a key target for treatment of inborn errors of metabolism, dyslipidemia and coagulation disorders. The development of potent expression cassettes is a critical target to improve the therapeutic index of gene transfer vectors. Here we evaluated 22 hepatocyte-specific expression cassettes containing a human apo A-I transgene following hydrodynamic transfer of plasmids or adenoviral transfer with E1E3E4-deleted vectors in C57BL/6 mice. The DC172 promoter consisting of a 890u2009bp human α1-antitrypsin promoter and two copies of the 160u2009bp α1-microglobulin enhancer results in superior expression levels compared to constructs containing the 1.5u2009kb human α1-antitrypsin promoter, the 790u2009bp synthetic liver-specific promoter or the DC190 promoter containing a 520u2009bp human albumin promoter and two copies of the 99u2009bp prothrombin enhancer. The most potent expression cassette consists of the DC172 promoter upstream of the transgene and two copies of the hepatic control region-1. Minicircles containing this expression cassette induce persistent physiological human apo A-I or human factor IX levels after hydrodynamic transfer. In conclusion, in this comparative study of 22 hepatocyte-specific expression cassettes, the DC172 promoter in combination with two copies of the hepatic control region-1 induces the highest expression levels following hydrodynamic and adenoviral transfer.

From preclinical to human – prediction of oral absorption and drug–drug interaction potential using physiologically based pharmacokinetic (PBPK) modeling approach in an industrial setting: a workflow by using case example

A case example is presented in which the physiologically based modeling approach has been used to model the absorption of a lipophilic BCS Class II compound predominantly metabolized by CYP3A4, and to assess the interplay of absorption related parameters with the drug–drug interaction (DDI) potential.

Evaluation of miR-122 and Other Biomarkers in Distinct Acute Liver Injury in Rats

The detection of drug-induced hepatotoxicity remains an important safety issue in drug development. A liver-specific microRNA species, microRNA-122 (miR-122), has recently shown potential for predicting liver injury in addition to the standard hepatic injury biomarkers. The objective of this study was to measure miR-122 together with several other liver markers in distinct settings of acute liver toxicity in rats to determine the value of miR-122 as a biomarker for liver injury in this species. Rats were exposed to 3 well-established liver toxicants (acetaminophen, allyl alcohol, and α-naphthyl isothiocyanate), a liver-enzyme inducer (phenobarbital), or a cardiotoxicant (doxorubicin). There was a clear increase in plasma miR-122 following administration of acetaminophen, allyl alcohol, and α-naphthyl isothiocyanate. The response of miR-122 paralleled that of other markers and was consistent with liver injury as indicated by histopathological evaluation. Furthermore, the changes in miR-122 were detected earlier than standard liver injury markers and exhibited a wide dynamic range. In contrast, miR-122 responses to phenobarbital and doxorubicin were low. Based on these findings, miR-122 shows significant promise and may provide added value for assessing liver toxicity in drug development.

Drug–Drug Interactions with the NS3/4A Protease Inhibitor Simeprevir

Simeprevir is an NS3/4A protease inhibitor approved for the treatment of hepatitis C infection, as a component of combination therapy. Simeprevir is metabolized by the cytochrome P450 (CYP) system, primarily CYP3A, and is a substrate for several drug transporters, including the organic anion transporting polypeptides (OATPs). It is susceptible to metabolic drug–drug interactions with drugs that are moderate or strong CYP3A inhibitors (e.g. ritonavir and erythromycin) or CYP3A inducers (e.g. rifampin and efavirenz); coadministration of these drugs may increase or decrease plasma concentrations of simeprevir, respectively, and should be avoided. Clinical studies have shown that simeprevir is a mild inhibitor of CYP1A2 and intestinal CYP3A but does not inhibit hepatic CYP3A. The effects of simeprevir on these enzymes are of clinical relevance only for narrow-therapeutic-index drugs that are metabolized solely by these enzymes (e.g. oral midazolam). Simeprevir does not have a clinically relevant effect on the pharmacokinetics of rilpivirine, tacrolimus, oral contraceptives and several other drugs metabolized by CYP enzymes. Simeprevir is a substrate and inhibitor of the transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and OATP1B1/3. Cyclosporine is an inhibitor of OATP1B1/3, BCRP and P-gp, and a mild inhibitor of CYP3A; cyclosporine causes a significant increase in simeprevir plasma concentrations, and coadministration is not recommended. Clinical studies have demonstrated increases in coadministered drug concentrations for drugs that are substrates of the OATP1B1/3, BRCP (e.g. rosuvastatin) and P-gp (e.g. digoxin) transporters; these drugs should be administered with dose titration and or/close monitoring.

Evaluation of various static in vitro-in vivo extrapolation models for risk assessment of the CYP3A inhibition potential of an investigational drug

Nine static models (seven basic and two mechanistic) and their respective cutoff values used for predicting cytochrome P450 3A (CYP3A) inhibition, as recommended by the US Food and Drug Administration and the European Medicines Agency, were evaluated using data from 119 clinical studies with orally administered midazolam as a substrate. Positive predictive error (PPE) and negative predictive error (NPE) rates were used to assess model performance, based on a cutoff of 1.25‐fold change in midazolam area under the curve (AUC) by inhibitor. For reversible inhibition, basic models using total or unbound systemic inhibitor concentration [I] had high NPE rates (46–47%), whereas those using intestinal luminal ([I]gut) values had no NPE but a higher PPE. All basic models for time‐dependent inhibition had no NPE and reasonable PPE rates (15–18%). Mechanistic static models that incorporate all interaction mechanisms and organ specific [I] values (enterocyte and hepatic inlet) provided a higher predictive precision, a slightly increased NPE, and a reasonable PPE. Various cutoffs for predicting the likelihood of CYP3A inhibition were evaluated for mechanistic models, and a cutoff of 1.25‐fold change in midazolam AUC appears appropriate.

Pharmacokinetic Interaction between Telaprevir and Methadone

ABSTRACT Hepatitis C virus (HCV) antibody is present in most patients enrolled in methadone maintenance programs. Therefore, interactions between the HCV protease inhibitor telaprevir and methadone were investigated. The pharmacokinetics of R- and S-methadone were measured after administration of methadone alone and after 7 days of telaprevir (750 mg every 8 h [q8h]) coadministration in HCV-negative subjects on stable, individualized methadone therapy. Unbound R-methadone was measured in predose plasma samples before and during telaprevir coadministration. Safety and symptoms of opioid withdrawal were evaluated throughout the study. In total, 18 subjects were enrolled; 2 discontinued prior to receiving telaprevir. The minimum plasma concentration in the dosing interval (Cmin), the maximum plasma concentration (Cmax), and the area under the plasma concentration-time curve from h 0 (time of administration) to 24 h postdose (AUC0–24) for R-methadone were reduced by 31%, 29%, and 29%, respectively, in the presence of telaprevir. The AUC0–24 ratio of S-methadone/R-methadone was not altered. The median unbound percentage of R-methadone increased by 26% in the presence of telaprevir. The R-methadone median (absolute) unbound Cmin values in the absence (10.63 ng/ml) and presence (10.45 ng/ml) of telaprevir were similar. There were no symptoms of opioid withdrawal and no discontinuations due to adverse events. In summary, exposure to total R-methadone was reduced by approximately 30% in the presence of telaprevir, while the exposure to unbound R-methadone was unchanged. No symptoms of opioid withdrawal were observed. These results suggest that dose adjustment of methadone is not required when initiating telaprevir treatment. (This study has been registered at ClinicalTrials.gov under registration no. NCT00933283.)