Current drug discovery strategies for treatment of hepatitis C virus infection.

OBJECTIVES: Hepatitis C virus (HCV) infection represents a major worldwide-health problem. The current standard of care is combination therapy with pegylated interferon and ribavirin, which achieves a successful response in only approximately 40% of genotype I patients. KEY FINDINGS: The biology of HCV infection has been under intensive research and important progress has been made in understanding the replication cycle of the virus. Several therapeutic targets have been under investigation, such as NS3 protease, NS4A replicase and NS5B polymerase. New potential targets, such as NS2 protease, as well as CD-81 and claudin-1 entry co-receptors, have also been identified. SUMMARY: Clinical evaluations of drug candidates targeting NS3 protease, NS4A cofactor, and NS5B polymerase have demonstrated the potential of developing small molecules that interfere with the replication of the virus. Additional issues, including genotype coverage, resistant mutations, and combination therapy represent major challenges for future drug discovery efforts.

Use of intrinsic clearance for prediction of human hepatic clearance.

IMPORTANCE OF THE FIELD: The use of intrinsic metabolic stability/clearance and other in vitro pharmacokinetic data for the selection of drug candidates for clinical evaluation during discovery lead optimization has become one of the primary focuses of research organizations involved in new drug discovery. Using intrinsic clearance determined from human liver microsomal preparations and/or hepatocyte to predict human clearance has become more acceptable. AREAS COVERED IN THIS REVIEW: This review focuses on the current methods for determining intrinsic clearance and scaling to predict human hepatic clearance, and novel physiologically-based models for improvement of human hepatic clearance prediction. Published microsomal metabolic stability data and in-house hepatocyte clearance data were compared with published in vivo human hepatic clearance data. Various scaling models and the effect of protein binding were examined. WHAT THE READER WILL GAIN: Use of a novel microfluidic model and other physiologically-based models are presented. Microsomal metabolic clearance requires correction for protein binding and in vitro microsomal binding in order to better predict in vivo hepatic clearance of compounds that are mainly eliminated by hepatic metabolism. TAKE HOME MESSAGE: Metabolic clearance obtained using hepatocytes may work well in combination with the well-stirred model. Novel models incorporating flow and protein binding in the system may be the most complete models for prediction of human in vivo metabolism.

The role of P-glycoprotein in the pharmacokinetics and tissue distribution of a hepatitis C virus protease inhibitor.

S5, a hepatitis C virus protease inhibitor, displays partially saturable efflux in the Caco-2 system. In addition, the efflux can be reversed by cyclosporine, indicating that S5 may be a human P-glycoprotein (P-gp) substrate. S5 can also activate the ATPase activity in vesicle membranes containing mouse P-gp 1a and 1b, suggesting that S5 may be a substrate for mouse P-gp. The pharmacokinetics and tissue distribution of S5 were evaluated after intravenous and oral administration to wild-type and 1a/1b knockout mice. Plasma and kidney levels of this compound in knockout mice were transiently higher than those in wild-type mice only after oral dosing, indicating effective P-gp efflux occurs in wild-type mice. The levels of S5 in brain samples from knockout mice were higher than those from wild-type mice after both intravenous and oral administration, but much more significantly after intravenous administration. The levels in liver were four time higher in knockout mice than in wild-type mice after oral administration, but were not different between knockout and wild-type mice after intravenous administration. These results suggest that P-gp efflux limits exposure to S5 in the brain and liver, and that the effect is dependent on the route of administration.

Co-induction of CYP3A12 and 3A26 in dog liver slices by xenobiotics: species difference between human and dog CYP3A induction.

The induction of dog CYP3A12 and CYP3A26 mRNA levels was evaluated in liver slices after treatment with 22 xenobiotics. Eleven of the 22 xenobiotics increased 3A12 mRNA by more than four-fold, while nine did the same for 3A26 mRNA. A four-fold increase in the mRNA level was used as the cut-off for indication of induction based on the noise level of the real time-PCR. A good correlation was found between the mRNA levels for 3A12 and 3A26 after treatment with compounds, suggesting that these two CYPs may be co-induced. Induction of CYP3A4 in human hepatocytes was evaluated after treatment with the same 22 compounds. Thirteen out of the 22 compounds increased the 3A4 mRNA levels by more than four-fold. When the mRNA levels of 3A4 and 3A12 were compared after treatment with compounds, no correlation was found. The regulation of CYP3A expression has been demonstrated to be controlled by pregnane X receptor (PXR). Upon examination of the sequence homology and the three-dimensional structures of human PXR and a dog PXR model, only two different amino acids (met323/val and arg410/lys) were found in the ligand-binding domain. This finding suggests that these two amino acids may play a role in the binding specificity of ligands.

Permeability evaluation of peptidic HCV protease inhibitors in Caco-2 cells-correlation with in vivo absorption predicted in humans.

The permeability of six peptidic hepatitis C virus (HCV) protease inhibitors, with molecular weights ranging from 500 to 780, was examined in the Caco-2 cell system. All six compounds permeated the cells transcellularly; paracellular permeability, evaluated in the Caco-2 cell system by reducing the calcium concentration in the media to increase the pore size of the tight junctions, most likely contributes only minimally to the oral absorption of the compounds. All six compounds were shown to be efflux substrates displaying concentration-dependent saturation of efflux. The efflux could be blocked by cyclosporine A, a specific P-glycoprotein (P-gp) inhibitor, suggesting that P-gp may be the responsible transporter. Oral absorption in rats was calculated using in vivo oral bioavailability and hepatic extraction ratios. Human oral absorption was projected to be similar to that of rats, as reported previously by comparing rat and human absorption values for 23 marketed drugs. Upon comparison of human oral absorption predicted by Caco-2 permeability and by rat pharmacokinetics, we show a better correlation with Caco-2 permeability obtained at higher compound concentrations, where efflux is saturated, than at lower concentrations. The higher concentrations are likely reflecting the lumen concentrations after in vivo oral dosing. The results presented in this study demonstrate that, when tested at relevant compound concentrations, Caco-2 permeability is useful for predicting the oral absorption of peptidic compounds.

Prediction of oral drug absorption in humans--from cultured cell lines and experimental animals.

BACKGROUND: Over the past decade, the use of drug metabolism and pharmacokinetic (DMPK) parameters to optimize selection of candidates for drug development has become one of the primary focuses of research organizations involved in new drug discovery. OBJECTIVE: This review will focus on the feasibility of predicting human absorption using data from a cultured Caco-2 cell system and non-clinical animals. METHODS: In-house Caco-2 permeability data were compared with human absorption data from the literature. Animal absorption data obtained from current literature were also compared with human data. RESULTS/CONCLUSION: Using a combination of rapid in-vivo and in-vitro DMPK screens on a large array of compounds during the lead optimization process has resulted in the streamlined development of compounds that have acceptable DMPK properties. Since it is well recognized that human intestinal absorption cannot be precisely predicted by a single screening assay, it is important to utilize various in-vitro and in-vivo non-clinical studies during lead optimization in drug discovery.

Correlation between PAMPA permeability and cellular activities of hepatitis C virus protease inhibitors.

Parallel artificial membrane permeability assay (PAMPA) and Caco-2 cells have been frequently used for the evaluation of in vitro permeability of new chemical entities. In this study we evaluated the correlation between permeability, assessed by both methods, and the cellular potency of 34 novel hepatitis C virus (HCV) protease inhibitors. Two types of assays were used to determine the potency of HCV protease inhibitors: a cell-free assay that evaluates the intrinsic affinity (K(i)) between the protease and the inhibitor and a cell-based replicon assay that determines the inhibitors' IC90. When the K(i)/IC90 ratios were compared with the PAMPA permeability and the Caco-2 permeability by linear regression analysis, a reasonable correlation was found between the K(i)/IC90 ratio and PAMPA permeability (r2=0.76) but not with Caco-2 permeability (r2=0.29). Correlations were also assessed between K(i)/IC90 ratios and the following physico-chemical properties: logP (r2=0.41), logD (r2=0.58), clogP (r2=0.13), and mlogP (r2=0.30). These results suggest that passive permeability may play a role in the uptake and cellular activity of these HCV protease inhibitors, and that PAMPA was more predictive of cellular activity than physico-chemical properties or Caco-2 permeability.

Development of in vitro pharmacokinetic screens using Caco-2, human hepatocyte, and Caco-2/human hepatocyte hybrid systems for the prediction of oral bioavailability in humans.

In this study, in vitro systems were used to build 2 pharmacokinetic models that predict human oral bioavailability: the Caco-2/hepatocyte combination model and the Caco-2/hepatocyte hybrid model. Data obtained in vitro on Caco-2 cell permeability and hepatocyte clearance are routinely used to predict the fraction of absorption after oral administration and the extent of first-pass metabolism, respectively. In the Caco-2/hepatocyte combination model, results from a Caco-2 cell permeability assay and a hepatocyte clearance assay were combined to project oral bioavailability. Comparison of oral bioavailabilities predicted by the combination model and reported oral bioavailabilities in humans for 30 marketed compounds resulted in a modest correlation (r(2) = 0.66). The Caco-2/hepatocyte hybrid model, as previously reported, joins the Caco-2 and hepatocyte clearance systems into 1 assay. Improvements to the previous model were made by incorporating an elimination phase into the Caco-2/hepatocyte hybrid model. In the new hybrid model, the compound was added to a Caco-2-containing donor compartment and allowed to permeate for 2 h to a hepatocyte-containing receiver compartment. Subsequently, to mimic an elimination phase, the donor compartment was removed, and permeated compound was incubated with hepatocytes alone for an additional 3 h. The area under the concentration versus time curve (AUC) was determined for each of the same 30 marketed compounds assessed by the combination model. A linear regression analysis comparing the in vitro AUCs and reported oral bioavailabilities in humans showed a reasonable correlation (r(2) = 0.73). This study demonstrates that the Caco-2/hepatocyte hybrid model is more favorable and further proves the potential and feasibility of using in vitro screenings for the prediction of in vivo pharmacokinetics in humans.