Substrate hydrolysis by human cytomegalovirus (HCMV) protease is essential to viral capsid assembly. The interaction of HCMV protease and the N-terminal cleavage products of the hydrolysis of R- and M-site oligopeptide substrate mimics (R and M, respectively, which span the P9-P1 positions) was studied by NMR methods. Protease-induced differential line broadening indicated that ligand binding is mediated by the P4-P1 amino acid residues of the peptides. A well-defined extended conformation of R from P1 through P4 when complexed to HCMV protease was evidenced by numerous transferred nuclear Overhauser effect (NOE) correlations for the peptide upon addition of the enzyme. NOE cross-peaks between the P4 and P5 side chains placing these two groups in proximity indicated a deviation from the extended conformation starting at P5. Similar studies carried out for the M peptide also indicated an extended peptide structure very similar to that of R, although the conformation of the P5 glycine could not be established. No obvious variation in structure between bound R and M (notably at P4, where the tyrosine of the R-site has been suggested to play a key role in ligand binding) could be discerned that might explain the observed differences in processing rates between R- and M-sequences. Kinetic studies, utilizing R- and M-site peptide substrates for which the P5 and P4 residues were separately exchanged, revealed that these positions had essentially no influence on the specificity constants (kcat/KM). In sharp contrast, substitution of the P2 residue of an M-site peptide changed its specificity constant to that of an R-site peptide substrate, and vice versa.

Pharmacokinetic-pharmacodynamic (PK-PD) data analyses from early hepatitis C virus (HCV) clinical trials failed to show a good correlation between the plasma inhibitory quotient (IQ) and antiviral activity of different classes of directly acting antiviral agents (DAAs). The present study explored whether use of the liver partition coefficient-corrected IQ (LCIQ) could improve the PK-PD relationship. Animal liver partition coefficients (Kp liver ) were calculated from liver to plasma exposure ratios. In vitro hepatocyte partition coefficients (Kp hep ) were determined by the ratio of cellular to medium drug concentrations. Human Kp liver was predicted using an in vitro-in vivo proportionality method: the species-averaged animal Kp liver multiplied by the ratio of human Kp hep over those in animals. LCIQ was calculated using the IQ multiplied by the predicted human T he inhibitory quotient (IQ) is a common pharmacological predictor of antiviral activity and generally represents the ratio between the trough plasma concentration (C min ) and an in vitro potency parameter such as 50% effective concentration (EC 50 ) (3). Several modifications have been proposed to improve the prognostic value of the plasma IQ-antiviral response relationship, including the use of serum-shifted EC 50 s, the consideration of intracellular drug concentrations, and the potential complication related to drug resistance mutations (1, 3), as well as the proposed instantaneous IQ model, which suggested that the slope of the dose-response curve is another critical fact to consider (34). Recently, several directly acting antivirals (DAAs) targeting HCV have been advanced into clinical trials, and the first DAAs have now been approved for use in combination therapy with pegylated interferon and ribavirin (2,18,21,32,35). The first DAA tested in chronic hepatitis C virus (HCV)-infected patients with potent antiviral effect was BILN 2061 (16, 20), which showed a clear dosedependent in vivo activity. Even at the low oral twice-daily (b.i.d.) dose of 25 mg, a mean of 2-log 10 IU/ml viral load reduction (VLR) was observed following a brief treatment (2 days) (16). The corresponding mean plasma IQ was 6.6 at this low oral dosage. A similar plasma IQ, but significantly greater antiviral effects, was observed for telaprevir dosed at 750 mg three times a day (t.i.d.), based on published clinical pharmacokinetic (PK) data (33) and the EC 50 s measured under the same experimental condition as used for BILN 2061 from our lab. Clinical trials for the polymerase inhibitor BILB 1941 (11) demonstrated that a mean plasma IQ of 21 was achieved with the t.i.d. oral dose of 300 mg (about 3.5-fold greater than for BILN 2061 at 25 mg b.i.d. or telaprevir at 750 mg t.i.d.). Yet this higher plasma IQ was associated with much lower antiviral effects in the clinic, with a mean viral load reduction of only 1 log 10 IU/ml. Correction of plasma IQ using serum-shifted EC 50 s, or plasma protein binding-corrected IQs, failed to improve the PK-pharmacodynamics (PD) relationshi...

The present study describes the cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV protease inhibitor currently in phase III clinical trials. BI 201335 showed a good Caco-II permeability (8.7 × 10(-6) cm/sec) and in vitro metabolic stability (predicted hepatic clearence (CL(hep)) <19% Q(h) in all species tested). Single dose PK revealed a clearance of 17, 3.0 and 2.6 mL/min/kg in rat, monkey and dog respectively, with a corresponding oral bioavailability of 29.1, 25.5 and 35.6%. Comparative plasma and liver PK profile in rodents showed a high liver Kp in the rat (42-fold), suggesting high target tissue distribution. Simple allometry based on animal PK predicted a human oral CL/F of 168 mL/min, within two-fold of the observed value (118 mL/min) at 240 mg in healthy volunteers. Allometry of volume of distribution generated a low exponent of 0.59, and a much lower predicted Vss/F (5-fold less than observed). Several different approaches of Vss/F prediction were evaluated and compared with the value observed in human. The averaged Vss/F from preclinical animals provides the best estimation of the observed human value (169 L vs. 175 L). Corresponding human "effective" t(1/2) values were also compared. The predicted human t(1/2) based on the CL from allometry with metabolic corrections and the averaged animal Vss represented the best estimation of the clinical data (12.1 vs. 17.2 hr). The present study demonstrated that the good preclinical ADMEPK profile of BI 201335 is consistent with that observed in the clinic. While preclinical data accurately predicted the human CL, the prediction of human Vss seems to be more challenging. The averaged Vss/F from all tested preclinical animals provided the best prediction of human Vss and the resulting "effective" t(1/2).

The IAE procedure coupled with VAMS is immune to HCT biases affecting sampling volume and recovery.

Aim: Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Materials & methods: Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Results: Both western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. Conclusion: The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.