Effect of itraconazole on the pharmacokinetics of faldaprevir in healthy subjects.

Faldaprevir (FDV), a substrate of CYP3A/P-glycoprotein (P-gp), is a selective inhibitor of the hepatitis C virus (HCV) NS3/4 protease. FDV is currently under clinical development for application in interferon-free treatment regimens for patients with chronic HCV infection. Understanding the drug-drug interaction potential of FDV is critical, as certain drug combinations may facilitate the more rapid achievement of steady-state-that is, the ideal drug concentration and balanced metabolic cycle of absorption and elimination that optimize drug efficacy. We thus conducted this study to investigate the effect of itraconazole (ICZ), a strong inhibitor of CYP3A and a moderate inhibitor of P-gp, on the pharmacokinetics (PK) of FDV. Eighteen healthy male and female volunteers participated in this open-label, fixed-sequence study. FDV 120 mg twice daily (BID) was administered on Day 1, followed by 120 mg once daily (QD) from Day 2 until the end of the 10-day study; after 6 days of FDV alone, ICZ 200 mg was added to FDV for an additional 4 days (BID on Day 7 and QD from Day 8 to Day 10). Intensive PK sampling was performed after 6 days of FDV treatment and again after 4 days of combined FDV/ICZ treatment. The adjusted geometric mean (gMean) ratios (%) of area under the concentration curve over dosing interval at steady-state (AUCτ, ss) and maximal concentration at steady-state (Cmax, ss) for combined FDV/ICZ treatment vs. FDV treatment alone were 198.6% and 180.6%, respectively, with 90% confidence intervals (CIs) of 182.4-216.1 and 165.7-196.9. Administration of FDV alone or in combination with ICZ was observed to be safe and well-tolerated. Co-administration with ICZ, however, resulted in an approximately two-fold increase in FDV steady-state exposure. Furthermore, FDV required no dosage adjustment when co-administered with ICZ.

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice.

Despite the tremendous potential of adenovirus (Ad) as a delivery vector for cancer gene therapy, its use in clinical settings has been limited, mainly as a result of the limited infectivity in many tumors and the wide tissue tropism associated with Ad. To modify the tropism of the virus, we have inserted the epidermal growth factor-like domain of the human heregulin-α (HRG) into the HI loop of Ad5 fiber. This insertion had no adverse effect on fiber trimerization nor did it affect incorporation of the modified fiber into infectious viral particles. Virions bearing modified fiber displayed growth characteristics and viral yields indistinguishable from those of wild-type (wt) virus. Most importantly, HRG-tagged virions showed enhanced infection of cells expressing the cognate receptors HER3/ErbB3 and HER4/ErbB4. This was significantly reduced in the presence of soluble HRG. Furthermore, HER3-expressing Chinese hamster ovary (CHO) cells were transduced by the HRG-modified virus, but not by wt virus. In contrast, CHO cells expressing the coxsackie-Ad receptor were transduced with both viruses. However, infection of an in vivo breast cancer xenograft model after intratumoral injection was similar with both viruses, suggesting that the tumor microenvironment and/or the route of delivery have important roles in infection of target cells with fiber-modified Ads.

Differential effects of tipranavir plus ritonavir on atorvastatin or rosuvastatin pharmacokinetics in healthy volunteers.

To identify pharmacokinetic (PK) drug-drug interactions between tipranavir-ritonavir (TPV/r) and rosuvastatin and atorvastatin, we conducted two prospective, open-label, single-arm, two-period studies. The geometric mean (GM) ratio was 1.37 (90% confidence interval [CI], 1.15 to 1.62) for the area under the concentration-time curve (AUC) for rosuvastatin and 2.23 (90% CI, 1.83 to 2.72) for the maximum concentration of drug in serum (Cmax) for rosuvastatin with TPV/r at steady state versus alone. The GM ratio was 9.36 (90% CI, 8.02 to 10.94) for the AUC of atorvastatin and 8.61 (90% CI, 7.25 to 10.21) for the Cmax of atorvastatin with TPV/r at steady state versus alone. Tipranavir PK parameters were not affected by single-dose rosuvastatin or atorvastatin. Mild gastrointestinal intolerance, headache, and mild reversible liver enzyme elevations (grade 1 and 2) were the most commonly reported adverse drug reactions. Based on these interactions, we recommend low initial doses of rosuvastatin (5 mg) and atorvastatin (10 mg), with careful clinical monitoring of rosuvastatin- or atorvastatin-related adverse events when combined with TPV/r.

Picornavirus internal ribosome entry site elements target RNA cleavage events induced by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. vhs displays weak amino acid sequence similarity to the fen-1 family of nucleases and suffices to induce accelerated RNA turnover through endoribonucleolytic cleavage events when it is expressed as the only HSV protein in a rabbit reticulocyte in vitro translation system. Although vhs selectively targets mRNAs in vivo, the basis for this selectivity remains obscure, since in vitro activity is not influenced by the presence of a 5' cap or 3' poly(A) tail. Here we show that vhs activity is greatly altered by placing an internal ribosome entry site (IRES) from encephalomyocarditis virus or poliovirus in the RNA substrate. Transcripts bearing the IRES were preferentially cleaved by the vhs-dependent endoribonuclease at multiple sites clustered in a narrow zone located immediately downstream of the element in a reaction that did not require ribosomes. Targeting was observed when the IRES was located at the 5' end or placed at internal sites in the substrate, indicating that it is independent of position or sequence context. These data indicate that the vhs-dependent nuclease can be selectively targeted by specific cis-acting elements in the RNA substrate, possibly through secondary structure or a component of the translational machinery.

The herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target RNAs in cell extracts.

The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5' cap or a 3' poly(A) tail in the RNA substrate, requires Mg(2+), and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5' quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system.

Sequence and sequence analysis of E1 and pIX regions of the BAV3 genome.

Bovine adenovirus type 3 (BAV3) is a DNA virus that causes respiratory and gastrointestinal disorders in cattle. We have sequenced the extreme left end of BAV3 genome (0-11.7 map units). Partial analysis of the nucleotide sequence revealed 19 potential open reading frames (ORFs) that could encode for polypeptides of 50 or more amino acids. Four of these ORFs show homology to known adenovirus polypeptides. The four BAV3 ORFs are located in approximately the same area as the Ad5 E1a, E1b, and pIX ORFs. ORF 1 has the potential to code for a 208 amino acid long polypeptide that is 75.5% homologous to the E1a conserved region III of Ad5. ORFs 2 and 3 encode 157 and 420 amino acid long polypeptide, respectively. The 157 amino acid polypeptides exhibits 69.3% homology to the Ad5 small T antigen, and the 420 amino acid polypeptide exhibits 73% homology to the large T antigen of Ad5. ORF4 has the potential to code for a 125 amino acid long polypeptide that has 73% homology to the hexon-associated pIX of Ad7.

Molecular cloning and restriction enzyme analysis of bovine adenovirus type 3.

Bovine adenovirus type 3 (BAV3) is a DNA virus that causes respiratory and gastrointestinal disorders in cattle. The viral genome consists of a linear double-stranded DNA molecule (35,000 base pairs) with inverted terminal repeats at each of its 5' molecular ends. We have subcloned 10 HindIII fragments spanning 4.9-96.0%, 5 EcoRI fragments spanning 3.4-89.5% and 2 XbaI fragments spanning 35.7-82.9% of the BAV3 (strain WBR-1) genome into the bacterial cloning vector pUC19. The subcloning of the viral genome facilitated the construction of linear restriction enzyme maps for BamHI, ClaI, EcoRI, HindIII, KpnI, NotI, NspV, PstI, PvuI, SalI, XbaI and XhoI. In this study we report on the molecular cloning and restriction endonuclease mapping of the BAV3 genome.