Edward A. Pham

Using Chimeric Mice with Humanized Livers to Predict Human Drug Metabolism and a Drug-Drug Interaction

Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.

Simplified RNA secondary structure mapping by automation of SHAPE data analysis

SHAPE (Selective 2′-hydroxyl acylation analysed by primer extension) technology has emerged as one of the leading methods of determining RNA secondary structure at the nucleotide level. A significant bottleneck in using SHAPE is the complex and time-consuming data processing that is required. We present here a modified data collection method and a series of algorithms, embodied in a program entitled Fast Analysis of SHAPE traces (FAST), which significantly reduces processing time. We have used this method to resolve the secondary structure of the first ∼900 nt of the hepatitis C virus (HCV) genome, including the entire core gene. We have also demonstrated the ability of SHAPE/FAST to detect the binding of a small molecule inhibitor to the HCV internal ribosomal entry site (IRES). In conclusion, FAST allows for high-throughput data processing to match the current high-throughput generation of data possible with SHAPE, reducing the barrier to determining the structure of RNAs of interest.

Sofosbuvir and simeprevir combination therapy in the setting of liver transplantation and hemodialysis

We report safety, tolerability, and 12‐week sustained virologic response with half‐standard dose sofosbuvir and standard‐dose simeprevir combination therapy in a hepatitis C virus genotype 1a‐infected liver transplant recipient on hemodialysis – uncharted territory for sofosbuvir‐based therapy. The patient was a non‐responder to prior treatment with pegylated interferon plus ribavirin. Sofosbuvir efficacy was maintained despite pill‐splitting and administration of half‐standard dose, 200 mg per day. No drug–drug interactions were noted with tacrolimus‐based immunosuppression. Laboratory tests remained stable or improved during therapy. Our observation, if reproduced in a larger study, may lead to significant improvement in clinical outcomes and cost savings in this patient population.

Structural map of a microRNA-122: hepatitis C virus complex.

ABSTRACT MicroRNA-122 (miR-122) enhances hepatitis C virus (HCV) fitness via targeting two sites in the 5′-untranslated region (UTR) of HCV. We used selective 2′-hydroxyl acylation analyzed by primer extension to resolve the HCV 5′-UTRs RNA secondary structure in the presence of miR-122. Nearly all nucleotides in miR-122 are involved in targeting the second site, beyond classic seed base pairings. These additional interactions enhance HCV replication in cell culture. To our knowledge, this is the first biophysical study of this complex to reveal the importance of ‘tail’ miR-122 nucleotide interactions.

Future Therapy for Hepatitis B Virus: Role of Immunomodulators

Although currently available therapies for chronic hepatitis B virus infection can suppress viremia and provide long-term benefits for patients, they do not lead to a functional cure for most patients. Advances in our understanding of the virus-host interaction and the recent remarkable success of immunotherapy in cancer offer new and promising strategies for developing immune modulators that may become important components of a total therapeutic approach to hepatitis B, some of which are now in clinical development. Among the immunomodulatory agents currently being investigated to combat chronic HBV are toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, and engineered T cells. The efficacy of some immune modulatory therapies is compromised by high viral antigen levels. Cutting edge strategies, including RNA interference and CRISPR/Cas9, are now being studied that may ultimately be shown to have the capacity to lower viral antigen levels sufficiently to substantially increase the efficacy of these agents. The current advances in therapies for chronic hepatitis B are leading us toward the possibility of a functional cure.

Nonalcoholic Fatty Liver Disease: Epidemiology, Natural History, and Diagnostic Challenges.

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A New Standard of Care? Standard Dose Sofosbuvir in an HCV-Infected Liver Transplant Recipient Undergoing Hemodialysis

A 54-year-old Hispanic man with history of Type 2 diabetes mellitus underwent liver transplantation (LT) in June 2012 for advanced liver disease complicating treatmentnaı̈ve hepatitis C virus (HCV) infection (genotype 4c/d); no preoperative evidence of renal dysfunction was noted. In October 2012, he was treated for Toxoplasma gondii infection of the brain and Cryptococcus neoformans infection of the lung, followed by prophylactic therapy. Two months later, he developed worsening renal function due to acute tubular necrosis in the setting of sepsis. Following LT, HCV infection recurred, accompanied by persistently abnormal liver enzymes and high HCV RNA levels with no evidence of fibrosing cholestatic hepatitis. In February 2013, hepatic decompensation with refractory ascites required weekly paracenteses and treatment of hepatic encephalopathy. A liver biopsy in April 2013 revealed lobular and portal mononuclear infiltrates consistent with recurrent HCV infection (Fig. 1a, b). In November 2013, he was treated for tuberculosis with isoniazid, pyrazinamide, ethambutol, and levofloxacin for Mycobacterium tuberculosis peritonitis. His renal function deteriorated rapidly to end-stage renal disease (ESRD) requiring hemodialysis (HD) 3/week by December 2013 due to the development of the hepatorenal syndrome. In February 2014, he was hospitalized for upper gastrointestinal bleeding due to esophageal varices. In March 2014, a 12-week course of sofosbuvir and simeprevir combination antiviral therapy was initiated, using a dose-escalation regimen of sofosbuvir initially with a half-standard dose of 200 mg daily for 4 weeks to assess tolerance, followed by standard dose of 400 mg daily for the remaining 8 weeks. Simeprevir was dosed without adjustment at 150 mg daily. His immunosuppressive therapy included tacrolimus with a goal trough level of 3–5 ng/ mL requiring no adjustments. His pretreatment HCV RNA level was 4.81 million international units/mL. His HCV RNA first became undetectable at week 3, with all subsequent assays remaining negative, including at weeks 12 and 24 after completion of therapy (Fig. 2). His liver enzymes normalized following completion of the course of antiviral therapy (Fig. 3). He remains HD-dependent. No adverse effects to HCV antiviral therapy were noted.

Reconstitution and Functional Analysis of a Full-Length Hepatitis C Virus NS5B Polymerase on a Supported Lipid Bilayer

Therapeutic targeting of membrane-associated viral proteins is complicated by the challenge of investigating their enzymatic activities in the native membrane-bound state. To permit functional characterization of these proteins, we hypothesized that the supported lipid bilayer (SLB) can support in situ reconstitution of membrane-associated viral protein complexes. As proof-of-principle, we selected the hepatitis C virus (HCV) NS5B polymerase which is essential for HCV genome replication, and determined that the SLB platform enables functional reconstitution of membrane protein activity. Quartz crystal microbalance with dissipation (QCM-D) monitoring enabled label-free detection of full-length NS5B membrane association, its interaction with replicase subunits NS3, NS5A, and template RNA, and most importantly its RNA synthesis activity. This latter activity could be inhibited by the addition of candidate small molecule drugs. Collectively, our results demonstrate that the SLB platform can support functional studies of membrane-associated viral proteins engaged in critical biological activities.