Hepatitis C vaccine signals landmark University of Alberta discovery

(Photo by Julianna Damer/The Gateway)

Last updated: February 29, 2012 5:23 pm

Vaccine could protect against all forms of virus

Andrew Jeffrey — The Gateway (University of Alberta)

EDMONTON (CUP) — A University of Alberta team has made a breakthrough in hepatitis C research, creating a vaccine that could potentially combat all forms of the liver-destroying virus.

The vaccine was developed by Michael Houghton, a U of A researcher who first discovered the hepatitis C virus (HCV) in 1989. The vaccine exposes the human body to a non-infectious sub-unit of the HCV so that it can begin developing antibodies to protect against the virus. These antibodies are able to cross-neutralize against the seven genotypes of the virus.

“Previously, many people believed that the virus was impossible to neutralize with just one type of vaccine because there are so many different genotypes,” explained John Law, a member of the U of A research team.

“This is a proof of principle finding — showing that we can make a vaccine that can allow cross protections of many different varieties of the hepatitis C virus.”

Houghton began developing a vaccine more than a decade ago, and was recruited by the U of A’s Li Ka Shing Institute of Virology three years ago to continue his research. Law attributes much of the success of their team to Houghton’s dedication.

“We didn’t really make anything special. Mostly it’s been the efforts of Dr. Houghton. We’ve persisted and been able to go through the hurdles of finally getting the samples, getting the trial, and eventually testing the results and being able to find out the answer,” Law said.

“He stayed with his idea and eventually carried it out to a point where now we can see there is a very good potential for things that are going to happen.”

Law said an obstacle in creating the vaccine is the HCV’s ability to mutate quickly and exists in a variety of genotypes, similar to AIDS.

Law predicts that it will still take another five to seven years before the vaccine is ready to be released. The research has only completed the first of three phases needed for the FDA to approve the vaccine. Although its safety has already been tested, the vaccine will require further testing in a clinical setting.

The vaccine has already been presented by the research team to various other members of the virology community.

Last weekend, the team travelled to Montreal to present their findings at a Canadian symposium for hepatitis C. Law is hopeful that the team can improve upon their vaccine further before releasing it to the public.

“We’re basically trying to understand those antibody responses, and trying to find out which part of the sub-unit the antibody recognizes. There might be some common area between the genotypes that the antibody can see that is therefore blocking infections of HCV,” Law explained.

“We can maybe learn the mechanism to increase the efficacy of the vaccine and be able to design a better vaccine and move forward.”

Source

Also See: The Scientist Who Discovered Hepatitis C Says He’s Now Discovered the Vaccine

Hepatitis C Virus Persistence After Sustained Virological Response to Antiviral Therapy in Patients With or Without Past Exposure to Hepatitis B Virus

From Journal of Viral Hepatitis

T. N. Q. Pham; C. S. Coffin; N. D. Churchill; S. J. Urbanski; S. S. Lee; T. I. Michalak

Posted: 02/28/2012; J Viral Hepat. 2012;19(2):103-111. © 2012 Blackwell Publishing

Abstract and Introduction

Abstract

Hepatitis C virus (HCV) and hepatitis B virus (HBV) frequently coinfect and persist long after clinical resolution. We assessed the incidence of low-level (occult) HCV infection (OCI) after sustained virological response (SVR) to standard anti-HCV therapy in individuals with or without past exposure to HBV to recognize whether HBV could influence the prevalence of OCI, HCV level and hepatic histology. Plasma and peripheral blood mononuclear cells (PBMC) were collected from 24 individuals at 6- to 12-month intervals for up to 72 months after SVR. Liver histology was available for nine patients. HCV and HBV genomes were detected with sensitivity <10 genome copies/mL. In individuals without HBV exposure (n = 15), comprehensive analyses of sequential plasma and PBMC samples revealed HCV RNA in all 15 cases (75% plasma and 61% PBMC). In the group with HBV exposure (n = 9), evidenced by circulating anti-HBc and/or HBV DNA detection by a highly sensitive assay, HCV RNA was identified in all cases (83% plasma and 59% PBMC), at levels similar to those in HBV nonexposed individuals. In both groups of patients, most liver biopsies included those reactive for viral genomes displayed low-grade inflammation (8 of 9) and fibrosis (7 of 9). Sequence polymorphisms at the 5`-UTR between PBMC and liver or plasma, as well as circulating HCV virion-like particles, were observed in patients with or without HBV exposure. In conclusion, the prevalence of OCI after SVR is comparable in individuals with or without past exposure to HBV. HCV loads and liver alterations in OCI appear to be unaffected by low-level HBV DNA carriage.

Introduction

There are at least 370 million people chronically infected with hepatitis B virus (HBV) and 170 million of those with chronic hepatitis C virus (HCV) infection worldwide. The chronic hepatitis induced by the viruses can lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC).^[1,2] However, while 90–95% of adults infected with HBV spontaneously resolve acute hepatitis, up to 85% of those infected with HCV develop chronic hepatitis C (CHC).^[3,4]

Although hepatocytes are major targets of HBV and HCV, both pathogens can also propagate in the cells of the immune system, as evidenced by the presence of their genomes and respective replicative intermediates and proteins in the lymphatic organs and peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) and C.^[5–10] The existence of small amounts of HBV and HCV genomes in plasma, PBMC or liver after clinical resolution of hepatitis highlighted the natural propensity for these two viruses to induce a persistent infection.^[11–15] Occult HBV infection (OBI) is defined as detection of HBV DNA in liver, plasma and/or PBMC in the absence of hepatitis B surface antigen (HBsAg) in serum. Occult HCV infection (OCI) is characterized by low-level HCV RNA persisting in plasma, liver or PBMC in anti-HCV antibody-reactive individuals who resolved hepatitis C either spontaneously or after antiviral treatment or seemingly anti-HCV antibody-negative individuals with infection of unknown aetiology.^[14–17] Although the existence of OBI has been generally accepted, that of OCI remains controversial in that HCV persisting in plasma, liver and/or immune cells after resolution of hepatitis C has not been observed by all investigators. This inconsistency is likely related to variations in the sensitivity of HCV RNA detection assays employed, processing of clinical samples, the number of samples tested and the amount of material used for analysis.^[18] Because of their shared modes of transmission, coinfection with HCV and HBV is common, particularly in high-risk populations and in areas considered to be endemic for both viruses. Molecular and pathological consequences of interactions between HCV and HBV in coinfected patients are not fully elucidated given the relatively contradicting data that are available. On one hand, acute HCV infection in the context of CHB and vice versa acute HBV infection or OBI in CHC have been associated with a more active liver disease and a greater likelihood of developing cirrhosis and HCC.^[19–24] Along this line, HBV superinfection has been correlated with an assumed complete clearance of serum HCV RNA, or even both HCV RNA and HBV DNA, in patients with CHC.^[24–26] The reverse was also true for HCV superinfection.^[22,24] In cell culture studies, HCV core or nonstructural proteins (e.g. NS5A) were found to repress HBV DNA synthesis.^[27–29] On the other hand, in arguing against the negative influence of one virus over the other, other investigations demonstrated not only a coexistence of hepatic HBV and HCV in coinfected patients,^[30,31] but also a simultaneous replication of HCV and HBV within the same hepatoma cell.^[32,33]

Nevertheless, given the lymphotropic propensity of HCV and HBV, what remained undefined was whether HCV and HBV could affect each other's ability to propagate in immune cells, especially in the context of occult infection. Such recognition would be important in furthering our understanding of the full extent of viral persistence. With this in mind, the current study examined whether HBV exposure would influence the prevalence of HCV occult infection, the level of persisting HCV and liver pathology in patients with clinical resolution of CHC.

Patients, Clinical Samples and Methods

Patients and Clinical Samples

Twenty-four patients determined to have resolved CHC according to clinical and laboratory assay criteria were randomly selected for the study. They were followed at the University of Calgary Liver Unit, Alberta, Canada. These individuals had achieved a sustained virological response (SVR) for duration of up to 6 years following treatment with interferon alpha (IFN) or pegylated IFN S1). None of them was subjected toand ribavirin (p-IFN/R) (Supplementary Table immunosuppressive or anticancerous therapy during follow-up. Liver function tests were repeatedly normal and serum HCV RNA negative by Roche Amplicor HCV v2.0 assay (sensitivity 500 IU/mL or 1000 virus genome equivalent (vge)/mL; Roche Molecular Diagnostics, Pleasanton, CA, USA). All patients were reactive for antibodies to HCV (anti-HCV) by immunoassays (Abbott Diagnostics, Mississauga, Canada) at the time of the first blood sample collection. Clinical charts revealed that eight of them were also positive for antibodies to HBV core S1), indicative of a past(HBV-C) antigen (anti-HBc) (Supplementary Table exposure to HBV, by the Abbot Corzyme assay (Abbott Laboratories, North Chicago, IL, USA). One individual was negative for anti-HBc while positive for antibodies to HBsAg (anti-HBs); however, plasma and PBMC from this person were HBV DNA reactive when tested by research polymerase chain reaction (PCR) assays employed in this study.

Plasma and corresponding PBMC were available from all 24 subjects (Supplementary Table S1). Up to three plasma and PBMC pairs were collected from 21 individuals every 6–12 months, while a single sample pair from the remaining three was obtained at 18 months post-SVR. Liver biopsies were available before and after therapy (up to 60 months post-SVR) for eight individuals and only after SVR for an additional patient. The study protocol was approved by local Human Investigation Committees. All patients provided written informed consent to participate in the study.

Preparation of Peripheral Blood Mononuclear Cells and Cell Cultures

Peripheral blood mononuclear cells were isolated by Ficoll-HyPaque (Pharmacia Biotech, Quebec, Canada) gradient fractionation and cultured, if required, for 72 h in the presence of phytohemagglutinin (PHA; 5 μg/mL; Sigma, Mississauga, Canada) and interleukin-2 (IL-2; 20 U/mL; Roche) prior to analysis for HCV RNA or HBV DNA.^[14]

Nucleic Acid Extraction

RNA was usually extracted from 250 μL of plasma using Trizol LS (Invitrogen Life Technologies, Burlington, Ontario, Canada) or 1 × 10⁷ naive (uncultured) PBMC using Trizol (Invitrogen).^[14] DNA was extracted^[34] from the same volume of plasma or number of PBMC. When necessary, extraction of 1-mL sample of test plasma was carried out and the nucleic acid analysed. If the sample remained virus nonreactive, 4 mL of plasma was ultracentrifuged at 40 000 rpm for 20 h at 4 °C and nucleic acid extracted from the resulting pellet was tested for HCV RNA or HBV DNA.

Hepatitis C Virus RNA-positive and Hepatitis C Virus RNA-negative Strand Detection

RNA from PBMC (1–2 μg RNA; equivalent of 1–2 × 10⁶ cells), plasma (equivalent of 250 μL, 1 mL or 4 mL) or liver tissue (2 μg RNA) was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) for HCV RNA-positive strand using HCV 5'-untranslated region (5'-UTR)-specific primers, cycling conditions and quantification standards reported earlier.^[14] In all instances, specificity of amplicons was confirmed by nucleic acid hybridization (NAH) using ³²P-labelled recombinant HCV 5'-UTR-E2 fragment as a probe.^[14] Sensitivity of this assay is <10 vge/mL or <2.5 vge/reaction. HCV RNA-negative strand was detected by strand-specific RT-PCR/NAH using rTth DNA polymerase.^[14] This assay detects ~100 copies of the correct (negative) strand, while nonspecifically identifying ≥10⁶ vge of the positive strand.^[14] Specificity of PCR amplicons and validity of the controls were confirmed by NAH. In every analysis, we included a number of negative and contamination controls, as reported.^[14] Briefly, for each group of samples subjected to nucleic acid extraction, a mock sample containing no patient material was extracted and analysed with test samples. In the PCR step, a water sample and a mock (no test cDNA) were amplified as controls.

Hepatitis B Virus DNA Detection

Hepatitis B virus DNA in plasma, PBMC or liver was detected by PCR/NAH using primers specific for HBV-C and X (HBV-X) genes (GenBank accession number X72702). For first-round amplification, the primers were as follows: 1847-TGTTCATGTCCCACTGTTCAAGC (HBV-C outer sense), 2274-AAGATAGGGGCATTTGGTGG (HBV-C outer antisense), 1266-CCATACTGCGGAACTCCTAGC (HBV-X outer sense) and 1779-ACAGACCAATTTATGCCTACAGCC (HBV-X outer antisense). For second-round amplification, the primers were 1893-TTTGGGGCATGGACATTGACC (HBV-C inner sense), 2301-ATAAGCTGGAGGAGTGCGAATCC (HBV-C inner antisense), 1310-CTGGAGCAAACATTATCGGG (HBV-X inner sense) and 1748-CAAAGACCTTTAACCTGATCTCC (HBV-X inner antisense). In all cases, amplifications were carried out for 40 cycles under the following conditions: 95 °C for 60 s (denaturation), 52 °C for 60 s (annealing) and 72 °C for 60 s (extension). Specificity of amplicons was confirmed by NAH using ³²P-labelled recombinant full-length HBV DNA as a probe.^[11] This assay detects ≤10 vge/mL or ≤2.5 vge/reaction. Negative, positive and contamination controls were routinely included, as reported.^[11,34]

Clonal Sequencing

To assess possible sequence variations and compartmentalization of HCV, 5'-UTR amplicons were cloned and at least 10 clones from each PCR product sequenced bidirectionally.^[8] Polymorphisms were analysed by Sequencher software version 4.7 (Gene Codes Corp., Ann Arbor, MI, USA).

Immunoelectron Microscopy

To examine the presence of HCV virions, 500 μL of plasma was incubated with anti-HCV E2 monoclonal antibody (mAb; AP33; provided by Dr. Arvind Patel, University of Glasgow, UK) or mAb isotype control, as reported.^[35,36] In the case of 16–45/M plasma, HCV RNA-positive fractions recovered after ultracentrifugation (4 mL plasma) over a 30% sucrose cushion^[36] were similarly incubated with anti-E2 mAb. HCV particles were detected with anti-mouse IgG conjugated with 12-nm gold particles (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and, after counterstaining with 1% phosphotungstic acid, examined under a JEM 120 EX microscope (JEOL Ltd., Tokyo, Japan).

Liver Histology

After routine processing, liver biopsies were blindly evaluated by a hepatopathologist according to the METAVIR scoring system.^[37]

Results

Occult Hepatitis C Virus Infection in Individuals With Clinical Sustained Virological Response Not Exposed to Hepatitis B Virus

Using the highly sensitive RT-PCR/NAH assays established earlier,^[14] 15 of 28 plasma samples from 13 of 15 individuals were found positive for HCV RNA when 250-μL samples were tested (Table 1). Of those samples that were virus nonreactive at 250 μL, further testing with one or 4 mL of plasma increased HCV RNA detection^[38] in an additional four and two samples, respectively. Overall, 21 of 28 (75%) of plasma samples were HCV RNA reactive (Table 1). HCV RNA levels varied considerably (2.5–400 vge/mL) at different time points within the same patient and also between different patients. For example, plasma HCV levels fluctuated from 400 vge/mL in the first sample to undetectable 6 months later (e.g. 5–45/F and 18–54/F), only to be detected again at 100 vge/mL 1 year thereafter (e.g. 18–54/F). In PBMC, 8 of 28 unstimulated samples were positive for HCV RNA, with ex vivo stimulation^[39,40] increasing virus identification in another nine (Table 1). As in plasma, viral genome levels in untreated or mitogen-treated PBMC varied between individuals, ranging between 5 and 500 vge/10⁶ cells, but not substantially in the same patient.

Overall, of the 11 cases with sequential samples, HCV RNA was detectable at least once in plasma in all cases and in unstimulated PBMC in six. Ex vivo stimulation of PBMC led to HCV RNA detection in the other five cases, making all cases ultimately positive for HCV RNA in PBMC. Hepatic HCV RNA was identified in one of three cases from this study group (Fig. 1).

Figure 1. Detection of hepatitis C virus (HCV) in liver biopsies from individuals years after clinical diagnosis of sustained virological response following treatment of chronic hepatitis C. Total liver RNA (4 μg) was amplified by nested reverse transcriptase-polymerase chain reaction (RT-PCR) with 5'-UTR-specific primers and amplicon specificity verified by nucleic acid hybridization (NAH). (a) Identification of HCV RNA-positive strand. (b) Detection of HCV RNA-negative strand using strand-specific RT-PCR/NAH. Tenfold serial dilutions of HCV sRNA-positive or HCV sRNA-negative strands were used as quantitative standards, respectively. Dilutions of HCV sRNA-positive strands were included in the negative strand assay as a specificity control. Contamination controls included water added instead of cDNA and amplified by direct (DW) and nested (NW) reactions and mock (M) treated as test RNA. Positive signals showed the expected 244-bp 5'-UTR sequence fragments.

The detection of HCV RNA-positive strand was unlikely due to viral adsorption to the cells because of the concurrent detection of the viral negative strand in liver and PBMC (Table 1 and Fig. 1), of viral sequence polymorphisms in PBMC relative to plasma or liver (2–53/M; Supplementary Table S2) and of circulating HCV-like particles (3–46/F; Fig. 2). The particles usually occurred singly and, occasionally, in small aggregates with estimated diameters between 50 and 75 nm. Collectively, these findings clearly indicated the persistence of authentic HCV replication at low level in persons with clinical resolution of CHC.

Figure 2. Immunoelectron microscopic identification of hepatitis C virus (HCV) particles in the plasma of individuals with clinical sustained virological response (SVR) as visualized by immunogold staining with anti-E2 mAb. (a–c) HCV particles in unfractionated plasma of 3–46/F obtained at 54 months after achieving SVR (d–f). HCV RNA-reactive particles recovered from fractions 3 and 4 after ultracentrifugation over a 30% sucrose cushion of 16–45/M plasma collected at 66 months after SVR (g–i). HCV virions in unfractionated plasma of 18–54/F obtained at 36 months following clinical SVR. (j) The unfractionated plasma from 3 to 46/F exposed to the isotype mAb control instead of anti-E2 mAb. Preparations were counterstained with 1% phosphotungstic acid. Bars indicate 50 nm.

Occult Hepatitis C Virus Infection in Patients With Clinical Sustained Virological Response and Past Exposure to Hepatitis B Virus

In the first instance, HBV DNA was identified, using our highly sensitive PCR/NAH assay,^[11,34] in 17 of 22 (78%) plasma, 9 of 18 (50%) PBMC and 2 of 2 (100%) liver samples tested (Table 1 and Fig. 3), giving a total case positivity of 8 of 9 for HBV (Table 2). However, the lack of available material prevented us from determining the status of HBV replication in these cases.

Figure 3. Detection of dual occult infection with hepatitis C virus (HCV) and hepatitis B virus (HBV) in individuals with clinically apparent sustained virological response (SVR) to anti-HCV therapy and past exposure to HBV. HCV RNA and HBV DNA were detected in plasma (Pl), peripheral blood mononuclear cells (P) and liver (L) by nested RT-PCR/NAH (for HCV RNA) or nested PCR/NAH (for HBV DNA) as described in Patients, Clinical Samples and Methods. Total RNA or DNA extracted from a patient with symptomatic chronic hepatitis C or serum hepatitis B surface antigen-positive chronic hepatitis B, respectively, was used as a positive control (PC). Contamination controls included water added instead of cDNA (HCV RNA analysis) or DNA (HBV DNA analysis) and amplified by direct (DW) and nested (NW) reactions and mock (M) treated as test RNA or DNA.

Using the same approach to virus detection as discussed in previous section, HCV RNA was ultimately identified in 83% plasma, 54.5% PBMC and 100% liver samples of patients with past exposure to HBV. As in the case of individuals without a past history with HBV, there was also evidence for the presence of HCV-negative strand (Table 1), circulating HCV-like particles (cases 16–45M and 18–54/F; Fig. 2) and single-nucleotide polymorphisms in PBMC (case 24–34F; Supplementary Table S2). Overall, the data as a whole lend support to the conclusion that past HBV exposure seems to have no identifiable influence on the frequency and virological properties of occult HCV persistence.

Liver Histology in Individuals With Occult Hepatitis C Virus Infection and With or Without Past Hepatitis B Virus Exposure

Histological examination of liver biopsies obtained before and after treatment (post-SVR) (Table 3; Supplementary Fig. S1) revealed (i) a decrease in necroinflammatory activity after SVR in five cases; (ii) no remarkable changes between pre- and post-treatment in two patients; and (iii) increased disease activity after SVR in two cases (1–61/F, 3–46/F). It was evident that despite histological improvement in the majority of cases investigated, most biopsies still displayed some degree (predominantly grades 1 and 2) of necroinflammation with piecemeal necrosis and lymphocytic infiltrations, as well as minimal to mild fibrosis (mainly stages 1 and 2) after SVR (Supplementary Fig. 1). But in the end, no remarkable differences were observed in liver histology between individuals with or without past exposure to HBV.

Discussion

In this study, we examined the prevalence of OCI in patients with clinically resolved hepatitis C who had or did not have past exposure to HBV. Our findings overall suggest that low-level HBV DNA carriage in plasma, PBMC or liver does not have a noticeable effect on the prevalence, virological characteristics and, most likely, features of liver histology encountered in the course of OCI. The data also indicate that regardless of the existence of past HBV exposure, the levels of HCV RNA, especially in plasma, could vary significantly over time in OCI. Given that both HCV and HBV genomes were detectable in the same compartments in many of the cases examined, it can be proposed that the reciprocal inhibition of HBV and HCV replication observed in highly viraemic hepatitis patients^{[22,24,26,41]} is not evident in low-level coinfection with these viruses.

Since its discovery in recent years,^[14] OCI continuing after clinical resolution of hepatitis C has been a subject of investigations by many groups. Although we and others have collectively documented the presence of low-level HCV persistence in plasma, PBMC and/or liver in individuals with spontaneous or treatment-induced recovery from CHC,^[13–15] studies from other groups came to rather opposite conclusions.^[42–44] As evidenced in this study, HCV RNA, in both plasma and PBMC, could fluctuate considerably over time in low-level HCV infection. This, in consequence, highlights the importance of testing multiple samples and, if required, using a larger amount of starting material for nucleic acid extraction. In addition, as ex vivo stimulation of lymphoid cells with mitogens could greatly enhance virus detection,^[14,39] we routinely adopt this approach to our investigation of OCI in PBMC. Nevertheless, this methodology was not used by most, if not all, studies that had refuted the existence of occult HCV persistence.^[42–44] Perhaps, a combination of the differences mentioned elsewhere, together with other factors discussed elsewhere,^[17,18] could help reconcile the discrepancies between the data that argued for^[13–15] and those that were against^[42–44] the notion of OCI persisting after clinically diagnosed recovery. Further, it is important to note that in this study, as well in those published elsewhere [8,13–15,36], low-level occult HCV persistence was not just about mere detection of HCV RNA. Indeed, not only the persistent expression of HCV replicative strand in PBMC and liver tissue was documented, but also HCV sequence polymorphisms in PBMC compared with plasma or liver, circulating virion-like particles and their replication competence were demonstrated.^[36]

In most cases evaluated, liver histology showed improvement after otherwise successful treatment. Nevertheless, there was also an indication of persistent low-grade liver inflammation and fibrosis (see Table 3), which was seemingly irrespective of whether the individuals were concurrently positive or not for HBV DNA. The question of whether OBI could contribute to such alterations after achieving SVR could not be conclusively addressed in this study and will require a similar investigation with a larger number of cases. At this point, our finding of hepatic changes after SVR is consistent with that reported by others.^[13,15,45]

Taken together, the current study provides new insights into characteristics of occult HCV persistence in general and in individuals with past exposure to HBV in particular. It also offers a standardized approach with greater uniformity and sensitivity in the identification of OCI. Our data indicate that a past encounter with HBV may not negatively influence the prevalence and characteristics of low-level HCV persistence continuing after resolution of CHC achieved following antiviral therapy.

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44. Maylin S, Martinot-Peignoux M, Moucari R et al. Eradication of hepatitis C virus in patients successfully treated for chronic hepatitis C. Gastroenterology 2008; 135: 821–829.
45. Hoare M, Gelson WT, Rushbrook SM et al. Histological changes in HCV antibody-positive, HCV RNA-negative subjects suggest persistent virus infection. Hepatology 2008; 48: 1737–1745.

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New Antivirals Show Promise for an Interferon-free Hepatitis C Treatment

From Journal Watch > Journal Watch Gastroenterology

Atif Zaman, MD, MPH

Posted: 02/28/2012; Journal Watch © 2012 Massachusetts Medical Society

Abstract and Introduction

Abstract

Patients with HCV genotype 1 and previous null response achieved a The current generation of direct acting antivirals (DAAs), telaprevir and boceprevir, in combination with peginterferon and ribavirin (PEG/RBV) have demonstrated limited efficacy in patients with hepatitis C virus (HCV) genotype 1 infection who have experienced a previous null response to PEG/RBV. Sustained virologic response (SVR) has been approximately 30%, with associated high rates of resistance. Now, researchers have explored the efficacy of two next-generation DAAs with and without PEG/RBV in this patient population.

This phase IIa, industry-sponsored, randomized study involved 21 patients with previous null response to PEG/RBV (defined as <2 log HCV RNA drop from baseline at 12 weeks). Eleven patients received 60 mg daily of daclatasvir (an NS5A replication complex inhibitor) and 600 mg twice daily of asunaprevir (an NS3 protease inhibitor) for 24 weeks (DAA-only group). Ten patients received both DAAs and PEG/RBV for 24 weeks (DAA+PEG/RBV group). Of note, 90% of the study cohort had IL28B genotypes CT or TT, which respond poorly to PEG/RBV. The primary endpoint was SVR at 12 weeks after stopping therapy.

The SVR in the DAA-only group was 36% (2 of 9 with genotype 1a and 2 of 2 with genotype 1b). Six patients, all with genotype 1a, had viral breakthrough that was associated with viral resistance mutations to both DAAs. Alternately, the SVR in the DAA+PEG/RBV group was 100% (90% with genotype 1a) at 12 weeks after therapy and 90% at 24 weeks. Most adverse events were mild to moderate and included diarrhea, fatigue, headache, and nausea.

Comment

These findings provide early evidence that the next generation of DAAs is more potent than the current generation in treating HCV genotype 1 infection. In this cohort of the most difficult-to-treat HCV patients — previous null responders with genotype 1 infection — a 24-week SVR of 90% was achieved after 24 weeks of quadruple therapy (2 DAAs and PEG/RBV). Even an interferon-free regimen of the 2 DAAs resulted in an SVR of 36%. Results are eagerly anticipated from the phase III trial currently under way.

References

• Lok AS et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N Engl J Med 2012 Jan 19; 366:216.
• Chung RT. A watershed moment in the treatment of hepatitis C. N Engl J Med 2012 Jan 19; 366:273.

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Serum Levels of Alanine Aminotransferase Decrease With Age in Longitudinal Analysis

Clinical Gastroenterology and Hepatology
Volume 10, Issue 3 , Pages 285-290.e1, March 2012

Mamie H. Dong, Ricki Bettencour, David A. Brenner, Elizabeth Barrett–Connor, Rohit Loomba

published online 24 October 2011.

Abstract

Background & Aims

An increased level of alanine aminotransferase (ALT) is a marker of liver injury. The mean ALT level has been reported to decrease with age; we performed a longitudinal analysis to determine whether serum levels of ALT changes with age among community-dwelling, older adults in the US.

Methods

We analyzed clinical data from 2 cohorts of individuals who participated in the Rancho Bernardo Study, in Southern CA. The first cohort comprised 1073 community-dwelling participants (59% women); clinical data was collected from 1984–1987 and 1992–1997. The second cohort comprised 416 participants (64% women); data was collected from 1984–1987, 1992–1997, and 1997–1999. Demographic, metabolic covariates, ALT, bilirubin, and albumin were measured. Changes in individual ALT over time were examined in unadjusted and multivariable-adjusted linear and logistic regression analyses.

Results

At the baseline visit, the patients' mean age was 65.7 years and body mass index was 24.9 kg/m². In cohort 1, the mean levels of ALT decreased with age by 10% (from 21 to 19 IU/L) between the time periods of 1984–1987 and 1992–1997 (P < .0001). In cohort 2, they decreased by 20% (from 20 to 16 IU/L) between the time periods of 1984–1987 and 1997–1999 (P < .0001). Categorically-defined increases in ALT also decreased with age (P < .0001). Results remained consistent in sex-specific analyses and after adjusting for metabolic syndrome components, alcohol use, bilirubin, and serum levels of albumin (P < .0001).

Conclusions

In a longitudinal analysis, we observed that levels of ALT decrease with age, independent of sex, metabolic factors, alcohol use, and results from commonly used liver function tests (bilirubin and albumin). When interpreting serum levels of ALT, physicians should consider patients’ age especially in the elderly.

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Hepatitis Viruses Activated By Stress In Cells

Article Date: 15 Feb 2012 - 1:00 PST

People who have received a donor organ need lifelong immunosuppressant drugs to keep their immune system from attacking the foreign tissue. However, with a suppressed immune system, many infectious agents turn into a threat. Infections such as with human cytomegalovirus and a certain type of human polyomavirus frequently cause complications in transplant recipients. For these patients it would therefore be particularly beneficial to have substances that suppress the immune system and exert an antiviral activity at the same time - thus killing two birds with one stone.

Jointly with colleges from Heidelberg University Hospital of Internal Medicine, researchers Professors Karin and Felix Hoppe-Seyler of the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) have now tested a number of drugs with such an activity profile. They also tested the substances in liver cells infected with hepatitis B viruses (HBV) in the culture dish. The result: Liver cells that had been treated even produced considerably more viral offspring than untreated ones.

The substances under investigation inhibit the synthesis of nucleotides, which are the basic building blocks of DNA. This is how they exert their immunosuppressive effect: They slow down multiplication of immune cells because these lack building material for duplicating their genetic material. "The lack of DNA building blocks can cause a kind of stress in specific cells, which shows in the activation of a stress protein called p38", says Felix Hoppe-Seyler. "In liver cells, p38 very effectively activates the replication of hepatitis B viruses. "

The findings of the DKFZ researchers are a definite indication that administering these drugs in transplant recipients bears risks. Liver transplants, in particular, often have to be done because the body's own organ has been destroyed by hepatitis B viruses. In such cases, drug-induced activation of remaining HBV in the body may lead to the donor organ being attacked by hepatitis B viruses again.

Felix Hoppe-Seyler suspects that besides the three substances the group has investigated there are other substances which also cause an activation of p38. "In cancer patients being treated by chemotherapy, there is often a reactivation of chronic HBV infections. This has always been put down to their weakened immune system. We will now investigate whether this may also be due to activation of stress protein p38," said the virologist outlining the goals of his further research.

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Ultrasound technology proves accurate in diagnosing cirrhosis from recurrent hepatitis C

February 29, 2012

Researchers from the Mayo Clinic confirm that ultrasound-based transient elastography (TE) provides excellent diagnostic accuracy for detecting cirrhosis due to recurrent infection with hepatitis C virus (HCV) infection following liver transplantation. Findings from the study published in the March issue of Liver Transplantation, a journal of the American Association for the Study of Liver Diseases, suggest that detection of significant fibrosis is more accurate when comparing patients with chronic HCV of the native liver

According to the World Health Organization (WHO), chronic HCV affects up to 170 million people worldwide and could lead to more severe liver diseases such as cirrhosis and liver cancer. Experts estimate that on average 6,000 liver transplants are performed in the U.S. each year. Medical evidence shows that following liver transplantation recipients who are HCV RNA-positive at the time of transplantation are at risk of reinfection with HCV. Moreover, studies have determined that fibrotic tissue can develop more quickly in the transplanted liver resulting in rapid progression of cirrhosis and graft failure.

"The current gold standard for determining liver disease severity and progression is liver biopsy," explains lead author Dr. Jayant Talwalkar with the Mayo Clinic in Rochester, Minnesota. "However, biopsy following liver transplantation may not accurately determine fibrosis severity and non-invasive imaging technology has advanced to more accurately assess the severity of liver injury which includes an indirect assessment of elevated portal pressure." A prior study reported liver biopsy can understage cirrhosis in up to 30% of cases.

For the present study researchers reviewed studies of the diagnostic accuracy of ultrasound-based TE, a non-invasive technology used to assess fibrosis by measuring liver stiffness. The team analyzed the performance of TE compared to liver biopsy in detecting sever hepatic fibrosis caused by recurrent HCV post-transplantation. Compared to liver biopsy, TE is a reproducible diagnostic technique that is quick and painless for patients.

Six studies were identified, with five studies that evaluated significant fibrosis and cirrhosis. Analysis of the pooled estimates showed TE had a sensitivity and specificity of 83%, respectively for detecting fibrosis. Of the five studies analyzing TE for detecting cirrhosis, sensitivity estimates were 98% and specificity at 84%. "Ultrasound-based TE provides excellent diagnostic accuracy for identifying cirrhosis caused by recurrent HCV following liver transplantation," concludes Dr. Talwalkar. "Further studies that confirm our results could highlight the importance of TE as a diagnostic tool for liver transplant recipients infected with HCV."

More information: "Ultrasound-based Transient Elastography for the Detection of Hepatic Fibrosis in Patients with Recurrent HCV after Liver Transplantation: Systematic Review and Meta-analysis." Corlan O. Adebajo, Jayant A. Talwalkar, John J. Poterucha, W. Ray Kim, and Michael R. Charlton. Liver Transplantation; (DOI: 10.1002/lt.22460) Published online: February 24, 2012; Print Issue Date: March 2012.

Provided by Wiley (news : web)

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Don't Count Vertex Out Just Yet

By Brian Orelli, PhD, The Motley Fool Posted 8:02PM 02/28/12

Vertex Pharmaceuticals' (NAS: VRTX) Incivek isn't even in the coffin yet, but its obituary has been written by me and others. Sales have peaked -- or will soon -- as hepatitis C patients wait for new medications that don't have to be administered with Roche's Pegasys or Merck's (NYS: MRK) Pegintron, injectable drugs with unpleasant side effects.

Vertex is hoping that one of those all-oral drug combinations will still include Incivek, even if it has to develop the cocktail itself. Last week the company announced phase 2 data for a combination of Incivek, ribavirin -- an oral medication currently taken with Incivek and Pegasys/Pegintron -- and a new medication, VX-222.

A respectable 83% of patients had undetectable virus levels after taking the three-drug combination for 12 weeks. Some of those patients are going to rebound -- two of 11 patients who reached four weeks post-treatment have relapsed -- but the data is solid enough to justify moving into a larger phase 2b trial, which will help guide the design of a phase 3 trial. If that's positive, Vertex hopes for approval by early 2015.

The worry here is that Vertex might be throwing good money into useless clinical trials if Incivek becomes obsolete. The blockbuster is a protease inhibitor, but there are plenty of next-generation protease inhibitors in development: Bristol-Myers Squibb's (NYS: BMY) asunaprevir, Johnson & Johnson's (NYS: JNJ) TMC435, Achillion Pharmaceuticals' (NAS: ACHN) ACH-1625, and others. None is on the market -- they'll all have to prove themselves in the clinic -- but considering how many there are, it seems likely that one will be able to beat Incivek on the efficacy or safety front.

Going forward, though, drugs' efficacies won't be measured individually, but as part of a cocktail. For an all-Vertex cocktail to work, the company needs VX-222 and other drugs in its pipeline to make up for any shortcomings Incivek might have versus other protease inhibitors in other cocktails.

It could happen, for sure -- I wouldn't count Vertex out just yet -- but the company might be better off ditching Incivek and partnering up with others to help get its pipeline drugs into whatever standard-of-care cocktail is eventually developed.

Vertex was recommended twice by the Fool's Rule Breakers newsletter with both handily beating the S&P 500. To see what the analysts like right now, grab a copy of their new free report, "Discover the Next Rule-Breaking Multibagger." You can get your copy for free by clicking here.

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Liver ‘kept alive’ outside body

The technique allows doctors to monitor how well the organ functions and means livers can be stored for far longer.

February 27 2012 at 10:22am
By JO MACFARLANE

London - Scientists have created a machine which can keep donor livers functioning outside the body.

The breakthrough - a British first - could save the lives of hundreds of people needing liver transplants every year.

Livers taken from organ donors are currently packed in ice for up to 12 hours to prevent them from decay while they are transported, but this technique can affect the blood vessels and cause excess fat on the organ to solidify.

It means many are considered unsuitable for use and are discarded, despite the wishes of patients and families. In other cases the livers fail because of the damage caused to them during the transplant process.

The new machine keeps the liver in a solution at body temperature and feeds it with blood, oxygen and nutrients.

The technique allows doctors to monitor how well the organ functions and means livers can be stored for far longer - up to 24 hours - before being transplanted.

Known as METRA, the machine was originally used on pig livers but a recent trial found that out of 13 human livers discarded by doctors, six would have been good enough to be transplanted.

The machine was developed by Organox, a company linked to Oxford University which was set up by Professor Peter Friend, director of the Oxford Transplant Centre. He said: “The results show the machine can keep a donor liver healthy as if it is still in the human body. You also can see if it is functioning well enough for transplant.

“Decisions on usability are made purely on visual judgment. That is all surgeons have at the moment. That is why this machine is so useful.”

Surgeon David Mayer, liver lead for NHS Blood and Transplant, described the research as “extremely exciting”.

He said: “If this machine comes into use then it will almost certainly increase the number of livers we can transplant.”

Further trials involving patients are set to be approved later this year. - Mail on Sunday

Source

New HCV Drug Promising for Difficult-to-Treat Genotype 1 Patients

Infectious Disease Special Edition

ISSUE: FEBRUARY 2012 | VOLUME: 1

Robust Responses, But Some Treatment Failures; Adverse Events Raise Questions

by Christina Frangou

San Francisco—A second Phase IIb study of a new oral therapy for hepatitis C, PSI-7977, taken once daily, increased sustained virologic response (SVR) rates up to 91% in patients with difficult-to-treat hepatitis C virus (HCV) genotype 1 infection—a substantial improvement from the 50% reported in patients who received standard peginterferon and ribavirin (Peg-IFN/RBV) therapy. The study also showed significant advantages of PSI-7977 in patients who typically have poor responses to interferon.

Investigators say the new drug has the potential to dramatically alter the treatment paradigm for patients with hepatitis C virus (HCV) infection. “That’s true for all genotypes and all patients,” said lead author Eric J. Lawitz, MD, medical director of Alamo Medical Research, in San Antonio.

Patients who have the interleukin-28B (IL28B) TT genotype generally have a much lower chance of responding to IFN than people with the CC or CT genotypes. Yet in this study, these patients achieved high SVR rates to PSI-7977. Of the 13 patients who carried the TT allele, all tested negative for HCV by the third week of treatment and achieved SVR at week 12.

“The high SVR of greater than 90% in HCV genotype 1 patients was independent of predictors of poor IFN response,” Dr. Lawitz said.

The trial—known as PROTON—was the second Phase IIb study of PSI-7977 presented at The Liver Meeting 2011. In the first study, the ELECTRON study, investigators reported that 100% of patients who received PSI-7977 without IFN achieved an SVR (see “New Polymerase Inhibitor Could Become Cornerstone of Interferon-free HCV Treatment Regimen”).

Results from the two studies sparked considerable excitement among attendees of The Liver Meeting about this investigational therapy.

“I think it’s proof of principle that the proper combination of direct-acting antivirals can, in fact, produce enough suppression of viral replication to result in extinction of infection without the benefit of a broadly acting antiviral like IFN,” said Raymond Chung, MD, chief of hepatology and vice-chief of gastroenterology at Massachusetts General Hospital, in Boston.

The PROTON study was designed to examine dose-dependent response rates for PSI-7977 in HCV genotype 1–infected patients. Investigators enrolled 121 treatment-naive patients with this genotype in a randomized, double-blind, placebo-controlled, dose-ranging fashion. All patients were at least 18 years old with an HCV RNA level of 50,000 IU/mL or greater, platelets greater than 90,000/mm³, neutrophils greater than 1,500/mm³, and a hemoglobin level of at least 11 g/dL, with no evidence of cirrhosis.

Trial participants were randomized in a 2:2:1 ratio to one of three treatment arms: PSI-7977 200 or 400 mg daily in combination with IFN and RBV (n=48 and n=47, respectively) or Peg-IFN/RBV alone (n=26). Patients in the PSI-7977 arms received triple therapy for 12 weeks, followed by an additional 12 weeks of Peg-IFN/RBV. All patients who achieved early rapid virologic response (RVR) discontinued therapy at 24 weeks, while all others continued therapy for a total of 48 weeks. Patients in the IFN and RBV arm received treatment for 48 weeks.

Analysis showed robust response rates among all PSI-7977 patients regardless of dose. Patients who received 200 mg daily showed an RVR rate of 98%, an early RVR rate of 98% and an end-of-treatment response rate of 91%. Patients receiving 400 mg showed a 98% RVR, 91% early RVR and 91% end-of treatment response through 24 weeks. In contrast, response rates among patients who received Peg-IFN/RBV alone were 19%, 50% and 50%, respectively.

In an as-treated analysis of patients who received at least eight weeks of PSI-7977, 88% of those in the 200-mg arm and 98% of those in the 400-mg arm achieved an SVR at 12 weeks.

Several failures occurred in the trial. Three patients in the 200-mg arm who had viral suppression during the first 12 weeks of treatment with PSI-7977 experienced a virologic breakthrough during the follow-up treatment period with Peg-IFN/RBV. No breakthroughs were observed among patients in the 400-mg arm, although one patient had a viral relapse before SVR at 4 weeks. “This suggests that [the] 400-mg dose achieved a deeper viral suppression,” said Dr. Lawitz.

The extent of viral suppression may be the key difference between the 200- and 400-mg doses of PSI-7977, he said. PSI-7977 at a 400-mg dose may provide a more thorough viral suppression with lower risk for virologic breakthrough. Patients in both groups quickly became negative for HCV RNA after PSI-7977 was started, but patients who received the 200-mg dose had more virologic breakthroughs after the PSI-7977 therapy was completed. No patient in either treatment arm developed an S282T mutation.

Investigators said they were pleased to see that the adverse events reported in the PROTON trial were typical of those seen with Peg-IFN/RBV treatment, except for a small increase in insomnia. Overall, 15% of patients in the 400-mg arm and 8% of those in the Peg-IFN/RBV–only arm reported insomnia.

More cases of neutropenia occurred with PSI-7977 than with Peg-IFN/RBV, but it was unlikely that the difference was significant, said the investigators. Three patients who received PSI-7977 had grade IV neutropenia compared with none in the Peg-IFN/RBV arm. However, the numbers of patients were too low to result in meaningful conclusions.

Still, experts say more patients must be studied to confirm that the new drug is safe.

“The numbers for neutropenia and anemia are confusing to me. They are small numbers but I’m still not certain about the safety of this compound,” said Paul Pockros, MD, head of gastroenterology and hepatology, and director of the Center for Liver Diseases, Scripps Clinic, in La Jolla, Calif.

The study was not powered to detect differences in neutropenia and anemia between the three groups, but differences would be statistically significant if 1,000 patients had participated in the trial, Dr. Pockros explained. “Is this drug going to be safe in 1,000 patients?” he asked.

Dr. Lawitz said investigators would expect to see a difference in adverse events between the 200- and 400-mg arms in this trial if PSI-7977 increased the risk for neutropenia, but that was not the case, with more neutropenia and anemia occurring in the patients receiving the lower dose.

“To me, that suggests that this is a trial with small numbers, and when we get larger numbers the difference is probably not going to be significant,” he said.

PSI 7977, in combination with RBV as dual therapy, is set to enter Phase III trials.

---

Dr. Lawitz reported relationships with Abbott Laboratories, Achillion Pharmaceuticals, Anadys Pharmaceuticals, Biolex Therapeutics, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, GlobeImmune, Idenix Pharmaceuticals, Idera Pharmaceuticals, Inhibitex Pharmaceuticals, Medarex, Medtronic, Merck & Co., Novartis, Pharmasset, Roche, Schering-Plough, Santaris Pharmaceuticals, Scynexis Pharmaceuticals, Tibotec, Vertex Pharmaceuticals, ViroChem Pharma and ZymoGenetics. Dr. Chung receives grant or research support from Gilead, Merck & Co., Pfizer and Romark. Dr. Pockros reported relationships with Abbott Laboratories, Bristol-Myers Squibb, Contus, Gilead Sciences, Novartis, Pfizer, Three Rivers Technologies, Tibotec and Vertex Pharmaceuticals.

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HCAB Position Statement: Hepatitis C Drug Development and Drug-Drug Interaction Studies

February 2012 - The Hepatitis C Community Advisory Board (HCAB) recognizes the value of more effective and less toxic treatment for hepatitis C virus (HCV). We believe that sponsors can conduct key drug-drug interaction (DDI) studies with direct-acting antivirals (DAAs) and other candidates in development and medications commonly used by people with hepatitis C and those coinfected with HIV/HCV prior to their approval, without delaying development of these important therapies.

DAAs may share metabolic pathways with drugs that are commonly used by populations with a high prevalence of hepatitis C, such as hormonal contraceptives, methadone, buprenorphine, lipid lowering agents, immunosuppressive drugs, herbal remedies, and commonly prescribed psychiatric medications.

In recognition of the suboptimal efficacy and tolerability of peginterferon and ribavirin, rapid trajectory of liver disease progression and increasing mortality from HCV-related complications among HIV/HCV coinfected patients, regulators in the US and the EU encourage sponsors to conduct trials in HIV/HCV coinfected patients prior to approval for HCV monoinfection. Sponsors have already opened, or plan to launch these trials.

The recent discovery of drug-drug interactions between boceprevir and boosted HIV protease inhibitors underscores the importance of DDI studies with DAAs. Although we commend the sponsor, Merck, for opening one of the first coinfection trials with a DAA, we were outraged that Merck chose not to conduct DDIs with commonly used antiretroviral agents prior to launching the trial, and prior to gaining approval for boceprevir. Vertex and Tibotec were able to bring telaprevir to market with a much fuller portfolio of DDI data, although both drugs were developed within the same timeframe.

HCAB asks FDA, EMA and pharmaceutical companies to work together to minimize potential harm to hepatitis C monoinfected and HIV/HCV coinfected patients from uncharacterized drug-drug interactions (DDIs). Furthermore, we call upon sponsors to perform DDI studies (as indicated by metabolic profile of their drug or drugs) with DHHS, EACS and WHO-recommended antiretroviral agents for first-line, and treatment-experienced HIV/HCV coinfected people prior to approval, and strongly encourage studies of hormonal contraceptives, methadone, buprenorphine, lipid lowering agents, immunosuppressive drugs, herbal remedies, and commonly prescribed psychiatric medications.

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FDA updates statin safety guidelines

In this June 14, 2011 photo, the drug Lipitor is displayed at Medco Health Solutions Inc., in Willingboro, N.J. (Matt Rourke - Associated Press)

Posted at 02:28 PM ET, 02/28/2012

By Jennifer LaRue Huget

The FDA Tuesday issued updated safety labeling information for the class of drugs known as statins. The agency noted those drugs’ utility in reducing “bad” cholesterol “when used with diet and exercise” but fine-tuned guidelines for their use.

The new guidelines, aimed at health-care professionals, make it easier for patients to stay on statin therapy by eliminating the need for routine monitoring of liver enzymes. Liver disease among statin users is too uncommon and unpredictable, the agency notes, for such routine screening to be practical. Instead, patients should be screened for liver disease before starting statin therapy and then only if symptoms of liver disease (such as unusual fatigue or weakness, loss of appetite, upper belly pain, dark-colored urine, or yellowing of the skin or the whites of the eyes) appear.

The statement also notes that some patients taking statins may experience memory loss or confusion, but that both those conditions resolve when statin use is discontinued. However, the FDA says, some studies show that statins slightly increase a patient’s risk of elevated blood-sugar levels and of developing type 2 diabetes.

Finally, the statement notes that one variety of statin drugs — lovastatin — may interfere with other medications and lead to muscle damage. Physicians should take care to read the new safety labels for those drugs when prescribing them, the statement says.

Statins include the following: Lipitor (atorvastatin), Lescol (fluvastatin), Mevacor (lovastatin), Altoprev (lovastatin extended-release), Livalo (pitavastatin), Pravachol (pravastatin), Crestor (rosuvastatin) and Zocor (simvastatin). Combination products include: Advicor (lovastatin/niacin extended-release), Simcor (simvastatin/niacin extended-release), and Vytorin (simvastatin/ezetimibe).

In other statin news, research published online Feb. 21 in the Journal of Clinical Psychiatry found that people with coronary heart disease who take statins are at reduced risk of depression.

By Jennifer LaRue Huget | 02:28 PM ET, 02/28/2012

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Is it Safe to Donate Part of Your Liver?

Provided by The AGA Journals Blog

Posted on February 27, 2012 

by Kristine Novak, PhD, Science Editor

Donating part of your liver is just as safe as donating a kidney—donors of these organs have survival rates similar to the rest of the population, according to an article in the February issue of Gastroenterology.

With organ shortages, live-donor liver transplantation (LDLT) is a lifesaving alternative to transplantation from deceased donors for patients with end-stage liver disease. Since the introduction of LDLT 20 years ago, thousands of children requiring liver transplants have benefited from left lateral segment grafts from live donors. Adults now also receive liver transplants from live donors. However, the numbers of livers transplanted from live donors has fallen short of the initial expectations over the past 8 years, due to safety concerns.

To determine the safety and side effects of LDLT, Abimereki Muzaale et al. followed 4111 live liver donors in the US from April 1994 to March 2011. They determined rates of deaths from the Social Security Death Master File and compared survival data with those from live kidney donors and healthy participants of the National Health and Nutrition Examination Survey (NHANES) III.

The authors found that the 90-day mortality of living liver donors (1.7 per 1000 donors or 0.17%) did not differ significantly from that of living kidney donors (0.05%). The long-term cumulative mortality risk was comparable to that of live kidney donors and NHANES participants (1.2%, 1.2%, and 1.4%, respectively) at 11 years.

Kaplan–Meier curves of cumulative mortalities for live donors of livers, matched live donors of kidneys, and matched controls (participants in NHANES III).

There were 4 cases of nonfatal acute liver failure in living liver donors; 3 of these patients required deceased-donor liver transplantation.

In an editorial that accompanies the article, James Trotter and James Everhart state that these results are likely the best estimation of donor mortality, and that this information will be useful for clinicians in counseling potential LDLT donors and recipients. They add that the format of cross-referencing 2 databases provides an important study design for future estimates of donor mortality.

Previous studies reported that physical well-being of donors decreases after surgery, but returns to pre-donation levels within 1 year. Out-of-pocket expenses and lost wages have been estimated to average about $5,000 for donors, although this amount varies greatly.

Nonetheless, Trotter and Everhart say it is important to consider the intangible benefits of an altruistic act. Other benefits include having a loved one remain alive and returned to function, as well as a public health benefit—living donation allows another patient to receive a deceased donor liver, thereby decreasing the demand for deceased donor transplantation.

Trotter and Everhart conclude that the findings of Muzaale et al. set the standard for estimating donor mortality. “Because the liver community has been unable to establish a national database for donor outcomes, and there are no real prospects for such, the rigorous methodology utilized in this study will allow the best means of prospectively reporting living liver donor mortality risk in the future.” However, further studies are needed to determine long-term outcomes of LDLTs.

Read the article online.
Muzaale AD, Dagher NN, Montgomery RA, et al. Estimates of early death, acute liver failure, and long-term mortality among live liver donors. Gastroenterology 2012;273–280.

Read the accompanying editorial.
Trotter JF and Everhart JT. Outcomes among living liver donors. Gastroenterology 2012;207–210.

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Gilead Hepatitis C Setback Opens Door to Rivals

By Deena Beasley

LOS ANGELES (Reuters) Feb 24 - Gilead Sciences Inc made its name by becoming the leader for drugs against HIV, but a setback for its hepatitis C treatment has opened the door to rivals, making that market tougher to dominate.

Top global drugmakers and tiny biotech companies have over 100 compounds in clinical development to treat the liver-destroying hepatitis C virus (HCV), according to Thomson Reuters Pharma.

The field has attracted broad interest with two new hepatitis C drugs reaching the U.S. market in the past year. Both represent a breakthrough in treatment for a global patient population of up to 170 million.

Gilead and its rivals plan to improve on those medicines with pills that do not need to be combined with injections of immune system boosters such as interferon, which have side effects that can deter patients. They are all vying for a market expected to reach around $10 billion a year, but they may each end up with a smaller slice of the pie.

"We don't forecast permanent dominance in any class of HCV antivirals by any participant, including Gilead," Sanford Bernstein analyst Geoff Porges said.

Gilead spent $11 billion in January to buy Pharmasset and its lead hepatitis C drug candidate in a new class known as nucleotide inhibitors, hoping to be the first to market with an all-oral hepatitis treatment.

But the enthusiasm of investors for the deal was doused last week after Gilead said some patients taking the drug saw the virus return after four weeks. The company's shares are off nearly 20% since the announcement, giving back some of the 50% gain in the wake of the Pharmasset purchase.

Gilead's stumble may make way for rivals such as Bristol-Myers Squibb Co, which recently bought Inhibitex for $2.5 billion, or a premium of 163%, to boost its HCV portfolio. It has already fueled additional interest in much smaller competitors Achillion Pharmaceuticals Inc and Idenix Pharmaceuticals Inc.

GOAL IS ALL-ORAL TREATMENT

Traditional hepatitis C therapy requires a year of treatment with a combination of interferon and ribavirin. It can cure about half of treated patients, but harsh flu-like side effects prevent many from reaching that goal.

Greater knowledge of genetics and molecular function has led to several new classes of direct acting antiviral drugs, which are expected to revolutionize treatment.

"The goal is to go interferon-free over the next few years, and have an all-oral regimen that basically can eradicate the virus in over 80% of people who are infected in 12 to 24 weeks," said Dr. Fred Poordad, chief of hepatology and liver transplantation at Cedars-Sinai Medical Center in Los Angeles.

Dr. Poordad is involved in "about 90%" of current hepatitis C trials and sees several viable treatment regimens becoming available.

"This is not going to be a winner takes all type of thing," he said.

Around 3.2 million Americans -- mainly baby boomers -- have chronic hepatitis C, according to the U.S. Centers for Disease Control and Prevention. The rate of new U.S. infections has slowed to around 17,000 a year following the introduction of blood and organ donor screening in the early 1990s and greater awareness of the risk from shared needles.

The virus is often undiagnosed and contracted through contact with blood from an infected person. There may be few initial symptoms, but it is the leading cause of liver transplants and can cause liver cancer.

Hepatitis C killed more than 15,000 Americans in 2007, compared with a little over 12,700 deaths related to HIV, according to recent CDC statistics.

New drugs include protease inhibitors, which aim to block enzymes used by the viruses to cling to certain proteins; nucleotide or nucleoside inhibitors, also known as "nukes," which interfere with enzymes needed for viral replication; and blockers of NS5A, a protein essential to viral replication.

Two new protease inhibitors launched last year -- Incivek from Vertex Pharmaceuticals Inc and Merck & Co's Victrelis -- are still taken with interferon and ribavirin.

Gilead's GS-7977, a nucleotide inhibitor, was supposed to work without interferon. But the latest data on patients who have not responded to earlier treatment showed the drug may need to be combined with additional antiviral medications.

Bristol-Myers acquired an experimental nucleotide inhibitor through the Inhibitex deal and it also has a promising NS5A inhibitor. The shares of Idenix, which has nucleotide inhibitors in development, have soared 64% so far this year.

Novartis AG said this week it would pay $34 million upfront and $406 million in milestone payments to license an NS5A inhibitor from Enanta Pharmaceuticals.

Other companies with second generation protease inhibitors include Johnson & Johnson and Merck. Roche Holding AG and Vertex are developing nukes and other all-oral hepatitis C drug regimens are being explored by Abbott Laboratories, Pfizer Inc and Boehringer Ingelheim.

Investor enthusiasm runs high, and on very early evidence. The shares of another small company, Biocryst Pharmaceuticals Inc, shot up 12% in a single day after it said experimental hepatitis C drug BCX5191 showed promise in rat testing.

NO WINNER TAKES ALL

Julie Hoggatt, an analyst with Wolters Kluwer's inThought, puts the total market for hepatitis C drugs in the United States, Europe and Japan at $10.4 billion in 2016, falling to $8.6 billion in 2021 as more patients are cured and new infection rates decline.

Hoggatt estimated that only about 60,000 U.S. hepatitis C patients are currently being treated.

"Over the next 10 years, we are going to treat the majority of (U.S.) patients," she said.

Current treatments run about $60,000 a year and analysts estimate the eventual cost of a course of therapy at between $40,000 and $100,000.

"It will be a fragmented market and everybody is going to be fighting over the $500- to $800-million-a-year range," she said. "It is hard for one player to get into billions a year."

Others are much more bullish. Pharmasset, in its regulatory filings, forecast that its own worldwide sales of HCV drugs would peak at $8.2 billion in 2017, falling to a run rate of $4.5 billion in 2025.

Its new owner Gilead has been more circumspect and forecasts a longer time frame to bring patients on board.

"We think there's a lot of patients that can be captured both in the United States, Europe, and other territories, which could provide for a very long and sustained revenue and cash flow stream for Gilead," Chief Operating Officer John Milligan said earlier this month.

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In 2015, Gilead's GS-7977 Plus Ribavirin Will Earn Decision Resources' Proprietary Clinical Gold Standard Status for the Treatment of Non-Responder Patients with Hepatitis C Virus

PRESS RELEASE

Feb. 28, 2012, 9:00 a.m. EST

GS-7977 Plus Ribavirin Will Displace the Current Proprietary Clinical Gold Standard, Telaprevir in Combination with Peg-IFNa/Ribavirin, According to Findings from Decision Resources

BURLINGTON, Mass., Feb 28, 2012 (BUSINESS WIRE) -- Decision Resources, one of the world's leading research and advisory firms for pharmaceutical and healthcare issues, finds that, based on clinical data and the opinions of interviewed thought leaders, telaprevir (Vertex's Incivek, Johnson & Johnson's Incivo) in combination with peg-IFNa (Roche's Pegasys or Merck's Victrelis) and ribavirin (Roche's Copegus; Merck's Rebetol; generics) has earned Decision Resources' proprietary clinical gold standard status for the treatment of non-responder patients with hepatitis C virus (HCV). Owing to its competitive advantages in safety and tolerability as well as delivery, the interferon-free combination of Gilead's GS-7977 (formerly PSI-7977) plus ribavirin will displace telaprevir plus peg-IFNa/ribavirin and earn proprietary clinical gold standard status for HCV non-responders in 2015, following its launch for the indication in 2014 in the United States.

Decision Resources' analysis of the hepatitis C virus drug market also finds that surveyed U.S. gastroenterologists and managed care organization (MCO) pharmacy directors agree that the percentage of genotype-1 null responders achieving sustained virologic response is one of the attributes that most influences their decisions regarding prescribing and formulary status determinations, respectively, in HCV non-responders.

"Clinical data and the opinions of interviewed thought leaders indicate that several emerging regimens utilizing novel, HCV-specific direct-acting antivirals have advantages over sales-leading telaprevir plus peg-IFNa/ribavirin on this attribute," said Decision Resources Analyst Seamus Levine-Wilkinson, Ph.D.

According to insights from surveyed U.S. gastroenterologists and MCO pharmacy directors, the absence of interferon-free treatment options for HCV is one of the greatest unmet needs in HCV. Clinical data and the opinions of interviewed thought leaders indicate that GS-7977 has demonstrated the potential to significantly fulfill this unmet need.

The findings also reveal that surveyed U.S. gastroenterologists indicate that they would prescribe the quadruple regimen of Bristol-Myers Squibb's NS5A inhibitor daclatasvir (BMS-790052) plus Bristol-Myers Squibb's protease inhibitor asunaprevir (BMS-650032) plus peg-IFNa/ribavirin to 41 percent of their HCV non-responder patients. Decision Resources' forecast for this quadruple regimen is more conservative due to anticipated reimbursement restrictions, positioning in later lines of therapy, competition from IFN-free regimens and competition asunaprevir will face from other protease inhibitors.

The launch of novel HCV-specific agents will increase the size of the drug-treated population mainly as a result of re-treatment of prior non-responders as well as increased referral and drug treatment rates. The overall HCV drug market will experience significant growth, expanding from almost $1.7 billion in 2010 to $14.4 billion in 2015 in the United States, France, Germany, Italy, Spain, the United Kingdom and Japan. Thereafter, the market will decrease to $11.2 billion in 2020, owing to a decline in the size of the treatment-eligible population due to declining prevalence and effective new regimens.

Decision Resources' Robust Market Forecast and Opportunities Analysis

Decision Resources provides a comprehensive view of what is happening in a specific drug market now and in the decade ahead. The research includes analysis of the unmet need and near-term drug development opportunities that exist within a drug market powered by primary research from physicians and payers. The robust market forecast and opportunities analysis is comprised of the Pharmacor 2012 advisory service and the DecisionBase 2012 report series.

About Decision Resources

Decision Resources ( www.decisionresources.com ) is a world leader in market research publications, advisory services and consulting designed to help clients shape strategy, allocate resources and master their chosen markets. Decision Resources is a Decision Resources Group company.

About Decision Resources Group

Decision Resources Group is a cohesive portfolio of companies that offers best-in-class, high-value information and insights on important sectors of the healthcare industry. Clients rely on this analysis and data to make informed decisions. Please visit Decision Resources Group at www.DecisionResourcesGroup.com .

All company, brand, or product names contained in this document may be trademarks or registered trademarks of their respective holders.

SOURCE: Decision Resources

Decision Resources 
Christopher Comfort, 781-993-2597 
ccomfort@dresources.com

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Math can save Tylenol overdose patients

SALT LAKE CITY, Feb. 27, 2012 – University of Utah mathematicians developed a set of calculus equations to make it easier for doctors to save Tylenol overdose patients by quickly estimating how much painkiller they took, when they consumed it and whether they will require a liver transplant to survive.

"It's an opportunity to use mathematical methods to improve medical practice and save lives," says Fred Adler, a professor of mathematics and biology and coauthor of a study that developed and tested the new method.

The study of acetaminophen – the generic pain and fever medicine sold as Tylenol and in many other nonprescription and prescription drugs – was set for publication within a week in Hepatology, a journal about liver function and disease.

Adler, math doctoral student Chris Remien and their colleagues showed that using only four common medical lab tests – known as AST, ALT, INR and creatinine – the equations can quickly and accurately predict which Tylenol overdose patients will survive with medical treatment and which will die unless they receive a liver transplant.

The researchers analyzed the records of 53 acetaminophen overdose patients treated at the University of Utah's University Hospital to test the equations and show they quickly and accurately predicted, in retrospect, which patients survived and which died.

Speed is essential in listing acute liver failure patients as candidates for transplant, says study coauthor Norman Sussman, a former University of Utah liver doctor now at the Baylor College of Medicine in Houston.

If a doctor is uncertain and starts to treat an acetaminophen-poisoning patient with the antidote to combat liver failure – even though the patient may not survive with such medicine – their odds for getting a new liver are reduced.

"If I wait another day until I list them for transplant, the chance of getting a liver is that much lower," Sussman says. "If you're going to get someone transplanted, you have to do it fast or you miss the boat. The patient may pass the window when transplants can be done. They become too sick and can't stand the transplant."

The new method using calculus equations will let doctors rapidly determine if a patient can survive with antidote treatment or will die unless they get a transplant.

The study urges another clinical trial to prove the new method's usefulness. Sussman plans to start a one-year prospective trial testing the method on 50 patients at the University of Utah and three hospitals in Houston.

If that trial proves the method can accurately predict ahead of time how Tylenol-poisoning patients will fare, "we believe we could create a tool available and immediately useful to clinicians," Sussman says. Adler foresees a smartphone application.

Adler, Remien and Sussman conducted the study with University of Utah hepatologist Terry Box and Lindsey Waddoups, clinical research coordinator for the University of Utah's gastroenterology division. The research was funded by a National Science Foundation grant to the University of Utah's program in mathematical biology.

Painkiller Can Be a Killer

Acetaminophen – the primary generic name for the drug also known generically as APAP and paracetemol – is found in prescription medicines such as Tylenol with Codeine, Percocet or Vicodin, and in dozens of over-the-counter medications, including Tylenol, Anacin, Pediacare, Triaminic and combination cold medications like Nyquil.

Many people don't realize the common analgesic can destroy the liver and kill at only about five times the recommended dosage – a narrow margin in medical terms.

"Acetaminophen is the leading cause of acute liver injury in the United States, accounting for some 56,000 emergency room visits, 26,000 hospital admissions and about 500 deaths annually," Adler and his coauthors write.

University of Utah mathematicians Chris Remien and Fred Adler with some of the calculus equations that are part of a new method they developed and tested to make it easier for doctors to save Tylenol overdose patients by quickly determining if they can be saved with an antidote or will require a liver transplant to survive.

(Photo Credit: Lee J. Siegel)

The current maximum dose of acetaminophen is 4 grams (4,000 milligrams or eight 500 milligram tablets, for example) in 24 hours. There is not a lot of room for error between that 4-gram maximum and the 6 grams that can cause liver damage or the 20 grams that are considered likely to destroy 70 percent of liver cells and cause death.

Acetaminophen overdoses can be treated successfully if an antidote named N-acetylcysteine (N-Ac or "nack") is administered within roughly 24 hours. After a certain time post-overdose, treatment becomes futile and the patient will die without a transplant.

Yet many overdose patients are confused or comatose, unable to say how much acetaminophen they took or when they took it, making it tough to predict their prognosis.

Life-Saving Calculus

The new method uses eight main "differential equations" – basic calculus equations that describe how changes in one variable affects changes in another variable over time. The equations simulate or "model," step-by-step, how acetaminophen is metabolized in the liver, including production of NAPQI, a liver-destroying substance.

That makes it the first known "dynamical" model based on real biology – a contrast to the existing "statistical" method for determining how overdose patients fare.

The statistical method – known as the King's College Criteria (KCC) – estimates who is likely to survive or die from acetaminophen toxicity using correlations between INR and creatinine lab tests and which patients actually did live or die in the past. The King's College Criteria predict liver failure if INR exceeds 6.4, creatinine exceeds 3.4 and there is confusion, altered consciousness or coma due to liver damage.

The problem, says Adler, is the criteria "look at the statistical relationship between lab test results and patient outcome without understanding what's happening inside the liver. It's just statistics."

The new method "tracks how the liver's health changes over time," he says.

The new equations use patients' measured levels of AST, ALT and INR to estimate when they consumed acetaminophen and how much they took. By also considering creatinine levels, the new method accurately predicts which Tylenol overdose patients will survive with treatment and which will require a liver transplant to avoid death.

AST (aspartate aminotransferase) and ALT (alanine aminotransferase) are enzymes that are released by dying liver cells, so higher levels indicate liver damage. INR (prothrombin time/international normalized ratio) measures how fast blood clots. Liver cells make clotting factors, so if the liver malfunctions, clotting is slower. Creatinine is a measure of kidney dysfunction, in this case secondary to liver damage.

Sussman says the King's College Criteria are outdated and have grown less useful over time. When a patient arrives with lab results indicating liver failure, "your first decision has to be, 'Do I list this patient for transplant?'" he says. "That was the purpose of the King's College Criteria. You need to make an immediate decision: Do I think this patient will live or die? If I think they'll live, I'm going to treat them [with the antidote]. If they're going to die, the next question is, are they a candidate for transplant?"

"Our goal was to try to trace it back to: when did the damage start?," he adds. "Once you know that and the peak damage reflected in the ALT, then you have the tools to predict survival or death."

Of people who are liver transplant candidates, those with acute liver failure – half due to acetaminophen poisoning – go straight to the top of the liver transplant list, ahead of the vast majority of candidates who have chronic liver failure, such as from alcoholism, Sussman says.

Predicting Overdose Outcomes

The 53 patients whose records were analyzed for the new study varied in alcohol use, malnutrition status and whether they took too much acetaminophen in a suicide attempt, an accidental single overdose or a chronic, multiple-day overdose.

Two patients got liver transplants and were excluded from the analysis "because we don't know if they would have died or recovered without transplant," Remien says.

Of the 51 remaining patients, eight died and 43 survived. The study showed that when AST, ALT and INR tests on admission were crunched through the equations, and when creatinine levels exceeded 3.4, the method was highly accurate in predicting, retrospectively, whether overdose patients lived or died. Specifically, the method had:

• 100 percent sensitivity, meaning the method correctly predicted the deaths of all eight patients who actually had died. By comparison, the King's College Criteria predicted only one of the eight deaths.
• 67 percent "positive predictive value," meaning eight patients died out of 12 deaths predicted by the method.
• 91 percent specificity, meaning the method predicted 39 patients would survive out of the 43 that really did survive.
• 100 percent "negative predictive value," meaning the method predicted 39 patients would survive and those 39 did survive.

Sussman says there were multiple reasons for the eight deaths. Some patients arrived too late to be treated, even by a transplant, and others didn't qualify as transplant candidates, perhaps due to serious drug or alcohol abuse and lack of family support.

About 16,000 people now are on the liver transplant waiting list in the United States. About 5,000 to 6,600 Americans get liver transplants each year.

Adler emphasizes the new method is based on a typical acute liver failure patient and may need refinement to better predict the prognoses of certain special patients, including those taking other drugs, with chronic alcohol use or suffering anorexia.

Source: University of Utah

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