Introduction

More than 100 scientists gathered at the Don CeSar Hotel in St. Pete, Florida on January 25-26 for the 5th IBC conference on the latest therapeutic developments in viral hepatitis B & C (HBV & HCV). These meetings have become important annual events for hearing updates and ideas regarding development- oriented research. They are characterized by their relatively small size and informal and often lively interactive nature. These are clearly historic times in viral hepatitis therapeutics, with the approval of the first orally available HBV therapeutic (Epivir-HBV, or lamivudine; Glaxo-Wellcome) and an increasing body of evidence that viral therapeutics for HBV and even HCV have clear clinical benefit for the patient. Needless to say, the development of HBV therapeutics is at least five to ten years ahead of that for HCV. The meeting results, however, suggest that HCV antiviral research is moving briskly and has benefited greatly from the HBV experience, from both clinical and basic science perspectives.

Meeting Highlights

• Interferon alpha is of clear value to an HCV patient minority, but species genotype is of great importance in predicting responsiveness
• Development of assays to detect anti-HCV compounds are up and running
• Histological benefit from lamivudine (Epivir-HBV) is now apparent from Phase III studies
• Resistant mutants to Epivir-HBV continue to cast a shadow on the long-term benefit from this drug
• Other nucleoside and non-nucleoside HBV therapies may be within a few years of launch
• Efficient transport of intact proteins across mammalian cell membranes is possible (surprise, off-beat presentation that generated a great deal of discussion!)

Presentation Overviews

HCV and the interferon (IF) experience. Dr. Gary Davis (University of Florida; Gainesville, FL) discussed the emerging threat of HCV, showing at least 4 million people chronically infected in the US alone, with an emphasis placed on the spread of infection worldwide. Clinically, the best correlate of outcome (as with HBV, see below) is the degree of liver damage in the presenting patient: those with mild to advanced cirrhosis proceed most rapidly to poorer outcomes, whilst those with limited liver disease are (perhaps not surprisingly) the farthest in time from complications. Since the majority of those chronically infected have limited liver damage, it is necessary to develop methods of predicting (a) which populations will deteriorate and (b) which will benefit from existing therapies. Sustained response rates to IF in combination with ribavirin exceed 20%, as defined by apparent clearance of serum HCV viral RNA. Moreover, decreases of viral titers of at least 1 log were associated with clear liver histological improvements. Although controversy surrounding the importance of viral genotype in influencing therapeutic decisions continues, there is a growing body of evidence that genotype 1a (the predominant species in the US) is associated with the greatest degree of IF resistance.

Interferon (IF) sensitivity/ resistance may be mediated at the viral level by a non-structural gene. Dr. Michael Katz (University of Washington) has shown that the NS5a polypeptide binds the dimerization domain of PKR. A region within NS5a (called the interferon sensitivity domain region, ISDR) has been genetically related to IF resistance, and inhibiting the PKR kinase in the IF pathway has been associated with viral antagonism of IF's antiviral function. Therefore, Dr. Katz provides a compelling argument that HCV variable genetic sensitivity to IF can, in part, be explained by the affinity of a quasi species NS5a region of IF PKR.

Current clinical options for HCV, in summary.

The current clinical options for HCV involve IFs and variations of them. IF, even as a parenteral therapy with numerous untoward side effects, will likely remain an important part of the antiviral HCV arsenal for the near term. However, its limitations underscore the need for alternatives. New HCV therapeutics are currently in their developmental infancy. The HCV genome specifies a handful of enzymatic functions essential to the virus life cycle; these are thought to be attractive targets for chemotherapy, and include the HCV proteases [e.g. NS2, a possible Zn protease, the serine protease/ RNA helicase (NS3), NS4A (NS3 protease cofactor), NS5A (interferon resistance) and NS5B (the RNA-dependent RNA polymerase)]. The infancy of the HCV antiviral programs was reflected in the emphasis upon assay development programs rather then discovery of new compounds. The next few reviews concern pursuit of antivirals directed against viral gene functions.

HCV protease as a target. Dr. Bruce Malcom (Schering-Plough Research Institute) reported systems permitting production of at least 10 mg/L recombinant histidine N-terminally tagged NS3/4 protease capable of cleaving NS5-like cleavage sites. Viral helicase activity can be added to the construct by inserting appropriate catalytic regions at the C-terminus. Continuous spectrophotometrically monitored assays provide the promise of a high-throughput screen. Dr. Chistian Steinkuhler (Instituto di Ricerche di Biologia Molecolare, Pomezia, Rome, Italy) continued the HCV protease theme by reporting about models of the NS3 protease with the NS4a/4b substrate sitting in the active site. Significantly, the DEMEEC-COOH product of the DEMEEC-ASHLPYK substrate causes inhibition of the protease, which can frustrate assays and must be considered in kinetic analysis. Also, the pockets of the protease within which the substrate sits are rather far apart, making the construction of small competitive inhibitors that span both sites a challenge. The solution, it was reasoned, might be to interfere with the active site by competing for only a single anchor site. The potential of a serine trap approach was discussed using N-Asp-Glu-Dap [Dansyl]-Glu-Cha-Cys-OH as a probe. Serine trap inhibitors may include compounds containing an activated carbonyl replacing the scissile bond that may form reversible, covalent adducts with the active site of serine proteases. Dr. Jeff Saunders (Vertex Pharmaceuticals) presented compelling physical and genetic evidence to support their proposed crystal structure of the helicase/NTPase component of NS3. This work helps set the stage for structure-assisted rationally designed inhibitors.

HCV RNA-dependent RNA polymerase as a classical target for antiviral therapy. Dr. Hicham Alaoui-Ismaili (Biochem Therapeutic, Inc.) described the development of HCV polymerase assays. The HCV polymerase (pol) function, specified by the NS5B gene, involves a 68 kDa membrane-associated phosphoprotein which may interact with other viral polypeptides such as NS3, 4A and 5A. A recombinant NS5B was produced using recombinant baculoviruses. 32-P-uridine incorporation assays showed that the purified recombinant polypeptide retained enzyme activity, using homo- and hetero-polymeric RNA templates. A "snap/copy back" priming mechanism was used with heteropolymeric templates. Histidine tagging at the N terminus decreased enzymatic activity, whilst C terminal tagging enhanced activity. The ability to generate recombinant polymerase polypeptide makes one optimistic that high-throughput screening for classical anti-pol compounds is a very realistic goal. These technologies were not routinely available in the pursuit of HBV pol inhibitors.

A variety of less conventional antiviral strategies for HCV infection were presented at the meeting and are discussed in the following section.

Internal ribosome entry site (IRES) as a target for antivirals. Protein translation from most cellular mRNAs is mediated by a 5' methyl cap resulting in initiation from an AUG near the 5' end of the message. Since internal initiation of translation is an unusual feature of some viral mRNAs, including HCV, OSI Pharmaceuticals scientist Dr. Weizhong Cai presented a strategy to pursue inhibitors of the HCV internal ribosome entry site (IRES) function. An innovative transfected cell-based assay was described in which plasmids directing both cap- and IRES- dependent translation of reporter luciferase functions were followed as a function of the presence or absence of test compounds. Selectivity is indicated by the ability of the inhibitor to not influence viability while inhibiting IRES -- but not cap -- dependent translation of the reported (luciferase) function. Lead compounds, from a screen of more than 120,000, were identified and are currently under investigation. Such compounds would certainly serve as interesting research tools, at a minimum, and could herald a new class of antivirals if real selectivity can be demonstrated.

Method to transport intact polypeptides into viable cells. In a show-stopping presentation, Dr. Steve Dowdy (Washington University) described a method in which virtually any polypeptide -- if kept in an unfolded state -- can be translocated across mammalian cell membranes if linked to the short N terminal region of HIV TAT (between 11 and 36 amino acids). Others had reported that the HIV TAT could be taken up by many cell types. The biological role of this property in the HIV life cycle (if any) is unknown. Dr. Dowdy has exploited this property, however, to create a vehicle in which it is claimed that virtually any polypeptide payload can be efficiently and rapidly (within seconds) taken up by cells. More than 60 different polypeptide -TAT chimeras were produced in vitro and incubated with target cells, and all chimeras tested were successfully transported. The polypeptides ranged in molecular weight from 10 to 100 kd; there seemed to be no "class" restrictions. The key, however, was that the payload be in an unfolded state. Once transported into the cell, salvage folding pathways, involving HSP90, appeared to restore functional topology to enzymatic payloads. Chimeras were administered to mice and said to be taken up "universally" by host tissues. The implications of this technology in the delivery of functional proteins, as drugs, are of course enormous.

Can HCiG, immunoglobulin (Ig) pool hyper specific for HCV, have a role in the control of HCV? Therapy with HBiG is highly effective in the control of post-exposure to sources of HBV. Nabi, Inc.'s Civacir, a human polyclonal antibody in preclinical development, is 5% Ig, derived from pooled serum from HCV-positive individuals. Dr. Kris Krawczynski (Hepatitis Branch, Centers for Disease Control) reported that the course of viremia in HCV-infected chimps could be influenced by multiple post-exposure infusions of Civacir. The number of animals studied was, of course, small, but trends were consistent. Although only the kinetics of infection seemed to be affected (not the ultimate outcome), taken at its most exciting interpretation, the data suggest that individuals infected with HCV (the source of the HCiG) do contain neutralizing antibodies. The implications of this for vaccine production and the eventual development of reagents that can neutralize a variety of HCV quasi species is very great.

Is a broad spectrum anti HCV/HBV compound possible? I (Timothy Block, Jefferson Center of Thomas Jefferson University at DVC) presented evidence that the extreme sensitivity of HBV secretion to imino sugar glucosidase inhibitors (see Block et al., 1998 Nat Med 4: 610-614) was also seen for bovine viral diarrhea virus (BVDV) production. Since BVDV is considered a good tissue culture surrogate of HCV, it is suggested that HCV will also be sensitive to these agents. The imino sugars described [notably, n-nonyl DNJ (nnDNJ)] prevent viral glycoprotein folding and trafficking by inhibiting the endoplasmic reticulum (ER) glucosidase. Although glucosidase is a host enzyme that mediates processing of cellular glycoproteins, it appears that the secretion of viruses which bud from internal membranes, such as HBV, BVDV and possibly HCV, are much more dependent. Evidence was presented that inhibiting as little as 10% of the ER glucosidase activity results in 99-100% inhibition of viral secretion (with only a modest effect upon secretion of host glycoproteins). Since nnDNJ is orally available and showed little animal toxicity, it is being pursued as a possible broad-spectrum antiviral drug. A new company, called IgX-Oxford (Summit, NJ), has been created to determine the feasibility of this approach.

HBV clinical overview. Dr. Eugene Schiff (University of Miami) reviewed the stunning epidemiology and natural history of HBV disease. Clearly, the immunopathogenesis of HBV and HCV share much in common. The limited usefulness of IFa for these two very different pathogens underscores their similar pathogenesis profiles. With HBV, however, the clinical and serological milestones to follow are better developed, and clearance of HBeAg, for example, with concomitant reductions in viral DNA, are almost always associated with more favorable outcomes. It is unclear, however, whether achieving this type of serological milestone with the new nucleoside analogues means the same thing as when it occurs spontaneously. There is now substantial experience with clinical trial designs for testing HBV drug efficacy and there is consensus that clearance of viral DNA and normalization of serum LFTs (liver function tests, such as ALT) are relevant goals. These serological markers, where supported by improved histological scores from biopsy, are certainly favorable outcomes of therapy.

Lamivudine (now called Epivir-HBV) is the first orally available FDA-approved drug for the treatment of HBV. Dr. Nat Brown (Glaxo-Wellcome, Research Triangle Park, NC) reviewed the impressive clinical performance of Epivir-HBV. The results of Phase III studies showed that Epivir-HBV, which targets the viral polymerase as a competitive inhibitor, consistently reduces serum viral DNA levels by at least two logs, within two weeks of treatment. eAg loss with eAb appearance and substantial trends to normalization of ALT levels were far more common in those receiving drug for one year, compared with those receiving placebo. After eAg loss, it may be possible to discontinue therapy (at least until virus reappears in the blood, which does happen). Clearly, the well-tolerated Epivir-HBV, at 100 mg/day, is likely to become the standard of care. Pediatric use studies are important and are underway.

On the downside, Epivir-HBV-resistant mutants haunt this model drug. Epivir-resistant mutants arise in at least 14% of those treated with the drug every year. However, as Dr. Lynn Condreay (Glaxo-Wellcome) reports, these viruses don't grow as well as the wild type, and individuals infected with the mutant may have better liver function test (LFT) profiles compared with their pre-dosing period. At present, the mutants' ultimate pathogenic potential is unclear. Since most Epivir-resistant mutants have 10 to 1,000 times less affinity for the drug than wild type, there is little rationale for increasing the daily dosage from the HBV (100 mg) to the HIV amount (150+ mg). An important experimental observation was made when Dr. Condreay pointed out that most Epivir-resistant mutants arising in woodchucks are within the polymerase "B" domain, unlike the YMDD motifs mutated in chronically infected people.

Other nucleoside analogues for HBV treatment are in clinical trials. Drs. Craig Gibbs and Alison Murray of Gilead Sciences presented the basic science of and clinical experiences with adefovir dipivoxil, and Dr. Phil Furman of Triangle, Inc. reported on FTC [5-fluoro-1(2-hydroxymethyl)-1,3-oxymethyl), 1,3-oxaththiolan-5-yl)cytosine] and L-FMAU [2-fluoro-5-methyl-5-methyl-b-L-arabinofuranosyl uracil].

Adefovir dipivoxil has good bioavailability (40%) when taken orally and is an effective and selective inhibitor of the HBV reverse transcriptase (pol) [IC-50 of 0.7 uM in HepG 2.2.15 cells; CC 50 of 150 uM]. Also used for HIV therapy, adefovir's most compelling property is its activity against the YMDD Epivir-resistant mutant viruses that emerge. Indeed, as Dr. Murray reports, adefovir resistance was not seen in patients after 12 weeks of therapy. However, although encouraging, it is noted that this is too short a time of treatment to make conclusions about the in vivo occurrence of resistance to this drug. Adefovir resistance can be engineered into the HIV reverse transcriptase and has been observed in HIV patients in therapy for six to twelve months. Nevertheless, the 12-week Phase 2 studies were encouraging (although some dietary supplements may be necessary to avoid specific adverse effects in some individuals). A global development program is underway and combination therapy with lamivudine is planned.

L-FMAU and FTC were shown in preclinical studies to be highly potent inhibitors of the HBV polymerase, with Hep G2.2.15 IC90 values of ~1 and 0.1 uM, respectively. CC50 values were in the 1-2 mM range for both compounds, consistent with excellent selectivity and low toxicity in small animals. The woodchuck studies of L-FMAU were particularly striking, showing the most rapid and potent anti-HBV activity seen yet: at least 3 log drops in viral serum viral DNA levels within two weeks of treatment and sustained reductions for 20 to 80 days post treatment. If the activity of L FMAU against Epivir-resistant virus and a favorable toxicity profile are also sustained, this may be the drug to watch in the future. Famvir (SmithKline-Beecham), lobucovir (Briston-Myers Squibb) and BMS 200,475 data were not presented at this meeting.

CTL therapy for HBV and possibly HCV. Dr. Kim Clary (Targeted Genetics Corp.) described a proprietary method of expanding T cells ex vivo, and the use of this technology to deliver CTLs made hyperimmune to HBV core into chronically infected chimpanzees. There appeared to be a modest but consistent CTL infusion-dependent drop in viremia, although lasting results were not achieved. Even sophisticated immunotherapies that are safe and effective will be welcome and logical complements to conventional antivirals. We look forward to improvements that simplify and enhance the activity of this method.

New reagents for HBV/ HCV antiviral drug discovery. Dr. Shlomo Dagan (XTL Biopharmaceuticals Ltd; Rehovot, Israel) described a chimeric (called "trimera") SCID mouse repopulated with human PBL and spleen and liver tissue following irradiation to eliminate the murine immunological cells. In addition to being a source of human MAb production, the mice were reported to accept human hepatocytes infected ex vivo with either HBV or HCV. The potential for use of these animals as systems for assessing anti-HBV or anti HCV compounds under in vivo conditions is clear.

Conclusions

The outlook is very promising for new treatments for viral hepatitis. Clearly, combination therapy is going to be the rule in order to avoid generating drug-resistant mutants and to possibly achieve therapeutic milestones that last long after discontinuation of drug.

This article was prepared by Dr. Timothy Block
Jefferson Center for Biomedical Research
Thomas Jefferson University College of Medicine
Delaware Valley College of Agriculture and Science
Doylestown, PA
Email: block@hendrix.jci.tju.edu

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