Chapter 1
Entecavir (Baraclude): A Carbocyclic Nucleoside for the Treatment for Chronic Hepatitis B

Jie Jack Li

1 Background

Chronic hepatitis B virus (HBV) infection is a major global cause of morbidity and mortality. An estimated 400 million people worldwide have chronic HBV infection and more than half a million people die every year because of complications from HBV-related chronic liver disease such as liver failure and hepatocellular carcinoma (HCC). In the United States, 12 million people have been infected at some time in their lives with HBV. Of those individuals, more than 1 million people have subsequently developed chronic hepatitis B infection. These chronically infected persons are at highest risk of death from liver scarring (cirrhosis) and liver cancer. In fact, more than five thousand Americans die from hepatitis B-related liver complications each year. In many Asian and African countries where the HBV is endemic, up to 20% of the population may be carriers, and transmission occurs primarily through perinatal or early childhood infection. In some of these areas, the perinatal transmission rate may be as high as 90%!1-4

During the last 10 years, hepatitis B treatment has made significant progresses. For example, two biologics have been approved by the FDA, namely, interferon-a (IFN-a) and Pegylated-interferon-a (PEG-IFN-a). Also on the market are five small molecule antiviral agents for the treatment of chronic HBV, namely, entecavir (1), lamivudine (2), telbivudine (3), adefovir dipivoxil (4), and tenofovir (5).

As a biologic, INF-a is effective only in a subset of patients, is often poorly tolerated, requires parenteral administration, and is expensive. Hence, there is a need for alternative therapies for chronic hepatitis B. The introduction of lamivudine (2) in 1995, the first oral treatment for chronic HBV, ushered in a new era in the treatment of chronic hepatitis B when safe, effective, and well-tolerated oral medications were made available. It is a nucleoside reverse transcriptase inhibitor (NRTI) with activity against both human immunodeficiency virus type 1 (HIV-1) and HBV. It has been used for the treatment of chronic hepatitis B at a lower dose than for the treatment of HIV, and it improves the seroconversion of e-antigen-positive hepatitis B and also improves histology staging of the liver. Unfortunately, long-term use of lamivudine (2) leads to emergence of a resistant HBV mutant (Tyr-Met-Asp-Asp, YMDD). Despite this fact, lamivudine (2) is still used widely as it is well tolerated.5

Telbivudine (3), a synthetic thymidine nucleoside analog, is the unmodified L-enantiomer of the naturally occurring D-thymidine. It prevents HBV DNA synthesis by acting as an HBV polymerase inhibitor. Within hepatocytes, telbivudine (3) is phosphorylated by host cell kinase to telbivudine-5´-triphosphate which, once incorporated into HBV DNA, causes DNA chain termination, thus inhibiting HBV replication. In this sense, telbivudine (3), like most nucleotide antiviral drugs, is a prodrug. Clinical trials have shown telbivudine (3) to be significantly more effective than lamivudine (2) or adefovir dipivoxil (4) and less likely to cause resistance.6

Adefovir dipivoxil (4) was initially developed as a treatment for HIV, but the FDA in 1999 rejected the drug due to concerns about the severity and frequency of kidney toxicity when dosed at 60 or 120 mg, respectively. However, 4 was effective at a much lower dose of 10 mg for the treatment of chronic hepatitis B in adults with evidence of active viral replication and either evidence of persistent elevations in serum alanine aminotransferases (primarily ALT) or histologically active disease. It works by blocking reverse transcriptase, an enzyme that is crucial for the HBV to reproduce in the body. Overall, the efficacy of 4 against wild-type and lamivudine (2)-resistant HBV and the delayed emergence of 4-resistance during monotherapy contribute to the durable safety and efficacy observed in a wide range of chronic hepatitis B patients.7

Tenofovir (5), a nucleotide analog closely related to adefovir dipivoxil (4) has been approved for the treatment of HBV in 2008, subsequent to its approval for the treatment of HIV infection in 2006. In vitro studies showed that it has activity against HBV with equimolar potency to 4. Clinical studies confirmed the efficacy of 5 in suppressing HBV replication, and it appears to be equally effective against both wild-type and lamivudine (2)-resistant HBV. The role of 5 in the rapidly expanding armamentarium of hepatitis B treatments will depend on the demonstration of long-term safety (renal and skeletal) and efficacy against wild-type HBV and HBV mutants that involve substitution of methionine within the YMDD motif, as well as a very low rate of resistance in NA-naïve as well as NA-experienced patients.8-10 NA stands for nucleos(t)ide analog.

The approval of the nucleotide and nucleoside analogs 1-5 marked a significant advance in the treatment of chronic hepatitis B. In comparison to compounds 2-5, entecavir (1) is a novel carbocyclic nucleoside analog with potent and highly selective activity against HBV, as well as a low rate of resistance. In this chapter, the pharmacological profile and syntheses of entecavir (1) will be profiled in detail.

2 Pharmacology

The hallmark of acute HBV infection is elevated alanine aminotransferase (ALT) levels. As a matter of fact, ALT levels are routinely screened during our annual physical exams where an elevated ALT level is a sign of a concern with regard to the liver function. For instance, long-term consumption of too much alcohol would cause liver to become hardened along with elevated ALT levels. Other telltale signs of acute HBV infection also include the presence of hepatitis B surface antigen (HBsAg), IgM antibody to hepatitis B core antigen (anti-HBc), and hepatitis B e-antigen (HBeAg), although the latter serological test is not routinely used. Chronic hepatitis B is defined as the presence of HBsAg or other viral markers in serum for more than 2 months.

Entecavir (1) is converted in mammalian cells in vitro to the 5´-triphosphate, which then acts as an inhibitor of hepadnaviral polymerase with an IC50 value for inhibition of HBV of 0.2-0.3 nM. The Ki value for binding of 1-triphosphate to HBV polymerase is 3.2 nM. In the HepG2 stably transfected cell line 2.2.15, 1 had an EC50 (50% effective concentration) value of 3.5 nM against HBV and an CC50 (50% cytotoxic concentration) value of ~30 µM against HBV as determined by analysis of secreted HBV DNA.11,12 This represents an excellent selectivity index of ~8,000 (toxicity dose is 8,000-fold greater than the concentration needed to inhibit HBV replication in the same cell line). Direct comparison with other nucleoside analogs in this cell line demonstrated that 1 is the most potent inhibitor of HBV replication, as shown in Table 1.13

Table 1 Potency of various nucleoside analogs for HBV inhibition based on the EC50 for inhibition of HBV replicase in HepG2.2.15 cell line.13

Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) were used as an in vivo model of HBV infection. During the first 4 weeks of study, 1 was administered at various doses and was found to suppress HBV DNA replication by approximately 3 log10 copies/mL regardless of the dose administered. After 12 weeks, most of the animals became HBV DNA-negative, reflecting greater than a 1,000-fold suppression in circulating HBV. Similar results were observed for 1 using ducks as the animal model.13

3 Structure-Activity Relationship (SAR)

The structure-activity relationship (SAR) around entecavir (1) was exhaustively investigated, and 1 was found to be the most potent member in the series as tested against HBV in HepG2.2.15 cells. As shown in Table 2 (next page), the enantiomer of 1 (ent-1) was inactive, while 1 was 6.6-fold more potent than lamivudine (2, entry 3).14 Similarly, the adenine analog 6 (entry 4) was 43-fold less potent than 1, while the thymine analog 7 (entry 5) and the 5-iodouracil analog 8 (entry 6) were much less potent in HepG2.2.15 cell culture.

Table 2 Activity of nucleoside analogs against HBV in HepG2.2.15 cells.