Entecavir T rade Name: Baraclude

1. EntecavirTrade Name: Baraclude Chronic Hepatitis B Virus Treatment November 25, 2008 Mr. Christe A. Marbbn

2. Table of Contents Part I. An Overview of Hepatitis B • What is Hepatitis B? • How is Hepatitis B transmitted? • Microbiological Aspect of Hepatitis B Virus • Structure • Genome • Pathogenesis • Symptoms and Complications related to Hepatitis B: Human Physiological Response to the Infection

3. Table of Contents Continued Part II. Treatment Option: Entecavir • Background Information • Chemical Data • Mechanism of Action at a Molecular/Cellular Level • Physiological Affect of Drug on Animals & Humans • Antiviral Activity • Absorption • Metabolism & Elimination • Side Effects Part III. References Surface Protein Receptors Figure 1. Computer-generated image of Hepatitis B Virion.

4. Part I: An Overview of Hepatitis B What is Hepatitis B? • Hepatitis B (HB) is a form of hepatitis derived from a DNA virus that causes liver infections in humans and animals. • Hepatitis B virus (HBV) belongs to a family of viruses called hepadnaviridae (hepa = liver, dna = deoxyribonucleotide, viridae = virus). • All known hepadnaviridae are hepatotropic (affecting liver cells) and contain double-stranded DNA genome.

5. Part I: An Overview of Hepatitis B • When a person is first infected with the HBV, it is called an acute infection. Most adults will recover and become immune to the virus without any problems. If the virus remains in the blood for six months, then the person is diagnosed as having a chronic infection (1). • Today, HB remains a major global concern (Figure 2) . • The portion of the world’s population currently infected with the virus is estimated at 3 to 6 percent, far greater than 0.6 percent of the world infected with HIV (2). What is Hepatitis B? Continued

6. Hepatitis B Surface Antigen Prevalence High >8% Intermediate 2%-7% Low <2% Figure 2. Geographical distribution of chronic HBV infection, by country, in 2006 (3). Part I: An Overview of Hepatitis B What is Hepatitis B? Continued

7. Part I: An Overview of Hepatitis B How is Hepatitis B transmitted? • HBV is spread by direct contact with blood or body fluids of an infected person, such as: • Unprotected sexual contact • Blood transfusion • Sharing of contaminated needles and syringes • Vertical transmission from mother to child during childbirth

8. Part I: An Overview of Hepatitis B Microbiological Aspect of Hepatitis B Virus Structure: • The HBV virus is a double-shelled spherical particle with a diameter of 42-47 nm (4). • It consists of icosahedral nucleocapsid (protein core - HBc) enclosing a circular, double-stranded DNA genome. • The outer lipid envelope contains embedded proteins (HB surface antigens) which are involved in viral binding of susceptible cells. 42-47 nm

9. Part I: An Overview of Hepatitis B Microbiological Aspect of Hepatitis B Virus Genome: • The HBV genome is a relaxed circular partially duplex DNA. • This genome is unique in that the minus strand is full length and the plus strand is less than full length, leaving 15-50% of the molecule single-stranded (5). • The genome is 3020-3320 nucleotides long (for the full length strand) and 1700-2800 long (for the shorter strand). Its circularity is maintained by 5’ cohesive ends linked to viral DNA polymerase (5).

10. Part I: An Overview of Hepatitis B Microbiological Aspect of Hepatitis B Virus Pathogenesis: Hepatitis B Virus Liver Cell (Hepatocyte) Receptors Nucleus Shortly after the virus enters the body of a new host, it’s initial response, if not already destroyed by the immune system, is to infect a liver cell. First the virus attaches to a liver cells membrane via receptors.

11. Part I: An Overview of Hepatitis B Microbiological Aspect of Hepatitis B Virus Pathogenesis: Hepatocyte Hepatocyte Nucleus Nucleus The core particle then translocates it's contents of virion DNA and DNA polymerase into the hepatocytes nucleus. This DNA is then organized to form a viral mini-chromosome (6). The virus is then transported into the liver cell by receptor-mediated endocytosis and uncoats in the cytoplasm (6).

12. Part I: An Overview of Hepatitis B Microbiological Aspect of Hepatitis B Virus Pathogenesis: Infected Hepatocyte Viral mRNA Nucleus HBc Transcription Assembly Translation HBs Once within the cell nucleus, the hepatitis B DNA causes the liver cell to produce, via messenger RNA: HBV surface proteins (HBs), viral core proteins (HBc), DNA polymerase, and HB e-antigen protein. The cell then assembles live copies of the virus. The copies of the virus are released from the liver cell membrane into the blood stream and from there can infect other liver cells and thus replicate effectively. Assembly within the endoplasmic reticulum is omitted for the sake of simplicity.

13. Part I: An Overview of Hepatitis B Symptoms and Complications related to Hepatitis B: Human Physiological Response to the Infection • After initial infection, HBV DNA, HBc antigen, HBs antigen, and HBe antigen are detectable in the blood stream. • If the person’s immune system is able to destroy the virus, HB antibodies can be detected one to six weeks following the infection (1). • Half of all people infected with HBV experience no symptoms. However, some people may experience: • Jaundice • Loss of appetite • Fatigue & weakness within the first six months of exposure. • HBV can eventually causes cirrhosis (liver scaring), leading to liver cancer called hepatocellular carcinoma, and other severe complications (6).

14. Part II. Treatment Option: Entecavir Background Information • Entecavir (ETV) is an FDA approved medicine used to treat for chronic HBV infection in adults who have active liver damage. • ETV belongs to the nucleoside analogue reverse transcriptase inhibitor class of drugs and is sold under the brand name Baraclude. It is a product of Bristol-Myers Squibb Pharmaceutical Research Institute. • ETV is not a cure for HB; however, using it may lower the amount of HBV in the body, lower the viruses ability to multiply and infect neighbouring hepatocytes, and improve the overall condition of one’s liver (7).

15. Part II. Treatment Option: Entecavir • Recently, many major research study have shown that ETV, a cyclopentyl guanosine nucleoside analogue, behaves far differently than most nucleoside reverse transcriptase inhibitors approved for HBV therapy in its class, namely lamivudine (LVD) and adefovir (ADV), in several ways: • ETV is more than 100-fold more potent against HBV in vitro (Table 1) (8). • ETV displays no significant activity counteracting HIV. In contrast, LVD is an analogue of cytosine while ADV is an analogue of adenine; both drugs can also select for HIV infected cells (9). • Finally, LVD and ADV are both obligate terminators of DNA chain elongation – meaning they lack a 3’hydroxyl required for nucleotide addition. On the contrary, ETV halts DNA elongation after incorporating a few additional bases (9). Background Information Continued

16. Part II. Treatment Option: Entecavir ETV shows superior potency in both the culture and enzyme assays (9).

17. Part II. Treatment Option: Entecavir Chemical Data Figure 5. Structural formula of entecavir. Created in ChemBioDraw Ultra 11. Figure 6. 3-dimensional active model depicting entecavirs structural formula. Created in ChemBio3D Ultra 11.

18. Part II. Treatment Option: Entecavir • The chemical name for ETV is 2-amino-1,9-dihydro-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]-6H-purin-6-one (7). • Its molecular formula is C12H15N5O3 which corresponds to a molecular weight of 277.12 g/mol. • ETV is a white to off-white powder. It is slightly soluble in water (2.4 mg/mL), and the pH of the saturated solution in water is 7.9 at 25°C ± 0.5°C. • ETV is administered orally in tablet form. It is available in strengths of 0.5 mg and 1 mg. Chemical Data Continued

19. Part II. Treatment Option: Entecavir Mechanism of Action at a Molecular/Cellular Level • Once ETV enters the cell, cellular kinases phosphorylate it into an active triphosphate compound. • It is important to note that other than the cells phosphorylation efficiency, a combination of intrinsic potency, exposure level, and genetic barriers to resistance also contribute to the initial and long-term effectiveness of ETV (9). HBV Infected Hepatocyte Cellular Kinases – P – P – P Nucleus Inactive Entecavir Membrane is permeable to uncharged nucleosides, including entecavir Active Entecavir Triphosphate (ETV-TP)

20. Part II. Treatment Option: Entecavir • ETV inhibits the only known enzymatic target of HBV, the viral polymerase (Pol). • The mRNA encoding POL serves as the template for synthesis of HBV genome through reverse transcriptase activity. • While cellular polymerases and reverse transcriptases use free hydroxyl groups of DNA or RNA as primers for DNA synthesis, the DNA polymerases of certain animal viruses, such as HBV, depend upon hydroxyl groups of amino acid residues within proteins (10). For instance, the primer for Pol DNA synthesis is a hydroxyl group of a tyrosine residue near the amino terminus of Pol. Viral mRNA encoding POL HBV Reverse Transcriptase (HBV RT) + – Viral Polymerase (POL) HBV Genome Mechanism of Action at a Molecular/Cellular LevelContinued

21. Part II. Treatment Option: Entecavir Viral mRNA encoding POL Reverse Transcriptase + – Viral Polymerase (POL) HBV Genome Tyrosine– OH RNA dissociates Pregenomic RNA (Serves as template) Covalent bond between Pol and newly formed viral DNA • ETV triphosphate (ETV-TP) displays activity against the synthetic activities of HBV polymerase: • The unique protein-linked priming activity • RNA-directed first-strand synthesis via reverse transcription • Second-strand DNA-directed DNA synthesis (Not Shown) • Recall that HBV genome is partially single-stranded (minus) and partially double-stranded (plus) Mechanism of Action at a Molecular/Cellular LevelContinued

22. Part II. Treatment Option: Entecavir • In order to understand the interaction of ETV with HBV at the molecular level, ETV-TP must be modeled in the catalytic site of the HBV RT as well as in the process of HBV DNA elongation. Mechanism of Action at a Molecular/Cellular LevelContinued

23. Part II. Treatment Option: Entecavir • Unlike conventional HB viral treatments, ETV is unique in having a D-configured cyclopentyl group. • During translation of viral genome, ETV fits directly into HBV RT’s hydrophobic pocket at the back dNTP binding site via hydrogen bonds. Normal dGTP cannot wholly occupy the pocket during replication since it does not contain a cyclopentyl group like ETV. Recall that ETV is a guanosine nucleoside analogue (9). • This property accounts for the higher affinity of ETV for HBV RT than that of dGTP, and higher potency compared to other drugs (9). • The absence of this small pocket in human polymerase correlates with the relative inactivity of ETV during host DNA replication. ETV-TP HBV RT Figure 7. Hydrogen bonding network between ETV-TP and HBV RT (9). Mechanism of Action at a Molecular/Cellular LevelContinued

24. Part II. Treatment Option: Entecavir • Since ETV possesses a potential 3’ hydroxyl group, it is important to establish the effect of ETV incorporation on HBV DNA chain extension. • The mechanism for chain termination by ETV likely involves incorporation and abortive extension of ETV-containing DNA. This can occur in any three instances (9,11): • Initial docking into the dNTP binding site and addition of ETV onto the 3’ end of the growing DNA • Upon addition of ETV in the +1 position, and • Upon further addition of nucleotides when ETV is elongated to the +2 position. • When ETV enters HBV RT, its cyclopentyl group causes steric strain on the HBV DNA, thereby distorting and partially blocking the dNTP binding site. As a result, this prevents the binding of any new substrate and resulting in chain termination (11). Mechanism of Action at a Molecular/Cellular LevelContinued

25. Part II. Treatment Option: Entecavir Physiological Affect of Drug on Animals & Humans Antiviral Activity: • The ETV EC50 of a laboratory HBV isolate in cell culture is 5.3 nM (7). This suggests that ETV is very responsible, even at low doses. • In two relevant animal models, daily or weekly ETV treatment significantly reduced viral levels. Long-term studies demonstrated that oral weekly dosing of 1 mg/kg maintained viral DNA levels at undetectable levels for up to 3 years (7). • No ETV resistance changes were detected in the HBV polymerase in any of the treated animals for up to 3 years of treatment.

26. Part II. Treatment Option: Entecavir Absorption: • Following oral administration in healthy subjects, ETV peak plasma concentrations occurred between 0.5 and 1.5 hours (7). • Following multiple daily doses ranging from 0.1 to 1.0 mg, Cmax and area under the concentration time curve at steady state increased in proportion to dose (7). • In healthy subjects, the bioavailability of the tablet was 100% relative to the oral solution. Metabolism & Elimination: • Following administration of ETV in humans and rats, no oxidative or acetylated metabolites were observed. • ETV is not a substrate, inhibitor, or inducer of the cytochrome P450 enzyme in the liver (7). Figure 8.3D rendition of mammalian cytochrome P450 enzyme (11). Physiological Affect of Drug on Humans Continued

27. Part II. Treatment Option: Entecavir Metabolism & Elimination: • After reaching peak concentration, ETV plasma concentrations decreased in a bi-exponential manner with a terminal elimination half-life of approximately 128-149 hours. The observed drug accumulation index is approximately 2-fold with once-daily dosing, suggesting an effective accumulation half-life of approximately 24 hours (7). • ETV is predominantly eliminated by the kidney with urinary recovery of unchanged drug at steady state ranging from 62% to 73% of the administered dose (7). • Renal clearance is independent of dose and ranges from 360 to 471 mL/min suggesting that ETV undergoes both glomerular filtration and net tubular secretion (7). Side Effects: • Headache, fatigue, dizziness, and nausea. Physiological Affect of Drug on Humans Continued

28. References • Hauser S., Pardi D., & Poterucha J. (2005). Mayo Clinic Gastroenterology and Hepatology Board Review. CRC Press. • WHO Hepatitis B Fact sheet N. 204. Accessed on: October 23, 2008. http://who.int/mediacentre/factsheets/fs204/en/print.html (3) Travelers’ health: Yellow Book. (2008). US Department of Health and Human Services, CDC. Accessed on: November 02, 2008. http://wwwn.cdc.gov/travel/yellowbookch4-HepB.aspx (4) Adachi Y., Fujita N., Horiike S., Ishida S., Kohara M., Konishi M., Tanaka H., Watanabe S. (2006). Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy. Medical Molecular Morphology, 39:63–71. (5) Büchen-Osmond C. (2006). Hepadnaviridae:The Universal Virus Database. ICTVdB Management. Accessed on: November 02, 2008. http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/ (6) Lemon S., Thomas H., & Zuckerman A. (2005). Viral Hepatitis. Blackwell Publishing. (7) Baraclude (Entecavir). (2007). Bristol-Myers Squibb Company. U.S. Food and Drug Administration. Accessed on: October 04, 2008. www.fda.gov/medwatch/safety/2007/Baraclude_PI_jul2407.pdf (8) Yao G. (2007). Entecavir is a potent anti-HBV drug superior to lamivudine: experience from clinical trials in China. Journal of Antimicrobial Chemotherapy, 60:201–205. (9) Baldick C., Colonno R., Eggers B., Kapur A., Langley D., Levine S., Rose R., Tenney D., & Walsh A. (2007). Inhibition of Hepatitis B Virus Polymerase by Entecavir. Journal of Virology, 81(8):3992-4001. (10) Seeger C., & Zoulim F. (1994). Reverse transcription in hepatitis B virus is primed by a tyrosine residue of the polymerase. Journal of Virology , 68(1):6-13. (11)Anderson K., Bailey C., Chloe L., Detorio M., Domaoal R., McMahon M, Obikhod A., Rapp K., Schinazi R., Siliciano F., Tirado-Rives J. (2007). Pre-steady-state Kinetic Studies Establish Entecavir 5'-triphosphate as a Substrate for Hiv-1 Reverse Transcriptase. Journal of Biological Chemistry, 283(9):5452-5459.