Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-06-13
2002-07-16
Prouty, Rebecca E. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S646000, C514S258100, C514S789000, C514S451000, C514S619000
Reexamination Certificate
active
06420338
ABSTRACT:
1. INTRODUCTION
The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target Src family kinases and components of the Src kinase family signal transduction pathways, including HBx activation of Src kinase family signal transduction pathways for the treatment and prevention of hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). The invention also relates to screening assays to identify potential antiviral agents which target HBx-mediated activation of Src kinase signaling cascades for the treatment of HBV.
2. BACKGROUND OF THE INVENTION
2.1 Hepatitis B Virus
Infection with HBV is an international public health problem of wide proportions. It has been estimated that at least 10% of the population of tropical Africa and Far-East Asia are chronic carriers of the virus (Tiollais et al., 1985, Nature 317:489-495). HBV is a hepatotropic virus whose course of infection can range from inapparent to acute hepatitis and severe chronic liver disease (Tiollais et al., 1985, Nature 317:489-495). Epidemiological studies have estimated that 250 million people are chronic carriers of HBV and serve as a reservoir for continued infections. Although the mechanism remains obscure, these HBV carriers have more than a 200 fold greater risk for development of hepatocellular carcinoma (HCC) (Beasley et al., 1981, Lancet 2:1129-1133).
HBV is a DNA-containing para-retrovirus that replicates by reverse transcription but comprises a separate family of viruses from retroviruses, known as hepadnaviruses. Human HBV is the prototype virus in a family that all possess a similar viral architecture and genetic arrangement, although only infection with the mammalian hepadnaviruses HBV (Tiollais et al., 1985, supra), woodchuck hepatitis B virus (WHV) (Popper et al., 1987, Proc. Natl. Acad. Sci. 84:866-870), and possibly ground squirrel hepatitis B virus (GSHV) (Marion et al., 1986, Proc. Natl. Acad. Sci. 83:4543-4546; Seeger et al., 1991, J. Virol. 65:1673-1679) cause both acute and chronic active hepatitis and HCC.
Acute hepatitis following a primary infection with HBV is usually self-limited in adults and often asymptomatic. Following acute hepatitis, 80-90% of infected adult individuals will clear viral antigens from liver and blood, resulting in clinical recovery and immunity to reinfection (Kumar et al., 1992, Basic Pathology, Fifth Edition (Philadelphia: W.B. Saunders Company)). However, 5-10% of individuals do not resolve the primary infection, instead developing a persistent hepatic infection (Ganem and Varmus, 1987, Ann. Rev. Biochem. 56:651-693). Chronic carriers represent a minority outcome following HBV infection, but constitute the majority of cases of HBV-related morbidity and mortality. Infection of infant and newborns results in a high carrier rate (approximately 90%), in contrast to infection of adults. Chronic carriers serve as the reservoir from which HBV is spread both horizontally (through blood and sexual contact) and vertically (from carrier mothers to newborns). Furthermore, chronic HBV infection frequently results in premature death from hepatic cirrhosis and liver failure (Ganem et al., 1987, Ann. Rev. Biochem. 56: 651-693). As previously noted, chronic carriers have a more than 200 fold increased risk for development of primary hepatocellular carcinoma (Beasley et al., 1981, Lancet 2:1129-1133). Because infection by HBV strongly correlates with development of HCC, considerable effort has been expended in identifying potential mechanisms for tumorigenicity by HBV (reviewed in Ganem et al. 1987, supra; Robinson, 1994, Ann. Rev. Med. 45:297-323; Rogler, 1991, Curr. Top. Micro. Immunol. 168:103-140). However, no clear mechanism has been described for the association between HCC and infection with HBV.
There are currently very limited therapeutics available for the treatment of HBV infection. Anti-HBV vaccines are currently being used to prevent HBV infection. However, the efficacy of these vaccines to treat chronic HBV infection and the availability of these vaccines to treat this worldwide health problem remains to be determined. Therefore, the need for an effective anti-HBV therapeutic still exists today.
2.2 HBx
The HBx protein is encoded by one of the four conserved open reading frames of the HBV genome. The L(+) (coding) strand encodes the four conserved open reading frames (ORFs) and codes for all the viral proteins (Ganem et al., 1987, supra). Four mRNAs have been identified. A 2.4 kb preS1 mRNA encodes the large surface antigen (pre-S1) and a 2.1 kb preS2/S mRNA encode the middle (pre-52) and small (major; S) surface antigens (Tiollais et al., 1985, supra). The 3.4 kb pregenome mRNA encodes the precore and core proteins, as well as the polymerase (P). The core protein is the principal structural component of the viral nucleocapsid and possesses nucleotide binding activity. The P protein, which has RNaseH activity, is the viral reverse transcriptase and the protein primer for synthesis of the L(−) strand (Robinson, 1994, Ann. Rev. Med. 45:297-323). The fourth mRNA is ~0.7 kb in size, and is thought to encode the transcriptional transactivator known as HBx. HBx is a conserved 154 amino acid polypeptide which corresponds to a protein of a molecular weight of ~17 kilodaltons.
The HBx protein is highly conserved within different mammalian HBV serotypes. However, in contrast to the other viral polypeptides, the role of HBx in the HBV life cycle is not yet understood. HBV-infected patient sera indicate that anti-HBx antibodies are produced (Elfassi et al., 1986, Proc. Natl. Acad. Sci. 83:2219-2222; Meyers et al., 1986, J. Virol. 57:101-109), demonstrating that expression of HBx does occur at some stage of HBV infection. HBx protein has also been detected in the livers of patients with chronic hepatitis (Haruna et al., 1991, Hepatol 13:417-421; Katayama et al., 1989, Gastroenterology 97:990-998). Patients testing positive for HBx expression have been found to have increased serum levels of HBV, thereby correlating HBx expression with increased viral replication (Haruna et al., 1991, Hepatol 13:417-421).
The precise role for HBx in the viral infectious process and in the development of HCC remains obscure. There are conflicting reports as to the role of HBx in the viral infectious process and in the development of HCC. It has been reported that there is a correlation between high levels of HBx expression and the development of HCC in transgenic mice. (Kim et al., 1991, Nature 353:317-320; Koike et al., 1994, Hepatol 19:810-819). However, these results remain controversial, as other groups have found no significant liver disease in HBx expressing mice (Balsano et al., 1994, J. Hepatol. 21:103-109; Dandri et al., 1996, J. Virol. 70; Lee et al., 1990, J. Virol. 64:5939-5947).
Several groups have shown HBx to be a largely if not entirely cytoplasmic protein, although 5-10% of HBx may reside in the nucleus (Doria et al. 1995, EMBO J. 14:4747-4757; Dandri et al. 1996 J. Virol. 70). HBx cannot be found to measurably associate with organelles, membrane vesicles or intermediate filaments, although some preferential accumulation near the cell surface can be observed (Doria et al., 1995, EMBO J. 14:4747-4757). HBx is a weak to moderately strong transcriptional transactivator. HBx has been shown to transactivate transcription of the interferon-&bgr; gene (Twu et al., 1987, J. Virol. 61:3448-3453) and of the HBV enhancer (Spandau et al., 1988, J. Virol. 62:427-434). Since those first reports, HBx has been shown to transactivate a wide variety of cellular and viral transcriptional elements (reviewed in Yen, 1996, J. Biomed. Sci. 3:20-30). Activation has been localized to specific binding sites for the transcription factors AP-1 (Benn & Schneider, 1994, Proc. Natl. Acad. Sci. 91; Natoli et al., 1994, Mol. Cell. Biol. 14:989-998; Seto et al., 1990, Nature 344:72-74), AP-2 (Seto et al., 1990, supra), NF-&kgr;B (Lucito & Schneider, 1992, J. Virol. 66:983-991; Mahe et al., 1991, J. Biol. Chem. 266:13759-13763; Su and Schneider, 1996 J. V
Klein Nicola
Schneider Robert J.
New York University Medical Center
Pennie & Edmonds LLP
Prouty Rebecca E.
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