Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
1999-11-12
2002-06-25
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C424S085100, C424S085100, C424S225100, C424S227100, C424S161100, C424S185100, C530S351000, C530S300000, C514S012200, C514S893000, C514S894000
Reexamination Certificate
active
06410009
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to pharmaceutical compositions for the treatment of hepatitis B virus (HBV) infection.
HBV infection in humans can cause chronic liver disease which will, in some cases, proceed to hepatocellular carcinoma. The initial steps of HBV attachment to cells and the targeting of the viral genome to the host cell nucleus have yet to be deciphered. The specific receptor for HBV has not so far been identified, even though various serum proteins and cellular membrane glycoproteins have been suggested as mediators of cell penetration or viral receptors. HBV envelope proteins were reported to contain residues which interact with polymerized albumin [P. Pontisso, et al.,
Journal of Virology
, Vol. 63, No. 1981-1, p. 988 (1981)] or with soluble transferring [M. Gagliardi, et al.,
Eur. J. Immunol
., Vol. 24, pp. 1372-1376 (1994)], enabling viral penetration of cells via their respective receptors, probably in a non-specific manner.
In a study reported by Neurath, et al. [A. Neurath, et al.,
J. Exp. Med
., Vol. 175, pp. 461-469 (1992)] hIL-6 was shown to bind the pS1 (aa 21-47) segment of the HBV envelope. Putative candidates for the HBV receptor were recently reported, including Annexin V (endohexin II) [K. Hertogs, et al.,
Virology
, Vol. 197, pp. 549-557 (1993)]; apolipoprotein H [H. Mehdi, et al.,
Journal of Virology
, Vol. 68, pp. 2415-2424 (1994)]; and asialoglycoprotein receptor [U. Treichel, et al.,
Journal of General Virology
, Vol. 75, pp. 3021-3029 (1994)].
Binding experiments have demonstrated that the pre-SI (pS1)region of the viral envelope protein contains a recognition site for the host cell [A.R. Neurath, et al.,
Cell
, Vol. 46, pp. 429-436 (1986); M. Petit, et al.,
Virology
, Vol. 180, pp. 483-491 (1990); M. Petit, et al.,
Virology
, Vol. 197, pp. 211-222 (1992)]. Although previous studies had suggested that HepG2 cells [R. Bchini, et al.,
Journal of Virology
, Vol. 64, pp. 3025-3032 (1991)] and human hepatocytes [P. Gripon, et al.,
Journal of Virology
, Vol. 62, pp. 4136-4143 (1988); T. Ochiya, et al.,
Proc. Natl. Acad. Sci. U.S.A
., Vol. 86, pp. 1875-1879 (1989); P. Gripon, et al.,
Virology
, Vol. 192, pp. 534-540 (1993); P. Galle, et al.,
Gastroenterology
, Vol. 106, pp. 664-673 (1994)] could support HBV infection in vitro, no cellular receptor has as yet been defined in either system, and these models were of low experimental reproducibility.
In current reports, it has been shown that a chimeric mouse, generated by using Beige/Nude/X linked immunodeficient (BNX) mice, preconditioned by total body irradiation (12Gy) and reconstituted with severe combined immunodeficient (SCID) mice bone marrow (BM) cells, is permissive for normal human T and B cells [I. Lubin, et al.,
Science
, Vol. 252, pp. 427-431 (1991)], as well as for normal human liver tissue [E. Galun, et al.,
Journal of Infectious Diseases
, Vol. 175, pp. 25-30 (1995)]. Hepatitis C virus (HCV) viremia was detectable for up to two months, after implantation under the kidney capsule of the BNX>SCID chimeric animals of either a human liver fragment with preexisting HCV infection, or normal human liver tissue following incubation ex-vivo of the transplanted liver fragment with HCV-positive sera [E. Galun, et al., ibid.].
Earlier studies have revealed that human interleukin 6 (hIL6) contains recognition sites for the hepatitis B virus (HBV). Chinese hamster ovary cells transfected with human IL-6 cDNA and Spondoptera frugiperdaovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS921-47) region of the HBV envelope protein, indicating that expression of IL-6 sequences encompasses a binding site for the HBV envelope protein. Thus, the possibility of developing antiviral compounds mimicking the receptor binding site for HBV on IL-6 but not displaying undesirable biological effects of the intact IL-6 molecule was raised, because it was found that the interaction between the preS1 region of the HBV envelope proteins and cells of hepatic origin was inhibited by IL-6 and by anti-IL-6 antibodies [A. Neurath, et al.,
J. Exp. Med
. Vol. 176, pp. 1561-1569 (1992)].
Heretofore, one of the major obstacles in elucidating the initial steps of HBV infection and the assessment of antiviral agents, has been the lack of a small animal model. Using the techniques referred to above, it was possible to develop SCID>BNX animals which sustain HBV viremia following the implantation of an ex-vivo HBV DNA-positive sera incubation with liver tissue. The method in which the animals were prepared for the experiments described herein, and the surgical technique for transplantation, are similar to those previously reported [E. Galun, et al., ibid.].
As described, e.g., in PCT/US94/05410, it has now been found, using a chimeric animal model, that human interleukin 6 (hIL6) is essential for HBV infection. Having identified that hIL6 serves as an essential bridge for HBV infection, the invention now provides a pharmaceutical composition for the treatment of hepatitis B virus infection, comprising an amount of a soluble active agent which interacts with at least one of the binding sites between hIL6 and pS1 and between hIL6 and hepatocytes and other HBV-permissive cells, said active agent being present in sufficient amount to competitively bind to at least one of said sites and thereby to prevent hIL6-mediated HBV infection of hepatocytes and other HBV-permissive cells.
In a first preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection, comprising an amount of soluble gp80 and/or gp130 receptor sites sufficient to inhibit the binding of hIL6 to hepatocytes and other HBV-permissive cells.
In a second preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of soluble amino acid sequences corresponding to amino acids 21 to 46 of pS1 to block the interaction of HBV with hIL6.
In a third preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising an amount of a soluble ligand selected from the group consisting of peptides LYS41-ALA56, GLY77-GLU95 and GLN153-HIS165 to block the interaction of hIL6 with hepatocytes and other HBV-permissive cells.
In a fourth preferred embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of HBV infection, comprising hIL6 conjugated with an anti-viral agent. In a preferred embodiment, the anti-viral agent conjugated to hIL6 comprises glycoprotein 80 or a portion thereof. In a preferred embodiment of the invention, the hIL6-anti-viral agent conjugate is Hyper-IL6 (HIL6).
In yet another preferred embodiment of the invention, there is provided a pharmaceutical composition for the treatment of hepatitis B virus (HBV) infection of hepatocytes, comprising a soluble active agent which competitively interacts with at least one of the binding sites between human interleukin 6 (hIL6) and hepatocytes, said soluble active agent being selected from the group consisting of glycoprotein 80 (gp80) having receptor sites which interact with hIL6, soluble glycoprotein 130 (gp130) having receptor sites which interact with hIL6, hIL6 derived peptide LYS41-ALA56, hIL6 derived peptide GLY77-GLU95, hIL6 derived peptide GLN153-HIS165, a combined &bgr;1 and &bgr;2 hIL6 mutant (mhIL6&bgr;1+&bgr;2), and mhIL6&bgr;1+&bgr;2 substituted with phe 171 to leu and ser 177 to arg, and mixtures of any of the foregoing. In certain preferred embodiments, such a pharmaceutical composition includes an effective amount of the soluble active agent to treat infection of the hepatocytes by HBV.
In yet another preferred embodiment of the invention, there is provided a pharmac
Blum Hubert E.
Galun Eithan
Nahor Orit
Davidson Davidson & Kappel LLC
Hadasit Medical Research Services & Development Co., Ltd.
Li Bao Qun
Salimi Ali R.
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