Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1999-06-03
2001-09-18
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S345000, C514S002600, C514S012200
Reexamination Certificate
active
06291638
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention refers to compounds with antiviral activity and, more particularly to conjugated compounds of antiviral drugs, with carriers, having hepatotropic activity.
2. Description of the Related Art
In the treatment of the infection caused by viruses, the side effects produced by antiviral drugs can be reduced with the adoption of the chemotherapeutic lysosomotropic approach (13Balboni, P. G., Minia, A., Grossi, M. P., Barbanti-Brodano, G., Mattioli, A., Fiume, L. Activity of albumin conjugates of 5-fluorodeoxyuridine and cytosine arabinoside on poxviruses as a lysosomotropic approach to antiviral chemotherapy. Nature 1976; 264: 181-183).
This consists in conjugating the drug to a macromolecule that is selectively captured from the infected cells and transported in the lysosomes therefrom.
If, as desired, the lysosomal enzymes break the linkage between the carrier and the drug, the latter results as being concentrated in a pharmacologically active form within the infected cells.
Glossary of Terms
LAC=lactosaminated
ACVHP=acyclovir monophosphate
AZTMP=3-azido-3-deoxythymidine
ACV=acyclovir
FUDR=5-fluoro-2′-deoxyuridine
dFdC=2′,2′-difluorodeoxycytidine
ASGP-R=asialoglycoprotein receptor
The chronic hepatitis caused by B (HBV) virus and by C (HCV) virus are proper targets for this chemotherapeutic approach because: (a) these viruses grow especially in the hepatocytes; (b) hepatocytes specifically bring inside and transport in the lysosomes some glycoproteins with galactose residues that can therefore function as hepatotropic vectors of drugs; (c) the conjugates of drug/glycoproteins can easily come into contact with the surface of the hepatocytes inasmuch as the hepatic sinusoids are not a barrier for proteins.
Following this approach and to reduce its neurotoxic side effects, the arabinoside adenine monophosphate antiviral drug (ara-AMP), active against HBV (Jacyna, M. R., Thomas, H. C. Antiviral therapy: Hepatitis B. Brit. Med. Bull. 1990; 46: 369-382) has been conjugated with asialofetuin (AF) (Fiume, L., Mattioli, A., Busi, C., Balboni, P. G., Barbanti-Brodano, G., De Vries, J., Altman, R., Wieland, Th. Selective inhibition of Ectromelia virus DNA synthesis in hepatocytes by adenine-9-&bgr;-D-arabinofuranoside (ara-A) and adenine-&bgr;-D-arabinofuranoside 5′-monophosphate (ara-AMP) conjugated to asialofetuin. FEBS Letters 1980; 116: 185-188) and with the lactosaminated albumin (L-SA) (iume, L., Busi, C., Mattioli, A., Balboni, P. G., Barbanti-Brodano, G. Hepatocyte targeting of adenine-9-&bgr;-D-arabinofuranoside 5′-monophosphate (ara-AMP) coupled to lactosaminated albumin. FEBS Letters 1981; 129: 261-264; Fiume, L., Bassi, B., Busi, C., Mattioli, A., Spinosa, G. Drug targeting in antiviral chemotherapy. A chemically stable conjugate of 9-&bgr;-D-arabinofiranosyladenine 5′-monophosphate with lactosaminated albumin accomplishes a selective delivery of the drug to liver cells. Biochem. Pharmacol. 1986; 35: 967-972). In mice, both these two carriers have brought about a hepatic targeting of the drug.
The L-SA has a great advantage on AF: In fact, the conjugates prepared with homologous lactosaminated albumin (that is, of the same species), when introduced intravenously, do not induce the formation of antibodies (Fiume, L., Mattioli, A., Busi, C., Spinosa, G., Wieland, Th. Conjugates of adenine-9-&bgr;-D-arabinofiuranoside monophosphate (ara-AMP) with lactosaminated homologous albumin are not immunogenic in the mouse. Experientia 1982; 38: 1087-1089; Fiume, L., Busi, C., Preti, P., Spinosa, G. Conjugates of ara-AMP with lactosaminated albumin: A study on their immunogeneticity in mouse and rat. Cancer Drug Delivery 1987; 4: 145-150). In woodchuck with hepatitis by WHV (Ponzetto, A., Fiume, L., Forzani, B., Song, S. Y., Busi, C., Mattioli, A., Spinelli, C., Marinelli, M., Smedile, A. Chiaberge, E., Bonino, F., Gervasi, G. B., Rapicetta, M.,. Verme, G. Adenine arabinoside monophosphate and acyclovir monophosphate coupled to lactosaminated albumin reduce woodchuck hepatitis virus viremia at doses lower than do the unconjugated drugs. Hepatology 1991; 14: 16-24) and in patients with chronic infection by HBV (Fiume, L., Torrani Cerenzia, M. R., Bonino, F., Busi, C., Mattioli, A., Brunetto, M. R., Chiberge, E., Verme, G. Inhibition of hepatitis B virus replication by vidarabine monophosphate conjugated with lactosaminated serum albumin. Lancet 1988; 2: 13-15; Torrani Cerenzia, M. R., Fiume, L., Busi, C., Mattioli, A., Di Stefano, G., Gervasi, G. B., Brunetto, M. R., Piantino, P., Verme, G., Bonino, F. Inhibition of hepatitis B virus replication by adenine arabinoside monophosphate coupled to lactosaminated albumin. Efficacy, minimal effective dose and plasma clearance of conjugate. J. Hepatol. 1994; 20: 307-309), the ara-AMP conjugated to the L-SA has inhibited the viral replication at doses 3-6 times lower than those of the free drug.
The conjugate with L-SA should be administered intravenously owing to the high volune needed for the injection and because, by other ways antibodies are produced, and this results in poor patient compliance in long-lasting treatments.
A hepatotropic carrier of ara-AMP and of other antiviral drugs allowing the intramuscular administration of the corresponding conjugates would therefore be a remarkable improvement from the therapeutic point of view.
It is also known that a basic polyaminoacid, the poly-L-lysine, with one-third of the amino-groups substituted for galactose residues, if intravenously administered, performs a hepatic targeting of ara-AMP (Fiume, L., Bassi, B., Busi, C., Mattioli, A., Spinosa, G., Faulstich, H. Galactosylated poly(L-lysine) as a hepatotropic carrier of 9-&bgr;-D-arabinofuranosyladenine 5′-monophosphate. FEBS Letters 1986; 203: 203-206). Moreover, the poly-L-lysine, when all or large part of its &egr;-amino groups are substituted, does not form antibodies even when administered by ways different from the intravenous injection (Levine, B. B. Studies on antigenicity. The effect of succinylation of &egr;-amino groups on antigenicity of benzylpenicilloyl-poly-L-lysine conjugates in random-bred and in strain 2 Guinea pig. Proc. Soc. Exptl. Biol. Med. 1964; 116: 1127-1131; Sela, M. Immunological studies with synthetic polypeptides. Advan. Immunol. 1966; 5: 29-129).
SUMMARY OF THE INVENTION
It has now been found that if in the poly-L-lysine most of the &egr;-amino groups are substituted with galactose and with one of the antiviral drugs known for performing their activity against hepatic viruses, three very important therapeutic effects are produced in mice:
(i) the conjugate loses the poly-L-lysine high toxicity which distinguished the previous galactosylated poly-L-lysine-ara-AMP conjugates (Fiume et al., FEBS Letters 1986; 203: 203-206) in which most the &egr;-amino groups remained unsubstituted;
(ii) the antiviral drug hepatic targeting is brought on even if the conjugate is not administered intravenously and, particularly, by intramuscular injection;
(iii) the repeated conjugate administration by intramuscular and intravenous injection does not produce antibodies.
The therapeutic importance of this property will be evident if it is considered that the possibility to achieve the hepatic targeting by intramuscular injection not only involves utilizing its interesting peculiar characteristics already previously mentioned in relation to the lysosomotropic approach (i.e., drastic reduction of the side effects relevant to the toxicity of the antiviral drugs), but also equally important to make it much more comfortable for the patient to undergo the chemotherapeutic treatment which, as a rule in the viral chronic hepatitis, is long lasting.
A further object of the present invention is represented by a conjugate of a basic polyaminoacid wherein more than 50% of the side chain amino groups (the &egr;-amino groups) are substituted with galactose or lactose residues and drug residues
Baldacci Massimo
Busi Corrado
Di Stefano Guiseppina
Fiume Luigi
Mattioli Alessandro
Anderson Kill & Olick P.C.
Laboratori Balducci S.p.A.
Low Christopher S. F.
Lukton David
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