Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Albumin
Patent
1994-09-30
1997-01-14
Weimar, Elizabeth C.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Albumin
530352, 530358, 530363, 530395, C07K 100, A61K 3816
Patent
active
055941109
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention refers to the preparation of conjugates of antiviral nucleosides and, more particularly to a conjugation process of phosphoric esters of antiviral nucleosides with lactosaminated human albumin.
ART RELATED TO THE INVENTION
It is known, that, to reduce the side effects of antiviral nucleosides in the treatment of chronic hepatitis B, two of these drugs, namely arabinoside adeninc (ara-A) and Acyclovir (ACV), have been conjugated with lactosaminated human albumin (L-HSA) (Fiume L, Bassi B., Busi C., Mattioli A. and Spinosa G., Drug targeting in antiviral chemotherapy. A chemically stable conjugate of 9-.beta.-D-arabinofuranosyl-adenine 5'-monophosphate with lactosaminated albumin accomplishes a selective delivery of the drug to liver cells, Biochem. Pharmacol., 35, 967, 1986;
Fiume L., Bassi B., Busi C., Mattioli A., Spinosa G. and Faulstich H., Galactosylated poly (L-lysine) as a hepatotropic carrier of 9-.beta.-D-arabinofuranosyladenine 5'-monophosphate, FEBS Letters, 203, 203, 1986).
L-HSA is a neoglycoprotein which penetrates selectively into the hepatic cells (Wilson G., Effect of reductive lactosamination on the hepatic uptake of bovine pancreatic ribonuclease A dimer, J. Biol. Chem., 253, 2070, 1978) where it is digested in the lysosomes (Ashwell G. and Harford J., Carbohydrate specific receptors of the liver Ann. Rev. Biochem, 51, 531, 1982).
Experiments conducted in mice with hepatitis caused by the virus of ectromelia have shown that the conjugates with the ara-A and with the ACV penetrate selectively in the liver where they release the drugs in an active form. In patients with cronic hepatitis induced by virus B, the ara-A bound to the L-HSA inhibited the viral replication at a dose 3-6 times lower than that of the free antiviral drug. (Fiume L., Torrani Cerenzia M. R., Bonino F., Busi C., Mattioli A., Brunetto M. R., Chiaberge E. and Verme G., Inhibition of hepatitis B virus replication by vidarabine monophosphate conjugated with lactosaminated serum albumin, Lancet, ii, 13, 1988). In order to bind the ara-A and the ACV with the L-HSA, these compounds have been phosphorilated at the OH primary group according to Yoshikawa's et al. method (Yoshikawa M., Kato T. and Takenishi T., A novel method for phosphorylation of nucleosides to 5'-nucleotides, Tetrahedron Lett. 50, 5065, 1967); the phosphorilated compounds have been further conjugated with the L-HSA with the use of 1-ethyl-3-(dimethylaminopropyl) carbodiimide (ECDI), adjusting the pH of the reaction medium to 7.5 before adding ECDI and prolonging the reaction for 24 hours at the temperature of 25.degree. C. (Biochem. Pharmacol. 35, 967, 1986; Fiume L., Bassi B., Busi C. and Mattioli A., Preparation of lactosaminated albumin-ara-AMP conjugate which remains soluble after lyophilization, Pharm. Acta Helv., 60, 318, 1985). The L-HSA-ara-AMP conjugates so obtained have molar ratios (MR) drug/L-HSA from 10 to 14. In these conjugates, both ara-A monophosphate (ara-AMP) and ACV monophosphate (ACVMP) are bound to L-HSA by means of phosphoamide bonds with lysine and histidine residues (Fiume L., Bassi B. and Bongini A., Conjugates of 9-.beta.-D-arabonofuranosyladenine 5'-monophosphate (ara-AMP) with lactosaminated albumin. Characterization of the drug-carrier bonds, Pharm. Acta Helv., 63, 137, 1988; Fiume L., Busi C., Mattioli A., Spinelli C. and Spinosa G., A conjugate of acyclovir monophosphate with lactosaminated albumin releases the phosphorylated drug in liver cells, Naturwissenschaften, 76, 74, 1989).
The conjugation with carbodiimides has the disadvantage of giving place to side reactions.
It causes a polymerisation of the L-HSA molecules that increases the potential immunogenicity of the conjugate.
The polymers originate from the formation (performed by the carbodiimide) of carboamide bonds linking the protein molecules (Sheehan J. C. and Hlavka J. J., The cross-linking of gelatin using a water-soluble carbodiimide, J. Am. Chem. Soc., 79, 4528, 1957).
Furthermore, by using ECDI, some carboxylic gro
REFERENCES:
patent: 4725672 (1988-02-01), Baldacci et al.
patent: 4794170 (1988-12-01), Fiume et al.
Fiume et al, Pharm. Acta. Helv., vol. 63, No. 4-5, pp. 137-139, 1988.
Pharm. Acta Helv,63(4) 137-139 (1988)L. Fiume et al. "Conjugates Of 9-.beta.-D-Arabinofuranosyladenine 5'-Monophosphate".
Analytic Biochemistry, 212(2) 407-411 L. Fuime et al. "Coupling of Antiviral Nucleoside . . . " (1993).
Computational Aspects, A. Polichetti et al. pp. 421-425 (1991) "Improvements In Resolution . . . ".
Database Biosis, A. Polichetti et al. "Prony-householder . . . " (1991).
Busi Corrado
Fiume Luigi
Mattioli Alessandro
Stefano Giuseppina D.
Laboratori Baldacci SpA
Meller Michael N.
Mohamed Abdel A.
Weimar Elizabeth C.
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