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Negatively charged albumins (NCAs, with the prototypes Suc-HSA and Aco-HSA), modified proteins with a potent anti-HIV-1 activity in the nanomolar concentration range, were studied for several aspects of their antiviral mechanism. In addition we investigated the antiviral activity of combinations and covalent conjugates of these NCAs and the reverse transcriptase inhibitor AZT.Addition of NCAs to HIV-l-infected target cells in time-of-addition experiments could be delayed for 30 min after infection before significant loss of activity occurred. Syncytium formation of HIV-infected and uninfected T-cells was inhibited by the NCAs at concentrations of 1-4 mu g/ml. The gp120-mediated virus/cell binding, however, was only inhibited by the NCAs at a 10-fold higher concentrations. The combined data are compatible with a preferential influence on virus/cell fusion.A subsynergistic activity against HIV-I was observed with the non-covalent mixture of Aco-HSA and AZT, When AZT was covalently coupled to the NCAs, and added one hour after infection of the target cells, the anti-HIV-l activity of NCA-AZTMP was diminished by only 2-6-fold, while this was diminished at least 120-fold for the NCAs, Furthermore the addition of NCA-AZTMP could be delayed up to at least 3h after infection without loss of antiviral activity. It is concluded that AZT that was coupled to the NCAs significantly contributes to the overall antiviral activity of the conjugates leading to complementary effects.These results highlight the potential of using NCA-AZTMP as dual-targeting preparations against HIV-I in which both the carrier and the coupled drug contribute to the therapeutic efficacy acting at a different level in the replication cycle.

Negatively charged albumins (NCAs, with the prototypes Suc-HSA and Aco-HSA), modified proteins with a potent anti-HIV-1 activity in the nanomolar concentration range, were studied for several aspects of their antiviral mechanism. In addition we investigated the antiviral activity of combinations and covalent conjugates of these NCAs and the reverse transcriptase inhibitor AZT. Addition of NCAs to HIV-l-infected target cells in time-of-addition experiments could be delayed for 30 min after infection before significant loss of activity occurred. Syncytium formation of HIV-infected and uninfected T-cells was inhibited by the NCAs at concentrations of 1-4 mu g/ml. The gp120-mediated virus/cell binding, however, was only inhibited by the NCAs at a 10-fold higher concentrations. The combined data are compatible with a preferential influence on virus/cell fusion. A subsynergistic activity against HIV-I was observed with the non-covalent mixture of Aco-HSA and AZT, When AZT was covalently coupled to the NCAs, and added one hour after infection of the target cells, the anti-HIV-l activity of NCA-AZTMP was diminished by only 2-6-fold, while this was diminished at least 120-fold for the NCAs, Furthermore the addition of NCA-AZTMP could be delayed up to at least 3h after infection without loss of antiviral activity. It is concluded that AZT that was coupled to the NCAs significantly contributes to the overall antiviral activity of the conjugates leading to complementary effects. These results highlight the potential of using NCA-AZTMP as dual-targeting preparations against HIV-I in which both the carrier and the coupled drug contribute to the therapeutic efficacy acting at a different level in the replication cycle.

Negatively charged albumins (NCAs, with the prototypes Suc-HSA and Aco-HSA), modified proteins with a potent anti-HIV-1 activity in the nanomolar concentration range, were studied for several aspects of their antiviral mechanism. In addition we investigated the antiviral activity of combinations and covalent conjugates of these NCAs and the reverse transcriptase inhibitor AZT. Addition of NCAs to HIV-l-infected target cells in time-of-addition experiments could be delayed for 30 min after infection before significant loss of activity occurred. Syncytium formation of HIV-infected and uninfected T-cells was inhibited by the NCAs at concentrations of 1-4 mu g/ml. The gp120-mediated virus/cell binding, however, was only inhibited by the NCAs at a 10-fold higher concentrations. The combined data are compatible with a preferential influence on virus/cell fusion. A subsynergistic activity against HIV-I was observed with the non-covalent mixture of Aco-HSA and AZT, When AZT was covalently coupled to the NCAs, and added one hour after infection of the target cells, the anti-HIV-l activity of NCA-AZTMP was diminished by only 2-6-fold, while this was diminished at least 120-fold for the NCAs, Furthermore the addition of NCA-AZTMP could be delayed up to at least 3h after infection without loss of antiviral activity. It is concluded that AZT that was coupled to the NCAs significantly contributes to the overall antiviral activity of the conjugates leading to complementary effects. These results highlight the potential of using NCA-AZTMP as dual-targeting preparations against HIV-I in which both the carrier and the coupled drug contribute to the therapeutic efficacy acting at a different level in the replication cycle.

Charge modification by succinylation or cis-aconitylation of the terminal epsilon NH2 functions of the amino acid lysine in human serum albumin, resulted in polyanionic compounds with an anti-HIV-1 activity in the low nanomolar concentration range. After iv injections in rats of the negatively charged albumins (NCAs), a dose dependent elimination pattern was observed indicating a saturable eliminations pathway. The Michaelis-Menten parameters V-max and K-m were 62 +/- 8 mu g.min(-1).k(-1) and 16 +/- 4 mu g.ml(-1) (Cl-intr 3.9 +/- 1.1 ml.min(-1).kg(-1)) and 74 +/- 6 mu g.min(-1).kg(-1) and 11 +/- 2 mu g.ml(-1) (Cl-intr 6.7 +/- 1.2 ml.min(-1).kg(-1)) for aconitylated-HSA (Aco-HSA) and succinylated-HSA (Suc-HSA) respectively, using I-125-labelled proteins. The volume of distribution (V) of both compounds was approximately 60 ml.kg(-1). Coadministration of poly-inosinic acid and formaldehyde treated albumin showed a marked inhibition of blood clearance indicating that the compounds are mainly cleared from the bloodstream by scavenger receptors on liver and spleen endothelial cells and macrophages.The Michaelis-Menten constant K-m was remarkably higher when the hydrophobic fluorophore fluorescein was covalently linked to the protein, indicating that the affinity for the scavenger receptors is largely decreased by FiTC conjugation. The latter observation may have implications for the kinetic behavior of drug-carrier preparations if antiviral drugs like AZT or PMEA are linked to these intrinsic active carriers.In contrast to other polyanionic compounds like heparins and dextran sulfate, these NCAs did not exhibit acute toxicity and had no effect on blood coagulation. They neither had an effect on the lymphocyte proliferation. Studies on immunogenicity of the homologous derivatized albumins in rats did not show a significant response.The present pharmacokinetic and toxicologic data of Suc-HSA and Aco-HSA show that both compounds are interesting preparations for studies in SIV infected monkeys and AIDS patients.