Synthesis and HIV-1 inhibitory activities of dicaffeoyl and digalloyl esters of quinic acid derivatives.

Twenty analogues of the anti-HIV-1 integrase (IN) inhibitors dicaffeoylquinic acids (DCQAs) were prepared. Their IC(50) values for 3'-end processing and strand transfer against recombinant HIV-1IN were determined in vitro, and their cell toxicities and EC(50) against HIV-1 were measured in cells (ex vivo). Acetylated or benzylated and/or with cyclohexylidene group compounds exhibited no inhibition of integration in biochemical assays or viral replication in HIV-infected cells, with the exception of 16 and 36. Removal of these groups, however, correlated with potent inhibition. Compounds 19, 31, and 38, all digalloyls, exhibited the most robust inhibitory performance in biochemical assays as well as in cell culture and less toxicity than other molecules in the current study.

Mismatched double-stranded RNA (polyI-polyC(12)U) is synergistic with multiple anti-HIV drugs and is active against drug-sensitive and drug-resistant HIV-1 in vitro.

Although highly active anti-retroviral therapy (HAART) is successful in the treatment of HIV infection, problems with toxicity, drug-resistant variants, and therapeutic failures have compromised the long-term utility of existing combination regimens. Mismatched double-stranded RNA (polyI-polyC(12)U) is an immune modulator with inherent anti-HIV activity. Cell toxicities and anti-HIV activities of fourteen anti-HIV agents were determined alone and in combination with polyI-polyC(12)U. Combination analyses for anti-HIV activity were performed at three drug ratios. Using Mixed Dose Effect analyses and the CalcuSyn for Windows software package, combination indeces were determined for all drug combinations. In general, polyI-polyC(12)U was synergistic in combination with abacavir, zidovudine, zalcitabine, didanosine, stavudine, efavirenz, indinavir, ritonavir, nelfinavir, and amprenavir. It was synergistic to antagonistic with lamivudine, delavirdine, nevirapine, and saquinavir. Thus, polyI-polyC(12)U is synergistic with most anti-HIV agents at most drug ratios and across most effective concentrations in vitro, although, certain members of each class were exceptions. PolyI-polyC(12)U alone was equally active against wild-type HIV and HIV resistant to nevirapine, protease inhibitors, or nucleoside analogue reverse transcriptase inhibitors. These results suggest that polyI-polyC(12)U should be re-evaluated as a potential adjunct therapy in patients who have failed current anti-retroviral therapeutic regimens.

Structure-activity relationships: analogues of the dicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of human immunodeficiency virus type 1 integrase and replication.

The dicaffeoylquinic acids (DCQAs) and dicaffeoyltartaric acids (DCTAs) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase. They also inhibit HIV-1 replication at nontoxic concentrations. Since integrase is an excellent target for anti-HIV therapy, structure-activity relationships were employed to synthesize compounds with: (1) improved potency against HIV-1 integrase, (2) improved anti-HIV effect in tissue culture, and (3) increased selectivity as indicated by low cellular toxicity. Thirty-four analogues of the DCTAs and DCQAs were synthesized and tested for cell toxicity, anti-HIV activity, and inhibition of HIV-1 integrase. Seventeen of the 34 analogues had potent activity against HIV-1 integrase ranging from 0. 07 to >10 microM. Seventeen analogues that were synthesized or purchased had no inhibitory activity against integrase at concentrations of 25 microM. Of the biologically active analogues, 7 of the 17 inhibited HIV replication at nontoxic concentrations. The most potent compounds were D-chicoric acid, meso-chicoric acid, bis(3,4-dihydroxydihydrocinnamoyl)-L-tartaric acid, digalloyl-L-tartaric acid, bis(3,4-dihydroxybenzoyl)-L-tartaric acid, dicaffeoylglyceric acid, and bis(3, 4-dihydroxyphenylacetyl)-L-tartaric acid. Anti-HIV activity of the active compounds in tissue culture ranged from 35 to 0.66 microM. Structure-activity relationships demonstrated that biscatechol moieties were absolutely required for inhibition of integrase, while at least one free carboxyl group was required for anti-HIV activity. These data demonstrate that analogues of the DCTAs and the DCQAs can be synthesized which have improved activity against HIV integrase.

Differences in humoral insulin-antibody response among inbred Lou/M rats and epitope presentation differences in ELISA and radioimmune titration.

Insulin antibodies were not detected in an insulin-capture enzyme-linked immunosorbent assay (ELISA), but they were easily detected in an insulin-copolymer-capture ELISA. Thus, there is a high degree of steric hindrance because of the proximity of the epitopes on the insulin monomer. This is circumvented by substituting an insulin copolymer for insulin in the capture ELISA. A regression analysis comparing the titers of 28 Lou/M rat insulin antiserums measured by liquid-phase radioimmune titration (RIT) with titers obtained in the direct insulin ELISA was not significant (P greater than .05). Thus, epitopes on insulin available and/or masked for antibody binding in the RIT differ from those available and/or masked in the direct insulin ELISA. As more of the epitopes become available when an insulin copolymer is substituted for monomeric insulin in the ELISAs, a significant positive correlation (P less than .05) with the RIT was observed with these 28 insulin antiserums. Twenty-five percent (7 of 28) of these antiserums contained more antibodies that bound to epitopes available in the ELISAs that were masked in the RIT. Conversely, two antiserums contained more antibodies that bound to epitopes that were available in the RIT but were masked in the ELISAs. Thus, the amount of insulin antibodies measured in a given antiserum can vary substantially, depending on which epitopes are made available or are masked in the particular antibody-titration method used. These results demonstrate that the humoral immune response to insulin among inbred Lou/M rats can vary in insulin-antibody levels as well as the epitopes on insulin to which the antibodies bind.

Effect of isologous and autologous insulin antibodies on in vivo bioavailability and metabolic fate of immune-complexed insulin in Lou/M rats.

The in vivo bioavailability, distribution, and metabolic fate of 125I-labeled insulin complexed to isologous and autologous antibodies were studied in inbred Lou/M rats. There was an impaired bioavailability of the 125I-insulin bound to the isologous and autologous antibodies. Very little of the 125I-insulin in these immune complexes could bind to insulin receptors on hepatocytes or renal tubular cells and be degraded, because the amounts of 125I from degraded 125I-insulin in the blood or secreted into the stomach were markedly attenuated in both cases for at least 30 min after injection. There was a simultaneous accumulation of 125I-insulin immune complexes in the liver and the kidneys of Lou/M rats injected with 125I-insulin complexed with isologous antibodies or when insulin-immunized Lou/M rats were injected with 125I-insulin during the same interval. The impaired bioavailability of immune-complexed insulin and altered distribution of radioactivity due to the accumulation of immune complexes in the liver and kidney were also observed in previous experiments in which Lewis rats were injected with xenogenic guinea pig and homologous insulin antibodies. These observations are therefore submitted as evidence that the Lou/M rat is a valid model in which to study the bioavailability of insulin immune complexed to isologous, homologous, and xenogenic antibodies and the metabolic fate of the respective insulin-antibody immune complexes.