Stability in human serum and plasma of the HIV peptide drug candidate CIGB-210 and improved variants.

The peptide CIGB-210 inhibits HIV replication, inducing a rearrangement of vimentin intermediate filaments. The assessment of the in vitro serum and plasma stability of this peptide is important to develop an optimal pharmacological formulation. A half-life of 17.68 ± 0.59 min was calculated for CIGB-210 in human serum by reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Eight metabolites of CIGB-210 were identified with this methodology, all of them lacking the N-terminal moiety. A previously developed CIGB-210 in-house competitive ELISA was used to compare the stability of CIGB-210 derivatives containing either D-amino acids, acetylation at the N-terminus, or both modifications. The half-life of CIGB-210 in serum was five times higher when measured by ELISA than by HPLC/MS, and twice higher in plasma as compared to serum. The substitution of D-asparagine on position 6 doubled the half-life, while D-amino acids on positions 8 and 9 did not improve the stability. The acetylation of the N-terminus resulted in a 24-fold more stable peptide in plasma. The positive effect of N-terminal acetylation on CIGB-210 serum stability was confirmed by the HPLC/MS method, as the half-life of the peptide was not reached after 2 h of incubation, which represents more than a 6.8-fold increase in the half-life with respect to the original peptide.

A competitive ELISA for the quantitative determination of the novel anti-HIV drug candidate CIGB-210 in biological fluids.

The synthetic peptide CIGB-210 is a promising anti-HIV drug candidate shown to inhibit HIV replication in MT4 cells at the nanomolar range by triggering the rearrangement of vimentin intermediate filaments. Sensitive and specific analytical methods are required for pharmacological studies of CIBG-210 in animals. In this study, we describe the development of a competitive ELISA for the quantitative determination of CIGB-210 using an anti-CIGB-210 hyperimmune serum. After optimization of all the steps, the assay exhibited a dynamic range from 11.87 to 0.0095 µg/mL. The intra-assay coefficient of variation (CV) was lower than or close to 5% for all the six concentrations of the calibrator, and the inter-assay CV was below 10% in five out of the six concentrations tested. No interference of either murine or human plasma was observed. The analyte was stable in plasma after five freeze-thaw cycles, while the hyperimmune serum maintained its binding capacity after 10 freeze-thaw cycles. Furthermore, the ELISA was able to detect the two main metabolites of CIGB-210, although with a tenfold decrease in sensitivity. Our results demonstrate the utility and feasibility of this analytical method for pharmacological experiments in animals as humans.

Identification of Vimentin as a Potential Therapeutic Target against HIV Infection.

A combination of antiviral drugs known as antiretroviral therapy (ART) has shown effectiveness against the human immunodeficiency virus (HIV). ART has markedly decreased mortality and morbidity among HIV-infected patients, having even reduced HIV transmission. However, an important current disadvantage, resistance development, remains to be solved. Hope is focused on developing drugs against cellular targets. This strategy is expected to prevent the emergence of viral resistance. In this study, using a comparative proteomic approach in MT4 cells treated with an anti-HIV leukocyte extract, we identified vimentin, a molecule forming intermediate filaments in the cell, as a possible target against HIV infection. We demonstrated a strong reduction of an HIV-1 based lentivirus expressing the enhanced green fluorescent protein (eGFP) in vimentin knockdown cells, and a noteworthy decrease of HIV-1 capsid protein antigen (CAp24) in those cells using a multiround infectivity assay. Electron micrographs showed changes in the structure of intermediate filaments when MT4 cells were treated with an anti-HIV leukocyte extract. Changes in the structure of intermediate filaments were also observed in vimentin knockdown MT4 cells. A synthetic peptide derived from a cytoskeleton protein showed potent inhibitory activity on HIV-1 infection, and low cytotoxicity. Our data suggest that vimentin can be a suitable target to inhibit HIV-1.