The Study Of Clinical Pharmacokinetics And The Interaction With Biomembrane Of The Novel Anti-HIV Fusion Inhibitor Sifuvirtide

Sifuvirtide is a newly developed HIV fusion inhibitor. As one of the new generation HIV fusion inhibitor, Sifuvirtide was designed based on the three-dimensional structure of core conformation of HIV-1 membrane fusion protein gp41 with good prospects for the development of HIV inhibitor. The aim of this paper is to make a comprehensive evaluation and in-depth research in clinical pharmacokinetics and pharmacological mechanisms at the molecular level of Sifuvirtide. First, we established a liquid chromatography-mass spectrometry based quantitative method for the pharmacokinetic studies in HIV-infected subjects. The results of complete and validated study for the LC-MS/MS method showed that the sensitivity of the method was up to 9.75ng/mL, the accuracy of intra-(and inter) batch was between-8.37- 2.53%, precision(CV) between 2.74 ~ 7.57%, and recoveries between 75.60- 80.35%. Under the various store conditions, the accuracy of the method for the plasma samples was between 4.51%-6.56, precision(CV) was between 3.86 ~ 10.41%, which indicated the developed LC-MS/MS method met the requirements of clinical pharmacokinetic studies of the new drug for the markets. Second, using the established method, the pharmacokinetic of Sifuvirtide on phase IIa clinical trial with two dose groups(10 mg, 20 mg) was studied. A total of 19 treatment naive HIV-infected subjects were analyzed with once daily subcutaneous administrated Sifuvirtide for 28 days continuously. The results indicated that Sifuvirtide showed a good pharmacokinetic behavior with rapid absorbance, short peak time, wide apparent distribution and long half-life time in both dose groups(10 mg, 20 mg). During 28 consecutive observation days, the two groups of subjects had stable blood concentration with the accumulation ratio 1.7 and 1.3 respectively.. In the phase IIb clinical trial study, 8 HAART treatment-experienced subjects were gave a 20 mg Sifuvirtide combined with HAART therapy for 168 days. The concentration-time profile of Sifuvirtide showed that the average trough drug concentration Cmin were much larger than EC50 in vitro efficacy test; the parameters of pharmacokinetics after administration of the drug for the first dose and the last dose showed no significantly statistical difference(P > 0.05), which means after 168 consecutive dosing, the pharmacokinetics parameters of the first dose and the last dose no significantly statistical difference; the pharmacokinetics of Sifuvirtide in HAART treatment-experienced subjects showed the significantly accelerated metabolism, the speeded up clearance rate, and the shorter elimination half-life.In the second part of this paper, we first established a peptide- single or bilayer membrane interaction system based on SPR. By using the data fitting and model analysis, we aquired the quantitative information of affinity constant of Sifuvirtide( and Enfuvirtide), the nature of the interaction force of the polypeptide binding with the biomembrane( electrostatic and hydrophobic interaction), the qualitative information of polypeptide-biomembrane binding mode( the binding surface of the membrane, membrane insertion, formation of pores) and the kinetics process information( one binding step, two state reaction), all of which made the beneficial information to understand the fusion inhibition mechanism of Sifuvirtide at the molecular level. The results showed Sifuvirtide was selectively adsorbed and enriched in the rigid regions of biomembrane where receptors are rich in with a two-state reaction. This reaction was drived by the hydrophobic interactions or electrostatic adsorption. There is only the membrane surface binding, no membrane insertion or the formation of pores during the process. Then, Sifuvirtide combined with the gp41 to play fusion inhibition efficacy. Whereas, Enfuvirtide combined with all the phospholipid membranes in some degree, not specifically adsorbed into single or bilayers of biomembrane constituted by the phospholipids with the higher saturation degree. This non-selectively binding capacity to phospholipid membrane was the way to play fusion inhibition efficacy of Enfuvirtide. The differences between Sifuvirtide and Enfuvirtide on the selecting behavior for biomembrane showed us not only the different pharmacological mechanism at the molecular level of two drugs but also the explanation of the stronger treatment efficacy of Sifuvirtide than that of the Enfuvirtide.This is the first report on Sifuvirtide with a long-term tracking and completed evaluation of pharmacokinetic, safety and efficacy for treatment naive and HAART treatment-experienced HIV-infected subjects. It provided the first-hand clinical reference for the Sifuvirtide into market, and the basis for the further improvement of clinical dosing regimen on different treatment experienced HIV-infected persons using Sifuvirtide alone or combination with HAART. The innovation in second part of the study is to establish a new type of peptide-single or bilayer membrane interaction SPR system, and applied it to the studies of peptide fusion inhibitors-biomembrane interaction, which overcame the defects that currently used methods can not resolve the real-time information of the interactions of peptide and biomembrane. Our study firstly fully expounded on the membrane tropism, force analysis and kinetics of the interaction of Sifuvirtide and Enfuvirtide at molecular level with the biomembrane, and further improved the understanding on fusion inhibition mechanism of Sifuvirtide and Enfuvirtide, made a reasonable explanation that the efficacy of Sifuvirtide was much better than that of Enfuvirtide, provided a new choice of method for the future interaction studies on the peptide drug and the biomembrane and a new strategy for screening HIV fusion inhibitor target.