Purpose: The purpose of this study is to develop an advanced stimulus-sensitive anti-retroviral drug delivery system for protection of women against HIV-1 infection. Women may be at a higher risk, especially in the developing countries, due to their biological susceptibility and lack of partner’s consent to have access to sexual health services. Also, a HIV-1 infected woman may give birth to a HIV-1 infected child – leading to an increased population suffering from HIV-1 infection. Thus, there is an urgent need to develop efficient drug delivery systems specific to women that can either prevent or treat HIV-1 infections.
An entry inhibitor of HIV-1 is known to be metabolized by the enzyme named ‘prostate specific antigen’ (PSA) which is present in seminal fluid. PSA is a serine protease and cleaves peptides into specific sequences. Those 6-10 amino acid long sequences will be incorporated in the drug delivery system and the release profiles of loaded candidate drugs will be examined. The anti-retroviral drug delivery systems currently available, such as vaginal rings and gels, include NRTIs, such as Tenofovir, which are not that efficient in prevention of HIV-1 infection by themselves, but merely lower the rates of HIV multiplication, after infection. Thus, the hydrogel-based advanced delivery system loaded with an entry inhibitor would be superior to currently available formulations in prevention of HIV-1 infection. Another major advantage of this system is to reduce potential toxicity associated with long term exposure of anti-retroviral drugs. A combined strategy of a hydrogel-based stimulus-sensitive delivery system incorporated with Tenofovir will efficiently lower the HIV-1 infection rate in women.
Methods: The material can be chemically differentiated into three moieties –
a) Polymeric support
b) PSA labile linker
c) Entry inhibitor
Polymeric support: Polymeric support serves as the base to which drug is attached via a PSA-labile linker. We intend to deliver the drug as a thermosensitive gel to have advantages – easy of application and handling, large scale production etc. Polymeric support can be selected based on the obtained outcomes and may either be purchased or synthesized from monomers from commercially available sources.
PSA-labile linker: A 6-10 amino acid sequence will link between candidate drugs and polymeric support. The following two sequences have been identified from literature – HSSKLQ and GISSFYSSK. These sequences were synthesized by Fmoc-solid phase peptide synthesis on a Rink amide resin. Both these sequences were hydrolyzed with PSA to determine an ideal sequence for the delivery system. Enzyme assay was performed in Tris- NaCl buffer at a pH of 7.8. A sequence with the faster hydrolysis outcomes were compared, as the sequence depends on kinetics of hydrolysis. The shorter sequence HSSKLQ was selected, as the rates of both candidates were almost same. An azide function was included at the C-terminus to allow them to be linked with polymeric support having alkyne function.
Entry inhibitor: Among two FDA approved entry inhibitors, maraviroc and enfuvirtide, enfuvirtide were chosen because of its peptidic nature. Linker and the drug enfuvirtide were either synthesized or purchased and linked to the polymeric support using copper free or copper catalyzed click chemistry.
Results: Peptide sequences YYHSSKLQ (modification of HSSKLQ for ease of detection by mass spectrometer and HPLC) and GISSFYSSK were synthesized using solid phase peptide synthesis and the final product was confirmed using mass spectrometry. The peptide GISSFYSSK was tested for hydrolysis by PSA and cleavage fragments were identified by mass spectrometry. The concentration of product linearly increased, as all the substrate was consumed. Enzyme kinetics were estimated using varying substrate concentrations. The similar results were obtained for YYHSSKLQ and compared with peptide GISSFYSSK.
Conclusion: In this study, the stimulus-sensitive delivery system incorporated with anti-retroviral drug has been developed for prevention of women against HIV-1 infection. The hydrolysis process of two peptide sequences was examined as a major stimuli for anti-retroviral drug release from the hydrogel-based advanced delivery system. Peptides were synthesized by Fmoc-solid phase peptide synthesis and assessed for the metabolic susceptibility to PSA, the entry inhibitors. Ideal sequence was chosen based on the best results to build the PSA-sensitive anti-retroviral drug delivery system.
Chi Lee– UMKC School of Pharmacy, Missouri