Evaluation of a Vitamin-Cloaking Strategy for Oligopeptide Therapeutics: Biotinylated HIV-1 Protease Inhibitors.

The outstanding limitations to the oligopeptide as a therapeutic agent are poor oral availability and rapid biliary clearance. To address these concerns a series of eight peptidic HIV-1 protease inhibitors containing the structural segment of the vitamin biotin have been prepared. These have been evaluated with regard to the hypothesis that this vitamin would cloak the peptidic character of these oligopeptides, and thus impart to these inhibitors the potential for absorption and distribution via biotin transporters and receptors. By iterative optimization about a -Cha$[CH(OH)CH(OH)IVal- core inLibitoryinsert, three particularly potent inhibitors (Ki I 10 nM) of the HIV-1 protease were obtained. Although excellent cell culture antiviral activity is observed for other peptidic protease inhibitors of comparable affinity, none in this series exhibited satisfactory antiviral activity. This failure is attributed to the incompatibility of the hydrophilic and hydrogenbonding biotin segment, with the facile membrane permeability and intracellular access presumably required for antiviral activity. The ability of the biotin to cloak the peptide, and thus render the overall appearance of the conjugate as that of a vitamin, was evaluated. Four of this series were evaluated for recognition by the Caco-2 cell intestinal biotin transporter. None inhibited competitively biotin uptake, indicating a lack of recognition. A vitamin may bind to a specific protein carrier, and thus attain an improved serum profile (by resistance to biliary clearance) and advantageous delivery to cells. Therefore, the serum concentrations of three were evaluated following an iv bolus in a rat model for serum clearance. One of the three protease inhibitors (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-2-(l-methylethyl)-5- [ [3-methyl-l-oxo-2- [ [5-(hexahydro-2-oxo-lHthieno[3,4-dlimidazol-4-yl)-l-oxopentyllaminol butyllaminol-N-[2-methyl-l-[ [ (2-pyridinylmethy1)aminol carbonyllbutyll-, [3aS-[3a(u,4~(1R*,2R*,3R*),6all-) sustained amore than 5-fold increase in serum concentration at all time points relative to the benchmark structure. The remaining two had serum concentrations at least equal to the benchmark, suggestive of improved resistance to clearance. One (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-5-[ [2-[[5-(hexahydro-2-oxo-lH-thieno[3,4-d]imidazol-4-yl)pentyl]thiolbenzoyl]amino]-2-( l-methylethyl)-N-[2-methyl-l-[ [ (2-pyridinylmethyl)aminolcarbonyllbutyll-, [3aS-[3~,4P(lR*,2R*),6a(u]]-) was prepared as a complex with the biotin-binding protein avidin. Avidin may resemble an endogenous serum biotin carrier protein. The antiviral activity (evaluated in an HS-HTLVIIB acute HIV-1 infection assay) of the inhibitor and the avidin complex was identical. This suggests that the avidin-inhibitor complex is capable of cell internalization. Although the weak antiviral activity of these biotinylated inhibitors precludes consideration as practical HIV therapeutics, the overall data remain suggestive of vitamin cloaking of oligopeptides as a strategy of potential value. The development of peptide-based therapeutic agents is limited by their low oral availability and their rapid and efficient removal from the serum by an hepatic active transporter.' In the particular example of inhibitors2 of the HIV-1 protease, these difficulties are exacerbated by the requirement (for antiviral activity) of a hydrophobic peptidic ~haracter.~ Two chemical strategies may be adopted in face of these difficulties. The first is liquidation and replacement, where each superfluous peptidic segment is expunged and each essential peptidic segment is replaced with a mimetic. The second strategy is that of cloaking. Here the peptidic character is preserved, but