Quantitation of a 36-amino-acid peptide inhibitor of HIV-1 membrane fusion in animal and human plasma using high-performance liquid chromatography and fluorescence detection.
Selective extraction of a 36-amino-acid peptide (DP-178, T20, pentafuside) from the protein matrices of animal and human plasma was achieved using acetonitrile containing 1% trifluoroacetic acid and 1% n-nonyl-beta-D-glucopyranoside. The peptide concentration of the extract was measured using reversed-phase high-performance liquid chromatography (RP-HPLC) and fluorescence detection. The eluent was excited at 280 nm and the intrinsic fluorescence signal was collected at 350 nm. Recovery of T20 from the plasma matrices was 75% (mouse), 60% (rat), 50% (cynomolgus monkey), and 55% (human) based on parallel-processed aqueous T20 standard solutions. The fluorescence peak area vs. concentration of T20 was linear in the range 4-160 ng/ml based on the final solute concentration in the HPLC vial, corresponding to original plasma concentrations of 100-4000 ng/ml. Experiments with truncated analogs of T20 demonstrate that this assay offers the advantage of detecting metabolites attributable to bio-transformation degradation processes differing by as little as one amino acid from the original peptide.
Lawless
M K
MK
Trimeris, Inc., Durham, NC 27707, USA.
Hopkins
S
S
Anwer
M K
MK
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000097:blood* / Q000493:pharmacokinetics; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D000077560:Enfuvirtide; D015700:HIV Envelope Protein gp41 / Q000097:blood*; D006801:Humans; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood*; D012015:Reference Standards; D013050:Spectrometry, Fluorescence
D000998:Antiviral Agents; D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments; D000077560:Enfuvirtide