During phase I the feasibility of a test kit for the fluorometric measurement of HIV protease inhibitors (PI) in human serum has been demonstrated. The overall objective of this phase II investigation is the development of a robust fluorometric diagnostic test kit, trade name Levoprin-ZG, for clinical laboratories, to be used for therapeutic dose monitoring (TDM). The test results should be similar in accuracy with those obtained by HPLC/mass spectrometry (LCMS) and have demonstrated clinical applications (for FDA approval). Principle of the method: in the well of a microtiter plate the HIV protease degrades a specific substrate attached to a solid phase, which releases a fluorescent signal. In the presence of diluted patient serum containing PI the activity of the enzyme is inhibited in proportion to the concentration of PI. Dosing of PI is based on the level of drug necessary to inhibit HIV. However, achievement of therapeutic levels is influenced by multiple factors, including 1) individual variability in levels; from sub-therapeutic trough to toxic peak levels, which reduces adherence. 2) Poor adherence leading to development of resistant mutants. 3) Resistant mutants require genotypic or phenotypic characterization in order to identify the drug level required to suppress the virus in individual patients; under these circumstances (rescue) the PI levels need to be substantially increased, monitored closely and adjusted. For these reasons, clinicians have expressed the need for a rapid, sensitive and inexpensive TDM test to be performed close to the point of care and able to determine accurately the plasma and/or intracellular level of PI. However, the only available test is based on LCMS available at a relatively high cost at unique specialized reference laboratories. Here we will perform the research and development necessary to complete the stable and robust kit for clinical laboratories. We will perform clinical studies to demonstrate utility for monitoring adherence and making dose adjustment and to obtain FDA pre-market approval. Moreover, we will investigate the compartment to be measured for best clinical utility: total drug (current state of the art), free drug (there is significant protein binding) or intracellular (there is large individual variability in this compartment). The test kit will then be manufactured for sale to clinical laboratories.
