The clinical care of patients having acquired immunodeficiency disease syndrome (AIDS) has been substantially improved by the introduction of compounds which function as potent and specific HIV protease inhibitors. It is believed that the human immunodeficiency virus (HIV) is the causative agent responsible for AIDS, and that the enzyme HIV protease is responsible for catalyzing specific cleavages in the gag and gag-pol polypeptides of the HIV. A virus that synthesizes a mutationally inactivated HIV protease does not generally form infectious virions. HIV protease is thus an important target for which drugs against AIDS can be designed. HIV protease inhibitors can cause a reduction or cessation of the activity of HIV protease.
Currently, there are six HIV protease inhibitors approved by the Food and Drug Administration (FDA) for treatment of AIDS patientsxe2x80x94amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir. Combination therapies involving HIV protease inhibitors and HIV reverse transcriptase inhibitors are the cornerstones of currently recommended therapies for HIV infection. Not all AIDS patients show the same optimal response to a combination therapy regimen. There can be a large variability in drug response between individual patients. Relationships between systemic exposure to protease inhibitors and antiviral effect have been supported by accumulating clinical information. When a combination therapeutic regimen is administered to a patient, potential pharmacokinetic drug-drug interactions can improve or weaken the treatment. Patient compliance, which directly relates to maintenance of adequate drug levels, may also affect the outcome of the treatment.
It is thus desirable to measure concentrations of HIV protease inhibitors in patients to ensure that drug exposure is sufficient to maintain antiviral activity in AIDS patients. In addition, the quantitative measurement of HIV protease inhibitors in biological samples from test subjects is crucial in the drug development process. Other measurements of interest in the treatment of HIV include the measurement of the metabolites of HIV protease inhibitors, and the measurement of anti-HIV protease antibodies. The presence and amount of metabolites can provide information regarding the effectiveness of the therapeutic treatment. The presence of anti-HIV protease antibodies indicates infection of a patient by HIV, and the detection of the antibodies can therefore be used to diagnose possible infection.
Typically, HIV protease inhibitors in patient samples have been quantified by assay methods which require sophisticated and expensive instrumentation, making the analysis difficult to perform in a clinical setting. For example, plasma samples can be analyzed for the presence of numerous HIV protease inhibitor compounds simultaneously using chromatographic methods such as high-performance liquid chromatography (HPLC) or HPLC coupled with dual mass spectrometry (HPLC/MS/MS). See, for example, Poirier, J. M. et al., Ther. Drug Monit. 22:465-473 (2000); Marzonlini, C., et al., J. Chromatogr. 740:43-58 (2000); and Remmel, R. P. et al., Clin. Chem. 46(1):73-81 (2000). The plasma samples are typically subjected to solid-phase extraction procedures prior to examination. Thus, the plasma is not analyzed directly but must be modified, adding complexity and expense to the analysis.
Radioimmunoassays typically involve competitive interaction between a radiolabeled conjugate containing an HIV protease inhibitor analog and any free HIV protease inhibitor in the sample. The receptor is generally an antibody for the particular HIV protease inhibitor. In an example of a radioimmunoassay, an analog of an HIV protease inhibitor labeled with iodine-125 is reported to compete with any HIV inhibitor compound in a sample of patient plasma for binding with an antibody. The precipitated antibody complexes are then analyzed for their level of radioactivity to determine the concentration of the HIV protease inhibitor compound. See, for example, Wiltshire, H. R. et al. Analyt. Biochem. 281:105-114 (2000). In an example of an assay which indirectly uses a radioimmunoassay, HIV protease can be added to a sample together with a substrate for HIV protease. Cleavage of the substrate can then be measured by a radioimmunoassay utilizing an antibody which specifically binds the cleavage products. An absence of cleavage products indicates the presence of anti-HIV protease antibodies. See, for example, U.S. Pat. No. 5,171,662.
All of these methods for measuring inhibitors and/or their metabolites have met with mixed success. There is thus a need for an improved method to quantify HIV protease inhibitors and/or their metabolites in a biological sample. It is desirable that such a method can be easily and rapidly carried out on currently available analytical instrumentation in clinical settings.
In one aspect of the invention, there is a method for quantifying an HIV protease inhibitor in a sample, comprising combining an HIV protease, a conjugate comprising an HIV protease inhibitor analog, and a sample suspected of containing an HIV protease inhibitor; wherein the HIV protease and the conjugate form a detectable complex; measuring the amount of the detectable complex, and relating the amount of said detectable complex to a concentration of the HIV protease inhibitor in the sample.
In another aspect of the invention, there is a method for quantifying an HIV protease inhibitor in a sample, comprising combining an HIV protease, a conjugate comprising a carrier and at least two HIV protease inhibitor analogs linked to the carrier, and a sample suspected of containing an HIV protease inhibitor; wherein the HIV protease is immobilized on a surface of a microparticle, the HIV protease and the conjugate together undergo agglutination; measuring the agglutination; and relating the amount of agglutination to a concentration of the HIV protease inhibitor in the sample.
In yet another aspect of the invention, there is a method for quantifying an HIV protease inhibitor in a sample, comprising combining a multivalent carrier comprising at least two HIV protease enzymes, a conjugate comprising a microparticle and an HIV protease inhibitor analog, and a sample suspected of containing an HIV protease inhibitor, wherein the HIV protease and the conjugate together undergo agglutination, and the sample is human serum or human plasma; measuring the agglutination; and relating the amount of agglutination to a concentration of the HIV protease inhibitor in the sample.
In yet another aspect of the invention, there is a method for quantifying an HIV protease inhibitor in a sample, comprising combining an HIV protease, a conjugate comprising a label and an HIV protease inhibitor analog linked to the label, and a sample suspected of containing an HIV protease inhibitor, wherein the HIV protease and the conjugate together form a detectable complex; measuring the amount of the detectable complex by monitoring a change in fluorescence polarization; and relating the amount of the detectable complex to a concentration of the HIV protease inhibitor in the sample.
In yet another aspect of the invention, there is a method for monitoring a concentration of an HIV protease inhibitor in an organism, comprising administering a dose of an HIV protease inhibitor to the organism; obtaining a fluid sample from the organism; combining the sample with an HIV protease and a conjugate comprising an HIV protease inhibitor analog, wherein the HIV protease and the conjugate together form a detectable complex; measuring the amount of the detectable complex in the sample; and relating the amount of the detectable complex to a concentration of the HIV protease inhibitor in the organism.
In yet another aspect of the invention, there is a reagent for quantifying an HIV protease inhibitor in a sample, comprising a particle comprising a surface, and HIV protease immobilized on the surface of the particle. The HIV protease forms a detectable complex with a conjugate, wherein the conjugate comprises an HIV protease inhibitor analog.
In yet another aspect of the invention, there is a kit for quantifying an HIV protease inhibitor in a sample, comprising HIV protease, and a conjugate comprising an HIV protease inhibitor analog.