Blood levels of selective antiretroviral drugs over a period of time, in Sprague-Dawley rats

Abstract

Selective antiretroviral (ARV) drugs are primarily metabolized by cytochrome P450 (CYP) enzymes, characteristically predisposed to variation, and are therefore primarily responsible for ARV pharmacokinetic variability and associated drug interactions. For the majority of ARV drugs, the therapeutic window is narrow and imminent toxicities due to CYP inhibition or sub-therapeutic drug levels as a result of CYP induction is inevitable. Animals provide a metabolism replica to conduct detailed investigations. We endeavored to establish a rat model to screen for variability in metabolism of selective ARV drugs responsible for treatment failure and drug interactions, over time in the liver and serum. Male Sprague-Dawley rats (n = 24) were divided into 6 groups: methylcellulose, 160mg/kg/day (n = 24) (control); efavirenz, 160mg/kg/day (n = 18); ritonavir, 20 mg/kg/day (n = 18); ritonavir, 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day, (n = 18); Kaletra® (ritonavir/lopinavir), 20 mg/kg/day and verapamil 5 mg/kg/day (n = 18). Treatment duration varied from one day (single dose), 7 or 21 days. Blood samples were collected after decapitation on days 1, 7 and 21. A sensitive and rapid liquid chromatograph (LC) interfaced to a quadrupoie mass spectrometer (MS) and coupled with electrospray ionization (ESI) method was employed for the blood sample determinations. One single injection was required to simultaneously quantify efavirenz, lopinavir and ritonavir within the linear concentration range of 78 - 5000 ng/ml. Efavirenz blood levels increased statistically significantly (p < 0.05) from day 1 to day 21 with distinct steady state achievement prior to day 7. The levels of ritonavir increased statistically significantly (p < 0.05) from day 7 to 21 when administered alone and statistically significantly (p < 0.01) from day 1 to 21 when administered as the ritonavir/lopinavir combination. The levels of lopinavir also increased statistically significantly (p<0.01) from day 1 and 21 in the ritonavir/lopinavir combination. However, the inclusion of a P-glycoprotein inhibitor, verapamil, increased both the ritonavir (administered alone) and lopinavir blood levels significantly (p < 0.05) at day 1. The ritonavir levels were also significantly increased on day 21 (p < 0.05). When verapamil was added to the ritonavir/lopinavir combination the levels of ritonavir increased statistically significantly (p < 0.01) from day 1 to 21. A rat model can be used to detect changes in metabolism over time as measured by blood levels. The influence of drug interactions, such as verapamil, on ARV drug metabolism can be investigated by this model. These results will be substantiated by PCR liver results in the future.