Circulating Metabolites of the Human Immunodeficiency Virus Protease Inhibitor Nelfinavir in Humans: Structural Identification, Levels in Plasma, and Antiviral Activities

Abstract

Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor (K i ‫؍‬ 2 nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. The current studies evaluated the presence of metabolites circulating in plasma following the oral administration of nelfinavir to healthy volunteers and HIV-infected patients, as well as the levels in plasma and antiviral activities of these metabolites. The results showed that the parent drug was the major circulating chemical species, followed in decreasing abundance by its hydroxy-t-butylamide metabolite (M8) and 3-methoxy-4-hydroxynelfinavir (M1). Antiviral assays with HIV-1 strain RF-infected CEM-SS cells showed that the 50% effective concentrations (EC 50 ) of nelfinavir, M8, and M1 were 30, 34, and 151 nM, respectively, and that the corresponding EC 50 against another HIV-1 strain, IIIB, in MT-2 cells were 60, 86, and 653 nM. Therefore, apparently similar in vitro antiviral activities were demonstrated for nelfinavir and M8, whereas an approximately 5-to 11-fold-lower level of antiviral activity was observed for M1. The active metabolite, M8, showed a degree of binding to human plasma proteins similar to that of nelfinavir (ca. 98%). Concentrations in plasma of nelfinavir and its metabolites in 10 HIV-positive patients receiving nelfinavir therapy (750 mg three times per day) were determined by a liquid chromatography tandem mass spectrometry assay. At steady state (day 28), the mean plasma nelfinavir concentrations ranged from 1.73 to 4.96 M and the M8 concentrations ranged from 0.55 to 1.96 M, whereas the M1 concentrations were low and ranged from 0.09 to 0.19 M. In conclusion, the findings from the current studies suggest that, in humans, nelfinavir forms an active metabolite circulating at appreciable levels in plasma. The active metabolite M8 may account for some of the antiviral activity associated with nelfinavir in the treatment of HIV disease.

Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir

“…Two metabolites of nelfinavir were detected after incubating with rat hepatocytes, previously identified as M1 by Zhang et al (2001). [Saquinavir]…”

“…The retention times were approximately 3.4 (verapamil) and 3.5 (saquinavir) min. Nelfinavir and its metabolite (M1) (Zhang et al, 2001) and ritonavir and its metabolites (M1, M2, M9, and M11) (Denissen et al, 1997) together with verapamil (internal standard) were separated using the same system as for saquinavir except that the initial mobile phase of 90% 0.001 M ammonium acetate/10% acetonitrile was ramped linearly to 90% acetonitrile/10% 0.001 M ammonium acetate from 1 to 4 min. The retention times were approximately 3.2 (verapamil), 3.3 (nelfinavir-M1), 3.4 (nelfinavir), 3.9 (ritonavir-M11), 4.1 (ritonavir-M2 and ritonavir-M9), and 4.6 (ritonavir and ritonavir-M1) min.…”

Effect of efavirenz treatment on the pharmacokinetics of nelfinavir boosted by ritonavir in healthy volunteers

“…Subjects received the following treatments: period 1 (days 1-10), 1875 mg nelfinavir plus 200 mg ritonavir to be taken once daily at 23.00 h with a light snack of around 300 kcal; period 2 (days [11][12][13][14][15][16][17][18][19][20], 1875 mg nelfinavir plus 200 mg ritonavir and 600 mg efavirenz to be taken once daily at 23.00 h with a light snack (around 300 kcal). Blood samples were collected throughout a 24-h period on days 10 and 20 following a light meal of 315 kcal.…”

“…CYP3A4 is mainly responsible for the metabolism of nelfinavir [10]. However, the formation of the virologically active [11] metabolite nelfinavir-hydroxy-t -butylamide (designated M8) is primarily dependent on CYP2C19 [12]. M8 is subsequently metabolized by CYP3A4 into inactive metabolites [12].…”

Twenty-six years of HIV science: an overview of anti-HIV drugs metabolism

“…Nelfinavir (NFV) (Figure 14) is biotransformed in the liver into three major metabolites, identified as nelfinavir-hydroxy-t-butyl-amide (66), 3,4-dihydroxy-nelfinavir (67) and 3-methoxy-4-hydroxy-nelfinavir (68) . Compound 66 shows antiretroviral activity in vitro comparable to that of NFV, contributing to its oral activity in anti-HIV therapy, while 68 showed low activity (Zhang et al, 2001;Hirani, Raucy, Lasker, 2004). It has been shown that CYP3A4 is involved in the formation of metabolites 67 and 68, while 66 is produced by CYP2C19 (Wu et al, 1998;Lillibridge et al, 1998;Khaliq et al, 2000).…”