Estimation of Serum-Free 50-Percent Inhibitory Concentrations for Human Immunodeficiency Virus Protease Inhibitors Lopinavir and Ritonavir

Abstract

Using measured free fraction and 50% inhibitory concentration (IC 50 ) values for the human immunodeficiency virus protease inhibitors lopinavir (LPV) and ritonavir (RTV) in tissue culture media with various protein concentrations ranging from 5 to 50%, we estimated serum-free IC 50 values for each drug. The range of serum-free IC 50 values (0.64 to 0.77 ng/ml for LPV and 3.0 to 5.0 ng/ml for RTV) did not exhibit a trend with increasing protein concentrations, despite a 10-fold difference in the free fraction value (0.006 to 0.063) for LPV and a 5-fold difference in the free fraction value (0.013 to 0.057) for RTV. The mean serum-free IC 50 by the MTT-MT4 assay (0.69 ng/ml for LPV and 4.0 ng/ml for RTV) may be the most accurate parameter for the estimation of the inhibitory quotient (IQ), a relative measure of in vivo potency defined as the ratio of the minimal free drug concentration in plasma (C trough,free ) for a specific patient population and the serum-free IC 50 . Using this approach, we calculated the average IQs for protease inhibitor-naïve patients for LPV and RTV to be 67 and 5.6, respectively.Regardless of the therapeutic category, drugs exist in plasma in a dynamic equilibrium between drug bound to plasma proteins and unbound or free drug. The two major binding proteins are serum albumin and alpha-1 acid glycoprotein (AGP), though emerging evidence suggests that drug binding to lipoproteins, immunoglobulins, complement, and other plasma proteins may also be important. In humans, serum albumin (66 kDa) is present at approximately 40 mg/ml (600 M) and AGP (40 kDa) is present at approximately 0.8 mg/ml (20 M). In general, albumin binds acidic drugs with a high capacity, whereas basic drugs are often selectively bound to AGP with a high affinity. Due to the higher relative abundance of albumin, binding to albumin is less easily saturable than AGP binding. Since a protein-bound drug is generally considered to be too large to pass through most cell membranes to exert pharmacological actions, protein binding can affect the potency of drugs that exert pharmacological actions intracellularly. The magnitude of this effect can be estimated by the reduction of in vitro potency of a compound in the presence or absence of exogenously added serum (19).Lopinavir (LPV) is a potent human immunodeficiency virus (HIV) protease inhibitor (PI) that is widely used for the therapeutic treatment of HIV infection as a coformulated combination with ritonavir (RTV), another PI that also enhances LPV levels in plasma by virtue of its inhibition of cytochrome P450-mediated metabolism (15,28). Previous studies have shown that LPV and RTV are extensively bound to both albumin and AGP

Novel Method To Assess Antiretroviral Target Trough Concentrations UsingIn VitroSusceptibility Data

“…Notably, lopinavir also has a relatively low PBCF, despite reports of high (98 to 99%) serum protein binding. This apparent discrepancy has been reported by others and may be attributed to differential binding properties of bovine serum proteins in the tissue culture medium (26,28).…”

Selection of Resistance in Protease Inhibitor-Experienced, Human Immunodeficiency Virus Type 1-Infected Subjects Failing Lopinavir- and Ritonavir-Based Therapy: Mutation Patterns and Baseline Correlates

“…Suitable genotypic breakpoints, based on the number of LPV-associated mutations, appear to be between five and six mutations and between seven and eight mutations, respectively. The phenotypic upper breakpoint is consistent with a pharmacological model for PI activity in vivo that predicts that the average inhibitory quotient (ratio of trough concentration of drug to human serum-adjusted IC 50 ) for LPV-RTV in patients with WT HIV is 67 (13). Patients with Ͼ60-fold reduced susceptibility would be likely to have plasma levels around the IC 50 for the mutant virus, and selective pressure for additional resistance would be expected to be reduced, particularly if further evolution requires a compromise in viral fitness.…”

Protein Binding of Lopinavir and Ritonavir During 4 Phases of Pregnancy

“…Only the C 12h at 30 weeks gestation prior to dose escalation (PK2) was significantly different from the postpartum C 12h [PK2: 0.10 (0.08–0.15) vs. PK4: 0.16 (0.14–0.27) µg/mL; p=0.05)]. Less than 2% of all concentrations were below the wild type IC 50 (50% inhibitory concentration) for unbound lopinavir of 0.00064 µg /mL(18). …”