title: Variation in oral clearance of saquinavir is predicted by CYP3A5*1 genotype but not by enterocyte content of cytochrome P450 3A5.
abstract: OBJECTIVE
  Saquinavir, a widely prescribed human immunodeficiency virus 1 protease inhibitor, has a low and variable oral bioavailability that has been attributed to extensive first-pass extraction mediated by hepatic or intestinal cytochrome P450 (CYP) 3A4 and intestinal P-glycoprotein (P-gp). The polymorphic CYP3A5 has also been shown to influence the saquinavir metabolite/parent urinary ratio, suggesting a role for CYP3A5.
  
  
  METHODS
  Twenty healthy subjects received a single oral dose of saquinavir (600 mg) with water (control) and, on a separate occasion, with Seville orange juice (a selective intestinal CYP3A4/5 inhibitor). Hepatic CYP3A4 activity was evaluated by use of the erythromycin breath test. Duodenal biopsy specimens were used to assess relative intestinal CYP3A4 and CYP3A5 protein contents. Relative P-gp content was also assessed in the biopsy specimens and in lymphocytes. Genetic polymorphisms in MDR1 (in exon 21 and 26), CYP3A5 (*1 and *3), and CYP3A4*1B were identified by direct sequencing. Saquinavir plasma concentrations were measured by tandem liquid chromatography-mass spectrometry. Pharmacokinetic parameter estimates (maximum concentration, time to reach maximum concentration, area under the concentration-time curve, apparent oral clearance [CL/F]) were computed by standard noncompartmental methods. Stepwise multiple regression analysis was used to identify the hepatic or intestinal variables that predicted variation in saquinavir pharmacokinetic measures.
  
  
  RESULTS
  Baseline saquinavir CL/F was not correlated with liver CYP3A4 activity (the erythromycin breath test result), intestinal CYP3A4 content, or intestinal P-gp content (r(2) = 0.08, 0.08, and 0.007, respectively; P > .2). MDR1 genotype and lymphocyte P-gp content were also not predictive. Among the 6 subjects expressing intestinal CYP3A5, the mean saquinavir CL/F was almost twice as high as for the 14 nonexpressors (36.7 L/h [95% confidence interval (CI), 18.7-54.6 L/h] and 19.3 L/h [95% CI, 11.2-27.4 L/h], respectively; P = .03). However, among the 6 CYP3A5 expressors, there was an unexpected negative correlation between CL/F and intestinal CYP3A5 content (r(2) = 0.58, P = .05). Seville orange juice decreased the mean CL/F in all 20 subjects from 24.5 L/h (95% CI, 16.7-32.3 L/h) to 14.7 L/h (95% CI, 8.4-20.6 L/h) (P = .05). The effect size did not appear to be influenced by CYP3A5 expression.
  
  
  CONCLUSIONS
  The CYP3A5*1 genotype is associated with increased saquinavir CL/F. This does not appear to reflect intestinal CYP3A5 expression and presumably reflects the contribution of hepatic CYP3A5. The interaction with Seville orange juice in subjects not expressing CYP3A5 supports a role for intestinal CYP3A4. However, the modest nature of the interaction, combined with the inability to detect a correlation between CL/F and CYP3A4 enterocyte content, supports our recent in vitro work suggesting a smaller contribution of intestinal CYP3A4 than has been assumed.
authors.affiliation: General Clinical Research Center, School of Pharmacy, and Department of Biostatistics, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Stéphane J
authors.initials: SJ
authors.lastname: Mouly
authors.forename: Chris
authors.initials: C
authors.lastname: Matheny
authors.forename: Mary F
authors.initials: MF
authors.lastname: Paine
authors.forename: Glenn
authors.initials: G
authors.lastname: Smith
authors.forename: Jatinder
authors.initials: J
authors.lastname: Lamba
authors.forename: Vishal
authors.initials: V
authors.lastname: Lamba
authors.forename: Susan N
authors.initials: SN
authors.lastname: Pusek
authors.forename: Erin G
authors.initials: EG
authors.lastname: Schuetz
authors.forename: Paul W
authors.initials: PW
authors.lastname: Stewart
authors.forename: Paul B
authors.initials: PB
authors.lastname: Watkins
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000284:Administration, Oral; D000328:Adult; D019540:Area Under Curve; D001628:Beverages; D001682:Biological Availability; D002250:Carbon Radioisotopes; D002957:Citrus; D018592:Cross-Over Studies; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D020895:Enterocytes / Q000201:enzymology* / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005838:Genotype; D017320:HIV Protease Inhibitors / Q000008:administration & dosage / Q000493:pharmacokinetics; D006207:Half-Life; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000201:enzymology; D008297:Male; D008657:Metabolic Clearance Rate; D011110:Polymorphism, Genetic; D019258:Saquinavir / Q000008:administration & dosage / Q000097:blood / Q000493:pharmacokinetics*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002250:Carbon Radioisotopes; D017320:HIV Protease Inhibitors; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D019258:Saquinavir