Grapefruit juice and the intestinal barrier in man: not pulp fiction

1. Grapefruit juice and the intestinal barrier in man: not pulp fiction Paul B. Watkins University of North Carolina Chapel Hill, N.C. December 2, 2003

2. [drug] time Effect of Grapefruit juice on plasma levels of a some drugs

4. Some drugs influenced by grapefruit juice Drug AUC increase felodipine ~ 3 fold cisapride ~ 1.4 fold cyclosporine~ 1.5 fold saquinavir ~ 2 fold terfenadine ~ 2.5 fold buspirone ~ 9 fold lovastatin/simvastatin ~ 10 fold

5. Med Watch Report (January 11, 1999) 60 y/o man with diabetes mellitus, coronary heart disease, and chronic renal insufficiency Ø5 years treatment with lovastatin (40 mg bid), gemfibrozil (600 mg bid) and others Started drinking grapefruit juice daily (8 ounces) for two weeks Presented with rhabdomyolysis (CPK:44,860)

6. First Pass Metabolism by CYP3A4

7. LOCATION OF INTESTINAL CYP3A4 Kolars et al. (1994) Pharmacogenetics 4:247-59

8. Screening HLPC fractions (Fraction C) for ability to inhibit CYP3A4 in human intestinal microsomes

9. Furocoumarins in Grapefruit Juice O H O O O H O O O O O O 6,7-dihydroxybergamottin (DHB) Bergamottin O H O O O H O H O O O O O O O O O O O O O O O O FC708 FC726

10. Basolateral medium insert apical medium culture dish Caco-2 cell monolayer Caco-2 Cells • Derived from a human colon adenocarcinoma • Upon differentiation resemble small intestinal enterocytes • In the presence of 1a,25-(OH)2-D3 express CYP3A4 • Schmiedlin-Ren et al. Mol Pharmacol 51: 741-754, 1997

11. FEL FEL FEL FEL FEL FEL FEL gut lumen enterocyte into the body

12. FEL FEL FEL FEL FEL FEL FEL* CYP3A4 Gut lumen enterocyte Into the body

13. FEL FEL FEL FC FC* FC* FC CYP3A4 Gut lumen enterocyte X Into the body

14. FEL FEL FEL FC FC* FC* FC Gut lumen enterocyte Into the body

15. Effect of a single glass of grapefruit juice on concentration of CYP3A4 in human small intestinal biopsies P-glycoprotein villin CYP3A4 Baseline 4 hours

16. FC’s in grapefruit juice reduce enterocyte CYP3A4 activity in 3 ways 1). Reversible inhibition 2). Irreversible inactivation 3). Actual loss of enzyme

17. Time course of the effect of DHB on CYP3A4 CYP3A4 2 hr 2 hr 4 hr 3 hr 4 hr 8 hr 0 hr 1 hr 1 hr 3 hr 8 hr 12 hr 12 hr 0.5 hr 0.5 hr 3A4 Standards DHB Vehicle

18. Is this decrease in CYP3A4 protein content due to decreased rate of synthesis or accelerated rate of degradation?

19. Pulse 35S 2 hours (methionine Free) 35S-Methionine 35S Chase 35S Medium with 6X cold Methionine 0-48 hours Degradation 35S Pulse Chase Culture Medium Protein Synthesis

20. CYP3A4 Vehicle Rsyn = 2.0 pmol/hr/insert DHB Rsyn = 1.9 pmol/hr/insert 0.5 D 0.5 V 0 hr V 1 hr V 2 hr V 1 hr D 0 hr D 2 hr D Effect of DHB on the Synthesis of CYP3A4 (Pulse 35S labeled Methionine)

21. Vehicle kdeg = 0.048h-1 t1/2 = 14.4h DHB kdeg = 0.21h-1 t1/2 = 3.1h 4 12 0.5 8 1 2 Effect of DHB on the Degradation of CYP3A4 (Pulse Chase 35S labeled Met/Cys) CYP3A4 0 4 12 24 48 0.5 8 1 2 DHB Vehicle

22. Summary • DHB accelerates the rate of CYP3A4 degradation while having no effect on the rate of its synthesis • This results in a fall in CYP3A4 half life from 14h to 3 h.

23. Modeling single administration GFJ effect for dose and time course Takanaga, et al, Br. J. Clin Pharmacol. 49,49, 2000.

24. Ub Ub Ubiquitination X Lactocystin Peptides Ubiquitin- Proteasome Pathway DDEP P450 Inactivation DDEP Proteasome

25. Physiologic DDEP inactivated DHB inactivated Ub Ubiquitinating Enzymes Ub CYP3A4 Inactivation Ubiquitination Peptides Summary Proteasome

26. Where is this research headed? 1). Development of new tools for human research 2). Improvements in oral drug delivery 3). New grapefruit juice

28. Saquinavir 1). Most widely used HIV protease inhibitor 2). Oral availability 4-14% 3). Very rapid metabolism by CYP3A4

29. Effect of SOJ on AUC of Saquinavir Unpublished data

30. Where is this research headed? 1). Development of new tools for human research 2). Improvements oral drug delivery 3). New grapefruit juice

31. Range of variation in enterocyte CYP3A4 content in adults 10 0

32. Variation in enterocyte CYP3A4 activity and the oral disposition of some substrates 10 0

33. Conclusion Grapefruit juice / drug interactions are of minimal importance because dramatic increases in oral availability only occur in those patients who have very low oral availability at baseline.

34. Effect of GFJ on Cisapride Gross et al, CPT 65:395, 1999

35. Reasons why grapefruit juice/drug interactions shouldn’t be very important: 1). Susceptible drugs must have excellent safety profile despite large interpatient variation in exposure. 2). People with very low intestinal CYP3A4 activity will be encountered in clinical trials.

36. Situations where grapefruit juice/drug interactions may rarely become clinically significant: 1). Patient is requiring higher than usual dose of “susceptible” drug and begins drinking juice for the first time. 2). Patient has severe liver disease. 3). Patient has a peculiar susceptibility to an adverse effect.

37. Where is this research headed? 1). Development of new tools for human research 2). Improvements oral drug delivery 3). New grapefruit juice

40. Elimination of Furanocoumarins from GFJ Retentate Juice Ultrafilration Ethyl Acetate Serum Pectin + Cellulose Furanocoumarins + Flavonoids Debitter Debittered Serum Absorbed Conc. + EtOH + Flash Chromatography Etute + EtOH +Flash Chromatography Flavonoids FC 6,7-DHB Flavonoids Clinical Test Juice Commercial FCF Flavor Package

41. AVERAGE PROFILE (n = 13 subjects) Felodipine (nM) Time (hours)

42. FELODIPINE PK (n = 13 subjects) *Significantly different from OJ and FC-free GFJ (p < 0.001, multiple pairwise comparisons with Bonferroni corrected level of significance).

43. Conclusion 1). It is possible to remove major FCs from grapefruit juice. 2). This may not remove all GFJ / drug interactions.

44. Fexo CsA Pgp OATP CYP3A4 gut lumen enterocyte into the body

45. Thanks Florida Dept of Citrus Bill Widmer, Ph.D. and Bill Stinson, Ph.D. Mary Paine, Ph.D. Shefali Malhotra Anne Criss Susan Pusek Stephane Mouly National Institutes of Health General Medical Sciences R37-38149 General Clinical Research Centers (NCRR).