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. time [drug] 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 and chronic renal insufficiency  5 years treatment with lovastatin (40 mg bid), gemfibrozil (600 mg bid) and others (600 mg bid) and others Started drinking grapefruit juice daily (8 ounces) for two weeks Presented with rhabdomyolysis (CPK:44,860)

6. Metabolism First Pass Metabolism by CYP3A4

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

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

9. Furocoumarins in Grapefruit Juice O O O O Bergamottin O O O O OH OH 6,7-dihydroxybergamottin (DHB) O O O O OH O O O O OH O FC708FC726 O O O O OH O O O O O

10. Caco-2 Cells Derived from a human colon adenocarcinoma Derived from a human colon adenocarcinoma Upon differentiation resemble small intestinal enterocytes Upon differentiation resemble small intestinal enterocytes In the presence of 1 ,25-(OH) 2 -D 3 express CYP3A4 In the presence of 1 ,25-(OH) 2 -D 3 express CYP3A4 Schmiedlin-Ren et al. Mol Pharmacol 51: 741-754, 1997 Schmiedlin-Ren et al. Mol Pharmacol 51: 741-754, 1997 culture dish insert Basolateral medium apical medium Caco-2 cell monolayer

11. gut lumen FEL into the body enterocyte FEL

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

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

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

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 0 hr 0.5 hr 1 hr 2 hr3 hr4 hr8 hr 12 hr 0.5 hr 1 hr 2 hr 3 hr 4 hr 8 hr 12 hr CYP3A4 3A4 Standards Vehicle DHB

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

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

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

21. Effect of DHB on the Degradation of CYP3A4 (Pulse Chase 35 S labeled Met/Cys) CYP3A4 0 0.5 12 4 8 1224 48 0.5 12 4 8 12 Vehicle DHB Vehicle k deg = 0.048h -1 t 1/2 = 14.4h Vehicle k deg = 0.048h -1 t 1/2 = 14.4h DHB k deg = 0.21h -1 t 1/2 = 3.1h DHB k deg = 0.21h -1 t 1/2 = 3.1h

22. Summary DHB accelerates the rate of CYP3A4 degradation while having no effect on the rate of its synthesisDHB 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.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. Ubiquitin- Proteasome Pathway P450 Inactivation Peptides Proteasome Ubiquitination Ub DDEP Lactocystin X

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

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 10 0

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

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

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

42. FELODIPINE PK (n = 13 subjects) Measure (Ave. ± SD) OJGFJ FC-free GFJ AUC (nM-h) 75 ± 36 149 ± 68 * 67 ± 33 C max (nM) 9.0 ± 3.1 26 ± 11 * 8.9 ± 4.0 T max (h) 2.8 ± 0.8 2.7 ± 0.6 2.7 ± 0.9 Cl/F (L/h) 420 ± 160 210 ± 100 * 500 ± 260 z (h -1 ) z (h -1 ) 0.09 ± 0.03 0.10 ± 0.03 * 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. gut lumen into the body enterocyte Pgp CYP3A4 OATP CsA Fexo

45. Thanks 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). Florida Dept of Citrus Bill Widmer, Ph.D. and Bill Stinson, Ph.D.