1. The role of furocoumarins in grapefruit juice/drug interactions. Paul B. Watkins University of North Carolina Chapel Hill, N.C.

2. “Grapefruit Effect On Drug Levels Has Sweeter Side” By SYLVIA PAGÁN WESTPHAL November 27, 2007; Page D1 Wall Street Journal

3. King's doctor prescribed Lipitor, along with continued diet and exercise. King obeyed. His Lipitor dose was gradually increased to a high dose of 60 milligrams a day. After four months, he'd brought his LDL cholesterol down to 104. He'd also lost 36 pounds. Later, King headed to his winter home in Florida. With a grapefruit tree on his patio, he drank two to three daily glasses of fresh grapefruit juice. But just two months after getting the good news about his cholesterol, King was in a Florida emergency room. His symptoms: muscle pain that had started suddenly, fatigue, and high fever. King was diagnosed with rhabdomyolysis, a severe muscle reaction that can cause death. Web MD - 2005

4. time [drug] Interpatient variation in pharmacokinetics

5. First Pass Metabolism

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

9. time [drug] Effect of Grapefruit juice on serum levels of a some drugs

10. 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

12. 8/30/02 email “….. our business outlook has been greatly challenged by the grapefruit - drug interaction and the way it is being communicated (often inaccurately) to the public… Jay Dravenstadt Manager R&D, Grapefruit Business Ocean Spray Cranberries Lakeville-Middleboro, MA 02349

13. Hypothesis Grapefruit juice interactions are not clinically important because intestinal CYP3A4 is important because intestinal CYP3A4 is upregulated (induced) in people who regularly upregulated (induced) in people who regularly drink the juice. drink the juice.

14. Clinical Study 10 healthy adults were admitted to the GCRC and received one glass of grapefruit juice (8 oz) and received one glass of grapefruit juice (8 oz) with each meal for 7 days with each meal for 7 days Endoscopy with “pinch biopsies” performed before and after. before and after. Biopsy content of CYP3A4 protein and mRNA measured mRNA measured

15. GFJ REDUCES INTESTINAL CYP3A4 PROTEIN GFJ reduced CYP3A4 protein content in all subjects Lown et al. (1997) JCI 99:2545-53

16. Conclusions Hypothesis is completely wrong. GFJ makes CYP3A4 go away!

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

18. 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

19. 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

20. 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 Unpublished data

21. 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?

22. 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

23. 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 Unpublished data

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. 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.

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

28. gut lumen FEL into the body enterocyte FEL

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

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

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

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

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

35. Effect of SOJ on AUC of Saquinavir JPET 308:941-948, 2004

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

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

38. 0 Variation in enterocyte CYP3A4 activity and the oral disposition of some substrates

39. 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

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

41. “In Florida, Bioavailability Systems LLC, a small biotechnology company, claims to have purified the grapefruit compounds responsible for the boosting effect and has been able to improve the blood levels of an anti-HIV drug. "This is definitely a lemons to lemonade story," says James Harris, founder and chief scientific officer of the company. ” November 27, 2007; Page D1 Wall Street Journal

42. 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.

43. 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.

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

47. 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

48. Felodipine study design 18 subjects brought to GCRC18 subjects brought to GCRC Three-way randomized crossover design.Three-way randomized crossover design. 10 mg sustained-release tablet of felodipine (Plendil) given with OJ, GFJ, or FC-free GFJ.10 mg sustained-release tablet of felodipine (Plendil) given with OJ, GFJ, or FC-free GFJ. Blood samples (10-mL) collected at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 hours.Blood samples (10-mL) collected at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 hours.

49. Felodipine interaction study Time (hours) Felodipine (nM) n = 18 Am. J. of Clin. Nutr. 83(5):1097-105, 2006

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

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

52. “ One such compound called naringin affects the efficacy of the popular allergy drug Allegra by blocking these transporters. "Even a normal glass of juice will reduce the effects of Allegra by half," says Dr. Bailey, whose team made the discovery last year. "It's the tip of the iceberg," he adds. "Big pharma is very interested." ” November 27, 2007; Page D1 Wall Street Journal

53. Thanks Mary Paine, PhD. 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. Bill Stinson,Ph.D.