The role of furocoumarins in grapefruit juice/drug interactions.

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

2. “Grapefruit EffectOn Drug LevelsHas Sweeter Side” By SYLVIA PAGÁN WESTPHALNovember 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. [drug] time Interpatient variation in pharmacokinetics

5. First Pass Metabolism

6. First Pass Metabolism

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

9. [drug] time 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 upregulated (induced) in people who regularly drink the juice.

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

15. GFJ REDUCESINTESTINAL 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 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

19. 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 Basolateral medium insert apical medium culture dish Caco-2 cell monolayer

20. 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 Unpublished data

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

22. 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

23. 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 Unpublished data

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. 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.

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

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

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

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

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

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 0

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

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 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

48. Felodipine study design • 18 subjects brought to GCRC • Three-way randomized crossover design. • 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.

49. Felodipine interaction study Felodipine (nM) Time (hours) 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.