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

2. 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

3. Interpatient variation in pharmacokinetics
time
[drug]

4. First Pass Metabolism

5. First Pass Metabolism

6. First Pass Metabolism

7. LOCATION OF INTESTINAL CYP3A4
This is a cross-section of actual human small intestinal tissue obtained from a duodenal pinch biopsy.
To orient you, this is the lumenal (or apical) side of the gut, and down here is the serosal (or basolateral or blood) side of the gut.
To remind you, a single layer of epithelial cells lines the entire villus, with the most mature cells residing at the tip, and immature cells residing in the crypts.
This tissue was examined by histochemistry using a polyclonal antibody to CYP3A4, with positive reaction indicated in red. As you can see, there is much staining in the mature cells, none in the goblet cells, and none in the crypt cells.
And so, at least for CYP3A4, the enzyme is conveniently located such that, for substrates that are absorbed transcellulary, the opportunity exists for first-pass metabolism.
Kolars et al. (1994) Pharmacogenetics 4:247-59

8. 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: , 1997
Caco-2 cells were used as a model for small intestine. Caco-2 cells are derived from human colon cancer cells and are usually grown as monolayers in a transwell such that there is an apical side and a basolateral side to the cells. DHB was added to the apical side to mimic grapefruit ingestion.
Upon differentiation resemble small intestinal enterocytes. Lee ren in our lab has shown a Vitamin D analog induces CYP3a4 expression. Therefore it is a good in vitro model to study intestinal CYP3A4
Basolateral medium
insert
apical medium
culture dish
Caco-2 cell monolayer

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

11. 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

13. 8/30/02
“….. 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

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

15. 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

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

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

18. 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: , 1997
Caco-2 cells were used as a model for small intestine. Caco-2 cells are derived from human colon cancer cells and are usually grown as monolayers in a transwell such that there is an apical side and a basolateral side to the cells. DHB was added to the apical side to mimic grapefruit ingestion.
Upon differentiation resemble small intestinal enterocytes. Lee ren in our lab has shown a Vitamin D analog induces CYP3a4 expression. Therefore it is a good in vitro model to study intestinal CYP3A4
Basolateral medium
insert
apical medium
culture dish
Caco-2 cell monolayer

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

20. Furocoumarins in Grapefruit JuiceH O O O H O O O O O O
6,7-dihydroxybergamottin
(DHB)
Furocoumarins have been identified as the active ingredients present in the juice. There are four major furocoumarins in the juice responsible for inhibition of CYP3A4. Data in literature suggests that DHB, the most abundant furanocoumarin is also a single major active ingredient in the juice, and was therfore used as a representative active ingredient of the juice to evaluate its effect on CYP3A4 protein.
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

21. Time course of the effect of DHB on CYP3A4
0 hr
0.5 hr
1 hr
2 hr
3 hr
4 hr
8 hr
12 hr
0.5 hr
1 hr
2 hr
3 hr
4 hr
8 hr
12 hr
3A4 Standards
Vehicle
DHB
First, we looked at the time course of the effect of DHB on CYP3A4. Cells were either treated with vehicle as control or with 30 uM DHB, the average concentration present in the juice. There was no time dependent effect of vehicle upto 12 hours. But DHB resulted in a time dependent loss of CYP3A4 protein and a new steady state level was achieved by 12 hours. The next question was, is this time dependent decrease in protein due to reduced rate of synthesis or accelerated degradation. To answer this question, rate of synthesis and degradation of 3A4 were measured using Pulse chase.
Unpublished data

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

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

24. Effect of DHB on the Degradation of CYP3A4 (Pulse Chase 35S labeled Met/Cys)
0.5 1 2 4 8 12 24 48
0.5 1 2 4 8 12
Vehicle
DHB
To measure the rate of degradation, cells were labeled for 2 hrs with S-35 methionine and then chased with cold methionine for upto 48 hours. There was a relatively slow degradation of 3A4 in the absence of DHB. Even at 48h, the labeled enzyme was clearly detectable. In contrast, with DHB treatment 3A4 rapidly degraded such that it was barely detectable at 12 h. The results from 4 such experiments are shown graphically below. Using these results, The half life of CYP3A4 was calculated to be about 14.4h in vehicle treated cells and 3.1 hrs in the presence of DHB suggesting that DHB accelerated the rate of degradation of CYP3A4.
Vehicle
kdeg = 0.048h-1
t1/2 = 14.4h
DHB
kdeg = 0.21h-1
t1/2 = 3.1h
Unpublished data

25. Ubiquitin- Proteasome Pathway
DDEP
Ubiquitination Ub
P450
Inactivation
DDEP
In the ubiquitin-proteasome pathway. After the protein is modified, it is identified by ubiquitinating enzymes that add one or more ubiquitin molecules on to the protein. This ubiquitinated protein is then targetted to the proteasome for degradation to small peptides. In order to determine if the proteasome is involved, we can inhibit the proteasome using a proteasome inhibitor such as lactacystin.
Lactocystin X
Peptides
Proteasome

26. Summary Physiologic DHB inactivated DDEP inactivated
Ubiquitinating Enzymes
Ubiquitination Ub
Physiologic
DDEP inactivated
DHB inactivated
CYP3A4 Inactivation
In summary, we propose that the physiological turnover of CYP3A4 and DDEP inactivated CYP3A4 undergo ubiquitination before being targeted to the proteasome for degradation. But DHB inactivated CYP3A4 appears to bypass the ubiquitin and is directly targeted to the proteasome for degradation.
Peptides
Proteasome

27. 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.

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

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

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

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

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

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

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

36. Effect of SOJ on AUC of Saquinavir
JPET 308: , 2004

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

38. Range of variation in enterocyte CYP3A4 content in adults10

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

40. 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

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

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
Retentate
Ultrafilration
Ethyl
Acetate
Serum
Pectin +
Cellulose
Furanocoumarins +
Flavonoids
Debitter
Absorbed
Debittered
Serum
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.
Our study design entailed enrolling 18 subjects to participate in a three-way randomized crossover, the number of subjects of which was based on statistical methods that accounted for 3 treatments, 3 periods, and 6 sequences of administration.
We gave each subject a 10 mg sustained-release tablet of felodipine with either OJ, GFJ, or FC-free GFJ
And collected blood samples over 24 hours.
And because felodipine is a calcium channel blocker, we also monitored various vital signs (specifically, blood pressure, heart rate, and respiratory rate) before and every 2-4 hours following ingestion of the drug with juice.
We allowed at least a one-week washout between admissions,
And asked each subject to abstain from alcohol, caffeine, and of course GFJ and grapefruit products for the duration of the study.

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

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

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

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