The role of furocoumarins in grapefruit juice/drug interactions.

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The role of furocoumarins in grapefruit
juice/drug interactions.

• Paul B. Watkins
• University of North Carolina
• Chapel Hill, N.C.

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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
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Interpatient variation in pharmacokinetics

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First Pass Metabolism
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First Pass Metabolism
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First Pass Metabolism
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LOCATION OF INTESTINAL CYP3A4
Kolars et al. (1994) Pharmacogenetics 4247-59
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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
9

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Effect of Grapefruit juice on serum levels of a
some drugs

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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

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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 RD, Grapefruit Business
Ocean Spray Cranberries Lakeville-Middleboro,
MA 02349

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• 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
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GFJ REDUCESINTESTINAL CYP3A4 PROTEIN
GFJ reduced CYP3A4 protein content in all
subjects
Lown et al. (1997) JCI 992545-53
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• Conclusions

Hypothesis is completely wrong. GFJ makes CYP3A4
go away!
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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
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Screening HLPC fractions (Fraction C) for ability
to inhibit CYP3A4 in human intestinal microsomes
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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
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Time course of the effect of DHB on CYP3A4
CYP3A4
0 hr
1 hr
2 hr
3 hr
4 hr
8 hr
1 hr
2 hr
3 hr
4 hr
8 hr
12 hr
12 hr
0.5 hr
0.5 hr
3A4 Standards
DHB
Vehicle
Unpublished data
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• Is this decrease in CYP3A4 protein content due to
  decreased rate of synthesis or accelerated rate
  of degradation?

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Pulse Chase
Culture Medium
Protein Synthesis
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Effect of DHB on the Degradation of CYP3A4
(Pulse Chase 35S labeled Met/Cys)
CYP3A4
0
0.5
1
2
4
8
12
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DHB
Vehicle
Unpublished data
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Ubiquitin- Proteasome Pathway
DDEP
P450 Inactivation
DDEP
Proteasome
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Summary
Proteasome
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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.

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Modeling single administration GFJ effect for
dose and time course
Takanaga, et al, Br. J. Clin Pharmacol.
49,49,2000.
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gut lumen  
 
enterocyte  
into the body  
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Gut lumen  
 
enterocyte  
Into the body  
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Gut lumen  
enterocyte  
 
X
Into the body  
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Gut lumen  
enterocyte  
 
Into the body  
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Where is this research headed?

• 1). Development of new tools for human research

2). Improvements in oral drug delivery
3). New grapefruit juice
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Saquinavir

• 1). Most widely used HIV protease inhibitor

2). Oral availability 4-14
3). Very rapid metabolism by CYP3A4
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Effect of SOJ on AUC of Saquinavir
JPET 308941-948, 2004
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Where is this research headed?

• 1). Development of new tools for human research

2). Improvements oral drug delivery
3). New grapefruit juice
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Range of variation in enterocyte CYP3A4 content
in adults

• 10

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

• 10

0
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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

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Effect of GFJ on Cisapride

Gross et al, CPT 65395,1999
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Reasons why grapefruit juice/drug interactions
shouldnt 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.
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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.
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Where is this research headed?

• 1). Development of new tools for human research

2). Improvements oral drug delivery
3). New grapefruit juice
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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
FC
Flavonoids
6,7-DHB
Flavonoids
Clinical Test Juice
Commercial FCF Flavor Package
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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.

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

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

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gut lumen  
 
enterocyte  
into the body  
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Thanks

• 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 R37-38149 General Clinical Research
Centers (NCRR).