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

The role of furocoumarins in The role of furocoumarins in grapefruit juice/drug grapefruit juice/drug

interactions. interactions.

Paul B. WatkinsPaul B. Watkins

University of North CarolinaUniversity of North Carolina

Chapel Hill, N.C.Chapel Hill, N.C.

“Grapefruit EffectOn Drug Levels

Has Sweeter Side”

By SYLVIA PAGÁN WESTPHALNovember 27, 2007; Page D1

Wall Street Journal

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

time

[drug]

Interpatient variation in pharmacokinetics

First Pass MetabolismFirst Pass Metabolism

First Pass MetabolismFirst Pass Metabolism

Kolars et al. (1994) Pharmacogenetics 4:247-59Kolars et al. (1994) Pharmacogenetics 4:247-59

LOCATION OF INTESTINAL LOCATION OF INTESTINAL CYP3A4CYP3A4

LOCATION OF INTESTINAL LOCATION OF INTESTINAL CYP3A4CYP3A4

time

[drug]

Effect of Grapefruit juice on serum levels of a some drugs

Some drugs influenced by Some drugs influenced by grapefruit juicegrapefruit juice

Drug Drug AUC increase AUC increase

felodipinefelodipine ~ 3 fold~ 3 fold

cisapridecisapride ~ 1.4 fold~ 1.4 fold

cyclosporinecyclosporine ~ 1.5 fold~ 1.5 fold

saquinavirsaquinavir ~ 2 fold~ 2 foldterfenadineterfenadine ~ 2.5 fold ~ 2.5 fold buspironebuspirone ~ 9 fold ~ 9 fold

lovastatin/simvastatinlovastatin/simvastatin ~ 10 fold~ 10 fold

8/30/02 email 8/30/02 email

“…“….. our business outlook has been greatly challenged by the .. our business outlook has been greatly challenged by the

grapefruit - drug interaction and the way it is being communicated grapefruit - drug interaction and the way it is being communicated

(often inaccurately) to the public…(often inaccurately) to the public…

Jay Dravenstadt Jay Dravenstadt Manager R&D, Grapefruit Business Manager R&D, Grapefruit Business Ocean Spray Cranberries Ocean Spray Cranberries Lakeville-Middleboro, MA 02349Lakeville-Middleboro, MA 02349

HypothesisHypothesis

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

Clinical StudyClinical Study

10 healthy adults were admitted to the GCRC 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 dayswith each meal for 7 days

Endoscopy with “pinch biopsies” performed Endoscopy with “pinch biopsies” performed before and after.before and after.

Biopsy content of CYP3A4 protein and Biopsy content of CYP3A4 protein and mRNA measured mRNA measured

GFJ REDUCESGFJ REDUCESINTESTINAL CYP3A4 INTESTINAL CYP3A4

PROTEINPROTEIN

GFJ REDUCESGFJ REDUCESINTESTINAL CYP3A4 INTESTINAL CYP3A4

PROTEINPROTEIN

GFJ reduced CYP3A4 protein content in all subjects

Lown et al. (1997) JCI 99:2545-53

ConclusionsConclusions

Hypothesis is completely wrong.Hypothesis is completely wrong.

GFJ makes CYP3A4 go away!GFJ makes CYP3A4 go away!

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

Furocoumarins in Grapefruit JuiceFurocoumarins in Grapefruit Juice

O O O

O

Bergamottin

O O O

OOH

OH

6,7-dihydroxybergamottin(DHB)

O

O

O

O

OH

O O O

OOH

O

FC708 FC726

O

O

O

O

OH

O O O

O

O

Caco-2 CellsCaco-2 Cells Derived from a human colon adenocarcinomaDerived from a human colon adenocarcinoma

Upon differentiation resemble small intestinal enterocytesUpon differentiation resemble small intestinal enterocytes

In the presence of 1In the presence of 1,25-(OH),25-(OH)22-D-D3 3 express CYP3A4 express CYP3A4 Schmiedlin-Ren et al. Mol PharmacolSchmiedlin-Ren et al. Mol Pharmacol 51: 741-754, 1997 51: 741-754, 1997

culture dish

insert Basolateral medium

apical medium

Caco-2 cell monolayer

0

40

80

120

0 4 8 12Time (hours)

% C

on

tro

l d

jfh

jfh

d

Vehicle

DHB Treated

Time course of the effect of DHB on CYP3A4Time course of the effect of DHB on CYP3A4

0 hr0.5 hr1 hr2 hr3 hr4 hr

8 hr12 hr0.5 hr1 hr2 hr

3 hr4 hr8 hr

12 hr

CYP3A4

3A4 Standards

Vehicle DHB

Unpublished data

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

Pulse ChasePulse Chase

Protein Synthesis 2 hours (methionine Free)

35S-Methionine

35S

35S

PulsePulse

35S

35S

Degradation 0-48 hoursMedium with 6X cold Methionine

ChaseChase

Culture Medium

Effect of DHB on the Degradation of CYP3A4 Effect of DHB on the Degradation of CYP3A4 (Pulse Chase (Pulse Chase 3535S labeled Met/Cys)S labeled Met/Cys)

CYP3A4

0 0.5 1 2 4 8 12 24 48 0.5 1 2 4 8 12

Vehicle DHB

Vehicle kdeg = 0.048h-1

t1/2 = 14.4h

Vehicle kdeg = 0.048h-1

t1/2 = 14.4h

DHBkdeg = 0.21h-1

t1/2 = 3.1h

DHBkdeg = 0.21h-1

t1/2 = 3.1h

1

10

100

0 8 16 24 32 40 48Time (hours)

% 3

A4

Rem

ain

ing

Vehicle

DHB Treated

Unpublished data

Ubiquitin- Proteasome Pathway

P450Inactivation

Peptides

Proteasome

Ubiquitination

Ub

UbDDEP

DDEP

Lactocystin

X

SummarySummary

CYP3A4 Inactivation

Peptides

Proteasome

Ubiquitinating Enzymes

Ubiquitination

Ub

Ub

PhysiologicDDEP inactivatedDHB inactivated

SummarySummary

• DHB accelerates the rate of CYP3A4 DHB accelerates the rate of CYP3A4 degradation while having no effect on the degradation while having no effect on the rate of its synthesisrate of its synthesis

• This results in a fall in CYP3A4 half life This results in a fall in CYP3A4 half life from 14h to 3 h.from 14h to 3 h.

Modeling single administration GFJ Modeling single administration GFJ effect for dose and time courseeffect for dose and time course

Takanaga, et al, Br. J. Clin Pharmacol. 49,49,2000.Takanaga, et al, Br. J. Clin Pharmacol. 49,49,2000.

gut lumen 

FEL

into the body 

enterocyte 

FEL

FEL

FEL

FELFEL

FEL

Gut lumen 

FEL

FELFEL*

CYP3A4

FEL

Into the body 

FEL

enterocyte 

FEL

FEL

Gut lumen 

CYP3A4

Into the body 

FEL

FEL

enterocyte  FC

FC

FC*FEL

FC*X

Gut lumen 

FEL

Into the body 

FEL

enterocyte  FC

FC

FC*FEL

FC*

Where is this research headed?Where is this research headed?

1). 1). Development of new tools for human Development of new tools for human researchresearch

2). Improvements in oral drug delivery2). Improvements in oral drug delivery

3). New grapefruit juice 3). New grapefruit juice

SaquinavirSaquinavir

1). 1). Most widely used HIV protease Most widely used HIV protease inhibitorinhibitor

2). Oral availability 4-14%2). Oral availability 4-14%

3). Very rapid metabolism by CYP3A4 3). Very rapid metabolism by CYP3A4

Effect of SOJ on AUC of SaquinavirEffect of SOJ on AUC of Saquinavir

JPET 308:941-948, 2004JPET 308:941-948, 2004

Effect of Seville orange juice on saquinavir AUC following a single dose of 600 mg (mesylate

formulation)

0

50

100

150

200

250

300

350

400

450

Water SOJ

AUC

(ng.h

/mL)

Where is this research headed?Where is this research headed?

1). 1). Development of new tools for human Development of new tools for human researchresearch

2). Improvements oral drug delivery2). Improvements oral drug delivery

3). New grapefruit juice3). New grapefruit juice

Range of variation in enterocyte Range of variation in enterocyte CYP3A4 content in adultsCYP3A4 content in adults

1010

0

1010

0

Variation in enterocyte CYP3A4 activityVariation in enterocyte CYP3A4 activityand the oral disposition of some substratesand the oral disposition of some substrates

ConclusionConclusion

Grapefruit juice / drug interactions are of Grapefruit juice / drug interactions are of minimal importance because dramatic minimal importance because dramatic increases in oral availability only occur in increases in oral availability only occur in those patients who have very low oral those patients who have very low oral availability at baselineavailability at baseline

Effect of GFJ on CisaprideEffect of GFJ on Cisapride

Gross et al, CPT 65:395,1999Gross et al, CPT 65:395,1999

“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 D1Wall Street Journal

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.

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.

Where is this research headed?Where is this research headed?

1). 1). Development of new tools for human Development of new tools for human researchresearch

2). Improvements oral drug delivery2). Improvements oral drug delivery

3). New grapefruit juice 3). New grapefruit juice

Elimination of Furanocoumarins from GFJ

Juice

FC

Serum

Absorbed DebitteredSerum

Retentate

Flavonoids

Pectin+

Cellulose

Furanocoumarins+

Flavonoids

6,7-DHB

Ultrafilration

Debitter

EthylAcetate

Etute+

EtOH +FlashChromatography

Conc.+

EtOH + FlashChromatography

Clinical Test Juice

Flavonoids

Commercial FCFFlavor Package

Felodipine study designFelodipine 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 10 mg sustained-release tablet of felodipine (Plendil) given with OJ, GFJ, or felodipine (Plendil) given with OJ, GFJ, or FC-free GFJ.FC-free GFJ.

• Blood samples (10-mL) collected at 0, Blood samples (10-mL) collected at 0, 0.5, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 hours.1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 hours.

Felodipine interaction studyFelodipine interaction study

Time (hours)

Felo

dip

ine (

nM

)

0

10

20

30

40

0 6 12 18 24

OJ

0

10

20

30

40

0 6 12 18 24

OJ

GFJ

0

10

20

30

40

0 6 12 18 24

OJ

GFJ

FC-free GFJ

n = 18Am. J. of Clin. Nutr. 83(5):1097-105, 2006

ConclusionConclusion

1). It is possible to remove major FCs from 1). It is possible to remove major FCs from grapefruit juice.grapefruit juice.

2). This may not remove all GFJ / drug 2). This may not remove all GFJ / drug interactions.interactions.

gut lumen 

into the body 

enterocyte 

Pgp

CYP3A4

OATP

CsA Fexo

“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 D1Wall Street Journal

ThanksThanksMary Paine, PhD.Mary Paine, PhD. Shefali Malhotra Shefali MalhotraAnne Criss Anne Criss Susan Pusek Susan PusekStephane MoulyStephane Mouly

National Institutes of HealthNational Institutes of Health General Medical Sciences R37-38149General Medical Sciences R37-38149General Clinical Research Centers (NCRR).General Clinical Research Centers (NCRR).

Florida Dept of CitrusFlorida Dept of CitrusBill Widmer, Ph.D. Bill Stinson,Ph.D.Bill Widmer, Ph.D. Bill Stinson,Ph.D.