Grapefruit juice and the Grapefruit juice and the intestinal barrier in man: intestinal barrier in man:

not pulp fictionnot pulp fiction

Paul B. WatkinsPaul B. Watkins

University of North CarolinaUniversity of North Carolina

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

December 2, 2003December 2, 2003

time

[drug]

Effect of Grapefruit juice on plasma 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

Med Watch Report (January 11, 1999)Med Watch Report (January 11, 1999)

 60 y/o man with diabetes mellitus, coronary heart disease,60 y/o man with diabetes mellitus, coronary heart disease, and chronic renal insufficiencyand chronic renal insufficiency  55 years treatment with lovastatin (40 mg bid), gemfibrozil years treatment with lovastatin (40 mg bid), gemfibrozil (600 mg bid) and others(600 mg bid) and others  Started drinking grapefruit juice daily (8 ounces) for two weeksStarted drinking grapefruit juice daily (8 ounces) for two weeks  Presented with rhabdomyolysis (CPK:44,860)Presented with rhabdomyolysis (CPK:44,860)  

First Pass MetabolismMetabolism by CYP3A4

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

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

 

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*

Effect of a single glass of grapefruit juice onEffect of a single glass of grapefruit juice onconcentration of CYP3A4 in human concentration of CYP3A4 in human

small intestinal biopsiessmall intestinal biopsies

P-glycoproteinP-glycoprotein

villinvillin

CYP3A4CYP3A4

Baseline 4 hoursBaseline 4 hours

FC’s in grapefruit juice reduce FC’s in grapefruit juice reduce enterocyte CYP3A4 activity in 3 waysenterocyte CYP3A4 activity in 3 ways

1). Reversible inhibition1). Reversible inhibition

2). Irreversible inactivation2). Irreversible inactivation

3). Actual loss of enzyme3). Actual loss of enzyme

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

• 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 Synthesis of CYP3A4 Effect of DHB on the Synthesis of CYP3A4 (Pulse (Pulse 3535S labeled Methionine)S labeled Methionine)

0 hr V

CYP3A4

0 hr D

0.5 V

0.5 D

1 hr V

1 hr D

2 hr V

2 hr D

Vehicle Rsyn = 2.0 pmol/hr/insert

Vehicle Rsyn = 2.0 pmol/hr/insert

DHBRsyn = 1.9 pmol/hr/insert

DHBRsyn = 1.9 pmol/hr/insert

0

50

100

150

0 1 2Time (h)

% c

on

tro

l

jfkd

jsfl

kd

jsd

Vehicle

DHB

Series3

Series4

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

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.

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

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

Unpublished dataUnpublished data

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 baseline.availability at baseline.

Effect of GFJ on CisaprideEffect of GFJ on Cisapride

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

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

AVERAGE PROFILE (n = 13 subjects)AVERAGE PROFILE (n = 13 subjects)

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

FELODIPINE PK (n = 13 subjects)FELODIPINE PK (n = 13 subjects)

MeasureMeasure(Ave. (Ave. ± ± SD)SD)

OJOJ GFJGFJFC-free FC-free

GFJGFJ

AUC (nM-h)AUC (nM-h) 75 75 ± 36± 36 149 149 ± 68± 68** 67 67 ± 33± 33

CCmaxmax (nM) (nM) 9.0 9.0 ± 3.1± 3.1 26 26 ± 11± 11** 8.9 8.9 ± 4.0± 4.0

TTmaxmax (h) (h) 2.8 2.8 ± 0.8± 0.8 2.7 2.7 ± 0.6± 0.6 2.7 2.7 ± 0.9± 0.9

Cl/F (L/h)Cl/F (L/h) 420 420 ± 160± 160 210 210 ± 100± 100** 500 500 ± 260± 260

z z (h(h-1-1)) 0.09 0.09 ±± 0.03 0.03 0.10 0.10 ± 0.03± 0.03 0.10 0.10 ± 0.03± 0.03*Significantly different from OJ and FC-free GFJ (p < 0.001, multiple pairwise comparisons with Bonferroni corrected level of significance).

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

ThanksThanksMary Paine, Ph.D.Mary Paine, Ph.D. 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. and Bill Stinson, Ph.D.Bill Widmer, Ph.D. and Bill Stinson, Ph.D.