Transcript No Slide Title

Grapefruit juice and the
intestinal barrier in man:
not pulp fiction
Paul B. Watkins
University of North Carolina
Chapel Hill, N.C.
December 2, 2003
Effect of Grapefruit juice on plasma levels of a some drugs
[drug]
time
Some drugs influenced by
grapefruit juice
Drug
AUC increase
felodipine
~ 3 fold
cisapride
cyclosporine
~ 1.4 fold
~ 1.5 fold
saquinavir
terfenadine
buspirone
~ 2 fold
~ 2.5 fold
~ 9 fold
lovastatin/simvastatin
~ 10 fold
Med Watch Report (January 11, 1999)
60 y/o man with diabetes mellitus, coronary heart disease,
and chronic renal insufficiency
5 years treatment with lovastatin (40 mg bid), gemfibrozil
(600 mg bid) and others
Started drinking grapefruit juice daily (8 ounces) for two weeks
Presented with rhabdomyolysis (CPK:44,860)
First Pass Metabolism by CYP3A4
LOCATION OF INTESTINAL
CYP3A4
Kolars et al. (1994) Pharmacogenetics 4:247-59
Screening HLPC fractions (Fraction C) for ability
to inhibit CYP3A4 in human intestinal microsomes
Furocoumarins in Grapefruit Juice
OH
O
O
OH
O
O
O
O
O
O
6,7-dihydroxybergamottin
(DHB)
Bergamottin
OH
O
O
O
O
O
O
OH
O
O
O
O
O
O
O
O
O
O
FC708
O
O
FC726
OH
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
culture dish
apical medium
Caco-2 cell monolayer
FEL
FEL
FEL
gut lumen
FEL
enterocyte
FEL
FEL
FEL
into the body
FEL
FEL
FEL
Gut lumen
FEL
FEL*
enterocyte
FEL
CYP3A4
FEL
Into the body
FEL
FC*
FEL
Gut lumen
enterocyte
FC
FC*
FC
X
CYP3A4
FEL
Into the body
FEL
FC*
FEL
Gut lumen
enterocyte
FC
FC*
FC
FEL
Into the body
Effect of a single glass of grapefruit juice on
concentration of CYP3A4 in human
small intestinal biopsies
P-glycoprotein
villin
CYP3A4
Baseline
4 hours
FC’s in grapefruit juice reduce
enterocyte CYP3A4 activity in 3 ways
1). Reversible inhibition
2). Irreversible inactivation
3). Actual loss of enzyme
Time course of the effect of DHB on CYP3A4
CYP3A4
DHB
Vehicle
% Control djfhjfhd
120
Vehicle
DHB Treated
80
40
0
0
4
8
Tim e (hours)
12
• Is this decrease in CYP3A4 protein content
due to decreased rate of synthesis or
accelerated rate of degradation?
Pulse Chase
Pulse
35S
Protein Synthesis
Culture Medium
(methionine Free)
2 hours
35S-Methionine
35S
Chase
35S
Medium with 6X cold Methionine
35S
Degradation
0-48 hours
Effect of DHB on the Synthesis of CYP3A4
(Pulse 35S labeled Methionine)
CYP3A4
150
% control
jfkdjsflkdjsd
Vehicle
DHB
100
Series3
Series4
50
0
0
Vehicle
Rsyn = 2.0 pmol/hr/insert
1
Time (h)
2
DHB
Rsyn = 1.9 pmol/hr/insert
Effect of DHB on the Degradation of CYP3A4
(Pulse Chase 35S labeled Met/Cys)
CYP3A4
0
0.5
1
2
4
8
12
24
48
0.5
1
2
4
8
DHB
Vehicle
100
% 3A4 Remaining
12
Vehicle
DHB Treated
10
1
0
8
16
Vehicle
kdeg = 0.048h-1
t1/2 = 14.4h
24
Time (hours)
32
40
48
DHB
kdeg = 0.21h-1
t1/2 = 3.1h
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.
Modeling single administration GFJ
effect for dose and time course
Takanaga, et al, Br. J. Clin Pharmacol. 49,49, 2000.
Ubiquitin- Proteasome Pathway
DDEP
Ub
Ub
P450
Inactivation
DDEP
Ubiquitination
Lactocystin
X
Proteasome
Peptides
Summary
Physiologic
DHB inactivated
DDEP inactivated
Ub
Ub
CYP3A4 Inactivation
Ubiquitination
Proteasome
Peptides
Where is this research headed?
1). Development of new tools for human
research
2). Improvements in oral drug delivery
3). New grapefruit juice
Saquinavir
1). Most widely used HIV protease
inhibitor
2). Oral availability 4-14%
3). Very rapid metabolism by CYP3A4
Effect of SOJ on AUC of Saquinavir
Effect of Seville orange juice on saquinavir AUC
following a single dose of 600 mg (mesylate
formulation)
450
400
350
AUC (ng.h/mL)
300
250
200
150
100
50
0
Water
SOJ
Unpublished data
Where is this research headed?
1). Development of new tools for human
research
2). Improvements oral drug delivery
3). New grapefruit juice
Range of variation in enterocyte
CYP3A4 content in adults
10
0
Variation in enterocyte CYP3A4 activity
and the oral disposition of some substrates
10
0
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.
Effect of GFJ on Cisapride
Gross 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?
1). Development of new tools for human
research
2). Improvements oral drug delivery
3). New grapefruit juice
Elimination of Furanocoumarins from GFJ
Retentate
Juice
Ultrafilration
Ethyl
Acetate
Serum
Pectin
+
Cellulose
Debitter
Absorbed
Etute
+
EtOH +Flash
Chromatography
6,7-DHB
Debittered
Serum
Furanocoumarins
+
Flavonoids
Conc.
+
EtOH + Flash
Chromatography
Flavonoids
Flavonoids
Commercial FCF
Flavor Package
Clinical Test Juice
FC
AVERAGE PROFILE (n = 13 subjects)
Felodipine (nM)
40
OJ
OJ
OJGFJ
GFJ
FC-free GFJ
30
20
10
0
0
6
12
Time (hours)
18
24
FELODIPINE PK (n = 13 subjects)
OJ
GFJ
FC-free
GFJ
AUC (nM-h)
75 ± 36
149 ± 68*
67 ± 33
Cmax (nM)
9.0 ± 3.1
26 ± 11*
8.9 ± 4.0
Tmax (h)
2.8 ± 0.8
2.7 ± 0.6
2.7 ± 0.9
Cl/F (L/h)
420 ± 160
210 ± 100*
500 ± 260
lz (h-1)
0.09 ± 0.03 0.10 ± 0.03 0.10 ± 0.03
Measure
(Ave. ± SD)
*Significantly
different from OJ and FC-free GFJ (p < 0.001, multiple pairwise comparisons with
Bonferroni corrected level of significance).
Conclusion
1). It is possible to remove major FCs from
grapefruit juice.
2). This may not remove all GFJ / drug
interactions.
CsA
Fexo
gut lumen
Pgp
OATP
enterocyte
CYP3A4
into the body
Thanks
Mary Paine, Ph.D.
Anne Criss
Stephane Mouly
Shefali Malhotra
Susan Pusek
Florida Dept of Citrus
Bill Widmer, Ph.D. and Bill Stinson, Ph.D.
National Institutes of Health
General Medical Sciences R37-38149
General Clinical Research Centers (NCRR).