Grapefruit Drug Interaction: Isolation, Biological Activities of Furocoumarins and Their Variation d

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Grapefruit -Drug Interaction Isolation,
Biological Activities of Furocoumarins and Their
Variation due to Pre- and Post-harvest Factors

• Bhimanagouda S. Patil

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What is Grapefruit Drug Interaction ?

• Concurrent intake of grapefruit juice and certain
  drugs increases the plasma concentrations of
  drugs from 1.5 to 15 times.
• GFJ Inhibits the drug metabolizing enzyme system
  cytochrome P450, P-glycoprotein and OATP.

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What is GFJ-Drug Interaction ?
Area Under Plasma
Time
Reference
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Drugs Influenced by Grapefruit Juice

• Drug Increase in Bioavailability
• Felodapine 3 Fold
• Cisapride 1.4 Fold
• Cyclosporin 1.5 Fold
• Saquinavir 2 Fold
• Terfenadine 2.5 Fold
• Buspirone 9 Fold
• Lovastatin/Simvastatin 10 Fold
• and....many more

Greenblatt et al., 2001
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Bioavailability of Drugs
J R Oesterheld Drug-Drug Interactions
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Bioavailability of Drugs

• ORAL DOSE
• CYP ----------? GRAPFRUITE JUICE

GI TRACT
PORTAL CIRCULATION
LIVER (CYP3A)
SYSTEMIC CIRCULATION
Greenblatt et. al. 2001
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• Flavonoids Naringin and Naringenin ???
• Inhibit CYP P450 enzymes in vitro but have little
  or no effect in vivo...
• --------------------------------------------------
  ----------------
• Bioactive Furocoumarins in Grapefruit
• 6, 7 Dihydroxybergamottin
• Bergamottin
• Paradisin A
• Paradisin B
• Paradisin C
• Epoxy-bergamottin
• Bergaptol
• Geranylcoumarin

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Extraction of Grapefruit Juice

• Extraction for isolation and purification.
• 300 L of juice was extracted with Ethyl acetate
  to get crude mixture, which was reconstituted in
  methanol.
• 3gms of extract was loaded on to Prep- HPLC
  column and eluted with aqueous methanol.

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Schematic Representation of Isolation
Girennavar et al 2006
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Isolation of Furocoumarins by PREP HPLC
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Analytical HPLC Profile of Crude mixture
DHB
Bergamottin
Bergaptol
Paradisin A
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Preparative HPLC separation
Bergamottin
Paradicin B
Paradicin A
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Crude Mixture
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Bergaptol
Geranylcoumarin
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NMR Profile of Paradisin A
H NMR
13C NMR
2D NMR
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Identification of furocoumarins by LC/MS
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• Inhibitory Property of GFJ and Furocumarins on
  Human Cytochrome P450 Enzymes

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CYP P450 3A4 enzymes

• Involved several reactions such as Phase I.
• Multiple forms Exists
• Isoforms interact with the phytochemicals
• Inhibited reversibly and/or irreversibly by
  grapefruit juice components.

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CYP P450 enzymes system

• 60 of the marketed drugs are metabolized by CYP
  3A4, 2C9 and 2D6

Rendic and Di Carlo 1997
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Inhibition of CYP3A4 by GFJ and pummelo
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Inhibition of CYP2C9 by GFJ and pummelo
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Inhibition of CYP2D6 by GFJ and pummelo
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Inhibition of CYP3A4 by furocoumarins
IC50
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Inhibition of CYP2C9 by furocoumarins
IC50
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Inhibition of CYP2D6 by furocoumarins
IC50
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IC50 values for different furocoumarins for major
drug metabolizing enzymes
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Levels of DHB in Different Varieties of GFJ and
Pummelo
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Levels of Paradisin A in Different Varieties of
GFJ and Pummelo
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Levels of Bergamottin in Different Varieties of
GFJ and Pummelo
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Influence of Location and Varieties on Levels of
DHB
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Influence of Location and Varieties on Levels of
Paradisin A
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Influence of Location and Varieties on Levels of
Bergamottin
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Variation of DHB During Season
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Variation of Paradisin A During Season
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Variation of Bergamottin During Season
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Effect of Storage and Processing on Furocoumarins

• Commercial GFJ production often involves
  processing at high temperature and mechanical
  peeling.
• Objective was to quantify the changes if any
  during processing.

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Harvesting Grapefruits at TAMUK- Citrus Center
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GFJ Processing Plant at Mission, Texas
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Levels of Furocoumarins in Hand Squeezed and
Processed GFJ
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Effect of Different Storage Temperatures on
Furocoumarins Level in Rio Red GFJ
aMean standard deviation, n15
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Effect of Different Storage Temperatures on
Furocoumarins Levels in Marsh White GFJ
aMean standard deviation, n15
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Cans
aMean standard deviation, n15
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Cardboards
aMean standard deviation, n15
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Cartoons
aMean standard deviation, n15
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Clinical applications of GFJ

• Grapefruit juice may provide a non-toxic and
  inexpensive alternative to drugs that are used to
  reduce cyclosporin dose. (Yee GC, Lancet. 1995
  Apr 15345(8955)955-6.)
• Grapefruit juice enhances the bioavailability of
  the HIV protease inhibitor in man. (Kupferschmidt
  et. al., Br. J. Clin. Pharmacol 1998 45 355-9.)
• Super pill ?

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Conclusions

• Various furocoumarins were isolated and
  characterized from GFJ
• GFJ and furocoumarins are potent inhibitors of
  CYP3A4, CYP2C9 and CYP2D6 isoenzymes. The order
  of inhibition poetntial is CYP2D6gtCYP3A4gtCYP2C9
• Furocoumarins levels are affected by pre- and
  post-harvest factors.

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Publications

• Girennavar, B., G.K. Jayaprakasha, Jadegouda,
  G.A. Nagana Gowda, and B.S. Patil. 2007. Radical
  scavenging and cytochrome P450 3A4 inhibitory
  activity of bergaptol and geranylcoumarin from
  grapefruit. Bioorganic and Medicinal Chemistry
  15 3684-3691.
• Girennavar, B., G. K. Jayaprakasha, J. L. Jifon
  and B.S. Patil. 2007. Variation of bioactive
  furocoumarins and flavonoids in different
  varieties of grapefruits and pummelo. European
  Food Research and Technology (in press).
  DOI10.1007/s00217.007.0654.4
• Girennavar, B., G. K. Jayaprakasha, and B.S.
  Patil. 2007. Potent Inhibition of Human
  Cytochrome P450 3A4, 2D6 and 2C9 Isoenzymes by
  Grapefruit Juice and its Furocoumarins. Journal
  of Food Science (in Print).
• Girennavar, B., S.M. Poulose, G.K. Jayaprakash,
  N.G. Bhat, B.S. Patil. 2006. Furocoumarins from
  Grapefruit Juice and their effect on Human CYP
  3A4 and CYP 1B1 Isoenzymes. Bioorganic and
  Medicinal Chemistry 142606-2612.
• Girennavar, B., G.K. Jayaprakash, B.S. Patil.
  2007. Influence of pre- and post- harvest factors
  and processing on the levels of furocoumarins in
  grapefruits (Citrus paradisi Macf.). Food
  Chemistry (under review).
• Girennavar, B., Simpkin, S.E., G.K. Jayaprakash,
  Maxim, J., B.S. Patil. 2007. Influence of
  Electron Beam Irradiation on Bioactive Compounds
  in Grapefruits (Citrus paradisi Macf.). Journal
  of Ag Food Chemistry (under review).

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