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© Oroxcell 2009
Percutaneous and intestinal absorption
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Table of contents
Percutaneous and intestinal absorption
1. Intestinal absorption• Permeability across in vitro epithelial monolayers• Ussing chambers• In situ intestinal perfusion
2. Percutaneous absorption• Reconstituted human epithelium• Human excised skin
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Intestinal absorption
Permeability across in vitro epithelial monolayers
Cellular models• Caco-2, TC7• HT29-MTX
Determination• Apparent permeability• Unidirectional flux• Effect of compounds, ingredients and formulations on monolayer integrity
Transport studies• Bidirectional transport studies• Revealing efflux• Assessment of P-gp mediated transport
Increase bioavailability• Formulation Search Engine
Exemple: Revealing efflux
Bidirectionnal transport of compounds across TC7 cell monolayers
Reference:
Pachot JI, Botham RP, Haegele KD, Hwang K.(2003)
Experimental estimation of the role of P-Glycoprotein in the pharmacokinetic behaviourof telithromycin, a novel ketolide, in comparisonwith roxithromycin and other macrolides usingthe Caco-2 cell model
J Pharm Pharm Sci. 6(1):1-12
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Intestinal absorption
Intestinal absorption
Co-incubation of the test compound withthe 3 modulators simultaneously
• Due to the existence of multiple active sitesin P-gp structure (Safa, Curr. Med. Chem.-Anti-Cancer Agents, 2004), it is recommendedto use at least 3 modulators.
• Verapamil is listed by US FDA as anacceptable P-gp modulator. Progesterone andnicardipine are two other P-gp modulatorswhich interact differently on P-gp, probably ondifferent sites (Orlowski et al., Biochem. J.,1996; Martin et al., Mol. Pharmacol., 2000).
• These 3 P-gp modulators have beensuccessfully used together (Pontier et al., J.Pharm. Sci., 2001).
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Exemple: Assessment of P-gp mediated transport
Bidirectionnal transport of digoxine with different P-gp modulators
Intestinal absorption
Reference:
Galenic applications of self-emulsifyingmixtures of lipidic excipients
WO 2006/018501 A1
F = formulation
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Selection of formulations forincreased bioavailability
Exemple: Formulation Search Engine
Transport of CpdX across TC7 cell monolayers with different formulations
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Intestinal absorption
Ussing chamber : principleThe active ingredient is added in the donor medium (either mucosal or serosal side).
Samples are collected in the receiver medium to determine flux and apparent permeability.
Electrical parameters are monitored during the transport study in order to assess a potentialeffect of the compound on intestinal physiology or intestinal integrity
Reference:
Boisset M, Botham RP, Haegele KD, Lenfant B, Pachot JI. (2000)
Absorption of angiotensin II antagonists in Ussing chambers, Caco-2, perfused jejunum loop and in vivo: importance of drug ionisation in the in vitro prediction of in vivo absorption
Eur J Pharm Sci. 10(3):215-224
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Intestinal absorption
Ussing chamber : scheme
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Intestinal absorption
Theabsorp+onmodelconsistsinanintes(nalsegmentoftheratperfusedinsitu:
Theformula+onsareperfused,withaperistal(cpump,ataconstantratethroughoutanintes+nalsegmentinratsunderanesthesia
Thesurfacesofcollectedintes+nalsegmentsarereported.ThepHoftheformula+onattheexitoftheintes+nalsegmentisreported
cm2
pH
Bloodissampledatregularintervalsinthecatheterizedjugularveinorthemesentericvein Plasmasamplesareanalyzedtomonitor
productconcentra(onvaria(ons
In situ intestinal perfusion
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Percutaneous absorption
Reconstituted human epithelium
Episkin™ model: skin culture and source• Reconstituted from adult human keratinocytes• Mammary/abdominal samples• From healthy consenting donors• Cultured on a collagen base in conditions which
permit terminal differentiation• Functional horny layer (stratum corneum)
Protocol outline• Formulated or not formulated test compound
(cream, liquid, patch…)• Preliminary solubility and stability of the test
compound in the different media or formulation• Percutaneous flux across human skin• LC-UV, LC-MS/MS, fluorescence, radioactivity Episkin™
Episkin™
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Percutaneous absorption
Exemple: percutaneous flux across Episkin™ model
TestoPatch® absorption through reconstituted human epithelium
Results were in agreement with bibliography:35 ± 3 µg/cm2 over 6hwhich corresponds to 7% of applied amount
Bibliographic data:5.7 to 7.5%(testosterone solution over 4h)
Cumulated amount in receiving mediumn = 3
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
Time (h)
Test
oste
rone
ng/
cm2
Test compound: Testosterone - TestoPatch
C1 36,9 7,1C2 31,1 5,9C3 37,4 7,3
Mean 35,1 6,8s.d. 3,5 0,8
EpiskinAmount in receiving medium
(µg/cm²/6h) Flux (µg.cm-2.h-1)
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Percutaneous absorption
Reconstituted human epithelium
Human excised skin• Fresh / Frozen• Calibrated thickness / Full thickness• Abdomen / Breast / Back• Animal / Human healthy consenting donors
Protocol outline• Formulated or not formulated test compound
(cream, liquid, patch…)• Franz cells• Split-thickness• Percutaneous flux• LC/UV, LC/MS or radiolabeled assay
Franz cell
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Percutaneous absorption
Human excised skin
Franz cell: principle
Magneticbar
Film
Skin
Formulation
Donor compartment
Receiving compartmentAppropriatemedium
Heated water
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Exemple: Percutaneous flux accros human skin using Franz cell
TestoPatch® absorption through real human skin
Time (h)
Test
oste
rone
ng/
cm2
Test compound: Testosterone patch
Amount in receiving medium (! g/cm?/24h)
Flux (! g/cm?/h)
C1 18,1 1,4
C2 17,6 1,6
C3 16,5 1,3
C4 32,5 2,0
C5 23,6 1,3
C6 23,7 1,3
Mean 22,0 1,5s.d. 6,0 0,3
Percutaneous absorption
Experimental in vitro flux : 1.5 ± 0.3 µg / h / cm2
Transdermal flux in human obtained with TestoPatch® : 1.7µg / h / cm2
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Percutaneous absorption
S.C.
Deposit
S.C. - Stratum Corneum E - Epidermis D -Dermis
Recovery (%) and repartition of compoundB using split-thickness human skin
Total Recovery : 90.8 ± 14.0 %
Exemple: Percutaneous absorption of compound B using Franz cell
E
D
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