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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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