Hepatic uptake transport in individual versus pooled ... - Inserm

Hepatic uptake transport inindividual versus pooledcryopreserved humanhepatocytes.Tom De Bruyn, Jasminder Sahi, Patrick Augstijns and Pieter AnnaertLab Pharmacotechnology and BiopharmacyDept. Pharmaceutical Sciences (KULeuven, Belgium)Tom De BruynHUG 2010Montpellier, 23Oct2010

Presentation outline• Background• Research questions• Methodology• Cryopreserved human hepatocytes to identify new transportersubstrates: individual versus pooled batches• Cryoprserved human hepatocytes to evaluate drug interactionpotential: individual versus pooled batches• Conclusion

Background: uptake transport in hepatocytesOrganicanions, cationsand bile saltsBile saltsNa+OrganiccationsOrganicanionsSLCOOATP 1B1 1B3 2B1SLCO10A1NTCPSLC22A1/3OCT1/3SLC22A7OAT2BILEhepatocytehepatocyte

Research Questions• Utility of cryopreserved human hepatocytes for identification oftransporter substrates ?– Evaluate transport activities of NTCP, OATP and OCT in different batches• Utility of cryopreserved human hepatocytes for identification oftransporter inhibitors ?– Evaluate the effect of different inhibitors on the uptake of known transportersubstrates in different batches.• Are individual and pooled batches equally suited to study hepaticuptake transport ?– Direct comparison of pooled batches and corresponding individual batches

Methodology: cryopreservation and resuscitation‣ Cryopreservationconducted at Life Technologies (Durham, NC) according to in-houseprocedures‣ Resuscitation of cryopreserved human hepatocytes• Thawing cryopreservation vials in waterbath @ 37°C• Transfer to recovery medium (CHRM ® medium)• Centrifugation ( 6 min, 76 g)• Re-suspension in uptake buffer (Krebs Henseleit Buffer pH 7.4)

Methodology: uptake experiments‣ Uptake experiments using “oil-spin” method200 µL SAMPLEoil mixtureNaOH 2 N14.000 rpm2 x 2 minPRE-INCUBATIONcell supsension + buffer/inhibitorINCUBATION+ substrate (1 min, 1µM)CENTRIFUGATION STEPto seperate cells fromuptake bufferQUANTIFICATIONtube bottoms were cut andanalysed by liquid scintillationspectrometry

Methodology: substrate overview

Research Q 1:Utility of cryopreservedhuman hepatocytes foridentification oftransporter substrates ?

Q 1:Utility of cryopreserved human hepatocytes foridentification of transporter substrates ?Taurocholate (NTCP)MPP + (OCT) Clear substrate accumulation in all batches Substrate accumulation values are variable among different batches

Q 1:Utility of cryopreserved human hepatocytesfor identification of transporter substrates ?Estrone-3-sulfate (OATP1B)Estradiol-17β-D-glucuronide (OATP1B1)Digoxin (OATP1B3)

Research Q 2:Utility of cryopreservedhuman hepatocytes foridentification oftransporter inhibitors ?

Q 2:Utility of cryopreserved human hepatocytes foridentification of transporter inhibitors ?

Research Q 3:Are individual andpooled batches equallysuited to study hepaticuptake transport ?

Q 3: Are individual and pooled batches equallysuited to study hepatic uptake transport ?

Q 3: Are individual and pooled batches equallysuited to study hepatic uptake transport ?Accumulation (% of control)

Conclusions‣ Cryopreserved hepatocytes can be used to identify transportersubstrates.‣ Cryopreserved hepatocytes can be used to identify transporterinhibitors.‣ Individual and pooled batches are equally suited to study hepaticuptake transport.

AcknowledgementsProf. Pieter AnnaertProf. Patrick AugustijnsDr. Jasminder Sahi (Life Technologies, Durham, NC)Funding:• Agency for Innovation by Science and Technology, Flanders.• 'Fonds voor Wetenschappelijk Onderzoek', Flanders• 'Onderzoeksfonds' of the Katholieke Universiteit Leuven, Belgium