Model systems and selected DME and DT
Both pooled human liver microsomes, S9 fractions and suspended human hepatocytes (2 pooled and at least 3 individual neonatal donors) should be used as model systems to evaluate the impact of hypothermia (33–37°C) on the activity of the most important hepatic DME, including CYP1A2, CYP2B6, CYP2C8/9/19, CYP2D6, CYP3A4, CYP2E1, Flavin- Containing Monooxygenase (FMO)1-3, uridine 5 ´-diphospho-glucuronosyltransferases (UGT's), aldehyde oxidases, and esterases; Suspended human hepatocytes (pooled and individual donors) should be used to determine the effect of hypothermia on the major drug uptake transporters [OATP1B1/3, sodium-taurocholate cotransporting polypeptide (NTCP), organic cation transporter (OCT)1, organic anion transporter (OAT)2] and the sinusoidal efflux transporter MRP3 (multidrug resistance-associated protein 3); Sandwich-cultured human hepatocytes (from at least 3 individual donors) and membrane vesicles to determine the effect of hypothermia on activity of key canalicular [MultiDrug Resistance protein (MDR)-1, Multidrug Resistance-associated Protein (MRP)-2, Breast Cancer Resistance Protein (BCRP)] efflux transporters; Similar in vitro experiments can be designed in at least 2 batches of minipig hepatocytes. Experiments with animal hepatocytes support unique insights into in vitro in vivo extrapolation (IVIVE) provided corresponding in vivo studies in the same species are also conducted.
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Study design and model compounds
To allow distinction between effects on protein stability versus catalytic activity, incubations with the in vitro model systems should be conducted either during hypothermia, or immediately after an in vitro hypothermia episode (60 min). Multiple cycles of hypothermia and warming may also be considered in an attempt to mimic the in vivo impact of hypothermia (for 72 h) and warming on DME and DT activity. Depending on the research question, a strategic selection of probe substrates should include:
...the hepatically metabolized model compounds of interest (e.g. midazolam, fentanyl, phenobarbital because of their clinical relevance in the neonatal population); ...established compounds for in vitro DME and DT phenotyping, and; ...compounds used as in vivo biomarkers of specific DME and DT activity, thus enabling more straightforward data interpretation as a well as scaling towards the in vivo context.
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Current challenges
Relatively high inter-donor and/or inter-occasion variability in the obtained in vitro data is to be expected. This may complicate the use of the data for a robust PBPK model. Mitigation strategies include more emphasis on the use of pooled microsomes/hepatocytes as well as the use of in vitro model systems expressing single heterologously expressed DME and DT. The relatively large number of conditions to be evaluated necessities a tiered approach, based on initial selections of DMET model systems and substrates.
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| Delayed effects of hypothermia on mRNA expression, protein abundance and activity of hepatic DME and DT |
Model systems and selected DME and DT
Sandwich-cultured human (and possibly animal) hepatocytes (from at least 3 individual donors) should be used to determine the delayed (>24 h after hypothermia episode) effect of hypothermia (72 h) on the mRNA expression and protein abundance of key DME (CYP1A2, CYP2C9, CY3A4) and DT (OATP1B1/3, MRP).
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Study design and model compounds
Sandwich-cultured hepatocytes offer the advantage that hepatocytes can be used in study designs covering more than 1 week. The activity assays described for studying immediate effects (see above) are also applicable in this model system.
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Current challenges
Altered protein expression and activity in cultured hepatocytes will complicate extrapolation to in vivo effects. Analogous to interpretation and extrapolation of in vitro induction response in cultured hepatocytes (Zhang et al., 2014), parallel evaluation of compounds exhibiting known in vivo impact of T° will be required.
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