Background The kidney has a critical role in disposition, efficacy and toxicity of drugs and xenobiotics. Developmental changes of renal membrane transporters have the potential to explain population variability in paediatric pharmacokinetics and -dynamics of drugs but data are missing. We aimed to further delineate the expression of human renal tubular transporters multidrug resistance-associated protein (MRP) 4 and MRP2 and study localization in paediatric kidney samples. Methods We planned to semi-quantify expression levels and to study the age-specific localization of the transporters MRP4 and MRP2 with immunohistochemistry on 44 human neonatal and paediatric kidney samples with age range of 24,00 - 40,00 weeks gestational age (GA) and 0,29 - 744 weeks post-natal age (PNA). The staining intensity was semi-quantitatively scored by two independent observers (MB and BG). Results MRP4 is found to be localized at the apical membrane of the renal proximal tubules at 27 weeks of GA (n=3, 1,29- 4 weeks PNA) and no age-related changes of expression levels were detected. In a premature neonate of 24 weeks GA (n=1), no MRP4 was detected. The MRP2 staining did not meet the requirements to be scored and was rejected. Conclusion MRP4 is expressed from at least 27 weeks GA onwards and does not show developmental changes. The localization was similar as in adults (Ritter et al., 2005). The half-life of the MRP4 substrate furosemide was found to be 6 to 20-fold longer in neonates than in adults (Pacifici, G.M., 2013). This could potentially be linked with the absence of MRP4 in a premature neonate with GA 24 weeks. However, these data should be confirmed as we only had 1 sample of ±24 weeks GA available. Moreover, our data help us in understanding altered disposition of transporter substrates in paediatrics. References Pacifici, G. M. ( 2013). Clinical pharmacology of furosemide in neonates: a review. Pharmaceuticals ;6(9):1094–1129. Ritter CA, Jedlitschky G, Meyer zu Schwabedissen H, Grube M, Köck K, & Kroemer HK. ( 2005). Cellular export of drugs and signaling molecules by the ATP-binding cassette transporters MRP4 (ABCC4) and MRP5 (ABCC5). Drug metabolism reviews ;37(1):253–278. Disclosure(s) Nothing to disclose
Background Alternative mRNA transcripts occur in >90% of human genes and may be triggered by developmental signals. The hepatic transporter OATP1B1 (gene name SLCO1B1 ) traffics substrates across the hepatic membrane, and shows age-related changes in protein expression. We aimed to predict novel isoforms of OATP1B1 by studying alternative splicing of SLCO1B1 in human paediatric post-mortem liver tissue, and the relationship of their mRNA expression with age. Methods mRNA expression of SLCO1B1 transcripts was determined using RNA sequencing (HISAT2/StringTie). Novel mRNA transcripts were considered of relevance when (1) the expression was >5% of the annotated isoform, (2) it was a SLCO1B7 and SCLO1B1 hybrid transcript, or (3) when the expression was associated with age. The software packages ORF-finder, TMpred and TOPO2 were used to predict the protein sequence and structure of the novel isoforms. Relationship of expression with age was studied with the Kruskal-Wallis test for age groups (fetal, 0–1.5 year, 1.5–6 year, 6–12 year and 12–18 year) and with Spearman correlation tests for age on continuous scale. Results In 97 hepatic post-mortem tissues (gestational age median 16.4 [range 14.7–41.3] weeks, postnatal age 0.36 [0 - 17] years) 27 novel mRNA transcripts were detected. Of these, 13 were relevant: 2 isoforms are predicted to translate into amino acid sequences similar to the annotated isoform for OATP1B1, 9 isoforms may translate into truncated versions, and the expression of 8 isoforms was associated with age. None of the isoforms had an ORF that covered the SLCO1B7 region. Conclusion We showed that novel SLCO1B1 mRNA isoforms potentially translate into OATP1B1 protein with unknown function, and that alternative splicing may well be a regulatory mechanism for SLCO1B1 expression during development. This data provides a better understanding of age-related changes in the expression of OATP1B1, and, with that, potentially improves prediction of disposition of endogenous and exogenous substrates. Disclosure(s) BG was supported, in part, by the Ter Meulen fund 2018 provided by the Royal Dutch Academy of Sciences. The National Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders at the University of Maryland is funded by the National Institutes of Health (NIH) contract HHSN275200900011C, reference number, N01-HD-9-0011 and the Liver Tissue Cell Distribution System is funded by NIH contract number N01-DK-7-0004/HHSN267200700004C.
Background Postnatal developmental changes of human renal membrane transporters, which are key players of disposition of renally cleared drugs and endogenous substrates, are largely unknown. This study aimed to characterize the ontogeny of 11 human renal transporters to understand changes in the renal clearance of substrate drugs in children. Methods mRNA levels of known renal transporters: BCRP, MATE1, MATE2-K, MDR1, MRP2, MRP4, URAT1, GLUT2, OAT1, OAT3 and OCT2, and the transcription factor PXR were measured with RT-qPCR in 184 human postmortem frozen renal cortical tissues (preterm newborns - adults; 1 day-75 yrs old) from individuals of European and African descent. Protein expression of all but MRP2, MRP4 and PXR was quantified with LC-MS/MS SRM in 62 of those samples (term newborns - adults; 1 day-29 yrs old). Localization of MRP4 was tested with immunohistochemistry. Results Expression levels of MDR1, URAT1, OAT1, OAT3, and OCT2 increased with age, but levels of MATE1 and GLUT2 were stable from birth. Protein levels of MATE2-K and BCRP showed no difference from newborns to adults despite age-related changes in mRNA expression. MRP2, MRP4 and PXR expression levels were stable. MRP4 localization in pediatric samples was similar to that in adult samples. Conclusion Renal drug transporters exhibited different rates and patterns of maturation, suggesting that renal handling of both endogenous and exogenous compounds may change with age. It is important to consider ontogeny of renal transporters during pediatric drug development. Disclosure(s) The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Views expressed in this paper are those of authors and do not necessarily reflect the official views or policies of the FDA; nor does any mention of trade names, commercial practices, or organization imply endorsement by the U.S. Government. *Contributed equally, **Contributed equally
Background In adults, the organic cation transporter 2 (protein name OCT2, gene name SLC22A2 ) is localised in the kidney proximal tubules where it mediates organic cation secretion. Hence, the transporter plays a role in the disposition and excretion of several drugs and drug-drug interactions. To better understand the disposition of OCT2 substrate drugs in children, we studied OCT2 localisation and expression in paediatric kidney tissue. Methods The expression of OCT2 was visualised in tissue using immunohistochemical staining. Tissues were derived post-mortem from children aged 0 -14 years. Gestational age varied between 24 and 40 weeks. Intensity of the staining at the basolateral membrane was scored by two individual observers using three categories; negative, detectible and high. Agreement between two observers was determined using Cohen’s kappa. Results 44 kidney samples (n=17 neonates, n=17 infants, n=7 children, n=3 adolescent) were analysed and scored. There was substantial agreement between two judgements with a kappa of 0.773 (p< 0.005). No age related pattern was observed in the expression of OCT2. Even in the youngest age group, the expression of OCT2 was clearly visible. Conclusion The kidney expression of OCT2 did not show an age-related pattern. In all age groups, expression levels were similar and OCT2 was properly localised at the basolateral membrane. These findings suggest that, with increasing age, OCT2 will not influence the renal excretion of its substrates. Disclosure(s) Nothing to disclose
Background Drug disposition in children may vary from adults due to age-related variation in drug metabolism, but paediatric pharmacokinetic (PK) studies are challenging. Microdose studies present an innovation to study PK in paediatrics, and can only be used when the PK of a microdose are dose-linear to a therapeutic dose. We aimed to assess dose-linearity of [ 14 C]midazolam (MDZ), a marker for the activity of the developmentally regulated CYP3A enzyme, by comparing the PK of an intravenous (IV) [ 14 C]MDZ microtracer given simultaneously with therapeutic MDZ, with the PK of a single IV [ 14 C]MDZ microdose. Methods Preterm to 2-year-old infants admitted to the intensive care unit received [ 14 C]MDZ IV either as a microtracer during therapeutic MDZ infusion or as an isolated microdose. Dense blood sampling was done up to 36 hours after dosing. Plasma concentrations of [ 14 C]MDZ and [ 14 C]1-OH-MDZ were determined by accelerator mass spectrometry. A population PK model was developed with NONMEM 7.4 to study whether there was a difference in the PK of the microtracer versus those of a microdose [ 14 C]MDZ. Results Of fifteen children (median gestational age 39.4 [range 23.9–41.4] weeks, postnatal age 11.4 [0.6–49.1] weeks), nine received a microdose and six a microtracer [ 14 C]MDZ (111 Bq/kg; 37.6 ng/kg). In a two-compartment PK model, bodyweight was the most significant covariate for volume of distribution. There was no statistically significant difference in any PK parameter between the [ 14 C]MDZ microdose or microtracer, suggesting the PK of MDZ to be linear within the range of the therapeutic doses and microdoses. Conclusion Our data supports the dose-linearity of an IV [ 14 C]MDZ microdose in children, thus a [ 14 C]MDZ microdosing approach can be used to study developmental changes in hepatic CYP3A activity. Disclosure(s) This project was funded by the ZonMw ERA-NET PRIOMEDCHILD programme (projectnumber 113205022). * both authors contributed equally
