Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project

: The value of pig as "large animal model" is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective

Case Report:Bosentan and Sildenafil Exposure in Human Milk - A Contribution From the ConcePTION Project

Introduction: Quantitative information on disposition of maternal medicines in human milk remains a major knowledge gap. This case report presents the clinical and pharmacokinetic data of a single mother-infant pair exposed to bosentan and sildenafil for the treatment of pulmonary arterial hypertension (PAH) during lactation. Case presentation: A 43-year old mother was treated with sildenafil (20 mg, 3x/day) and bosentan (125 mg, 2x/day) for PAH. Her 21-months old infant received breastfeeding in combination with adequate complementary foods. Milk samples were collected over 24 h, at day 637 and 651 after delivery. The observed average steady-state concentrations of sildenafil (2.84 μg/L) and bosentan (49.0 μg/L) in human milk were low. The Daily Infant Dosage ingested by the nursing infant through human milk was 0.02 μg/kg/day for sildenafil and 0.29 μg/kg/day for bosentan at day 637, and 0.03 μg/kg/day and 0.60 μg/kg/day at day 651. The Relative Infant Dose calculated for an exclusively breastfed infant with an estimated milk intake of 150 ml/kg/day, was 0.06% for sildenafil and 0.24% for bosentan. General health outcome of the infant, reported by the mother, was uneventful until the sampling days. Conclusion: Low medicine concentrations were found in human milk expressed 21 months after delivery after maternal intake of 20 mg sildenafil three times daily and 125 mg bosentan twice daily. General health of the nursing infant until sampling was reported as optimal by the mother

A comprehensive review on non-clinical methods to study transfer of medication into breast milk – A contribution from the ConcePTION project

open17siBreastfeeding plays a major role in the health and wellbeing of mother and infant. However, information on the safety of maternal medication during breastfeeding is lacking for most medications. This leads to discontinuation of either breastfeeding or maternal therapy, although many medications are likely to be safe. Since human lactation studies are costly and challenging, validated non-clinical methods would offer an attractive alternative. This review gives an extensive overview of the non-clinical methods (in vitro, in vivo and in silico) to study the transfer of maternal medication into the human breast milk, and subsequent neonatal systemic exposure. Several in vitro models are available, but model characterization, including quantitative medication transport data across the in vitro blood-milk barrier, remains rather limited. Furthermore, animal in vivo models have been used successfully in the past. However, these models don't always mimic human physiology due to species-specific differences. Several efforts have been made to predict medication transfer into the milk based on physicochemical characteristics. However, the role of transporter proteins and several physiological factors (e.g., variable milk lipid content) are not accounted for by these methods. Physiologically-based pharmacokinetic (PBPK) modelling offers a mechanism-oriented strategy with bio-relevance. Recently, lactation PBPK models have been reported for some medications, showing at least the feasibility and value of PBPK modelling to predict transfer of medication into the human milk. However, reliable data as input for PBPK models is often missing. The iterative development of in vitro, animal in vivo and PBPK modelling methods seems to be a promising approach. Human in vitro models will deliver essential data on the transepithelial transport of medication, whereas the combination of animal in vitro and in vivo methods will deliver information to establish accurate in vitro/in vivo extrapolation (IVIVE) algorithms and mechanistic insights. Such a non-clinical platform will be developed and thoroughly evaluated by the Innovative Medicines Initiative ConcePTION.openNauwelaerts N.; Deferm N.; Smits A.; Bernardini C.; Lammens B.; Gandia P.; Panchaud A.; Nordeng H.; Bacci M.L.; Forni M.; Ventrella D.; Van Calsteren K.; DeLise A.; Huys I.; Bouisset-Leonard M.; Allegaert K.; Annaert P.Nauwelaerts N.; Deferm N.; Smits A.; Bernardini C.; Lammens B.; Gandia P.; Panchaud A.; Nordeng H.; Bacci M.L.; Forni M.; Ventrella D.; Van Calsteren K.; DeLise A.; Huys I.; Bouisset-Leonard M.; Allegaert K.; Annaert P

A comprehensive review on non-clinical methods to study transfer of medication into breast milk – A contribution from the ConcePTION project

Breastfeeding plays a major role in the health and wellbeing of mother and infant. However, information on the safety of maternal medication during breastfeeding is lacking for most medications. This leads to discontinuation of either breastfeeding or maternal therapy, although many medications are likely to be safe. Since human lactation studies are costly and challenging, validated non-clinical methods would offer an attractive alternative. This review gives an extensive overview of the non-clinical methods (in vitro, in vivo and in silico) to study the transfer of maternal medication into the human breast milk, and subsequent neonatal systemic exposure. Several in vitro models are available, but model characterization, including quantitative medication transport data across the in vitro blood-milk barrier, remains rather limited. Furthermore, animal in vivo models have been used successfully in the past. However, these models don't always mimic human physiology due to species-specific differences. Several efforts have been made to predict medication transfer into the milk based on physicochemical characteristics. However, the role of transporter proteins and several physiological factors (e.g., variable milk lipid content) are not accounted for by these methods. Physiologically-based pharmacokinetic (PBPK) modelling offers a mechanism-oriented strategy with bio-relevance. Recently, lactation PBPK models have been reported for some medications, showing at least the feasibility and value of PBP

Challenges Related to Acquisition of Physiological Data for Physiologically Based Pharmacokinetic (PBPK) Models in Postpartum, Lactating Women and Breastfed Infants—A Contribution from the ConcePTION Project

Physiologically based pharmacokinetic (PBPK) modelling is a bottom-up approach to predict pharmacokinetics in specific populations based on population-specific and medicine-specific data. Using an illustrative approach, this review aims to highlight the challenges of incorporating physiological data to develop postpartum, lactating women and breastfed infant PBPK models. For instance, most women retain pregnancy weight during the postpartum period, especially after excessive gestational weight gain, while breastfeeding might be associated with lower postpartum weight retention and long-term weight control. Based on a structured search, an equation for human milk intake reported the maximum intake of 153 mL/kg/day in exclusively breastfed infants at 20 days, which correlates with a high risk for medicine reactions at 2–4 weeks in breastfed infants. Furthermore, the changing composition of human milk and its enzymatic activities could affect pharmacokinetics in breastfed infants. Growth in breastfed infants is slower and gastric emptying faster than in formula-fed infants, while a slower maturation of specific metabolizing enzymes in breastfed infants has been described. The currently available PBPK models for these populations lack structured systematic acquisition of population-specific data. Future directions include systematic searches to fully identify physiological data. Following data integration as mathematical equations, this holds the promise to improve postpartum, lactation and infant PBPK models.</p

Determining the exposure of maternal medicines through breastfeeding:the UmbrelLACT study protocol - a contribution from the ConcePTION project

Introduction:Breastfeeding is beneficial for the health of the mother and child. However, at least 50% of postpartum women need pharmacotherapy, and this number is rising due to the increasing prevalence of chronic diseases and pregnancies at a later age. Making informed decisions on medicine use while breastfeeding is often challenging, considering the extensive information gap on medicine exposure and safety during lactation. This can result in the unnecessary cessation of breastfeeding, the avoidance of pharmacotherapy or the off-label use of medicines. The UmbrelLACT study aims to collect data on human milk transfer of maternal medicines, child exposure and general health outcomes. Additionally, the predictive performance of lactation and paediatric physiologically based pharmacokinetic (PBPK) models, a promising tool to predict medicine exposure in special populations, will be evaluated. Methods and analysis:Each year, we expect to recruit 5-15 breastfeeding mothers using pharmacotherapy via the University Hospitals Leuven, the BELpREG project (pregnancy registry in Belgium) or external health facilities. Each request and compound will be evaluated on relevance (ie, added value to available scientific evidence) and feasibility (including access to analytical assays). Participants will be requested to complete at least one questionnaire on maternal and child's general health and collect human milk samples over 24 hours. Optionally, two maternal and one child's blood samples can be collected. The maternal medicine concentration in human milk will be determined along with the estimation of the medicine intake (eg, daily infant dose and relative infant dose) and systemic exposure of the breastfed child. The predictive performance of PBPK models will be assessed by comparing the observed concentrations in human milk and plasma to the PBPK predictions. Ethics and dissemination:This study has been approved by the Ethics Committee Research UZ/KU Leuven (internal study number S67204). Results will be published in peer-reviewed journals and presented at (inter)national scientific meetings. Trial registration number NCT06042803.</p

Generic PBPK template for predicting drug concentration time profiles in human breast milk (D3.6)

This deliverable described in the present report reports on the development, evaluation and application of a generic Physiologically-based pharmacokinetic modelling (PBPK) template: To predict medicine concentration time profiles in human milk; To calculate milk-associated medicine doses ingested by neonates and infants

ConcePTION WP3 task 3.2. Isolation and characterization of Mammary Epithelial Cells from Göttingen minipig (mpMECs): an in vitro study to compare mpMECs vs pMECs (porcine Mammary Epithelial Cells)

This data set contains data related to the characterization of an appropriate in vitro animal model based on primary culture of Göttingen Minipig Mammary Epithelial Cells (mpMECs). The objective of the research was to verify the accuracy of mpMECs as solid translational model for the study of mammary epithelial barrier and compare mpMECs results with those obtained from Mammary Epithelial Cells isolated from hybrid commercial pig (pMECs). The results showed that it was possible to isolate, culture and expand three pure epithelial cell lines obtained from three different animals. The mpMECs maintained until P10 showed a typical cobblestone morphology and a similar doubling time profile (MG9 32.6 ± 4 h, MG11 30.5 ± 3 h and MG12 27.4 ± 2.8 h, as reported in the data file CONCEPTION_WP3_mpMECs_DoublingTime_09032023.xlsx). DNA index was normal for all the three cell populations with a mean value of 0.98 ± 0.01. Regarding the cell cycle, the cell populations MG9, MG11 and MG12 showed the three distinct phases that could be recognized in a proliferating cell population: G0/G1, S and G2/M (see data file CONCEPTION_WP3_mpMECs_CellCycle_DNAIndex_09032023.xlsx). All the three mpMECs cell lines expressed epithelial markers Epithelial-Cadherin (E-Cad) and Cytokeratin 18 (CK18), confirming their epithelial origin (see data file CONCEPTION_WP3_mpMECs_FlowCytometer_E-Cad_CK18_09032023.xlsx). In particular in MG12, the contour of CK18 positive peak showed a shoulder suggesting the presence of a cellular subpopulation with a particularly high positivity. The barrier function of mpMECs was evaluated via TEER and fluorescein sodium (SF) transport, the formation of the monolayer integrity was evaluated in all the three primary mpMECs and in the pre-mixed pool of mpMECs and pMECs. MG9, MG11 and MG12 resulted in a similar TEER profile at the 0.15×10^6 cells, achieving higher TEER and lower SF values at the day 3 and 4 of culture, which was also confirmed in the pre-mixed pool case. Moreover, by comparing the maximum values of TEER reached at the different seeding density tested, a significative difference resulted in MG9 with respect to MG11, MG12 and pool. From a similar comparison with minimum values of SF transport, no difference between the groups was showed. Finally, in pre-mixed pools of mpMECs and pMECs a difference in the ability to form the epithelial barrier was shown: mpMEC barrier resulted less tight compact then that formed by pMECs. (see data file CONCEPTION_WP3_mpMECs_TEER_SF_09032023.xlsx). On pre-mixed pool of mpMECs and pMECs we evaluated the bioenergetic metabolism: no difference in energy production under basal cell culture condition was observed but under stressed state pMECs made more efficient use of mitochondrial oxidative metabolism than mpMECs, conversely, these last ones could more efficiently increased the glycolytic activity (see data file CONCEPTION_WP3_mpMECs_BioenergerticMetabolism_09032023.xlsx). The transcriptional profile of drug transporters evaluated in pre-mixed pool of mpMECs or pMECs cultured MECGM medium. Among the 84 genes, 66 genes were detectable, 18 genes were not detectable or higher than 35 threshold cycle, so considered as negative according to the handbook, in both cell lines. No difference between mpMECs and pMECs drug transporter gene expression levels was observed (see data file CONCEPTION_WP3_mpMECs_RT2ARRAY_09032023.xlsx)

Urinary CC16, a potential indicator of lung integrity and inflammation, increases in children after short-term exposure to PM/PM and is driven by the CC16 38GG genotype.

Particular matter (PM) exposure is a big hazard for public health, especially for children. Serum CC16 is a well-known biomarker of respiratory health. Urinary CC16 (U-CC16) can be a noninvasive alternative, albeit requiring adequate adjustment for renal handling. Moreover, the SNP CC16 G38A influences CC16 levels. This study aimed to monitor the effect of short-term PM exposure on CC16 levels, measured noninvasively in schoolchildren, using an integrative approach. We used a selection of urine and buccal DNA samples from 86 children stored in an existing biobank. Using a multiple reaction monitoring method, we measured U-CC16, as well as RBP4 (retinol binding protein 4) and β2M (beta-2-microglobulin), required for adjustment. Buccal DNA samples were used for CC16 G38A genotyping. Linear mixed-effects models were used to find relevant associations between U-CC16 and previously obtained data from recent daily PM&nbsp;≤&nbsp;2.5 or 10&nbsp;μm exposure (PM, PM) modeled at the child&#8217;s residence. Our study showed that exposure to low PM at the child&#8217;s residence (median levels 18.9&nbsp;μg/m³ (PM) and 23.6&nbsp;μg/m³ (PM)) one day before sampling had an effect on the covariates-adjusted U-CC16 levels. This effect was dependent on the CC16 G38A genotype, due to its strong interaction with the association between PM levels and covariates-adjusted U-CC16 (P&nbsp;=&nbsp;0.024 (PM); P&nbsp;=&nbsp;0.061 (PM)). Only children carrying the 38GG genotype showed an increase of covariates-adjusted U-CC16, measured 24h after exposure, with increasing PM and PM (β&nbsp;=&nbsp;0.332; 95% CI: 0.110 to 0.554 and β&nbsp;=&nbsp;0.372; 95% CI: 0.101 to 0.643, respectively). To the best of our knowledge, this is the first study using an integrative approach to investigate short-term PM exposure of children, using urine to detect early signs of pulmonary damage, and taking into account important determinants such as the genetic background and adequate adjustment of the measured biomarker in&nbsp;urine.</p

A comprehensive review on non-clinical methods to study transfer of medication into breast milk - A contribution from the ConcePTION project.

Breastfeeding plays a major role in the health and wellbeing of mother and infant. However, information on the safety of maternal medication during breastfeeding is lacking for most medications. This leads to discontinuation of either breastfeeding or maternal therapy, although many medications are likely to be safe. Since human lactation studies are costly and challenging, validated non-clinical methods would offer an attractive alternative. This review gives an extensive overview of the non-clinical methods (in vitro, in vivo and in silico) to study the transfer of maternal medication into the human breast milk, and subsequent neonatal systemic exposure. Several in vitro models are available, but model characterization, including quantitative medication transport data across the in vitro blood-milk barrier, remains rather limited. Furthermore, animal in vivo models have been used successfully in the past. However, these models don't always mimic human physiology due to species-specific differences. Several efforts have been made to predict medication transfer into the milk based on physicochemical characteristics. However, the role of transporter proteins and several physiological factors (e.g., variable milk lipid content) are not accounted for by these methods. Physiologically-based pharmacokinetic (PBPK) modelling offers a mechanism-oriented strategy with bio-relevance. Recently, lactation PBPK models have been reported for some medications, showing at least the feasibility and value of PBPK modelling to predict transfer of medication into the human milk. However, reliable data as input for PBPK models is often missing. The iterative development of in vitro, animal in vivo and PBPK modelling methods seems to be a promising approach. Human in vitro models will deliver essential data on the transepithelial transport of medication, whereas the combination of animal in vitro and in vivo methods will deliver information to establish accurate in vitro/in vivo extrapolation (IVIVE) algorithms and mechanistic insights. Such a non-clinical platform will be developed and thoroughly evaluated by the Innovative Medicines Initiative ConcePTION