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

Drug-induced cholestasis: in vitro detection and mechanistic insights

Drug-induced liver injury (DILI) is the primary cause for the discontinuation of clinical trials and post-marketing withdrawal of drugs, leading to considerable losses for the pharmaceutical industry. Despite the immense financial losses that are related to DILI, its impact on patient healthcare is of much greater importance. Indeed, numerous cases have been reported in literature that illustrate the potentially fatal consequences of DILI. As DILI presents itself in multiple clinical forms (e.g., acute hepatitis, cholestasis and steatosis), the plethora of possible underlying mechanisms is an immense hurdle for the development of next generation prediction tools. Therefore, a better understanding of these mechanisms is an absolute priority for the pharmaceutical industry. During the last decade, both academia and industry have put a great deal of effort into unravelling these underlying mechanisms. Based on this, it has become more and more apparent that the pathological disturbance of bile acid homeostasis plays a key role in the onset of liver injury, making cholestasis one of the major causes of DILI. However, the early detection of drug-induced cholestasis (DIC) remains challenging during the drug development process. Indeed, the majority of currently developed in vitro tools that evaluate DIC are based on the assumption that assessing a drug's potency to interfere with certain hepatobiliary transporters suffices to accurately predict the drug's in vivo cholestatic potential. However, as these tools do not adequately mimic the in vivo situation where various types of cellular responses (both adaptive and adverse) regulate bile acid homeostasis, they are unable to thoroughly assess human DIC risks. This shortcoming can be overcome by using more in vivo-relevant in vitro models such as sandwich-cultured human hepatocytes (SCHH). Indeed, our group recently developed a SCHH-based in vitro assay which allows for the detection of cholestatic compounds based on their ability to modulate bile acid homeostasis. Although the assay is able to distinguish cholestatic compounds from both non-hepatotoxic and non-cholestatic but hepatotoxic compounds, it was not designed to yield mechanistic insights into drug-induced changes in bile acid homeostasis. Such information is extremely valuable, especially when trying to make an informed decision regarding the cholestatic potential of a given drug. Consequently, the goal of the current doctoral thesis was to investigate human DIC by profiling in vitro bile acid disposition in SCHH and by evaluating these data using both non-compartmental analysis (NCA) techniques as well as a more advanced cellular mechanistic bile acid disposition model. In vitro data were obtained using an improved experimental setup that consisted of incubating 10 μM of the prototypical bile acid chenodeoxycholic acid (CDCA) in absence and presence of therapeutically relevant bosentan concentrations. As cryopreserved human hepatocytes were used throughout this thesis, the aim of the first study was to evaluate whether cryopreserved cells are a viable alternative to freshly-isolated cells. The results from this study clearly showed that cryopreservation does not affect the hepatocyte's biochemical integrity nor its application potential for drug disposition studies. Moreover, transporter studies, which were conducted using fluorescent probes, indicated that the organic anion transporting polypeptide (OATP) and the multidrug resistance-associated protein 2 (MRP2) activity levels remained unaltered following cryopreservation. In the same study, disposition of telmisartan and telmisartan-glucuronide was evaluated using an in-house developed mechanistic cellular disposition model which allowed us to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation. The model predicted that the relative contribution of uptake, metabolism and efflux of telmisartan and telmisartan-glucuronide remained unchanged following cryopreservation, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes. This result also represents an inspiring example of how optimization of hepatocyte cryopreservation protocols can support implementation of the 3R concept for animal experimentation. In addition to assessing the effects of cryopreservation on hepatocyte longevity and functionality, the first study conducted in this doctoral thesis aimed to establish a generic modelling framework that could be used in future studies. Indeed, in a second study, the framework was utilized to develop a mechanistic model that was able to quantitatively evaluate the effects of bosentan on CDCA and GCDCA disposition in SCHH originating from five different human liver tissue donors. The model consisted of seven compartments to which CDCA and GCDCA could distribute using both linear and non-linear kinetics. Model predictions showed that the amidation of CDCA as well as biliary efflux clearance of GCDCA decreased in presence of bosentan, in line with the non-compartmental analysis results and reports from other research groups. Interestingly, not all tested donors were affected by in vitro bosentan treatment, underlining the importance of using an in vivo-relevant in vitro model when assessing human DIC. Indeed, human hepatocytes, that originate from various donors, allow for the evaluation of interindividual differences in susceptibility to cholestatic compounds, something which cannot be achieved with for instance hepatic cell lines as they do not reflect the interindividual variability. In a last study, we aimed to expand on our study regarding bosentan-mediated cholestasis, by pursuing new insights into the role of Ro 47-8634 (O-demethylation of the phenolic methyl ether of bosentan), Ro 48-5033 (hydroxylation at the t-butyl group of bosentan) and Ro 64-1056 (combination of hydroxylation and O-demethylation of bosentan), the three known phase I metabolites of bosentan. To do so, disposition of CDCA and GCDCA in presence of bosentan was evaluated using SCHH originating from three donors which showed distinct capacities in terms of enzymes that play a role in the metabolic pathways of bosentan. As expected, the donor with greatest metabolic capacity showed increased formation of all metabolites as compared to the other donors. However, this was also the only donor which showed a significant decrease in CDCA uptake and its subsequent conjugation to GCDCA following bosentan treatment, suggesting that formation of Ro 47-8634, Ro 48-5033 and/or Ro 64-1056 could drive the observed effects. Indeed, linear regression analysis indicated that inhibition of CDCA's uptake could (at least in part) be attributed to formation of bosentan's metabolites, while inhibition of CDCA conjugation most likely resulted from interaction with the parent. In summary, this doctoral thesis provided new insights into human DIC using an alternative experimental setup that consisted of incubating SCHH with CDCA in presence and absence of therapeutically relevant bosentan concentrations. By quantifying subtle changes in CDCA and GCDCA disposition using NCA techniques and an in-house developed cellular mechanistic disposition model, quantitative insights into the interplay of bosentan's mechanisms of toxicity were gained. More specifically, based on our results, it has become apparent that bosentan's metabolites play a role in the inhibition of bile acid uptake, leading to the prehepatic accumulation of bile acids, while, in addition, bosentan decreases the further conjugation of unconjugated bile acids to their conjugated forms.status: publishe

Modeling of telmisartan disposition in sandwich-cultured rat hepatocytes. Does cryopreservation change disposition kinetics?

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A mechanistic cellular disposition model of bile acid handling in sandwich-cultured human hepatocytes

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Extra collagen overlay prolongs the differentiated phenotype in sandwich-cultured rat hepatocytes

INTRODUCTION: Sandwich-cultured rat hepatocytes (SCRH) have become an invaluable in vitro model to study hepatic drug disposition. SCRH are maintained between two layers of extracellular matrix. In this configuration, culture periods of 4days are typically applicable. The aim of the present study was to modify conventional SCRH by applying an additional collagen overlay to prolong the hepatic phenotype in SCRH and thus to extend the applicability of the model. METHODS: The cultures receiving an extra top layer ('SCRH-plus' cultures) were compared with the conventional SCRH by testing the morphology, cell functionality, metabolic capacity and Mrp2-activity. RESULTS: In the SCRH-plus cultures, cell functionality, evaluated by measuring urea production, was increased from day 5 onwards, compared to conventional cultures. Furthermore, these cells retained the appearance of typical hepatocytes, in contrast with conventional sandwich cultures which showed rapid dedifferentiation. SCRH-plus exhibited significantly improved metabolic clearance mediated by cytochrome P450 3A compared to conventional SCRH whereas UDP-glucuronosyltransferase-mediated metabolism was unaffected. Both conventional SCRH and SCRH-plus showed extensive biliary networks at day 4 of culture. However, from day 4 onwards, a decline in biliary excretion index (BEI) was observed in the conventional SCRH, while BEI values in SCRH-plus cultures did not decrease until day 7. DISCUSSION: The application of an extra top layer of collagen on the SCRH prolongs their useful life-span to 7days. Therefore, SCRH-plus cultures will broaden the applications of SCRH in terms of long-term toxicity evaluation and when determining metabolism of low turnover compounds.status: publishe

Mechanisms and in vitro models of drug-induced cholestasis

Cholestasis underlies one of the major manifestations of drug-induced liver injury. Drug-induced cholestatic liver toxicity is a complex process, as it can be triggered by a variety of factors that induce 2 types of biological responses, namely a deteriorative response, caused by bile acid accumulation, and an adaptive response, aimed at removing the accumulated bile acids. Several key events in both types of responses have been characterized in the past few years. In parallel, many efforts have focused on the development and further optimization of experimental cell culture models to predict the occurrence of drug-induced cholestatic liver toxicity in vivo. In this paper, a state-of-the-art overview of mechanisms and in vitro models of drug-induced cholestatic liver injury is provided

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

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