Integrated Approaches in Pediatric Pharmacotherapy: Applications of the Neonatal Asphyxia Göttingen Minipig Model

In the context of supporting drug development and pharmacotherapy for human neonates, this study was undertaken to enhance our understanding of the effects of perinatal asphyxia (PA) and therapeutic hypothermia (TH). The overarching goal of this research, using the neonatal Göttingen Minipig model, was to disentangle the effects of systemic hypoxia and TH on the pharmacokinetics (PK) of four drugs used in the neonatal intensive care unit (NICU), and on drug enzyme functionality. Such insights can subsequently inform the development of a physiologically-based pharmacokinetic (PBPK) model, which is instrumental for dose precision in human neonates. The data below are a summary of the PhD research of Marina Stroe [1].

Introduction

The neonatal population presents challenging dose predictions due to the pronounced growth and development during early life stages, affecting drug exposure. These complexities are further amplified in neonates affected by perinatal asphyxia (PA), a condition that impacts multiple organ systems and often necessitates intensive care interventions such as therapeutic hypothermia (TH). TH, the standard treatment for hypoxic-ischemic encephalopathy, improves survival and neurodevelopmental outcomes by modulating metabolic processes and reducing oxygen consumption. However, TH also alters drug disposition through its effects on cardiac output, organ blood flow, and enzymatic activity, necessitating tailored pharmacotherapy to optimize safety and efficacy.

Pediatric drug therapy faces significant challenges due to limited clinical data and ethical barriers to conducting trials in neonates, often resulting in the off-label use of medications without robust evidence. In this context, animal models, such as the Göttingen Minipig, could provide a critical solution for understanding the pharmacokinetics (PK) in this population. Göttingen Minipigs exhibit physiological and anatomical similarities to humans, including comparable gastrointestinal, hepatic, and cardiovascular development, and their larger size facilitates sampling without significantly impacting physiology [2]. Additionally, their genetic consistency and high homology to human drug-metabolizing enzymes, such as Cytochrome P450 (CYP), make them an ideal model for neonatal studies [3,4].

The I-PREDICT project: Innovative physiology-based pharmacokinetic (PBPK) model to predict drug exposure in neonates undergoing cooling therapy (Senior research grant from the Research Scientific Foundation-Flanders (FWO), Belgium - G0D0520N) aimed to refine dosing strategies for neonates with PA undergoing TH by developing a PBPK model using the Göttingen Minipig model [5]. The first hypothesis stated in this project was that systemic hypoxia and TH reduce metabolic drug clearance, necessitating dosing adjustments. To test this, an experimental study was conducted at the research facility of Ellegaard Göttingen Minipigs A/S, Dalmose, Denmark [6], examining the PK of midazolam, fentanyl, phenobarbital, and topiramate under four conditions: control, hypoxia (H), TH, and combined hypoxia and TH (H+TH). Neonatal Göttingen Minipigs, within 24 hours of birth, were subjected to these conditions, with drug administration and blood sampling performed under anesthesia (Figure 1). The gene and protein expression, as well as the activity of specific CYP enzymes were evaluated using in vitro methods. Hepatic CYP expression levels and activity were assessed in both adult and neonatal Göttingen Minipigs, in addition to the 24 experimental Göttingen Minipigs.

In Vivo: Drug Disposition in Neonatal Göttingen Minipigs with Asphyxia and Hypothermia

Midazolam, fentanyl, phenobarbital and topiramate were selected as model drugs based on their different physicochemical and/or PK characteristics and clinical relevance: midazolam (CYP3A4; intermediate extraction ratio (ER)), fentanyl (CYP3A4; high ER); phenobarbital (CYP2C19; low ER), and topiramate (largely renally excreted unchanged). The PK was investigated under four conditions i.e., hypoxia (group H), therapeutic hypothermia (group TH), hypoxia and therapeutic hypothermia (group H+TH) and controls (group C) (Figure 2). 

A. Impact of therapeutic hypothermia on drug disposition
In this study [7], a statistically significant decrease in fentanyl clearance by 66% was observed, leading to an approximately 3-fold longer half-life in the TH group compared to the C group. Additionally, the 24-hour plasma concentrations were statistically significantly higher in the TH group compared to the three other groups investigated. According to thermopharmacological principles, CYP activity decreases at lower body temperatures [8]. As summarized by Tortorici et al (using non-clinical data), TH can decrease the systemic clearance of drugs metabolized by CYPs, by 7-22% per degree Celsius below 37 °C [9]. Therefore, the TH impact on fentanyl metabolism could be attributed to the reduced CYP3A activity. However, it could also be associated with the diminished liver blood flow [10,11]. Trends towards lower clearance and longer half-life of midazolam and phenobarbital due to TH, and the inability to reach a steady state, provide evidence of the limited drug removal capacity in neonatal Göttingen Minipigs. Regarding phenobarbital, only the H+TH group demonstrated a potential trend, with a 30% decrease in clearance with a 35% longer half-life compared to the C group. A possible reason for the discrepancy between the TH and H+TH groups may lie in the small sample size, which limits the ability to draw robust conclusions. Future studies should include a larger sample size and a longer duration of the experiment to better assess these trends and clarify the effects of TH on drug disposition. 

Concerning drugs undergoing little or no hepatic metabolism (i.e., topiramate), existing evidence suggests that TH following hypoxic-ischemic injury does not influence the PK [12,13]. This hypothesis was confirmed in our study since we could not detect a significant effect of TH on topiramate PK in neonatal Göttingen Minipigs. In our study a decrease in topiramate clearance of 28% for the H group was detected compared to the C group. These PK changes induced by hypoxia are expected since the hypoxic-ischemic injury itself can induce renal impairment (e.g., gentamicin in neonatal pigs [13] and human neonates [12]). 

B. Impact of therapeutic hypothermia on cardiovascular processes
In addition to the potential direct impact on drug disposition, TH also has an impact on cardiovascular parameters [14,15]. Firstly, high ER drugs, such as fentanyl, are flow limited drugs meaning that upon IV administration, the systemic clearance is highly dependent on the liver blood flow. Data on organ blood flow, cardiac index, or cardiac output are typically needed to draw such conclusions. Although our study did not directly measure these parameters, we recorded the heart rate (HR) and detected statistically significant lower values in the hypothermic groups: statistically significant lower HR (P<0.0001) and body temperature (P=0.0051) were detected for both TH and H+TH groups compared to C and H groups. As HR impacts cardiac output, the observed lower HR in our study could be linked to the lower capacity of drug removal from the body, as a possible mechanistic hypothesis. This trend was observed for fentanyl (high ER), while drugs with intermediate (midazolam) and low (phenobarbital) ER were less impacted. Therefore, this might explain the more pronounced and statistically significant TH impact on fentanyl PK compared to midazolam, even though both drugs share the same metabolic pathway (i.e., hepatic CYP3A22, CYP3A29, CYP3A46 Göttingen Minipig metabolism [16]). Future experiments measuring organ blood flow, cardiac index, or cardiac output would provide more comprehensive insights, especially in the context of PBPK modelling.

In Vitro: Cytochrome P450 Drug Metabolism in Göttingen Minipigs

It is crucial to acknowledge that CYPs can be evaluated at various levels, including gene expression, protein abundance, and enzyme activity. All these levels are important particularly in distinguishing between acute and prolonged temperature-induced modifications: while alterations in expression levels require a period for manifestation, changes in enzymatic activity can manifest promptly. Therefore, this study [17] aimed to investigate the impact of age, hypothermia, and hypoxia on hepatic CYP expression, abundance, and activity in Göttingen Minipigs. 

Significantly lower CYP3A22, CYP3A29, CYP3A46, CYP2C42, and higher CYP2E1 expressions were observed in neonates compared to adult Göttingen Minipigs and no differences were noted for CYP1A2, CYP2C33, and CYP2D25 (Figure 3). Additionally, the CYP activity in neonatal Göttingen Minipig liver microsomes was 75% lower than in adults (Figure 4). 

During hypothermia, most cellular processes are slowed down, including the activity of drug metabolizing enzymes [18]. Our results showed that general CYP activity in adult Göttingen Minipig liver microsomes decreased significantly (by 36%) when exposed to in vitro hypothermia (33 °C) (Figure 4). Our in vivo drug disposition data in neonatal Göttingen Minipigs sustain this [7].

It is well known that acute systemic hypoxia in humans down-regulates specific CYP isoforms and up-regulates CYP3A4 and P-glycoprotein, changing the drug clearance and the kinetics [19]. Our study results, resumed in Table 1, align with these findings regarding hypoxia-induced changes in CYP3A. We observed a statistically significant up-regulation of CYP3A29, the only CYP3A Göttingen Minipig isoform that showed significant changes in gene expression in response to hypoxia.

Our study revealed increased gene expression of CYP3A29 and CYP2C33 in the H group, with hypoxia combined with TH further elevating CYP2C33 and CYP2C42 expression compared to in vivo controls. However, these changes were not consistently reflected at the protein level. Similar variability has been observed in other studies, such as in rats, where the impact of hypoxia on CYP2C protein abundance and activity depended on the severity and duration of hypoxia [20]. These findings highlight that the effects of hypoxia on CYP2C enzymes vary with the isoform, the experimental conditions, and the type and intensity of hypoxia.

Lastly, CYP2E1, recognized for its high oxidase activity, plays a significant role in hypoxic injury, with hypoxia leading to a marked increase in its expression, catalytic activity, reactive oxygen species production, and associated cell death [21]. Our results indicated a statistically significant increase in CYP2E1 protein abundance in the H group compared to controls, aligning with these previous findings. These results show that CYP2E1 in the liver may be involved in hypoxic injury.

Conclusion

In this study, a neonatal Göttingen Minipig model for dose precision in PA was developed in which the impact of systemic hypoxia and TH on drug disposition and enzyme functionality could be studied separately. TH increased fentanyl plasma levels by reducing hepatic clearance and prolonging half-life, likely due to impaired liver blood flow and decreased CYP3A metabolism. Midazolam showed similar trends, with increased plasma levels and reduced clearance under TH. Additionally, TH lowered heart rate, particularly impacting high ER drugs like fentanyl. In vitro studies revealed differences in CYP gene expression and activity between neonatal and adult Göttingen Minipigs, highlighting one more time the role of maturation in drug metabolism. The exposure to in vitro hypothermia reduced CYP activity in adult liver microsomes by 36%, while hypoxia in neonates altered CYP3A29 expression and CYP2E1 abundance, though no gene expression changes were observed with in vivo TH. Despite the success of several challenging techniques, the model has limitations, such as the 24-hour survival period, which excludes a rewarming phase and limits the PK profile. Enhanced diagnostic tools would improve the evaluation hypoxic-ischemic encephalopathy in this setting. This research provides valuable insights, difficult to obtain clinically, into the different effects of hypoxia and TH on drug metabolism. These findings can inform PBPK models to improve precision dosing in human neonates.

ACKNOWLEDGEMENTS
This research was funded by a Senior research grant from the Research Scientific Foundation-Flanders (FWO) - G0D0520N, I-PREDICT project: Innovative physiology-based pharmacokinetic model to predict drug exposure in neonates undergoing cooling therapy, project number 41889. We are grateful to Ellegaard Göttingen Minipigs A/S for the collaboration and for allowing us to perform the study on their site in Dalmose, Denmark. This research would also not have been possible without the active contribution of several people who are explicitly listed in the papers and PhD thesis of Marina Stroe: 

1. Stroe M-S. Integrated approaches in pediatric pharmacotherapy: the neonatal Göttingen minipig model for drug disposition in perinatal asphyxia and therapeutic hypothermia: University of Antwerp; 2024, doi:10.63028/10067/2100480151162165141.
2. Stroe M-S, Van Bockstal L, et al. Development of a neonatal Göttingen Minipig model for dose precision in perinatal asphyxia: technical opportunities, challenges, and potential further steps. Front. Pediatr. Sec. Children and Health. 2023 (11), doi:10.3389/fped.2023.1163100
3. Stroe M-S, Huang M-C, et al. Drug disposition in neonatal Göttingen Minipigs: Exploring Effects of Perinatal Asphyxia and Therapeutic Hypothermia. Drug Metab Dispos (2024), doi:10.1124/dmd.124.001677
4. Stroe M-S, De Clerck L, et al. In vitro assessment of the effects of hypothermia and hypoxia on cytochrome P450-mediated drug metabolism in neonatal Göttingen Minipigs, Basic Clinical Pharmacology & Toxicology, (2024) doi:10.1111/bcpt.14081 
5. Leys Ka# & Stroe M-Sb#, et al. Pharmacokinetics during therapeutic hypothermia in neonates: from pathophysiology to translational knowledge and physiologically-based pharmacokinetic (PBPK) modeling. Expert opinion on drug metabolism & toxicology, (2023) 19(7), 461–477, doi:10.1080/17425255.2023.2237412
6. Ayuso M, Buyssens L, Stroe M-S, et al. The Neonatal and Juvenile Pig in Pediatric Drug Discovery and Development. Pharmaceutics. 2020;13(1):44. Published 2020 Dec 30. doi:10.3390/pharmaceutics13010044

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