Can Göttingen Minipigs Claim a Spot in CNS Drug Research & Development?

This study evaluates the Göttingen minipig as a pharmacokinetic (PK) model for central nervous system (CNS) Research & Development (R&D). It aimed to address the limited knowledge on brain penetration and prediction of systemic clearance, based on in vitro to in vivo extrapolation (IVIVE) techniques, in minipig. Presented are in vivo data on brain drug penetration and PK. Despite a potential higher P-glycoprotein expression in the porcine blood-brain barrier (BBB), in vivo minipig drug brain penetration was comparable to other non-rodent species and human, supporting its suitability for brain drug research. The minipig also appeared to be a suitable PK model and is considered a useful addition to the species used to assess performance of human clearance prediction by IVIVE. This work was performed as part of the PhD project by Kristine Langthaler in a joint collaboration between School of Pharmaceutical Sciences at University of Copenhagen and H. Lundbeck A/S.

1. Introduction

In the field of pharmaceutical CNS R&D, animal models play a crucial role in predicting drug safety and PK behavior of drugs in the human body. One such species gaining popularity is the Göttingen minipig due to its size, ease of handling, anatomical and physiological resemblances to human etc. (Achour et al. 2011; Swindle et al. 2012).

CNS disorders are a global burden, and their increasing prevalence over the last few decades has not been accompanied by a similar increase in new medicines. In most cases, to effectively treat a CNS disease it is essential the drug penetrates into the brain. To predict this with confidence necessitates preclinical testing in appropriate animal models. Knowledge of brain penetration of drugs in minipigs is limited compared to other species such as rats (Friden et al. 2009; Summerfield. et al. 2016). Before reaching the BBB a drug has to be absorbed into the systemic circulation. For oral drugs it is therefore important to minimize clearance (CL) in order to maximize circulating concentration available to penetrate the BBB; given CL influences a drugs systemic half-life as well as bioavailability (fraction of dose reaching the systemic circulation). Animal PK models that can support accurate prediction of human systemic PK, particularly clearance (CL), are therefore very important. Prediction of CL from in vitro metabolic stability data is a well-established approach for prediction of human CL (Obach 1999; Sohlenius-Sternbeck et al. 2010; Lignet et al. 2016) and animal PK models are commonly used to verify performance of the IVIVE technique applied to the human data (Obach 1999; Ito and Houston 2004; Jones et al. 2022). Hence, to establish Göttingen minipigs as a non-rodent PK species for CNS research, it requires the animal to be characterized, using reference compounds that have been tested in other species, in terms of its systemic disposition, brain penetration as well as various absorption-metabolismdistribution-excretion (ADME) parameters (Figure 1).

A concern regarding utility of minipig as a model of brain penetration for translation to humans is the relatively high expression levels of key efflux transporters, such as P-glycoprotein, in the porcine BBB. This transporter expels harmful substances from the brain but can also restrict entry of therapeutic drugs intended to treat brain diseases. Notably, P-glycoprotein is expressed at 4-times higher levels in pigs than in humans (Figure 2) (Zhang et al., 2017), raising concerns of potentially lower brain penetration in the Göttingen minipig (calculated as the unbound brain concentration to unbound plasma concentration ratio, Kp,uu,brain). This could impact its utility in CNS R&D as a non-rodent toxicology/safety pharmacology species or for translation of human brain PK (extent of brain penetration). Therefore, our study aimed to address this knowledge gap and evaluate the suitability of the Göttingen minipig for brain drug research and as an additional PK species for evaluating performance of IVIVE techniques supporting prospective human CL predictions in drug research.

2. Materials and methods

A total of 20 compounds with diverse physiochemical properties and rat Kp,uu,brain values were selected. Of these, 17 were used in minipig brain penetration studies and 18 in protein binding studies. Compounds were combined (up to 4 compounds per cassette) then administered at low doses, to increase throughput and reduce animal usage.

Binding to human, rat and Göttingen minipig whole plasma and rat or minipig brain homogenate was quantitatively assessed by equilibrium dialysis. The free fractions in these matrix were used to correct the total plasma and brain compound concentrations to unbound concentrations for calculation of the in vivo unbound brain to plasma drug partition coefficient (Kp,uu,brain). This coefficient is recognized as the key parameter for evaluating the extent of brain penetration (Loryan et al. 2022) and is calculated according to equation 1.

Compounds with Kp,uu,brain values < 0.3 are considered to have restricted brain penetration, whilst those approximating a value of 1 are considered to have unrestricted brain penetration.

PK parameters were calculated using non-compartmental analysis of plasma concentration-time profiles using Phoenix WinNonlin software. The plasma elimination half-life, volume of distribution (Vss), and total clearance were determined, using equations 2-4.

3. Results

Binding of compounds to Göttingen minipig plasma proteins and brain homogenate.
To evaluate brain penetration, firstly the compound free fraction in whole plasma and brain homogenate was determined by equilibrium dialysis. A 300-fold range in binding affinity to brain homogenate and a 125-fold range in binding affinity to plasma proteins was observed for the reference set. Comparison of rat and Göttingen minipig brain tissue homogenate, as well as human and Göttingen minipig plasma, revealed a strong level of agreement across the reference set (with majority of individual compound data points falling close to the line of unity, as depicted in Figure 3).

Compound brain penetration using Göttingen minipig.
Kp,uu,brain was calculated from the unbound fractions and the in vivo brain to plasma ratios and provided an overview of the distribution into the brain for the reference set. Values ranged from 0.02 for cimetidine to 2.40 for fluoxetine (Figure 4A), demonstrating a high dynamic range covering restricted, partially restricted and unrestricted brain penetration as well as possible active uptake in minipigs (e.g. Kp,uu,brain >>1). A subset of 9 reference compounds were compared with published data for other non-rodent species often used in R&D – namely dog and non-human primate (NHP). Acknowledging that human brain exposure data is sparce, nonetheless a comparison was possible for 4 of the reference compounds (Figure 4B).

Systemic PK in Göttingen minipig – determination of volume of distribution and liver clearance for the reference set.
Plasma PK parameters were calculated for the reference set ofcompounds (n=20) from mean IV plasma concentration-time profiles. The CL and Vss values have been summarized in Table 1 (additional PK-parameters have been included in Langthaler et al. 2022). The total CL and Vss data found for antipyrine, atenolol, cimetidine, diazepam, and verapamil in female Göttingen minipigs (this study) were in good agreement with previously published female Göttingen minipig data; falling within 2-fold for total CL and 2.5-fold for Vss (Figure 5A-B) (Lignet et al. 2016; Patel et al. 2016; Ding et al. 2021). Indicating that robust PK data can be generated in this species. In previously published studies, reference compounds were dosed discretely (one compound per animal). The good agreement shown between cassette and discretely dosed studies was very encouraging and provided greater confidence that a cassette dosing paradigm can be used to characterize PK; which is particularly attractive from a cost and 3R’s perspective in non-rodent species.
 

Prediction of systemic CL based on IVIVE.
The IVIVE model was established using a more diverse reference set comprising of acids, zwitterions, and a broader coverage of human drug-metabolizing enzymes. As such, the reference set was supplemented with an additional 13 compounds (n=33) where published CL values were available for each. IVIVE of CL using the well-stirred liver model was established. The predicted CL scaled from in vitro metabolic stability data (CLint) in hepatocyte was compared to the in vivo CL derived from the PK experiments. The IVIVE had a low prediction bias (AFE = 0.9) and high precision (AAFE = 1.4 and RMSE = 1.5). The strength of relationship was good (R2 = 0.70) with a high percentage of predicted CL values falling within 2-fold of the observed (96%).

4. Discussion

The utilization of Göttingen minipigs in non-rodent PK studies and drug safety evaluations has grown in momentum in recent years (Suenderhauf and Parrott 2013; Suenderhauf et al. 2014; Lignet et al. 2016; Poulin et al. 2019; Ding et al. 2021). In light of potential interspecies variations in drug transporter expression at the BBB, there is an urgent need to verify the extent of brain penetration in minipigs to qualify the Göttingen minipig as an alternative species in brain drug research. To our knowledge this is the first work to be published determining the brain penetration in this species for a diverse set of reference compounds. Included were CNS and non-CNS acting drugs as well as known human P-glycoprotein substrates. As demonstrated in Figure 4A the reference set also spanned a relevant dynamic range (restricted to unrestricted and active brain uptake) providing a solid basis on which to assess brain penetration in Göttingen minipig.

Drugs identified as potential human P-glycoprotein substrates, such as cimetidine, risperidone, and its 9-hydroxyrisperidone metabolite, were shown to have restricted brain penetration (Kp,uu,brain values <0.3). A restricted Kp,uu,brain was also observed for drugs such as atenolol and gabapentin, which are often considered in literature as having low passive membrane permeability (measured in vitro in MDCKII-cells; Langthaler et al. 2024).

Species differences in brain penetration in vivo have been reported by several laboratories (Doran et al. 2012; Sato et al. 2021; Kido et al. 2022). The current study provides an expansive new dataset in Göttingen minipig to facilitate a comparison with other species (Figure 4A-B). A drug’s brain penetration can be influenced by active processes such as uptake or efflux (controlled by transporters pumping drug in or out of the brain, respectively). These processes can differ across species due to transporter substrate affinity as well as transporter expression levels at the BBB. Therefore, brain penetration data was compiled from the literature for a subset of reference compounds allowing a broader comparison across species. Given published porcine P-glycoprotein expression levels were relatively high compared to other species we had initially anticipated that brain penetration could be much lower in minipig. However, there appeared no overall significant difference in the minipig ‘brain penetration signature’ (e.g. trend in Kp,uu,brain values across reference compounds) compared to NHP, dog and human.

The brain penetration for human P-glycoprotein nonsubstrates, such as antipyrine and propranolol, was consistent across NHP and dog as depicted in Figure 4B (also for rat, but data not shown here, see Langthaler et al. 2023). For known human P-glycoprotein substrates the brain penetration across species appeared more variable and compound dependent – for metoclopramide a relatively high Kp,uu,brain was observed in minipig versus other species whereas for risperidone a relatively low value was observed in minipig.

Based on these data the minipig does not present itself as an outlier species for assessment of brain penetration. As such, it could be considered an alternative to dog or NHP in safety studies for development of CNS, as well as non-CNS, acting drugs. In-line with previous findings (Di et al. 2011) brain homogenate binding in the current study (Figure 3B) clearly showed that non-specific binding to brain components was consistent across species (Langthaler et al. 2023). This indicated that rat and minipig binding can be used interchangeably, eliminating the need for testing in multiple species. Drug binding to in plasma can often show species differences (Colclough et al. 2014), influenced by different binding affinities for different plasma proteins. For the current reference set, drug binding in minipig and human plasma was closely aligned.

In pharmaceutical R&D IVIVE of CL across several species is often undertaken to build confidence that CL is predictable from scaled in vitro metabolic stability data and that is appropriate to apply the same scaling approach to make prospective prediction of human CL (Obach 1999; Ito and Houston 2004; Riley et al. 2005; Sohlenius-Sternbeck et al. 2010; Poulin 2013; Francis et al. 2021; Jones et al. 2022). Combining the CL data from this project with existing literature data, we have successfully established an in vitro scaling method to predict in vivo CL in Gottingen minipig for a diverse set of reference compounds. As such minipig can be considered a useful addition to the PK species typically used to assess IVIVE performance underpinning robust prediction of human PK (Langthaler et al. 2022).

5. Conclusions

This PhD research project achieved several important milestones. For the first time, brain penetration has been characterized in minipig for a broad set of reference compounds. We have published an extensive new PK dataset for female Göttingen minipigs and demonstrated the beneficial impact of a cassette dosing paradigm (administering multiple compounds simultaneously at low doses to maximize the reference set tested whilst minimizing the number of animals used). Furthermore, we demonstrated that in vitro free
fraction in rat brain homogenate or minipig could be used with free fraction in Göttingen minipig plasma and in vivo total brain to plasma ratios to estimate the Kp,uu,brain (extent of brain penetration).

Despite noticeable differences in transporter expression levels across species (Figure 2), these variations did not translate into notable species differences in brain penetration in vivo (Figure 4B). In addition to the successful approach for IVIVE of CL (Figure 6) it highlights the potential of the Göttingen minipig as a valuable non-rodent species for evaluating extent of brain penetration and performance of CL scaling methodologies; two important parameters underpinning human PK predictions for CNS drug candidates.

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