What is the study about?
To stay compliant with Lundbeck’s high standards of animal ethics and welfare, we wanted to explore the possibility of oral dosing of a gelatin formulation as an alternative to conventional oral gavage. The basic thought was to “gelatinize” the solution for oral gavage and investigate the feasibility of dosing this to minipigs by voluntarily ingestion. The study was planned and conducted successfully as a cross-functional effort.
Why is it important?
Oral gavage is a stressful procedure for pigs as the pig must be caught, fixated in order for the oral gavage tube to be placed, and restrained throughout the procedure. The oral gavage procedure may lead to physiological stress (Balcombe et al. 2004; Ellegaard et al. 2010) and the animals often become more aversive towards humans. It can also be a stressful procedure for the animal technicians as they must use force to restrain the animals, which in the long run lowers their motivation to do the work, with the risk of compassion fatigue. A typical oral gavage procedure requires at least three persons for fixating, placing the tube and dosing the animal. This procedure takes a few to five minutes.
From a practical point of view, the Danish work environment legislation does not allow heavy lifting (>12.5 kgs) (Dansk AT vejledning), which in practice limits the use of the minipigs for PO procedures for longer periods, as by approximately five months of age the minipigs usually weigh around 12.5 kg (Ellegaard minipig background data).
To improve animal welfare, improve working environment, and save resources, the usage of gelatin formulation was proposed as a new approach for oral administration.
Study setup
Five marketed therapeutic compounds were chosen as model compounds to compare the pharmacokinetics of dosing an oral solution via oral gavage and as an oral gelatin formulation. Selection of the compounds was based on their properties in terms of intestinal solubility and permeability, according to the Biopharmaceutics Classification System. The study was set up in a cross-over design in which the minipigs were given similar doses (mg/kg) of each of the compounds as both an oral solution and an edible gelatin formulation with a short wash-out period between dosing. In total, the study was conducted over six weeks of dosing both gelatin and oral gavage every week (Table 1).
The cross-over study design for assessing peroral gavage vs. peroral gelatin formulation with listing of test compounds and doses.
The gelatin formulations were prepared by gelatinizing the solution used for dosing via oral gavage. Due to solubility, one compound (griseofulvin) had to be formulated as a suspension with methylcellulose. The solutions and the suspension were mixed with a high-concentration (15% W/V) bovine gelatin solution that was liquified by melting (max 60 deg. C). Subsequently, the gelatin-containing mixtures were portioned out in 30 mL plastic-cups and solidified overnight at 5-10°C. A blue colorant was also added to the gels to facilitate visual inspection of the surroundings and the mouths of the minipigs for any gel formulation not swallowed (Figure 1). If some of the gel was not ingested, it was collected and weighed to correct the actual given dose.
Three oral gelatin formulations evaluated for potential precipitation of compound on a light-box.
Training the pigs for dosing
As the minipigs were not used to eating gelatin, they had to be trained to eat the gelatin with minimal chewing to ensure that they ate most – and preferably all - of the gelatin.
To reduce the time needed for training, the animals were ordered as habituated to human contact and touching at Ellegaard Göttingen Minipigs A/S. Furthermore, the least fearsome pigs, willing to interact with humans, were selected. This is something Ellegaard provide as a service.
When the animals arrived at our facility, the transport cages were situated at the entry of the pig pen and opened so the pig could enter the pen when ready. Feed was scattered on the floor in the pen as a teaser.
Socialization to our staff was initiated on the first day. Contact was attempted by going into the pen, kneeling, and waiting for the pig to come forward, but in such a way as to make sure the minipig did not feel cornered without an option to escape. However, as pigs are curious, they usually make contact either right away or within the first few days.
Working with positive reinforcement training, goodies were provided when the minipigs were doing as desired. It was important to select goodies that the minipigs liked and which most likely would not interfere with the study purpose. Also, the goodies should not provide too much energy, as the pigs might otherwise gain too much weight. We tried yoghurt, apples, and juice but after a bit of habituation, carrots seemed to best fulfil the criteria for goodies that the minipigs liked.
Thereafter, the positive reinforcement training was started by using a whistle to bridge a wanted behaviour to the carrot reward. When the pig had learned this, and was able to follow a target-stick, a vascular access surgery was performed to allow for easier repeated painless blood sampling from the minipig. This may also be achieved by a per-cutaneous catheter for single dosing. In our experience, under these circumstances the pigs are ready to participate in a study 21 days after arrival in our facility.
The pigs were trained to swallow the gelatin formulation before getting a piece of carrot. Current experience with training and dosing with the gelatin formulation was highly positive. Up to five gels could be administered within 10 min, and dosing and blood sampling was handled by one animal technician, in contrast to the at least three technicians required for oral gavage.
Results from proof-of-Concept study
Data from the comparison of dosing formulations to the minipigs, showed that when corrected for the actual doses (mg/kg), the two formulations performed similarly with regards to tmax, Cmax, and area-under-the-curve (AUC). One compound (griseofulvin) was dosed as a suspension at low concentration and here the two formulations also showed similar PK profiles (see Figure 2A and 2B for plasma profiles for paracetamol and griseofulvin, respectively).
Plasma concentration profiles for paracetamol (A) and griseofulvin (B) following oral dosing as either gavage (red symbols) or with gelatin formulation (blue symbols).
Which challenges have you met during the study?
Formulation aspects
One challenge we experienced was regarding poorly soluble compounds, where it is often required to add solubility enhancing excipients to dissolve the entire dose. Not all solubility enhancing excipients were found to be compatible with the gelatin. One excipient, PEG400, was found to disrupt the integrity of the gelatin formulation and cause precipitate. Therefore, development of a gelatin formulation is not limited to identifying the most appropriate solubility enhancing excipient, but also to thoroughly assess the effect of excipients on the gelatin formulation, including precipitation of the drug compound and viscosity changes of the gelatin.
A further challenge was that the gelatin formulations had to be prepared a day in advance and thus the animal bodyweights had to be assessed based on last measured weight and growth curves. This required detailed communication between gel manufacturer and animal facility.
In some instances, it was observed that not all the gelatin formulation was ingested by the pig, and any uneaten gelatin was collected and weighed to correct for the actual dose given. This entailed some extra requirements for documentation in the animal facility during dosing and appropriate corrections for actual doses when doing pharmacokinetic calculations.
Minipig habituation and training
Initially, the pigs were not willing to eat the gelatin, nor did they find it intersting. Several attempts were made to increase the curiosity of the pigs by providing frozen gelatin or adding artificial flavours (strawberry, caramel, and vanilla), but we eventually succeeded by adding shredded carrots into the gelatin. In the beginning, a lot of carrot was added, but for each training session the amount was decreased, and nothing was actually needed when fully trained.
Due to the large cross-over study conducted over 6 weeks, and the pigs not being allowed a weight of more than 12.5 kgs, we ordered the pigs quite young (4 kg ~1.5 months old). The younger minipigs were more apprehensive, compared to the older minipigs that we normally order (8 kg~4 months old), and the training had to be adapted with more time needed for socialization.
Due to the young age, the housing area was required to be warmer (28-30 oC). By coincidence, we found out that the increased room temperature caused the gelatin to melt after some hours. This could be one reason why at first the pigs did not seem interested in the gelatin, as the gelatin was provided on the floor, thinking that the pigs would eat it by themselves. But either the gel melted before they got to eat it, or they just played with the (melted) gel. We actually do not know, but if they start perceiving the gelatin as a toy, it seems difficult to re-train them to eat it if they first consider the gel as enrichment.
Whether it is the younger age or the method for habituation to the gel, that made the pigs eating the gelatin more troublesome than expected in the beginning, is yet to be elucidated.
What is next step?
It is the plan to proceed with investigating the feasibility of using gelatin formulations as an alternative to oral gavage. It seems necessary to get more experience with training the minipigs and continuously refine the positive reinforcement training. Especially, best practices for training pigs to eating the gelatin is important to explore further.
Furthermore, investigating the pharmaceutical limitations of the gelatin formulation is essential. Our next step is to enhance the knowledge of formulating test compounds with different physico-chemical properties, and to explore excipients which may be used to aid in these formulations. For instance, stabilization of a suspension in a gelatin formulation may be challenged by lack of homogeneity. If compound particles sediment at the bottom, it hinders dose-adjustment to the animal bodyweight by dividing the gelatin formulation ( Figure 3).
Compound dispersed as solid particles (non-dissolved) in a gelatin matrix. The illustration demonstrates the sedimentation of compound at the bottom, i.e., as an inhomogeneous suspension.
Application of gelatin formulation mainly seems relevant for PK studies and single dose studies. The gelatin formulation is currently not considered relevant for toxicology studies as during these studies, with higher doses and sometimes longer duration, the pigs will expectedly experience adverse effects of the test compounds and therefore may not feel as hungry or curious as normal, which can affect the willingness of the pigs to eat the gelatin.
Any learnings you would like to share?
We found that it paid off to be creative if the pigs did not like what was presented to them. As described above, adding carrots to the gel seemed to be a solution to train the pigs to eat the gelatin. Also, in the beginning, the gel was cut into smaller pieces and presented, and over time the gelatin pieces could be made increasingly larger.
The gelatin for training purposes may be manufactured in larger batches and kept in the freezer to prolong durability and shape. However, if drugs or compounds are added to the gelatin, stability analysis must be made to ensure the compound is not affected by the freezing and thawing processes.
REFERENCES
- Ellegaard Göttingen Minipigs A/S, Göttingen Minipigs background data minipigs.dk/about-gottingen-minipigs/background-data
- Balcombe, Jonathan P.; Barnard, Neal D.; Sandusky, Chad. Laboratory Routines Cause Animal Stress. Journal of the American Association for Laboratory Animal Science, Volume 43, Number 6, November 2004, pp. 42-51(10) ingentaconnect.com/content/aalas/jaalas/2004/00000043/00000006/art00009
- Danish Working Environment Authority, WEA guideline D3.1 Lifting, Pulling and Pushing - Arbejdstilsynet (at.dk) at.dk/en/regulations/guidelines/lifting-pushing-and-pulling-d-3-1/ (In English) / at.dk/regler/at-vejledninger/loeft-traek-skub-d-3-1/ (In Danish)
- Göttingen Minipigs Magazine | #66 | Autumn 2023. Page 22. minipigs.dk/about-us/gottingen-minipigs-magazine
- Lars Ellegaard, Andrew Cunningham, Sandra Edwards, Nanna Grand, Timo Nevalainen, Mark Prescott, Teun Schuurman, and under the auspices of the Steering Group of the RETHINK Project. Welfare of the minipig with special reference to use in regulatory toxicology studies. Journal of Pharmacological and Toxicological Methods 62 (2010) 167–183
