The need for more and better research in pediatric pharmacotherapy and the regulation of authorizations for pediatric drugs have noticeably increased the amount of pediatric nonclinical testing. In this section, we present the most recent advances on pediatric research in pigs as well as ongoing investigations in our group.
3.2.1. Nonclinical Safety Models for Small and Large Molecules
Small molecule drugs have traditionally dominated the pharmaceutical panorama, accounting for 67% of all new drugs approved between 1999 and 2008 [
121]. They include any compound with a low molecular weight (less than ~1000 g/mol) manufactured by chemical synthesis. Large molecules or biopharmaceuticals such as (monoclonal) antibodies, recombinant proteins and gene and cell therapies comprise a wide field with respect to the structure, PK and function of these compounds. Even though they represent still a smaller percentage of new drugs approved, the field is rapidly advancing. The pig can potentially be used in nonclinical studies for both types of molecules aiming at pediatric use.
An extensive survey of 24 pharma companies on the use of juvenile animal models in nonclinical trials (1999 to 2008) showed that only 2 out of the 241 drugs included in the survey were tested in minipigs, both of them with dermal preparations for Impetigo and Eczema. In general, findings of this survey depict results that are comparable to adult data for most of the drugs included, novel toxicity in 16 studies and greater exposure (leading to potential toxicity) in about 35% of the cases. More recently, statistics presented by the EMA on PIPs from 2008 to 2016 revealed that 26% of the PIPs contain juvenile animal studies (mostly rat) and none of them used pigs [
122]. Rats are the most commonly used species in juvenile animal studies as they show several advantages, of which some are described below (for a more complete list of (dis)advantages, see [
36]). Rats are in most cases used in earlier repeated dose and developmental toxicity studies during drug development. This facilitates comparisons of toxicity and systemic exposure between juvenile and adult animals. As this is a widely-used species, extensive historical control data are available. They have a large litter size, pups can be easily fostered, they require a small amount of test compound, they are easily housed, transported and managed. However, there are also several disadvantages to this model species. Several organ systems are less developed at birth compared to man, e.g., ADME characteristics often relate poorly to humans preweaning due to the immaturity of the gastrointestinal tract; blood sampling is often a terminal procedure, especially for the youngest age groups; they are usually not suitable for biopharmaceuticals; and their maturation is much faster than that of humans, which makes the detection of distinct windows of vulnerability difficult. The dog was often used in earlier PIPs, especially when a rodent and nonrodent species were required, but nowadays, often only one nonclinical species, usually the rat, is requested by regulatory authorities, and as such, the number of juvenile dog studies has decreased. Its advantages and disadvantages are also extensively discussed in the ICH S11 guidelines [
36]. In brief, the juvenile dog also has the advantage of comparability with adult animals, as this species is often used in adult repeated dose toxicity studies. Pups are relatively large at birth, can be easily handled and postnatal development of most organ systems is relatively similar to that of human infants. However, pups cannot be fostered, they require a larger amount of test compound than rat pups, dogs are seasonal breeders and litter size varies, they are prone to vomiting and drug metabolizing capacity varies with humans. Historical control data are also much more limited compared with rats. Nonhuman primates, and especially cynomolgus and rhesus monkeys, are often considered to be the best representative model for humans, but although they show many similarities with man, they also have some disadvantages as nonclinical model [
36]. Nonhuman primates have a protracted development, which makes studying all developmental stages impossible, and they only have single offspring. Additionally, neonatal NHPs are precocious for some organs compared to humans, e.g., musculoskeletal, respiratory, endocrine and CNS system. With regard to the pig/Göttingen Minipig, most advantages have been addressed before (see
Section 2). Disadvantages are limited and will be discussed in
Section 4. Despite the fact that the piglet appears to be a good translational model for pediatric drug development, only two PIPs including neonatal (2-iminobiotin) and juvenile (concentrate of proteolytic enzymes in bromelain, NexoBrid) minipigs in the nonclinical studies were processed after 2016. Despite the limited use of juvenile pig testing in PIPs, their potential translational value and their advantages over other models are translated into several independent studies collecting information and improving the knowledge on the applicability of the pig in pediatric drug research. For example, among small molecules, studies have been published in recent years addressing the PK of antibiotics, anesthetic drugs and NSAIDs in juvenile pigs. Rifampin was selected as a model compound to provide preliminary proof-of-concept results supporting the use of the juvenile porcine model for nonclinical pediatric PK testing. The pig showed higher similarities in ontogenic changes in PK than other species, including the dog [
123]. The distribution and PK of another antibiotic, cefpodoxime, was successfully studied, in this case focusing on cerebrospinal fluid, in juvenile (10–20 days of age) pigs. The neonatal pig model has been widely used in the field of anesthetic drugs to study different aspects from PK and blood-barrier crossing [
124] to hemodynamic changes after halothane administration [
125] or side effects derived from anesthesia. Some of these effects include neurotoxicity observed in immature patients after anesthesia with propofol, where a pig model was used to identify the underlying mechanisms [
126]. In fact, a pig model for the study of anesthetic-induced developmental neurotoxicity has recently been developed [
127]. As another example of the usefulness of juvenile pig models in drug development, a study on developmental PK and safety of ibuprofen was published in 2019. Authors demonstrated an effect of age on several parameters including clearance and volume of distribution that is consistent with reports in children, although data are limited and somehow inconsistent. Thus, the pig would be a good model for PK of ibuprofen and potentially other NSAIDs in the pediatric population [
128]. Similarly, age-related differences were observed in a desmopressin population PK model. These results contrast with data from human pediatric trials, probably because of a wider age range (neonates to 6 months) in the pig population. Moreover, a different model in pigs (i.e., two compartments, similarly to what was reported in adults) than in children (one compartment) was described. This, together with the limited number of children used in pediatric trials, led the authors to conclude that studies aimed at testing one vs. two-compartments models in children are necessary [
129]. This is, in our opinion, good evidence that the pig can be superior than other model species in representing the human pediatric population, allowing for more accurate dosing in children. Also, results obtained in nonclinical studies with pigs should be combined with data from clinical trials and the data should be revisited when conflicting results are obtained. Despite the efforts to better characterize the neonatal and juvenile pig PK for different drugs, more research is necessary for the development of more accurate models.
Pig models have also potential as nonrodent species in the development of biopharmaceuticals, Europe being particularly interested in further exploration of the model [
130]. So far, most of the data available relate to toxicology studies and nontraditional application routes, such as dermal or intra-ocular. Recently, the pig is gaining interest from a PK point of view: a variety of different molecules have been tested on pigs, including growth hormones, vaccines and several approaches of gene therapy. For example, the efficacy of growth hormone and analogues has been proved in pigs with compromised growth [
131,
132], although PK studies are lacking. The high similarities between the pig and human genome may make the pig a good model for hormonal therapies and thus, research in this field should be encouraged. In agreement with this, a recent study on somapacitan, a growth hormone derivative, showed similar PK and PD behaviors in minipigs, monkeys and hypophysectomized rats [
133], the golden standard animal model for studying growth hormone pharmacology. Pigs have been more extensively used in the development of vaccines. A tuberculosis vaccine was tested in neonatal pigs and a similar immunological response by means of T cells and monocytes to that described in previous studies in infants was observed. This supports further development of the pig as a model with which to test the efficacy of pediatric vaccines [
134], although the level of protection against
Mycobacterium tuberculosis could not be determined in this study. Similarly, juvenile pigs (7-week-old) have been used for efficacy testing of a different (oral) administration route for Influenza A, with promising results [
135]. The pig is a good model for influenza vaccine testing for obvious reasons: the same influenza viruses are endemic in pigs and humans and a similar clinical disease and pathogenesis have been reported [
136], conferring the model an important target (i.e., similar molecular mechanisms participating in the pathogenesis of the disease) and predictive (i.e., response of the model to the pharmacological effects of treatments) validity [
137]. Juvenile pigs were also used in the nonclinical evaluation of a new buccal form of Measles vaccine, with positive results [
138].
A novel research topic within therapeutic research is genetic therapy, which includes a variety of approaches to treat disease by regulating, repairing, replacing, adding or deleting a genetic sequence, as defined by the EMA. Pigs have been used to test toxicity of a high dose of an adeno-associated vector expressing human survival motor neuron protein, together with NHPs. Both species showed neuron toxicity, which raised concerns regarding the potential side effects of this kind of therapy [
139]. Some other works on gene therapy using neonatal and juvenile pigs use specific models of disease, and will therefore be discussed in
Section 3.2.2. Our group aims to fully characterize a slightly different kind of gene therapy, consisting of the use of antisense oligonucleotides (ASOs). ASOs are a promising drug modality that, amongst other mechanisms, inhibit the translation of currently undruggable disease-causing proteins. ASOs are typically oligomers of 12–24 modified nucleic acids designed to target specific mRNA sequences [
140]. Currently, there are no specific guidelines that regulate the authorization of ASO therapy candidates. For this reason, the nonclinical testing strategy of new chemical entities is followed for the safety assessment of ASOs [
141]. Nonhuman primates have been a popular nonrodent choice because of their PK similarities and high genetic homology with humans. However, with the sequencing of the minipig genome [
83,
142], designing cross-reactive ASOs and evaluating pharmacology-related adverse effects became feasible in this porcine strain. The adult Göttingen Minipig has already been characterized to be a suitable alternative model for NHPs in the adult safety assessment of ASO drugs [
143]. Target binding by ASOs was demonstrated and the toxicokinetic behavior in plasma, kidney and liver was similar compared to NHPs [
143]. ASOs are well-absorbed in the circulation following subcutaneous administration. Transiently, they bind to plasma proteins (primarily albumin) across all species and this supports tissue distribution and hinders renal filtration [
144,
145]. Evaluating interactions of future ASO drug candidates with plasma proteins (as well as interactions with other competing drug substrates) in the juvenile minipig is essential since their plasma albumin levels may undergo maturation [
63] and low plasma protein in younger animals can cause changes in drug distribution [
97]. Nonetheless, as observed in different mammalian species, ASOs usually accumulate primarily in the kidney and liver, followed by a long elimination phase. Accumulation-related histopathological changes in these organs, causing nephrotoxicity and hepatotoxicity, are commonly observed in nonclinical testing of ASOs [
146,
147,
148]. Aside from accumulation-related toxicities, nonspecific binding of ASOs to platelet-related proteins (i.e., platelet factor 4 and platelet collagen receptor VI) with corresponding platelet activation induces thrombocytopenia [
149]. Similar effects have been observed in the adult minipig [
143]. As fluctuations in platelet counts and coagulation parameters (prothrombin time, activated partial thromboplastin time) occur in the juvenile conventional pig and Göttingen Minipig [
150,
151], these factors may be of practical importance during pediatric safety testing of ASO drug candidates. Lastly, ASOs do not cross the intact blood-brain barrier (BBB) [
152]. However, no data is available regarding the functional maturation of the BBB in the pig or the extent of access of ASOs into the developing brain of neonatal and juvenile pigs. The BBB undergoes functional postnatal modulation to provide a suitable microenvironment for the developing brain [
153], and this may allow brain penetration of ASOs, resulting in neurotoxicity that is not relevant for humans. Therefore, BBB function in the neonatal and juvenile minipig is currently being investigated in our group in order to ultimately qualify the juvenile Göttingen Minipig as a pediatric safety testing model for ASOs.
Despite the numerous advantages of the pig over other models and the recommendations on selecting the best species based on scientific principles [
29], statistics show a limited inclusion of the pig in PIPs during pediatric drug research. A still-developing characterization of the pig on the one hand and pharmaceutical companies being more used to working with traditional nonrodent species such as dogs and NHP on the other hand, may explain the limited use of pigs in pediatric drug development programs. However, efforts are continuously being made in our and other groups, to better characterize the physiological and pharmacological parameters of the developing pig. A recent review on vehicle systems and excipients tolerated by adult minipigs may also help investigators to more frequently consider the minipig as a nonclinical species [
154], although this information is still lacking for neonatal and juvenile pigs. We believe that more training and facilitating the use of this species in pharmaceutical companies would also help to spread their adoption as nonclinical species in pediatric drug safety assessment. However, the pig occupies a privileged place when it comes to others aspects of welfare in the pediatric population, as will be discussed in the following sections.