Animal Utilization in Drug Development: Clinical, Legal,
7.5 THE EFFECT OF SPECIES DIFFERENCES ON STUDY RESULTS
Animal models have been applied in the study of the anatomical, physiolog- ical, and biochemical differences in the gastrointestinal (GI) tract of humans.
However, interspecies differences significantly influence the interpretation and accuracy of the results [10].
The following are typical cases.
7.5.1 Pain Cognition/Recognition or Expression
The level of pain tolerance or pain threshold varies across the various ani- mal species. Those that are more resilient express pain at higher thresholds.
Horses have low thresholds for pain when compared to a resilient ass with a high threshold [11]. Intuitively, data from these animal species are not comparable and have been taken less seriously. Dogs have been classified as resilient and as such were not given analgesics after major surgery. But the passing of the Laboratory Animal Welfare Act of 1966, P.L. 89-544 stimu- lated the acceptance and administering of pain relieving medications for dogs [12]. The study of conditions such as colic can proceed to advanced irreversible stages before they are detected. Due to lack of a clear indicator of pain in fish, the effect of pain relieving drugs is judged by the ease of handling of fish [13]. The proposals concerning the use or theories that are fundamental to the handling of fish have not been scientifically justified.
There is a belief that fish have fewer C fibers (pain sensors) than mammals,
which are thought to transmit longer signals due to injury. This proposition has led to a conclusion that the aquatic adaptation would respond differ- ently to pain when compared to other mammals [14,15].
7.5.2 Effect of Dosage Regimen
Doses expressed by body weight could result in marked variation in dosage between species. For example, the dosage for aspirin is approximately 40 times higher in cattle than in cats, whereas the effective dose for morphine is 10-fold lower in cats than in dogs. A depolarizing neuromuscular blocker, named suxamethonium (succinylcholine), is 40 times lower in cattle than in cats. As a consequence, extra caution is expected when interspecies com- parison is made. Genetic variation in similar mouse models leads to a subtle divergence in physical and physiological traits.
7.5.3 Effects of Genetic Modification
Intraspecies variation, referred to as variation within the same species, is present in genetically modified animals. Intraspecies genetic manipulation could lead to variable phenotypes as there are reasonable differences in the genetic makeup. Certain phenotypic changes resulting from genetic modi- fication are indirect effects and these might not be easily determined. For example, certain physical and physiological (or phenotypic) traits could sur- face due to genetic manipulation, which are not the same in their human counterparts. A tumor gene knockout could lead to a separate range of tumors in comparison to that of humans [16,17].
7.5.4 Cytochrome P450 (CYP)-Mediated Drug Metabolism and Inhibition
Cytochrome P450 (CYP) enzymes are widely recognized due to their role as drug metabolizing enzymes. They act by binding two atoms of oxygen resulting in the formation of a water molecule and a metabolite with a higher polarity than the parent drug. There are subtle differences in amino acid composition and sequence arrangement, which contribute to differ- ences in function of the CYP isoforms and, consequently, differences in drug metabolism for the highly metabolized drugs. For drugs that are not metabolized at all, interspecies differences are minimal and could be ex- trapolated to another species through allometry scaling.
In comparison to humans, rat and mouse genomes possess higher num- bers of Phase I metabolism genes while other model organisms have rela- tively similar numbers [18,19].
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Out of the 57 human cytochrome P450 genes, 23 are associated with ADMET, whereas the minipig and Duroc pig contain 19 and 18 ADMET- related CYPs, respectively, with minipigs showing a comparable level of abundance and activity of drug metabolizing enzymes as humans [20], but total P450 activity is lower for beagles relative to humans.
Body weight plays a major role in the abundance of hepatic enzymes.
For instance, CYP/gram body weight is higher in small animals than in humans [21]. It also directly correlates with the metabolic rate; higher in small animals than in humans. There are variable CYP expression patterns in humans, rats, mice, dogs, and monkeys. CYP1A1 and -1A2 are strongly conserved among the species but CYP1A1 is minimally expressed in the liver of all species. In human and animal species, CYP1B1 is the only gene product of the CYP1B subfamily. In humans and rodents, CYP2A is ex- pressed in liver and extrahepatic tissues. The substrate specificity of human CYP2A6 and CYP2A enzymes in animals are different. CYP2C is the larg- est and most complicated subfamily across the laboratory animal models, such as, rat and mouse; and humans. CYP2C is present in the liver of rodent and nonrodent species, and its expression in extrahepatic tissue is isoform specific but human forms have different substrate specificities. The CYP2D family displays a genetic polymorphism that plays a role in human drug metabolism. In addition such a trait was observed in rats [22,23]. CYP2E1 is expressed in the liver and in many extrahepatic tissues of numerous animal species. CYP2E1 is only involved in the metabolism of a handful of drugs.
A remarkable degree of conservation suggests success in the extrapolation between species. The rat shows superiority as a potential animal model for humans for CYP2E1. CYP3A is the isoform most commonly known to exist in the species; however, various CYP3A isoforms expressed show dif- fering substrate specificities making their suitability for extrapolation to hu- mans impracticable. No animal species possesses all the qualities of a perfect laboratory model for drug development. Thus, in vitro studies with liver microsomes and hepatocytes should inform the selection of animals to be used in each case [24].
Similarities in the CYP isoforms are important when choosing animal models for preclinical studies. In the model animal, a candidate drug should show an acceptable range of bioavailability, a comparable metabolic profile (similarity of the cytochrome P450), and systematic exposure more than that of humans. Also the distribution and elimination mechanism should be sufficiently similar. It is important that the phylogenetic status in relation to humans is often backed up with justifiable physiological processes.