Wistar and Sprague Dawley are the most common strains of rat used in pharmaceutical research and are used interchangeably in pre-clinical drug development. No studies have assessed whether Wistar and Sprague Dawley rats are equivalent in the gastrointestinal factors that influence oral drug absorption, specifically in relation to intestinal transporters. Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) are two reliable methods for quantifying intestinal protein levels with their own distinct advantages and limitations. In this study, P-glycoprotein (P-gp), a key efflux transporter, was quantified using ELISA and LC-MS/MS along the complete intestinal tract of male and female Wistar and Sprague Dawley rats. This work presents that Sprague Dawley rats have innately higher baseline P-gp expression than Wistar rats. Significant sex differences in P-gp expression were identified in the jejunum, ileum and colon between male and female Wistar rats using both techniques, with males exhibiting higher P-gp levels. Sprague Dawley rats showed no sex differences in P-gp expression through ELISA and LC-MS/MS. Both methods demonstrated similar trends for P-gp quantification, but ELISA could offer faster data acquisition. Our findings report significant sex differences between the strains and highlight that Wistar and Sprague Dawley rats are not equivalent in their P-gp expression. As humans exhibit distinct sex differences in intestinal P-gp levels, Wistar rats may therefore be a more suitable pre-clinical animal strain to model oral drug absorption of P-gp substrates in male and female subjects.
This chapter focuses on special populations often overlooked in drug development to better understand why drug absorption may change in these populations. Ethnicity can influence the variability in pharmacokinetics, pharmacodynamics, safety and efficacy of a drug. Fatty food will induce the secretions of bile salts and phospholipids, which together with the products of lipolysis can form micelles with drugs, improving the drug solubility of lipophilic drugs. The changes that occur along the length of the gastrointestinal tract in celiac disease are also seen to correlate with disease severity and can impair normal patterns of drug absorption. Reduced drug bioavailability could also be caused by fat malabsorption syndromes prevalent in the majority of HIV patients. Drug development sometimes falls short in considering normal circumstances, such as sex differences, the state of pregnancy, ethnic differences, the impact of ageing and comorbidities on oral drug performance.
Intestinal efflux transporters affect the gastrointestinal processing of many drugs but further data on their intestinal expression levels are required. Relative mRNA expression and relative and absolute protein expression data of transporters are commonly measured by real-time polymerase chain reaction (RT-PCR), Western blot and mass spectrometry-based targeted proteomics techniques. All of these methods, however, have their own strengths and limitations, and therefore, validation for optimized quantification methods is needed. As such, the identification of the most appropriate technique is necessary to effectively translate preclinical findings to first-in-human trials. In this study, the mRNA expression and protein levels of the efflux transporter P-glycoprotein (P-gp) in jejunal and ileal epithelia of 30 male and female human subjects, and the duodenal, jejunal, ileal and colonic tissues in 48 Wistar rats were quantified using RT-PCR, Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A similar sex difference was observed in the expression of small intestinal P-gp in humans and Wistar rats where P-gp was higher in males than females with an increasing trend from the proximal to the distal parts in both species. A strong positive linear correlation was determined between the Western blot data and LC-MS/MS data in the small intestine of humans (R2 = 0.85). Conflicting results, however, were shown in rat small intestinal and colonic P-gp expression between the techniques (R2 = 0.29 and 0.05, respectively). In RT-PCR and Western blot, an internal reference protein is experimentally required; here, beta-actin was used which is innately variable along the intestinal tract. Quantification via LC-MS/MS can provide data on P-gp expression without the need for an internal reference protein and consequently, can give higher confidence on the expression levels of P-gp along the intestinal tract. Overall, these findings highlight similar trends between the species and suggest that the Wistar rat is an appropriate preclinical animal model to predict the oral drug absorption of P-gp substrates in the human small intestine.
The older population represents a growing and heterogeneous subset of the population. Developing formulations for geriatric patients involves multiple challenges considering the prevalence of multi-morbidities requiring treatment with multiple therapies. Advancing age causes physiological changes, which can impact the drug pharmacokinetic performance of administered drugs. Dysphagia, malnutrition and impairments in cognition, dexterity, and vision contribute to lower medication adherence. Dose-flexible, easy-to-swallow formulations with accessible packaging are required that are acceptable to the patients. Researchers are designing patient-centric dosage forms such as orodispersibles, mini-tablets, and oral liquids to meet the needs of the ageing population. Furthermore, the digital revolution provides tools such as wearable devices and artificial intelligence to tailor delivery of drugs to our ageing population.
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