Crohn's Disease Is Associated with Decreased CYP3A4 and P

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Crohn's disease is associated with decreased CYP3A4 and P-glycoprotein protein expression Aze Wilson, Bradley L Urquhart, Terry Ponich, Nilesh Chande, James C. Gregor, Melanie Beaton, and Richard B. Kim Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00459 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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Molecular Pharmaceutics

Crohn's disease is associated with decreased CYP3A4 and P-glycoprotein protein expression Aze Wilson§¥γ, Bradley L. Urquhartγ, Terry Ponich¥, Nilesh Chande¥, James C. Gregor¥, Melanie Beaton¥, Richard B. Kim§γ* Divisions of Clinical Pharmacology§ and Gastroenterology¥, Department of Medicine; Department of Physiology and Pharmacologyγ; Western University, London, Ontario, Canada

KEYWORDS CYP3A4, P-glycoprotein, Crohn's disease, intestinal protein expression

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Cytochrome P450 (CYP) 3A4 and P-glycoprotein (P-gp) have broad substrate overlap and are involved in the metabolism and transport of nearly 50% of currently prescribed medications. In the intestine, CYP3A4 and P-gp are co-expressed in the enterocytes at the intestinal villous tip, and act in a coordinated manner to limit drug and xenobiotic oral bioavailability, prior to further metabolism and disposition in the liver. Crohn's disease (CD), a form of inflammatory bowel disease (IBD), introduces a transmural intestinal insult that disrupts the intestinal barrier function; it therefore has the potential to affect intestinal drug metabolism and transport. We hypothesized that individuals with CD have reduced intestinal expression of CYP3A4 and P-gp. We obtained intestinal biopsy samples from individuals with and without CD, and quantified the expression of CYP3A4 and P-gp. When we carried out Western analysis for protein expression, we observed a significant reduction in ileal (45% decrease) and colonic (78% decrease) CYP3A4 protein expression in subjects with CD compared to those without. Similarly, an 85%-reduction in colonic P-gp protein expression was seen in the CD patients. Our data highlight important and novel findings pertaining to CD-associated changes to the intestinal expression of CYP3A4 and P-gp expression of relevance to better predicting substrate drug dosing for patients with CD.


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ABSTRACT GRAPHIC



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INTRODUCTION The intestinal mucosa plays a vital role in human health: it is the key interface for the absorption of nutrients including electrolytes and water as well as an effective barrier against host exposure to environmental pathogens and toxins. It is composed of a monolayer of columnar epithelial cells known as enterocytes, as well as goblet cells, paneth cells and enteroendocrine cells. Cells are arranged in villi, superimposed over the lamina propria and inferiorly bound by the muscularis mucosae 1, 2. The enterocytes, overlaying the lamina propria, are tightly joined by a series of junctional proteins (tight junctions, adherens junctions, desmosomes) and are arranged in a folded pattern, producing crypts and villi on a cytoskeleton framework 3. The lamina propria, the bedrock of the epithelium, houses a number of cell types that carry out a host of important functions for maintaining the integrity of the mucosal layer and its immunological role in barrier function. Fibroblasts, lymphocytes, plasma cells, granulocytes, macrophages and dendritic cells form part of the rich cellular fabric of the lamina propria amidst an extensive vascular network 4. An extension of the barrier function of the intestine, is its contribution to first-pass metabolism5 6, 7. The cytochrome P450 enzyme CYP3A4 is an integral part of the intestinal as well as hepatic first-pass effect. Moreover, among CYP enzymes, it is the most abundantly expressed CYP isoform and accounts for up to 40% and 80% of CYP enzymes in the liver and intestine, mediating the metabolism of nearly 50% of drugs used in clinical practice 8-10. Within the intestine, CYP3A4 is localized to enterocytes found at the tips of villi and is expressed in highest concentrations within the jejunum and ileum 7, 11. P-glycoprotein (P-gp), a member of the ATP-binding cassette protein (ABC) superfamily, is also highly expressed in the GI-tract 12. The coordinated expression of P-gp and CYP3A4 in the GI-tract regulates an individual's 4 ACS Paragon Plus Environment

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systemic exposure to shared substrate drugs through intracellular metabolism and efflux membrane transport. The impact of chronic liver and kidney disease on the expression of hepatic CYP3A4 has been evaluated and reductions in CYP3A4 expression have been observed13-16. Interestingly, studies of the intestinal expression of CYP3A4 and P-gp among patients with intestinal diseases have been limited 17-25. Crohn's disease (CD) is a primary, chronic illness of the intestinal tract that produces transmural, discontinuous inflammatory lesions along the gut's longitudinal access. It is often confined to the small and large intestines, but can be found anywhere from the oral cavity to the most distal colon 26. Hallmarks of CD include disruption of the epithelial cell tight junctions, epithelial cell apoptosis, expansion of the lamina propria with pro-inflammatory cells and the recruitment and activation of pro-fibrotic fibroblasts 27-29. Such changes in barrier function lead to the trademark clinical profile of CD including abdominal pain, profuse diarrhea, intestinal stricturing and fistulization. The impact of CD on expression of both CYP3A4 and Pgp has only had limited evaluation, particularly in an adult population. The aim of this study was to evaluate the intestinal expression of CYP3A4 and P-gp in adult patients with CD compared to non-CD controls.



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EXPERIMENTAL SECTION Subjects: The study protocol was approved by the Western University Health Sciences Research Ethics Board and was carried out in accordance with the Declaration of Helsinki. Written and informed consent was obtained from all participants. Twenty three subjects with CD and 37 nonCD controls were recruited from a single centre in London, Ontario between August 2009 and December 2014. Eligible CD subjects were 18 years of age or older with a history of CD confirmed by previous histopathologic examination. Eligible controls were 18 years of age or older with no clinical history of gastrointestinal pathology or other illness. Subjects in either group taking CYP3A4 or P-gp inducers or inhibitors (with the exception of budesonide) were excluded. Additionally, control subjects were excluded if there was a history of co-morbid illness or surgery requiring any drug therapy. Clinical data pertaining to CD subject age, sex, weight, smoking history, medication history and drug response, diagnosis, disease location, disease activity (based on the clinical scoring index, the Harvey-Bradshaw Index, HBI , where active disease was indicated by a score of 4 or more)30. Demographic data were collected for each non-CD control. All participant data is summarized in Table 1. Study Objectives and Outcomes: The objective of this study was to test the hypothesis that individuals with CD have reduced expression of intestinal CYP3A4 and P-gp protein relative to a healthy population. The primary endpoints were the density ratios of ileal CYP3A4 and P-gp protein to villin in subjects with CD compared to non-CD controls. Secondary endpoints included the density ratios of colonic CYP3A4 and P-gp protein to villin in CD versus non-CD cohorts, as well as the relative


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densities of ileal and colonic CYP3A4 and P-gp protein stratified by CD activity respectively for the CD cohort. Tissue Collection: Tissues samples from the terminal ileum and ascending colon were obtained from all subjects at the time of colonoscopy. Two adjacent biopsies, each 5mm2,were taken from each location and pooled for further analysis. Tissue sampling from the CD cohort was obtained exclusively from non-inflamed areas (based on endoscopic examination) in the presence or absence of active disease. All tissue samples were immediately frozen on dry ice after excision and stored at -80ºC until further use. Protein Quantification: Tissue lysates from flash frozen ileal and colonic biopsies were prepared by sonication in Pierce IP Lysis buffer (Thermoscientific, Rockford, IL) with protease inhibitor cocktail (SigmaAldrich, St. Louis, MO) on ice. Total protein was quantified using the Pierce bicinchoninic acid (BCA) assay (Thermoscientific, Rockford, IL) with bovine serum albumin as an internal standard, normalized to 1000μg/mL. Homogenates were diluted in NuPage LDS sample buffer (Invitrogen, Carlsbad, CA) with 5% β-mercaptoethanol and heated to 95◦C for 5 minutes. Twenty micrograms of isolated protein was separated on a 4-12% tris-acetate gel and transferred to a nitrocellulose membrane. P-gp-over-expressing Caco-2 cells and human liver samples were used as controls for P-gp and CYP3A4, respectively. Blots were blocked overnight at 4ºC in 10% non-fat dried milk in Tris buffer containing 1% Tween 20. Blots were incubated with the primary antibody, 3H8 directed against CYP3A4 (Thermoscientific, Rockford IL, USA; dilution 1:500) and C219 directed against P-gp (Covance, Dedham MA, USA; dilution: 1:500) respectively. Villin (CWWB1) was used as the internal loading control to account for variation 7 ACS Paragon Plus Environment


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in sample preparation and loading (Santa Cruz Biotechnology; dilution 1:500). Villin is a constitutively-expressed intestinal cytoskeleton protein found in the brush border epithelium of the small intestine and colon31, 32. Horse radish peroxidase conjugate-labeled goat anti-mouse IgG was used as a secondary antibody (dilution 1:10,000). Detection was achieved by chemiluminescence with enhanced chemiluminescence western blot detecting reagents (GE Healthcare, Chicago Illinois, USA). Quantification by densitometry was performed using Image J version 1.48 software. CYP3A4 and P-gp protein expression were represented as the ratios of densities of the target protein to villin. Statistical Analysis: All statistical analyses were carried out in Graphpad Prism 5 and SPSS, version 21. The distribution of the protein expression data was assessed using the D'Agostino & Pearson omnibus normality test. The Mann-Whitney test was used to assess any statistically significant differences in protein expression between two groups, while the Kruskal-Wallis test was used to assess any statistically significant differences in protein expression between three or more groups. Dunn's multiple comparison test was used to conduct a post-hoc analysis of data evaluated by the Kruskal-Wallis test. The coefficient of variation (CV) was used to calculate inter-individual variability in CYP3A4 and P-gp expression. A multiple linear regression model was used to evaluate the effect of multiple covariates (age, sex, disease presence, disease activity) on inter-individual variation in ileal and colonic CYP3A4 and P-gp relative densities. Additionally, the Spearman Rank test was used to assess correlations between the expression of CYP3A4 and P-gp among the CD patients and controls respectively 33. A Kruskall-Wallis test with Dunn's Multiple comparison test was used to define the p-value for statistical significance.


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RESULTS The effect of disease presence and activity on intestinal CYP3A4 protein expression Demographic data are presented in Table 1. All non-CD controls were non-smokers, without co-morbid illnesses or history of prior surgery. Additionally, they were not taking any medication. The expression of intestinal CYP3A4 protein is presented as the density of CYP3A4 protein on western blot relative to the density of villin (Figure 1a and c). The protein expression patterns of CYP3A4 relative to villin in ileum and ascending colon are presented for CD and non-CD populations in Figure 1b. CYP3A4 content was higher in the ileum than in the colon (CD, 5-fold higher, p

Crohn's Disease Is Associated with Decreased CYP3A4 and P

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