Katrina L. Mealey

Because P‐glycoprotein (P‐gp) influences the disposition of substrate drugs, regulatory agencies recommend ascertaining the P‐gp substrate status of active pharmaceutical ingredients (API's) intended for humans. Transwell‐based assays using P‐gp‐overexpressing human cell lines are routinely used to predict the API's P‐gp substrate status. Although ABCB1‐1Δ‐mediated P‐gp deficiency contributes to serious canine adverse drug reactions, assessment of canine P‐gp status is not currently recommended by regulatory agencies for canine API's. We have previously employed a competitive efflux assay to assess drugs as canine P‐gp substrates as it affords some advantages over transwell assays. Our objective was to preliminarily compare results generated from these two methods using historical data for transwell assays. Eleven drugs were assessed as canine P‐gp substrates by both methods, with two identified as non‐P‐gp substrates (erythromycin, propranolol) and nine identified as canine P‐gp substrates (clarithromycin, daunorubicin, digoxin, etoposide, paclitaxel, quinidine, ritonavir, verapamil, vinblastine). Erythromycin was identified as a canine P‐gp substrate by competitive efflux assay at roughly 10‐fold higher concentrations than was used for transwell assays. Concordance of these methods assessing canine P‐gp substrate status using historical data supports the need for head‐to‐head studies comparing these two methods for identifying canine P‐gp substrates.

Objective: Because dogs with P-glycoprotein (P-gp) deficiency experience enhanced susceptibility to adverse reactions caused by P-gp substrate drugs, this study’s purpose was to validate and employ a competitive efflux assay to assess clinically relevant drugs as P-gp substrates. Methods: A competitive efflux assay employing a cell line expressing canine P-gp was used in these studies. Intracellular fluorescence was detected flow cytometrically following the coincubation of experimental drugs with 2 intrinsically fluorescent P-gp substrates (rhodamine 123 or calcein AM) that bind different sites within the P-gp binding pocket. The fluorescence intensity was determined for cells treated with each experimental drug and fluorescent P-gp substrate combination and compared to the fluorescence intensity of cells treated with rhodamine 123 and calcein AM alone. Additionally, assay validation parameters were assessed. Results: Mean fluorescence intensity (MFI) ratios < 2 indicate non–P-gp substrates [cisplatin (negative control), ropinirole, doxycycline, and trilostane]; MFI ratios from 2 to 5 indicate weak canine P-gp substrates (apomorphine, capromorelin, and enrofloxacin); MFI ratios > 5 but < 10 indicate moderate P-gp substrates (trazodone); and MFI ratios > 10 indicate strong P-gp substrates [loperamide (positive control), and moxidectin]. Assay specificity, precision, and linearity were within established acceptance criteria. Conclusions: Several clinically relevant drugs were identified as P-gp substrates. The competitive efflux assay is a useful tool for screening drugs as canine P-gp substrates. Clinical Relevance: These results imply that P-gp–deficient dogs may be at increased risk of adverse effects if treated with apomorphine, capromorelin, enrofloxacin, or moxidectin, depending on the dose administered.

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This mini‐review describes the pathophysiology of the disrupted hypothalamic–pituitary–adrenal axis in dogs, and probably cats, with the MDR1 (ABCB1) mutation, ABCB1‐1Δ in dogs and ABCB1 1930‐1931del TC in cats. Clinical consequences in affected animals are discussed and therapeutic interventions to consider are presented.

P‐glycoprotein (P‐gp) greatly impacts substrate drug disposition, so much so that regulatory agencies recommend ascertaining the P‐gp status of active pharmaceutical ingredients (APIs) intended for human use. Arguably, the P‐gp status of drugs intended for canine patients is equally, if not more, important. Our research objectives were to assess whether human P‐gp substrate data can predict canine P‐gp substrate status and to explore the three previously reported binding sites within the P‐gp binding pocket, the H‐, R‐, and P‐sites. Competitive efflux assays employing cell lines expressing canine or human P‐gp were used to compare the degree of overlap or independence of the three binding sites in canine versus human P‐gp using site‐specific fluorescent P‐gp substrates rhodamine 123, calcein AM and Hoechst 33342. Because calcein AM can also be transported by multidrug resistance protein 1 (MRP1), experiments were performed to assess its potential influence on calcein AM efflux studies. Results indicate that: (i) MRP1 is either a non‐factor or negligible factor for cells expressing canine or human P‐gp respectively; (ii) determining an API's P‐gp binding site may provide clinically relevant information; and (iii) use of human P‐gp substrate data as a proxy for canine P‐gp substrate data will often prove inaccurate.

Introduction: The drug transporter P-glycoprotein (P-gp) influences drug disposition by playing key roles in limiting brain penetration and enhancing biliary excretion of substrate drugs. Guidance documents from U.S. and European regulatory agencies recommend that manufacturers determine the P-gp substrate status of new medications intended for human patients. The rationale is that P-glycoprotein-mediated drug–drug interactions may cause serious adverse drug events. Unfortunately, the same regulatory guidance does not encompass new feline drugs even though a P-gp knockout mutation (ABCB11930_1931del TC) is present in a subpopulation of cats. Recent reports of a novel macrocyclic lactone, eprinomectin, causing neurological toxicosis in cats homozygous for ABCB11930_1931del TC, imply it is a feline P-gp substrate, but definitive data is lacking, It is intriguing that neurological toxicity has also been reported in a small number of cats treated with amlodipine, capromorelin, and cisapride, however their MDR1 genotypes are unknown. Methods: A competitive efflux assay and feline P-gp expressing cell line were used to assess the P-gp substrate status of thirteen clinically important drugs used in cats. Results: Ten drugs, including eprinomectin, were determined to be substrates for feline P-gp while three drugs were not. Discussion: This information will help improve drug safety for cats with intrinsic (ABCB11930_1931del TC) and acquired P-gp deficiency. Further, this type of assay may be useful for screening feline drug candidates during the drug approval process.

The feline MDR1 mutation (ABCB11930_1931delTC) has been associated with neurological toxicosis after topical application of eprinomectin products labeled for feline use. Information was collected from veterinarians who submitted samples for ABCB11930_1931delTC genotyping. In most cases, the submission form indicated an adverse event involving eprinomectin, in other cases submitting veterinarians were contacted to determine whether the patient had experienced an adverse drug event involving eprinomectin. If so, additional information was obtained to determine whether the case met inclusion criteria. 14 cases were highly consistent with eprinomectin toxicosis. Eight cats were homozygous for ABCB11930_1931del TC (3 died; 5 recovered). Six cats were homozygous wildtype (2 died; 4 recovered). The observed ABCB11930_1931delTC frequency (57%) was higher than the expected frequency (<= 1%) in the feline population (Fisher Exact test, p < .0001). Among wildtype cats, four were concurrently treated with potential competitive inhibitors of P-glycoprotein. Results indicate that topical eprinomectin products, should be avoided in cats homozygous for ABCB11930_1931delTC. This is a serious, preventable adverse event occurring in an identifiable subpopulation treated with FDA-approved products in accordance with label directions. Acquired P-glycoprotein deficiency resulting from drug interactions may enhance susceptibility to eprinomectin-induced neurological toxicosis in any cat, regardless of ABCB1 genotype.

Phytocannabinoid‐rich hemp extracts containing cannabidiol (CBD) and cannabidiolic acid (CBDA) are increasingly being used to treat various disorders in dogs. The objectives of this study were to obtain preliminary information regarding the in vitro metabolism of these compounds and their capacity to inhibit canine cytochrome P450 (CYP)‐mediated drug metabolism and canine P‐glycoprotein‐mediated transport. Pure CBD and CBDA, and hemp extracts enriched for CBD and for CBDA were evaluated. Substrate depletion assays using pooled dog liver microsomes showed CYP cofactor‐dependent depletion of CBD (but not CBDA) and UDP‐glucuronosytransferase cofactor‐dependent depletion of CBDA (but not CBD) indicating major roles for CYP and UDP‐glucuronosytransferase in the metabolism of these phytocannabinoids, respectively. Further studies using recombinant canine CYPs demonstrated substantial CBD depletion by the major hepatic P450 enzymes CYP1A2 and CYP2C21. These results were confirmed by showing increased CBD depletion by liver microsomes from dogs treated with a known CYP1A2 inducer (β‐naphthoflavone) and with a known CYP2C21 inducer (phenobarbital). Cannabinoid‐drug inhibition experiments showed inhibition (IC₅₀ = 4.6–8.1 μM) of tramadol metabolism via CYP2B11‐mediated N‐demethylation (CBD and CBDA) and CYP2D15‐mediated O‐demethylation (CBDA only) by dog liver microsomes. CBD and CBDA did not inhibit CYP3A12‐mediated midazolam 1′‐hydroxylation (IC₅₀ > 10 μM). CBD and CBDA were not substrates or competitive inhibitors of canine P‐glycoprotein. Results for cannabinoid‐enriched hemp extracts were identical to those for pure cannabinoids. These in vitro studies indicate the potential for cannabinoid–drug interactions involving certain CYPs (but not P‐glycoprotein). Confirmatory in vivo studies are warranted.

"Investigate, discover, solve, repeat" published on 10 Jun 2023 by American Veterinary Medical Association.

The P‐glycoprotein (P‐gp) substrate status of antineoplastic drugs intended for veterinary patients is an important characteristic to define for two reasons. First, neoplastic cells expressing P‐gp can actively efflux drugs that are P‐gp substrates curtailing their efficacy. Second, antineoplastic drugs tend to have a narrow therapeutic index. Antineoplastic drugs that are P‐gp substrates can cause severe adverse reactions in animals with P‐gp dysfunction such as dogs with ABCB1‐1Δ and cats with ABCB11930_1931del TC. Animals with P‐gp dysfunction experience greater overall exposure to P‐gp substrate drugs due to mechanisms such as increased intestinal absorption, decreased biliary clearance and greater central nervous system penetration compared with animals with normal P‐gp function. Accordingly, knowing the P‐gp substrate status of antineoplastic drugs is an important safety consideration prior to use in canine or feline cancer patients. This study used a cell line overexpressing canine P‐gp to assess the P‐gp substrate status of verdinexor. Based on both a cytotoxicity assay and a competitive flow cytometry assay verdinexor is not a substrate for canine P‐gp.