A functional model for feline P-glycoprotein.

P-gp (ABCB1) belongs to the group of export transporters that is expressed in various species at biological barriers. Inhibition of P-gp can lead to changes in pharmacokinetics of drugs (drug-drug interactions), which can lead to toxicity and adverse side effects. This study aimed to establish a functional assay to measure the inhibitory potential of veterinary drugs on feline P-gp by means of fluorescence-associated flow cytometry of feline lymphoma cells. In this model, PSC833 and ivermectin potently inhibited P-gp function; cyclosporine and verapamil moderately inhibited P-gp function, whereas ketoconazole, itraconazole, diazepam, and its metabolites had no effect on P-gp function. This model can be used for testing the inhibitory potency of (new) drugs on feline P-gp.

A longitudinal study of ABC transporter expression in canine multicentric lymphoma.

Canine lymphoma is typically treated with a doxorubicin-based multidrug chemotherapy protocol. Although this is often initially successful, tumour recurrence is common and frequently refractory to treatment. Failure to respond to chemotherapy is thought to represent drug resistance and has been associated with active efflux of cytostatic drugs by transporter proteins of the ATP-binding cassette (ABC) family, including P-glycoprotein (ABCB1), MRP1 (ABCC1) and BCRP (ABCG2). In this study, ABC transporter mRNA expression was assessed in 63 dogs diagnosed with multicentric lymphoma that were treated with a doxorubicin-based chemotherapy protocol. Expression of ABCB1, ABCB5, ABCB8, ABCC1, ABCC3, ABCC5 and ABCG2 mRNA was quantified in tumour samples (n = 107) obtained at the time of diagnosis, at first tumour relapse and when the tumour was no longer responsive to cytostatic drugs while receiving chemotherapy. Expression data were related to patient demographics, staging, treatment response and drug resistance (absent, intrinsic, acquired). ABC transporter expression was independent of sex, weight, age, stage or substage, but T cell lymphoma and hypercalcaemia were associated with increased ABCB5 and ABCC5 expression, and decreased ABCC1 mRNA expression. Drug resistance occurred in 35/63 (55.6%) dogs and was associated with increased ABCB1 mRNA expression in a subset of dogs with B cell lymphoma, and with increased ABCG2 and decreased ABCB8, ABCC1 and ABCC3 mRNA expression in T cell lymphomas. ABC transporter expression in the pre-treatment sample was not predictive of the length of the first disease-free period or overall survival. Glucocorticoids had no effect on ABC transporter mRNA expression. In conclusion, drug resistance in canine multicentric lymphoma is an important cause of treatment failure and is associated with upregulation of ABCB1 and ABCG2 mRNA.

Multi-drug resistance in a canine lymphoid cell line due to increased P-glycoprotein expression, a potential model for drug-resistant canine lymphoma.

Canine lymphoma is routinely treated with a doxorubicin-based multidrug chemotherapy protocol, and although treatment is initially successful, tumor recurrence is common and associated with therapy resistance. Active efflux of chemotherapeutic agents by transporter proteins of the ATP-Binding Cassette superfamily forms an effective cellular defense mechanism and a high expression of these transporters is frequently observed in chemotherapy-resistant tumors in both humans and dogs. In this study we describe the ABC-transporter expression in a canine lymphoid cell line and a sub-cell line with acquired drug resistance following prolonged exposure to doxorubicin. This sub-cell line was more resistant to doxorubicin and vincristine, but not to prednisolone, and had a highly increased P-glycoprotein (P-gp/abcb1) expression and transport capacity for the P-gp model-substrate rhodamine123. Both resistance to doxorubicin and vincristine, and rhodamine123 transport capacity were fully reversed by the P-gp inhibitor PSC833. No changes were observed in the expression and function of the ABC-transporters MRP-1 and BCRP. It is concluded that GL-40 cells represent a useful model for studying P-gp dependent drug resistance in canine lymphoid neoplasia, and that this model can be used for screening substances as potential P-gp substrates and their capacity to modulate P-gp mediated drug resistance.

Inhibition of P-glycoprotein by psychotherapeutic drugs in a canine cell model.

Drug-drug interactions related to long-term therapies are of increasing concern. Psychotherapeutic drugs, licensed for the use in dogs for the management of separation anxiety and other behavioural disorders, are examples of drugs used in long-term therapies. In an in vitro system with canine P-glycoprotein (P-gp) expressing cell lines, three psychotherapeutic drugs with a different mode of action were tested for their ability to inhibit the canine multidrug transporter P-gp. At 10 µm, the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant clomipramine inhibited P-gp for 41% and 59%, respectively. In contrast, selegeline did not inhibit the function of the canine P-gp.

Comparing the glucuronidation capacity of the feline liver with substrate-specific glucuronidation in dogs.

This study aimed to assess the overall glucuronidation capacity of cats, using prototypic substrates identified for human UDP-glucuronosyltransferases (UGTs). To this end, Michaelis-Menten kinetics were established for the substrates using feline hepatic microsomal fractions, and results were compared with similar experiments carried out with dog liver microsomes. Cats are known for their low capacity of glucuronide formation, and UGT1A6 was found to be a pseudogene. However, functional studies with typical substrates were not performed and knowledge of the enzymology and genetics of other glucuronidation enzymes in felidae is lacking. The results of this study showed extremely low formation of naphthol-1-glucuronide (1.7 ± 0.4 nmol/mg protein/min), estradiol-17-glucuronide (<0.7 nmol/mg protein/min), and morphine-3-glucuronide (0.2 ± 0.03 nmol/mg protein/min), suggesting a lack of functional UGT1A6 and UGT2B7 homologues in the cat's liver. Dog liver microsomes were producing these glucuronides in much higher amounts. Glucuronide capacity was present for the substrates 17ß-estradiol (estradiol-3-glucuronide, 2.9 ± 0.2 nmol/mg protein/min) and 4-methylumbelliferone (31.3 ± 3.3 nmol/mg protein/min), assuming that cats have functional homologue enzymes to at least the human UGT1A1 and probably other UGT1A isozymes. This implies that for new drugs, glucuronidation capacity has to be investigated on a substance-to-substance base. Knowledge of the glucuronidation rate of a drug provides the basis for pharmacokinetic modeling and as a result proper dosage regimens can be established to avoid undesirable drug toxicity in cats.

Masitinib reverses doxorubicin resistance in canine lymphoid cells by inhibiting the function of P-glycoprotein.

Overexpression of ABC-transporters including Pgp, MRP1, and BCRP has been associated with multidrug resistance (MDR) in both human and canine oncology. Therapeutic interventions to reverse MDR are limited, but include multidrug protocols and the temporary concomitant use of inhibitors of ABC-transporters. Recently, the use of tyrosine kinase inhibitors has been proposed to overcome MDR in human oncology. One of the tyrosine kinase inhibitors, masitinib, is licensed for veterinary use in the treatment of canine mast cell tumors. Therefore, this study aimed to assess the potential of masitinib to revert MDR in canine malignant lymphoma using an in vitro model with canine lymphoid cell lines. Masitinib had a mild antiproliferative effect on lymphoid cells, inhibited Pgp function at concentrations equal to or exceeding 1 µm and was able to reverse doxorubicin resistance. The current findings provide the rationale for a combined use of masitinib with doxorubicin in the treatment of dogs with doxorubicin-resistant malignant lymphoma but await confirmation in clinical trials.

Danofloxacin-mesylate is a substrate for ATP-dependent efflux transporters.

BACKGROUND AND PURPOSE: Next to its broad antimicrobial spectrum, the therapeutic advantages of the fluoroquinolone antimicrobial drug Danofloxacin-Mesylate (DM) are attributed to its rapid distribution to the major target tissues such as lungs, intestines and the mammary gland in animals. Previous analyses revealed that effective drug concentrations are achieved also in luminal compartments of these organs, suggesting that active transport proteins facilitate excretion into the luminal space. Members of the ATP-Binding Cassette (ABC) superfamily, including P-gp, BCRP and MRP2 are known to be expressed in many tissue barriers and in cell-membranes facing luminal compartments. Hence we hypothesized that DM is a substrate for one of these efflux-transporters. EXPERIMENTAL APPROACH: Confluent monolayers of Caco-2 cells, grown on microporous membranes in two-chamber devices were used. DM concentrations were measured by fluorimetric assay after HPLC of the culture media. KEY RESULTS: DM transport across Caco-2 cells was asymmetric, with a rate of secretion exceeding that of absorption. The P-gp inhibitors PSC833 and GF120918 and the MRP-inhibitor MK571 partially decreased the secretion of DM and increased its absorption rate. The BCRP inhibitor, Ko143, decreased secretion only at a concentration of 1 microM. When DM was applied together with ciprofloxacin, secretion as well as absorption of DM decreased. CONCLUSIONS AND IMPLICATIONS: DM is a substrate for the efflux transporters P-gp and MRP2, whereas the specific role of BCRP in DM transport needs further evaluation. These findings provide a mechanistic basis for the understanding of the pharmacokinetics of DM in healthy and diseased individuals.