Expression of P-glycoprotein in the intestinal epithelium of dogs with lymphoplasmacytic enteritis.

Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory disease of the stomach, the small intestine and/or the large intestine. Loss of integrity of the intestinal barrier may be an important factor in the pathogenesis of IBD. In dogs, lymphoplasmacytic enteritis (LPE) is one of the recognized forms of IBD. P-glycoprotein (P-gp) is a membrane-bound efflux pump constituting an important component of the intestinal barrier. Changes in P-gp expression at the level of the intestinal barrier may be important in the pathogenesis of canine LPE, as this may lead to variable protection against xenobiotics and bacterial products in the intestine. The aim of the present study was to evaluate the expression of epithelial P-gp in the intestine in dogs with LPE compared with disease-free animals. Formalin-fixed intestinal biopsy samples from 57 dogs with histopathological evidence of LPE were immunolabelled with anti-P-gp antibodies (C494 and C219). Endoscopic biopsy samples of the duodenum and colon from 16 healthy beagles were used as controls. None of the control dogs had P-gp expression in the apical membrane of duodenal enterocytes, but all had P-gp labelling at the colonic epithelial surface. Twenty out of 57 dogs with LPE had P-gp expression at the apical surface membrane of villus epithelial cells in the duodenum, jejunum and/or ileum. Six out of 16 colonic samples from dogs with LPE had decreased P-gp expression at the epithelial surface compared with controls. It is unclear whether these changes in P-gp expression in dogs with LPE are a cause or a consequence of the inflammation. The observed changes could affect bioavailability of therapeutic drugs used in LPE.

The influence of modulation of P-glycoprotein and /or cytochrome P450 3A on the pharmacokinetics and pharmacodynamics of orally administered morphine in dogs.

The influence of pretreatment with ketoconazole [cytochrome P450 3A (CYP3A) + P-glycoprotein (P-gp) inhibitor], elacridar (selective P-gp inhibitor) and rifampicin (CYP3A + P-gp inducer) on oral morphine pharmacokinetics and pharmacodynamics was investigated in experimental dogs. Seven beagles were used in a four-way crossover design. Morphine hydrochloride was administered orally (2.5 mg/kg) alone (control group CON) or after pretreatment with ketoconazole (group KETO), elacridar (group ELA) or rifampicin (group RIF). Morphine plasma concentrations were analysed by liquid chromatography-tandem mass spectrometry. Sedation scores (none, mild, moderate or severe) were evaluated subjectively. Dogs were significantly (P < 0.05) more sedated after ketoconazole pretreatment. There were no significant differences between group CON and the other pretreatment groups in pharmacokinetic parameters taking both sexes into account. Sex differences were apparent in some pharmacokinetic parameters of morphine. The area under the plasma concentration time curve (AUC(0-∞) ) was significantly higher, and the total body clearance was significantly lower in male compared to female dogs in all treatment groups. Ketoconazole, rifampicin and elacridar pretreatment had no significant effects on morphine pharmacokinetics, although dogs in the ketoconazole group showed higher sedation scores.

Expression of multidrug resistance-associated P-glycoprotein in feline tumours.

Permeability glycoprotein (P-gp) is a membrane-bound efflux pump that mediates the active transmembrane transport of a variety of substrates. Several studies in human and canine normal and neoplastic tissues indicate that P-gp is involved in resistance to chemotherapy and in the development of multidrug resistance (MDR). The purpose of this study was to evaluate P-gp expression with the monoclonal antibody C494 in common feline tumours from 88 patients not pretreated with chemotherapy. Tumours arising from tissues with intrinsic P-gp expression consistently showed positive labelling for P-gp in a cellular pattern identical to that described for the normal feline tissues. Both P-gp positive and negative tumour cells, however, were found in mammary gland tumours, lymphomas, mastocytomas and squamous cell carcinomas. Feline mammary gland tumours in particular showed strong membranous P-gp labelling, especially in areas with infiltrative growth and atypical cells, although this was not correlated with the grade of malignancy. These findings might have implications for future response to chemotherapy.

Tissue distribution of p-glycoprotein in cats.

Permeability glycoprotein (P-gp) is a membrane-bound efflux pump that exports various substances out of the cell. Variations in P-gp expression play an important role in susceptibility to toxic substances, drug efficacy and disease risk. In the present study, the distribution of the MDR1-gene product P-gp was determined in normal tissues of domestic shorthair cats using immunohistochemistry. Two monoclonal antibodies C494 and C219 were used, recognizing a different epitope on the human P-gp molecule. A consistent positive immunolabelling was obtained. The tissue distribution and cellular locations with antibody C494 were similar to those in man and dogs; with liver, colon, adrenal cortex and brain capillaries being consistently and intensely labelled. However, the immunolabelling in the kidney was in contradiction to man and dogs. The C219 antibody seems to react with a specific form of P-gp, only expressed in feline tissues with a barrier function, i.e. endothelia of the brain, testes and ovaries, and intestinal epithelial cells in contact with the lumen.

Bluetongue virus serotype 8-associated congenital hydranencephaly in calves.

Hydranencephaly, the almost complete absence of the cerebral parenchyma, induced by infection with modified live bluetongue virus (BTV) crossing the placenta has previously been reported in sheep and rarely in cattle in the USA and in South Africa. The current study describes 29 cases of hydranencephaly in bovine foetuses and 'dummy' calves up to 3 months of age in Belgium associated with natural BTV serotype 8 infection very early in gestation. Histological examination of the remaining cerebral parenchyma showed moderate to severe atrophy of the neural tissue. The lesions observed support the hypothesis of BTV-induced destruction of precursor cells. However, in several calves a slight infiltration of the walls of venules and arterioles with T lymphocytes (vasculitis) was observed as well, which seems to be responsible for at least some of the lesions. Bluetongue viral RNA was detected in 15 animals using a BTV-specific real-time RT-PCR with a much higher success rate in brain tissues compared with blood and spleen samples. Virus isolation in embryonated eggs was unsuccessful. In conclusion, hydranencephaly in calves can be associated with natural wild-type BTV-8 intra-uterine infection.

Concurrent atypical myopathy and equine dysautonomia in two horses.

This report concerns 2 horses that suffered typical clinical signs of atypical myopathy (AM) and equine grass sickness (EGS) concurrently. Clinical details and pathological lesions of the cases are described. EGS and AM are relatively rare diseases and the concurrency of the diseases in the same animals is therefore considered unlikely to be a coincidence. However, it is not suggested that the evidence shows a common aetiology but rather the existence of common predisposing causes.

Pharmacokinetics and bioavailability of oral 5'-deoxy-5-fluorouridine in cancer patients.

AIMS: Oral administration of 5-fluorouracil (FUra), an important cytotoxic agent, is limited by a wide variation in bioavailability. 5'-deoxy-5-fluorouridine (dFUrd), a masked form of FUra, has shown promise clinically when given intravenously or orally as a solution or tablet. This study investigates the efficacy of an oral capsule formulation of dFUrd in generating continuous systemic levels of this compound in cancer patients. METHODS: Six patients with advanced intestinal or ovarian malignancies were given three cycles of dFUrd, days 1-5, at intervals of 4 weeks. The doses of dFUrd were 600 mg m-2 three times daily, 800 mg m-2 three times daily, and 1000 mg m-2 three times daily, on cycles one, two and three, respectively (total dose 36 g m-2 ). The initial dose in each cycle was given as a slow intravenous injection over 10 min, and the remainder orally. Plasma and urine levels of dFUrd and two of its metabolites, FUra and 5,6-dihydro-5-fluorouracil (FUraH2 ), were monitored in six patients at each dose level. RESULTS: All six patients completed the study, receiving three different doses over a 3 month period, following which one had achieved a partial response, one had stable disease, and four had developed progressive disease. Side-effects were negligible, and only two instances of transient diarrhoea WHO grade 1 were seen. Total body clearance (CLtot) of intravenous dFUrd decreased with increasing dose; 2.7, 2.0 and 1.3 l min-1 m-2, following doses of 600, 800 and 1000 mg m-2, respectively. The mean elimination half-life of intravenous dFUrd increased with the dose from 15 to 22 min. Oral dFUrd was rapidly absorbed with a lag time of less than 20 min. The mean elimination half-life (t1/2, z ) of oral dFUrd was 32-45 min in the dose range 600-1000 mg m-2. The AUC of FUra and FUraH2 increased overproportionally with increasing intravenous doses of dFUrd. The mean systemic bioavailability of oral dFUrd was 34-47%. CONCLUSIONS: dFUrd, which selectively releases the antimetabolite FUra in tumour cells, can be given orally at doses of 600-1000 mg m-2 three times daily for 5 days. The systemic levels achieved are equivalent to those seen following continuous infusions of dFUrd or FUra. Toxicity is tolerable, and further clinical investigation of oral dFUrd is warranted.

Comparison of daunorubicin and Fluo-3 for detection of multidrug resistance in human tumor cells.

The detection of multidrug resistance (MDR) in clinical samples is still a topic for discussion. One method, proven extremely useful for detection of membrane proteins in patients with hematological malignancies is the flow cytometrical analysis of individual tumor cells. Recently an assay was described based on the labeling of the P-glycoprotein (P-gp) with the monoclonal antibody MRK16, combined with detection of active daunorubicin (DNR) extrusion. In order to improve the specificity of the assay, on line with the results obtained by Wall et al., we exploited staining with Fluo-3. Both assays prove to be able to discriminate between drug-resistant and drug-sensitive cells. A major drawback of labeling with Fluo-3 in combination with the monoclonal antibody MRK16 is the important overlap of emission spectra of both fluorochromes. Moreover, using Fluo-3 for the detection of MDR might be complicated by the fact that differences in fluorescence intensities are not solely dependent on the presence of P-gp, but also on the activity of cytosolic esterases and the intracellular calcium concentration. Combination of the detection of structural and functional aspects of the MDR-associated protein may lead to a more precise detection of the MDR-positive patient.

[In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions)]. / Effets in vitro du piracetam sur la radiosensibilité des cellules hypoxiques (adaptation du test au MTT aux conditions d'hypoxie).

This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass versus reference polystyrene plates were comparable and confirm the validity of using special glass plates. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. However, this does not preclude a potential in vivo effect of piracetam on the radiosensitivity owing to its action on microcirculation and its rheologic properties. The adaptation of the MTT assay to hypoxic irradiation conditions yields the easy screening of radiosensitizing drugs: shorter incubation, semi-automatic method and simultaneous analysis with different serial concentrations thanks to the special 48-well glass plates.

Lack of correlation between mdr-1 expression and volume-activation of cloride-currents in rat colon cancer cells.

Correlation between expression of the mdr-1 genes (a and b) at the mRNA and protein level and volume-activation of chloride-channels was studied in rat colon cancer CC531 cells by means of RT-PCR, Western blotting and patch clamp, respectively. Three different kinds of cell lines were used: CC531-PAR, CC531-COL and CC531-REV. At the mRNA level, the parental cell line CC531-PAR showed significantly less mdr-1a expression in comparison with CC531-COL, a drug-resistant cell line induced from the parental CC531 cells by growth in the presence of colchicin. The third cell line, CC531-REV, was a spontaneous revertant of the drug-resistant cell line to a drug-sensitive one, but with a maintained level of mdr-1a mRNA. In none of the three cell lines, mdr-1b mRNA could be detected. At the protein level, a clear difference in mdr1 expression between CC531-PAR/REV and CC531-COL was observed. Although the amount of mdr-1a mRNA detected in CC531-REV was comparable to that found in CC531-COL, the amount of mdr-1 encoded protein in CC531-REV was remarkably reduced. In all three cell types, cell swelling activated chloride-currents which could be blocked by NPPB.(ABSTRACT TRUNCATED AT 250 WORDS)

P-glycoprotein: clinical significance and methods of analysis.

Multidrug resistance (MDR) is responsible for a decrease in sensitivity of tumor cells tumor cells to unrelated, naturally occurring anticancer drugs. This resistance is correlated with expression and activity of a membrane protein, P-gp 170, functioning as a drug-extruding pump. It has been well described in in vitro situations; however, the clinical detection and implications are not yet clear. Multiple detection assays have been developed based on the discovery of the MDR gene family and the corresponding protein. Southern, Northern, or Western blot analysis, S1 nuclease protection or PCR-based assays, immunohistochemical detection or functionality tests by flow cytometry have been used extensively. However, by use of these techniques on clinical material, both normal and malignant, contradictory results have emerged. The sensitivity and specificity of a certain technique are always limited by unavoidable parameters, for example, skill of the technician. Moreover, the complexity of the development of resistance against anticancer agents (external determinants), such as the diversity of tumor tissues, the simultaneous presence of other resistance mechanisms, and the low expression level, make MDR detection equivocal and can lead to contradictory results. Previous treatment influencing the MDR profile and inappropriate timing of the test make a possible correlation between MDR expression and chemotherapeutic resistance difficult to establish and can lead to discordant results. In this review, the need for proper criteria is stressed. No single detection technique provides the ideal test to detect MDR. Tandem testing could give more certainty, although small sample size limit this application. Formulation of a standard assay with better definition of a positivity is essential before clinical trials are started.

Cytotoxic activity of 7-N-(2-((2-(-gamma-L-glutamylamino)- ethyl)dithio)ethyl)-mitomycin C and metabolites in cell lines with different resistance patterns.

In this study the activity of KW-2149 and two of its metabolites, M-16 and M-18, was measured against cell lines with different types of resistance. The influence of these metabolites and of the exposure time on the cytotoxic efficacy of KW-2149 was investigated. Against the human ovarian carcinoma cell lines, AOvC and A2780, KW-2149 was more active than mitomycin C (MMC), with an IC50 of, respectively, 3.4 nM and 9.82 microM for KW-2149 and 18.2 nM and 67.71 microM for MMC. Activity of M-18 was significant against both cell lines and was comparable with that of KW-2149. Against an MMC-resistant cell line, AOvCMMC, the resistance factor (RF) for KW-2149 was 3.1 versus 8.0 for MMC. Tested against a cisplatin-resistant cell line, AOvCCDDP, KW-2149 had a RF of 7.7 versus 2.4 for MMC. Increasing the exposure time from 1 to 8 h decreased the RF for KW-2149 from 7.7 to 3.0. In an MDR mediated resistant cell line, A2780mdr+, prolongation of exposure time increased RF for KW-2149 and MMC but decreased RF for M-18 from 7.0 at 1 h to 5.3 at 8 h. Tested against a rat colon carcinoma cell line CC531, KW-2149 and M-18 again demonstrated superior or equal activity compared with MMC, IC50 being, respectively, 0.6, 2.1 and 2.6. Here again M-18 showed an aberrant sensitivity pattern, as its activity decreased with mdr-1 expression in contrast to the other mitomycins. Our data confirm the activity of KW-2149 as an agent with equal or superior activity as compared with MMC. It is concluded that the metabolite M-18 can contribute to the activity of KW-2149. Efficacy of both KW-2149 and its metabolites increases with increasing exposure times. The increments of exposure time appeared even as a means to overcome resistance in some instances.

5'-Deoxy-5-fluorouridine increases daunorubicin uptake in multidrug-resistant cells and its activity is related with P-gp 170 expression.

Most anticancer agents fail to induce clear responses in the treatment of colorectal cancer. This can be explained by involvement of overexpression of the membrane glycoprotein, P-gp 170, which is associated with multidrug resistance (MDR), and/or with involvement of ras. Fluoropyrimidines are amongst the few options in the chemotherapeutic treatment of colorectal cancers. 5'-Deoxy-5-fluorouridine (dFUrd) needs intracellular activation via 5-fluorouracil into 5-fluoro-2'-deoxyuridine-5'-monophosphate and 5-fluorouridine-5'-triphosphate. In the present study, the cytotoxic activity of dFUrd in vitro and dFUrd combined with daunorubicin (DNR) was assessed with the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) bromide assay in cells with increased P-gp 170 expression versus controls. Surprisingly, dFUrd was most active in cells with high P-gp 170 expression, a finding which can not be explained by intracellular metabolic activity only. The results also show that dFUrd improves the DNR uptake in MDR-positive cells, and this is related with increased cytotoxicity of the anthracycline.

Multidrug resistance in rat colon carcinoma cell lines CC531, CC531mdr+ and CC531rev.

A rat colon carcinoma cell line, CC531, was exposed to stepwise increasing concentrations of colchicine. A cell line, CC531mdr+, which grows in the presence of 0.2 microM of colchicine was obtained. A revertant cell line, CC531rev was isolated upon colchicine withdrawal. The CC531mdr+ displayed a multidrug-resistant phenotype. Marked resistance to the selecting agent colchicine, was found (RF = 37.5) as well as to vinblastine (RF = 11.3) and actinomycin D (RF = 2.6). Cross resistance to doxorubicin (RF = 8) and daunorubicin (RF = 13.3) was demonstrated. Verapamil was able to reverse drug resistance to colchicine and daunorubicin. The revertant cell line, CC531rev, showed increased sensitivity to colchicine (RF = 0.43), vinblastine (RF = 0.13), doxorubicin (RF = 0.28) and daunorubicin (RF = 0.56). Marked cross resistance to cis-platinum (RF = 6.9) was also induced in CC531mdr+ and was maintained in CC531rev. We conclude that CC531 displays an intrinsic low-level multidrug-resistant phenotype, which was amplified in the CC531mdr+ variant. This correlates with a higher level of expression of P-glycoprotein. CC531rev lacks the multidrug-resistant phenotype and can be used as the drug-sensitive counterpart of the latter two cell lines. Furthermore, it has been shown that in these cell lines cis-platinum resistance is mediated through a mechanism independent of the multidrug-resistant phenotype, since the revertant cell line CC531rev has lost the multidrug-resistant phenotype while retaining the concomitantly induced cis-platinum resistance of the multidrug-resistant variant CC531mdr+.

Drug resistance in rat colon cancer cell lines is associated with minor changes in susceptibility to cytotoxic cells.

The development of resistance to anticancer drugs urges the search for different treatment modalities. Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility. We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS). In a 4-h 51Cr-release assay we found no difference in susceptibility to NK cell lysis. No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay. In a prolonged 20-h 51Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS). None of the cell lines was completely resistant to lysis by aLAK cells. Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.

Fluorouracil catabolism in the combination treatment of cyclophosphamide, methotrexate and fluorouracil.

The CMF-regimen is amongst the most effective chemotherapeutic approaches in the treatment of breast cancer. It is generally accepted that the efficacy of the combination of the three agents used in the regimen, i.e., cyclophosphamide (CY), methotrexate (MTX) and fluorouracil (FUra), is based on interactions between the drugs at the intratumoral level. In WAG/Rij rats we previously demonstrated that change of FUra clearance at the first day of the CMF-regimen occurs owing to concomitant CY + MTX. In the present study clearance of FUra and the first product of FUra catabolism, FUraH2, were monitored at day 1 and day 8 of the regimen upon treatment with single agent FUra (F), MTX + FUra (MF), CY + FUra (CF), and CY + MTX + FUra (CMF). At the first day of treatment, FUra and FUraH2 systemic exposure was demonstrated to be increased in CMF-treated rats owing to concomitant CY+MTX. At the eighth day of treatment it was found that repeated CY administration during the previous seven days in CF-treated rats resulted in increased FUra and FUraH2 systemic exposure and therefore increased the dose of FUra artificially. It is concluded that altered FUra clearance owing to extratumoral interactions by concomitant CY and MTX contributes to the efficacy of the CMF-regimen.