Health management program: factors influencing completion of therapy with high-dose interferon alfa-2b for high-risk melanoma.

The goal of the 1-year observational, multicentre, open-label study reported here was to identify factors influencing adherence to high-dose interferon alfa-2b adjuvant therapy in patients at high risk of recurrence following surgical excision of malignant melanoma. The study was carried out in 23 tertiary-care centres across Canada.The 225 patients enrolled in the study all had malignant melanoma that was surgically excised and that required adjuvant treatment with interferon alfa-2b. Of these patients, 64% were men. Mean age was 51.7 years. All patients received interferon alfa-2b treatment during a 4-week induction phase (20 MU/m(2) intravenously 5 days per week) followed by a 48-week maintenance phase (10 MU/m(2) subcutaneously 3 days per week).Oncology nurses reviewed side-effect management with the patients before the induction and maintenance phases. Patients were provided with daily diaries, comprehensive educational materials, and ongoing nursing support. Data on side effects and discontinuations were obtained from patient interviews and diaries. THE MAIN OUTCOME MEASUREMENTS WERE RELATED TO TREATMENT DISCONTINUATION: rate, timing, reason, and prevention. Of the 225 patients, 75 (33.3%) discontinued interferon during the induction phase, and 58 (25.8%) discontinued during the maintenance phase. The main reasons for discontinuation were adverse events (58%) and disease progression (26%). Patients with a daily fluid intake greater than 1.5 L were more likely to complete therapy than were those with an intake less than 1.5 L (64% vs. 36%, p < 0.0001).Of 225 patients enrolled in the interferon alfa-2b health management program, 41% completed the 1-year treatment course. Higher fluid intake (>1.5 L daily) was associated with increased adherence to therapy.

Effectiveness of a nursing support program for patients with recurrent ovarian cancer receiving pegylated liposomal doxorubicin (Caelyx/Doxil).

Pegylated liposomal doxorubicin (PLD) has become the preferred alternative for ovarian cancer patients who have failed platinum-based therapy, but side effects, such as palmar-plantar erythrodysesthesia (PPE), may lead to sub-optimal drug exposure and treatment discontinuation. A prospective Canadian multicentre open-label study evaluated the effects of a nurse-administered education and support program on treatment adherence and tolerability in 112 women with recurrent ovarian cancer. Subjects received an average of four four-week PLD cycles, the recommended number of courses required to evaluate the efficacy from PLD. Side effects were common, but 75% of patients were able to complete > 3 cycles and 59% completed > 4 cycles of PLD chemotherapy. With proactive nursing intervention, the incidence of PLD-associated grade three-four toxicities such as PPE and mucositis was substantially decreased. Nursing intervention may allow more patients to receive chemotherapy on schedule, thus reproducing the conditions of the clinical study in which the efficacy of the drug has been established.

Phase II study of pegylated liposomal doxorubicin HCl (Caelyx) in combination with cyclophosphamide and vincristine as second-line treatment of patients with small cell lung cancer.

BACKGROUND: Despite chemotherapy and radiotherapy for small cell lung cancer (SCLC), most patients die within 2 years. Response rates for second-line chemotherapy are 15-25%, with a median survival of 5 months. Caelyx, a pegylated liposomal formulation of doxorubicin, may be better tolerated and has activity in SCLC. PATIENTS AND METHODS: Thirty-two patients were enrolled in a phase II study of intravenous Caelyx (35 mg/m2), cyclophosphamide (750 mg/m2) and vincristine (1.2 mg/m2) every 21 days as second-line therapy in SCLC for up to six cycles. RESULTS: Thirty patients were evaluable for response, with a response rate of 10%. Another two had an unconfirmed response. Stable disease (SD) for >or=2 cycles was seen in an additional 53%. Grade 3 or 4 non-hematologic toxicity was seen in 17 (55%) patients (26 [22%] cycles) and included fatigue, mucositis, plantar-palmar erythrodysesthesia, rash and neuropathy. Twelve patients required transfusions. All patients on study have now expired, with a median survival of 28 weeks (7 months). For patients with SD or partial response, median time to progression was 15 weeks. CONCLUSION: The combination of Caelyx, cyclophosphamide and vincristine, despite cyclophosphamide and Caelyx dose reductions, has modest activity in relapsed SCLC with acceptable toxicity.

Haemonchus contortus: selection at a glutamate-gated chloride channel gene in ivermectin- and moxidectin-selected strains.

Anthelmintic resistance in nematode parasites of livestock is a serious problem worldwide. Ivermectin, an avermectin, and moxidectin, a milbemycin, are potent endectocides commonly used to control these parasites. The proposed mode of action of avermectins and possibly the milbemycins involves the binding of the drug to the alpha-subunit of a glutamate-gated chloride channel, which opens or potentiates gating of the channel and leads to the hyperpolarization of the target neuromuscular cell. Glutamate gates the channel by binding to the beta-subunit. We have cloned a fragment of a putative alpha-subunit gene from Haemonchus contortus. The sequence of the beta subunit is available from GenBank. Genetic variability of this fragment was analysed by single-strand conformation polymorphism in five strains of H. contortus: two strains passaged without drug selection, two strains selected with ivermectin, and one strain selected with moxidectin. One allele of the putative alpha-subunit gene appeared to be associated with resistance to the drugs, increasing in frequency in the three drug-selected strains relative to the unselected strains. Another allele appeared to be associated with susceptibility, decreasing in frequency in the three drug-selected strains relative to the unselected strains. A similar analysis of the beta-subunit gene showed no significant differences in allele frequencies between the unselected and drug-selected strains. Our findings suggest a correlation between changes in allele frequencies of the putative alpha-subunit gene and resistance to ivermectin and moxidectin.

Reversal of P-glycoprotein-associated multidrug resistance by ivermectin.

P-Glycoprotein (P-gp) causes a multidrug resistance (MDR) phenotype in tumour cells. In some cancers, the expression of P-gp has been correlated with low clinical response to chemotherapy and survival of patients. Previous studies have shown that certain lipophilic drugs bind to P-gp and reverse the MDR phenotype of tumour cells. In this study, we extend that list of compounds and present evidence for the capacity of a potent and clinically safe anthelmintic, ivermectin (IVM), as an MDR-reversing drug. Using a highly drug-resistant human cell line, we compared IVM with other MDR-reversing agents and showed that IVM is 4- and 9-fold more potent than cyclosporin A and verapamil, respectively. The capacity of IVM to inhibit iodoaryl-azidoprazosin photolabeling of P-gp is consistent with direct binding to P-gp. Studies showed that [3H]IVM binding to membranes from resistant cells is specific and saturable with KD and Bmax values of 10.6 nM and 19.8 pmol/mg, respectively. However, while cyclosporin A or vinblastine inhibited [3H]IVM binding to membranes from drug-resistant but not drug-sensitive cells, neither verapamil nor colchicine had any effect. Furthermore, both IVM and cyclosporin A and, to a lesser extent, verapamil also inhibited [3H]vinblastine binding to membranes from drug-resistant cells. Drug transport studies showed that [3H]IVM is a substrate for the P-gp drug efflux pump. However, it was transported less efficiently by P-gp than [3H]vinblastine. Moreover, only cyclosporin A was effective in potentiating the accumulation of [3H]IVM in drug-resistant cells. Taken together, the high efficiency of MDR reversal by IVM combined with its low toxicity are consistent with the properties of an ideal MDR-reversing agent.

BIBW22 BS, potent multidrug resistance-reversing agent, binds directly to P-glycoprotein and accumulates in drug-resistant cells.

The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.

Palmitoylation of the glucose transporter in blood-brain barrier capillaries.

Palmitoylation of GLUT1 was investigated in brain capillaries. The glucose transporter was shown to be palmitoylated using [3H]palmitate labeling and immunoprecipitation. The labeling was sensitive to methanolic KOH or hydroxylamine hydrolysis, indicating the presence of an ester or thioester bond. The released fatty acid was analyzed by reverse-phase HPLC and was identified as [3H]palmitate. Specificity of the immunoprecipitation was assessed by competitive inhibition of anti-GLUT1 binding with a synthetic C-terminal peptide against which the antibody was raised. In vivo studies were performed using capillaries isolated from control rats, streptozotocin-induced diabetic rats and diet-induced hyperglycemic rats. Glycemia was increased 2- and 5-fold in the hyperglycemic and diabetic groups, respectively. GLUT1 expression was evaluated in the three groups by Western blot analysis. A 36% decrease in GLUT1 expression was observed in the diabetic group, while there was no significant variation in GLUT1 expression in the hyperglycemic group. Palmitoylation of GLUT1 was increased in both diet-induced hyperglycemic and diabetic groups. These results suggest that palmitoylation may be involved in the regulation of glucose transport activity in hyperglycemia.

Isoform I (mdr3) is the major form of P-glycoprotein expressed in mouse brain capillaries. Evidence for cross-reactivity of antibody C219 with an unrelated protein.

P-glycoprotein (P-gp) is expressed in various non-cancerous tissues such as the endothelial cells of the blood-brain barrier. We used several monoclonal antibodies (mAbs) and isoform-specific polyclonal antibodies to establish which P-gp isoforms are expressed in isolated mouse brain capillaries. P-gp class I isoform was detected in capillaries with a Western immunoblotting procedure using a specific antiserum. No immunoreactivity was observed with either class II- or class III-specific antisera. Immunoreactivity was observed with mAb C219. However, this antibody detected two distinct immunoreactive proteins (155 and 190 kDa) in the isolated brain capillaries. These two proteins comigrated as a broad band when the samples were submitted to heat prior to gel electrophoresis. The glycoprotein nature of these two antigens was evaluated by their sensitivity to N-glycanase treatment. Following this treatment, the size of the proteins was reduced from 190 and 155 kDa to 180 and 120 kDa, respectively. Triton X-114 phase-partitioning studies showed that the 190 kDa immunoreactive protein was poorly solubilized by Triton X-114, while the 155 kDa protein was partitioned in the detergent-rich phase. In labelling experiments, only the 155 kDa protein was photolabelled with [125I]iodoarylazidoprazosin. These results show that a 190 kDa protein detected by antibody C219 is an antigen unrelated to the three P-gp isoforms presently known. Cross-reactivity of C219 with an unrelated protein emphasizes the fact that more than one antibody should be used in the assessment of P-gp expression in cell lines and tissues.

P-glycoprotein of blood brain barrier: cross-reactivity of Mab C219 with a 190 kDa protein in bovine and rat isolated brain capillaries.

P-glycoprotein (P-gp), an active efflux pump of antitumor drugs, is strongly expressed in endothelial cells of the blood brain barrier (BBB). Two proteins (155 and 190 kDa) were detected by Western blot analysis of beef and rat capillaries with the monoclonal antibody (MAb) C219. In order to characterize the nature of these proteins, their profile of solubilization by different detergents was established and compared with that of P-gp from the CHRC5 tumoral cell line. The 155 kDa protein (p155) of capillaries and the P-gp of CHRC5 cells were well solubilized by deoxycholate and Elugent, whereas the 190 kDa kDa protein (p190) was only solubilized by sodium dodecylsulfate (SDS). Both proteins have different patterns of extraction by Triton X-114, p155 partitioning as a membrane protein, while p190 was insoluble. Deglycosylation of capillary proteins resulted in a 27-28 kDa decrease in the apparent molecular weight of p155, similar to that observed for the P-gp of CHRC5 cells, but a decrease of only 7-8 for p190. Only p155 was immunoprecipitated by MAb C219. These results suggest that only p155 is the P-gp in BBB and that MAb C219 cross-reacts with a 190 kDa MDR-unrelated glycosylated protein. Consequently, the use of this antibody, which is frequently used to detect P-gp in tumors, could be a pitfall of immunohistochemistry screening for cancer tissues and lead to false positive in the diagnosis of MDR.

Extraction of brain capillary membrane proteins using Triton X-114.

Brain capillaries contain a great variety of membrane proteins involved in the transport of hydrophilic nutrients or in the reception of hormonal signals. The use of Triton X-114 fractionation to purify membrane proteins according to their degree of hydrophobicity was investigated. Analysis by polyacrylamide gel electrophoresis showed a distinct polypeptide composition for each fraction. Most of the proteins (68%) were solubilized by Triton X-114 and, of these proteins, the majority (74%) was found in the detergent-poor phase. Alkaline phosphatase which possesses a glycosyl-phosphatidylinositol anchor partitioned in the pellet of insoluble proteins where it was enriched 2.3-fold. In contrast, gamma-glutamyltranspeptidase, the GLUT1 glucose transporter and P-glycoprotein, three integral membrane proteins, and p21ras and a 42 kDa G protein alpha subunit, both covalently modified by lipids, were efficiently solubilized and fractionated in the detergent-rich fraction where they were enriched 3.5-, 4.8-, 4.4-, 4.5- and 4.7-fold, respectively. Triton X-114 fractionation could therefore be used as a first step in the purification of many blood-brain barrier membrane proteins.

Brush border membrane proteins in experimental Fanconi's syndrome induced by 4-pentenoate and maleate.

Fanconi's syndrome was investigated using brush border membrane (BBM) vesicles isolated from dog kidney. Sodium-dependent uptake of glucose, phosphate, and amino acids and protein phosphorylation were studied in BBM isolated from normal and from 4-pentenoate- and maleate-treated animals. The time course of D-glucose and phosphate uptake, in BBM vesicles, remained unchanged, indicating that both treatments had no effect on carrier properties, and that permeabilities to these substrates and to sodium were not modified. Furthermore, sodium-dependent transport of alanine, phenylalanine, proline, glycine, and glutamate into vesicles remained unaltered by either treatment. 4-Pentenoate treatment caused modifications of the phosphorylation pattern of BBM proteins: the phosphorylation of two proteins (61 and 74 kDa) was increased and that of two others (48 and 53 kDa) was decreased. Maleate treatment caused an increase in the phosphorylation for the same 61-kDa protein, which was also affected by 4-pentenoate treatment, suggesting that phosphorylation of this protein could be related to a mechanism involved in both 4-pentenoate- and maleate-induced Fanconi's syndrome. These changes were also observed in the presence of sodium fluoride and L-bromotetramisole, indicating that the modification of phosphorylation was not due to a difference in phosphatase activities. These results suggest that Fanconi's syndrome induced by 4-pentenoate or maleate is not caused by an inhibition of BBM Na(+)-dependent transport systems. Our results also suggest that protein phosphorylation may play an important role in the molecular defect involved in Fanconi's syndrome.

Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.

Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.