Increased susceptibility of a triclabendazole (TCBZ)-resistant isolate of Fasciola hepatica to TCBZ following co-incubation in vitro with the P-glycoprotein inhibitor, R(+)-verapamil

SUMMARY A study was carried out to investigate whether the action of triclabendazole sulphoxide (TCBZ.SO) against the liver fluke, Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for this in vitro study and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. For experiments with the Oberon isolate, flukes were incubated for 24 h with either R(+)-VPL (1×10-4 m) on its own, TCBZ.SO (15 µg mL-1) alone, a combination of R(+)-VPL (1×10-4 m) plus TCBZ.SO (15 µg mL-1), TCBZ.SO (50 µg mL-1) on its own, or a combination of TCBZ.SO (50 µg mL-1) plus R(+)-VPL (1×10-4 m). They were also incubated in TCBZ.SO (50 µg mL-1) alone or in combination with R(+)-VPL (1×10-4 m) until they became inactive; and in TCBZ.SO (50 µg mL-1) alone for a time to match that of the combination inactivity time. Flukes from the Cullompton isolate were treated with either TCBZ.SO (50 µg mL-1) alone or in combination with R(+)-VPL (1×10-4 m) until they became inactive, or with TCBZ.SO (50 µg mL-1) alone time-matched to the combination inactivity time. Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R(+)-VPL alone had a minimal effect on either isolate. TCBZ.SO treatment had a relatively greater impact on the TCBZ-susceptible Cullompton isolate. When R(+)-VPL was combined with TCBZ.SO in the incubation medium, however, the surface disruption to both isolates was more severe than that seen after TCBZ.SO treatment alone; also, the time taken to reach inactivity was shorter. More significantly, though, the potentiation of drug activity was greater in the Oberon isolate; also, it was more distinct at the higher concentration of TCBZ.SO. So, the Oberon isolate appears to be particularly sensitive to efflux pump inhibition. The results of this study suggest that enhanced drug efflux in the Oberon isolate may be involved in the mechanism of resistance to TCBZ.

Effect of the P-glycoprotein inhibitor, R(+)-verapamil on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by the inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R-VPL]. In the first experiment, flukes were initially incubated for 2 h in R-VPL (100 µM), then incubated for a further 22 h in R-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 µg/ml, or 0.1327 µM). For controls, flukes were incubated for 24 h in R-VPL and TCBZ.SO on their own. In a second experiment, flukes were removed from the incubation media following cessation of movement. In the third experiment, Sligo flukes were incubated in lower concentrations of R-VPL (10 µM) and TCBZ.SO (15 µg/ml, or 0.0398 µM). Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R-VPL alone had minimal effect on either isolate. After treatment with TCBZ.SO alone, there was greater surface disruption to the Cullompton than Sligo isolate. However, combined treatment of R-VPL+TCBZ.SO led to more severe surface changes to the Sligo isolate than with TCBZ.SO on its own; this potentiation of drug activity was not seen with the Cullompton isolate. The phenomenon was evident at both concentrations of TCBZ.SO. Inclusion of R-VPL in the incubation medium also reduced the time taken for the flukes to become inactive; again, this effect was more distinct with the Sligo isolate. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Children with stable asthma have reduced airway matrix metalloproteinase-9 and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio

Background Childhood asthma is characterized by inflammation of the airways. Structural changes of the airway wall may also be seen in some children early in the course of the disease. Matrix metalloproteinases (MMPs) are key mediators in the metabolism of the extracellular matrix (ECM). Objective To investigate the balance of MMP-8, MMP-9 and tissue inhibitor of metalloproteinases (TIMP)-1 in the airways of children with asthma. Methods One hundred and twenty-four children undergoing elective surgical procedures also underwent non-bronchoscopic bronchoalveolar lavage (BAL). MMP-8, MMP-9 and TIMP-1 were measured by ELISA. Results There was a significant reduction in MMP-9 in atopic asthmatic children (n=31) compared with normal children (n=30) [median difference: 0.57 ng/mL (95% confidence interval: 0.18–1.1 ng/mL)]. The ratio of MMP-9 to TIMP-1 was also reduced in asthmatic children. Levels of all three proteins were significantly correlated to each other and to the relative proportions of particular inflammatory cells in BAL fluid (BALF). Both MMP-8 and MMP-9 were moderately strongly correlated to the percentage neutrophil count (r=0.40 and 0.47, respectively, P

The inactivation of bovine cathepsin B by novel N-chloro-acetyl-dipeptides. Application of the Houghten 'tea bag' methodology to inhibitor synthesis

In this study, a series of N-chloro-acetylated dipeptides were synthesised by the application of Houghten's methodology of multiple analog peptide syntheses. The peptides, all of which contain a C-terminal free acid, were tested as inactivators of bovine cathepsin B, in an attempt at exploiting the known and, amongst the cysteine proteinases, unique carboxy dipeptidyl peptidase activity of the protease. We have succeeded in obtaining a number of effective inactivators, the most potent of which-chloroacetyl-Leu-Leu-OH, inactivates the enzyme with an apparent second-order rate constant of 3.8 x 10(4) M-1 min(-1). In contrast, the esterified analog, chloroacetyl-Leu-Leu-OMe, inactivates the enzyme some three orders of magnitude less efficiently, lending credence to our thesis that a free carboxylic acid moiety is an important determinant for inhibitor effectiveness. This preliminary study has highlighted a number of interesting features about the specificity requirements of the bovine proteinase and we believe that our approach has great potential for the rapid delineation of the subsite specificities of cathepsin B-like proteases from various species. (c) 2005 Elsevier Inc. All rights reserved.

Epidermal Growth Factor Receptor Activity Determines Response of Colorectal Cancer Cells to Gefitinib Alone and in Combination with Chemotherapy.

Purpose: Up to now, there have been no established predictive markers for response to epidermal growth factor receptor (EGFR/HER1/erbB1) inhibitors alone and in combination with chemotherapy in colorectal cancer. To identify markers that predict response to EGFR-based chemotherapy regimens, we analyzed the response of human colorectal cancer cell lines to the EGFR-tyrosine kinase inhibitor, gefitinib (Iressa, AstraZeneca, Wilmington, DE), as a single agent and in combination with oxaliplatin and 5-fluorouracil (5-FU). Experimental Design: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays and analyzed by ANOVA. Apoptosis was measured by flow cytometry, poly(ADP-ribose) polymerase, and caspase 3 cleavage. EGFR protein phosphorylation was detected by Western blotting. Results: Cell lines displaying high constitutive EGFR phosphorylation (a surrogate marker for EGFR activity) were more sensitive to gefitinib. Furthermore, in cell lines exhibiting low constitutive EGFR phosphorylation, an antagonistic interaction between gefitinib and oxaliplatin was observed, whereas in cell lines with high basal EGFR phosphorylation, the interaction was synergistic. In addition, oxaliplatin treatment increased EGFR phosphorylation in those cell lines in which oxaliplatin and gefitinib were synergistic but down-regulated EGFR phosphorylation in those lines in which oxaliplatin and gefitinib were antagonistic. In contrast to oxaliplatin, 5-FU treatment increased EGFR phosphorylation in all cell lines and this correlated with synergistic decreases in cell viability when 5-FU was combined with gefitinib. Conclusions: These results suggest that phospho-EGFR levels determine the sensitivity of colorectal cancer cells to gefitinib alone and that chemotherapy-mediated changes in phospho-EGFR levels determine the nature of interaction between gefitinib and chemotherapy.

A Phase I and Pharmacokinetic Study of OSI-7904L, a Liposomal Thymidylate Synthase Inhibitor in Combination with Oxaliplatin in Patients with Advanced Colorectal Cancer.

OSI-7904L is a liposomal formulation of a potent thymidylate synthase (TS) inhibitor. This phase I study evaluated the safety, tolerability and pharmacokinetics (PK) of OSI-7904L administered in combination with oxaliplatin every 21 days in patients with advanced colorectal carcinoma. METHOD: A 3+3 study design was utilized at predefined dose levels. Polymorphisms in the TS enhancer region and XPD enzyme were investigated as potential predictors of efficacy and toxicity. RESULTS: Fourteen patients received 76 cycles of treatment. At the highest dose level (OSI-7904L 9 mg/m(2), oxaliplatin 130 mg/m(2)) investigated, one of nine patients experienced dose-limiting toxicity of grade 3 oral mucositis with cycle 1 and five further patients required dose reductions. The toxicity profile of stomatitis, diarrhea, nausea, fatigue, sensory neuropathy and skin rash was consistent with that expected for a TS inhibitor/oxaliplatin combination regimen. PK analysis showed high interpatient variability with no detectable interaction between OSI-7904L and oxaliplatin. Partial radiological responses were documented in two patients. CONCLUSIONS: The recommended regimen for further investigation is OSI-7904L 9 mg/m(2) and oxaliplatin 130 mg/m(2).

Functional domains of the human epididymal protease inhibitor, eppin

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin’s domains are involved in the protein’s antibacterial activity, only the Kunitz domain is required for selective protease inhibition.

Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

Effective inhibitors of osteopontin (OPN)-mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced beta-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates beta-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G(2) phase of cell cycle.