Pharmacogenetics of anticancer drugs in non-Hodgkin lymphomas

The variability of tumour responses to chemotherapeutic agents is a topic of major interest in current oncology research. Advances in the knowledge of molecular pathology of cancer make available strategies by which tumour cells can be profiled for their genetic background in order to select anticancer agents that might selectively kill cells in a molecular context that matches the mechanism of action of drugs. The next generation of anticancer treatments might thus be tailored on the basis of the numerous molecular alterations identified in tumour cells of a particular patient. However, to exploit these alterations, it is necessary to understand how they influence the cellular pathways that control the sensitivity or, conversely, resistance to chemotherapeutic agents. The aim of this article is to outline major genetic abnormalities in non-Hodgkin lymphomas that can be used to streamline anticancer drug selection and to underscore the major role of pharmacogenetics, which studies the interactions between genetic background and drug activity, to the prediction of likelihood of response and identification of potential new targets for pharmacological intervention. © 2001 Cancer Research Campaign  http://www.bjcancer.co

Fructose-1,6-diphosphate reduces acute ECG changes due to doxorubicin in isolated rat heart

Doxorubicin (DXR) (0.17 x 10(-4) M) induces an acute cardiotoxicity in isolated rat heart; there is a progressive widening of the S alpha T segment, with a decrease in force derivatives and in the coronary flow. Concurrent perfusion with fructose-1,6-diphosphate (FDP) (10(-5)-10(-4) M) dose-dependently reduces the S alpha T enlargement but fails to affect the reduction in force derivatives and coronary flow. The target of cardiac protection by FDP might be the ionic mechanisms underlying the action potential configuration

Synergistic cytotoxicity and pharmacogenetics of gemcitabine and pemetrexed combination in pancreatic cancer cell lines

PURPOSE: Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA synthesis and is an effective agent in the treatment of pancreas cancer. The present study investigates whether the multitargeted antifolate pemetrexed would be synergistic with gemcitabine against MIA PaCa-2, PANC-1, and Capan-1 pancreatic cancer cell lines. EXPERIMENTAL DESIGN: Cells were treated with gemcitabine and pemetrexed, and the type of drug interaction was assessed using the combination index. Cytotoxicity of gemcitabine was examined with inhibitors of (a) deoxycytidine kinase (dCK), which activates gemcitabine by phosphorylation, and (b) 5'-nucleotidase (drug dephosphorylation) and cytidine deaminase (drug deamination), the main inactivating enzymes. The effects of gemcitabine and pemetrexed on cell cycle were analyzed by flow cytometry, and apoptosis was examined by fluorescence microscopy. Finally, quantitative, real-time PCR was used to study the pharmacogenetics of the drug combination. RESULTS: Synergistic cytotoxicity and enhancement of apoptosis was demonstrated, mostly with the sequence pemetrexed-->gemcitabine. Pemetrexed increased cells in S phase, the most sensitive to gemcitabine, and a positive correlation was found between the expression ratio of dCK:RR and gemcitabine sensitivity. Indeed, pemetrexed significantly enhanced dCK gene expression (+227.9, +86.0, and +135.5% in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively), and the crucial role of this enzyme was confirmed by impairment of gemcitabine cytotoxicity after dCK saturation with 2'-deoxycytidine. CONCLUSIONS: These data demonstrate that the gemcitabine and pemetrexed combination displays schedule-dependent synergistic cytotoxic activity, favorably modulates cell cycle, induces apoptosis, and enhances dCK expression in pancreatic cancer cells

Suramin inhibits bFGF-induced endothelial cell proliferation and angiogenesis in the chick chorioallantoic membrane

The effects of suramin, an inhibitor of growth factor mitogenic activity, were evaluated on basic fibroblast growth factor (bFGF)-induced proliferation of bovine aortic endothelial cells and on angiogenesis in the chorioallantoic membrane (CAM) of chick embryos. The role of bFGF gene expression in endothelial cell growth was also investigated by using an antisense oligodeoxynucleotide to bFGF. The 4-fold increase in [3H]-thymidine uptake in endothelial cells in vitro upon stimulation with 10 ng ml-1 of bFGF was inhibited by suramin 300 micrograms ml-1. bFGF antisense oligomer (10 microM) reduced [3H]-thymidine incorporation in exponentially growing cells by 76%; this effect was reversed by bFGF 10 ng ml-1. In the CAM of chick embryos suramin 50 micrograms was a more potent inhibitor of angiogenesis than the combination of heparin 60 micrograms/hydrocortisone 50 micrograms; the mean value of the area with reduced vascularity was significantly larger in suramin-treated CAMs (2.4 cm2) than in heparin/hydrocortisone (0.6 cm2), while the reduction of vascular density was similar (- 35 and - 29% compared to controls, respectively), In conclusion, the effects of treatments with bFGF and bFGF antisense oligomer demonstrate that bFGF plays a relevant role in endothelial cell proliferation and may be the target of suramin since the drug is able to suppress basal and bFGF-induced endothelial cell growth; in addition to this, suramin is a more potent angiogenesis inhibitor in the CAM than the combination of heparin/hydrocortisone

Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small cell lung cancer cells

Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5'-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations

REDUCED CARDIOTOXICITY AND INCREASED CYTOTOXICITY IN A NOVEL ANTHRACYCLINE ANALOG, 4'-AMINO-3'-HYDROXY-DOXORUBICIN

The acute and chronic cardiotoxicity and cytotoxicity of the novel doxorubicin (DXR) derivative 4'-amino-3'-hydroxy-DXR were compared with those of 4'-deoxy-DXR and DXR. In the acute cardiotoxicity study, the ECG and hemodynamic changes recorded in anesthetized rats that had been treated i.v. with 10 mg/kg 4'-amino-3'-hydroxy-DXR or 8.6 mg/kg 4'-deoxy-DXR were significantly less severe than those caused by 13 mg/kg DXR. In the chronic cardiotoxicity study, rats received 3 weekly i.v. injections of 3 mg/kg DXR, 3 mg/kg 4'-amino-3'-hydroxy-DXR, or 2 mg/kg 4'-deoxy-DXR during the first 14 days of the study and were observed for an additional 35-day period. DXR induced severe cardiomyopathy that was characterized by ECG changes in vivo (S-alpha-T-segment widening and T-wave flattening) and by impairment of the contractile responses (F(max), +/- dF/dt(max)) to adrenaline of hearts isolated from treated animals. 4'-Deoxy-DXR caused a progressive enlargement of the S-alpha-T segment in vivo and a significant impairment of the - dF/dt(max) value in vitro, which were less severe than those produced by DXR. The least cardiotoxic drug was 4'-amino-3'-hydroxy-DXR, which induced minor ECG changes without causing significant alterations in the contractile responses of isolated hearts to adrenaline. On the basis of the drug concentration required to inhibit 50% of the colony formation (IC50) of cell lines in vitro, 4'-amino-3'-hydroxy-DXR was less active than 4'-deoxy-DXR but at least twice as active as DXR against human cancer and murine transformed cell lines. These data indicate that 4'-amino-3'-hydroxy-DXR is significantly less cardiotoxic and more cytotoxic than DXR

Involvement of basic fibroblast growth factor in suramin-induced inhibition of V79/AP4 fibroblast cell proliferation.

The V79/AP4 Chinese hamster fibroblasts were densely stained with the anti-basic fibroblast growth factor (bFGF) antibody demonstrating an endogenous production of the peptide. The in vitro proliferation of these cells was stimulated by exogenous bFGF and the maximum growth (259% increase in 3H-thymidine incorporation into DNA) was reached with bFGF 10 ng ml-1. Inhibition of bFGF-mediated mitogenic pathway was obtained with a 15-mer antisense oligodeoxynucleotide targeted against bFGF mRNA and with suramin, a drug which blocks the biological activity of heparin-binding growth factors. bFGF antisense oligomer reduced the synthesis of DNA by 79.5 and 89.5% at 20 and 60 microM, respectively; this effect was reversed by the addition of exogenous bFGF to the culture medium. A short-term exposure to suramin 300 micrograms ml-1 produced a modest reduction in 3H-thymidine incorporation but suppressed the mitogenic effect of bFGF on V79/AP4 cells. In cells treated with suramin 300 micrograms ml-1 the drug concentration increased linearly over 3 days, reaching 13.15 micrograms mg-1 of protein; cell proliferation was inhibited in a dose-related manner as evaluated by the colony formation assay (IC50: 344.22 micrograms ml-1) and by the number of mitoses observed in culture. Furthermore, the drug induced ultrastructural alterations, consisting of perinuclear cisternae swelling, chromatin condensation, nucleolar segregation and cytoplasmic vacuolations. These findings demonstrated that the endogenous production of bFGF plays an important role in V79/AP4 fibroblasts proliferation, and the inhibition of bFGF-mediated mitogenic signalling with bFGF antisense oligomer or suramin is an effective mean of reducing cell growth

INVOLVEMENT OF BASIC FIBROBLAST GROWTH-FACTOR IN SURAMIN-INDUCED INHIBITION OF V79/AP4 FIBROBLAST CELL-PROLIFERATION

The V79/AP4 Chinese hamster fibroblasts were densely stained with the anti-basic fibroblast growth factor (bFGF) antibody demonstrating an endogenous production of the peptide. The in vitro proliferation of these cells was stimulated by exogenous bFGF and the maximum growth (259% increase in H-3-thymidine incorporation into DNA) was reached with bFGF 10 ng ml-1. Inhibition of bFGF-mediated mitogenic pathway was obtained with a 15-mer antisense oligodeoxynucleotide targeted against bFGF mRNA and with suramin, a drug which blocks the biological activity of heparin-binding growth factors. bFGF antisense oligomer reduced the synthesis of DNA by 79.5 and 89.5% at 20 and 60 muM, respectively; this effect was reversed by the addition of exogenous bFGF to the culture medium. A short-term exposure to suramin 300 mug ml-1 produced a modest reduction in H-3-thymidine incorporation but suppressed the mitogenic effect of bFGF on V79/AP4 cells. In cells treated with suramin 300 mug ml-1 the drug concentration increased linearly over 3 days, reaching 13.15 mug mg-1 of protein; cell proliferation was inhibited in a dose-related manner as evaluated by the colony formation assay (IC50: 344.22 mug ml-1) and by the number of mitoses observed in culture. Furthermore, the drug induced ultrastructural alterations, consisting of perinuclear cisternae swelling, chromatin condensation, nucleolar segregation and cytoplasmic vacuolations. These findings demonstrated that the endogenous production of bFGF plays an important role in V79/AP4 fibroblasts proliferation, and the inhibition of bFGF-mediated mitogenic signalling with bFGF antisense oligomer or suramin is an effective mean of reducing cell growth

Inhibition of protein farnesylation enhances the chemotherapeutic efficacy of the novel geranylgeranyltransferase inhibitor BAL9611 in human colon cancer cells

Proteins belonging to the ras superfamily are involved in cell proliferation of normal and neoplastic tissues. To be biologically active, they require post-translational isoprenylation by farnesyl-transferase and geranylgeranyl-transferase. Enzyme inhibition by drugs may thus represent a promising approach to the treatment of cancer. Therefore, the combined effect of BAL9611, a novel inhibitor of geranylgeranylation, and manumycin, a farnesyl-transferase inhibitor, was evaluated on the SW620 human colon cancer cell line which harbours a mutated K-ras gene. BAL9611 and manumycin dose-dependently inhibited SW620 cell growth with 50% inhibitory concentration (IC 50) of 0.47 ± 0.03 and 5.24 ± 1.41 μM (mean ± SE), respectively. The isobologram analysis performed at the IC 50 level revealed that the combined treatment was highly synergistic with respect to cell growth inhibition. BAL9611 and manumycin were able to inhibit the geranylgeranylation of p21rhoA and farnesylation of p21ras; both drugs inhibited p42ERK2/MAPK phosphorylation, but their combination was more effective than either drug alone. Moreover, the enhanced inhibition of cell growth in vitro by the BAL9611-manumycin combination was also observed in vivo in CD nu/nu female mice xenografted with SW620 tumours. Finally, both drugs were able to induce cell death by apoptosis in vitro and in vivo, as demonstrated by perinuclear chromatin condensation, cytoplasm budding and nuclear fragmentation, and interoligonucleosomal DNA digestion. In conclusion, the inhibition of protein farnesylation enhances the chemotherapeutic effect of BAL9611 in vitro and in vivo in a synergistic fashion, as a result of the impairment of post-translational isoprenylation of proteins and phosphorylation of p42ERK2/MAPK, whose activation is associated with post-translational geranylgeranylation and farnesylation of p21rhoA and p21ras. © 2001 Cancer ResearchCampaign http://www.bjcancer.co

Comparative pharmacokinetic and pharmacodynamic evaluation of branded and generic formulations of meloxicam in healthy male volunteers

PURPOSE: The primary aim of the present study was to assess the pharmacokinetic bioequivalence between a generic formulation of meloxicam 15 mg tablets (Meloxicam Hexal) and its respective brand product (Mobic), in order to verify whether the generic product conforms to the regulatory standards of bioequivalence in the postmarketing setting. As a secondary exploratory aim, the pharmacodynamic effects of the two formulations were also evaluated by means of rating scales following hyperalgesia induced by cutaneous freeze injury. SUBJECTS AND METHODS: A single 15 mg dose of generic or branded meloxicam tablets was administered to 24 healthy male volunteers in a crossover fashion. Plasma samples, collected for 24 hours after dosing, were assayed for meloxicam concentration by a validated highperformance liquid chromatography method. RESULTS: THE ANALYSIS OF PHARMACOKINETIC PARAMETERS DID NOT SHOW ANY SIGNIFICANT DIFFERENCE BETWEEN THE TWO MELOXICAM FORMULATIONS: the 90% confidence intervals fell within the acceptance range of 80%-125% (0.84-1.16 for area under the curve [0-24], and 0.89-1.23 for peak concentration). No difference in the pharmacodynamic end point was observed between the two groups. CONCLUSION: The pharmacokinetic profiles of the two meloxicam formulations confirm the regulatory criteria for bioequivalence; pharmacodynamic data indicate a similar antihyperalgesic effect. The two formulations can be used interchangeably in the clinical setting