Gunther Guetens

Chronic imatinib mesylate exposure leads to reduced intracellular drug accumulation by induction of the ABCG2 (BCRP) and ABCB1 (MDR1) drug transport pumps

Imatinib mesylate is a selective tyrosine kinase inhibitor that is successfully used in the treatment of Philadelphia-positive chronic and acute leukaemias, and gastrointestinal stromal tumours. We investigated whether the intended chronic oral administration of imatinib might lead to the induction of the intestinal ABC transport proteins ABCB1, ABCC1 (MRP1), ABCC2 (MRP2) and ABCG2. Using Caco-2 cells as an in vitro model for intestinal drug transport, we found that continuous exposure (up to 100 days) with imatinib (10 ?M) specifically upregulates the expression of ABCG2 (maximal ~17-fold) and ABCB1 (maximal ~5-fold). The induction of gene expression appeared to be biphasic in time, with a significant increase in ABCG2 and ABCB1 at day 3 and day 25, respectively, and was not mediated through activation of the human orphan nuclear receptor SXR/NR1I2. Importantly, chronic imatinib exposure of Caco-2 cells resulted in a ~50% decrease in intracellular accumulation of imatinib, probably by enhanced ABCG2- and ABCB1-mediated efflux, as a result of upregulated expression of these drug pumps. Both ABCG2 and ABCB1 are normally expressed in the gastrointestinal tract and it might be anticipated that drug-induced upregulation of these intestinal pumps could reduce the oral bioavailability of imatinib, representing a novel mechanism of acquired pharmacokinetic drug resistance in cancer patients that are chronically treated with imatinib.

Association of enzyme and transporter genotypes with the pharmacokinetics of imatinib

Our objective was to explore the relationships between imatinib pharmacokinetics and 9 allelic variants in 7 genes coding for adenosine triphosphate‐binding cassette transporters (ABCB1 and ABCG2) and enzymes (cytochrome P450 [CYP] 2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) of putative relevance for imatinib.

Oxidative DNA damage: biological significance and methods of analysis.

All forms of aerobic life are subjected constantly to oxidant pressure from molecular oxygen and also reactive oxygen species (ROS), produced during the biochemical utilization of O2 and prooxidant stimulation of O2 metabolism. ROS are thought to influence the development of human cancer and more than 50 other human diseases. To prevent oxidative DNA damage (protection) or to reverse damage, thereby preventing mutagenesis and cancer (repair), the aerobic cell possesses antioxidant defense systems and DNA repair mechanisms. During the last 20 years, many analytical techniques have been developed to monitor oxidative DNA base damage. High-performance liquid chromatography-electrochemical detection and gas chromatography-mass spectro-metry are the two pioneering contributions to the field. Currently, the arsenal of methods available include the promising high-performance liquid chromatography-tandem mass spectrometry technique, capillary electrophoresis, 32P-postlabeling, fluorescence postlabeling, 3H-postlabeling, antibody-base immunoassays, and assays involving the use of DNA repair glycosylases such as the comet assay, the alkaline elution assay, and the alkaline unwinding method. Recently, the use of liquid chromatography-mass spectrometry has been introduced for the measurement of a number of modified nucleosides in oxidatively damaged DNA. The bulk of available chromatographic methods aimed at measuring individual DNA base lesions require either chemical hydrolysis or enzymatic digestion of oxidized DNA, following extraction from cells or tissues. The effect of experimental conditions (DNA isolation, hydrolysis, and/or derivatization) on the levels of oxidatively modified bases in DNA is enormous and has been studied intensively in the last 10 years.

Nanotechnology in bio/clinical analysis

Nanotechnology is being exploited now in different fields of analytical chemistry: Single cell analysis; in chip/micro machined devices; hyphenated technology and sampling techniques. Secretory vesicles can be chemically and individually analyzed with a combination of optical trapping, capillary electrophoresis separation, and laser induced fluorescence detection. Attoliters (10(-18) l) can be introduced into the tapered inlets of separation capillaries. Chip technology has come of age in the field of genomics, allowing faster analyses, and will fulfil an important role in RNA and peptide/protein analysis. The introduction of nanotechnology in LC-MS and CE-MS has resulted in new findings in the study of DNA adduct formation caused by carcinogenic substances, including anticancer drugs. Sample handling and introduction also can benefit from nanotechnology: The downscaling of sample volumes to the picoliter level has resulted in zeptomole (10(-21)) detection limits in the single-shot mass spectrum of proteins.

Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-α-based isolated hepatic perfusion

Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF-α) is currently performed in clinical trials in patients with hepatic metastases. Previous studies led to the hypothesis that the use of TNF-α in isolated limb perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature. However, whether TNF-α contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-α. IHP with melphalan alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-α resulted in a strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-α, ruling out a direct interaction of TNF-α with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-α was coadministrated with melphalan was strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and TNF-α mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-α in this setting.

Human Red Blood Cells: Rheological Aspects, Uptake, and Release of Cytotoxic Drugs

The shape of a normal human red blood cell (RBC) is well known: under resting conditions it is that of a biconcave discocyte. However, RBCs can easily undergo transformation to other shapes with stomatocytes and echinocytes as extremes. Various anticancer agents, generally reactive and labile substances, e.g., oxazaphosphorines and fluoropyrimidines, can induce severe deformation of shape. Shape changes in erythrocytes can induce rheological disturbances, which occasionally have pathophysiological consequences. It is difficult to estimate the impact of shape changes on the in vivo behavior of agents of biological interest. However, it has been demonstrated for various anticancer agents that erythrocytes fulfill an important role in their uptake, transport, and release. Moreover, some anticancer agents are capable of influencing important transporters such as MRP and GLUT-1. Monitoring of erythrocyte concentrations of certain cytotoxic agents is therefore of interest as the data generated can have a predictive outcome for therapeutic efficacy. This is true for cyclophosphamide, ifosfamide, lometrexol, and 6-mercaptopurine, as well as MRP and GLUT-1 mediated agents.

Isolated lung perfusion with gemcitabine in a rat: pharmacokinetics and survival.

BACKGROUND Toxicity and pharmacokinetics of gemcitabine (GCB) were evaluated in a rat model of isolated lung perfusion (ILuP) and compared to intravenous (iv) infusion. MATERIALS AND METHODS CC531S adenocarcinoma cells were incubated in vitro for 24 h with GCB. Cell survival was determined 4 days after GCB treatment with the sulforhodamine B test. In a first in vivo experiment, Wag/Rij rats underwent left ILuP with 20 mg/kg (n = 3), 40 mg/kg (n = 6), 80 mg/kg (n = 6), 160 mg/kg (n = 6), or 320 mg/kg (n = 6) of GCB and a control group (n = 6) with buffered starch. After 3 weeks, right pneumonectomy was performed. Furthermore, survival was determined for rats treated with iv infusion of 40 mg/kg (n = 10), 80 mg/kg (n = 10), 160 mg/kg (n = 10), or 320 mg/kg (n = 6) of GCB and a control group (n = 6) treated with saline (0.9% NaCl). In a second experiment lung and serum GCB levels were determined for rats treated with iv infusion (160 mg/kg, n = 6) and rats which had ILuP (160 mg/kg, n = 6; 320 mg/kg, n = 6). RESULTS Incubation of the CC531S adenocarcinoma cells with GCB led to a 50% decrease (P < 0.05) in the number of cells compared to controls at a dose of 23.6 nM. After 90 days, the mortality for rats treated with 320 mg/kg iv GCB was 100% compared to 17% after ILuP for the same dose. ILuP with 160 and 320 mg/kg resulted in significantly higher lung levels of GCB compared to iv therapy without any systemic leakage. CONCLUSIONS GCB ILuP is well-tolerated to a maximum dose of 320 mg/kg and results in significantly higher GCB lung levels with undetectable serum levels compared to iv treatment.

Single-pass isolated lung perfusion versus recirculating isolated lung perfusion with melphalan in a rat model

BACKGROUND Isolated lung perfusion (ILuP) with melphalan (MN) is superior to intravenous infusion for the treatment of pulmonary carcinoma and sarcoma metastases. However, it is unknown whether a bolus injection of MN into the perfusion circuit or ILuP with a fixed concentration of MN will result in the highest lung levels. METHODS ILuP with 0.5 mg MN was performed in Wag-Rij rats for 30 minutes either by a single-pass system (SP) (fixed concentration) (n = 10) or by reperfusion (RP) (bolus injection) (n = 10). In a separate experiment, rats were perfused with blood as the perfusate. In a third experiment, tumor levels were compared between SP, RP, or intravenous therapy with a dose of 0.5 mg. For induction of pulmonary metastases, 0.5 x 10(6) single adenocarcinoma cells were injected intravenously and therapy was given on day 30. For comparison of drug concentrations, unpaired Students t test was applied. Statistical significance was accepted at p less than 0.05. RESULTS Lung perfusion studies were succesfully performed without systemic leakage. Temperature of perfusate and rats was 34 degrees C to 37 degrees C. A significantly higher hematocrit (mean 27.9) compared with buffered starch (mean 2.5) did not result in higher MN lung levels or lower wet-to-dry ratio. Tumor levels were significantly higher after ILuP compared with intravenous therapy. However, no difference in tumor and lung levels was seen between single-pass and reperfusion. CONCLUSIONS Both ILuP techniques resulted in significantly higher MN lung levels than after intravenous therapy. Because no difference was seen between single-pass and recirculating perfusion, MN can be injected as a bolus into the closed perfusion circuit.

Pharmacokinetics after pulmonary artery perfusion with gemcitabine

BACKGROUND Isolated lung perfusion (ILuP) proved to be superior for the treatment of lung metastases compared with intravenous (i.v.) injection. However its invasive character limits repetitive treatment. Blood flow occlusion (BFO) as a regional therapy with gemcitabine (GCB) was evaluated in a rat model. Lung levels of GCB were examined with different exposure times and flow rates and compared with ILuP and i.v.. Cell kill was studied in vitro. METHODS In vitro survival of CC531 adenocarcinoma cells was determined after 10, 20, and 40 minutes of exposure to GCB. In vivo 48 Wag/Rij rats underwent BFO with GCB at a rate of 0.2 mL/min and 0.5 mL/min during 10, 20, 30, and 40 minutes. Statistical analysis was performed using Students t test. RESULTS In vitro, the dose of GCB resulting in 50% growth inhibition was 9.1 microg/mL, 7.2 microg/mL, and 2.2 microg/mL after 10, 20, and 40 minutes exposure respectively. In vivo, no significant difference in lung levels of GCB was observed between a flow rate of 0.2 mL/min compared with 0.5 mL/min at any exposure time point (p < 0.05). Lung tissue was saturated after 20 minutes. Blood flow occlusion resulted in a lower plasma levels and higher lung levels of GCB compared with i.v. injection of the maximal tolerated dose of 40 mg. CONCLUSIONS Growth inhibition of CC531 cells in vitro increased with exposure time while lung tissue was saturated after 20 minutes of BFO. No difference in GCB lung levels were seen after BFO compared with ILuP. Systemic exposure after i.v. injection was higher compared with BFO but did not result in higher lung levels.

Erythrocytes and the transfer of anticancer drugs and metabolites: A possible relationship with therapeutic outcome

Blood functions as a mobile tissue in an exchange system, with the remaining body tissue as a stationary phase. The equilibrium among plasma water, plasma proteins, and blood cells is described by models, but little consideration has been given to the substance-binding capacity of erythrocytes. There are numerous reasons for this, including bioanalytical limitations (ie, it has been difficult to study erythrocytes in the laboratory in their natural state). Erythrocyte monitoring requires accurate blood sampling and quantitative isolation of erythrocytes without disturbing the equilibrium of substances of interest between erythrocytes and plasma or other blood constituents. This became possible with the advent of the measurement of sediment device. The mass of a given substance available in blood can be described by M(Blood) = M(Plasma) + M(ERY) (+ M(REM)). M(ERY) is the mass of a substance present in erythrocytes and it is shown that for several oxazaphosphorines, such as iphosphoramide mustard, that M(ERY) determines M(Blood) with great superiority over M(Plasma). The impact of erythrocyte monitoring on therapeutic outcome has to be defined, but is an important area of research.