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A reversed phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the simultaneous determination of imatinib (Gleevec, Glivec, STI571) and AMN107 in cultured tumour cells, using clozapine as an internal standard. The compounds of interest were extracted by liquid-liquid extraction using TOXI-TUBES((R)) A extraction tubes. Chromatographic separation was performed on a Phenomenex Gemini C18 reversed phase column (150 mm x 2.0 mm, 5 microm particle size), using a mixture of 65% CH(3)OH (methanol) and 35% NH(4)Ac (Ammonium acetate) buffer (20mM, pH 10). Separation was achieved under isocratic conditions at a flow rate of 0.5 ml/min. Imatinib, clozapine and AMN107 are detected by UV detection at 260 nm. Calibration curves were linear from 50 to 7500 ng/ml with correlation coefficients (r(2)) better than 0.998. The limit of quantitation (LOD) was 50 ng/ml. The method has been successfully applied to a cellular kinetics study.

Recent years have seen the development of powerful technologies that have provided forensic scientists with new analytical capabilities, unimaginable only a few years ago. With liquid chromatography-mass spectrometry (LC-MS) in particular, there has been an explosion in the range of new products available for solving many analytical problems, especially for those applications in which non-volatile, labile and/or high molecular weight compounds are being analysed. The aim of this article is to present an overview of some of the most recent applications of LC-MS (/MS) to forensic analysis. To this end, our survey encompasses the period from 2002 to 2005 and focuses on trace analysis (including chemical warfare agents, explosives and dyes), the use of alternative specimens for monitoring drugs of abuse, systematic toxicological analysis and high-throughput analysis. It is not the intention to provide an exhaustive review of the literature but rather to provide the reader with a 'flavour' of the versatility and utility of the technique within the forensic sciences.

Background: Isolated lung perfusion (ILuP) is an experimental technique for the treatment of pulmonary metastases. We hypothesized that part of the drug taken up by the lung during ILuP is washed out during the flush procedure. Therefore, we investigated gemcitabine uptake at different inflow concentrations, and the effect of delayed clamp release after ILuP on lung levels was studied., Methods: Thirty rats had ILuP during 30 minutes using gemcitabine perfusate levels of 1.3, 2.7, 4.0, 5.3, and 6.7 mg/mL. Another 37 rats underwent ILuP with gemcitabine perfusate levels of 6.7 mg/mL during 6 minutes followed by a 5-minute flush and 30 or 60 minutes of reperfusion, while two other groups had ILuP and delayed clamp release for 30 or 60 minutes followed by a 5-minute flush. All effluent and lung samples were stored for later analysis. Results were evaluated using Friedmann two-way analysis and two-way analysis of variance., Results: At 6 minutes, steady-state of gemcitabine uptake was achieved for all inflow concentrations and a linear relation (r = 0.933, p < 0.0001) between effluent and lung levels was observed. Delayed clamp release resulted in significantly higher lung levels compared with immediate restoration of blood circulation after ILuP (456% at 30 minutes and 828% at 60 minutes)., Conclusions: Effective gemcitabine lung levels are already achieved after 6 minutes of ILuP with 6.7 mg/mL followed by delayed clamp release during 30 minutes instead of the clinically applied 30 minutes ILuP.

Background: Oral administration of irinotecan (CPT-11) should allow sustained exposure to the drug without the inconvenience of intravenous delivery and with fewer side-effects., Patients and Methods: The present phase I trial of CPT-11, administered orally as a powder-filled capsule for 5 consecutive days every 3 weeks at doses ranging from 30 to 90 mg/m(2)/day, was conducted in 47 patients for whom a satisfactory standard treatment option was no longer available (24 males/23 females; median age 51 years, range 26-85). Tumour types included melanoma (11), colorectal (4), urinary tract (3), lung/pleura (4), thyroid (3), liver (3), gallbladder (2), cervix/uterus (3), breast (2), pancreas (2), carcinoma and other cancer types (10)., Results: A total of 171 cycles were administered (median 3, range 1-11). Dose limiting toxicities (DLTs) occurred during the first cycle in five of 31 patients in the dose-escalation part of the study: one patient at the 50 mg/m(2)/day dose level (diarrhoea grade 4); one patient at the 80 mg/m(2)/day dose level (prolonged neutropenia grade 4 and diarrhoea grade 3); and three patients at the 90 mg/m(2)/day dose level (diarrhoea, vomiting and neutropenia). The 80 mg/m(2)/day dose level was expanded, as a feasibility study, to include 16 additional patients, five of whom had received extensive prior pelvic irradiation. A further three patients in this cohort experienced DLTs, two of whom had received extensive prior pelvic irradiation. One patient died on study day 15 during the first cycle of oral CPT-11 following grade 3 diarrhoea, febrile neutropenia and a necrotic enterocolitis. Overall the grade 3/4 toxicities in 47 patients were asthenia (19%), anorexia (17%), neutropenia (14.9 %), diarrhoea (13%), nausea (12.7%), vomiting (8.5%) and thrombocytopenia (8.5%). Partial responses were observed in two melanoma patients and disease stabilisation was noted in 17 (36.1%) patients. Pharmacokinetic parameters were recorded for 46 patients., Conclusions: At the maximum tolerated dose, defined as 80 mg/m(2)/day for 5 days every 3 weeks, oral CPT-11 was shown to be well tolerated and safe with few of the haematological toxicities associated with the intravenous formulation.

With the human genome sequence now determined, the field of molecular medicine is moving beyond genomics to proteomics, the large-scale analysis of proteins. It is now possible to examine the expression of more than 1000 proteins using mass spectrometry technology coupled with various separation methods. Microarray technology is a new and efficient approach, for extracting relevant biomedical data and has a wide range of applications. It provides a versatile tool to study protein-protein, protein-nucleic acid, protein-lipid, enzyme-substrate and protein-drug interactions. This review paper will explore the key themes in proteomics and their application in clinical cancer research.

Aims: To review the development and current status of balloon catheter mediated hypoxic perfusion of abdomen, pelvis and liver for treatment of locally advanced malignancies. Within this context we focus on the addition of tumour necrosis factor-alpha (TNF) to these minimal invasive perfusion procedures., Methods: A literature search on these topics was carried out in PubMed for indexed articles and in all issues of Regional Cancer Treatment. The findings were related to our own experiences., Results: Hypoxic abdominal (HAP) and hypoxic pelvic perfusion (HPP) using balloon catheters, are currently applied modalities for treatment of a wide variety of abdominal and pelvic tumours, yet scientific validation of these procedures is poor. Following the results of several Phase I-II trials, both treatments are associated with severe systemic toxicity, significant morbidity and even mortality. The degree of systemic leakage associated with these procedures prohibits addition of TNF. For leakage free liver perfusion surgery is still required, as with current balloon catheter techniques it is not possible to perform leakage free isolated hypoxic hepatic perfusion (IHHP), using either orthograde or retrograde hepatic flow. Experimental and clinical observations suggest that within any perfusion setting, the utilization of TNF is only indicated for treatment of highly vascularised tumours and not for treatment of colorectal tumours., Conclusion: Balloon catheter technology in its present form does not provide adequate leakage control in any of these settings and is therefore associated with considerable toxicity. It is associated with poor response rates and cannot be considered in any setting as a standard of care.

Aims: To investigate the feasibility of hypoxic pelvic perfusion (HPP), using balloon catheter techniques as treatment modality for locally advanced pelvic malignancies., Methods: In a phase I--II study, 16 patients with various non-resectable pelvic tumours were treated with two HPP with MMC and melphalan, followed by radiotherapy (25 Gy) and surgical resection if feasible. Toxicity and procedure related complications were documented. Tumour responses were assessed by MRI or CT. Pain reductive effects were assessed by evaluation of pain registration forms., Results: HPP resulted in augmented regional drug concentrations with relatively low systemic levels. Some severe systemic toxicity was observed. One procedure related death occurred. Pain reduction effects were short-lived. Ten patients had radiological NC, two PD and one PR. In 11 patients surgical resection was performed, which was microscopically radical in six cases. Mean survival was 26.8 months (range 1--86)., Conclusion: The seemingly favorable pharmacokinetic profiles observed with HPP in this and other studies can still lead to severe systemic toxicity. In terms of survival, local (re-)recurrence and pain reduction there seems no benefit of addition of HPP to pre-operative radiotherapy. HPP with MMC and melphalan, does not seem a therapeutic option in patients with locally advanced pelvic tumours.

The development of an on-column focusing gradient capillary LC method coupled to tandem mass spectrometry (quadrupole-linear ion trap) for the quantitative determination of the anticancer agent ZD1839 (Gefitinib, Iressa) in blood plasma is described. Plasma samples (0.2 ml) were extracted with methyl tert-butyl ether. The analytes of interest, ZD1839 and the internal standard [(2)H8]ZD1839 (ZD1839-d8) were eluted on a 50 mm x 1 mm, 5 microm particle size, capillary ODS Hypersil column using an aqueous ammonium acetate gradient at 40 microl/min. Mass spectrometric detection was performed by a Q-Trap tandem mass spectrometer with electrospray positive ionisation, and monitored in the multiple reaction monitoring transitions 447 >128 and 455 >136, respectively. The limit of quantification of ZD18395 was 0.1 ng/ml. The method proved to be robust, allowing quantification of ZD1839 with sufficient precision, accuracy and sensitivity.

We have developed a rapid method that enables the simultaneous analysis of gamma-hydroxybutyrate (GHB) and its precursors, i.e. gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in urine. The method comprised a simple dilution of the urine sample, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation was achieved using an Atlantis dC18 column, eluted with a mixture of formic acid and methanol. The method was linear from 1-80 mg/L for GHB and 1,4-BD and from 1-50 mg/L for GBL. The limit of quantification was 1 mg/L for all analytes. The procedure, which has a total analysis time (including sample preparation) of less than 12 min, was fully validated and applied to the analysis of 182 authentic urine samples; the results were correlated with a previously published GC-MS procedure and revealed a low prevalence of GHB-positive samples. Since no commercial immunoassay is available for the routine screening of GHB, this simple and rapid method should prove useful to meet the current increased demand for the measurement of GHB and its precursors.

Background: Addition of tumour necrosis factor-alpha (TNF) to hypoxic abdominal perfusion (HAP) and hypoxic pelvic perfusion (HPP) with chemotherapeutic agents for treatment of un-resectable malignancies may lead to similar enhanced anti-tumour effects as are observed when TNF is added to isolated limb perfusions (ILP) with Melphalan. Here, we validate the methodology of HAP and HPP using balloon catheter techniques, and investigate the distribution of TNF, Melphalan and Mitomycin C (MMC) over the regional and systemic blood compartments when applying these techniques., Materials and Methods: Twelve pigs underwent HAP or HPP with TNF, Melphalan and MMC for 20 min. Throughout and after the procedures blood samples were obtained from hepatic, portal and systemic blood compartments and plasma concentrations of perfused agents were determined., Results: We demonstrated that HAP and HPP result in temporary loco-regional concentration advantages of all perfused agents, although from start of perfusion significant systemic leakage occurred., Conclusion: On basis of these results it seems that the advantage in terms of regional plasma concentration of TNF may be insufficient for TNF-mediated effects to occur, making future addition of this cytokine to these procedures in the clinical setting questionable. The observed regional concentration advantages of MMC and Melphalan, however, warrant further studies on clinical application of these agents in both settings.

Background: Developments in balloon catheter methodology have made hypoxic abdominal perfusion (HAP) with anti-tumour agents possible with only minimal invasive surgery. The initial reports on this modality and celiac axis stop-flow infusion for treatment of pancreatic cancer were very promising in terms of tumour response, median survival and pain reduction. Recent reports, however, have not been able to confirm these results and some have disputed the efficacy of these currently still applied treatment modalities., Methods: Twenty-one patients with advanced pancreatic carcinoma were included in a phase I-II trial of HAP with MMC and Melphalan followed by celiac axis infusion (CAI) with the same agents six weeks later. Tumour response was assessed by abdominal-CT and by determining tumour markers. Effect on pain reduction was assessed by evaluation of pain registration forms., Results: HAP resulted in augmented regional drug concentrations. One patient died after CAI due to acute mesenterial ischaemia. One agent-toxicity related death was observed in the phase-I study. Significant hematological toxicity was observed after HAP and CAI at MTD. No patients were considered resectable after treatment. Median survival after HAP was 6 months (range 1-29). Pain reduction was experienced by only 5/18 patients and was short-lived., Conclusion: In contrast to earlier reports HAP and CAI with MMC and Melphalan did not demonstrate any benefit in terms of tumour response, median survival and pain reduction, compared to less invasive treatment options. As this treatment was associated with significant toxic side-effects and even one procedure related death, we do not consider this a therapeutic option in patients with advanced pancreatic cancer.

An isocratic high-performance liquid chromatographic method coupled to tandem mass spectrometry for the quantification of the revolutionary and promising anticancer agent STI-571 (tradenames Gleevec, Glivec, Imatinib) in blood plasma and red blood cells (RBCs) is described. The method involves measurement of sediment technology for RBCs and a subsequent single protein precipitation step by the addition of acetonitrile to both the RBC isolate and plasma. The sample mixture was centrifuged (10 min, 3600 g), and the supernatant filtered through a HPLC filter (0.45 microm). The analytes of interest, STI-571 and the internal standard [2H8]STI-571 were eluted on a Waters Symmetry C18 column (50x2.1 mm I.D., 3.5 microm particle size) using a methanol-0.05% ammonium acetate (72:28, v/v) mixture. STI-571 and [2H8]STI-571 were detected by electrospray tandem mass spectrometry in the positive mode, and monitored in the multiple reaction monitoring transitions 494>394 and 502<394, respectively. The lower limit of quantitation of STI-571 was 2.1 ng/ml in RBCs and 1.8 ng/ml in plasma. The recovery from both plasma and RBCs was between 65 and 70%. The method proved to be robust, allowing simultaneous quantification of STI-571 in RBCs and plasma with sufficient precision, accuracy and sensitivity and is useful in monitoring the fate of this signal transduction inhibitor in whole blood of cancer patients.

Most anticancer agents are relatively unstable substances and are subjected to intensive metabolism in vivo and radiation during sample pretreatment. Hyphenated techniques including a separation technique and, most frequently, mass spectrometry are therefore chosen to obtain insight into the in vivo behavior of anticancer agents. Once established, simpler assays can be derived from those based on hyphenation, which are less expensive. Capillary gas chromatography (cGC)-mass spectrometry (MS) is amongst the most frequently applied hyphenated analytical technologies in anticancer drug monitoring. Here a selection has been made of: (i) cGC-MS applied to the analysis of agents frequently used in clinical oncology (e.g. tamoxifen, oxazaphosphorines); (ii) cGC-MS applied to the development of new agents (Swainsonine and Brefeldin); (iii) cGC-MS applied to the analysis of agents for which comparisons with other frequently applied hyphenation technologies are possible (see Part I of this series). cGC-MS played a key role in the elucidation of the in vivo behavior of the oxazaphosphorine cyclophosphamide, historically the most frequently applied anticancer agent. cGC-MS appeared to be of special interest in the analysis of cyclophosphoramide and congeners in human erythrocytes by coupling of the hyphenated technique with a measurement of sediment technique. This resulted in the quantitative and qualitative analysis of oxaphosphorine-related mustard gas moieties in human erthrocytes for the first time.

High-performance liquid chromatography has become the separation technique of choice for the monitoring of generally thermolabile anticancer agents. With the introduction of electrospray mass spectrometry, the coupling of liquid chromatogaphy and mass spectrometry has opened the way to widely and routinely applied anticancer drug monitoring. Real-time metabolism versus degradation can now be distinguished, since derivatization is no longer obligatory. This is important for the monitoring of the anabolic and catabolic pathways of the same agent, such as 5-fluorouracil. Detection limits almost equal to those obtained with capillary gas chromatography-mass spectrometry are realistic with the latest generation of mass spectrometers, enabling quantitative analysis of various anticancer agents and their metabolites down to the low ng/ml level. Furthermore, sample clean-up and chromatography can be downscaled markedly using the latest column technologies, such as the generally applied 10 cm x 2.8 mm I.D. RP 18 columns. The coupling of capillary electrophoresis to mass spectrometry is today far from a routine application in anticancer drug monitoring. Nevertheless, interesting applications have been reported and are selected for the present review.

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 Student's 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.

A gradient high-performance liquid chromatographic (HPLC) method is described for the quantification of KW-2149 and its two major metabolites in plasma. The method involves a sample clean-up by solid-phase extraction on C18 columns, separation of the respective compounds by HPLC on a YMC ODS-AQ column (5-microm particle size, 150x6 mm I.D.), using a methanol-water gradient system as an eluent, and measurement by UV absorbance detection at 375 nm. The limits of quantitation were 10 ng/ml for KW-2149 and M-16, and 15 ng/ml for M-18. Recoveries from plasma were higher than 92% on C18 extraction columns. Intra-day precision, expressed as %C.V., was between 1.4 and 6.5%. Intra-day accuracy ranged from 94 to 107%. Precision and accuracy of variability of inter-assays increased somewhat; however, were still within acceptable ranges. The ability of the method to quantify KW-2149 and two major metabolites simultaneously, with precision, accuracy and sensitivity, make it useful in monitoring the fate of this new mitomycin in cancer patients.

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.

Background: Serum VEGF levels are elevated in cancer patients and are used as a tumor marker in different malignancies. We have measured VEGF levels in different blood compartments in cancer patients and healthy volunteers in order to assess the most suitable way of processing blood for measuring VEGF as a marker of tumor-angiogenesis., Patients and Methods: VEGF concentrations were analyzed by an enzyme-linked immunosorbent assay in serum (VEGFS), EDTA plasma (VEGFEDTA), citrated plasma (VEGFC), CTAD-plasma (VEGFCTAD), platelet poor plasma (VEGFPPP), platelet rich plasma after induction of platelet activation (VEGFPRP). Platelet activation was assessed by measuring PF4 concentrations in different plasma samples., Results: We observed higher VEGFS (P = 0.0027), VEGFEDTA (P = 0.003) and VEGFPPP (P = 0.0007) levels in cancer patients than in volunteers; VEGFPRP concentrations showed no significant difference (P = 0.208). Analysis of the correlation between VEGFplt and VEGFS in cancer patients showed a similar correlation in a comparable VEGFS concentration range as in the volunteers. When comparing VEGFC to VEGFCTAD, we find significantly higher VEGF and PF4 levels in citrated plasma (VEGF: P = 0.00019; PF4: P = 0.00023)., Conclusions: It is likely that VEGFS in cancer patients encompass platelet-delivered VEGF and VEGF from other sources, notably from (neo)-angiogenesis in tumoral tissue. The best discrimination between volunteers and cancer patients was observed in PPP. As generating plasma can induce platelet activation, with consequent VEGF release from platelets, we suggest that to assess free circulating VEGF, CTAD plasma should be used.

Aim of the Study: Cisplatin (CDDP) and radiotherapy are frequently used concomitantly in the treatment of various malignant conditions. Because of its toxicity, cisplatin tends to be replaced by carboplatin (CBDCA) in several indications. Available data regarding the combined effects of cisplatin and carboplatin with ionising radiation are contradictory., Materials and Methods: Various concentrations of cisplatin and carboplatin and various timing of association with radiation have been tested in vitro in a human ovarian cancer cell line. The parental cell line (AOvC-0) and a cisplatin-resistant stable subline (AOvC-CDDP/0) (De Pooter et al., Canc Res, 1991) were exposed to carboplatin (2.5, 5 and 10 M) and to CDDP (1, 2.5 and 5 M), 16 h and 4 h before and 4 h and 16 h after irradiation, respectively. Cell survival was evaluated by a classical clonogenic assay., Results: Exposure of AOvC-0 to 5 M CBDCA and of AOvC-CDDP/0 to 10 M CBDCA, before or shortly after radiation exposure, increased cell lethality in a clear supra-additive way, with the highest DEF in the shoulder region of the survival curve and at radiation doses relevant to clinical radiotherapy. In the sensitive cell line, 5 M carboplatin resulted in an additional lethality equivalent to 4.5 Gy; in the resistant cells, 10 M carboplatin was equivalent to 3.6 Gy. Replacing carboplatin by cisplatin in an identical set-up demonstrated exclusively simple additivity (DEF = 1)., Conclusion: These data suggest that carboplatin and cisplatin delivered at equitoxic doses interact with radiation in a different way and that, in the present set-up, only carboplatin enhanced the effects of radiation. Carboplatin might consequently be a better candidate than cisplatin in some concomitant combinations with radiotherapy.

Purpose: Oxazaphosphorines are metabolised by a variety of pathways, one of which leads to activation and the formation of alkylating compounds. However, the transport forms conveying activated oxazaphosphorines to the tumour cell have not been fully characterised. There is increasing recognition of the importance of the erythrocyte as a carrier of compounds in the circulation, and we have recently described higher concentrations of 4-hydroxycyclophosphamide within the erythrocyte compartment compared to plasma. We have now determined the concentrations of ifosfamide and seven of its metabolites in the plasma and erythrocytes of patients receiving a six-hour intravenous infusion of ifosfamide., Patients and Methods: Red cells from five patients, receiving a total of eight cycles of ifosfamide, were separated from plasma using the MESED instrument, and analysis of red cells and plasma performed using Gas Chromatography-Mass Spectrometry (GC/MS)., Results: The concentration of all compounds in the erythrocyte compartment was higher than or equal to those in plasma, and isophosphoramide mustard and carboxyifosfamide showed a particular affinity for the erythrocyte. The red cell fraction can contain as much as 77% of the total blood concentration of isophosphoramide mustard., Conclusions: Erythrocyte associated isophosphoramide mustard is an important transport form of activated ifosfamide. Red cells may have a role in the delivery of activated oxazaphosphorines to tissues.

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.

A recently developed GC-MS analytical method for the quantitative determination of oxazaphosphorines and their metabolites in blood plasma, using stable trifluoroacetyl derivatives and electron capture negative chemical ionization detection, was applied to measure the partitioning of the antitumor drug ifosfamide and its metabolites between plasma and red blood cells for four cancer patients. The separation of a constant volume of red blood cells was performed using a special instrument, MESED, through centrifugation of blood samples. The measured compounds were ifosfamide, 2- and 3-dechloroethylifosfamide, 4-ketoifosfamide, carboxyifosfamide, ifosfamide mustard, 2-chloroethylamine and 1,3-oxazolidin-2-one. Concentration-time profiles for the metabolites in the two blood fractions and partitioning factors between erythrocytes and plasma were obtained. For ifosfamide itself, and metabolites with an intact ring system, a partitioning factor between 1 and 2 was observed for the concentration ratio between red blood cells and plasma in the patients studied. However, for the compounds with an open structure, carboxyifosfamide and ifosfamide mustard, partitioning factors higher than 3 were obtained. The active antitumor metabolite ifosfamide mustard showed a strong preference for the red blood cells in the measured patient samples. This means that erythrocytes may play an important role in the transport and the subsequent release of the active alkylating agent to the tumor cells.

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.

Background: Thymidine incorporation studies performed in animal tumour models, revealed major differences in endothelial cell proliferation when tumour tissue was compared with normal tissue. The fraction of proliferating endothelial cells is reported to be increased by a factor of 30 to 40 in tumour tissue., Patients and Methods: To make it possible to analyze the endothelial cell proliferation in human tumours, an immunohistochemical double staining technique comprising CD31, an endothelial cell marker, and Ki-67, a proliferation marker, was developed. Endothelial cell proliferation was analysed in 21 primary human colorectal adenocarcinomas and in the adjacent mucosa., Results: Proliferating endothelial cells were found throughout the entire carcinoma. The mean overall endothelial cell labeling index (ECLI) was 9.9% (range, 5.4-18.0), and the labeling index of endothelial cells in areas of intense neovascularisation was even higher. Mean ECLI in the vascular hot spots was 21.0% (range, 6.8-35.0), and the mean tumour cell labeling index (TCLI) in the maximally Ki-67 immunostained areas was 78.3% (range 47.0-89.7). In 14 of 21 carcinomas, these areas were predominantly found at the luminal margin of the tumour, as were the vascular hot spots. A significant positive correlation was found between tumour vascularity, measured in the vascular hot spots, and tumour cell proliferation, measured in the maximally Ki-67 immunostained areas (p < 0.05). To analyse this relation in more detail, microvessel density (MVD), TCLI and ECLI were determined per x400 microscopic field by scanning in sequence from the luminal tumour margin to the invasive tumour base. In all tumours, the pattern of the MVD per x400 field, from the luminal margin to the tumour base, was similar to that of the TCLI and ECLI., Conclusions: These findings confirm that the fraction of cycling endothelial cells is higher in human colorectal carcinoma than in the adjacent mucosa which suggests that endothelial cells are proliferating in most of the individual capillaries in tumour tissue. Regional differences in MVD correlate with differences in tumour cell proliferation in these tumours.

The new mitomycin 7-N-[2-[2-(gamma-L-glutamylamino)ethyl)dithio)ethyl] mitomycin C (KW 2149) (I) proved to be active against a wide variety of experimental tumours. In order to perform pharmacokinetic studies with the new drug in Phase I sessions, a fast and reliable method has been developed based on the data of previous assays for mitomycin C. XAD-2 was preferred for isolation of I from blood plasma. The recovery of I was 50% whereas that of mitomycin C was 85%. Optimal separation was obtained on octadecyl silica columns with methanol-water (45:55, v/v) as mobile phase, while ultraviolet absorbance detection was performed at 375 nm. The assay enabled determination of I in a plasma concentration range of 20-1000 ng/ml using porfiromycin as internal standard.

The present review on the methods for the analysis of anticancer drugs should be seen as an addition to the excellent work of Eksborg and Ehrsson published half a decade ago in this journal (Vol. 340, p.31). The style and format have been followed closely, with the focus again on chromatographic techniques. We felt it important to add a list of compound (group) structures as a service to the reader. Methods have been reviewed for alkylating agents, platinum compounds, antitumour antibiotics, antimetabolites, alkaloids, suramin, 1-hydroxy-3-amino-propylidene-1,1-bisphosphonate and tamoxifen.

A liquid chromatographic method is described that can be used for the determination of suramin in plasma samples from cancer patients treated with this drug. The chromatographic system is based on the use of tetrabutylammonium bromide as an ion-pairing agent, while ultraviolet detection is applied. The sample pretreatment is a simple deproteination step by an organic solvent. The same counter-ion as used in the phase system is added in order to increase the recovery of the almost complete protein-bound suramin. The minimum detectable concentration in plasma is ca. 0.1 microgram/ml, thus allowing the monitoring of patients treated with this drug. One example of a plasma concentration-time course after administration of suramin is given.

Information about the extent and persistence of cytostatic activity in blood plasma after administration of a cytostatic drug into the body is needed for a better evaluation of the inter-individual variations in drug metabolism and disposition. As an assay for cytotoxic activity, a test system was chosen in which Chinese hamster ovary cells (CHO) were incubated with plasma containing active metabolites of cyclophosphamide (from human patients or rats), after which the frequencies of induced sister-chromatid exchanges per cell were determined. The treatment with plasma increased the frequencies of SCEs very effectively at concentrations of metabolites that were negative in the Salmonella typhimurium back-mutation test with strain TA100. The results obtained indicate that the SCE test system offers the possibility to follow the cytotoxic activity of plasma at various time intervals after administration of cyclophosphamide.

The performance of a number of normal phase and reversed-phase systems, with ultraviolet detection at 360 nm, has been investigated with respect to their applicability to pharmacokinetic studies of mitomycin C (MMC). The reversed-phase system developed was also combined with a polarographic detector in order to compare the sensitivity and selectivity of ultraviolet and electrochemical detection. A simple isolation procedure, based on the adsorption of MMC on a non-ionogenic resin, has been developed. The developed assay is applied to a pharmacokinetic study from which some examples are given.

A gas chromatographic assay for the determination of 5-fluorouracil (5-FU) and 5,6-dihydrofluorouracil (FDHU) is described. The selectivity and sensitivity of the method allows the determination of both 5-FU and FDHU in 200 microliters of plasma. Diphenylsuccinimide and chlorouracil were used as external and internal standard, respectively. The assay including the extraction shows a good linearity in the range 0-5000 ng/ml plasma for 5-FU as well as for FDHU. 5-FU and FDHU plasma concentrations of a number of patients with breast cancer treated with 5-FU were determined in order to demonstrate the usefulness of the method.

Although several pharmacological data of intravesical mitomycin C (MMC) are available now, data about resorption following subsequent intravesical instillations with different instillation times are lacking. We have analyzed MMC concentrations in blood plasma and urine following eight subsequent instillations, with 0.5- and 1.0-h instillation times. The relationships between urinary pH, urinary MMC concentrations, and MMC blood plasma concentrations were determined, as well as the stability of MMC in urine at pH 5-8 at 37 degrees C. An average of 40.3 and 46.4% of the total parent drug was recovered for the 0.5- and 1.0-h instillations, respectively. Blood plasma concentrations of MMC could be measured in nearly all patients and were independent of instillation times, urine concentration, or urinary pH. Resorption of MMC and recovery was stable during eight subsequent instillations. It was demonstrated that MMC can be degradated in urine at physiological conditions (pH less than 6; 37 degrees C). However, neither an influence of prolongation of the instillation time on MMC recovery from urine, nor a significant correlation between urinary pH and urinary MMC concentrations could be demonstrated. Since MMC can be degradated at pH less than 6 within 0.5 h, buffering of instillation fluids containing MMC is recommended. Reuse of instilled MMC, therefore, cannot be advised.

A fully automated liquid chromatographic system for the bioanalysis of mitomycin C has been described. The isolation of the analyte from the biological matrix (plasma, ascites and urine) is performed using a continuous-flow system equipped with a dialysis membrane in order to remove proteins. The samples are concentrated on a reversed-phase pre-column and subsequently introduced on to a reversed-phase analytical column by applying column-switching techniques. The drug is detected by absorbance measurements at 360 nm. Using the described system up to 100 samples a day can be analysed with determination limits of the order of 1 ng/ml, with a linear dynamic range of at least three decades for plasma and urine samples. The procedure was applied to pharmacokinetic studies of ovarian cancer patients treated intraperitoneally with mitomycin C.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is an antiviral drug that is experimentally used for modulation of the antitumour effect of fluoropyrimidines, such as ftorafur and 5-fluorouracil. The isolation of the analyte, in the presence of 5-fluorouracil, from the matrix is performed either by means of a simple protein precipitation (plasma) or by means of a liquid-liquid extraction with ethyl acetate (urine). Following pretreatment, the analyte is analysed by reversed-phase chromatography and quantified by absorbance detection at 307 nm. The minimum detectable concentration in plasma and urine samples is ca. 6 ng/ml. The recovery after deproteination of plasma samples is 75%, while after liquid-liquid extraction of urine the recovery amounts 92%. The degree of protein binding of the analyte, measured by ultrafiltration, is found to be 97%. These data allow the bioanalysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine for pharmacokinetic studies.