Multicenter Phase II Clinical Trial of Isolated Lung Perfusion in Patients With Lung Metastases.

BACKGROUND: Up to 66% of patients show local pulmonary disease progression after pulmonary metastasectomy. Regional treatment with isolated lung perfusion (ILuP) may improve local control with minimal systemic adverse effects. The aims of this study were to evaluate local and distant control after ILuP, determine the effect on overall survival compared with historical controls, and confirm the safety and feasibility of ILuP. METHODS: A total of 107 patients with resectable pulmonary metastases of colorectal carcinoma, osteosarcoma, and soft-tissue sarcoma were included in a prospective phase II study of pulmonary metastasectomy combined with ILuP with 45 mg melphalan at 37°C. Local and distant control, overall survival, lung function, and 90-day mortality and morbidity were monitored. RESULTS: We report 0% mortality, low morbidity, and no long-term pulmonary toxicity. For colorectal carcinoma, median time to local pulmonary progression, median time to progression, and median survival time were 31, 14, and 78 months, respectively. Median time to local progression was not reached for sarcoma, whereas median time to progression and median survival time were 13 and 39 months, respectively. The 5-year disease-free rate and pulmonary progression-free rate were 26% and 44% for colorectal carcinoma and 29% and 63% for sarcoma, respectively. CONCLUSIONS: ILuP with melphalan combined with metastasectomy is feasible and safe. Compared with historical controls, favorable results were obtained in this phase II study for local control. Further evaluation of locoregional lung perfusion techniques with other chemotherapeutic drugs is warranted.

Selective pulmonary artery perfusion with melphalan is equal to isolated lung perfusion but superior to intravenous melphalan for the treatment of sarcoma lung metastases in a rodent model.

OBJECTIVES: Isolated lung perfusion (ILuP) and selective pulmonary artery perfusion (SPAP) are experimental surgical techniques to deliver high-dose chemotherapy selectively to the lung for the treatment of lung metastases. ILuP with melphalan (MN) has shown to be feasible in clinical studies but can only be used once because it is invasive. SPAP as an endovascular technique can be repeated several times, but no results have been reported so far. Pharmacokinetics and efficacy of SPAP with MN were studied in a rodent lung metastasis model and compared it with ILuP and intravenous (IV) therapy. METHODS: Pharmacokinetics: forty-five Wag-Rij rats were randomized into three groups: IV 0.5 mg MN, ILuP 0.5 mg MN and SPAP 0.5 mg MN. Every treatment group was again randomized in three groups: 15 min treatment, 30 min treatment and 30 min treatment with 30 min reperfusion. Blood and tissue samples were taken for MN concentrations. EFFICACY: twenty-five Wag-Rij rats were randomized into five groups: control, sham thoracotomy, IV 0.5 mg MN, ILuP 0.5 mg MN and SPAP 0.5 mg MN. At day 0, bilateral lung metastases were induced, and treatment followed at day 7. At day 28, rats were sacrificed and pulmonary metastases counted. Survival: thirty Wag-Rij rats were randomized into five groups: control, sham ILuP, IV 0.5 mg MN, ILuP 0.5 mg MN, SPAP 0.5 mg MN. At day 0, left-sided lungmetastases were induced with treatment at day 7. Endpoints were death due to disease or survival up to 90 days. RESULTS: Pharmacokinetics: SPAP and ILuP resulted in significantly higher left lung MN concentrations compared with IV (P = 0.05). EFFICACY: SPAP (30 ± 22 nodules) and ILuP (20 ± 9 nodules) resulted in significantly less nodules compared with IV (113 ± 17 nodules; P < 0.01). Survival: median survival of SPAP (74 ± 8 days) was equal to ILuP MN (71 ± 10 days) but significantly longer compared with IV (54 ± 7 days; P < 0.01 both). CONCLUSIONS: SPAP with MN for the treatment of sarcoma lung metastases in rats is equally effective to ILuP but resulted in a significantly better survival compared with IV MN. As SPAP can be applied as a minimally invasive endovascular procedure, continued research with this technique is warranted.

Entomotoxicology, experimental set-up and interpretation for forensic toxicologists.

Forensic entomotoxicology studies the usefulness of insects as alternative toxicological samples. Use of insects as alternative matrix for drug detection is well documented and recommended when conventional matrices such as blood, urine or internal organs are no longer available. However, several limitations of entomotoxicology have been highlighted, especially concerning interpretation of the drug concentrations in insects on human forensic cases. In addition, the lack of knowledge in pharmacokinetic of drugs in insects, large variability of experimental set-up and toxicological analysis compromise the utility of this science. This review focuses on the current knowledge of factors influencing drug detection in insects. Reasons for the current limitations, but also recommendations for future research are discussed and proposed in this paper.

Conventional and alternative matrices for driving under the influence of cannabis: recent progress and remaining challenges.

In the past decade much research concerning the impact of cannabis use on road safety has been conducted. More specifically, studies on effects of cannabis smoking on driving performance, as well as epidemiological studies and cannabis-detection techniques have been published. As a result, several countries have adopted driving under the influence of drugs (DUID) legislations, with varying approaches worldwide. A wide variety of bodily fluids have been utilized to determine the presence of cannabis. Urine and blood are the most widely used matrices for DUID legislations. However, more and more publications focus on the usability of oral fluid testing for this purpose. Each matrix provides different information about time and extent of use and likelihood of impairment. This review will focus on the practical aspects of implying a DUID legislation. The pros and cons of the different biological matrices used for Δ(9)-tetrahydrocannabinol screening and quantification will be discussed. In addition, a literature overview concerning (roadside) cannabinoid detection, as well as laboratory confirmation techniques is given. Finally, we will discuss important issues influencing interpretation of these data, such as oral fluid collection, choice of cut-offs, stability and proficiency testing.

Quantification of methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in third instar larvae of Lucilia sericata (Diptera: Calliphoridae) using liquid chromatography-tandem mass spectrometry.

Entomotoxicology studies the application of toxicological analysis on necrophageous insects present on human remains. This paper describes the development and validation of a sensitive liquid chromatography tandem mass spectrometry method for quantification of methadone and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in developmental stages of Lucilia sericata. One single larva was pulverized in a disposable vial and then extracted with 1-chlorobutane. After evaporation of the organic layer, samples were reconstituted in the mobile phase. Chromatographic separation was achieved on a NUCLEODUR Sphinx RP column with a liquid chromatographic gradient (0.1% formic acid and methanol), ensuring the elution of methadone and EDDP within 15 min. The method was fully validated according to international guidelines. The use of liquid liquid extraction was demonstrated to be effective (matrix effect < 27% and recovery > 66%). The method was linear over the dynamic range (10-400 pg/mg larva) with excellent within- and between-run precision and bias (CV% < 5%). The lower limit of quantification was fixed at 10 pg/mg larva. No instability of the extracted samples was observed in the autosampler after three freeze/thaw cycles and after two months at -20 degrees C. The validated method was applied to third instar larvae of Lucilia sericata reared on beef heart spiked with 4 microg/g methadone and on a postmortem methadone overdose case. The validation and actual sample analysis showed that the method is sensitive, rugged, precise, accurate, and well-suited for routine analysis of methadone and EDDP in a single larva obtained from forensic cases.

Evaluation of on-site oral fluid screening using Drugwipe-5(+), RapidSTAT and Drug Test 5000 for the detection of drugs of abuse in drivers.

Driving under the influence of drugs is a major problem worldwide. At the moment, several countries have adopted a 'per se' legislation to address this problem. One of the key elements in the enforcement process is the possibility of rapid on-site screening tests to take immediate administrative measures. In this study, the reliability of three oral fluid screening devices (Mavand RapidSTAT, Securetec Drugwipe-5(+), and Dräger DrugTest 5000) was assessed by comparing their on-site results with confirmatory GC-MS plasma analysis. Our results demonstrate that for amphetamine screening, the oral fluid on-site devices on the market today are certainly sensitive enough. RapidSTAT, Drugwipe-5(+), and DrugTest 5000 demonstrated respectively a sensitivity of 93%, 100% and 92% for amphetamine/MDMA. For cocaine screening, sensitivities of 75%, 78% and 67% were obtained for the RapidSTAT, Drugwipe-5(+), and DrugTest 5000 devices, respectively. The studied devices were able to detect about 70% of all cannabis users in a roadside setting. However, a newer version of the DrugTest 5000 test cassette demonstrated a sensitivity of 93%, indicating an increased detection of Delta(9)-tetrahydrocannabinol using 'new generation' oral fluid screening tests with lowered cut-offs. Due to these promising results police officers and judicial experts are keen to use oral fluid screening devices. They believe that their ease of use and diminished amount of false positive results in comparison with urine screening will lead to more roadside tests and more appropriate juridical measures.

High-throughput analysis of amphetamines in blood and urine with online solid-phase extraction-liquid chromatography-tandem mass spectrometry.

An automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of amphetamines in blood and urine was developed and validated. Chromatographic separation was achieved on a Nucleodur Sphinx RP column with an LC gradient (a mixture of 10 mM ammonium formate buffer and acetonitrile), ensuring the elution of amphetamine, methamphetamine, MDMA, MDA, MDEA, PMA, and ephedrine within 11 min. The method was fully validated, according to international guidelines, using only 100 and 50 microL of blood and urine, respectively. The method showed an excellent intra- and interassay precision (relative standard deviation < 11.2% and bias < 13%) for two external quality control samples (QC) for both matrices and three and two 'in house' QCs for blood and urine, respectively. Responses were linear over the investigated range (r(2) > 0.99, 2.5-400 microg/L for blood and 25-1000 microg/L for urine). Limits of quantification were determined to be 2.5 and 25 microg/L for blood and urine, respectively. Limits of detection ranged from 0.05 to 0.5 microg/L for blood and 0.25 to 2.5 microg/L for urine, depending on the compound. Furthermore, the analytes and the processed samples were demonstrated to be stable (in the autosampler for at least 72 h and after three freeze/thaw cycles), and no disturbing matrix effects were observed for all compounds. Moreover, no carryover was observed after the analysis of high concentration samples (15,000 microg/L). The method was subsequently applied to authentic blood and urine samples obtained from forensic cases, which covered a broad range of concentrations. The validation results and actual sample analyses demonstrated that this method is rugged, precise, accurate, and well-suited for routine analysis as more than 72 samples are analyzed non-stop in 24 h with minimum sample handling. The combination of the high-throughput online SPE and the well-known sensitivity and selectivity assured by MS-MS resulted in the elimination of the bottleneck associated with the sample preparation requirements and provided increased sensitivity, accuracy, and precision.

On-line solid-phase extraction combined with liquid chromatography-tandem mass spectrometry for high throughput analysis of 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in urine.

A simple, rapid and highly sensitive method for the analysis of THC-COOH in urine, using automated on-line solid-phase extraction (SPE) combined with liquid chromatography (LC)-mass spectrometry (MS/MS), is developed and fully validated according to international guidelines. Chromatographic separation was achieved on an Atlantis dC(18) column with an isocratical gradient, ensuring the elution of THC-COOH within 4.1 min. The total process time was 6 min and 500 microL of sample was required. SPE using C(8) cartridges was highly effective, reproducible and led to significant decreases in the interferences present in the matrix. The method showed an excellent intra- and inter-assay precision (relative standard deviation (RSD) <7% and bias <13%) for four external quality control (QC) samples and three 'in house' QCs. Responses were linear over the investigated range (r(2)>0.99, 5-200 microg/L). Limits of quantification (LOQ) and detection (LOD) were determined to be 5 micro/L and 0.25 microg/L, respectively. Furthermore, the analyte and the processed samples were demonstrated to be stable. Moreover, no carryover was observed after the analysis of high concentrated urine samples (5000 microg/L THC-COOH)). The method was subsequently applied to authentic samples previously screened by a routine immunoassay method.

Simultaneous analysis of THC and its metabolites in blood using liquid chromatography-tandem mass spectrometry.

Cannabis is considered to be the most widely abused illicit drug in Europe. Consequently, sensitive and specific analytical methods are needed for forensic purposes and for cannabinoid pharmacokinetic and pharmacodynamic studies. A simple, rapid and highly sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy- Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy- Delta(9)-tetrahydrocannabinol (THC-COOH) in blood is presented. The method was fully validated according to international guidelines and comprises simultaneous liquid-liquid extraction (LLE) of the three analytes with hexane:ethyl acetate (90:10, v/v) into a single eluant followed by separation and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved using a XBridge C(18) column eluted isocratically with methanol:0.1% formic acid (80:20, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the LLE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using 250 microL of blood. The method was linear over the range investigated (0.5-40 microg/L for THC, 1-40 microg/L for 11-OH-THC, and 2-160 microg/L for THC-COOH) with excellent intra-assay and inter-assay precision; relative standard deviations (RSDs) were <12% for THC and 11-OH-THC and <8% for THC-COOH for certified quality control samples. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments. No instability was observed after repeated freezing and thawing or in processed samples. The method was subsequently applied to 63 authentic blood samples obtained from toxicology cases. The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well suited for routine analysis.

Celecoxib inhibits growth of tumors in a syngeneic rat liver metastases model for colorectal cancer.

INTRODUCTION: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce the risk of colorectal cancer in cyclooxygenase-2 (COX-2) overexpressing colorectal cancers. The present study was designed to evaluate the inhibitory effects of the COX-2 inhibitor celecoxib on the growth of colorectal cancer liver metastases in a syngeneic rat model, CC531. MATERIALS AND METHODS: The effects of celecoxib on cell viability in vitro were evaluated by treatment of CC531 tumor cell cultures with celecoxib. In vivo, Wag/Rij rats were inoculated with CC531 tumor cells at two sites in the liver and treated with celecoxib starting one week before, or directly after tumor inoculation. Control rats were inoculated without treatment. Three weeks after tumor inoculation rats were sacrificed. Tumor size, immune cell infiltration, caspase-3 activity, PGE(2) and celecoxib levels were determined. RESULTS: CC531 tumors did not show COX-2 expression. Tumor growth was significantly inhibited by celecoxib treatment in a dose dependent manner. Immune cell infiltration was decreased after celecoxib treatment, indicating that the immune system was not involved in preventing tumor growth. Tumor caspase-3 levels were only significantly increased if treatment was started before tumor inoculation. Celecoxib serum concentration starting at 0.84 microg/ml significantly inhibited the outgrowth of CC531 liver tumors. In contrast, in vitro concentrations of celecoxib of at least 12 microg/ml were needed to affect tumor cell viability. CONCLUSION: These results suggest that the inhibitory effects of celecoxib on tumor growth are not by direct cytotoxicity, but by creating an unfavorable environment for tumor growth.

Isolated hypoxic hepatic perfusion with retrograde outflow in patients with irresectable liver metastases; a new simplified technique in isolated hepatic perfusion.

BACKGROUND: Isolated hepatic perfusion with high-dose chemotherapy is a treatment option for patients with irresectable metastases confined to the liver. Prolonged local control and impact on survival have been claimed. Major drawbacks are magnitude and costs of the procedure. We developed an isolated hypoxic hepatic perfusion (IHHP) with retrograde outflow without the need for a heart-lung machine. PATIENTS AND METHODS: Twenty-four consecutive patients with irresectable metastases of various origins were treated. IHHP inflow was via the hepatic artery, outflow via the portal vein with occlusion of the retrohepatic caval vein. Radiolabeled albumine was used for leakage monitoring. Melphalan was used at 1-2 mg/kg. A 25-minute perfusion period was followed by a complete washout. Local and systemic melphalan concentrations were determined. RESULTS: Compared with oxygenated classical IHP, the IHPP procedure reduced operation time from >8 h to 4 hours, blood loss from >4000 to 900 cc and saved material and personnel costs. Leakage was 0% with negligible systemic toxicity and 0% perioperative mortality. Tumor response: complete response (CR) in 4%, partial response (PR) in 58%, and stable disease (SD) in 13%. Median time to progression was 9 months (2-24 months); pharmacokinetics demonstrated intrahepatic melphalan concentrations more than 9 fold higher than postperfusion systemic concentrations. CONCLUSIONS: IHPP is a relatively simple procedure with reduced costs, reduced blood loss, no mortality, limited toxicity, and response rates comparable to classic IHP. The median duration of 9 months of tumor control should be improved. Hereto, vasoactive drugs, will be explored in further studies.

Pharmacokinetics of gemcitabine when delivered by selective pulmonary artery perfusion for the treatment of lung cancer.

Lung cancer represents a major health problem. Cytostatic and radiotherapeutic treatment is limited because of dose-limiting systemic toxicity and surgery as a result of its invasive nature. Therefore, we developed a catheterization model of selective pulmonary artery perfusion (SPAP) combining the properties of isolated lung perfusion and i.v. treatment to achieve higher local drug levels and equivalent systemic exposure. Sixteen pigs underwent SPAP using a clinically applied dose of gemcitabine (1 g/m(2)). They furthermore underwent thoracotomy for tissue sampling. Three groups were treated with SPAP for 2 min with normal pulmonary blood flow, 50 and 90% flow reduction. Another group had SPAP for 10 min with normal blood flow. All the SPAP groups underwent catheterization of the left pulmonary artery. An additional group (n = 4) was infused i.v. for 30 min using the same dose. Concentrations were analyzed with analysis of variance. Pulmonary peak concentrations (p = 0.01) and areas under the curve (AUC) (p = 0.001) of SPAP for 2 and 10 min were significantly higher compared with i.v., whereas SPAP for 10 min resulted in the highest AUC (p = 0.045) compared with SPAP for 2 min. Flow reduction during SPAP resulted in inhomogeneous distribution. Liver levels, AUC (serum), and wet-to-dry ratios of all the SPAP groups were not significantly different compared with i.v. SPAP resulted in higher lung concentrations, whereas systemic exposure was comparable with i.v. Therefore, we advocate SPAP as a new method to be tested clinically to achieve down-staging of the tumor and lymph node status in lung cancer.

Liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and metabolites, in urine.

A validated method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and several metabolites is presented. The procedure comprises a sample clean-up step, using mixed-mode solid-phase extraction followed by liquid chromatography (LC)-tandem mass spectrometry analysis. Chromatographic separation was achieved using a Sunfire C(8) column eluted with a mixture of formate buffer, methanol, and acetonitrile. The applied LC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated analogues. Validation of the method was performed using 500 microL of urine. The limits of quantification (LOQ) for LSD and 2-oxo-3-hydroxy-LSD were 0.05 and 1 ng/mL, respectively, and ranged, for the other hallucinogens, from 0.5 to 10 ng/mL. Linear and quadratic regression was observed from the LOQ of each compound to 12.5 ng/mL for LSD, 50 ng/mL for 2-oxo-3-hydroxy-LSD and 500 ng/mL for the others (r(2) > 0.99). Precision for the QC samples, spiked at a minimum of two concentrations, was calculated [%CV and %bias < 20% for most of the compounds, except for bufotenine and cathinone (%bias < 24%), and ibogaine (%bias < 30%)]. Extraction was found to be both reproducible and efficient with recoveries > 87% for all the analytes. Furthermore, the processed samples were demonstrated to be stable in the autosampler for at least 24 h. Finally, the validated method was applied to the determination of chlorpheniramine, ketamine, LSD, and psilocin in authentic urine samples.

Detection of diazepam in urine, hair and preserved oral fluid samples with LC-MS-MS after single and repeated administration of Myolastan and Valium.

Sedative agents are used to facilitate sexual assault due to their ability to render the victim passive, submissive and unable to resist. The primary pharmacological effect of the benzodiazepine tetrazepam is muscle relaxation, whereas the benzodiazepine diazepam acts on the central nervous system (CNS) exerting mainly sedation effects. Therefore, contrary to tetrazepam, diazepam is an often-abused drug, which can potentially be used as a date-rape drug. In this study, we describe the detection of low amounts of diazepam in Myolastan (Sanofi-Synthelabo S.A., Brussels, Belgium) and Epsipam (Will-Pharma, Wavre, Belgium) 50 mg tablet preparations by LC-MS-MS, GC-FID and HPLC-DAD. Considering the important forensic implication of this finding, a study was conducted with volunteers receiving a single or repeated dosage of Myolastan. Urine, hair and preserved oral fluid samples were analysed using a previously described sensitive and specific LC-MS-MS detection method allowing for the simultaneous quantification of tetrazepam, diazepam, nordiazepam, oxazepam and temazepam. This study demonstrates that diazepam can be observed in urine samples even after a single dose of Myolastan. In addition, maintaining therapy for 1 week results in the detection of both diazepam and nordiazepam in urine samples and of diazepam in the first hair segment. Importantly, comparing urine and hair samples after a single intake of diazepam versus the single and 1 week administration of Myolastan shows that the possible metabolic conversion of tetrazepam to diazepam is a more plausible explanation for the detection of diazepam in biological samples after the intake of Myolastan. As such, these results reveal that the presence of diazepam and/or nordiazepam in biological samples from alleged drug-facilitated assault cases should be interpreted with care.

Bioanalytical procedures for determination of drugs of abuse in oral fluid.

Recent advances in analytical techniques have enabled the detection of drugs and drug metabolites in oral fluid specimens. Although GC-MS is still commonly used in practice, many laboratories have developed and successfully validated methods for LC-MS(-MS) that can detect a large number of compounds in the limited sample volume available. In addition, several enzyme immunoassays have been commercialized for the detection of drugs of abuse in oral fluid samples, enabling the fast screening and selection of presumably positive samples. A number of concerns are discussed, such as the variability in the volume of sample collected and its implications in terms of quantitative measurements, and the drug recoveries of the many different specimen collection systems on the market. Additional considerations that also receive attention are the importance of providing complete validation data with respect to analyte stability, matrix effect, and the choice of collection method.

High-throughput on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of 14 antidepressants and their metabolites in plasma.

A rapid, sensitive and fully automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma. Integration of the sample extraction and LC separation into a single system permitted direct injection of the plasma without prior sample pre-treatment. The applied gradient ensured the elution of all the examined drugs within 14 min and produced chromatographic peaks of acceptable symmetry. The total process time was 20 min and only 50 microL of plasma was required. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated compounds. The use of SPE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Extraction was found to be both reproducible and efficient with recoveries >99% for all the analytes. The method showed excellent intra-assay and inter-assay precision (relative standard deviation (RSD) and bias <20%) for quality control (QC) samples spiked at a concentration of 40, 200 and 800 microg/L and the r2>0.99 over the range investigated (10-1000 microg/L). Limits of quantification (LOQs) were estimated to be 10 microg/L. Furthermore, the processed samples were demonstrated to be stable for at least 48 h, except for clomipramine and norclomipramine, where a slight negative trend was observed, but did not compromise the quantification. The method was subsequently applied to authentic samples previously screened by a routine HPLC method with diode array detection (DAD).

Simultaneous determination of AMN107 and Imatinib (Gleevec, Glivec, STI571) in cultured tumour cells using an isocratic high-performance liquid chromatography procedure with UV detection.

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.

Docetaxel and gemcitabine combination therapy in advanced transitional cell carcinoma of the urothelium: results of a phase II and pharmacologic study.

Our objective was to determine the response to gemcitabine plus docetaxel in advanced urothelial transitional cell carcinoma in a phase II trial, and gemcitabine distribution between plasma and erythrocytes, following docetaxel administration. Patients with locally advanced or metastatic transitional cell carcinoma, following a maximum of one prior chemotherapy regimen, were given gemcitabine 800 mg/m on days 1 and 8 plus docetaxel 85 mg/m on day 8, every 21 days. Gemcitabine was measured in the plasma and erythrocytes of nine patients before and after docetaxel administration. Thirty-four patients (median 63 years; range 49-79 years), of whom seven had prior chemotherapy and 27 were chemotherapy-naive, received a median of six cycles (range 1-6). Complete and partial remissions were observed in two and 16 (including three pretreated) patients, respectively, for an overall response rate of 53%. Median response duration was 5 months (range 1-39+). Haematoxicity was manageable, despite grade 3 infections in 24% of patients, but other toxicities were mostly mild. An apparent shift of gemcitabine from plasma to erythrocytes occurred after docetaxel in five of six patients evaluable for this analysis. We conclude gemcitabine plus docetaxel is tolerable and highly active in treated and untreated patients with advanced transitional cell carcinoma.

Modified approach of administering cytostatics to the lung: more efficient isolated lung perfusion.

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.

Correlation of Delta9-tetrahydrocannabinol concentrations determined by LC-MS-MS in oral fluid and plasma from impaired drivers and evaluation of the on-site Dräger DrugTest.

Oral fluid (collected with the Intercept((R)) device) and plasma samples were obtained from 139 individuals suspected of driving under the influence of drugs and analyzed for Delta(9)-tetrahydrocannabinol (THC), the major psychoactive constituent of cannabis, using a validated quantitative LC-MS-MS method. The first aim of the study was to investigate the correlation between the analytical data obtained in the plasma and oral fluid samples, to evaluate the use of oral fluid as a 'predictor' of actual cannabis influence. The results of the study indicated a good accuracy when comparing THC detection in oral fluid and plasma (84.9-95.7% depending on the cut-off used for plasma analysis). ROC curve analysis was subsequently used to determine the optimal cut-off value for THC in oral fluid with plasma as reference sample, in order to 'predict' a positive plasma result for THC. When using the LOQ of the method for plasma (0.5 ng/mL), the optimal cut-off was 1.2 ng/mL THC in oral fluid (sensitivity, 94.7%; specificity, 92.0%). When using the legal cut-off in Belgium for driving under the influence in plasma (2 ng/mL), an optimal cut-off value of 5.2 ng/mL THC in oral fluid (sensitivity, 91.6%; specificity, 88.6%) was observed. In the second part of the study, the performance of the on-site Dräger DrugTest for the screening of THC in oral fluid during roadside controls was assessed by comparison with the corresponding LC-MS-MS results in plasma and oral fluid. Since the accuracy was always less than 66%, we do not recommend this Dräger DrugTest system for the on-site screening of THC in oral fluid.