Development of a Multichannel Dialysis Microchip for Bioassay of Drug Efficacy and Retention.

Here, we developed a multichannel dialysis microchip having three sets of dialysis systems, which consisted of three independent circulation channels with a common micropump. Each dialysis system was composed of a pneumatic micropump (heart), dialysis unit (renal corpuscle), and cell culture chamber (drug target) as well as circulation and dialysate channels to mimic the circulation system. Small molecules were successfully separated in parallel from macromolecules at the dialysis components. Anticancer tests using this microchip showed results that considered both serum protein-binding nature and drug activity. In the anticancer bioassay, the multichannel chip showed similar reproducible and reliable results as those of the single-channel system but with higher throughput.

Simultaneous quantification of cyclophosphamide, 4-hydroxycyclophosphamide, N,N',N"-triethylenethiophosphoramide (thiotepa) and N,N',N"-triethylenephosphoramide (tepa) in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

The alkylating agents cyclophosphamide (CP) and N, N', N"-triethylenethiophosphoramide (thiotepa) are often co-administered in high-dose chemotherapy regimens. Since these regimens can be complicated by the occurrence of severe and sometimes life-threatening toxicities, pharmacokinetically guided administration of these compounds, to reduce variability in exposure, may lead to improved tolerability. For rapid dose adaptations during a chemotherapy course, we have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of CP, thiotepa and their respective active metabolites 4-hydroxycyclophosphamide (4OHCP) and N, N', N"-triethylenephosphoramide (tepa) in plasma. Because of the instability of 4OHCP in plasma, the compound is derivatized with semicarbazide (SCZ) immediately after sample collection and quantified as 4OHCP-SCZ. Sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl of plasma. Chromatographic separation was performed on an Zorbax Extend C18 column (150 x 2.1 mm i.d., particle size 5 microm), with a quick gradient using 1 mM ammonia solution and acetonitrile, at a flow-rate of 0.4 ml min(-1). The analytical run time was 10 min. The triple quadrupole mass spectrometer was operating in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 200-40,000 ng ml(-1) for CP, 50-5000 ng ml(-1) for 4OHCP-SCZ and 5-2500 ng ml(-1) for thiotepa and tepa, using 100 microl of human plasma. These dynamic concentration ranges proved to be relevant in daily practice. Hexamethylphosphoramide was used as an internal standard. The coefficients of variation were <12% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/- 15%). This robust and rapid LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring of CP, thiotepa and their metabolites in our hospital.

Determination of N,N',N"-triethylenthiophosphoramide in biological samples using capillary gas chromatography.

A sensitive assay for the determination of N,N',N"-triethylenthiophosphoramide (thioTEPA) in microvolumes of human plasma and urine has been developed. ThioTEPA was analysed using gas chromatography with selective nitrogen-phosphorus detection, after extraction with ethyl acetate from the biological matrix. Diphenylamine is the internal standard. The limit of quantitation was 0.1 ng/ml, using only 100 microl of sample; recoveries ranged between 85 and 100% and both accuracy and precision were less than 10%. Using a flame ionisation nitrogen-phosphorus detector, the assay was not linear over the concentration range of 2-1000 ng/ml for plasma and 10-1000 ng/ml for urine. Linearity was accomplished in the range of 1-1000 ng/ml for plasma and urine when a thermionic nitrogen/phosphorous detector was used. The stability of thioTEPA in plasma proved to be satisfactory over a period of 3 months, when kept at -20 degrees C, whereas it was stable in urine for at least 1 month at -80 degrees C. ThioTEPA plasma concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay.

Metabolism and alkylating activity of thio-TEPA in rat liver slice incubation.

Precision-cut rat-liver slices were used to study the metabolism of the alkylating agent N,N',N''-triethylenethiophosphoramide (thio-TEPA). Exposure to high concentrations (1-10 mM) of thio-TEPA for 6 h did not prove to be toxic to the liver slices as indicated by insignificant leakage of potassium from the cells. The time course of the disappearance of thio-TEPA (initial concentration, 5.2 microM) from the buffer during incubation followed first-order kinetics. Formation of N,N'N''-triethylenephosphoramide (TEPA) apparently accounted for the elimination of thio-TEPA. Pretreatment of the rats with phenobarbital significantly increased the reaction rate. Conversely, pretreatment with the cytochrome P-450 inhibitor allylisopropylacetamide significantly reduced the metabolic rate. The elimination of thio-TEPA and formation of TEPA occurred independently of thio-TEPA concentration, which ranged from 5.2 to 104 microM. Thio-TEPA's oxo-analogue TEPA, which was not further metabolized, was the only metabolite identified. However, a significantly time-related increase in 4-(nitrobenzyl)-pyridine (NBP) alkylating activity was observed following incubation of liver slices with thio-TEPA but not after their incubation with TEPA. This may possibly indicate the formation of unknown active metabolites.

Supercritical fluid chromatography/chemical ionization/mass spectrometry of some anticancer drugs in a thermospray ion source.

A packed-column supercritical-fluid chromatograph was interfaced with a mass spectrometer via a modification of a thermospray probe. This modification allowed a capillary restrictor for the supercritical fluid (CO2) and reagent gas for chemical ionization to be introduced directly into a thermospray source. Chemical ionization conditions were observed when either the filament or discharge electrode was used and the source pressure was above 0.5 torr. The discharge electrode produced more efficient ionization, resulting in approximately a tenfold larger signal than that observed in the filament mode. The usefulness of this instrumentation was demonstrated on several anticancer drugs. Methanol positive ion chemical ionization (PICI) spectra were recorded for cyclophosphamide, diaziquone, mitomycin C and thiotepa. Methane PICI spectra of thiotepa were obtained in the absence of methanol as a mobile-phase modifier. A 50 ng on-column injection of diaziquone produced approximately a 6:1 signal to noise ratio in the scanning mode.

Liquid chromatography-thermospray mass spectrometry of DNA adducts formed with mitomycin C, porfiromycin and thiotepa.

High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reaction of mitomycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography-mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated.

Preoperative one-shot intra-arterial infusion chemotherapy for bladder cancer.

We have performed preoperative one-shot intra-arterial chemotherapy since 1976. However, in some cases, the results have not been satisfactory. Experimental studies were conducted to choose the drugs most suitable for this procedure. Drugs that were considered to be effective against bladder cancer, i.e., thio-TEPA, adriamycin (ADM), and cis-platinum (CDDP), were separately administered to groups of dogs via the common iliac artery or cephalic vein, and the concentrations of these drugs in the serum, bladder (mucosa and muscular layer), ileum, kidneys, and liver were measured 1 h later. The results revealed significantly high concentrations of intra-arterially injected ADM and CDDP in the bladder mucosa, suggesting that these drugs may be suitable for intra-arterial injection. It also appeared that thio-TEPA is unsuitable for this procedure. In clinical studies of 29 cases, preoperative one-shot intra-arterial injections were performed prior to total cystectomy or segmental resection of the bladder, and the effectiveness of the treatment was evaluated in terms of the histological effect, survival, and the relationship with the characteristics of the tumor. The results showed that the prognosis for cases showing therapeutic histological effectiveness (grade-IIb according to the classification of Shimosato et al.) was extremely good. Many patients in the grade-IIb group had stage-pT2 tumor or a tumor in the lateral wall. However, there seemed to be no significant differences between the drugs with respect to their histological effects.

Analysis of insect chemosterilants.

Background information concerning the concept of insect chemosterilization and analytical methods for determining trace levels of 19 P- and/or S- containing chemicals of current interest are presented. GC retention times of the compounds on 6 different columns and their p-values in 11 solvent systems are tabulated. The utility of the flame photometric detector for determining subnanogram levels of the sterilants in biological substrates is illustrated.

Persistence of thiotepa and tepa in pupae and adults of Culex pipiens fatigans Wiedemann.

Thiotepa and its oxygen analogue tepa, used to chemosterilize males of Culex pipiens fatigans for genetic control purposes, are toxic and mutagenic. An investigation showed that adult mosquitos that had been treated as pupae showed no detectable chemosterilant in their tissue 24 hours after emergence from the pupal stage.