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Cyclophosphamide (CY) was administered to 22 breast cancer patients treated routinely according to the CMF regimen: 75 mg/m3/d x 14 d p.o. CY, 30 mg/m2 days 1 and 8 i.v. methotrexate (MTX) and 500 mg/m2 days 1 and 8 i.v. 5-Fluorouracil (5-FU). The sequence of drug administration was always the same: 1) CY, 2) MTX; and 3) 5-FU. Capillary gas chromatography was performed for determination of CY in blood. Bioavailability (F) could be determined in 14 patients since CY was also administered intravenously in the same dose. The data of systemic exposure of oral CY in the other 8 patients were matched to those of the first 14 in whom bioavailability could be determined. Mean F was 0.85 +/- 0.22 (85% +/- 22%); in 1 patient F was 0.43 (43%). Furthermore, 3 patients treated with only p.o. CY had low estimated F values: 0.45, 0.49 and 0.50. In comparing patient characteristics with pharmacokinetic data, it was concluded that age might have a predictive value for elimination half-life t 1/2 z of i.v. CY. The youngest patients showed shortest t1/2 z and were also amongst those with the lowest F. This indication requires an extension of the study as well as monitoring of CY metabolism as a function of age. For the premenopausal patients this might be of particular importance, since this group is known to be prone to benefit from chemotherapeutic treatment according to the CMF regimen.

Influence of the 2 antimetabolites used in the CMF-regimen, methotrexate (MTX, M) and fluorouracil (FUra, F) on in vivo pharmacokinetics of orally administered cyclophosphamide (CY, C), were studied in WAG/Rij rats. Blood plasma concentrations of CY following oral administration were monitored in single-agent CY, in CY + MTX (CM), in CY + FUra (CF) and in CY + MTX + FUra (CMF) treatments. Each treatment group consisted of at least 10 rats. CY was determined in 50 microliters of plasma by capillary gas chromatography on the first day of chemotherapy. Statistical analysis of blood plasma concentration data revealed a significant influence of both MTX and FUra on CY input/output function (p:0.01). MTX and FUra significantly increased the area under the plasma concentration time-curve, whereas tmax was significantly prolonged in CF and CMF treatment groups (p:0.01). It is suggested that MTX and FUra interact at the site of CY pre-systemic metabolism, including first-pass metabolism, subsequently resulting in prolonged absorption.

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.

Forty-one clinical patients were studied on the first day of the first course of the CMF-regimen; administered by a fixed dosage scheme depending solely on body surface area of the patient in question. Parmacokinetic parameters were calculated for each drug: the data were analysed in conformity with the usual pharmacokinetic models. The results indicate a large pharmacokinetic variability, especially for cyclophosphamide (C). The large variability in plasma concentrations of C is presumed to be substantially attributable to the drug formula used.

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.

Several in vivo methods have been assessed for their capacity to predict sensitivity for anticancer agents in humans. Standard strategies have been developed for screening purposes. Adjustments of these strategies are frequently suggested in reports in which the correlation between assay results and clinical therapeutic efficacy is analysed. Low predictivity and high costs of these assays are important reasons for changing the screening strategy. In vivo methods which predict the clinical response in the individual patient, are under investigation. Only the results of the subrenal capsule assay (in normal mice) have been correlated with the clinical response in a larger study. The criticism of the method and the low predictivity for sensitivity in a prospective study provide no reason for optimism. Methods which study changes predicting the clinical response in patients are still in a developmental phase.

The pharmacokinetics of the new fluoropyrimidine 5'-deoxy-5-fluorouridine (5'-dFUrd) was investigated in twelve patients. The kinetics of the main metabolite 5-fluorouracil (5-FUra) was studied in eight patients and those of 5,6-dihydro-5-fluorouracil (5-FUraH2) in five patients. The patients participated in a Phase II study performed to investigate the response rate of 5'-dFUrd in advanced ovarian cancer. The pharmacokinetic data were compared with the clinical effects of the drug. The parent drug and 5-FUra were measured in both plasma and urine by high performance liquid chromatography. 5-FUraH2 concentrations in plasma were determined by capillary gas chromatography using electron capture detection and nitrogen-phosphorus specific detection. Several pharmacokinetic parameters such as elimination half-life, mean residence time, and steady state volumes of distribution are presented for 5'-dFUrd, 5-FUra, and 5-FUraH2. Two patients showed a partial response, four had stable disease, and five had progressive disease; one patient died due to myelotoxicity. The severity of the side effects correlated with the mean residence times of 5'-dFUrd and 5-FUra. Low pretreatment serum creatinine clearance (due to renal impairment) correlated with low renal excretion of 5'-dFUrd and a long mean residence time of 5'-dFUrd with the sum of observed toxicity. However, the extent of degradation of 5-FUra to 5-FUraH2 may also be related to the severity of the toxicity of 5'-dFUrd.

Flow cytometric determination of time dependent changes of numbers of reticulocytes in peripheral blood were investigated as a parameter for changes in erythropoiesis induced by radiation- or chemotherapy. Rats irradiated or treated with drugs (such as e.g. cyclophosphamide 100 mg/kg, vincristin 0.2 mg/kg, or mitomycin C 1.0 mg/kg) showed clear changes in erythropoietic activity. Reticulocyte numbers decreased rapidly until day 3-4 after treatment; this period was followed by a gradual increase and normal control values were seen at day 8-11. Radiation effects of doses as low 0.5 Gy could be detected in such a way. Similar studies were performed with patients with ovarian tumors treated with cis-platinum, a drug that may cause non-immune haemolysis. During prolonged treatment some patients showed increasing numbers of reticulocytes, measured at the first day of each hospitalization period, whereas leucocyte and platelet counts stayed more or less constant. Increasing numbers of reticulocytes generally indicates stimulation of erythropoietic activity of the bone marrow (due to increased blood loss); in this study increasing numbers often preceeded a decrease in hemoglobin values later on. Flow cytometric analysis of reticulocytes is therefore a potentially useful tool to detect changes in erythropoiesis, and considered more sensitive for the early recognition of patients that develop anemia, than hemoglobin measurements only.

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.

A new concept in the natural history of gastrointestinal (GI) cancer suggests that recurrence of this malignancy can be separated into two types. Hematogenous and lymphatic metastases occur before surgical removal of the primary cancer. The spread of cancer to the resection site and to peritoneal surfaces occurs at the time of surgical removal of the primary tumor. Surgical trauma leads to a dispersal of malignant tumor emboli, which then implant within the raw tissue surfaces of the resection site and abraded peritoneal surfaces. Instillation of chemotherapy directly into the peritoneal cavity, as part of GI surgery, provides cytotoxic levels of drug that may change the natural history of GI cancer. The most common sites of disease recurrence have been, in the past, at the resection site and on peritoneal surfaces. With the optimal use of intraperitoneal chemotherapy, these sites of surgical treatment failure should no longer occur. Early phase I and II and pharmacologic studies suggest that an effective dose and schedule have been achieved, that toxicity is at reasonable levels, and that responses with small volumes of intra-abdominal cancer are exceptionally high. Chemotherapy that has an impact on the surgical event by decreasing cancer spread to the resection site and to peritoneal surfaces may significantly improve survival and quality of life in patients with GI cancer.

Cyclophosphamide was administered to 12 breast cancer patients in combination with methotrexate and fluorouracil. Doses prescribed were cyclophosphamide 75 mg/m2, methotrexate 30 mg/m2 and fluorouracil 500 mg/m2 (per square meter body surface). Cyclophosphamide was administered intravenously and orally in aqueous solutions and in tablets in a randomized cross-over trial. Methotrexate and fluorouracil were administered intravenously, methotrexate was given first and then fluorouracil. Assays of cyclophosphamide in blood plasma were performed by capillary gas chromatography. Data of mean bioavailability of cyclophosphamide administered by tablets were suggestive of sufficient absorption. In 2 patients, however, a lower bioavailability of cyclophosphamide was demonstrated. Intra-individual differences in the terminal slope of the plasma decay curves after intravenous and oral administration in some patients decreased the calculated bioavailability of cyclophosphamide, if these values were included in the calculation of cyclophosphamide bioavailability. Compared with the administration of the solutions peak times, lag-times and mean absorption times of cyclophosphamide given in tablets were markedly prolonged. It is concluded that interactions between cyclophosphamide and methotrexate and/or fluorouracil after oral dosing as tablets are different from interactions observed after intravenous administration of cyclosphosphamide.

The present study is concerned with the investigation of the potentials and limitations of fast atom bombardment (FAB) and laser microprobe mass spectrometry (LAMMA) for the structural characterization of a series of cisplatin analogues. The limiting factors for obtaining good quality FAB spectra are the solubility and the stability of the organometallic platinum complexes in the FAB matrix. In the case of a suitable matrix being found, molecular weight information is derived from the (M + H)+ and/or (M - H)- ions. Drawbacks of the application of FAB are (i) the low signal intensities of the molecular ion-like species as compared to the matrix signals and (ii) the scarcity of fragmentation necessary for structure determination. Combination of FAB with tandem mass spectrometry was used to overcome these problems. LAMMA provides a valuable alternative for the direct mass spectral analysis of cisplatin analogues. For some compounds, LAMMA results in useful mass spectra, whereas FAB fails. The abundant fragmentation yields structural information which is complementary for positive and negative ions. The laser power density applied to the sample is of critical importance for the quality of the spectra.

In two human ovarian tumor cell lines, resistance to cis-diamminedichloroplatinum(II) (CDDP) was induced by continuous exposure of the parental lines to an increasing CDDP concentration in the culture medium. In contrast, a six times repeated pulse exposure of 6.7 microM or 16.7 microM CDDP for 1 h did not result in a cell line that showed a higher survival in CDDP-containing medium. The ID50 value for CDDP was seven to eight times higher for the resistant lines and those lines were able to grow at 3.3 microM CDDP. The induced resistance was stable during at least 25 doubling times. The CDDP-resistant sublines showed cross-resistance to the CDDP-analogues cis-diammine-1,1-cyclobutane-dicarboxylateplatinum(II) and cis-dichlorobis(isopropylamine)-trans-dihydroxyplatinum(IV) indicating that resistance to the different platinum compounds is generated by a common mechanism. The resistant sublines were also cross-resistant to mitomycin C and melphalan. The degree of cross-resistance for the tested drugs varied widely between the two cell lines. Resistance to CDDP was clearly correlated to decreased amounts of platinum in the resistant cells as compared to the sensitive cells. The amplification and expression of genes encoding proteins that had been shown to be involved in multidrug resistance, e.g., the Mr 170,000 P-glycoprotein was also studied. No amplification or overexpression of these genes could be shown in the resistant cell lines.

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.

In tumor-bearing WAG/Rij rats the interaction of cyclophosphamide and/or 5-fluorouracil (5-FU) with methotrexate as manifested at the pharmacokinetic level was studied. Four groups were formed of at least ten animals. The control group, which received single-agent methotrexate, was compared with groups that received methotrexate plus cyclophosphamide, methotrexate plus 5-FU, and methotrexate plus cyclophosphamide plus 5-FU. There appeared to be an increase of 40% in the clearance of methotrexate by the triple combination. Cyclophosphamide especially diminished the terminal part of the concentration-time curve of methotrexate.

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.

5-Fluorouracil is at present one of the most administered cytostatic drugs in cancer chemotherapy. However, due to its high toxicity, local administration of the drug, e.g. by isolated liver perfusion, may be advantageous. A bioanalytical procedure, suitable for the routine analysis of 5-fluorouracil in pig bile, liver homogenates, plasma and urine is described using reversed-phase ion-pair chromatography with absorbance detection at 268 nm. Using a protein precipitation procedure with acetonitrile, a determination limit of 3500 ng g-1 was achieved for liver samples, and 5 ng ml-1 for plasma samples using a liquid-liquid extraction step.

(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.

An in vivo method of isolated rat liver perfusion was developed with true vascular isolation and recirculating perfusate. This new surgical technique to temporarily isolate the liver vascularly, and the perfusion procedure are described in depth. Twelve inbred WAG/RIJ rats were subjected to 25 min of normothermic liver perfusion without chemotherapy, and all rats survived the procedure. Hepatic functional and histological integrity were not significantly altered during perfusion. To determine the role of isolated liver perfusion (ILP) as a means of improved targeting of antitumor agents, 5-fluorouracil (5-FU) concentrations were monitored in hepatic tumor and liver tissues and in systemic plasma using high-performance liquid chromatography. Fifty-one rats with hepatic tumors of colonic origin were randomly assigned to one of three dosage groups (20, 40, or 80 mg/kg) receiving 5-FU by ILP, hepatic artery infusion (HAI), or jugular vein infusion (JVI). ILP resulted in significantly increased 5-FU concentrations in liver tissue. However, no significant differences were found in tumor tissue concentrations of 5-FU between the three treatment modalities. 5-FU concentrations in tumor tissue increased as a function of the dose with ILP, HAI, and JVI. ILP was associated with the lowest systemic drug concentrations. The low systemic 5-FU concentrations with ILP suggest a higher maximum tolerable dose. This mode of treatment deserves to be studied further in our model before conclusions can be drawn regarding its therapeutic potential.

Capillary Gas Chromatography (CGC) is capable of determining underivatized cyclophosphamide (CPA) using SCOT OV 275 columns. Then CPA is subjected to in situ degradation resulting in formation of a cyclization product which can be determined selectively in biological fluids. In routine bioanalysis however cyclization products of CPA metabolites might interfere, e.g. 4-keto CPA. In the present study possible formation of cyclization products of 4-keto CPA similar to CPA was monitored by Mass Spectrometry. Cyclization of 4-keto CPA in situ was demonstrated to occur, resulting in a product similar to that of CPA. Both cyclization products could be determined selectively and it appeared that in situ cyclization of 4-keto CPA was negligible (less than 5%), probably owing to extra stabilization of the CPA metabolite by keto-enol tautomerism as has been demonstrated by NMR.

The interaction of methotrexate and/or cyclophosphamide with the pharmacokinetics of 5-fluorouracil (5-FU) was studied in tumor-bearing WAG/Rij rats. Four groups were formed including treatment with single-agent 5-FU (eight rats); 5-FU plus methotrexate (11 rats); 5-FU plus cyclophosphamide (12 rats); and 5-FU, cyclophosphamide, and methotrexate (13 rats). The area-under-the-plasma-concentration/time curve, total-body clearance, elimination half-life, mean residence time, and steady-state volume of distribution were computed and compared. The mean residence time and elimination half-life of 5-FU increased when methotrexate was included in the combination. The increase was significant (P less than 0.05) for 5-FU, cyclophosphamide, and methotrexate versus 5-FU and cyclophosphamide.