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The purpose of the study was to determine toxicity, efficacy and immunologic effects of concurrent subcutaneous injections of low-dose interleukin-2 (LD-IL-2), granulocyte-monocyte colony-stimulating factor (GM-CSF) and interferon-alpha 2b (IFNalpha) in progressive metastatic renal cell carcinoma. In a multicentre phase II study, 59 evaluable patients received two to six cycles of subcutaneous IL-2 (4 mIU m(-2)), GM-CSF (2.5 microg kg(-1)) and IFNalpha (5 mIU flat(-1)) for 12 days per 3 weeks with evaluation after every two cycles. Cycles were repeated in responding or stable patients. Data were analysed after a median of 30 months follow-up (range 16-48 months). In 42 patients, the immunologic response was studied and related to response and survival. The main toxicity were flu-like symptoms, malaise and transient liver enzyme elevations, necessitating IL-2 reduction to 2 mIU m(-2) in 29 patients, which should be considered the maximal tolerable dose. The response was 24% (eight out of 34, three complete response (CR), five partial response (PR)) in patients with metachronic metastases and 12% (three out of 25, 2CR, 1PR) in patients with synchronic metastases. Overall response was 19% (11 out of 59). Median survival was 9.5 months. All tested patients showed expansion and/or activation of lymphocytes, T cells and subsets, NK cells, eosinophils and monocytes. Pretreatment HLA-DR levels on monocytes and number of CD4(+)HLA-DR(+) cells correlated with response. Pretreatment number of CD4(+)HLA-DR(+) cells and postimmunotherapy levels of lymphocytes, CD3(+), CD4(+) and CD8(+) T cells, but not of NK or B cells, correlated with prolonged survival. Immunotherapy with concurrent subcutaneous GM-CSF, LD-IL-2 and IFNalpha has limited toxicity, can be given as outpatient treatment and can induce durable CR. Response and survival with this form of immunotherapy seem to be more dependent on expansion/activation of T cells than of NK cells. [ABSTRACT FROM AUTHOR]

The purpose of this study is to determine the toxicity and efficacy of temozolomide (TMZ) p.o. followed by subcutaneous (s.c.) low-dose interleukin-2 (IL2), granulocyte-monocyte colony stimulating factor (GM-CSF) and interferon-alpha 2b (IFN alpha) in patients with metastatic melanoma. A total of 74 evaluable patients received, in four separate cohorts, escalating doses of TMZ (150-250 mg m(-2)) for 5 days followed by s.c. IL2 (4 MIU m(-2)), GM-CSF (2.5 microg kg(-1)) and IFN alpha (5 MIU flat) for 12 days. A second identical treatment was scheduled on day 22 and cycles were repeated in stable or responding patients following evaluation. Data were analysed after a median follow-up of 20 months (12-30 months). The overall objective response rate was 31% (23 out of 74; confidence limits 20.8-42.9%) with 5% CR. Responses occurred in all disease sites including the central nervous system (CNS). Of the 36 patients with responding or stable disease, none developed CNS metastasis as the first or concurrent site of progressive disease. Median survival was 252 days (8.3 months), 1 year survival 41%. Thrombocytopenia was the primary toxicity of TMZ and was dose- and patient-dependent. Lymphocytopenia (grade 3-4 CTC) occurred in 48.5% (34 out of 70) fully monitored patients following TMZ and was present after immunotherapy in two patients. The main toxicity of combined immunotherapy was the flu-like syndrome (grade 3) and transient liver function disturbances (grade 2 in 20, grade 3 in 15 patients). TMZ p.o. followed by s.c. combined immunotherapy demonstrates efficacy in patients with stage IV melanoma and is associated with toxicity that is manageable on an outpatient basis. [ABSTRACT FROM AUTHOR]

Three metabolites of the cytotoxic drug paclitaxel (Taxol) were isolated and purified from the feces of cancer patients receiving the agent as an intravenous infusion. The procedures involved sample homogenization in water followed by liquid-liquid extraction with diethyl ether and high-performance liquid chromatography (HPLC). Approximately 1-3.5 mg of each metabolite was obtained from 100 g of feces. As judged from the chromatographic traces of analytical HPLC with ultraviolet (UV) detection at 227 nm, the purity of each compound was > 97%. On-line photodiode-array detection demonstrated that the UV spectrum of the isolated compounds closely resembles that of the parent drug. Mass spectrometry provided evidence that these metabolites are mono- and dihydroxy-substituted derivatives, namely, 6 alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel, and 6 alpha, 3'-p-dihydroxypaclitaxel. The two 6 alpha-hydroxy-substituted metabolites were shown to have lost their cytotoxicity in in vitro clonogenic assays using the A2780 human ovarian carcinoma and the CC531 rat colon-carcinoma tumor cell lines. In addition, the metabolites showed reduced myelotoxic effects as compared with paclitaxel in an in vitro hemopoietic progenitor toxicity assay. Our procedure for the isolation and purification of paclitaxel metabolites in milligram quantities should be useful for testing the biological activities of these compounds and for the preparation of calibration standards essential for pharmacokinetics studies. [ABSTRACT FROM AUTHOR]

We investigated the pharmacokinetics of tetrahydrofolates following the administration of [6S,R]-folinic acid and 5-flurouracil delivered i.v., i.p., and by a combination of both routes in patients with colon cancer. The concentrations of the biologically active tetrahydrofolates ([6S]-folinic acid and 5-methyltetrahydrofolate) and the relatively inert diastereomer [6R]-folinic acid were monitored using a selective on-line coupled achiral-chiral high-performance liquid chromatographic method. In plasma, a target concentration of 5 microM active tetrahydrofolates, which is considered necessary for an optimal synergistic effect, could be achieved after i.v. or combined i.v. and i.p. administration but was not reached in a patient receiving i.p. [6S,R]-folinic acid alone. In three patients receiving i.p. [6S,R]-folinic acid a high level of [6S]-folinic acid was observed in ascites, suggesting that the peritoneal cavity may act as a storage site for tetrahydrofolates after i.p. administration. In these patients, only a trace level of the active metabolite 5-methyltetrahydrofolate was detected in ascites, which may indicate that tetrahydrofolate derivatives penetrate only minimally, if at all, into the peritoneal cavity from the central compartment. These data would indicate that a combination of i.p. and i.v. administration may, from the pharmacological point of view, indeed contribute to an improved treatment of minimal residual disease persisting in the peritoneal cavity. [ABSTRACT FROM AUTHOR]

We investigated the pharmacokinetics of the experimental semisynthetic vinca alkaloid vinleucinol (VileE; O4-deacetyl-3-de(methoxycarbonyl)-3-[[[1-ethoxycarbonyl-2- methylbutyl]amino]carbonyl]-vincaleukoblastine). The study was performed in male FVB mice receiving 10.5 mg/kg VileE i.v. or p.o. Plasma, urine, faeces and tissue samples were analysed by a selective method based on ion-exchange normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection and liquid-liquid extraction for sample clean-up. Apart from the parent drug, two other metabolic compounds were detected. One of these metabolites is vinleucinol acid (VileA; O4-deacetyl-3-de(methoxycarbonyl)-3-[[[1-carboxyl-2- methylbutyl]amino]carbonyl]-vincaleukoblastine), which possesses no cytotoxic activity. The structure proposed for the second metabolite (VileX) was based on tandem mass spectrometry (MS-MS) and infrared (IR) spectroscopy data. Metabolization of VileE to VileX must occur in the amino acid moiety of the molecule, with a (beta- or gamma-) lactone ring being formed after oxidation of the (beta- or gamma) carbon of the amino acid. VileX is a major metabolite, which is excreted in faeces and urine after i.v. administration and accounting for up to 23% of the administered dose. The activity of VileX against cultured L1210 cells is four times that of the parent drug VileE and comparable with that of vinblastine (VBL). At 48 h after administration of VileE, the concentration of VileX exceeds that of the parent drug in many tissues. These findings indicate that the metabolite VileX may be at least largely responsible for the activity observed against xenografts in mice after administration of the parent drug, VileE. [ABSTRACT FROM AUTHOR]

We have developed and validated a selective analytical procedure, based on ion-exchange normal-phase liquid chromatography with fluorescence detection and liquid-liquid extraction, for the analysis of vinblastine (VBL) in biological matrices. The assay is suitable for the determination of the parent compound and its metabolites in plasma, tissue, faeces and urine specimens. Pharmacokinetics studies were performed in male FVB mice receiving VBL by intravenous (i.v.) bolus injection at a dose of 6 mg/kg. Plasma concentrations were monitored until 48 h after drug administration. Urine and faeces samples were collected in 24-h portions for up to 72 h and tissue samples were obtained at 4, 24, 72 and 168 h after drug administration. To facilitate a comparison between the findings we obtained by high-performance liquid chromatography (HPLC) and the results of previous studies using radiolabeled drug monitoring, some of the animals were also given radiolabeled drug. Large discrepancies were observed between the results obtained by the two methods. Excretion of the radiolabel in faeces and urine was 85% of the dose within 72 h. HPLC revealed that only 18% of the dose was excreted as unchanged drug and 19%, as measurable metabolites [O4-deacetylvinblastine (DVBL) and two unknown compounds]. In most of the tissues taken at 4 h after drug administration, virtually all of the radioactivity represented VBL or DVBL. In all tissues taken at 72 h after drug administration, however, only very little of the radioactivity remained in the form of these compounds. Following the administration, VBL and DVBL were distributed extensively to most tissues. Many tissues appeared to possess effective means of extruding the cytotoxic drug with decreasing plasma levels. However, in some organs, including those from the genital tract and lymphatic tissues, VBL and DVBL were retained for prolonged periods. Our studies confirm previous indications that selective retention may be the basis of the activity of VBL against malignant transformations derived from these tissues. [ABSTRACT FROM AUTHOR]

Previous studies in mice with disrupted mdr1a P-glycoprotein genes have shown that the oral bioavailability of paclitaxel is very low because of the presence of this drug-transporting protein in the intestinal wall. Additional studies with cyclosporin A have shown that this P-glycoprotein-inhibiting agent is able to increase the bioavailability of paclitaxel in mouse models and in patients. However, the potential immune-suppressive side effects of cyclosporin A renders this compound less suitable for chronic use in cancer patients. In this paper we present the results obtained with GF120918, an experimental P-glycoprotein inhibitor, on the oral bioavailability of paclitaxel in both wild-type and mdrlab knockout mice. GF120918 (25 mg/kg) was administered p.o. by gavage 15 min or 2 h before oral or i.v. dosing of paclitaxel, respectively. Paclitaxel plasma levels were quantified by high-performance liquid chromatography. GF120918 increased the plasma values for areas under the concentration-time curve of oral paclitaxel in wild-type mice by 6.6-fold from 408 to 2701 ng x ml(-1) h. Calculated relative to their respective values for area under the concentration-time curve after i.v. administration, GF120918 increased the oral bioavailability of paclitaxel in wild-type mice from 8.5 to 40.2%. The plasma pharmacokinetics of paclitaxel in mdr1ab knockout mice was not altered by GF120918, whereas the pharmacokinetics of paclitaxel in wild-type mice receiving GF120918 became comparable with mdr1ab knockout mice. This result indicates that GF120918 at this dose-level selectively and completely blocks P-glycoprotein in the intestines and does not notably interfere in the elimination of paclitaxel by metabolism or other transporters. On the basis of this result, GF120918 has been selected for additional study in humans.

The purpose of our study was to determine the maximally tolerated dose (MTD) and DLT of combined administration of granulocyte macrophage colony-stimulating factor (GM-CSF), low-dose interleukin 2 (IL-2) and IFN-alpha in patients with progressive metastatic melanoma or renal cell carcinoma (RCC). In addition, the activation and expansion of effector cells were measured. Cohorts of three patients were treated with increasing doses of IL-2 (1, 4, and 8 MIU/m2) and GM-CSF (2.5 and 5 microg/kg) with a constant dose of IFNalpha (5 million units) s.c. for 12 days every 3 weeks. An additional six patients were treated at the MTD. Immune activation was monitored during the first cycle. Response was evaluated after two cycles. The MTD was found to be 2.5 microg/kg GM-CSF, 4 MIU/m2 IL-2, and 5 mega units of IFNalpha. DLT was grade 4 fever, chills with hypotension, grade 3 fatigue/malaise, and fluid retention. Dose reduction of IL-2 to 2 MIU/m2 was necessary in three of nine patients who initially received the MTD. Treatment was initiated in the hospital but could be continued at home after 3-4 days. Significant increases in lymphocytes, (activated) T cells (CD4+ and CD8+), NK cells, monocyte DR expression, neutrophils, and eosinophils were found. CD8+ T-cell activation (sCD8) and NK cell expansion was mainly present in patients receiving 2 or 4 MIU/m2 IL-2. Of eight patients with progressive metastatic RCC after nephrectomy, three achieved a complete remission, and 1 of 7 patients with metastatic melanoma achieved a partial remission. In our study, the MTD of combined immunotherapy with GM-CSF, IL-2, and IFNalpha was established; DLT was: (a) grade 4 fever with hypotension needing i.v. fluid support; and (b) grade 3 fluid retention and/or fatigue/malaise. The scheme resulted in considerable expansion and/or activation of various effector cells. The complete responses in RCC patients are promising but need to be confirmed in Phase II studies.

We have developed and validated an analytical methodology for the quantification of docetaxel and its four major human oxidation metabolites in mouse plasma. We have used this procedure to study the pharmacokinetics and metabolism of docetaxel in female FVB mice, receiving 2.5, 10, or 33 mg/kg of docetaxel by i.v. injection. We have also studied the pharmacokinetics of polysorbate 80, because it was shown previously that the vehicle substance Cremophor EL, which is used in the formulation of paclitaxel, exerts a profound effect on the pharmacokinetics of this compound. Linear pharmacokinetics of docetaxel was observed at dose levels between 2.5 and 10 mg/kg, where plasma levels corresponded to those in patients receiving the maximum tolerated dose. At the highest dose level of 33 mg/kg, a deviation from the linear kinetics was observed. Compared with humans, mice could tolerate much higher plasma levels, suggesting that the toxic side effects are related to a certain plasma threshold concentration instead of area under the curve or Cmax. At the highest dose level, three docetaxel metabolites could be detected in the plasma samples of mice for up to 4 h after drug administration. The hydroxy metabolite of the tert-butoxy group (metabolite II) was the major metabolite, followed by the two epimeric hydroxyoxazolone-type compounds (metabolites I and III). A fourth putative metabolite (e.g., the cyclic oxazolidinedione derivative) was not detected. Because of rapid degradation of polysorbate 80 by esterases in plasma, the concentration of this vehicle substance declined very rapidly. Consequently, this substance was not able to interfere in the disposition of docetaxel.

Background: After high-dose chemotherapy with autologous stem-cell support long hospital stays in the aplastic phase are expensive, lead to increased risk of hospital infections and to increasing pressure on available hospital beds. We developed a home care regimen that allows patients to be at home for most of the aplastic period, without daily hospital visits., Patients and Methods: Between October 1995 and December 1997, transfer of supportive care to the home setting took place in three phases for patients undergoing high-dose chemotherapy with stem-cell transplant for malignant lymphoma (one course of BEAM), breast cancer or germ-cell cancer (three courses of tCTC). In the inpatient cohort, the supportive care designed for at home use was administered in the hospital until neutrophile recovery to 0.5 x 10(9)/l. In the second, outpatient cohort, patients were discharged the day after stem-cell reinfusion but the supportive care was delivered daily in hospital. The third, home care cohort, consisted of patients who were discharged the day after stemcell reinfusion, after which specialized home care professionals delivered all supportive care including transfusions and parenteral antibiotics at home, with once weekly check-up in hospital by the transplant physician., Results: Forty-two patients were treated with 81 cycles of high-dose chemotherapy (11, 18 and 13 patients and 17, 40 and 24 courses in the inpatient, outpatient and home care cohorts respectively). Inpatients were hospitalized in the aplastic phase for a median of 14 days. Patients in the outpatient cohort were at home in the aplastic phase for a median of six days (with a median of six days in hospital), and in the home care cohort for a median of 10 days (with a median of 1.5 days in hospital). Unscheduled readmissions and hospital visits were frequent in the outpatient and home care cohorts, mostly due to fever, central indwelling catheter malfunctioning or chemotherapy-related toxicity. However, patients could usually be discharged again after observation and treatment. No infectious deaths or unexpected emergencies occurred in the outpatient or home care cohort. Neither was there any suggestion of an increased number of fevers, infections, or other complications., Conclusions: At home management in the aplastic phase after high-dose chemotherapy and stemcell transplant by community-based professionals is feasible without signs of increased toxicity or infections.

We have developed a rapid, sensitive and selective method for the determination of the cyclosporin analog PSC 833 in human and mouse plasma using cyclosporin A as internal standard. The assay uses liquid-liquid extraction with diethyl ether for sample clean-up followed by reversed-phase high-performance liquid chromatography with UV detection at 210 nm. Good peak shapes were obtained using a NovaPak Phenyl column operating at 72 degrees C. Good selectivity from endogenous compounds was achieved using a mobile phase composed of methanol-acetonitrile-water (34:34:32). The retention times of cyclosporin A and PSC 833 were approximately 7.8 and 11.7 min, respectively, with two major endogenous peaks at 9.2 and 16.7 min. Selective decreasing of the retention times of cyclosporin A and PSC 833 relative to these interferences occurring upon aging of the column was balanced by increasing the percentage of methanol relative to acetonitrile. No other late eluting peaks were present, resulting in a total analysis time of 20 min per sample. The assay performance in human plasma was good. The absolute recovery of PSC 833 after the sample clean-up step was 48+/-6%. The lower limit of quantitation was 0.05 microM using 500 microl of sample. Within the linear dynamic range of the assay (0.10-5.0 microM) the accuracy was close to 100% and within-day and between-day variation less than 7%. Because of the limited availability of blank mouse plasma, the concentration in samples from mice were determined using calibration curves constructed in human plasma. The lower limit of quantitation in mouse was 0.25 microM using 200 microl of sample. Overall, the performance of the assay in mouse plasma was somewhat less than in human plasma but accuracy and precision were within the ranges that are considered acceptable for bio-analytical assays.

Background: Uncontrolled studies suggest that high-dose chemotherapy is beneficial in patients with breast cancer and multiple metastases to the axillary lymph nodes. Many physicians accept this treatment as standard care. We aimed to assess adjuvant high-dose chemotherapy in breast cancer in a phase II randomised trial., Methods: 97 women aged younger than 60 years, who had breast cancer with extensive axillary-node metastases (confirmed by a tumour-positive infraclavicular lymph-node biopsy), received three courses of up-front chemotherapy (FE120C). This regimen consisted of cyclophosphamide 500 mg/m2, epirubicin 120 mg/m2, and 5-fluorouracil 500 mg/m2 once weekly for 3 weeks. After surgery, stable patients or those who responded to chemotherapy were randomly assigned conventional therapy (fourth course of FE120C, followed by radiation therapy and 2 years of tamoxifen [40 patients]) or high-dose therapy (identical treatment but an additional high-dose regimen and peripheral-blood progenitor-cell [PBPC] support after the fourth FE120C course [41 patients]). This high-dose regimen comprised cyclophosphamide 6 g/m2, thiotepa 480 mg/m2, and carboplatin 1600 mg/m2. The primary endpoint was overall and disease-free survival. All analyses were by intention to treat., Findings: No patients died from toxic effects of chemotherapy. With a median follow-up of 49 (range 21-76) months, the 4-year overall and relapse-free survivals for all 97 patients were 75% and 54%, respectively. There was no significant difference in survival between the patients on conventional therapy and those on high-dose therapy., Interpretation: High-dose therapy is associated with substantial cost and acute toxic effects, but also has potentially irreversible long-term effects. Until the benefit of this therapy is substantiated by large-scale phase III trials, high-dose chemotherapy should not be used in the adjuvant treatment of breast cancer, apart from in randomised studies.

Carzelesin is a novel cyclopropylpyrroloindole prodrug analogue that has recently been tested in Phase I clinical trials. To increase our understanding in the pharmacology of this new class of cytotoxic drugs, we have compared the pharmacology of this drug in mice, rats, and humans. The mouse was the most tolerant [10% lethal dose (LD10), 500 microg/kg], the rat was intermediate (LD10, 40 microg/kg), and humans were the least tolerant species in this series (maximum tolerated dose, 300 microg/m2 corresponding to 7.5 microg/kg). In both mice and humans, bone marrow toxicity was the primary toxic side effect. Pharmacokinetic studies, using a validated high-performance liquid chromatographic procedure, revealed that differences in drug clearance and conversion to the active drug (U-76,074) could not explain the substantial interspecies differences. The area under the plasma concentration time curve (AUCs) of carzelesin in mice and rats at their LD10s were about 80- and 20-fold higher, respectively, than in humans receiving the maximum tolerated dose, whereas the respective AUCs of U-76,074 in mice and rats were 50- and 10-fold higher. By using a colony-forming assay with bone marrow stem cells from mice and humans, we observed only a 3-fold higher toxicity in the latter. Although some of this discrepancy may be explained by the fact that the in vitro and the in vivo assays probably reflect the toxicity on different populations of colony-forming units, the tolerance of the mouse bone marrow in vivo against the very high drug levels in plasma suggest the presence of a protective mechanism, which is less active in humans. An important consequence of the much higher susceptibility of the human bone marrow for carzelesin is that the target plasma levels in humans are much below active concentrations achieved in mice, and it is clear that this may compromise the successful use of this agent in the clinic. Ultimately, however, the efficacy of this drug will be established in Phase II clinical trials.

The taxanes paclitaxel and docetaxel represent a novel class of antineoplastic agents. A major problem of both drugs is their low aqueous solubility and the design of suitable formulations has been a difficult step in the process of therapeutic development. The formulations currently used are mixtures of Cremophor EL:ethanol for paclitaxel (Taxol) and Tween 80:ethanol for docetaxel (Taxotere), but many new approaches have been tested or are under investigation. Paclitaxel and docetaxel have a similar mechanism of action, which is based on promotion of tubulin assembly and inhibition of microtubule disassembly. Pharmacokinetic studies revealed a marked non-linearity of paclitaxel in mice, which appeared to result exclusively from Cremophor EL, the major component present in the pharmaceutical formulation. An almost linear pharmacokinetic behavior was observed in the case of docetaxel. The reported plasma protein binding of both compounds ranged from 76 to 97% in different animal species. Paclitaxel and docetaxel widely distribute into most tissues of mice and rats, including tumor tissue, but only low concentrations were detected in the central nervous system. Despite the great similarity in the chemical structures of paclitaxel and docetaxel, their metabolic profile is very distinct. Furthermore, whereas paclitaxel metabolism is largely species dependent, docetaxel metabolism is similar across species in both isolated hepatic microsomal fractions and in vivo models. For both taxanes, hepatobiliary excretion is the major pathway of elimination and a major fraction of the dose is excreted in feces as parent drug or hydroxylated metabolites.

In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(-/-) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(-/-) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in mdr1a(-/-) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(-/-) mice. The cumulative fecal excretion (0-96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(-/-) mice. Biliary excretion was not significantly different in wt and mdr1a(-/-) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(-/-) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen.

After a pre-operative 1-h i.v. infusion of 150 mg/m2 of teniposide (Vumon; VM26), the drug levels were determined in resected brain tumor specimens from three patients with malignant glioma and from three patients with brain metastases. Tissue dissections were performed within 0-2.5 h after drug administration in three patients and after 24 h in the other three patients. Teniposide was quantified by high-performance liquid chromatography and the levels of albumin in the resected tissue samples were quantified by radial immunodiffusion. In addition, albumin levels were quantified in normal brain tissue, in malignant glioma and in metastatic brain tumor tissue obtained post mortem from deceased patients. The albumin levels indicated that a substantial fraction (range: 0.16-0.50) of the resected brain tumor specimens consisted of blood. As the plasma concentration of teniposide during the first hours after infusion is high, the major part of the drug measured in the tumor specimens collected within 2.5 h after drug administration originated from the blood compartment. At 24 h after drug administration, when the plasma level of teniposide had declined to approximately 0.20 microgram/ml, we could discern a real tissue uptake of teniposide ranging from 0.15-0.27 microgram/g wet tissue weight in the resected tumor. Although the number of patients in this study is small, this work clearly illustrates that an accurate determination of the tissue concentration of teniposide is hindered by the high concurrent plasma levels. It is therefore essential that future tissue distribution studies also include a suitable procedure that establishes the contribution of drug originating from the blood compartment.

Background: P-glycoprotein (Pgp) is a membrane protein that acts as an extrusion pump for many cytotoxic drugs. Pgp is expressed in normal tissues, and its (over)expression in tumor cells contributes to their drug resistance. Human Pgp is encoded by the MDR1 gene, In mice, two Pgps (encoded by the mdr1a and mdr1b genes) appear to perform the same function as the single human protein. The simultaneous use of cytotoxic drugs and agents that block Pgp function has raised questions of safety, since a blockade of Pgp in normal tissues could alter drug pharmacokinetics and change the spectrum of toxic side effects. Analysis of the consequences of Pgp blockade has been facilitated by the generation of mice with disrupted mdr1a genes [mdr1a(-/-)]., Purpose: We studied the plasma pharmaco-kinetics, tissue distribution, and excretion of the cytotoxic drug vinblastine (VBL) and its metabolites in mdr1a (-/-) mice and in wild-type [mdr1a(+/+)] mice., Methods: VBL was administered to mice in bolus doses of either 1 or 6 mg/kg body weight by intravenous injection. VBL and its metabolites were quantified in tissue specimens, plasma, feces, and urine by use of high-performance liquid chromatography. Liquid scintillation counting was used to measure radioactivity in specimens from animals that had received [3H]VBL. Pharmacokinetic parameters were calculated by use of noncompartmental methods. Only two-sided P values are reported., Results: The half-life (t1/2) of VBL during its terminal phase of elimination was longer in mdr1a (-/-) mice than in wild-type mice. The t1/2 values with a 1-mg/kg dose were 3.6 hours +/- 0.3 hour (mean +/- standard error) and 2.1 hours +/- 0.3 hour, respectively (P < .05); with a 6-mg/kg dose, the values were 8.6 hours +/- 1.8 hours and 4.2 hours +/- 0.2 hour, respectively (P = .058). Fecal excretion of nonmetabolized VBL was reduced from 20%-25% of the administered dose (either 1 or 6 mg/kg) in wild-type mice to 9.3% (1-mg/kg dose) or 3.4% (6-mg/kg dose) in mdr1a(-/-) mice (both P < .05); the cumulative urinary excretion of VBL was low (< 6% of the administered dose) and not substantially different in the two types of mice. The metabolism of VBL to hydrophilic compounds, a primary mechanism involved in its elimination, was not altered in mdr1a(-/-) mice. The brains of mdr1a(-/-) mice accumulated substantially more VBL than the brains of wild-type mice. In mdr1a(-/-) mice, a few other tissues, such as the heart and the liver, accumulated increased amounts of VBL, but the relative levels of accumulation were lower than those found in the brain., Conclusions: Mice lacking the Pgp encoded by the mdr1a gene exhibit reduced fecal excretion of VBL, leading to a prolonged elimination t1/2 for this drug. Intact mdr1a function appears to protect the brain against high plasma levels of VBL, but most other tissues are not similarly protected., Implications: Enhanced drug accumulation in nonmalignant tissues after Pgp blockade should be carefully considered in future clinical trials of Pgp modulation.

Studies in humans and mice have demonstrated a nonlinear pharmacokinetic behavior of paclitaxel. Because of its poor water solubility, the drug is formulated in a mixture of Cremophor EL and ethanol (1:1, v/v; Taxol). We hypothesized that the substantial amounts of concurrently administered Cremophor EL on the disposition of paclitaxel, female FVB mice received paclitazel by i.v. injection at does levels of 2, 10, and 20 mg/kg by appropriate (standard) dilution of the commercially available formulation of paclitaxel (Taxol) with saline. The drug was also given at 2 mg/kg with supplemental Cremophor EL-ethanol to achieve the same amount of vehicle as by standard administration of 10 mg/kg. Furthermore, paclitaxel formulations in Tween 80-ethanol (1:1, v/v) and dimethylacetamide were tested. Plasma samples were collected between 5 min and 48 h, and tissue specimens were sampled at 1, 4, and 8 h after drug administration. Paclitaxel and metabolites were quantified by high-performance liquid chromatography. Cremophor EL levels were determined by a novel high-performance liquid chromatography procedure. For comparative reasons, Cremophor EL was also assayed in plasma samples from three patients receiving a 3-h i.v. infusion of 175 mg/m2 of paclitaxel. A marked nonlinear pharmacokinetic behavior of paclitaxel was observed when the drug was formulated in Cremophor EL-ethanol. The clearance of 2.37 L/h/kg at 2 mg/kg was reduced to 0.33 and 0.15 L/h/kg at 10 and 20 mg/kg, respectively. When 2 mg/kg were given with an amount of Cremophor EL-ethanol matching that of the 10-mg/kg dose level, the clearance was 0.56 L/h/kg. If administered at 10 mg/kg in Tween 80-ethanol or at 2 and 10 mg/kg in dimethylacetamide, the clearances were 2.66, 2.57, and 2.62 L/h/kg, respectively. Despite the fact that much higher plasma levels of paclitaxel are reached when given in the Cremophor EL-ethanol formulation, the tissue levels were essentially similar with all tested drug preparations. The Cremophor EL levels in patients were in the same order of magnitude as those observed in mice after administration of 2 and 10 mg/kg. These data demonstrate that Cremophor EL has a profound effect on the pharmacokinetics of paclitaxel im mice. Because Cremophor EL also contributes substantially to the nonlinear pharmacokinetic behavior of paclitaxel observed in humans.

We investigated the pharmacokinetics of etoposide given to a patient suffering from multifocal liver metastases from an unknown primary tumor. The drug was given either by i.v. infusion or by hepatic arterial infusion (HAI). The calculated pharmacokinetic parameters (mean values +/- SD) were similar after i.v. infusion and HAI, viz., 6.4 +/- 0.7 versus 6.5 +/- 0.2 h for the terminal elimination half-life (t1/2 beta), 98.5 +/- 1.3 versus 101.3 +/- 5.9 mg l(-1) h for the area under the plasma concentration-time curve (AUC), 21.2 +/- 0.3 versus 20.6 +/- 1.2 ml min-1 m-2 for clearance (Cl), 17.7 +/- 1.9 versus 18.1 +/- 2.6 mg/l for the peak concentration, and 11.7 +/- 1.3 versus 11.6 +/- 1.01/m2 for the volume of distribution (Vd), respectively. We therefore conclude that administration of etoposide by HAI does not result in a significantly higher liver extraction. Hepatic extraction of etoposide is determined by the fraction of non-protein-bound (free) drug present. The lack of a difference between the two administration routes suggests that under in vivo conditions the equilibrium between free and bound drug is established before the drug reaches the hepatic arterioles. Consequently, administration by HAI does not lead to an increased exposure of the tumor in the liver to free (active) etoposide. Furthermore, the overall exposure of the liver to total (bound + free) etoposide is increased only from about 100 to 120 mg l-1 h. These results do not favor the use of this more complex route of drug administration in the treatment of (metastatic) cancer located in the liver.

So far, all animal pharmacokinetic studies of paclitaxel, which used analytical procedures based on HPLC, have not been sensitive enough to quantify drug levels below 500 ng/ml. Consequently, the interpretation of the results is restricted because drug levels of paclitaxel as low as at least 50 nM (43 ng/ml) are relevant for the pharmacology of this drug. We recently described an accurate and very sensitive method based on HPLC for the determination of paclitaxel and the metabolites 3'-p-hydroxypaclitaxel (I), 6 alpha-hydroxypaclitaxel (II) and 6 alpha,3'-p-dihydroxypaclitaxel (III) in a wide variety of biological matrices. We have now implemented this methodology in a comprehensive pharmacokinetic study in female FVB mice. Previous pharmacokinetic studies in humans demonstrated a large steady-state volume of distribution, indicating that the drug is widely distributed into tissues. Comprehensive tissue distribution studies may, therefore, be helpful in providing more insight into possible relationships between plasma levels, drug levels in tissues and toxicity. Paclitaxel, formulated in Cremophor EL and ethanol (1:1, v/v), was given as a single i.v. bolus dose of 2, 10 and 20 mg/kg to female FVB mice. Except for the brain, the distribution of paclitaxel to all other tissues in the female mice was substantial and maximum drug levels were achieved within 0.5 or 1 h. A marked non-linear increase in the area under the concentration-time curve (AUC) in plasma was observed, which was not paralleled by a proportional increase in the tissue AUC levels. It is postulated that this effect may be related to the substantial amounts of Cremophor EL administered concurrently. The recovery of paclitaxel in the feces (0-96 h) was reduced from 58% at the 2 mg/kg dose level to 44% at the 20 mg/kg dose level. Small amounts of metabolites I and II were detected in the gut, liver and gall bladder, but not in the systemic circulation or any other tissue. Metabolite III was not detected. Metabolites I and II are likely excreted directly into the bile, and since their recovery in the feces accounts for about 25% of the administered dose, their formation thus represents an important pathway of detoxification.

In 29 chemotherapy-naive patients with stage II-III breast cancer, peripheral blood stem cells (PBSCs) were mobilised following fluorouracil 500 mg m-2, epirubicin 90-120 mg m-2 and cyclophosphamide 500 mg m-2 (FEC) and granulocyte colony-stimulating factor (G-CSF; Filgrastim) 300 microgram s.c. daily. In all but one patient, mobilisation was successful, requiring three or fewer leucocytopheresis sessions in 26 patients; 28 patients subsequently underwent high-dose chemotherapy consisting of carboplatin 1600 mg m-2, thiotepa 480 mg m-2 and cyclophosphamide 6 g m-2 (CTC) followed by PBSC transplantation. Haemopoietic engraftment was rapid with a median time to neutrophils of 500 x 10(6) l(-1) of 9 days (range 8-10) in patients who received G-CSF after PBSC-transplantation; platelet transfusion independence was reached within a median of 10 days (range 7-16). Neutropenic fever occurred in 96% of patients. Gastrointestinal toxicity was substantial but reversible. Renal, neural or ototoxicity was not observed. Complications related to the central venous catheter were encountered in 64% of patients, with major vein thrombosis occurring in 18%. High-dose CTC-chemotherapy with PBSC-transplantation, harvested after mobilisation with FEC and G-CSF, is reasonably well tolerated without life-threatening toxicity and is a suitable high-dose strategy for the adjuvant treatment of breast cancer.

Background: Peripheral blood progenitor cell transplantation is rapidly replacing autologous bone marrow transplantation as hematological support after high-dose chemotherapy for lymphoma or solid tumors. Controversy exists concerning the number of progenitor cells required for rapid and sustained bone marrow recovery, and as to which of the widely available methods for estimating this number should be employed., Methods: Forty consecutive patients with solid tumors or lymphomas received high-dose chemotherapy followed by autologous peripheral stem cell reinfusion. All stem cell harvests had been performed after mobilization with standard-dose chemotherapy followed by 300 micrograms G-CSF daily. Hematopoietic reconstitution was studied in relation to pertinent patient characteristics, to the size of the graft (in terms of the total number of mononuclear cells (MNC), the number of granulocyte/macrophage colony-forming units (CFU-GM) and the number of CD34+ cells, and to the use of G-CSF after stem cell reinfusion., Results: Both the numbers of CFU-GM and CD34+ cells reinfused, but not those of the MNC, correlated with granulocyte and platelet recovery. Patients who received at least 5 x 10(6) CD34+ cells/kg body weight achieved platelet transfusion independence on day 12 after reinfusion (range: day 7-37), significantly earlier than patients who had received less (p = 0.001). Thirty patients who received G-CSF (300 micrograms s.c. daily) after reinfusion achieved granulocyte recovery (> or = 500 x 10(6)/l) on day 9 (range: day 8-12), while this took a median of 15 days (range: day 10-28) in 10 consecutive patients not receiving G-CSF (p = 0.0003). In one patient who had received 1.4 x 10(6) CD34+ cells/kg, secondary bone marrow failure developed 3 months after transplantation. Reinfusion of cryopreserved autologous bone marrow was followed by prompt recovery., Conclusion: Peripheral stem cells, mobilized by moderate-dose chemotherapy and G-CSF, lead to rapid and durable engraftment after high-dose chemotherapy when at least 3-5 x 10(6) CD34+ cells/kg are reinfused. Lower numbers may also be satisfactory, but are associated with slower granulocyte and platelet recoveries. A moderate dose of G-CSF after reinfusion significantly hastens granulocyte recovery without interfering with platelet recovery.

Carzelesin (U80,244, I) is a cyclopropylpyrroloindole prodrug analog. The compound exerts its cytotoxic activity after conversion, via U-76,073 (II) to U-76,074 (III) by binding to the DNA minor groove in a sequence selective fashion. In pre-clinical investigations the drug displayed a broad spectrum activity against human xenografts in mice. To enable pharmacokinetic monitoring during the phase I clinical trials, we have developed a selective and sensitive assay for the parent compound and its two metabolites. Sample pre-treatment has been automated by using the ASPEC system and involves solid-phase extraction of the diluted plasma sample (1:3 in 20% v/v acetic acid) on an SPE-C18 precolumn followed by two consecutive washings with water and acetonitrile. The compounds are eluted with 600 microliters of dimethylacetamide and 500 microliters is injected on a Spherisorb-CN column. The sample is chromatographed using a linear gradient from 24% to 60% (v/v) acetonitrile in 20 mM phosphate buffer (pH 6.5). The eluate fraction containing the three compounds is heart-cutted in a 2-ml sample loop, switched onto a Spherisorb-ODS column and separation is accomplished using a mobile phase of acetonitrile-20 mM phosphate buffer pH 6.5 (64:36, v/v). UV detection is used with absorbance monitored at 360 nm. This highly selective method offers a lower limit of detection of less than 1 ng/ml for each of the compounds using 1000 microliters of plasma and enables the quantification of the analytes with an acceptable precision and accuracy over a range of 1 to 50 ng/ml. The assay has been implemented in a phase I clinical trial.

We have investigated the pharmacokinetics of the investigational semi-synthetic vinca alkaloid vinorelbine (navelbine, NVB). The analyses have been performed by using a sensitive and selective method based on ion-exchange normal phase high-performance liquid chromatography with fluorescence detection combined with liquid-liquid extraction for sample clean-up. Pharmacokinetic studies were performed in male FVB mice receiving 12 mg/kg NVB through intravenous injection. The results have been compared to those obtained for vinblastine (VBL). The plasma pharmacokinetics of NVB can be described by a three compartment model. The elimination half-life is significantly longer and the plasma AUC values higher for NVB compared to VBL. This is reflected in tissues, where, 24 hr after drug administration, the concentration of NVB is 5 to 10-fold higher compared to VBL. Qualitatively, the tissue distribution and retention of the drugs is very similar. The drug concentrations in most tissues decline parallel with the circulating plasma levels, whereas prolonged retention is found in tissues of lymphatic and testicular origin. Deacetylation yielding deacetylnavelbine (DNVB) is the primary metabolic route for NVB. This cytotoxic metabolite accounts for a substantial part of the overall disposition of drug. Only 58% of the administered dose is excreted in the urine (17%) and faeces (41%) as NVB or DNVB. No other metabolites have been detected.

The plasma pharmacokinetics of vinblastine and N-(deacetyl-O-4-vinblastoyl-23)-L-ethyl isoleucinate (VileE) in mice have been studied as part of the preclinical investigations of VileE, a new investigational semisynthetic Vinca alkaloid. Groups of animals received the test compounds through i.v. bolus injection at LD10, 0.5 x LD10, and 0.1 x LD10 doses. VileE has also been administered p.o. Drug plasma levels have been analyzed with a sensitive and selective method using liquid-liquid extraction for sample clean-up and high-performance liquid chromatography combined with fluorescence detection for quantification. Following i.v. injection, plasma kinetics of both vinblastine and VileE can be described adequately by a three-compartment open model. VileE demonstrates nonlinear pharmacokinetics with decreasing clearance and increasing terminal half-lives at increasing doses. Comparison of the plasma concentration versus time curves for vinblastine in humans and mice indicates that the toxicity of these compounds may not be directly related to the drug exposure expressed by the area under curve in plasma but by the terminal half-life and the time that a toxic threshold level is attained. Pharmacokinetically guided dose escalation in coming phase I trials of VileE is, therefore, discouraged.

Life-style has a major influence on the incidence of breast cancer. To evaluate the effects of life-style related metabolic-endocrine factors on breast cancer risk we conducted a case-control study comparing 223 women aged 38 to 75 years presenting with operable (stage I or II) breast cancer and 441 women of the same age having no breast cancer, who participated in a population-based breast cancer screening program. Women reporting diabetes mellitus were excluded. Sera from 110 women of the same age group presenting with early stage melanoma, lymphoma or cervical cancer were used as a second 'other-cancer control group'. Serum levels of C-peptide were significantly higher in early breast cancer cases compared to controls. The same was found for the ratios C-peptide to glucose or C-peptide to fructosamine, indicating insulin resistance. Sex hormone binding globulin was inversely, triglycerides and available estradiol were positively related to C-peptide. Serum C-peptide levels were related to body mass index (BMI), and to waist/hip ratio (WHR), in particular in controls. However, the relative increase of C-peptide, C-peptide to glucose or C-peptide to fructosamine in cases was independent of BMI or WHR. The log relative risk was linearly related to the log C-peptide levels. Relative risk according to quintiles, and adjusted for age, family history, BMI and WHR, for women at the 80% level was 2.9 as compared with those at the 20% level for C-peptide. Elevated C-peptide or C-peptide to fructosamine values were not observed in the sera from women belonging to the 'other-cancer control group'. This study suggests that hyperinsulinemia with insulin resistance is a significant risk factor for breast cancer independent of general adiposity or body fat distribution.

Vinca alkaloids have been used as chemotherapeutic agents now for over three decades. Especially during recent years, the development of a considerable number of semi-synthetic derivatives, showing attractive properties in preclinical or clinical investigations, has been reported. In this paper we shall give a critical review of the investigations presented on this matter.

Thyroid function was measured in 81 patients who had been curatively irradiated on a mantle field for Hodgkin's disease 10-18 years ago. 47 patients (58%) had elevated levels of thyroid stimulating hormone, indicating hypofunction of the thyroid gland, compared with 4.6% of controls (hospital visitors) matched for age and sex. The mean free thyroxine index (FTI) was significantly lower in patients than in controls, but all FTI values were still normal. Age at the time of irradiation, sex, time since irradiation and administration of chemotherapy were not significant factors in the development of thyroid dysfunction. A life-long awareness of the possibility of insidiously developing myxedema in these patients is strongly advocated.

Episialin, a mucus glycoprotein, is a well-known tumor-associated antigen used in a variety of tests to detect the presence of adenocarcinoma. With the introduction of the microparticle-captured enzyme immunoassay (MEIA), a new technique was introduced. We compared this assay with our standard method to detect adenocarcinomas, the measurement of carcinoembryonic antigen (CEA). In breast cancer, the breast cancer mucin (BCM) assay was more often positive in metastatic disease but was not better than CEA in stages I-III. In lung carcinomas, BCM and CEA gave similar results while in colorectal carcinoma, CEA was superior. BCM gave similar results to CA 15.3 in a group of breast cancer patients.