A phase I dose-finding clinical pharmacokinetic study of an oral formulation of irinotecan (CPT-11) administered for 5 days every 3 weeks in patients with advanced solid tumours.

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

Balloon catheter hypoxic pelvic perfusion with mitomycin C and melphalan for locally advanced tumours in the pelvic region: a phase I-II trial.

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

Sensitive and specific quantification of the anticancer agent ZD1839 (Gefitinib) in plasma by on-column focusing capillary liquid chromatography-tandem mass spectrometry.

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

Bio-chemotherapeutic strategies and the (dis) utility of hypoxic perfusion of liver, abdomen and pelvis using balloon catheter techniques.

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

Differential protein expression profile in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) arise from the interstitial cells of Cajal through gain of function mutations of the oncogene KIT. Imatinib offers the first effective treatment for patients with GISTs, but the therapeutic outcome strongly depends on the type of KIT mutation. We used ProteinChip technology to investigate whether GISTs with different KIT mutations express different proteins. In total, 154 proteins were significantly differentially expressed in GISTs with exon 9 KIT mutation compared to GISTs with exon 11 KIT mutation.

Balloon catheter hypoxic abdominal perfusion with Mitomycin C and Melphalan for locally advanced pancreatic cancer: a phase I-II trial.

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

Balloon catheter hypoxic abdominal and pelvic perfusion with tumour necrosis factor-alpha, Melphalan and Mitomycin C: a pharmacokinetic study in pigs.

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

Combretastatin A-4 phosphate enhances CPT-11 activity independently of the administration sequence.

We evaluated the effect of different intervals and sequences of the vascular targeting agent combretastatin A-4 disodium phosphate (CA4DP) and CPT-11 administration on tumour growth delay and intratumoral uptake of CPT-11 using a syngeneic rhabdomyosarcoma tumour model. Irrespective of the administration sequence, the combination of CA4DP and CPT-11 significantly increases tumour growth delay in comparison with both drugs alone (P<0.001). Intratumoral CPT-11 concentration generally decreased (up to 5-fold) in the combination groups, while SN-38, the active metabolite of CPT-11, increased up to 9-fold. However, the increased amount of intratumoral SN-38 trapping after CA4DP injection did not correlate with the observed tumour growth delay. In conclusion, CA4DP significantly enhances the antitumour effect of CPT-11, which is not greatly influenced by the administration sequence, and which lacks a correlation with the intratumoral trapping of CPT-11 or SN-38. Mechanisms other than trapping are likely to be involved in the chemosensitising capacity of CA4DP.

Development of a rapid and sensitive method for the quantitation of benzodiazepines in Calliphora vicina larvae and puparia by LC-MS-MS.

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is emerging as the tool of choice for rapid analysis and the detection of biologically active compounds in complex mixtures. We describe the development of a sensitive method for the simultaneous quantitation of 10 benzodiazepines in Calliphora vicina (Diptera: Calliphoridae) larvae and puparia. The use of larvae for toxicological analyses offers some technical advantages over putrefied tissue. Four sample pretreatment methods for isolating the benzodiazepines out of larvae were evaluated. A simple homogenization, followed by acetonitrile precipitation yielded the highest recoveries. Puparia were pulverized and extracted by ultrasonification in methanol. All extracts were subsequently analyzed using reversed-phase LC-MS-MS. Larvae and puparia calibrators containing benzodiazepines at concentrations ranging from 25 to 750 pg/mg and 50 to 500 pg/mg, respectively, were prepared and analyzed. The method was demonstrated to be linear over the ranges investigated. Limits of detection were from 1.88 to 5.13 pg/mg larva and from 6.28 to 19.03 pg/mg puparium. The developed method was applied to the determination of nordiazepam and its metabolite oxazepam in larvae and puparia of the Calliphora vicina fly that had been reared on artificial foodstuff (beef heart) spiked with 1 microg/g nordiazepam. The larvae were harvested at day 5 for analysis of drug content. The method was sufficiently sensitive to allow the detection of nordiazepam and oxazepam in a single larva or puparium.

Effect of antivascular endothelial growth factor treatment on the intratumoral uptake of CPT-11.

Promising preclinical activity with agents blocking the function of vascular endothelial growth factor (VEGF) has been observed in various cancer types, especially with combination therapy. However, these drugs decrease microvessel density, and it is not known whether this reduced vessel density (VD) results in decreased delivery of concomitantly administered classical anticancer drugs. We designed an in vivo study to investigate the relation between VEGF-blocking therapy, tumoral blood vessels, and intratumoral uptake of anticancer drugs. Nude NMRI mice bearing colon adenocarcinoma (HT29) were treated with the anti-VEGFmAb A4.6.1 or placebo. After 1 week, CPT-11 was administered 1 h prior to killing the animals. In A4.6.1 treated tumours, there was a significant decrease in VD, more pronounced with potentially functional large vessels than endothelial cords. Interestingly, a trend to increased intratumoral CPT-11 concentration was observed (P=0.09). In parallel, we measured an increase in tumour perfusion, as estimated by high-performance liquid chromatography determination of intratumoural Hoechst 33342 concentration. In the growth delay study, CPT-11 was at least equally effective with or without pretreatment with A4.6.1. These data suggest that tumour vascular function and tumour uptake of anticancer drugs improve with VEGF-blocking therapy, and indicate the relevance for further investigations.

Development of a rapid and sensitive method for the quantitation of amphetamines in human plasma and oral fluid by LC-MS-MS.

Target analysis of amphetamines in biological samples is of great importance for clinical and forensic toxicologists alike. At present, most laboratories analyze such samples by gas chromatography-mass spectrometry. However, this procedure is labor-intensive and time-consuming, particularly as a preliminary extraction and derivatization are usually unavoidable. Here we describe the development of an alternative method. Amphetamines were isolated from human plasma and oral fluid using a simple methanol precipitation step and subsequently analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. Quantitation of the drugs was performed using multiple reaction monitoring. The developed method, which requires only 50 microL of biological sample, has a total analysis time of less than 20 min (including sample preparation) and enables the simultaneous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine, methamphetamine, and ephedrine in a single chromatographic run. Limits of detection of 2 microg/L or better were obtained. The method has been validated and subsequently applied to the analysis of plasma and oral fluid samples collected from current drug users.

Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-alpha-based isolated hepatic perfusion.

Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF-alpha) is currently performed in clinical trials in patients with hepatic metastases. Previous studies led to the hypothesis that the use of TNF-alpha in isolated limb perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature. However, whether TNF-alpha contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-alpha. IHP with melphalan alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-alpha resulted in a strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-alpha, ruling out a direct interaction of TNF-alpha with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-alpha was coadministrated with melphalan was strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and TNF-alpha mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-alpha in this setting.

Variations in flow, duration, and concentration do not change the final lung concentration of melphalan after isolated lung perfusion in rats.

OBJECTIVE: The influences of flow, perfusion time, and concentration on melphalan (MN) lung levels in a rat model of isolated lung perfusion (ILuP) were studied. METHODS: ILuP was performed in WAG/Rij rats by using a single-pass system with 0.5, 0.05, 0.01, and 0.005 mg of MN. Subsequently ILuP with 0.05 mg MN was performed during 30 min with a flow rate of 0.5 ml/min, during 60 min with a flow rate of 0.5 ml/min, and during 30 min with a flow rate of 1.0 ml/min. RESULTS: The lung MN levels of 0.05 mg were significantly lower as compared with 0.5 mg (p = 0.02). In the second experiment, no significant differences were seen in lung levels of MN between the different groups. CONCLUSION: The final lung MN levels were only determined by the absolute amount administered.

Hyphenated techniques in anticancer drug monitoring. I. Capillary gas chromatography-mass spectrometry.

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

Hyphenated techniques in anticancer drug monitoring. II. Liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry.

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

Erythrocytes and the transfer of anticancer drugs and metabolites: a possible relationship with therapeutic outcome.

Blood functions as a mobile tissue in an exchange system, with the remaining body tissue as a stationary phase. The equilibrium among plasma water, plasma proteins, and blood cells is described by models, but little consideration has been given to the substance-binding capacity of erythrocytes. There are numerous reasons for this, including bioanalytical limitations (ie, it has been difficult to study erythrocytes in the laboratory in their natural state). Erythrocyte monitoring requires accurate blood sampling and quantitative isolation of erythrocytes without disturbing the equilibrium of substances of interest between erythrocytes and plasma or other blood constituents. This became possible with the advent of the measurement of sediment device. The mass of a given substance available in blood can be described by M(Blood) = M(Plasma) + M(ERY) (+ M(REM)). M(ERY) is the mass of a substance present in erythrocytes and it is shown that for several oxazaphosphorines, such as iphosphoramide mustard, that M(ERY) determines M(Blood) with great superiority over M(Plasma). The impact of erythrocyte monitoring on therapeutic outcome has to be defined, but is an important area of research.

Isolated hypoxic hepatic perfusion (IHHP) using balloon catheter techniques: from laboratory to the clinic towards a percutaneous procedure.

BACKGROUND/AIMS: The success of and our extensive experience with TNF alpha-based isolated limb perfusions in patients with unresectable extremity soft tissue sarcomas made us explore the possibilities for a similar approach for the treatment of hepatic metastases. After experience with the classic surgical isolated hepatic perfusion in pigs and in patients, we concluded that the classic surgical approach was associated with serious drawbacks i.e., magnitude of the procedure with morbidity, lack of repeatability of the procedure, complexity and costs. These problems were addressed in a program aimed at developing a repeatable method of isolated perfusion of the liver with minimally invasive techniques. METHODOLOGY: We validated the methodology of isolated hypoxic hepatic perfusion using balloon catheter techniques in pigs. RESULTS: The excellent pharmacokinetic profile of the procedure, resulting in very high regional drug concentrations and negligible systemic drug concentrations, allowed us to move on to the clinic and start to study and further develop this method in a phase I-II study using the drug melphalan, in patients with irresectable hepatic metastases. CONCLUSIONS: We aim to develop step by step a fully percutaneous approach for isolated hypoxic hepatic perfusion.

Nanotechnology in bio/clinical analysis.

Nanotechnology is being exploited now in different fields of analytical chemistry: Single cell analysis; in chip/micro machined devices; hyphenated technology and sampling techniques. Secretory vesicles can be chemically and individually analyzed with a combination of optical trapping, capillary electrophoresis separation, and laser induced fluorescence detection. Attoliters (10(-18) l) can be introduced into the tapered inlets of separation capillaries. Chip technology has come of age in the field of genomics, allowing faster analyses, and will fulfil an important role in RNA and peptide/protein analysis. The introduction of nanotechnology in LC-MS and CE-MS has resulted in new findings in the study of DNA adduct formation caused by carcinogenic substances, including anticancer drugs. Sample handling and introduction also can benefit from nanotechnology: The downscaling of sample volumes to the picoliter level has resulted in zeptomole (10(-21)) detection limits in the single-shot mass spectrum of proteins.

Simultaneous determination of the peptide-mitomycin KW-2149 and its metabolites in plasma by high-performance liquid chromatography.

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

Tumour necrosis factor alpha increases melphalan concentration in tumour tissue after isolated limb perfusion.

Several possible mechanisms for the synergistic anti-tumour effects between tumour necrosis factor alpha (TNF-alpha) and melphalan after isolated limb perfusion (ILP) have been presented. We found a significant sixfold increase in melphalan tumour tissue concentration after ILP when TNF-alpha was added to the perfusate, which provides a straightforward explanation for the observed synergism between melphalan and TNF-alpha in ILP.