Population pharmacokinetics of carboplatin, etoposide and melphalan in children: a re-evaluation of paediatric dosing formulas for carboplatin in patients with normal or mild impairment of renal function.

AIMS: Carboplatin dosage is calculated by using the estimated glomerular filtration rate (GFR) to achieve a target plasma area under the plasma concentration-time curve (AUC). The aims of the present study were to investigate factors that influence the pharmacokinetics of carboplatin in children with high-risk neuroblastoma, and whether target exposures for carboplatin were achieved using current treatment protocols. METHODS: Data on children receiving high-dose carboplatin, etoposide and melphalan for neuroblastoma were obtained from two study sites [European International Society for Paediatric Oncology (SIOP) Neuroblastoma study, Children's Hospital at Westmead; n = 51]. A population pharmacokinetic model was built for carboplatin to evaluate various dosing formulas. The pharmacokinetics of etoposide and melphalan was also investigated. The final model was used to simulate whether target carboplatin AUC (16.4 mg ml-1 ·min) would be achieved using the paediatric Newell formula, modified Calvert formula and weight-based dosing. RESULTS: Allometric weight was the only significant, independent covariate for the pharmacokinetic parameters of carboplatin, etoposide and melphalan. The paediatric Newell formula and modified Calvert formula were suitable for achieving the target AUC of carboplatin for children with a GFR <100 ml min-1 1.73 m-2 but not for those with a GFR ≥100 ml min-1 1.73 m-2 . A weight-based dosing regimen of 50 mg kg-1 achieved the target AUC more consistently than the other formulas, regardless of renal function. CONCLUSIONS: GFR did not appear to influence the pharmacokinetics of carboplatin after adjusting pharmacokinetic parameters for weight. This model-based approach validates the use of weight-based dosing as an appropriate alternative for carboplatin in children with either mild renal impairment or normal renal function.

Investigating the heterogeneity of alkylating agents' efficacy and toxicity between sexes: A systematic review and meta-analysis of randomized trials comparing cyclophosphamide and ifosfamide (MAIAGE study).

BACKGROUND: A marginal interaction between sex and the type of alkylating agent was observed for event-free survival in the Euro-EWING99-R1 randomized controlled trial (RCT) comparing cyclophosphamide and ifosfamide in Ewing sarcoma. To further evaluate this interaction, we performed an individual patient data meta-analysis of RCTs assessing cyclophosphamide versus ifosfamide in any type of cancer. METHODS: A literature search produced two more eligible RCTs (EICESS92 and IRS-IV). The endpoints were progression-free survival (PFS, main endpoint) and overall survival (OS). The hazard ratios (HRs) of the treatment-by-sex interaction and their 95% confidence interval (95% CI) were assessed using stratified multivariable Cox models. Heterogeneity of the interaction across age categories and trials was explored. We also assessed this interaction for severe acute toxicity using logistic models. RESULTS: The meta-analysis comprised 1,528 pediatric and young adult sarcoma patients from three RCTs: Euro-EWING99-R1 (n = 856), EICESS92 (n = 155), and IRS-IV (n = 517). There were 224 PFS events in Euro-EWING99-R1 and 200 in the validation set (EICESS92 + IRS-IV), and 171 and 154 deaths in each dataset, respectively. The estimated treatment-by-sex interaction for PFS in Euro-EWING99-R1 (HR = 1.73, 95% CI = 1.00-3.00) was not replicated in the validation set (HR = 0.97, 95% CI = 0.55-1.72), without heterogeneity across trials (P = 0.62). In the pooled analysis, the treatment-by-sex interaction was not significant (HR = 1.31, 95% CI = 0.89-1.95, P = 0.17), without heterogeneity across age categories (P = 0.88) and trials (P = 0.36). Similar results were observed for OS. No significant treatment-by-sex interaction was observed for leucopenia/neutropenia (P = 0.45), infection (P = 0.64), or renal toxicity (P = 0.20). CONCLUSION: Our meta-analysis did not confirm the hypothesis of a treatment-by-sex interaction on efficacy or toxicity outcomes.

A phase I/II trial of AT9283, a selective inhibitor of aurora kinase in children with relapsed or refractory acute leukemia: challenges to run early phase clinical trials for children with leukemia.

Aurora kinases regulate mitosis and are commonly overexpressed in leukemia. This phase I/IIa study of AT9283, a multikinase inhibitor, was designed to identify maximal tolerated doses, safety, pharmacokinetics, and pharmacodynamic activity in children with relapsed/refractory acute leukemia. The trial suffered from poor recruitment and terminated early, therefore failing to identify its primary endpoints. AT9283 caused tolerable toxicity, but failed to show clinical responses. Future trials should be based on robust preclinical data that provide an indication of which patients may benefit from the experimental agent, and recruitment should be improved through international collaborations and early combination with established treatment strategies.

Tumour cell retention of rucaparib, sustained PARP inhibition and efficacy of weekly as well as daily schedules.

BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP) inhibitors (PARPi) exploit tumour-specific defects in homologous recombination DNA repair and continuous dosing is most efficacious. Early clinical trial data with rucaparib suggested that it caused sustained PARP inhibition. Here we investigate the mechanism of this durable inhibition and potential exploitation. METHODS: Uptake and retention of rucaparib and persistence of PARP inhibition were determined by radiochemical and immunological assays in human cancer cell lines. The pharmacokinetics and pharmacodynamics of rucaparib were determined in tumour-bearing mice and the efficacy of different schedules of rucaparib was determined in mice bearing homologous recombination DNA repair-defective tumours. RESULTS: Rucaparib accumulation is carrier mediated (Km=8.4±1.2 µM, Vmax=469±22 pmol per 10(6) cells per 10 min), reaching steady-state levels >10 times higher than the extracellular concentration within 30 min. Rucaparib is retained in cells and inhibits PARP ≥50% for ≥72 h days after a 30-min pulse of 400 nM. In Capan-1 tumour-bearing mice rucaparib accumulated and was retained in the tumours, and PARP was inhibited for 7 days following a single dose of 10 mg kg(-1) i.p or 150 mg kg(-1) p.o. by 70% and 90%, respectively. Weekly dosing of 150 mg kg(-1) p.o once a week was as effective as 10 mg kg(-1) i.p daily for five days every week for 6 weeks in delaying Capan-1 tumour growth. CONCLUSIONS: Rucaparib accumulates and is retained in tumour cells and inhibits PARP for long periods such that weekly schedules have equivalent anticancer activity to daily dosing in a pre-clinical model, suggesting that clinical evaluation of alternative schedules of rucaparib should be considered.

Genetics of cisplatin ototoxicity: confirming the unexplained?

Replication of findings in clinical pharmacogenetic studies is essential but often neglected. The observation that TPMT and COMT genotypes, in combination with ABCC3 genotype, are predictive of ototoxicity following cisplatin treatment has been confirmed. However, translating this observation into a useful preventive strategy requires more mechanistic insight.

Thiothymidine combined with UVA as a potential novel therapy for bladder cancer.

BACKGROUND: Thiothymidine (S(4)TdR) can be incorporated into DNA and sensitise cells to DNA damage and cell death following exposure to UVA light. Studies were performed to determine if the combination of S(4)TdR and UVA could be an effective treatment for bladder cancer. METHODS: Uptake and incorporation of S(4)TdR was determined in rat and human bladder tumour cell lines. Measures of DNA crosslinking and apoptosis were also performed. In vivo activity of the combination of S(4)TdR and UVA was investigated in an orthotopic model of bladder cancer in rats. RESULTS: Thiothymidine (200 µM) replaced up to 0.63% of thymidine in rat and tumour bladder cancer cells. The combination of S(4)TdR (10-200 µM) and UVA (1-5 kJ m(-2)) caused apoptosis and cell death at doses that were not toxic alone. Addition of raltitrexed (Astra Zeneca, Alderley Edge, Cheshire, UK) increased the incorporation of S(4)TdR into DNA (up to 20-fold at IC(5)) and further sensitised cells to UVA. Cytotoxic effect was associated with crosslinking of DNA, at least partially to protein. Intravenous administration of S(4)TdR, in combination with UVA delivered directly to the bladder, resulted in an antitumour effect in three of five animals treated. CONCLUSION: These data indicate that the combination of S(4)TdR and UVA has potential as a treatment for bladder cancer, and give some insight into the mechanism of action. Further work is necessary to optimise the delivery of the two components.

A Phase I clinical study of cisplatin-incorporated polymeric micelles (NC-6004) in patients with solid tumours.

BACKGROUND: On the basis of preclinical studies of NC-6004, a cisplatin-incorporated micellar formulation, we hypothesised that NC-6004 could show lower toxicity than cisplatin and show greater anti-tumour activity in phase I study. METHODS: A total of 17 patients were recruited in a range of advanced solid tumour types. NC-6004 was administered intravenously (i.v.) every 3 weeks. The dose escalation started at 10 mg m(-2) and was increased up to 120 mg m(-2) according to the accelerated titration method and modified Fibonacci method. RESULTS: One dose-limiting toxicity (DLT) occurred in a patient who was given 90 mg m(-2) of NC-6004, otherwise any significant cisplatin-related toxicity was not observed or generally mild toxicity was observed. Despite the implementation of post-hydration and pre-medication regimen, renal impairment and hypersensitivity reactions still developed at 120 mg m(-2), which led to the conclusion that the maximum tolerated dose was 120 mg m(-2), and the recommended dose was 90 mg m(-2), although DLT was not defined as per protocol. Stable disease was observed in seven patients. The maximum concentration and area under the concentration-time curve of ultrafilterable platinum at 120 mg m(-2) NC-6004 were 34-fold smaller and 8.5-fold larger, respectively, than those for cisplatin. CONCLUSION: The delayed and sustained release of cisplatin after i.v. administration contributes to the low toxicity of NC-6004.

Characterization of the metabolism of fenretinide by human liver microsomes, cytochrome P450 enzymes and UDP-glucuronosyltransferases.

BACKGROUND AND PURPOSE: Fenretinide (4-HPR) is a retinoic acid analogue, currently used in clinical trials in oncology. Metabolism of 4-HPR is of particular interest due to production of the active metabolite 4'-oxo 4-HPR and the clinical challenge of obtaining consistent 4-HPR plasma concentrations in patients. Here, we assessed the enzymes involved in various 4-HPR metabolic pathways. EXPERIMENTAL APPROACH: Enzymes involved in 4-HPR metabolism were characterized using human liver microsomes (HLM), supersomes over-expressing individual human cytochrome P450s (CYPs), uridine 5'-diphospho-glucoronosyl transferases (UGTs) and CYP2C8 variants expressed in Escherichia coli. Samples were analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry assays and kinetic parameters for metabolite formation determined. Incubations were also carried out with inhibitors of CYPs and methylation enzymes. KEY RESULTS: HLM were found to predominantly produce 4'-oxo 4-HPR, with an additional polar metabolite, 4'-hydroxy 4-HPR (4'-OH 4-HPR), produced by individual CYPs. CYPs 2C8, 3A4 and 3A5 were found to metabolize 4-HPR, with metabolite formation prevented by inhibitors of CYP3A4 and CYP2C8. Differences in metabolism to 4'-OH 4-HPR were observed with 2C8 variants, CYP2C8*4 exhibited a significantly lower V(max) value compared with *1. Conversely, a significantly higher V(max) value for CYP2C8*4 versus *1 was observed in terms of 4'-oxo formation. In terms of 4-HPR glucuronidation, UGTs 1A1, 1A3 and 1A6 produced the 4-HPR glucuronide metabolite. CONCLUSIONS AND IMPLICATIONS: The enzymes involved in 4-HPR metabolism have been characterized. The CYP2C8 isoform was found to have a significant effect on oxidative metabolism and may be of clinical relevance.

Central nervous system penetration and enhancement of temozolomide activity in childhood medulloblastoma models by poly(ADP-ribose) polymerase inhibitor AG-014699.

BACKGROUND: Temozolomide shows activity against medulloblastoma, the most common malignant paediatric brain tumour. Poly(ADP-ribose) polymerase (PARP) inhibitors enhance temozolomide activity in extracranial adult and paediatric human malignancies. METHODS: We assessed the effect of AG-014699, a clinically active PARP inhibitor, on temozolomide-induced growth inhibition in human medulloblastoma models. Pharmacokinetic, pharmacodynamic and toxicity assays were performed in tumour-bearing mice. RESULTS: Sensitivity to temozolomide in vitro was consistent with methylguanine methyltransferase (MGMT) and DNA mismatch repair (MMR) status; MGMT(+) MMR(+) D384Med cells (temozolomide GI(50)=220 µM), representative of most primary medulloblastomas, were sensitised fourfold by AG-014699; MGMT⁻ MMR(+) D425Med cells were hypersensitive (GI(50)=9 µM) and not sensitised by AG-014699, whereas MGMT(+) MMR⁻ temozolomide-resistant D283Med cells (GI50=807 µM) were sensitised 20-fold. In xenograft models, co-administration of AG-014699 produced an increase in temozolomide-induced tumour growth delay in D384Med xenografts. Consistent with the in vitro data, temozolomide caused complete tumour regressions of D425Med xenografts, whereas D283Med xenografts were relatively resistant. AG-014699 was not toxic, accumulated and reduced PARP activity ≥75% in xenograft and brain tissues. CONCLUSION: We show for the first time central nervous system penetration and inhibition of brain PARP activity by AG-014699. Taken together with our in vitro chemosensitisation and toxicity data, these findings support further evaluation of the clinical potential of AG-014699-temozolomide combinations in intra-cranial malignancies.

Influence of pharmacogenetics on response and toxicity in breast cancer patients treated with doxorubicin and cyclophosphamide.

BACKGROUND: Doxorubicin and cyclophosphamide (AC) therapy is an effective treatment for early-stage breast cancer. Doxorubicin is a substrate for ABCB1 and SLC22A16 transporters. Cyclophosphamide is a prodrug that requires oxidation to 4-hydroxycyclophosphamide, which yields a cytotoxic alkylating agent. The initial oxidation is catalysed by cytochrome P450 enzymes including CYP2B6, CYP2C9, CYP2C19 and CYP3A5. Polymorphic variants of the genes coding for these enzymes and transporters have been identified, which may influence the systemic pharmacology of the two drugs. It is not known whether this genetic variation has an impact on the efficacy or toxicity of AC therapy. METHODS: Germ line DNA samples from 230 patients with breast cancer on AC therapy were genotyped for the following SNPs: ABCB1 C1236T, G2677T/A and C3435T, SLC22A16 A146G, T312C, T755C and T1226C, CYP2B6*2, *8, *9, *3, *4 and *5, CYP2C9*2 and *3, CYP3A5*3 and CYP2C19*2. Clinical data on survival, toxicity, demographics and pathology were collated. RESULTS: A lower incidence of dose delay, indicative of less toxicity, was seen in carriers of the SLC22A16 A146G, T312C, T755C variants. In contrast, a higher incidence of dose delay was seen in carriers of the SLC22A16 1226C, CYP2B6*2 and CYP2B6*5 alleles. The ABCB1 2677A, CYP2B6*2, CYP 2B6*8, CYP 2B6*9, CYP 2B6*4 alleles were associated with a worse outcome. CONCLUSION: Variant alleles in the ABCB1, SLC22A16 and CYP2B6 genes are associated with response to AC therapy in the treatment of breast cancer.

Therapeutic monitoring of carboplatin dosing in a premature infant with retinoblastoma.

INTRODUCTION: Carboplatin dosing based on renal function and therapeutic monitoring have been previously shown to be beneficial in the treatment of children with cancer. However, the applicability of such approaches to the treatment of premature or newborn infants, where kidney function may change markedly with advancing gestational and postnatal age, is unknown. Diagnosis of retinoblastoma in a preterm infant provided a rare opportunity to carry out adaptive carboplatin dosing in a patient with immature renal function. CASE REPORT: A preterm female infant born at a gestational age of 32 weeks was diagnosed with bilateral retinoblastoma at 35 weeks. Carboplatin treatment with real-time pharmacokinetic monitoring was initiated on day 26 of life at an initial dose of 6.6 mg/kg. Plasma samples were obtained at specified time points and carboplatin levels quantified by atomic absorption spectrometry. Additional doses of carboplatin were determined by pharmacokinetic monitoring based on the achievement of carboplatin AUC values of 5.2-7.8 mg/ml min on three courses of treatment. Increased carboplatin doses administered on successive courses of treatment reflected a greater than twofold increase in drug clearance, from 3.4-7.1 ml/min over a 7-week period. Pharmacokinetically-guided carboplatin dosing led to the attainment of AUCs within 10% of target values on each course of treatment. The patient completed five courses of carboplatin with both tumours defined as inactive after this treatment period. CONCLUSIONS: Data obtained from studying this patient suggests that adaptive carboplatin monitoring represents a feasible and beneficial clinical approach in preterm infants or neonates.

Estimation of renal function and its potential impact on carboplatin dosing in children with cancer.

Renal function-based carboplatin dosing is used routinely in paediatric oncology clinical practice. It is important that accurate assessments of renal function are carried out consistently across clinical centres, a view supported by recently published British Nuclear Medicine Society (BNMS) guidelines for measuring glomerular filtration rate (GFR). These guidelines recommend the use of a radioisotope method for GFR determination, with between two and five blood samples taken starting 2 h after radioisotope injection and application of the Brochner-Mortensen (BM) correction factor. To study the likely impact of these guidelines, we have investigated current practices of measuring GFR in all 21 Children's Cancer and Leukaemia Group (CCLG) paediatric oncology centres in the United Kingdom. This information was used to evaluate the potential impact on renal function-based carboplatin dosing using raw 51Cr-EDTA clearance data from 337 GFR tests carried out in children with cancer. A questionnaire survey revealed that between two and four samples were taken after isotope administration, with BM and Chantler corrections used in 38% (8/21) and 28% (6/21) of centres, respectively. A change from Chantler to BM correction, based on the BNMS guidelines, would result in a > 10% decrease in carboplatin dose in at least 15% of patients and a > 25% decrease in 2% of patients. A greater proportion of patients would have an alteration in carboplatin dose when centres not using any correction factor implement the BM correction. The increase in estimated 51Cr-EDTA half-life observed by omitting the I h sample decreases carboplatin dose by > 10% in 23-52% of patients and by > 25% in 3% of patients. This study highlights current variations in renal function measurement between clinical centres and the potential impact on carboplatin dosing. A standard methodology for estimating GFR should be followed to achieve uniform dosing in children with cancer.

Molecular targeting of retinoic acid metabolism in neuroblastoma: the role of the CYP26 inhibitor R116010 in vitro and in vivo.

Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg(-1)) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA.

Adaptive dosing and platinum-DNA adduct formation in children receiving high-dose carboplatin for the treatment of solid tumours.

A pharmacokinetic-pharmacodynamic study was carried out to investigate the feasibility and potential importance of therapeutic monitoring following high-dose carboplatin treatment in children. High-dose carboplatin was administered over 3 or 5 days, with the initial dose based on renal function, to achieve target area under the plasma concentration-time curve (AUC) values of 21 or 20 mg ml(-1).min, respectively. Dose adjustment was carried out based on observed individual daily AUC values, to obtain the defined target exposures. Platinum-DNA adduct levels were determined in peripheral blood leucocytes and toxicity data were obtained. Twenty-eight children were studied. Based on observed AUC values, carboplatin dose adjustment was performed in 75% (21 out of 28) patients. Therapeutic monitoring resulted in the achievement of carboplatin exposures within 80-126% of target AUC values, as compared to estimated exposures of 65-213% of target values without dose adjustment. The carboplatin AUC predicted with no dose modification was positively correlated with pretreatment glomerular filtration rate (GFR) values. Higher GFR values were observed in those patients who would have experienced AUC values >25% above the target AUC than those patients attaining AUC values >25% below the target AUC, following renal function-based dosing. Platinum-DNA adduct levels correlated with observed AUC values on day 1 of carboplatin and increased over a 5-day course of treatment. Real-time monitoring of carboplatin pharmacokinetics with adaptive dosing is both feasible and necessary for the attainment of consistent AUC values in children receiving high-dose carboplatin treatment. Pharmacodynamic data suggest a strong correlation between carboplatin pharmacokinetics and the drug-target interaction.

Pharmacokinetics and metabolism of 13-cis-retinoic acid (isotretinoin) in children with high-risk neuroblastoma - a study of the United Kingdom Children's Cancer Study Group.

The administration of 13-cis-retinoic acid (13-cisRA), following myeloablative therapy improves 3-year event-free survival rates in children with high-risk neuroblastoma. This study aimed to determine the degree of inter-patient pharmacokinetic variation and extent of metabolism in children treated with 13-cisRA. 13-cis-retinoic acid (80 mg m(-2) b.d.) was administered orally and plasma concentrations of parent drug and metabolites determined on days 1 and 14 of courses 2, 4 and 6 of treatment. Twenty-eight children were studied. The pharmacokinetics of 13-cisRA were best described by a modified one-compartment, zero-order absorption model combined with lag time. Mean population pharmacokinetic parameters included an apparent clearance of 15.9 l h(-1), apparent volume of distribution of 85 l and absorption lag time of 40 min with a large inter-individual variability associated with all parameters (coefficients of variation greater than 50%). Day 1 peak 13-cisRA levels and exposure (AUC) were correlated with method of administration (P<0.02), with 2.44- and 1.95-fold higher parameter values respectively, when 13-cisRA capsules were swallowed as opposed to being opened and the contents mixed with food before administration. Extensive accumulation of 4-oxo-13-cisRA occurred during each course of treatment with plasma concentrations (mean+/-s.d. 4.67+/-3.17 microM) higher than those of 13-cisRA (2.83+/-1.44 microM) in 16 out of 23 patients on day 14 of course 2. Extensive metabolism to 4-oxo-13-cisRA may influence pharmacological activity of 13-cisRA.

A study to determine the minimum volume of blood necessary to be discarded from a central venous catheter before a valid sample is obtained in children with cancer.

BACKGROUND: The objective of this study was to determine the minimum volume of blood that should be discarded from a range of different types of central venous catheter (CVC), such that the subsequent blood sample was not diluted or contaminated by the residual intra-luminal fluid. PROCEDURE: Seventy children aged 1-19 years with central venous access inserted as part of their standard clinical treatment were recruited to this prospective study. Statistical comparison of the extent of variation in biochemical and haematological parameters observed between two blood samples taken following routine 5 ml discard blood volumes, as compared to the extent of variation between samples drawn following a 5 ml discard volume and <5 ml volumes, was carried out. RESULTS: Data indicate that the measurement error in a clinical sample obtained following a 3 ml discard volume is no different to the measurement error obtained when using a standard 5 ml discard volume. Comparable results were obtained from patients with various different types of CVC or portacath access. CONCLUSIONS: The withdrawal of a 3 ml discard volume is sufficient to ensure that the subsequent blood sample is not diluted or contaminated by residual intra-luminal fluid. This may have a significant clinical impact in paediatric oncology, where patients frequently require blood transfusions due to the haematological toxicities associated with chemotherapy. It is hoped that these results will impact on hospital policies concerning specified discard volumes taken from CVCs prior to the withdrawal of blood samples for research purposes and routine clinical analysis.

Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor.

NRH:Quinone Oxidoreductase 2 (NQO2) has been described as having no enzymatic activity with nicotinamide adenine dinucleotide (NADH) or NADPH as electron donating cosubstrates. Mitomycin C (MMC) is both a substrate for and a mechanistic inhibitor of the NQO2 homologue NQO1. NRH:quinone oxidoreductase 2 catalysed the reduction of MMC at pH 5.8 with NADH as a co-factor. This reaction results in species that inhibit the NQO2-mediated metabolism of CB1954. In addition, MMC caused an increase in DNA cross-links in a cell line transfected to overexpress NQO2 to an extent comparable to that observed with an isogenic NQO1-expressing cell line. These data indicate that NQO2 may contribute to the metabolism of MMC to cytotoxic species.

Pharmacokinetics and metabolism of ifosfamide in relation to DNA damage assessed by the COMET assay in children with cancer.

The degree of damage to DNA following ifosfamide (IFO) treatment may be linked to the therapeutic efficacy. The pharmacokinetics and metabolism of IFO were studied in 19 paediatric patients, mostly with rhabdomyosarcoma or Ewings sarcoma. Ifosfamide was dosed either as a continuous infusion or as fractionated doses over 2 or 3 days. Samples of peripheral blood lymphocytes were obtained during and up to 96 h after treatment, and again prior to the next cycle of chemotherapy. DNA damage was measured using the alkaline COMET assay, and quantified as the percentage of highly damaged cells per sample. Samples were also taken for the determination of IFO and metabolites. Pharmacokinetics and metabolism of IFO were comparable with previous studies. Elevations in DNA damage could be determined in all patients after IFO administration. The degree of damage increased to a peak at 72 h, but had returned to pretreatment values prior to the next dose of chemotherapy. There was a good correlation between area under the curve of IFO and the cumulative percentage of cells with DNA damage (r2=0.554, P=0.004), but only in those patients receiving fractionated dosing. The latter patients had more DNA damage (mean+/-s.d., 2736+/-597) than those patients in whom IFO was administered by continuous infusion (1453+/-730). The COMET assay can be used to quantify DNA damage following IFO therapy. Fractionated dosing causes a greater degree of DNA damage, which may suggest a greater degree of efficacy, with a good correlation between pharmacokinetic and pharmacodynamic data.

Phase I study of temozolomide plus paclitaxel in patients with advanced malignant melanoma and associated in vitro investigations.

The purpose of this study was to determine activity of temozolomide combined with paclitaxel or epothilone B in vitro, and to investigate the combination of temozolomide with paclitaxel in a Phase I clinical trial. Melanoma cell lines A375P and DX3 were treated with temozolomide and either paclitaxel or epothilone B. Combination indices were determined to assess the degree of synergism. In a clinical study, 21 patients with malignant melanoma were treated with increasing doses of temozolomide (orally, days 1-5), in combination with a fixed dose of paclitaxel (i.v. infusion day 1), followed by dose escalation of the latter drug. Cycles of treatment were repeated every 3 weeks. Pharmacokinetics of both agents were determined on day 1, with temozolomide pharmacokinetics also assessed on day 5. All three compounds were active against the melanoma cell lines, with epothilone B being the most potent. There was a strong degree of synergism between temozolomide and either paclitaxel or epothilone B. In the clinical study, no pharmacokinetic interaction was observed between temozolomide and paclitaxel. Dose escalation of both drugs to clinically active doses was possible, with no dose-limiting toxicities observed at 200 mg m(-2) day(-1) temozolomide and 225 mg m(-2) day(-1) paclitaxel. There were two partial responses out of 15 evaluable patients. One patient remains alive and symptom-free at 4 years after treatment. Temozolomide and paclitaxel may be administered safely at clinically effective doses. Further evaluation of these combinations in melanoma is warranted.

Increasing the intracellular availability of all-trans retinoic acid in neuroblastoma cells.

Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.