Pharmacokinetics and Safety of a Novel Oral Liquid Formulation of 13-cis Retinoic Acid in Children with Neuroblastoma: A Randomized Crossover Clinical Trial.

(1) Background: 13-cis-retinoic acid (13-CRA) is a key component of neuroblastoma treatment protocols. This randomized crossover study compares the pharmacokinetics (PK), safety and palatability of a novel oral liquid formulation to the current method of extracting 13-CRA from capsules. (2) Methods: Pharmacokinetics was evaluated in two consecutive treatment cycles. Patients were randomized to receive either liquid or capsule formulation on cycle 1 and then crossed over to the alternative formulation on cycle 2. The daily dose was 200 mg/m2, reduced to 160 mg/m2 in patients with weight ≤ 12 kg. (3) Results: A total of 20 children, median (range) age 4.3 (1-11.6) y were recruited. Pharmacokinetic data were pooled and a population model describing the disposition of 13-CRA and 4-oxo-13-CRA was developed. Bioavailability of the liquid formulation was estimated to be 65% higher (95% CI; 51-79%) than the extracted capsule. CmaxSS and AUC(0-12)SS estimates were also significantly higher; mean (95% CI) differences were 489 (144-835) ng/mL and 3933 (2020-5846) ng/mL·h, respectively (p < 0.01). There were no significant differences in reported adverse effects. Parents found dosing considerably easier with liquid formulation. (4) Conclusions: The pharmacokinetics, safety and palatability of a new liquid formulation of 13-CRA compares favorably to 13-CRA extracted from capsules. Clinical Trial Registration: clinicaltrial.gov NCT03291080.

Pharmacokinetics and Pharmacogenetics of Cyclophosphamide in a Neonate and Infant Childhood Cancer Patient Population.

Infants and young children represent an important but much understudied childhood cancer patient population. The pharmacokinetics and pharmacogenetics of the widely used anticancer prodrug cyclophosphamide were investigated in children <2 years of age. Concentrations of cyclophosphamide and selected metabolites were determined in patients administered cyclophosphamide at doses ranging from 100-1500 mg/m2 (5-75 mg/kg), with various infusion times as determined by the standard treatment regimen that each patient was receiving. Polymorphisms in genes including CYP2B6 and CYP2C19 were investigated. Data generated for cyclophosphamide were analysed using a previously published population pharmacokinetic model. Cyclophosphamide pharmacokinetics was assessed in 111 samples obtained from 25 patients ranging from 4-23 months of age. The average cyclophosphamide clearance for the patients was 46.6 mL/min/m2 (ranging from 9.4-153 mL/min/m2), with marked inter-patient variability observed (CV 41%). No significant differences in cyclophosphamide clearance or exposure (AUC) were observed between patient groups as separated by age or body weight. However, marked differences in drug clearance and metabolism were noted between the current data in children <2 years of age and recently published results from a comparable study conducted by our group in older children, which reported significantly lower cyclophosphamide clearance values and metabolite exposures using the same population pharmacokinetic model for analysis. Whilst this study demonstrates no significant differences in cyclophosphamide clearance in patients <2 years, it highlights large differences in dosing protocols across tumour types. Furthermore, the study suggests marked differences in cyclophosphamide clearance in children less than two years of age as compared to older patients.

Development of a LC-MS/MS method for the quantification of toxic payload DM1 cleaved from BT1718 in a Phase I study.

Background: BT1718 is a novel bicyclic peptide anticancer drug targeting membrane type I matrix metalloproteinase to release its toxic payload DM1. A LC-MS/MS method was validated to quantify DM1 generated from BT1718 in a Phase I/IIa clinical trial. Materials & methods: Plasma samples underwent a reduction reaction to artificially cleave BT1718 into DM1 and its bicycle components. An alkylation step was carried out to stabilize the reaction products, and plasma proteins extracted using acetonitrile. LC-MS/MS analysis utilized a C18 column and Agilent 6460 triple quadrupole mass spectrometer (Agilent, Cheshire, UK). Results: The method was fully validated over a linear range of 200-50,000 ng/ml BT1718, with overall precision ≤10% and accuracy 89-102%. Conclusion: A novel method for quantifying DM1 yielded from BT1718 has been validated and is now being utilized clinically.

Impact of dose and duration of therapy on dexamethasone pharmacokinetics in childhood acute lymphoblastic leukaemia-a report from the UKALL 2011 trial.

INTRODUCTION: The use of dexamethasone in acute lymphoblastic leukaemia therapy contributes to short- and long-term toxicities. The UKALL 2011 randomised trial investigated whether a more intense dexamethasone dose (10 mg/m2/d x 14d, short vs 6 mg/m2/d x 28d, standard) would lead to a more rapid cytoreduction and reduced adverse effects associated with longer durations of steroids in induction. The impact of dose and duration on dexamethasone pharmacokinetics was investigated. METHODS: Blood samples were obtained on one of the first three and last three days of induction dexamethasone dosing at time points up to 8 h after oral administration. Plasma dexamethasone levels were quantified in 1084 plasma samples obtained from 174 children and a population pharmacokinetic model developed. RESULTS: Drug exposure varied significantly between patients, with a >12-fold variation in AUC0-12h values and a marked overlap in dexamethasone exposures between dose levels. Intuitively, AUC0-12h was significantly higher with short dosing (10 mg/m2/d), but cumulative exposure was significantly higher with standard dosing over 28 days, after a higher cumulative dose. Concomitant rasburicase administration was associated with a 60% higher dexamethasone clearance. Day 8 bone marrow response was comparable between dosing arms, but those with <5% blast count exhibited a greater mean dexamethasone exposure than those with >5%. No statistical differences were observed between arms in terms of steroid-related toxicity or minimal residual disease at the end of induction. CONCLUSION: The potential significance of dexamethasone AUC0-12h on early response and higher cumulative exposure on the standard arm suggest that duration of therapy and exposure may be more important factors than absolute dose from a clinical pharmacology perspective.

The use of pharmacokinetically guided carboplatin chemotherapy in a pre-term infant with neuroblastoma-associated spinal cord compression.

Neonatal neuroblastoma may require chemotherapy either due to mass effect or unfavourable cytogenetics. This case focuses on using pharmacokinetic (PK) guided chemotherapy to treat neonatal neuroblastoma. A newborn baby was noted to have left leg immobility. Imaging showed a retroperitoneal tumour with spinal canal extension causing spinal cord compression. PK-guided carboplatin was given after conventionally dosed chemotherapy demonstrated no improvement. After initiation of PK therapy, clinical and radiological improvement was seen. We discuss our decision to use PK-guided chemotherapy despite guidelines recommending weight-based dosing and discuss the benefits in terms of clinical efficacy without increased toxicity.

Investigating the Experiences of Childhood Cancer Patients and Parents Participating in Optional Nontherapeutic Clinical Research Studies in the UK.

BACKGROUND: While the majority of childhood cancer clinical trials are treatment related, additional optional research investigations may be carried out that do not directly impact on treatment. It is essential that these studies are conducted ethically and that the experiences of families participating in these studies are as positive as possible. METHODS: A questionnaire study was carried out to investigate the key factors that influence why families choose to participate in optional nontherapeutic research studies, the level of understanding of the trials involved, and the experiences of participation. RESULTS: A total of 100 participants from six UK centers were studied; 77 parents, 10 patients >16 years, and 13 patients aged 8-15 years. Ninety-seven percent of parents and 90% of patients felt that information provided prior to study consent was of the right length, with 52% of parents and 65% of patients fully understanding the information provided. Seventy-four percent of parents participated in research studies in order to "do something important", while 74% of patients participated "to help medical staff". Encouragingly, <5% of participants felt that their clinical care would be negatively affected if they did not participate. Positive aspects of participation included a perception of increased attention from medical staff. Negative aspects included spending longer periods in hospital and the requirement for additional blood samples. Ninety-six percent of parents and 87% of patients would participate in future studies. CONCLUSIONS: The study provides an insight into the views of childhood cancer patients and their parents participating in nontherapeutic clinical research studies. Overwhelmingly, the findings suggest that participation is seen as a positive experience.

Adaptive dosing of anticancer drugs in neonates: facilitating evidence-based dosing regimens.

PURPOSE: Selection of the most appropriate chemotherapy dosing regimens for neonates treated within the first weeks of life represents a significant clinical dilemma. Due to a lack of information relating to the clinical pharmacology of anticancer drugs in these challenging patients, current dosing guidelines are based on limited scientific rationale. In the current study, we investigate the utilisation of therapeutic drug monitoring approaches in neonates with localised hepatoblastoma, Wilms' tumour and stage 4S neuroblastoma, being treated with widely used anticancer drugs. METHODS: Plasma concentrations of cisplatin, vincristine, etoposide and carboplatin were quantified in two neonates being treated within the first 3 weeks of life and in a 32-week preterm infant treated at a gestational age of 40 weeks. Therapeutic drug monitoring was carried out where appropriate, based on the pharmacokinetic data obtained in conjunction with clinical response and toxicity. RESULTS: Treatment of a child aged 2 weeks with a recommended cisplatin dose reduction for weight to 1.8 mg/kg resulted in achievement of unbound cisplatin plasma concentrations of 0.01-0.08 µg/mL, markedly lower than exposures previously reported in infants and older children. A dose increase to 2.7 mg/kg was implemented, leading to the achievement of levels more in-line with those previously reported. This increased dose level was well tolerated over six courses of treatment, resulting in a good response to cisplatin monotherapy and the patient remains in remission at 3.5 years. In contrast, a 50 % vincristine dose reduction for weight in a 3-week-old neonate resulted in plasma concentrations comparable to levels observed in older children, leading to successful treatment and continued remission at 2 years. In a third patient, etoposide and carboplatin clearance values normalised to body weight were comparable to those reported in older children, resulting in comparatively lower exposures following reduced dosing. CONCLUSIONS: The current report provides unique data on the pharmacokinetics of several widely used anticancer drugs in neonates treated within the first few weeks of life. The provision of these data acts as a useful reference point to support future dosing decisions to be made by clinicians in the treatment of these challenging patients.

Cyclophosphamide pharmacokinetics and pharmacogenetics in children with B-cell non-Hodgkin's lymphoma.

INTRODUCTION: Variation in cyclophosphamide pharmacokinetics and metabolism has been highlighted as a factor that may impact on clinical outcome in various tumour types. The current study in children with B-cell non-Hodgkin's lymphoma (NHL) was designed to corroborate previous findings in a large prospective study incorporating genotype for common polymorphisms known to influence cyclophosphamide pharmacology. METHODS: A total of 644 plasma samples collected over a 5 year period, from 49 B-cell NHL patients ≤ 18 years receiving cyclophosphamide (250 mg/m(2)), were used to characterise a population pharmacokinetic model. Polymorphisms in genes including CYP2B6 and CYP2C19 were analysed. RESULTS: A two-compartment model provided the best fit of the population analysis. The mean cyclophosphamide clearance value following dose 1 was significantly lower than following dose 5 (1.83 ± 1.07 versus 3.68 ± 1.43 L/h/m(2), respectively; mean ± standard deviation from empirical Bayes estimates; P < 0.001). The presence of at least one CYP2B6*6 variant allele was associated with a lower cyclophosphamide clearance following both dose 1 (1.54 ± 0.11 L/h/m(2) versus 2.20 ± 0.31 L/h/m(2), P = 0.033) and dose 5 (3.12 ± 0.17 L/h/m(2) versus 4.35 ± 0.37 L/h/m(2), P = 0.0028), as compared to homozygous wild-type patients. No pharmacokinetic parameters investigated were shown to have a significant influence on progression free survival. CONCLUSION: The results do not support previous findings of a link between cyclophosphamide pharmacokinetics or metabolism and disease recurrence in childhood B-cell NHL. While CYP2B6 genotype was shown to influence pharmacokinetics, there was no clear impact on clinical outcome.

Carboplatin therapeutic monitoring in preterm and full-term neonates.

INTRODUCTION: Administration of the most appropriate dose of chemotherapy to neonates is particularly challenging and frequently not standardised based on any scientific rationale. We report the clinical utility of carboplatin therapeutic drug monitoring in preterm and full-term neonates within the first month of life. METHODS: Carboplatin therapeutic monitoring was performed to achieve target drug exposures area under the plasma concentration-time curve (AUC values) in nine preterm and full-term neonates diagnosed with retinoblastoma or neuroblastoma treated over an 8 year period. Carboplatin was administered over 3 days with therapeutic drug monitoring utilised to target cumulative AUC values of 5.2-7.8 mg/ml min. RESULTS: AUC values achieved were within 15% of target values for the individual courses of treatment in all but one patient (12/13 courses of treatment), with dose modifications of up to 215% required to achieve target AUC values, based on initial mg/kg dosing schedules. Carboplatin clearance determined across three consecutive chemotherapy courses in two patients increased from 3.4 to 7.1 ml/min and from 7.2 to 16.5 ml/min, representing increases of 210-230% over several weeks of treatment. Complete remission was observed in 8/9 patients, with no renal toxicity reported and only one patient experiencing ototoxicity. CONCLUSION: The study highlights the benefits of utilising therapeutic drug monitoring to achieve target carboplatin AUC values in preterm and full-term neonates treated within the first few weeks of life, particularly in view of marked increases in drug clearance observed over consecutive chemotherapy courses. In the absence of therapeutic drug monitoring, body-weight based dosing is recommended, with dosing guidance provided for both approaches to inform future treatment.

Characterisation of the clinical pharmacokinetics of actinomycin D and the influence of ABCB1 pharmacogenetic variation on actinomycin D disposition in children with cancer.

BACKGROUND AND OBJECTIVE: Despite its important role in cancer treatment, there is currently very limited available information concerning the clinical pharmacology of actinomycin D (Act D). The study was designed to characterise Act D pharmacokinetics and investigate the impact of pharmacogenetic variation on Act D disposition in children with cancer. METHODS: A total of 650 plasma samples collected over an 8 year period from 117 patients ≤21 years receiving Act D (0.4-1.6 mg/m(2)) were used to characterise a population pharmacokinetic model. Polymorphisms in ABCB1 were analysed in 140 patients. RESULTS: A 3-compartment model provided a good fit to the data. Median values for Act D clearance and volume of distribution in the central compartment (V 1) obtained from the model were 5.3 L/h and 1.9 L (13.9 L/h/70 kg and 7.5 L/70 kg), respectively. There was substantial inter-subject variation in all pharmacokinetic parameters (coefficients of variation 53-81 % for non-normalised values). Body weight was a major determinant of Act D clearance, such that dose capping at 2 mg in larger children at a protocol dose of 1.5 mg/m(2) resulted in significantly lower area under the plasma concentration-time curves (mean AUC values: 9.3 versus 12.8 mg·min/L; P < 0.0001). No significant relationships were found between ABCB1 genetic variants and Act D pharmacokinetic parameters, nor between CL, V 1 or dose and incidence of grade 3 or 4 toxicity. CONCLUSION: We have defined the pharmacokinetics of Act D in a paediatric patient population, providing robust estimates of key pharmacokinetic parameters. Pharmacokinetic data bring into question the current clinical practice of dose capping at 2 mg in larger patients. Pharmacogenetic variation in candidate drug transporter genes identified from preclinical studies does not significantly impact on Act D exposure in a clinical setting.

Adaptive dosing approaches to the individualization of 13-cis-retinoic acid (isotretinoin) treatment for children with high-risk neuroblastoma.

PURPOSE: To investigate the feasibility of adaptive dosing and the impact of pharmacogenetic variation on 13-cis-retinoic acid (13-cisRA) disposition in high-risk patients with neuroblastoma. EXPERIMENTAL DESIGN: 13-cisRA (160 mg/m(2) or 5.33 mg/kg/d) was administered to 103 patients ages 21 years or less and plasma concentrations of 13-cisRA and 4-oxo-13-cisRA quantitated on day 14 of treatment. Seventy-one patients were recruited to a dose adjustment group, targeting a 13-cisRA C(max) of 2 µmol/L, with dose increases of 25% to 50% implemented for patients with C(max) values less than 2 µmol/L. A population pharmacokinetic model was applied and polymorphisms in relevant cytochrome P450 genes analyzed. RESULTS: 13-cisRA C(max) values ranged from 0.42 to 11.2 µmol/L, with 34 of 103 (33%) patients failing to achieve a C(max) more than 2 µmol/L. Dose increases carried out in 20 patients in the dose adjustment study group led to concentrations more than 2 µmol/L in 18 patients (90%). Eight of 11 (73%) patients less than 12 kg, receiving a dose of 5.33 mg/kg, failed to achieve a C(max) of 2 µmol/L or more. Significantly, lower C(max) values were observed for patients treated with 5.33 mg/kg versus 160 mg/m(2) (1.9 ± 1.2 vs. 3.1 ± 2.0 µmol/L; mean ± SD; P = 0.023). C(max) was higher in patients who swallowed 13-cisRA capsules as compared with receiving the drug extracted from capsules (4.0 ± 2.2 vs. 2.6 ± 1.8 µmol/L; P = 0.0012). The target C(max) was achieved by 93% (25/27) versus 55% (42/76) of patients in these 2 groups, respectively. No clear relationships were found between genetic variants and 13-cisRA pharmacokinetic parameters. CONCLUSIONS: Dosing regimen and method of administration have a marked influence on 13-cisRA plasma concentrations. Body weight-based dosing should not be implemented for children less than 12 kg and pharmacologic data support higher doses for children unable to swallow 13-cisRA capsules.

Pharmacokinetics of cyclophosphamide and its metabolites in paediatric patients receiving high-dose myeloablative therapy.

INTRODUCTION: In order to better understand the impact of high-dose on the pharmacokinetics and metabolism of cyclophosphamide, a pharmacological study was performed in children with malignant mesenchymal tumours with metastatic disease. METHODS: Patients received four courses of chemotherapy including two courses of cyclophosphamide. Plasma concentrations of cyclophosphamide and the metabolites 4-ketocyclophosphamide, dechloroethylcyclophosphamide and carboxyphosphamide were determined on days 1, 2 and 3 of each course. A population pharmacokinetic model for cyclophosphamide was developed using non-linear mixed effects modelling and metabolite AUC values were compared between days and courses. RESULTS: Data were available on 21 cyclophosphamide courses from 15 patients. A one compartment model, incorporating a term in surface area for both CL and V, best described cyclophosphamide pharmacokinetics. Typical CL and V on day 1 of treatment for a patient with a SA of 1.4m(2) were 4.3 L/h and 28.5L, respectively. On days 2 and 3 CL increased by 88% (95% CI, 72-105%) and 125% (95% CI, 108-145%) over day 1 levels; V increased by 14% (95% CI, 5-23%) on days 2 and 3. V tended to be larger for males than similarly sized females but no effect of age was found upon CL or V. Significant increases in metabolite AUCs were observed on days 2 and 3 compared to day 1 and a significant increase in CXCP AUC from course 1 to course 3. CONCLUSION: Administration of high-dose cyclophosphamide over several days results in an increase in metabolism, possibly by induction of the activation pathway. This induction is effectively reversed following a four week period between cyclophosphamide doses. The degree of intersubject variation in cyclophosphamide elimination is largely accounted for by body surface area and is less than previously reported.

Pharmacokinetics of carboplatin and etoposide in infant neuroblastoma patients.

PURPOSE: Carboplatin and etoposide are commonly used chemotherapeutics for the treatment of many paediatric cancers. However, there are very limited published data concerning the pharmacokinetics of these agents in infants and very young children, for whom dose reductions are frequently implemented. METHODS: Etoposide (5 mg/kg; 2 h i.v. infusion) was co-administered with carboplatin (6.6 mg/kg; 1 h i.v. infusion) on each of 3 days of treatment and samples were taken between 0.5 and 4 h after the start of administration, from a total of 19 neuroblastoma patients aged <1 year at diagnosis and weighing <12 kg at treatment. Pharmacokinetic analysis was carried out using a non-linear mixed effects modelling approach. RESULTS: Two compartment structural models were selected for both carboplatin and etoposide analysis. Body weight (BW) was strongly associated with carboplatin clearance (Cl), with a slightly weaker relationship observed with etoposide Cl. Carboplatin Cl values ranged from 12.8 to 33.6 ml/min, with total AUC values of 4.2-9.3 mg/ml.min achieved over the 3 days of treatment. Cl values normalized to BW were significantly higher in patients <12 kg than in children >12 kg, with mean +/- SD values of 2.9 +/- 0.4 and 2.5 +/- 0.4 ml/min/kg, respectively (P < 0.05). Etoposide exhibited a median half-life of 4.6 h (range 4.1-6.6), a median AUC of 7.1 mg/ml.min (range 3.4-11.0) and a median Cl of 6.6 ml/min (range 3.2-13.0). CONCLUSION: Results suggest that prediction of absolute carboplatin Cl values may be difficult in infant patients <12 kg, with a small but significant difference in Cl values normalized to BW observed in this patient group. Etoposide pharmacokinetic data support previous findings that question the utility of modified dosing in infants. The current study demonstrates the feasibility of generating informative pharmacokinetic data in infants and young children.

Biliary excretion of etoposide in children with cancer.

PURPOSE: Two children with soft tissue sarcomas receiving etoposide as part of their standard clinical treatment had external biliary drainage due to obstruction of the bile duct. These unusual cases provided an opportunity to investigate the biliary clearance of etoposide by determining etoposide concentrations in bile and plasma samples obtained during chemotherapy. PATIENTS AND METHODS: Etoposide was administered to patient 1 at a dose of 150 mg/m(2), as a 4 h infusion, on each of three days of treatment. Patient 2 received a daily etoposide dose of 800 mg/m(2) as a 24 h continuous infusion, also over a 3-day treatment period. Bile and plasma samples were obtained at regular intervals from both patients and etoposide levels quantified by LC/MS analysis. RESULTS AND DISCUSSION: Biliary etoposide clearance was approximately equal to the flow of bile, with an average clearance of 0.32 ml/min determined in patient 1. Less than 2% of the etoposide dose administered was excreted in the bile in either patient studied, indicating that biliary clearance of etoposide is relatively minor. These results suggest that etoposide dose adjustment is unnecessary in patients with biliary obstruction.

Pharmacokinetics of dactinomycin in a pediatric patient population: a United Kingdom Children's Cancer Study Group Study.

PURPOSE: Dactinomycin (actinomycin D) is an antitumor antibiotic used routinely to treat certain pediatric and adult cancers. Despite concerns over the incidence of toxicity, little is known about the pharmacology of dactinomycin. A study was done to investigate dactinomycin pharmacokinetics in children. EXPERIMENTAL DESIGN: Dactinomycin was administered to 31 patients by bolus i.v. infusion, at doses of 0.70 to 1.50 mg/m2. Plasma concentrations were determined by liquid chromatography-mass spectrometry up to 24 hours after drug administration and National Cancer Institute Common Toxicity Criteria was assessed. RESULTS: Pharmacokinetic data analysis suggested that a three-compartment model most accurately reflected dactinomycin pharmacokinetics. However, there was insufficient data available to fully characterize this model. A median peak plasma concentration (Cmax) of 25.1 ng/mL (range, 3.2-99.2 ng/mL) was observed at 15 minutes after administration. The median exposure (AUC0-6), determined in 16 patients with sampling to 6 hours, was 2.67 mg/L.min (range, 1.12-4.90 mg/L.min). After adjusting for body size, AUC0-6 and Cmax were positively related to dose (P = 0.03 and P = 0.04, respectively). Patients who experienced any level of Common Toxicity Criteria grade had a 1.46-fold higher AUC0-6, 95% confidence interval (1.02-2.09). AUC0-6 was higher in patients <40 kg, possibly indicating a greater toxicity risk. CONCLUSIONS: Data presented suggest that dosing of dactinomycin based on surface area is not optimal, either in younger patients in whom the risk of toxicity is greater, or in older patients where doses are capped.

Determination of anti-cancer drug actinomycin D in human plasma by liquid chromatography-mass spectrometry.

Actinomycin D is an anti-cancer drug commonly used in the treatment of paediatric malignancies such as Wilms' tumour, Ewing's sarcoma and rhabdomyosarcoma. Despite its long history of clinical use, little is known about the pharmacokinetics of actinomycin D in humans, largely due to problems in developing an analytical assay with the required sensitivity to measure relevant clinical concentrations. As actinomycin D treatment in children with cancer is associated with veno-occlusive disease (VOD), and as the dose intensity of actinomycin D treatment has been defined as a significant risk factor for the development of this potentially life-threatening hepatic toxicity, pharmacokinetic studies of actinomycin D may be beneficial in optimizing treatment with this drug. In order to investigate this issue, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) method for the determination of actinomycin D in human plasma samples. Extraction of analytical samples was carried out with acetonitrile and analysis performed on an API 2000 LC/MS/MS using an internal standard of 7-aminoactinomycin D. A limit of quantitation of 1.0 ng/ml was determined, allowing the reliable measurement of actinomycin D in plasma samples obtained from patients receiving this drug clinically. The method demonstrated good reproducibility, over the calibration curve range of 1.0-100 ng/ml, with intra- and inter-assay precision CVs of 2.7-11.3 and 2.3-7.8%, respectively. Accuracy data showed relative errors of 2.0-16.4 and 10.4-15.2% for intra-assay (n=10) and inter-assay (n=7) experiments, respectively. Initial results of actinomycin D pharmacokinetics in paediatric patients are shown.

Influence of isomerisation on the growth inhibitory effects and cellular activity of 13-cis and all-trans retinoic acid in neuroblastoma cells.

Treatment with 13-cis retinoic acid (13-cis RA) has been shown to significantly improve the clinical outcome of children with high-risk neuroblastoma. Despite the large number of studies investigating the cellular effects of retinoids in neuroblastoma cells, the influence of RA isomerisation and the factors that determine the extent of RA isomerisation and uptake are unknown. The aim of this study was to establish the extent of extra- and intracellular isomerisation of 13-cis RA and all-trans retinoic acid (ATRA) in neuroblastoma cell lines, and to investigate the influence of isomerisation on their growth inhibitory effects and on the regulation of expression of cellular retinoic acid binding protein II (CRABP II) and RAR-beta. Limited extracellular isomerisation was observed up to 72 hr after incubation of four neuroblastoma cell lines with 10 microM 13-cis RA or ATRA. The retinoic acid isomer present initially in the medium accounted for >75% of extracellular retinoid exposure. By contrast, incubation with 13-cis RA resulted in intracellular levels of ATRA comparable to those of 13-cis RA. This degree of intracellular isomerisation was not observed after ATRA incubations, with 13-cis RA accounting for <10% of total intracellular retinoids. No differences were observed in the sensitivity of three N-type neuroblastoma cell lines to either 13-cis RA (IC(50): 11.2-13.9 microM) or ATRA (IC(50): 12.9-14.4 microM), despite 10-fold differences in intracellular retinoid levels. A decrease in sensitivity to 13-cis RA (IC(50)=137 microM), as compared to ATRA (IC(50)=41 microM), was observed in the S-type cell line SH S EP. RAR-beta was induced in a dose-dependent manner in SH SY 5Y cells following incubation with ATRA, whereas a weaker and delayed induction was observed with 13-cis RA. Similarly, incubation with ATRA resulted in a greater induction of CRABP II in these cells. In summary, these results indicate either an intracellular conversion of 13-cis RA to ATRA or a selective uptake of ATRA and suggest that this may mediate the differential activity of 13-cis RA in neuroblastoma cell subtypes.