Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype.

Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.

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.

Characterisation of the roles of ABCB1, ABCC1, ABCC2 and ABCG2 in the transport and pharmacokinetics of actinomycin D in vitro and in vivo.

Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI(50) was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7 nM vs. 745 nM, p<0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI(50) 25.7 and 40.4 nM respectively, p<0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98 nM vs. 0.1 nM, p<0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2 h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b(-/-) as compared to WT mice following administration of 0.5 mg/kg actinomycin D (AUC(0-6 h) 242 vs. 152 µg/Lh respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b(-/-) and Abcc2(-/-) mice, concentrations in the brain were significantly higher at 6h following drug administration in Abcb1a/1b(-/-) mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.

DNA as a target for anticancer compounds screening directly by resonance light scattering technique.

Anticancer drugs which selectively interact with DNA can change DNA's conformation and inhibit the duplication or transcription of DNA. An instrument-based assay for directly screening DNA-targeted anticancer drugs using resonance light scattering (RLS) technique with a common spectrofluorometer was proposed. To monitor the proposed screening method, the interactions between three anticarcinogens (Adriamycin (ADM), Bleomycin A (BLMA), Actinomycin D (ACTD)) and DNA were studied. The sequence of binding constants for the three anticarcinogens obtained from RLS spectra is: K(RLS) (ACTD, 9.43 × 10(5) L mol(-1)) > K(RLS) (ADM, 6.67 × 10(5) L mol(-1)) > K(RLS) (BLMA, 8.88 × 10(3) L mol(-1)) and of binding numbers is: N(RLS) (ACTD, 3.36 mmol g(-1)) < N(RLS) (ADM, 3.81 mmol g(-1)) < N(RLS) (BLMA, 57.44 mmol g(-1)). From the results we got the sequence of combination intensity between these three drugs and DNA as follows: ACTD-DNA > ADM-DNA > BLMA-DNA, which was completely consistent with drug activity. The conclusion indicated that the present method was direct, rapid, reliable and was another important innovation of the application of RLS technique.

Application of dried blood spots combined with high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of vincristine and actinomycin-D.

A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile-methanol-water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50 x 2.1 mm ID Xbridge C(18) column using elution with 1 mM ammonium acetate-acetonitrile (70:30, v/v) adjusted to pH 10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycin-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.

Validated assay for the simultaneous quantification of total vincristine and actinomycin-D concentrations in human EDTA plasma and of vincristine concentrations in human plasma ultrafiltrate by high-performance liquid chromatography coupled with tandem mass spectrometry.

A sensitive, specific and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin-D concentrations in human plasma and an assay for the determination of unbound vincristine are presented. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and heated electrospray ionization (H-ESI) were tested as ionization interfaces. For reasons of robustness ESI was chosen followed by tandem mass spectrometry (ESI-MS/MS). For the plasma assay a 30 microL aliquot was protein precipitated with acetonitrile/methanol (50:50, v/v) containing the internal standard vinorelbine and 10 microL volumes were injected onto the HPLC system. To determine unbound vincristine, ultrafiltrate was produced from plasma using 30 kDa centrifugal filter units. The plasma ultrafiltrate was mixed with methanol (50:50, v/v), internal standard vinorelbine was added and 20 microL aliquots were injected onto the HPLC system. Separation was achieved on a 50x2.1 mm i.d. Xbridge C18 column using 1 mM ammonium acetate/acetonitrile (30:70, v/v) adjusted to pH 10.5 with ammonia, run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies in plasma vincristine from 0.25 to 100 ng/mL and actinomycin-D from 0.5 to 250 ng/mL using plasma sample volumes of only 30 microL. Vincristine in plasma ultrafiltrate can be quantified from 1 to 100 ng/mL. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human plasma and plasma ultrafiltrate with the presented methods. The assays are now in use to support clinical pharmacological studies in children treated with vincristine and actinomycin-D.

Population pharmacokinetic investigation of actinomycin-D in children and young adults.

Actinomycin-D is an antineoplastic agent that inhibits RNA synthesis by binding to guanine residues and inhibiting DNA-dependent RNA polymerase. Although actinomycin-D has been used to treat rhabdomyosarcoma and Wilms tumor for more than 40 years, the dose/exposure relationship is not well characterized. The objective of this study was to develop an initial population pharmacokinetic model to describe actinomycin-D disposition in children and young adults from which a prospective study could be designed. A total of 165 actinomycin-D plasma concentration measurements from 33 patients, aged 1.6 to 20.3 years, were used for the analysis. The data were analyzed using nonlinear mixed-effects modeling with the NONMEM software system. Age, weight, and gender were examined as covariates for the ability to explain interindividual variability in actinomycin-D pharmacokinetics. The final model was qualified via predictive check and nonparametric bootstrap procedures. A 3-compartment model with first-order elimination was chosen as the structural model. Allometric expressions incorporating weight were used to describe the effects of body size on actinomycin-D pharmacokinetics. Age and gender had no discernible effects on actinomycin-D pharmacokinetics in the population studied. The predictive check showed that the developed model was able to simulate data in close agreement with the actual study observations. The availability of an initial population pharmacokinetic model to describe actinomycin-D pharmacokinetics will facilitate the development of a large-scale clinical trial to study the actinomycin-D dose/exposure relationship in pediatric patients with rhabdomyosarcoma and Wilms tumor. The covariate analysis described by the current data set suggests that indices of body size captured via allometric expressions improve the partition of variation in actinomycin-D pharmacokinetics from this pilot data set. Relationships between pharmacokinetics and toxicity will be examined in future prospective studies in which children less than 1 year old will be enrolled.

A sensitive and selective liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of actinomycin-D and vincristine in children with cancer.

We describe a selective and a highly sensitive assay for actinomycin-D (Act-D) and vincristine (VCR) in plasma employing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection. The intraday precision (as defined by the coefficient of variation, CV) based on the standard deviation of replicates of quality control samples ranged from 4.9 to 7.5% and 6.5 to 11.3% with accuracy ranging from 90.7 to 98.1% and 91.2 to 103% for Act-D and VCR, respectively. The interday precision ranged from 7.2 to 10.0% and 11.3 to 13.0% and the accuracy ranged from 94.3 to 102% and 90.7 to 91.6% for Act-D and VCR, respectively. Stability studies showed that Act-D and VCR were stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) for both Act-D and VCR was 0.05 ng/ml. The analytical method showed excellent sensitivity, precision, and accuracy. This method is robust and is being successfully employed in a pharmacokinetic study of these agents in children with cancer, and is expected to support several ongoing and future pediatric trials.

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of actinomycin-D and vincristine in children with cancer.

Actinomycin-D (Act-D) and vincristine (VCR) are cytotoxic agents commonly used in the treatment of pediatric cancers. To date, there are few published methods on quantifying Act-D or VCR and no published methods on quantifying the two drugs together. We present a methodology for the simultaneous quantification of Act-D and VCR in human plasma using liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection. Following solid phase extraction, plasma samples were separated and analyzed using electrospray ionization (ESI). The lower limit of quantitation (LLOQ) for both Act-D and VCR was 0.5 ng/ml. The analytical accuracy for detection of both Act-D and VCR was > or = 90%. The analytical precision, as estimated by the coefficient of variation was < or = 6% for Act-D and < or = 11% for VCR. Given the prevalence of the use of the two drugs as combination therapy in a variety of pediatric oncological indications, the small sample volume requirements and the assay sensitivity, this methodology is expected to support several ongoing and future pediatric trials.

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.

Granulocyte storage and antigen stability.

BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time-consuming, costly, and technically difficult. STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4 degrees C for 7 days under three conditions: 1 -percent formaldehyde-fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI-1640); and cells were fixed and stored with a commercial white cell-storage solution (Cyto-Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis. RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI-1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell-storage solution remained. All antigens were detectable by the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)-conjugated secondary antibody hindered interpretation of test results on Day 4. Non-specific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC-conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7-aminoactinomycin-D. CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of non-specific staining due to enhanced membrane permeability of dying cells.

A phase I trial of dactinomycin intravenous infusion in patients with advanced malignancies.

Eighteen patients with advanced malignancies refractory to other forms of treatment were given dactinomycin (Act D) as continuous intravenous infusions. Their median age was 51 years (range, 36-67); their median performance status was 50 (range, 40-90) on the Karnofsky scale. Act D was administered continuously for 5 days, utilizing a central venous line and a perfusion pump. The starting dose was 0.1 mg/m2/24 hours X 5 days (total dose, 0.5 mg/m2) and was escalated according to a modified Fibonacci scale to 0.2, 0.33, and 0.5 mg/m2/24 hours X 5 days, respectively. Three, three, four, and eight patients were entered, respectively, in each dose level. Toxicities observed were: leukopenia in four patients (nadir leukocyte count less than 1000 cells/nm3 in one patient and 2000-3000 cells/mm3 in 3 patients); thrombocytopenia, with nadir platelet counts between 50,000 and 100,000 platelets/mm3 in 2 patients; stomatitis in four patients; and nausea in three patients. Vomiting was not observed during the infusions. Two patients may have had a radiation recall phenomenon. Blood count depression, nausea, and mucositis were transient, resolving after a few days. One patient at level IV died of sepsis, which was diagnosed on the fourth day of the infusion, before leukopenia intervened. No objective responses were seen. It was concluded that a higher dose of Act D can be given by continuous infusion than by a bolus injection; the authors recommended 0.5 mg/m2/day X 5 days (total dose, 2.5 mg/m2) for further studies.

Development of an antibody to actinomycin D and its application for the detection of serum levels by radioimmunoassay.

An antibody specific for actinomycin D (Act D) has been developed and used in a rapid, sensitive radioimmunoassay for detection of this anticancer drug in serum. The 2-amino group of the heterocyclic chromophore of Act D was covalently coupled to available free carboxyl groups of bovine serum albumin with carbodiimide. The resulting complex was then used for the production of a specific antibody to Act D in two male New Zealand rabbits. Antibody production was of sufficient titer in both rabbits to allow the development of a radioimmunoassay for the free drug which is rapid and sensitive enough to accurately measure 0.1 pmol of Act D. The antibody produced was characterized to be immunoglobulin G by virtue of its ability to bind to Protein A:Sepharose columns. With the use of Act-D analog, actinomine, the antibody was characterized to be specific for the pentapeptide portion of the molecule. Pharmacokinetic analysis of serial serum samples obtained from a patient who received the drug i.v. revealed a biphasic response with an alpha-serum half-life of 1.78 and a beta serum half-life of 34 min. An i.v. injection of Act D into a dog and assay of serum concentration revealed a similar biphasic response with an alpha serum half-life of 0.78 min and a beta-serum half-life of 208 min.

Disposition of [3H]actinomycin D in tumor-bearing mice.

A single, nonlethal dose of actinomycin D will cause total regression and cure of Ridgway osteogenic sarcoma in mice. A cure is not obtained with a single dose daily for 7 days, a dose regimen which kills 10% of normal C57BL/6 X DBA/2 mice. This suggests that actinomycin D is more effective on a single high-dose schedule than on chronic daily therapy. Analysis of drug exposure in Ridgway osteogenic sarcoma and normal mouse tissues following the single and multiple-dose regimens suggests the difference in therapeutic response is due to drug exposure at a higher concentration in Ridgway osteogenic sarcoma after the single high dose than after the multiple-dose regimen. This may be related to the higher drug concentration attained in blood following the single-dose regimen than is attained with the multidose regimen.