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A liquid chromatography–tandem mass spectrometry method was developed and validated to quantify the small-molecule inhibitors (SMIs) brigatinib, lorlatinib, pralsetinib and selpercatinib, which are used in patients with oncogenic-driven non-small cell lung cancer. Chromatographic separation was performed on a HyPURITY® C 18 analytical column with a gradient elution using ammonium acetate in water and in methanol, both acidified with formic acid 0,1%. Detection and quantification were performed using a triple quad mass spectrometer with an electrospray ionization interface. The assay was validated over a linear range of 50–2,500 ng/ml for brigatinib, 25–1,000 ng/ml for lorlatinib, 100–10,000 ng/ml for pralsetinib and 50–5,000 ng/ml for selpercatinib. All four SMIs were stable for at least 7 days under cool conditions (2–8°C), and at least 24 h at room temperature (15–25°C) in K2-EDTA plasma. Under freezing conditions (−20°C), all SMIs were stable for at least 30 days, except for the lowest quality control (QC LOW) of pralsetinib. The QC LOW of pralsetinib was stable for at least 7 days at −20°C. This method provides an efficient and simple way to quantify four SMIs with a single assay in clinical practice.

A liquid chromatography-tandem mass spectrometry method was developed and validated to quantify alectinib, crizotinib, erlotinib and gefitinib. This assay can be combined with our method for osimertinib, allowing quantification of the most used ALK- and EGFR-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer with a single-assay setup. Chromatographic separation was performed on a HyPurity (R) C-18 analytical column using an elution gradient of ammonium acetate in water and in methanol, both acidified with formic acid 0.1%. Detection and quantification were performed using a triple quad mass spectrometer with an electrospray ionization interface. This method led to robust results, as the selectivity, carryover, precision and accuracy met all pre-specified requirements. The assay was validated over a linear range of 100-2,000 ng/ml for alectinib and erlotinib and 50-1,000 ng/ml for crizotinib and gefitinib. Alectinib, crizotinib, erlotinib and gefitinib were all stable for at least 4 h in whole blood (at room temperature and at 4 degrees C) and for at least 1 month in EDTA plasma when stored at -80 degrees C, while osimertinib proved to be unstable at room temperature. Although high-performance liquid chromatography was used, the run time was short and comparable with other methods using ultra-high performance liquid chromatography.

Dried blood spot (DBS) analysis has been introduced more and more into clinical practice to facilitate Therapeutic Drug Monitoring (TDM). To assure the quality of bioanalytical methods, the design, development and validation needs to fit the intended use. Current validation requirements, described in guidelines for traditional matrices (blood, plasma, serum), do not cover all necessary aspects of method development, analytical- and clinical validation of DBS assays for TDM. Therefore, this guideline provides parameters required for the validation of quantitative determination of small molecule drugs in DBS using chromatographic methods, and to provide advice on how these can be assessed. In addition, guidance is given on the application of validated methods in a routine context. First, considerations for the method development stage are described covering sample collection procedure, type of filter paper and punch size, sample volume, drying and storage, internal standard incorporation, type of blood used, sample preparation and prevalidation. Second, common parameters regarding analytical validation are described in context of DBS analysis with the addition of DBS-specific parameters, such as volume-, volcano- and hematocrit effects. Third, clinical validation studies are described, including number of clinical samples and patients, comparison of DBS with venous blood, statistical methods and interpretation, spot quality, sampling procedure, duplicates, outliers, automated analysis methods and quality control programs. Lastly, cross-validation is discussed, covering changes made to existing sampling- and analysis methods. This guideline of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology on the development, validation and evaluation of DBS-based methods for the purpose of TDM aims to contribute to high-quality micro sampling methods used in clinical practice.

Aim Direct-acting oral anticoagulants (DOACs) have become available for the prevention of stroke in patients with atrial fibrillation (AF). Conflicting results have been published on the risk of acute myocardial infarction (AMI) with the use of DOACs in comparison with vitamin K antagonists (VKAs). The objective of the present study was to evaluate the risk of AMI in patients with AF who are exposed to either VKAs, DOACs or low-dose (< 325 mg) aspirin. Methods We conducted a population-based cohort study using data from the Clinical Practice Research Datalink (2008–2014). The study population (n = 30 146) consisted of all patients ≥18 years with a diagnosis of AF who were new users of VKAs, DOACs (rivaroxaban and dabigatran) or aspirin. Cox proportional hazards models were used to estimate the hazard ratio (HR) of AMI for users of DOACs or aspirin vs. VKA. Adjustments were made for age, gender, lifestyle, risk factors, comorbidity and other drugs. Results The risk of AMI was doubled when we compared current use of DOACs with current use of VKAs [adjusted HR 2.11; 95% confidence interval (CI) 1.08, 4.12] and for current users of aspirin vs. current VKA users (adjusted HR 1.91; 95% CI 1.45, 2.51). Conclusions There is a twofold increase in the risk of AMI for users of DOACs, in comparison with VKAs, in AF therapy. In addition, the results suggested that in patients with AF, the incidence of AMI is higher during aspirin monotherapy than during the use of VKAs.

Contains fulltext : 218134.pdf (Publisher’s version ) (Closed access) A new method for quantification of osimertinib (OSIM) in human plasma using a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated. Methanol was used for protein precipitation and pazopanib as internal standard. Separation was performed on a HyPURITY(R)C18 analytical column (50 x 2.1 mm; 3 mum) using a gradient elution of ammonium acetate in water and ammonium acetate in methanol, both acidified with formic acid 0.1%. Detection and quantification of OSIM and pazopanib was performed using a triple quadruple mass spectrometer after electrospray ionization. This method led to robust results, as the selectivity, carryover, precision and accuracy all met pre-specified requirements. OSIM was stable in human serum when stored at -80 degrees C. Reduced stability was found when stored at 2-4 degrees C or room temperature. Degradation of OSIM slowed down in EDTA-plasma and acidified human serum. The limited stability of OSIM at room temperature should be considered for transport and sample preparation. Plasma samples should be frozen as soon as possible and sample preparation should be performed on dry-ice. In the future, EDTA-plasma and sample acidification may be used to improve OSIM stability at room temperature. However, more research and validation of such an approach are required.

Background: Particularly in the pediatric clinical pharmacology field, data-sharing offers the possibility of making the most of all available data. In this study, we utilize previously collected therapeutic drug monitoring (TDM) data of term and preterm newborns to develop a population pharmacokinetic model for phenobarbital. We externally validate the model using prospective phenobarbital data from an ongoing pharmacokinetic study in preterm neonates.Methods: TDM data from 53 neonates (gestational age (GA): 37 (24-42) weeks, bodyweight: 2.7 (0.45-4.5) kg; postnatal age (PNA): 4.5 (0 - 22) days) contained information on dosage histories, concentration and covariate data (including birth weight, actual weight, post-natal age (PNA), postmenstrual age, GA, sex, liver and kidney function, APGAR-score). Model development was carried out using NONMEM (R) 7.3. After assessment of model fit, the model was validated using data of 17 neonates included in the DINO (Drug dosage Improvement in NeOnates)-study.Results: Modelling of 229 plasma concentrations, ranging from 3.2 to 75.2 mg/L, resulted in a one compartment model for phenobarbital. Clearance (CL) and volume (V-d) for a child with a birthweight of 2.6 kg at PNA day 4.5 was 0.0091 L/h (9%) and 2.38 L (5%), respectively. Birthweight and PNA were the best predictors for CL maturation, increasing CL by 36.7% per kg birthweight and 5.3% per postnatal day of living, respectively. The best predictor for the increase in Vd was actual bodyweight (0.31 L/kg). External validation showed that the model can adequately predict the pharmacokinetics in a prospective study.Conclusion: Data-sharing can help to successfully develop and validate population pharmacokinetic models in neonates. From the results it seems that both PNA and bodyweight are required to guide dosing of phenobarbital in term and preterm neonates.

Observational studies have shown conflicting results on the potential protecting effect of biguanide use with the risk of colorectal neoplasms. In addition, the cellular mechanism can either support or oppose biguanides influence on colorectal carcinoma. Our objective was to evaluate the association between biguanide use and colorectal carcinoma. A population-based cohort study using healthcare data from the Danish National database (1996-2007), was conducted. Oral antidiabetic drug users (n = 177,281) were matched 1:3 with a population-based reference group. Cox proportional hazard models estimated hazard ratios (HRs) of colorectal carcinoma. Stratification was performed to analyse the risk of colorectal cancer in current biguanide users. Two sub-analyses were performed, to investigate the risk of colorectal cancer associated with discontinuous and prolonged use of biguanides. Instead of a protective effect, we found that current biguanide users had a 1.2-fold increased risk of colorectal cancer (HR = 1.19, 95% CI = 1.08-1.30) as compared with the non-diabetes reference group. Prolonged use was not inversely associated with colorectal cancer either. When studying colorectal risk with biguanides, the underlying T2DM should be taken into account since a 1.3-1.6-fold increased risk was found in oral antidiabetic drug users compared to controls unexposed to diabetic medication. This study could not detect a protective effect of biguanide use with colorectal cancer. Therefore, this study does not support a further investigation of the effectiveness of biguanides to prevent colorectal carcinoma in clinical studies.

This journal suppl. entitled: Abstracts of the 26th Annual Meeting of the European Society of Human Reproduction and Embryology, Rome, Italy, 27-30 June 2010

This article reviews dried blood spot (DBS) sampling in therapeutic drug monitoring. The DBS method involves applying whole blood obtained via a fingerprick to a sampling paper. After drying and transportation, the blood spot is extracted and analyzed in the laboratory. Assays of many medicines in DBS have already been reported in the literature and are reviewed here. The technique involved in and factors that may influence the accuracy and reproducibility of DBS methods are also discussed. DBS sampling ultimately seems to be a useful technique for therapeutic drug monitoring that could have many advantages in comparison with conventional venous sampling. However, its benefits must be weighed against the degree of potential errors introduced via the sampling method; there is evidently a need for more standardization, quality assurance, basic research, and assay development.

The development of a new drug is characterized by distinct developmental stages, usually described as phases I to IV. Dose tolerance and dose response exploration studies are undertaken in phase II or III. Pharmacokinetic studies are often involved in these phases, but frequently only as an objective of minor importance. The usefulness of therapeutic drug monitoring (TDM) is not consequently investigated for new drugs. Usually the need for TDM is only discovered much later, when the drug is already on the market. TDM is particularly valuable under the following circumstances: (i) if there is a stronger relationship between the drug concentration and effect than between the dose and effect; (ii) if there is no simple and clear clinical parameter available to evaluate the clinical efficacy of the drug; (iii) if the therapeutic window is small; (iv) to document interactions; (v) to monitor drug compliance; and (vi) if there is large intra- and interindividual variability and unpredictability in pharmacokinetic parameters. Our recommendation is that randomized concentration controlled trials should be performed during the early stages of drug development and that it should be obligatory for drug licensing.

OBJECTIVE: In literature, a great diversity of limited sampling strategies (LSS) have been recommended for tacrolimus monitoring, however proper validation of these strategies to accurately predict the area under the time concentration curve (AUC0-12) is limited. The aim of this study was to determine whether these LSS might be useful for AUC prediction of other patient populations. METHODS: The LSS from literature studied were based on regression equations or on Bayesian fitting using MWPHARM 3.50 (Mediware, Groningen, the Netherlands). The performance was evaluated on 24 of these LSS in our population of 37 renal transplant patients with known AUCs. The results were also compared with the predictability of the regression equation based on the trough concentrations C0 and C12 of these 37 patients. Criterion was an absolute prediction error (APE) that differed less than 15% from the complete AUC0-12 calculated by the trapezoidal rule. RESULTS: Thirteen of the 18 (72%) LSS based on regression analysis were capable of predicting at least 90% of the 37 individual AUC0-12 within an APE of 15%. Additionally, all but three LSS examined gave a better prediction of the complete AUC0-12 in comparison with the trough concentrations C0 or C12 (mean 62%). All six LSS based on Bayesian fitting predicted

The isoxazolyl penicillins, including flucloxacillin, have the highest levels of plasma protein binding among the semisynthetic penicillins. Because only the free fraction of the penicillin is pharmacologically active, it would be useful to measure both protein-bound and free flucloxacillin to determine its protein binding. Until now, flucloxacillin protein binding in newborn infants has been investigated in only two studies with relatively small populations. In the present study, flucloxacillin protein binding was investigated in 56 (preterm) infants aged 3 to 87 days (gestational age, 25-41 weeks). Surplus plasma samples from routine gentamicin assays of each infant were collected and combined to obtain a sufficiently large sample for analysis. Free flucloxacillin was separated from protein-bound flucloxacillin using ultrafiltration. Reversed-phase high-performance liquid chromatography with ultraviolet detection was used to measure free flucloxacillin concentrations in ultrafiltrate and total flucloxacillin concentrations in pooled plasma. Flucloxacillin protein binding was 74.5% +/- 13.1% (mean +/- standard deviation) with a high variability among the infants (34.3% to 89.7%). High Pearson correlations were found between protein binding and the covariates-plasma albumin concentration (r = 0.804, P < 0.001, n = 18) and plasma creatinine concentration (r = -0.601, P < 0.001, n = 45). Statistically significant but less striking correlations were found between protein binding and gestational age, postconceptional age, body weight, and triglyceride concentration. Because of the high variability of protein binding among infants, it is difficult to devise a flucloxacillin dosage regimen effective for all infants. Individualized dosing, based on free flucloxacillin concentrations, might help to optimize treatment of late-onset neonatal sepsis, but practical obstacles will probably prevent analysis of free flucloxacillin concentrations in newborn infants on a routine basis.

The pharmacokinetic parameters of amoxicillin were determined in 32 newborn infants aged 10 to 52 days (mean postnatal age, 24.7 +/- 12.4 days) to improve amoxicillin dosing in this age group. Amoxicillin plasma concentrations were determined using reversed-phase high-performance liquid chromatography in surplus plasma samples from routine gentamicin assays. Amoxicillin pharmacokinetic parameters (mean +/- SD) were as follows: first-order elimination constant (K(el)) = 0.27 +/- 0.10 h(-1), volume of distribution corrected for body weight (V/W) = 0.66 +/- 0.27 L/kg, total body clearance corrected for body weight (CL/W) = 0.18 +/- 0.10 Lkg(-1)h(-1), and elimination half-life (t(1/2)) = 3.0 +/- 1.3 hours. Amoxicillin body clearance was approximately twofold greater in our patients compared with published values in younger neonates (mean postnatal age, 0.76 +/- 1.57 days). Simulation studies using the observed amoxicillin pharmacokinetic data suggest an amoxicillin dose of 40 mg/kg administered every 8 hours in infants older than 9 days postnatal age, independent of gestational age and postconceptional age, to achieve satisfactory target plasma amoxicillin concentrations less than 140 mg/L and time above minimum inhibitory concentration of at least 40%. Prospective evaluation of this suggested new dosage regimen is necessary before implementation in the care of ill neonates.

Tacrolimus, an immunosuppressant used after organ transplantation, has a narrow therapeutic range and its pharmacokinetic variability complicates its daily dose assessment. P-glycoprotein (P-gp), encoded by the adenosine triphosphate-binding cassette B1 (ABCB1) and the cytochrome (CYP) 3A4 and 3A5 enzymes appears to play a role in the tacrolimus metabolism. In the present study, two different renal transplant recipient groups were used to examine the influence of ABCB1 and CYP3A polymorphisms on the daily tacrolimus dose and several pharmacokinetic parameters. In total 63 Caucasian renal transplant recipients divided into 26 early [median (range) of the days since transplantation - 16 (3-74)] and 37 late [median (range) of the days since transplantation - 1465 (453-4128)] post-transplant recipients were genotyped for ABCB1 and CYP3A polymorphisms. The pharmacokinetic parameters of tacrolimus were determined for all renal transplant recipients and correlated with their corresponding genotypes. A significant difference in allele frequencies of the CYP3A4*1B (P = 0.028) and CYP3A5*1 (P = 0.022) alleles was observed between the early and late post-transplant recipient groups. Significantly higher dose-normalized trough levels (dnC(0)), dose-normalized area under the curve (dnAUC(0-12)), and dose-normalized maximum concentration (dnC(max)) were observed for carriers of the CYP3A5*3 variant allele in both renal transplant patient groups. Except for the daily tacrolimus dose (P = 0.025) no significant differences were observed for carriers of the CYP3A4*1B variant allele. Neither the individual ABCB1 polymorphisms nor the ABCB1 haplotypes were associated with any pharmacokinetic parameter. We noticed that patients carrying a CYP3A5*1 allele require a twofold higher tacrolimus dose compared with homozygous carriers of the CYP3A5*3 variant allele to maintain the target dnAUC(0-12). Therefore, genotyping for the CYP3A5*3 variant allele can contribute to a better and more individualized immunosuppressive therapy in transplant patients.

Simple and rapid reversed-phase high-performance liquid chromatographic assays with ultraviolet detection have been developed and validated for the determination of amoxicillin, flucloxacillin and rifampicin in neonatal plasma. Plasma samples were either precipitated with perchloric acid (amoxicillin) or methanol (rifampicin) or extracted with methylene chloride (flucloxacillin). Precision coefficients of variation and inaccuracy were less than 15% for all three assays. Only small sample volumes (20-40 microL) were required, making the assays suitable for therapeutic drug monitoring and pharmacokinetic studies in preterm and term neonates. The assays have successfully been applied to analysis of amoxicillin, flucloxacillin and rifampicin in previously published pharmacokinetic studies in neonates.

Few reports have addressed neonatal rifampicin plasma concentrations and data on neonatal rifampicin pharmacokinetics are completely lacking. Therefore, plasma concentrations of rifampicin and its main metabolite 25-O-desacetylrifampicin (DES) were measured in 123 surplus plasma samples from routine vancomycin monitoring in 21 neonates using reversed-phase HPLC. Rifampicin peak and trough plasma concentrations were 4.66 +/- 1.47 mg/L and 0.21 +/- 0.20 mg/L, respectively, after a dose of 8.5 +/- 2.1 (mean +/- SD) mg/kg per day. A significant linear relationship between rifampicin dose and peak plasma concentrations was found, but inter-patient variability was high. Pharmacokinetic parameters of rifampicin were calculated according to a one-compartment open model with iterative two-stage Bayesian fitting (MW\PHARM 3.60, Mediware, The Netherlands). First-order elimination constant, volume of distribution corrected for weight, total body clearance corrected for weight (CL/W), and elimination half-life were 0.16 +/- 0.06 h(-1), 1.84 +/- 0.59 L/kg, 0.28 +/- 0.11 Lkg(-1) h(-1), and 4.9 +/- 1.7 h, respectively. A high Pearson correlation was found between CL/W rifampicin and the covariates plasma creatinine and CL/W gentamicin of a preceding gentamicin treatment course, r = 0.728 (n = 17) and r = 0.837 (n = 12), respectively. DES was detected in each plasma sample. Therefore, rifampicin seems to be eliminated by both renal and metabolic pathways in neonates. In 8 study patients, plasma concentrations of rifampicin and DES were measured again after two weeks of therapy. CL/W rifampicin was significantly higher (67 +/- 50%). The authors suggest maintaining the current dose regimen of 10 mg/kg once a day. Because of the large inter-patient variability in rifampicin plasma concentrations and CL/W increase during therapy, the authors suggest monitoring rifampicin peak and trough plasma concentrations to avoid low plasma concentrations. More research is needed to determine well-founded dosing guidelines.

In total 235 flucloxacillin total (free+protein bound) plasma concentrations were determined in 55 neonates (gestational age 26 to 42 weeks, postnatal age 0 to 44 days) with reversed-phase HPLC in surplus plasma samples from routine gentamicin assays. Population pharmacokinetic parameters were calculated according to an one compartment open model with iterative two-stage Bayesian fitting (MWPHARM 3.50, Mediware, The Netherlands). Mean clearance corrected for weight was 0.18+/-0.10 L kgh and volume of distribution corrected for weight was 0.54+/-0.17 L/kg. Pearson correlations between the individual pharmacokinetic parameters and covariates, like gestational age, plasma creatinine, and gentamicin clearance, were low and therefore not relevant for use in clinical practice. Total plasma concentrations above 200 mg/L were considered toxic and T>MIC (time above minimum inhibitory free plasma concentration) of more than 40% was considered effective. Protein binding was assumed to be 86.3% in all neonates, based on literature. The current dosage regimen, 25 or 50 mg/kg every 8 or 12 hours, did not result in effective plasma concentrations for the treatment of Staphylococcus aureus in 17 (31%) of the 55 neonates. Therefore, the authors suggest an initial dose of 25 mg/kg/4 h for all neonates, irrespective of their age, based on the breakpoint MIC value of flucloxacillin for Staphylococcus aureus (2.0 mg/L). After isolation of the causative agent of infection, flucloxacillin administration ought to be reconsidered based on the expected susceptibility pattern of the isolate. When oxacillin sensitive coagulase negative staphylococci are isolated, the initial dose should be reduced to 10 mg/kg/6 h, based on the breakpoint MIC value of 0.25 mg/L. Simulation with these new dosage regimens indicated that satisfactory plasma concentrations were reached in 52 of the 55 neonates. However, the regimens need prospective verification. Moreover, the exact role of neonatal protein binding needs to be further investigated.

Aim: This review surveys the different classes of drugs. Method: We searched electronic databases and selected articles about pharmacotherapy with anti-retroviral drugs. Results: We discuss anti-retroviral drugs: the nucleotide reverse transcriptase inhibitors (NRTIs), the non-nucleoside reverse transcriptase inhibitors (NNRTIs), and the protease inhibitors (PIs). The combination of a PI and two NRTIs is very effective and lowers the viral load to below the limit of detection. However, it is not yet possible to eliminate the virus completely. Recently, the first member of a brand new class of drugs has become available, the fusion inhibitor enfuvirtide. We also discuss the problems associated with combining anti-retroviral drugs, such as compliance, resistance and serious side effects. Discussion. It is important to investigate new combinations with new classes of drugs, like the fusion inhibitors, for better effectiveness and fewer side effects.

We have demonstrated that imaging of programmed cell death (PCD) in patients is possible using 99mTc-Annexin A5. Because of the short half-life of the technetium label it is important to limit the time span between the preparation of 99mTc-Annexin A5 and its administration into the patient. Therefore methods of quality control that determine the biological active fraction in the 99mTc-Annexin A5 should be not only accurate and precise but also rapid. We report the development and validation of a rapid, simple assay measuring the biological active fraction of 99mTc-Annexin A5. The assay is based on a solid phase of paramagnetic beads which are coated with phospholipids. Annexin A5 binds to these beads with high affinity if phosphatidyl serine is present within the phospholipid coat. Furthermore the binding depends on Ca2+ ions and functional Ca2+/phospholipid binding sites of Annexin A5. The bead assay is specific, stability-indicating, repeatable, and reproducible. It allows one to determine within 25 min the biological active fraction of a 99mTc-Annexin A5 preparation. We dubbed this assay the ApoCorrect assay.

Recently a small number of patients were observed in two psychiatric hospitals in the Netherlands with clozapine intoxications that complicate or mimic infections. These patients were on chronic medication and normally had stable clozapine blood plasma levels. This article presents four of these cases. Medline was searched for reports of similar cases. A hypothesis was formulated and tested by literature study. Immune modulatory and toxic effects of clozapine protein reactive metabolites or haptens, may play a role in the development of inflammation. Clozapine has a direct influence on different cytokines resembling an inflammatory reaction. Infection or inflammation could induce bioactivation of clozapine into its nitrenium ion that can exert a toxic reaction that induces apoptosis and gives rise to elevated cytokine levels. Clozapine can function as a hapten and induce an IgG, IgM or IgE mediated hypersensitivity reaction. The cytokines released during infection or inflammation downregulate the clozapine metabolism in the P450 system through CYP 1A2. Clozapine plasma levels should be monitored closely if an inflammatory or infectious process is suspected.

Increase in tacrolimus trough levels after steroid withdrawal.van Duijnhoven EM, Boots JM, Christiaans MH, Stolk LM, Undre NA, van Hooff JP.Department of Internal Medicine, University Hospital of Maastricht, P.O. Box 5800, 6202 AZ, Maastricht, The Netherlands. evd@groupwise.azm.nlAlthough there are experimental reports of cytochrome P450 3A4 iso-enzyme (CYP3A4) induction by glucocorticoids, there are no clinical reports about an interaction between tacrolimus and steroids. Therefore, tacrolimus trough level and dose were compared after dose-normalization before and after withdrawal of prednisolone. After withdrawal of 5 mg prednisolone, the median tacrolimus dose-normalized level increased by 14% in the retrospective ( n=54), and by 11% in the prospective ( n=8) part of the study. After withdrawal of 10 mg, this increase was 33% ( n=30) and 36% ( n=14), respectively. An additional pharmacokinetic part of the study ( n=8) revealed an 18% increase in AUC ( P=0.05) after withdrawal of 5 mg prednisolone, which is compatible with a reduced metabolism after steroid withdrawal. The significant increase in tacrolimus exposure after steroid withdrawal may on the one hand counteract the reduction in immunosuppression intended by steroid withdrawal, and, on the other hand, may result in an increase of serum creatinine which could be misinterpreted as rejection

Annexin A5 (AnxA5) is a protein with high affinity for phosphatidyl serine, a phospholipid exposed on the cell surface during apoptosis. This phenomenon has been used for determination of cell death after myocardial infarction. To evaluate the potential of (99m)Tc-AnxA5 for in vivo scintigraphy of apoptotic cells, the pharmacokinetics and imaging properties of two radiopharmaceuticals, (99m)Tc-(n-1-imino-4-mercaptobutyl)-AnxA5 (I-AnxA5) and (99m)Tc-(4,5-bis(thioacetamido)pentanoyl)-AnxA5 (B-AnxA5), were studied. I-AnxA5 was administered intravenously to seven patients and one healthy volunteer, and B-AnxA5 was administered to 12 patients. All patients in the pharmacokinetic study had myocardial disease. Additionally, imaging was performed in a patient with acute myocardial infarction, as well as in three patients with different malignancies. The plasma concentration, excretion and biodistribution of (99m)Tc-AnxA5 were measured, as well as levels of AnxA5 antigen. The kinetic data of both radiopharmaceuticals in plasma fitted a two-compartment model. Both preparations had similar half-lives, but a different distribution over the two compartments. Plasma levels of AnxA5 antigen showed a broad variation. Both radiopharmaceuticals accumulated in the kidney, liver and gut. B-AnxA5 was excreted significantly faster than I-AnxA5. Both compounds can be used for imaging of the head/neck region, the thorax and the extremities. B-AnxA5 has a faster clearance and a lower radiation dose. Imaging of apoptosis in the abdomen will be difficult with both radiopharmaceuticals, and especially with B-AnxA5 because of its faster appearance in the gut.

Cavity ring down spectroscopy (CRDS) was used for measuring absolute concentration profiles of the CN (cyano) radical during oxyacetylene flame deposition of diamond. Profiles were measured for three different nitrogen additions to the flame. Novel ways for the determination of the lateral position of the laser beam and its height above the deposition substrate are presented. Measured column density profiles show that the influence of added nitrogen is limited to a central area within a radius of 2.7 mm. Outside this area the profiles are hardly influenced by the added nitrogen flow. Comparison with work of S. J. Firchow and K. L. Menningen [J. Phys. D: Appl. Phys. 32, 937 (1999)] shows a good correspondence. Absolute concentration profiles were obtained by applying Abel inversion processing and using a temperature distribution resulting from numerical simulations. The resulting profiles show an off-axis maximum for all three nitrogen additions. With an increasing nitrogen addition the maximum and central concentrations also increased. Upon inspection of the profiles it follows that the gas phase above the deposition area can be divided into three sections: a central one in which the added nitrogen is the dominant nitrogen source, an outer one where the nitrogen from the ambient plays a dominant role, and an intermediate zone where both nitrogen sources have a significant influence. The latter is the area in which the maximum of the CN concentration is situated. Previous laser-induced fluorescence (LIF) profiles of CN, measured under very similar experimental conditions, show similar distributions. Differences between the CRDS and LIF results are discussed.

Population pharmacokinetic parameter estimates were calculated from 725 routine plasma gentamicin concentrations obtained in 177 neonates of 24 to 42 weeks' gestational age in their first week of life. Kel increases and V/W decreases with increasing gestational age. Almost identical results were obtained with iterative two-stage Bayesian fitting (MW\PHARM 3.30) as with a non-parametric maximization algorithm (NPEM2). The effect of various covariates on drug disposition was investigated retrospectively using multiple regression analysis. Predictive power for Kel increases with rising gestational age. For neonates/=28.5 weeks and neonates28.5 weeks and/=30.9 weeks, the predictive power of the regression equation for Kel was relatively low (r2 respectively 0.270 and 0.364). Better predictivity was found for Kel at gestational ages30.9 weeks (r2 = 0.482), with gestational age, postnatal age, and Apgar score at 5 minutes being predictors. A very strong correlation existed between volume of distribution and weight (r2 = 0.83). Volume as a function of weight could be described with low predictivity by gestational age and to a lesser degree by Apgar score at 5 minutes (r2 = 0.298). The developed models need appropriate prospective clinical validation.

Measurements of absolute concentrations of C2 (A 1Πu) in an atmospheric oxyacetylene flame are presented. Cavity ring down spectroscopy (CRDS) was applied to measure lateral column density profiles at a number of vertical positions. By means of Abel inversion processing and estimated radial temperature profiles, the column densities were converted to absolute concentration profiles, which have central maxima with peak concentrations between 8×1014 and 2.5×1015 m−3. Comparison of the measured A state concentration profiles with calculated ground state C2 profiles supports an earlier suggestion that the gas-phase mechanism used in the calculations needs improvement. This work demonstrates the applicability of CRDS for measuring absolute concentrations of electronically excited species in atmospheric flames, a result which can be of importance to combustion research.

The pharmacodynamic (PD) efficacy target of amoxicillin is 40% time above the minimal inhibition concentration (40%T > MIC). Recent studies of other antibiotics have shown that PD-efficacy targets are not always reached. The aim of this study was to evaluate the percentage of hospitalised patients, using amoxicillin/clavulanic acid intravenously (iv), that reach the pharmacodynamic efficacy target 40%T > MIC. Additionally, the association of demographic anthropomorphic and clinical parameters with the pharmacokinetics and pharmacodynamics of amoxicillin were determined. In serum of 57 hospitalised patients amoxicillin concentrations were measured using high performance liquid chromatography. Patients were older than 18 years and most patients had an abdominal infection. The standard amoxicillin/clavulanic acid dose was 4 times a day 1000/200 mg iv. Pharmacokinetic parameters were calculated with maximum a posteriori Bayesian estimation (MW\Pharm 3.60). A one-compartment open model was used. Individual dosing simulations were performed with MW\Pharm. In our study population, the mean (±SD) age was 67 (±16) years and the mean clearance corrected for bodyweight was 0.17 (±0.07) L/h/kg. Only, 65% of the patients reached the proposed amoxicillin 40%T > MIC with amoxicillin/clavulanic acid for bacterial MICs of 8 mg/L. A computer simulated increase of the standard dose to 6 times daily, increased this percentage to 95%. In this small study group 40%T > MIC was not associated with clinical or microbiological cure. A substantial proportion of the hospitalised patients did not reach the 40%T > MIC with the standard dose amoxicillin/clavulanic acid for a bacterial MIC of 8 mg/L. Therefore, we suggest increasing the standard dose of amoxicillin/clavulanic acid to 6 times a day in patients with severe Enterobacteriaceae infections. Trial registration number: NTR1725 16th march 2009.

Controlled amounts of nitrogen were added during oxyacetylene flame deposition of diamond to investigate the possible role of the CN radical in the effects of nitrogen addition. CN radical distributions were visualized using two-dimensional laser induced fluorescence (LIF) and compared with nitrogen incorporation into the layer and with the diamond growth rate, which were measured by means of cathodoluminescence (CL) and optical microscopy, respectively. For the studied range of nitrogen flows, it was found that the CN LIF signal in the center of the flame is linearly dependent on the added amount of nitrogen. Diamond deposition in the central region is mainly influenced by the deposition parameters, whereas deposition in the outer zone is largely determined by the interaction of the flame with the ambient; the annulus of enhanced growth is affected by both the deposition parameters and the ambient. By a simple consideration, in which the growth rate is separated in nitrogen dependent and independent cont...

The influence of nitrogen addition on oxyacetylene flame deposition of diamond has been investigated by controlled addition of nitrogen to the combustion gases. Heretofor, the nitrogen incorporation into the diamond layer, the growth rate, and the diamond morphology were determined as a function of the nitrogen flow and compared to gas-phase distributions of the CN radical close to the deposition substrate. Two-dimensional laser induced fluorescence (LIF) was applied for the gas-phase measurements: cathodoluminescence (CL), scanning electron microscopy (SEM), and optical microscopy were used for the determination of the nitrogen incorporation, morphology and growth rate, respectively. In the studied range of nitrogen flows, a linear relation was observed between the added amount of nitrogen and the CN LIF signal in the center of the flame. It was found that that diamond deposition in the central region is largely influenced by the deposition parameters, whereas deposition in the outer zone depends mainly on the interaction with the ambient air: the annulus of enhanced growth marks the transition between those areas. In the central area, a correlation was observed between the onset of quenching of the CL signal and the occurrence of secondary nucleation on the diamond {111} facets. The observation that both the CN LIF signal and the nitrogen incorporation in the central region increase with the added amount of nitrogen indicates, together with the changes in central growth rate and morphology that are observed upon nitrogen addition, that CN or a closely related species plays an important role in the influence of nitrogen addition on flame deposition of diamond.

The application of Cavity Ring Down Spectroscopy for the quantitative determination of CH densities in atmospheric flames has been investigated. Two different atmospheric flames have been studied: a premixed burner stabilised flat CH4/air flame and an oxyacetylene flame from a welding torch, which is used for diamond deposition. For the methane flame density profiles of CH are obtained and compared with densities calculated by De Goey and Van Oijen (1998). The temperature is derived from the measured Boltzmann distribution. The data from the oxyacetylene flame are compared to LIF profiles measured by Klein-Douwel et al. (1995).

Cavity ring down spectroscopy was applied as a laser diagnostic tool to measure CH radical concentrations in a diamond depositing oxyacetylene flame. CH column densities were determined during diamond growth at about 0.4 mm above the deposition substrate. By applying Abel inversion processing absolute concentration profiles are obtained which are compared to diamond growth rate profiles. A correlation is found between the CH density distribution and the local growth rate. From this it can be concluded that the CH radical possibly plays a role in the formation of an annulus of enhanced diamond growth.

A simple method is proposed for analysis of stimulant laxatives and metabolites of laxatives in urine. All stimulant laxatives commercially available in Germany, Begium and the Netherlands, the diphenylmethane derivatives and the anthraquinones, were included. Chromatography was performed with a standardized isocratic HPLC system with diode array detection ('STIP'), which is commonly used in the Netherlands for toxicological screening. The method was validated by ingestion of a normal dose of the laxatives by human volunteers. In all cases the expected laxative metabolite could be detected in urine twelve hours after intake. Also urine samples of patients, suspected of laxative abuse, were analyzed.

The pharmacokinetics, efficacy and safety of metoprolol tartrate 25 mg fatty suppositories were studied in 5 healthy volunteers and in 8 patients suffering from instable angina pectoris. Metoprolol 25 mg capsules were used as a control oral dosage form. Metoprolol showed a considerable rectal bioavailability (AUC, C max) and was absorbed quickly from the rectum (T max). In both groups rectal bioavailability was comparable. However, oral bioavailability was much lower in the volunteer group than in the patient group. Furthermore, ratios of metoprolol/a‐OH‐metoprolol concentrations in plasma and urine gave an indication for a partial avoidance of the first pass effect after rectal administration. Further research is necessary to define an exact rectal dosage of metoprolol. In all patients, a substantial drop in heart rate, systolic and diastolic blood pressure was seen after administration of the first suppository. Metoprolol suppositories appear to be an effective, safe and suitable alternative for patients who are in need for beta blocking medication and who are unable to take oral medication for a certain amount of time.

Background: To compare vancomycin pharmacokinetic parameters in patients with and without neutropenia. Methods: Patients >= 18 years admitted on general wards were included. Routinely vancomycin trough and peak plasma concentrations were measured with a fluorescence polarization immunoassay. Pharmacokinetic parameters of individual patients were determined with maximum a posterior Bayesian estimation (MW Pharm 3.60). Neutropenia was defined as neutrophils = 400 mgxh/L. Furthermore, 15 initially neutropenic patients in our study group received vancomycin for a second administration period. Ten patients received the second administration period during another neutropenic period and 5 patients during a non-neutropenic phase. All 5 patients with vancomycin during both neutropenic and non-neutropenic phase had higher CLva (91 (+/- 26) mL/min) during the neutropenic period and lower CLva (45 (+/- 10) mL/min) during the non-neutropenic phase (p = 0.009). Conclusion: This study shows that most patients with neutropenia have augmented CLva. In a small group of patients that received vancomycin during two episodes, the augmented CLva seems to be reversible in the non-neutropenic period. Our data indicate that it is important to increase the daily dose with one third in patients with neutropenia (from 15 mg/kg twice daily to 13 mg/kg three times daily). Frequent performance of therapeutic drug monitoring in patients with neutropenia may prevent both therapy failure due to low AUCs and overcomes toxicity due to high vancomycin trough concentrations during recovery from neutropenia.

A 67-year-old man accidentally ingested 75 g of sodium nitrate. He had instant gastrointestinal symptoms. On physical examination, he was respiratorily and haemodynamically stable and there were no signs of central or peripheral cyanosis. Repeated methaemoglobin levels were normal and he made an uneventful recovery. Sodium nitrate intoxication is rare. Serious effects can occur, mainly through formation of nitrite and nitric oxide, which can cause methaemoglobinaemia and vasodilation. Even if the presenting symptoms are mild, it is important to remain cautious since more serious symptoms can occur later. Monitoring of respiratory and haemodynamic status and repeated blood gas analysis in order to detect methaemoglobinaemia are recommended.

Methods for analysis of methadone and its principal metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in meconium, based on fluorescence polarization immunoassay (FPIA) and high-performance liquid chromatography (HPLC) and diode array detection were developed. Meconium and urine samples of 16 neonates from 15 methadone-using mothers were analyzed. Because of the lower detection limit and the possibility of coanalyzing EDDP, meconium analysis with HPLC for detecting methadone use is very much preferable to FPIA. Identical results were obtained with HPLC analysis for both matrices: methadone or EDDP or both could be detected in the urine and meconium samples from 15 children. The amount of EDDP in meconium was much higher than the amount of methadone (ratio, 9.6). EDDP only was detected in eight of the meconium samples. A positive correlation was found between the methadone dose of the mothers and the methadone concentration in meconium, but not with the EDDP concentration in meconium.

Dried blood spot (DBS) sampling and high-performance liquid chromatography tandem-mass spectrometry have been developed in monitoring tacrolimus levels. Our center favors the use of limited sampling strategy and abbreviated formula to estimate the area under concentration-time curve (AUC(0-12)). However, it is inconvenient for patients because they have to wait in the center for blood sampling. We investigated the application of DBS method in tacrolimus level monitoring using limited sampling strategy and abbreviated AUC estimation approach. Duplicate venous samples were obtained at each time point (C(0), C(2), and C(4)). To determine the stability of blood samples, one venous sample was sent to our laboratory immediately. The other duplicate venous samples, together with simultaneous fingerprick blood samples, were sent to the University of Maastricht in the Netherlands. Thirty six patients were recruited and 108 sets of blood samples were collected. There was a highly significant relationship between AUC(0-12), estimated from venous blood samples, and fingerprick blood samples (r(2) = 0.96, P < 0.0001). Moreover, there was an excellent correlation between whole blood venous tacrolimus levels in the two centers (r(2) = 0.97; P < 0.0001). The blood samples were stable after long-distance transport. DBS sampling can be used in centers using limited sampling and abbreviated AUC(0-12) strategy as drug monitoring.

Several commercial DNA isolation kits are available for extracting the genomic DNA from the ethylene diamine tetra-acetic acid (EDTA) whole blood samples. To obtain DNA from whole blood these DNA isolation procedures require quite some hands on time and are rather expensive. An alternative technique could be dried blood spot (DBS) sampling, with which DNA isolation is faster, cheaper and logistics are easier. We have developed a non-commercial DBS method and examined its performance in practice. DNA isolation from EDTA blood samples and made blood spots on filter paper from 106 renal transplant recipients were compared. Additionally, DNA isolation with a column method and two different DBS method was performed for 10 healthy volunteers and compared. Also DNA isolation with only capillary blood using both DBS methods from another 100 healthy volunteers has been investigated. Real-time PCR FRET assays for the CYP3A4 A-392G, CYP3A5 A6986G, ABCB1 C1236T, G2677T/A and C3435T polymorphisms were used and the melting curves of both DNA isolation methods were compared. In all cases DNA extracted with the column method corresponded completely with the results of the DNA isolated with the DBS procedure. Hence, DNA isolation from filter paper appears to be a useful alternative for the commercially available DNA isolation kits.

In a preliminary investigation an assay for tacrolimus based on fingerprick sampling and consecutive application as a blood spot on sampling paper has been developed. The dried blood spot was analysed by HPLC-tandem mass spectrometry. The validated range was 1-30 microg/l. Intra- and inter-assay variability for precision and accuracy was

The usefulness of dried blood spot (DBS) sampling for therapeutic drug monitoring of tacrolimus was investigated with renal transplant patients. There was no significant difference between the concentrations (ranging 3.33-53.9 mug/l) of 34 samples of 26 stable renal transplant outpatients, measured both after venous and DBS sampling. DBS sampling is easy to perform because concentrations with and without nurse assistance did not significantly differ. No significant difference was found between tacrolimus concentrations in 20 duplicate DBS samples before and after postal transport. DBS seems promising for routine patient monitoring.

Amoxicillin plasma concentrations, pharmacokinetic parameters, and the influence of demographic, anthropometric, and clinical covariates were investigated in 150 neonates. Gestational age (GA) ranged from 25 to 42 weeks and mean postnatal age (PNA) was 0.8 days. Amoxicillin concentrations were measured with reversed-phase HPLC in surplus plasma from routine assays of coadministered gentamicin. Mean total body clearance corrected for body weight (CL/W) was 0.096 +/- 0.036 L/kg(-1)h(-1), mean elimination half-life (t(1/2)) was 5.2 +/- 1.9 hours, and mean volume of distribution corrected for body weight (V/W) was 0.65 +/- 0.13 L/kg. Multiple regression equations were calculated for the prediction of CL/W amoxicillin. CL/W gentamicin, V/W gentamicin, and GA were significant predictors of CL/W amoxicillin. Amoxicillin peak and trough concentrations after the second dose and the time the concentration exceeds the minimum inhibitory concentration (TMIC), reached with the current dosage regimen, were evaluated. Toxic plasma concentrations were reached in several patients. Therefore, the authors have proposed a lower dosage regimen, based on GA, population pharmacokinetic parameters, bacterial susceptibility (TMIC), and possible toxicity: 15 mg/kg per 8 hours and 20 mg/kg per 8 hours for neonates with GAor = 34 and GA34 weeks, respectively. Simulation with this new dosage regimen indicated that satisfactory plasma concentrations were reached in all 150 neonates. Therefore, use of therapeutic drug monitoring and pharmacokinetic calculations for dosage adjustment is generally not necessary.

In this article, we review the clinical aspects of imaging with the programmed cell-detecting protein annexin A5 (anxA5). AnxA5 binds to phosphatidylserine, which is one of the "eat me" signals at the surface of the apoptotic cell. This biologic property forms the basis for the development of anxA5 as a diagnostic tool. Within this context, the clinical relevance, limitations, and future perspectives of this approach of visualizing cell death are discussed in this article, as are other potential applications of anxA5. Furthermore, the biologic properties and the radiopharmaceutical, pharmacologic, and biodistribution aspects of anxA5 are reviewed and discussed in this article. Radiolabeled anxA5 bears the promise of becoming a clinically applied radiopharmaceutical with potential applications in cardiology and oncology. Visualization of cell death is important in pathologies such as myocardial infarction, atherosclerosis, and cancer. Furthermore, radiolabeled anxA5 may be developed as a tool for monitoring cell death-inducing or cell death-preventing therapies. In addition, experiences with radiolabeled anxA5 open novel avenues for drug targeting with anxA5 as a biologic "cruise missile."

The influence of nitrogen addition on the properties of oxyacetylene flame-deposited diamond has been studied by means of X-ray diffraction (XRD), micro-Raman spectroscopy and cathodoluminescence spectroscopy (CL). A morphological transition reported in earlier work was found to have a major influence on the diamond properties. XRD indicated the presence of a molybdenum carbide layer in between the diamond film and the molybdenum substrate. Thermal stress proved to be the dominant source of layer stress, which in turn was found to be the main cause for the width of the diamond Raman line. In the fraction of the Raman signal that is not due to the diamond Raman peak a drop was observed that can be ascribed to a decrease in the sp2 carbon-related luminescence. The CL measurements showed that, for the present series of samples, blue band A luminescence is stronger from {111} than from {001} facets. Diamond film with the optimal combination of fast growth rate, good surface coverage, high crystalline quality, rather good 〈001〉 texture and zero net stress was grown with 10 sccm of nitrogen.

The effect of nitrogen addition on oxyacetylene flame deposition of diamond has been investigated. Two-dimensional laser-induced fluorescence measurements of CN radical distributions in the flame during deposition have been performed. These measurements show that nitrogen added to the source gases and nitrogen from the ambient air dominate the CN formation in the central and outer-flame area, respectively. Both sources of nitrogen have a significant influence in the area between the other two. Most of the observed film morphologies, which were studied by scanning electron microscopy, can be understood from the recently developed deterioration-gradient (DG) model for chemical vapor deposition of diamond, reported by J. J. Schermer et al. [J. Cryst. Growth 243, 302 (2002)]. It was found that the nitrogen range in which the α-parameter, defined by C. Wild et al. [Diamond Relat. Mater. 2, 158 (1993)], changes from slightly below or equal to 1.5 to a value of 3 or more, is extremely narrow. This implies that u...