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Tamoxifen is still an important antihormonal treatment option for patients with breast cancer and estrogen receptor-positive tumors. More than 20% of patients relapse despite treatment. The drug is usually dosed 20 mg/d irrespective of interindividual variation in drug clearance. To study interindividual and intraindividual variation in plasma levels we measured tamoxifen and metabolite levels in plasma on 2 occasions, with at least 4 weeks in between, of 39 women (19 premenopausal and 20 postmenopausal women) on adjuvant treatment (20 mg/d) of early breast cancer.We used an ultra-performance liquid chromatography with a mass spectrometry detection method for identification and quantification of tamoxifen, N-desmethyltamoxifen, 4-OH-tamoxifen, and endoxifen. Follicle-stimulating hormone, luteinizing hormone, and estradiol levels were also measured.The plasma concentrations of tamoxifen and its metabolites showed a pronounced interindividual variation, whereas intraindividual concentrations were rather stable. Despite the same dosage, interindividual tamoxifen concentrations varied from 51 to 307 ng/mL (124 ± 57, mean ± SD) and endoxifen values showed a range from 3.2 to 19 ng/mL (10.4 ± 5.2, mean ± SD), that is, 6-fold variation for both.Large interindividual variation of tamoxifen and endoxifen with stable intraindividual levels, and too low levels of endoxifen in a considerable proportion of patients strongly support that therapeutic drug monitoring and individualized dosing could lead to optimal exposure and hopefully better outcome. A randomized outcome study between conventional dosing and therapeutic drug monitoring-guided dosing is needed to show whether this approach works.

ViscoGel, a chitosan-based hydrogel, has earlier been shown to improve humoral and cell-mediated immune responses in mice. In this study, a Phase I/IIa clinical trial was conducted to primarily evaluate safety and secondarily to study the effects of ViscoGel in combination with a model vaccine, Act-HIB to Haemophilus influenzae type b, administered as a single intramuscular injection. Healthy volunteers of both sexes, ages 22-50 and not previously vaccinated to HIB, were recruited. The trial had two phases. In Phase A, three ascending dose levels of ViscoGel (25, 50 and 75mg) were evaluated for safety in 3×10 subjects. Phase B had a single-blind, randomised, parallel-group design evaluating safety and efficacy in five groups, 20 subjects/group, comparing vaccination with 0.2μg or 2μg Act-HIB alone or combined with ViscoGel (50mg) and one group receiving the standard Act-HIB dose (10μg). No safety or tolerability concerns were identified. Local, transient reactions at the injection site were the most common adverse events. These were more frequent in groups receiving Act-HIB+ViscoGel, while other AEs were recorded at similar frequency in Act-HIB and Act-HIB+ViscoGel groups. Efficacy was evaluated by measuring serum anti-HIB antibodies and cellular responses in peripheral blood mononuclear cells (PBMC). There was a large variation in baseline anti-HIB antibody titres and no adjuvant effect was observed on the anti-HIB antibody production in groups vaccinated with Act-HIB+ViscoGel. ELISpot analyses revealed increased interferon-γ (IFN-γ) responses to Act-HIB in PBMCs from subjects vaccinated with Act-HIB in combination with ViscoGel, compared to groups receiving Act-HIB alone. Moreover, ViscoGel counteracted an inhibitory effect of Act-HIB vaccination on the IFN-γ response to both the vaccine itself and an irrelevant influenza antigen. In summary, ViscoGel was found to be safe and well-tolerated, supporting further examination of ViscoGel as a new innovative vehicle for vaccine development.

Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic inflammatory disease and to date few treatments or tools for investigating the activity of the disease are available. This study evaluated whether luminal nitric oxide (NO) could be used as a marker for evaluation of therapeutic outcome in BPS/IC type 3C treated with the immunosuppressive agent cyclosporine A (CsA).Ten patients with BPS/IC type 3C were given CsA for 16 weeks, initially at 3 mg/kg/day, and after 12 weeks the dose was scaled down. Formation of NO was measured in the urinary bladder with a silicone catheter, and symptom and bother score related to the disease were evaluated with the Interstitial Cystitis Symptom and Problem Index, every second week.All patients had elevated NO levels in the bladder initially and NO levels decreased during treatment with CsA. When the dose of CsA was lowered NO formation increased and after 2 weeks without medication, the NO formation was the same as before the study began.The results indicate that measurement of NO is a tool for evaluating the response to anti-inflammatory treatment in patients with BPS/IC type 3C. NO could serve as a marker for assessing the activity of the inflammation.

center dot In vitro studies show that daunorubicin (DNR) cytotoxicity decreases with increasing cell density because of a high cellular uptake and depletion of drug in the medium. center dot It i ...

Anthracyclines like daunorubicin (DNR) are important drugs in the treatment of acute myeloid leukaemia (AML). In vitro studies have shown that cellular metabolism of antrhacyclines could play a role in drug resistance. Currently, it is not known what enzyme is responsible for anthracycline metabolism in leukemic cells. To study C-13 reduction of DNR to daunorubicinol (DOL) in leukemic cells isolated from patients with AML and to determine the most important enzyme involved. Mononuclear blood cells from 25 AML patients were isolated at diagnosis and used in a metabolic assay to determine the % DOL formed. MRNA and western blot analysis were performed on the 2 most likely candidates for anthracycline metabolism; carbonyl reductase 1 (CR1) and aldoketoreductase 1A1 (AKR1A1). DNR and DOL concentrations were determined by HPLC. We found a large interindividual variation (up to 47-fold) in leukemic cell DNR metabolism. The specific CR1 inhibitor zeraleone analogue 5 significantly inhibited DNR metabolism with a mean inhibitory effect of 68 %. No correlation between mRNA levels of the enzymes and metabolism were found. Cellular DNR metabolism correlated significantly with CR1 protein expression, determined by western blot, (p < 0.05, R2 = 0,229) while no significant correlation was found with AKR1A1 protein expression. DNR metabolism in AML cells shows a pronounced interindividual variability. Our results support that CR1 is the most important enzyme for conversion of DNR to DOL in AML cells. This information could in the future be used to genotype CR1 and possibly help to individualise dosing.

Objectives: The aim of this study was to evaluate the efficacy of the natural antioxidant astaxanthin in functional dyspepsia in different doses and compared with placebo. Design: The study was a controlled, prospective, randomized, and double blind trial. Participants: Patients with functional dyspepsia, divided into three groups with 44 individuals in each group (placebo, 16 mg, or 40 mg astaxanthin, respectively). Interventions: Participants were asked to accept gastroscopy before treatment, together with questionnaires: GSRS and SF-36. Urea breath test (UBT) was done before the treatment. Main outcome: The primary objective was to test the hypothesis that the antioxidant astaxanthin at two doses regimens compared to placebo should ameliorate gastrointestinal discomfort measured as GSRS in patients with functional dyspepsia, who were either positive or negative for Helicobacter pylori, after 4 weeks of treatment. Results: At the end of therapy (week 4) no difference between the three treatment groups was observed regarding mean Gastrointestinal Symptom Rating Scale (GSRS) scores of abdominal pain, indigestion and reflux syndromes. The same results were observed at the end of follow-up. However reduction of reflux syndrome before treatment to week 4 was significantly pronounced in the higher (40 mg) dose compared to the other treatment groups (16 mg and placebo, p = 0.04). Conclusion: In general, no curative effect of astaxanthin was found in functional dyspepsia patients. Significantly greater reduction of reflux symptoms were detected in patients treated with the highest dose of the natural antioxidant astaxanthin. The response was more pronounced in H. pylori-infected patients. (c) 2008 Elsevier GmbH. All rights reserved. (Less)

To evaluate the effect of a single injection of 500 U of botulinum toxin A (BTX-A; Dysport) on use of oral rescue medication, bladder compliance, continence and quality of life in a randomized, placebo-controlled, double-blind study in patients with incontinence due to neurogenic detrusor overactivity. As this group of patients often have severe symptoms, oral tolterodine was allowed as rescue medication and the amount of tolterodine consumed was our primary endpoint.A total of 31 patients with urinary leakage due to spinal cord injury, myelomeningocele, trauma at birth, multiple sclerosis and myelitis of another cause were randomized to intravesical injections of either 500 U of BTX-A or placebo. Intake of tolterodine and episodes of urinary leakage were registered. Cystometry was performed after 6, 12 and 26 weeks and quality of life was assessed.Patients in the BTX-A group had a significantly lower intake of tolterodine throughout the study compared to those in the placebo group (p=0.003). Cystometric capacity was significantly higher at 6 (p0.001) and 12 weeks (p=0.026) and maximum detrusor pressure and frequency of urinary leakage were significantly (p0.01) lower during follow-up in the BTX-A group compared to the placebo group. In addition, many quality-of-life parameters were significantly improved in the BTX-A group compared to the placebo group.Intravesical injection of 500 U of BTX-A in patients with neurogenic detrusor instability was shown to be an effective treatment which reduced use of oral medication, high detrusor pressure and frequency of urinary leakage during the overall study period of 26 weeks. Quality of life was also significantly improved.

The present legal definition of the term research creates problems with what can be considered for ethical vetting by the Research Ethical Review Board. The Ethical Review Act should be revised in order for student projects involving patients or quality assurance in healthcare to be accepted for ethical vetting by the Board.

National health care registries in the Nordic countries share many attributes, but different legal and ethical frameworks represent a challenge to promoting effective joint research. Internationally, there is a lack of knowledge about how ethical matters are considered in Nordic registry-based research, and a lack of knowledge about how Nordic ethics committees operate and what is needed to obtain an approval. In this paper, we review ethical aspects of registry-based research, the legal framework, the role of ethics review boards in the Nordic countries, and the structure of the ethics application. We discuss the role of informed consent in registry-based research and how to safeguard the integrity of study participants, including vulnerable subjects and children. Our review also provides information on the different government agencies that contribute registry-based data, and a list of the major health registries in Denmark, Finland, Iceland, Norway, and Sweden. Both ethical values and conditions for registry-based research are similar in the Nordic countries. While Denmark, Finland, Iceland, Norway, and Sweden have chosen different legal frameworks, these differences can be resolved through mutual recognition of ethical applications and by harmonizing the different systems, likely leading to increased collaboration and enlarged studies.

A better education and training of clinical investigators and their teams is one of the factors that could foster the development of clinical research in Europe, a key objective of the Innovative Medicines Initiative (IMI). PharmaTrain (an IMI programme on training in medicines development), and European Clinical Research Infrastructures Network (ECRIN) have joined forces to address this issue. An advisory group composed of representatives of universities, pharmaceutical companies and other organisations met four times between June 2011 and July 2012. This resulted in a position paper proposing a strategy to improve and harmonize clinical investigator training in Europe, and including a detailed syllabus and list of learning outcomes. Major recommendations are the establishment of minimal and mutually recognized certification requirement for investigators throughout the EU and the creation of a European platform to provide a suitable course and examination infrastructure.

The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1μM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug.

Aims: The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge about uptake mechanisms could be used to increase the selectivity of the drugs. We compared the uptake patterns of, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by cultured leukemic cells and investigated possible involvement of specific carriers. Methods: HL-60 cells were incubated with anthracyclines for 1 hour in the absence or presence of transport inhibitors, suramin, or nucleosides and cellular drug uptake was determined. Cell survival was also determined. MCF-7 breast cancer cells were used as a negative control for concentrative nucleoside transporters (CNTs). Anthracycline concentration was determined with HPLC and fluorometric detection and apoptosis was determined with propidium iodide and flow cytometry. Results: DNR, IDA, and PIRA had higher uptake than DOX and EPI with a prominent increase in uptake at concentrations > 1 µM. Uptake of all anthracyclines was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and increased cell survival. Dipyridamole, an equilibrative NT (ENT) inhibitor, significantly inhibited the uptake of DNR only. The addition of nucleosides significantly inhibited the uptake of DNR, IDA, and PIRA but not in MCF-7 cells lacking functional CNTs. Conclusion: Our results suggest different uptake mechanisms for the anthracyclines studied. We found evidence for carrier mediated uptake mechanisms, supporting involvement of NTs in transmembrane transport of DNR, IDA, and PIRA. The results also showed a strong inhibition of suramin on anthracycline uptake by so far unknown mechanisms.

Different analytical procedures for quantitation of total morphine and codeine in urine were evaluated. Hydrolysis of urine with hydrochloric acid and four different enzymes was compared. In order to effect complete hydrolysis of glucuronide conjugates, hydrolysis with 6.5M HCl containing bisulphite and heating at 100 degrees C for 20 min was required. Results using this hydrolysis procedure agreed well with results from direct analysis of free and conjugated forms by liquid chromatography. Use of more dilute HCl but at a higher temperature was not sufficient. Some enzymatic procedures appeared to give effective hydrolysis with a higher variability, but these procedures were more time-consuming. There was a good quantitative agreement between FID gas chromatography and ion-trap gas chromatography/mass spectrometry. The validated analytical procedure was applied to authentic patient samples.

Background: Ketobemidone is often used as an alternative to morphine in children in the Scandinavian countries. The aim of this clinical trial was to explore the pharmacokinetics of ketobemidone in children because these properties have not been reported previously. Methods: Thirty children, newborn to 10 years, scheduled for elective surgery were included in the trial. Ketobemidone hydrochloride was administered as a single intravenous bolus dose and ketobemidone and norketobemidone concentrations were measured by LC-MS over 8 h. Pharmacokinetic parameters were determined using compartmental methods. Results: Six children were excluded from pharmacokinetic analysis because of incomplete blood sampling. The values of ketobemidone clearance (l/h/kg) given as median (range) were 0.84 (0.29–3.0) in Group A (0–90 days), 0.89 (0.55–1.35) in Group B (1–2.5 years) and 0.74 (0.50–0.99) in Group C (7–10 years). The corresponding values for apparent volume of distribution (l/kg) were 4.4 (3.7–6.9) (Group A), 2.6 (2.0–5.6) (Group B) and 3.9 (2.7–5.0 (Group C), and for elimination half-life (h) 3.0 (1.4–8.9) (Group A), 2.0 (1.2–4.7) (Group B) and 3.7 (2.4–6.9) (Group C), respectively. In the two neonates the elimination half-life was almost 9 h. The metabolite norketobemidone did not reach levels above the limit of quantification (0.07 ng/ml) in any of the patients. Conclusion: The pharmacokinetic parameters of ketobemidone in children older than 1 month appear to be similar to those in adults. Because of the large interindividual variability of the pharmacokinetics in neonates, further studies especially in this age group are warranted.

We report here a simple method involving urine creatine measurements for testing authenticity and reducing false-negative results in urine testing for drugs of abuse. Urinary creatinine in consecutive patient samples (n = 176) ranged between 0.1 and 31.9 mmol/L (mean 9.8 +/- SD 6.2) and the osmolality in these urines ranged between 49 and 1183 mOsm/kg (mean 595 +/- SD 276). With other consecutive samples in which creatinine was (arbitrarily chosen) less than 4.3 mmol/L (n = 85), the correlation with osmolality was lower. In 10 randomly selected urine samples from different patients, all "clean" for all drugs of abuse in initial immunological drug testing with approved methodology (in which creatinine was less than 4.3 mmol/L and osmolality was less than 200 mOsm/kg), five patients turned out to be drug positive after a simple concentration by volume. In a formerly heavy smoker of cannabis, the excretion of cannabinoids and creatinine was monitored for 93 days. The substances showed very good correlation throughout this period (r = 0.93, P less than 0.001), whereas simple measurements of cannabinoid concentrations would have falsely indicated several relapses of cannabis abuse. Urine samples used in drug-abuse testing should be tested for creatinine; if creatinine is less than 4.0 mmol/L, negative results for drugs may not be valid.

Determination of plasma methadone is essential in connection with dose adjustments for patients participating in methadone maintenance programs. We successfully adapted the existing fluorescence polarization immunoassay (FPIA) kit intended for urinary methadone to plasma assays. A concentration interval of 50-900 ng/ml could be covered. The coefficient of variation was less than 7%, and the limit of detection below 50 ng/ml. The intercorrelation between the immunoassay and a specific gas chromatographic-mass spectrometric (GC-MS) method was studied in samples from 19 heroin addicts in methadone maintenance treatment. A total number of 97 plasma samples with a concentration range of 31-842 ng/ml were used. The slope and intercept of the regression line (CFPIA = 0.93 X CGC-MS + 15) was in good agreement with the theoretical relation (CFPIA = CGC-MS), with a coefficient of correlation of 0.978. The mean ratio, in quantitative result, between the techniques (CFPIA/CGC-MS) was 1.03 +/- 0.01 (SEM). We conclude that the immunoassay proposed in this study can be safely used in patients participating in methadone maintenance programs.

Background: Tramadol is used as an analgesic in post-operative pain treatment. Intravenous tramadol is often combined with morphine to achieve better pain relief and less side-effects after orthopaedic surgery. However, the available evidence is insufficient to support this combination. For this reason, we conducted the present non-commercial, randomized, double-blind clinical trial. Method: Sixty-three patients with osteoarthritis of the knee, selected for primary total knee arthroplasty (TKA), were randomized to receive saline or tramadol 100 mg/ml intravenously every 6 h during the first post-operative day (total, 400 mg/24 h). All patients had access to morphine via a patient-controlled analgesia (PCA) pump. Results: Neither during the 6 h after the first dose nor during the first post-operative day could we detect any statistically significant difference with regard to pain intensity, sedation and nausea between patients treated with tramadol and the placebo group. However, the withdrawal rate caused by insufficient pain relief was greater in the tramadol group (7/31) than in the saline group (2/32). This difference did not reach statistical significance. In the group of patients who remained in the study for 24 h (‘per protocol’), those randomized to receive tramadol had a significantly (P < 0.05) lower morphine consumption (20 mg or 31%) than the placebo group. Conclusion: Our study does not support the combination of tramadol and morphine via PCA for post-operative pain relief after primary TKA. In addition, our study indicates that morphine via PCA as the sole means of post-operative analgesia does not provide sufficient pain relief after TKA. Thus, other means of post-operative analgesia should be used following TKA.

Successful biomedical scientists learn throughout their careers. The LifeTrain project aims to optimize investments in learning to support the development of medicines in Europe and to encourage stronger interactions between industry and academia.

Clopidogrel is a new antiplatelet agent that offers increased protection over aspirin in preventing vascular ischaemic events in patients with symptomatic atherosclerosis. In a large, randomized, international study of clopidogrel and aspirin (n = 19,185 patients) clopidogrel was associated with a lower incidence of gastrointestinal adverse events, including gastrointestinal haemorrhage and hospitalizations because of gastrointestinal haemorrhage. The aim of the study was to determine whether macroscopic differences in the gastric mucosa between aspirin- and clopidogrel-treated subjects could be detected by gastroscopy after short-term treatment.Thirty-six healthy volunteers were randomized in a double-blind, double-dummy, parallel design, to 75 mg/day of clopidogrel or 325 mg/day of aspirin for 8 days. Gastroscopy was performed at base line before administration of study drug and directly after treatment completion. Gastroduodenal effects were measured in accordance with a modified Lanza scale.At base line no difference between the groups was detected (median Lanza score, 0.0 in both groups). At the end of treatment the aspirin group showed a median score of 7.5, and the clopidogrel group showed an unchanged median score of 0.0 (P0.001). In the aspirin group 13 individuals reported 19 adverse events versus 8 individuals and 13 adverse events for clopidogrel, with approximately half of the adverse events being gastrointestinal in each group. No serious adverse events were reported.In contrast to aspirin, short-term treatment with clopidogrel does not induce macroscopic changes in the gastroduodenal mucosa. The study results show that in patients without gastroduodenal disease clopidogrel, but not aspirin, does not induce any gastroscopically evident erosions during short-term treatment.

Thirteen healthy volunteers participating in an open and randomized study received two single doses (25 and 50 mg) of codeine orally two weeks apart. Urine concentrations of opiates were studied for 96 h, and plasma concentrations of codeine and the metabolites codeine-6-glucuronide (C6G), morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) were monitored for 24 h. Plasma was analyzed by high-performance liquid chromatography. Measurements of urine were made with the EMIT opiate-screening assay and with gas chromatography-mass spectrometry for total (conjugates liberated by acid hydrolysis) codeine, morphine, and norcodeine. In urine, the ratio between total recovered morphine and codeine as expressed in percent ranged from 2.3 to 23.3% with a mean value of 9.8%. This ratio increased with time, and, in all but three subjects, rose to greater than 1 after 22-36 h. In 58% of cases, this occurred within the detection time in the EMIT assay. The detection time in the EMIT screening assay was found to be 20-39 h after the 25-mg dose and 30-52 h after the 50-mg dose. Elimination rates calculated from urine data corrected for creatinine concentration showed that morphine was eliminated more slowly than codeine. In plasma, the highest concentrations and area-under-curve values were observed for C6G, followed by codeine and M3G. All compounds had peak plasma values 1-2 h after dosing. The elimination of M3G was slower than that of C6G. We concluded that the relative proportion of codeine and morphine varies both between individuals and as a function of time and that morphine may be present in concentrations above those of codeine even after moderate and single doses of codeine. This must be taken into consideration when interpreting the presence of opiates during drugs-of-abuse testing.

Two rating scales, which were originally developed for measurements of objective and subjective signs of opiate withdrawal, were used to evaluate potential estimates (correlates) of methadone effects in relation to plasma methadone concentrations. Patients participating in our regular methdone maintenance treatment project were studied during 24 h after the intake of the daily methadone dose. Methadone concentrations in plasma were compared to the subjective (estimated by the patients) and objective (estimated by the investigator) signs of the drug effects before, and 2.5, 5, 9 and 24 h after intake of methadone. Some new items possibly related to rising methadone concentrations were added to the subjective scale. Results indicated that, for subjective ratings, the majority of the items investigated corresponded well with the plasma methadone concentrations. The most significant associations were found for the following items: low psychomotor speed, alertness, running nose, yawning and anxiety. For objective ratings, only the items rhinorrhea, piloerection and signs of anxiety were significantly associated with the methadone concentrations. These rating scales may, together with plasma methadone determinations, be of considerable value when making dose adjustments for methadone maintenance patients. Further work is, however, needed to establish concentration — effect relationships.

We evaluated the EMIT (enzyme-multiplied immuno technique) and FPIA (fluorescence polarization immunoassay) urine screening systems for detection of benzodiazepine intake. Healthy male volunteers were given single oral therapeutic doses of alprazolam (2 mg), chlordiazepoxide (25 mg), flunitrazepam (1 mg), lorazepam (3.75 mg), nitrazepam (5 mg), and triazolam (0.25 mg), after which urine was collected for the next 32 h. The EMIT method failed to detect the intake of flunitrazepam, lorazepam, and nitrazepam. FPIA did not detect the intake of chlordiazepoxide, flunitrazepam, lorazepam, nitrazepam, and triazolam. Modification of the EMIT method to include enzymatic hydrolysis did not significantly alter the results obtained with this method. A modification of the FPIA method to include enzymatic hydrolysis and a lower cutoff value improved the results considerably, so that we reliably detected all studied substances but flunitrazepam. We conclude that (a) both EMIT and FPIA techniques, when used as intended by the manufacturers, are unreliable for the detection of intake of therapeutic doses of these benzodiazepines, and (b) the described modification of the FPIA should provide a much improved tool for detection of benzodiazepine intake.

A method for the chiral high-performance liquid chromatographic analysis of methadone in plasma has been developed. The method employed organic solvent extraction, enantiomeric separation on a Chiral AGP column, and ultraviolet absorption detection at 212 nm. The intra-day variation in the quantification of methadone enantiomers was less than 9% at the 100 ng/ml level, and the values obtained correlated well with those from a gas chromatographic-mass spectrometric method. Results from patients indicate inter- and intra-individual differences in the ratio between l- and d-methadone in plasma during therapy with racemic methadone. In one patient, a higher level of d-methadone in plasma was caused by both faster elimination and lower bioavailability of l-methadone.

Intraindividual variation in 6-mercaptopurine (6-MP) kinetics has been little studied. It has now been examined in 18 children with acute lymphoblastic leukaemia (ALL). On 2 to 4 occasions in each patient drug concentrations in plasma and red cells were followed for 4 h after administration by means of HPLC. The mean individual coefficient of variation (C.V.) in AUC was 57.9% and it was not related to dose or concentration. The variation was the same in plasma and in red cells. It is concluded that regular monitoring of 6-mercaptopurine concentration would identify periods when a patient deviates strongly from the mean range. Both undertreatment and concentration-dependent toxicity could then be corrected.

Two specific high-performance liquid chromatography methods for determining plasma concentrations of azathioprine and 6-mercaptopurine after oral administration of azathioprine are presented. It was shown that azathioprine is unstable in the blood samples unless immediately cooled in ice water. The 2-amino analog, guaneran, was used as internal standard for azathioprine, which was extracted from plasma with ethylacetate. A Nucleosil C18 column was used for the separation. The detection limit was 6 nM. For quantification of 6-mercaptopurine, 6-thioguanine was used as internal standard. Plasma was deproteinized with HClO4 and the sample was purified on mercurial cellulose. A Beckman ODS column was used and the detection limit was 5 nM. Pharmacokinetic data from two patients are presented. Unchanged azathioprine was seen until 6 h after an oral dose of 32 mg/m2.

Immunological screening analysis of benzodiazepines in urine using the EMIT (enzyme multipled immuno-technique) and FPIA (fluorescence polarization immunoassay) techniques does not reliably detect the intake of therapeutic doses of oxazepam. In 23 patient urine samples, in which the presence of oxazepam could be verified chromatographically, only about 50% were detected as positive in the immunoassay systems. However, when the screening procedure was modified to include a simple step of hydrolysis of urine using the enzyme β-glucuronidase to liberate conjugated oxazepam, improved detection of oxazepam intake was achieved. With EMIT 95% and with FPIA 100% of the samples were detected as positive. Since oxazepam arises in vivo also as a metabolite of other common benzodiazepines, the modification will most likely contribute to the generally improved detection of benzodiazepines.

Intrathecal cytarabine (cytosine arabinoside) is included in many protocols for the treatment of acute lymphoblastic leukaemia of childhood. We report here the accidental administration of 200mg cytarabine intrathecally to a 4-year-old boy with CNS relapse. After the overdose the patient had dilated pupils during the first hour. One month later an unsteady gait and mild intention tremor in the hands were noted. By the exchange of cerebrospinal fluid with isotonic saline started 1 hour after overdose through a lumbar needle, about 27% of the administered dose was recovered. The estimated recovery in view of the time elapsed between overdose and start/end of the exchange procedure was 36%. This indicates that this procedure is of value in managing patients with heavy overdose of intrathecal cytarabine in hospitals without neurosurgical facilities.

The concentrations of 6-mercaptopurine were studied in plasma and red blood cells from 10 children with acute leukaemia or non-Hodgkin lymphoma receiving oral maintenance therapy. The doses varied between 25 and 79 mg/m2 body surface once daily. Large interindividual differences were found in the concentrations both in plasma and in red blood cells. There was no clear relationship between the dose administered and the concentrations obtained. However, in each patient the concentrations in plasma and in red blood cells were very similar. In most patients, the peak concentrations were reached 1-2 hours after dose intake and the concentrations then declined with half-lives less than 1 hour. Further studies are necessary to evaluate the potential value of drug level monitoring in optimizing treatment with oral 6-mercaptopurine.

Plasma levels of 6-mercaptopurine were determined in 22 consecutive children with acute lymphoblastic leukemia on oral remission maintenance therapy during the time period of August 1984 to January 1988. Each child received the drug once daily for up to 3 years and was studied repeatedly (1-12 times). An HPLC method was used for drug analysis. We found large interpatient variations in the mean peak plasma concentration (range of 50-424 ng/ml) and in the mean area under the concentration vs. time from 0-4 h curve (range of 82-637 ng ml-1 h). There were also pronounced variations between different sampling occasions in the same patient. Nine of the 22 patients had complications during the maintenance therapy. Five children with a mean peak plasma level below 135 ng/ml and a mean area under the curve (AUC) value below 251 ng ml-1 h relapsed (three in the central nervous system and two in the bone marrow). Both children with a bone marrow relapse died. Relapse risk was related to the AUC (p less than 0.05). Four children with a mean peak plasma level above 166 ng/ml and a mean AUC value above 363 ng/ml/h developed severe myelotoxicity, which necessitated a temporary cessation of the maintenance therapy. In addition, two patient relapsed 6 and 11 months after termination of maintenance therapy. Their mean peak and AUC values were not low but the concentrations decreased markedly towards the end of the maintenance period. The results indicate that the plasma levels of 6-mercaptopurine, when determined repeatedly, might be of significance for the outcome of the remission maintenance treatment.

In three children receiving oral remission maintenance therapy for acute lymphoblastic leukemia, the concentrations of 6-mercaptopurine (6-MP) in cerebrospinal fluid (CSF), plasma and red blood cells were compared. CSF samples were obtained from an Ommaya reservoir previously inserted for treatment of CNS relapse. At the time of the study, the children were all in remission and had been on oral 6-MP (42–63 mg m−2) once daily for at least 24 weeks. Immediately before dose intake on the day of study (about 24 h after last dose), the concentrations of 6-MP in CSF, plasma and red blood cells were rather similar and below 20 ng ml−1 in all patients. After dose intake, the concentrations in plasma and in red blood cells increased to 40–200 ng nl−1 within 0.5–4 h. In contrast, the concentration of 6-MP in the CSF remained fairly constant around 4–10 ng ml−1 throughout the time period studied (up to 4 h). It is concluded that 6-MP can be detected in CSF during oral maintenance therapy and that the drug has different pharmacokinetic profile in CSF to that in plasma and red blood cells. Further studies are necessary to evaluate the significance of the 6-MP concentrations obtained in CSF for the prevention of CNS relapse.

The effects of some environmental and genetic factors on the inter- and intraindividual variations of 6-mercaptopurine (6-MP) pharmacokinetics were studied in children on oral remission maintenance therapy for acute lymphoblastic leukemia or non-Hodgkin's lymphoma. Blood samples were obtained 0-4 h after drug intake. 6-MP concentrations were determined in plasma and in erythrocyte concentrates. The influence of food on the pharmacokinetics was examined in a prospective study of 15 children. Each child was examined four times, twice in the fasted state and twice after intake of a standardized, milky, breakfast. There were pronounced inter- and intraindividual variations. Food intake seemed to reduce these variations but there were no significant changes in peak concentrations and area under the plasma concentration vs time curves (AUC) between the fasted and fed states. Food intake reduced the time to peak concentration both in plasma, from 1.8 h to 1.1 h (P less than 0.01) and in red blood cells, from 1.8 h to 1.3 h (P less than 0.01). Retrospective subdivision of the patients indicated a tendency for different pharmacokinetic patterns according to dose; five out of seven patients receiving greater than 70 mg m-2 had a higher AUC in the fasting state, while five out of eight patients receiving less than 70 mg m-2 had a higher AUC in the fed state. The cytochrome P-450-dependent hydroxylation capacity was evaluated with debrisoquine but no correlation was found to the pharmacokinetics of 6-MP.