Pronounced Interindividual But Not Intraindividual Variation in Tamoxifen and Metabolite Levels in Plasma During Adjuvant Treatment of Women With Early Breast Cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Ethical aspects of registry-based research in the Nordic countries.

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.

[Ethical review of research in the grey area. The Ethical Review Act should be widened and case management more efficient]. / Forskningsetisk prövning i gråzonen--Etikprövningslagen bör vidgas och ärendehanteringen effektiviseras.

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.

Evaluation of safety and efficacy as an adjuvant for the chitosan-based vaccine delivery vehicle ViscoGel in a single-blind randomised Phase I/IIa clinical trial.

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.

A European approach to clinical investigator training.

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.

Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells.

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.

Nitric oxide as a marker for evaluation of treatment effect of cyclosporine A in patients with bladder pain syndrome/interstitial cystitis type 3C.

OBJECTIVE: 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). MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Comparison of uptake mechanisms for anthracyclines in human leukemic cells.

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.

Inverse relationship between leukaemic cell burden and plasma concentrations of daunorubicin in patients with acute myeloid leukaemia.

AIMS: It has been shown that the cellular uptake and cytotoxicity of anthracyclines decrease with increasing cell density in vitro, an event termed 'the inocculum effect'. It is not known whether such an effect occurs in vivo. In this study the relationships between white blood cell (WBC) count, plasma and cellular concentrations of daunorubicin (DNR) in patients with acute myeloid leukaemia were investigated. METHODS: Plasma and mononuclear blood cells were isolated from peripheral blood from 40 patients with acute myeloid leukaemia at end of infusion (time 1 h), 5 and 24 h following the first DNR infusion. DNR concentrations were determined by high-pressure liquid chromatography and related to the WBC count at diagnosis. A population pharmacokinetic model was used to estimate the correlations between baseline WBC count, volume of distribution and clearance of DNR. RESULTS: A clear but weak inverse relationship between the baseline WBC count and plasma concentrations of DNR (r(2)=0.11, P<0.05) at time 1 was found. Furthermore, a clear relationship between baseline WBC count and DNR central volume of distribution using population pharmacokinetic modelling (dOFV 4.77, P<0.05) was also noted. Analysis of plasma DNR and the metabolite daunorubicinol (DOL) concentrations in patients with a high WBC count support that the low DNR/DOL concentrations are due a distribution effect. CONCLUSION: This study shows that the leukaemic cell burden influences the plasma concentrations of anthracyclines. Further studies are needed to explore if patients with high a WBC count may require higher doses of anthracyclines.

Daunorubicin metabolism in leukemic cells isolated from patients with acute myeloid leukemia.

BACKGROUND: Anthracyclines like daunorubicin (DNR) are important drugs in the treatment of acute myeloid leukaemia (AML). In vitro studies have shown that cellular metabolism of anthracyclines could play a role in drug resistance. Currently, it is not known what enzyme is responsible for anthracycline metabolism in leukemic cells. AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Efficacy of the natural antioxidant astaxanthin in the treatment of functional dyspepsia in patients with or without Helicobacter pylori infection: A prospective, randomized, double blind, and placebo-controlled study.

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, 16mg, or 40mg 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 (40mg) dose compared to the other treatment groups (16mg 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.

Efficacy and impact of botulinum toxin A on quality of life in patients with neurogenic detrusor overactivity: a randomised, placebo-controlled, double-blind study.

OBJECTIVE: 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. MATERIAL AND METHODS: 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. RESULTS: 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 (p<0.001) and 12 weeks (p=0.026) and maximum detrusor pressure and frequency of urinary leakage were significantly (p<0.01) 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. CONCLUSIONS: 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.