V.-P. Harjola¹, L. Oikarinen², L. Toivonen², R. Jurkko², J. Puttonen³, T. Sarapohja⁴, S. Sundberg⁵ and M.S. Nieminen^{2 1}Division of Emergency Care, ²Division of Cardiology, Department of Medicine, Helsinki University Central Hospital, Helsinki, ³Orion Pharma, Clinical R&D, Kuopio and ⁴,⁵Department of Biostatistics and Data Management, Orion Pharma, Clinical R&D, Espoo, Finland Objective: Levosimendan is a calcium-sensitizing drug for the treatment of heart failure. The aim of this exploratory study was to assess the hemodynamic and pharmacokinetic interactions between digoxin and oral levosimendan as well as the proarrhythmic potential of this combination in patients with chronic heart failure. Materials: Male or female patients (n = 24) with chronic heart failure of NYHA Classes II Correspondence to:
V.-P. Harjola, MD, PhD
Division of Emergency Care, Department of Medicine, Helsinki University Central Hospital, P.O. Box 340, 00290 Helsinki, Finland
Email: veli-pekka.harjola@hus.fi

A. Sutton¹, P. Gouws² and A. Ropo^{3 1}Guildford Clinical Pharmacology Ltd., The Technology Center, Guildford, Surrey, ²Conquest Hospital, The Ridge, St. Leonards-on-Sea, East Sussex, UK and ³Santen Oy Clinical Research, Tampere, Finland Objective: Prostaglandin receptor analogs lower intraocular pressure (IOP) and are used for the treatment of glaucoma. This study aimed to compare the safety, tolerability and pharmacodynamics of four doses of the new, selective-prostanoid receptor agonist, tafluprost (AFP-168) in a Phase I placebo-controlled study. Methods: Healthy volunteers (n = 16) received sequentially ascending doses of tafluprost (0.0001%, 0.0005%, 0.0025% and 0.005%) in one eye, and placebo in the other. Each treatment period consisted of 2 days of treatment, with 5 days between the treatment periods. Safety and tolerability assessments, as well as IOP measurements, were performed at defined intervals. Results: Tafluprost was generally well tolerated and no volunteer discontinued due to adverse events (AEs). The most common ocular AE was ocular hyperemia, which was mild-to-moderate, and highly concentration-dependent. All doses of tafluprost decreased IOP, with the maximum effect occurring 12 hours after treatment. The decrease in IOP relative to placebo was significantly more effective with tafluprost 0.0025% and 0.005%, compared with tafluprost 0.0001% (p £ 0.005). Conclusion: Tafluprost was well tolerated and effective in lowering IOP. These data support further testing of tafluprost 0.0025% and 0.005%.Correspondence to:
A. Sutton, MD
Cedars Vanzell Road, Midhurst, West Sussex, UK GU29 9BA, UK
Email: asutton@gcpl.co.uk

J.M. Leitner¹, F.B. Mayr¹, A.O. Spiel¹, C. Firbas¹, C. Savulsky², R. Mis², M.E. Corrado³ and B. Jilma^{1 1}Department of Clinical Pharmacology, Division of Immunohematology, Medical University of Vienna, Austria, ²GP-Pharm S.A., Barcelona, Spain and ³Cross S.A., Arzo, Switzerland Objective: The aim of this study was to compare the efficacy of Lutrate^® 3.75 and 7.5 mg depot to marketed references Lucrin^® 3.75 mg and Procrin^® 7.5 mg depot. Methods: 20 healthy male volunteers were randomly assigned to receive 1 of 4 active single dose treatments in this double-blind, parallel-group pilot study. Leuprolide acetate and testosterone levels were quantified by radioimmunoassays. Results: The pharmacokinetic profile of leuprolide could be well-described by a 4-step release curve. Leuprolide levels were detectable 14 days longer after injection of the test formulations as compared to the reference products. The total AUC observed with 3.75 and 7.5 mg of the test product were approximately 1.5- and 2.2-fold higher, compared to the reference products, respectively. After the expected testosterone “flare-up” effect, castration was achieved in 4 of 4 subjects with the test formulations, 4 of 5 subjects with Procrin and 2 of 5 subjects with Lucrin. On average, castration lasted more than 1 month with both test formulations compared to 2 weeks with the reference products. Conclusion: Sustained release of leuprolide from this new depot formulation suppressed testosterone levels at least as effectively and for a longer period of time than the reference products.Correspondence to:
B. Jilma, MD
Department of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel
18 – 20, 1090 Vienna, Austria
Email: bernd.jilma@meduniwien.ac.at

K. Saito¹, Y. Kodama², S. Ono³, C. Maida¹, A. Fujimura⁴ and E. Miyamoto^{1 1}Department of Pharmacy, Faculty of Pharmaceutical Sciences, Hokuriku University, Ishikawa, ²Laboratory of Bio-Pharmaceutics, Faculty of Pharmaceutical Sciences, Josai International University, Chiba, ³Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo and ⁴Department of Pharmacology, Jichi Medical School, Tochigi, Japan To describe the reliability of Japanese clinical trials, we compared the results of a Good Clinical Practice (GCP) audit conducted between April 1997 and March 2000 (fiscal year (FY) 1997 – 1999) with those from April 2004 – March 2005 (FY2004). The number and proportion of various types of deficiencies described in GCP audit reports were compared between the 2 periods. The audit findings in the former period were based on official audits that covered 331 hospitals and 775 trials. The audits in the latter period targeted 114 hospitals and 189 trials. The inspection of former period was undertaken by the Organization for Pharmaceuticals Safety and Research (OPSR). On the other hand, the latter period was undertaken by the Pharmaceuticals and Medical Devices Agency (PMDA). The total number of deficiencies detected in GCP audits was 1,529 in the former 3-year period (FY1997 – 1999) and 819 in the latter period (FY2004). The total number of deficiencies detected and reported was more than 1.5-fold on an annual basis in the latter period. By category of deficiencies, the proportion of protocol deviations increased from 14.7 (225/1,529) to 45.7% (374/819), while the proportion of errors in case report forms (CRFs) decreased from 43.6 (666/ 1,529) to 27.1% (222/819). There were two remarkable changes in audit findings between FY1997 – 1999 and FY2004; the increase in the proportion of protocol deviations and the decrease in the proportion of CRF-related deficiencies. We think that in Japan the improvement of research environments is needed to provide reliable clinical data responsible for the regulatory standard of GCP.Correspondence to:
K. Saito, PhD
Department of Pharmacy, Faculty of Pharmaceutical Sciences, Hokuriku University, ho-3 Kanagawa-machi, Kanazawa, Ishikawa, 920-1181, Japan
Email: k-saito@hokuriku-u.ac.jp

T.J. Cleophas and A.H. Zwinderman Albert Schweitzer Hospital, Dordrecht, The Netherlands and European Inter-University College of Pharmaceutical Medicine, Lyon, France Background: In clinical trials a fixed effects research model assumes that the patients selected for a specific treatment have the same true quantitative effect and that the differences observed are residual error. If, however, we have reasons to believe that certain patients respond differently from others, then the spread in the data is caused not only by the residual error but also by between-patient differences. The latter situation requires a random effects model. Objective: To explain random effects models in analysis of variance and to give examples of studies qualifying for them. Results: If in a particular study the data are believed to be different from one assessing doctor to the other, and if we have no prior theory that 1 or 2 assessing doctors produced the highest scores, but rather expect there may be heterogeneity in the population of doctors at large, then a random effects model will be appropriate. For that purpose between-doctor variability is compared to within-doctor variability. If the data of 2 separate studies of the same new treatment are analyzed simultaneously, it will be safe to consider an interaction effect between the study number and treatment efficacy. If the interaction is significant, a random effects model with the study number as random variable, will be adequate. For that purpose the treatment effect is tested against the interaction effect. In a multicenter study the data are at risk of interaction between centers and treatment efficacy. If this interaction is significant, a random effects model with the health center as random variable, will be adequate. The treatment effect is tested not against residual but against the interaction. If in a crossover study a treatment difference is not observed, this may be due to random subgroup effects. A post-hoc random effects model, with patients effect as random variable, testing the treatment effect against treatments × patients interaction, will be appropriate. Discussion: Random effects research models enable the assessment of an entire sample of data for subgroup differences without need to split the data into subgroups. Clinical investigators, in general, are hardly aware of this possibility and, therefore, wrongly assess random effects as fixed effects leading to a biased interpretation of the data.Correspondence to:
T.J. Cleophas, MD
Department of Medicine, Albert Schweitzer Hospital, Box 444, 3300 AK Dordrecht, The Netherlands
Email: a.j.m.cleophas@asz.nl

D. Díaz-Carballo¹, S. Malak², M. Freistühler³, A. Elmaagacli⁴, W. Bardenheuer² and H.P. Reusch^{1 1}Abteilung für Klinische Pharmakologie, Ruhr-Universität Bochum, ²Innere Klinik und Poliklinik (Tumorforschung), Zentrum für konservative Onkologie, ³Klinik für Augenheilkunde and ⁴Klinik für Knochenmarktransplantation, Universitätsklinikum Essen, Germany Objective: This work is aimed at characterizing nemorosone, isolated from Clusia rosea, as a potential antileukemic agent. In addition, we analyzed its influence on hematopoiesis in a mouse model. Materials and methods: The isolation of nemorosone was carried out employing the RP-HPLC (reversed phase high-performance liquid chromatography) technique. Cytotoxicity was assessed in human leukemia cell lines including parental and chemotherapy-refractory sublines based on the MTT compound. Its effects on the cell cycle were analyzed using FACS (fluorescence-activated cell sorting) and Western blot techniques. Studies on the drug-induced early apoptotic process were carried out by means of fluorescence microscopy. Major signal transducers and the enzymatic inhibition of immunoprecipitated Akt/PKB were detected by Western blot. Hematopoiesis was analyzed in NMRI nu/nu mice after chronic nemorosone treatment, measuring hematological parameters by conventional laboratory techniques. Results: Nemorosone proved cytotoxic in both parental and chemoresistant leukemia cell lines with IC₅₀ values between 2.10 and 3.10 mg/ml. No cross-resistances could be detected. Cell cycle studies showed apoptosis induction accompanied by an increase in the G0/G1 population in both cell lines studied, whereas a significant decrease in the S-phase was found in Jurkat cells. Nemorosone induced a down-regulation of cyclins A, B1, D1, and E as well as a dephosphorylation of cdc2. Major signal transduction elements such as ERK1/2 and p38 MAPK, as well as important oncoproteins such as c-Myb and BCR/ABL were also found down-regulated. The enzymatic activity of immunoprecipitated Akt/PKB was substantially inhibited in vitro. Moreover, subchronic nemorosone treatment induced reversible monocytosis and thrombocytosis in the mouse model examined. Conclusions: Here, we demonstrate for the first time that nemorosone exerts cytotoxicity in leukemia cells, partly by targeting the Akt/PKB signal transducer, affecting protein levels and cell cycle progression. Finally, in vivo studies suggest that nemorosone significantly affects hematopoiesis in mice.Correspondence to:
D. Díaz-Carballo, PhD
Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, Universitätsstraße 150, 44801 Bochum, Germany
Email: david.diaz-carballo@rub.de

S. Kant, N.K. Natu and V. Mahajan Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University, Lucknow, Uttar Pradesh, India Drug-induced thrombocytopenia is an uncommon but serious side effect of many drugs including antituberculosis drugs. It is difficult to diagnose but easy to prevent just by stopping the exposure to the same drug again. In some cases, it can prove fatal if not taken care of urgently. Here, we report a case of thrombocytopenia due to rifampicin, ethambutol and pyrazinamide all in an adult male, which we believe is the first to be reported.Correspondence to:
V. Mahajan, MD
Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University, 226003, Lucknow, Uttar Pradesh, India
Email: drvineetmahajan14@yahoo.co.in