Evaluation of the efficacy and safety of pamapimod a p38 MAP kinase inhibitor in a double-blind methotrexate-controlled study of patients with active rheumatoid arthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 60, No. 2, February 2009, pp 335–344 DOI 10.1002/art.24266 © 2009, American College of Rheumatology Evaluation of the Efficacy and Safety of Pamapimod, a p38 MAP Kinase Inhibitor, in a Double-Blind, Methotrexate-Controlled Study of Patients With Active Rheumatoid Arthritis Stanley B. Cohen,1 Tien-Tsai Cheng,2 Vishala Chindalore,3 Nemanja Damjanov,4 Ruben Burgos-Vargas,5 Patricia DeLora,6 Kathleen Zimany,6 Helen Travers,7 and John P. Caulfield8 Objective. To determine the efficacy and safety of pamapimod (a selective inhibitor of the ␣-isoform of p38 MAP kinase) as monotherapy in comparison with methotrexate (MTX) treatment in adult patients with active rheumatoid arthritis (RA). Methods. Patients were randomly assigned to 1 of 4 treatment groups and received 12 weeks of doubleblind treatment. One group received MTX (7.5 mg/week with planned escalation to 20 mg/week), and 3 groups received pamapimod (50, 150, or 300 mg) once daily. The primary efficacy end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at 12 weeks. Secondary end points included ACR50 and ACR70 responses, change from baseline in the Disease Activity Score in 28 joints (DAS28), categorical analyses of DAS28/European League Against Rheumatism response, and change from baseline in each parameter of the ACR core set of measures. Safety monitoring included recording of adverse events (AEs), laboratory testing, immunology assessments, administration of electrocardiograms, and assessment of vital signs. Results. Patients assigned to receive MTX and pamapimod had similar demographics and baseline characteristics. At week 12, fewer patients taking pamapimod had an ACR20 response (23%, 18%, and 31% in the 50-, 150-, and 300-mg groups, respectively) compared with patients taking MTX (45%). Secondary efficacy end points showed a similar pattern. AEs were typically characterized as mild and included infections, skin disorders, and dizziness. Pamapimod was generally well tolerated, but the 300-mg dose appeared to be more toxic than either the 2 lower doses or MTX. Conclusion. The present results showed that pamapimod was not as effective as MTX in the treatment of active RA. ClinicalTrials.gov identifier: NCT00303563. Supported by Hoffman-La Roche, Nutley, New Jersey. 1 Stanley B. Cohen, MD: Metroplex Clinical Research, Dallas, 2 Texas; Tien-Tsai Cheng, MD: Chang Gung Memorial Hospital– Kaohsiung Medical Center, and Chang Gung University College of Medicine, Kaohsiung, Taiwan; 3Vishala Chindalore, MD: Pinnacle Research Group, Anniston, Alabama; 4Nemanja Damjanov, MD, PhD: Institut za Reumatologiju, Belgrade University School of Medicine, Belgrade, Serbia and Montenegro; 5Ruben Burgos-Vargas, MD: Clı́nica para el Diagnostico y Tratamiento de las Enfermedades Reumáticas and Hospital General de México, Mexico City, Mexico; 6 Patricia DeLora, BA, Kathleen Zimany, MS: Hoffman-La Roche, Nutley, New Jersey; 7Helen Travers, PhD: Roche Products Ltd., Welwyn Garden City, UK; 8John P. Caulfield, MD: Roche Palo Alto, LLC, Palo Alto, California. Dr. Cohen has served as a clinical investigator and research consultant for Genentech, Biogen Idec, Roche, Procter & Gamble, Pfizer, Centocor, Amgen, Scios, and Wyeth-Ayerst, and has received consulting and/or speaking fees from these companies (less than $10,000 each). Dr. Chindalore has received speaking fees from Roche, Pfizer, and GlaxoSmithKline (less than $10,000 each). Dr. BurgosVargas has received consulting fees, speaking fees, and/or honoraria from Roche, Schering-Plough, Wyeth, and Abbott (less than $10,000 each). Ms DeLora owns stock or stock options in Hoffman-La Roche. Dr. Caulfield owns stock or stock options in Roche. Address correspondence and reprint requests to Stanley B. Cohen, MD, Metroplex Clinical Research Center, 5939 Harry Hines Boulevard, Suite 441, Dallas, TX 75235. E-mail: firstname.lastname@example.org. Submitted for publication June 6, 2008; accepted in revised form October 22, 2008. Parenteral biologic therapies that selectively neutralize proinflammatory cytokines such as tumor necrosis factor ␣ (TNF␣) and interleukin-1␤ (IL-1␤) or their receptors, such as the IL-6 receptor, substantially improve signs and symptoms of rheumatoid arthritis (RA), reduce the number of swollen and tender joints, and 335 336 slow radiographic progression of erosive disease. However, there are no marketed orally active, safe, and effective agents that act primarily to inhibit TNF␣ or IL-1␤, despite attempts to develop such compounds (1–3). The p38␣ MAP kinase (MAPK) has been a major molecular target for the development of an anti– proinflammatory cytokine small-molecule oral therapy for RA. The ␣-isoform is an enzyme important to the intracellular signaling pathway for the generation of TNF␣ or IL-1␤. Multiple extracellular stimuli, including stress signals (lipopolysaccharide), osmotic or heat shock, and proinflammatory cytokines (TNF␣ or IL-1␤), stimulate the p38 pathway (4). The p38␣ MAPK also regulates the expression of cyclooxygenase 2, the enzyme that regulates prostanoids (e.g., prostaglandin E2) in inflammation (5). Activation of the p38␣ pathway causes both transcriptional and translational modulation of gene expression of TNF␣ and IL-1␤ that is both cell type and signal specific. Inhibitors of p38␣ block the production of TNF␣ and IL-1␤ in monocytes and in animal models of arthritis (6). The evidence for a role of the other 3 isoforms, ␤, ␥, and ␦, in TNF generation is not as strong as that for the ␣-isoform, although the ␣ and ␥ isoforms have been localized to the rheumatoid synovium (7–9). Pamapimod is a novel small molecule of the pyridopyrimidine class that selectively inhibits the ␣-isoform of p38 MAPK (10). Its mean ⫾ SD 50% inhibition concentration against recombinant p38␣ is 0.014 ⫾ 0.002 M, while that against TNF␣ release from cultured monocytic leukemia-derived THP-1 cells is 0.025 ⫾ 0.001 M, and that against IL-1␤ production from human whole blood is 0.10 ⫾ 0.03 M. In a variety of in vitro and in vivo preclinical models, pamapimod inhibited TNF␣, IL-1␤, and IL-6 production; it also reduced inflammation and provided joint protection in collagen-induced arthritis in mice. In studies in healthy volunteers, pamapimod was well tolerated and was associated with mild dizziness, mild infections (such as folliculitis, bronchitis, and urinary tract infections), and mild, reversible elevations of creatinine phosphokinase (CPK) (Zhang X, Huang Y, Caulfield JP: unpublished data). Therefore, phase II development of pamapimod as an oral agent to treat RA was initiated. In the study reported here (PA18604), patients not currently receiving methotrexate (MTX) were given either pamapimod once daily at 1 of 3 dosage levels or MTX weekly with appropriate increase of the MTX dosage. In a companion study (PA18439), patients with an incomplete response to MTX were given once-daily or twice-daily COHEN ET AL dosing regimens of pamapimod or were given placebo (Alten RE, Zerbini C, Jeka S, Irazoque F, Khatib F, Emery P, et al: unpublished observations). PATIENTS AND METHODS Patients. Eligible patients were age ⱖ18 years, had active RA according to the 1987 revised criteria of the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) (11), received antiinflammatory therapy for RA on an outpatient basis, had ⱖ6 swollen joints (of 66 joints assessed) and ⱖ8 tender joints (of 68 joints assessed), and had a high-sensitivity C-reactive protein (hsCRP) level ⱖ0.6 mg/dl or an erythrocyte sedimentation rate (ESR) ⱖ28 mm/hour or morning stiffness ⱖ45 minutes. Patients were excluded if they were unable to get out of bed without assistance or required use of a wheelchair, had a history of or current inflammatory joint disease other than RA, or had a rheumatic autoimmune disease other than RA or significant systemic involvement secondary to RA; Sjögren’s syndrome with RA was allowed. Other exclusion criteria included discontinuation of previous MTX therapy due to clinically important toxicity or insufficient efficacy, previous treatment with alkylating agents, evidence of serious uncontrolled disease, history or evidence of tuberculosis, active infections, history of recurrent bacterial infections, history of hereditary or acquired immune deficiency disorder, pregnancy or breast-feeding, or neuropathies or other painful conditions that had the potential to interfere with pain evaluations. Treatment with disease-modifying antirheumatic drugs (DMARDs) and biologic agents had to be stopped prior to baseline as follows: infliximab and adalimumab, 8 weeks; leflunomide, 8 weeks, or after cholestyramine, 4 weeks; intraarticular or parenteral corticosteroids, 4 weeks; sulfasalazine, azathioprine, cyclosporine, D-penicillamine, hydroxychloroquine, chloroquine, and gold, 3 weeks; etanercept and anakinra, 2 weeks. Study protocol. This 12-week, double-blind, doubledummy, parallel-group, active-controlled study was conducted at 61 centers in the US, Canada, Mexico, Europe, South Africa, and Taiwan. All participating sites received approval from their governing institutional review board (or equivalent), all patients provided informed written consent, and the study was performed in accordance with the Declaration of Helsinki. A list of investigators who recruited patients for this study is shown in Appendix A. At screening, medical history was recorded, and information was collected regarding swollen and tender joints and duration of morning stiffness. Vital signs and physical measurements were obtained, and a complete physical examination, electrocardiography (EKG), chest radiography, tuberculin skin test, clinical laboratory tests (hematology, serum chemistry, nonfasting lipid panel, and urinalysis), and a serum pregnancy test (as required) were performed. Blood samples were obtained to test for hepatitis B surface antigen and hepatitis C virus and to determine the hsCRP level and ESR. Patients were randomly assigned (1:1:1:1) to 1 of 4 treatment regimens in a blinded manner and stratified by geographic region and previous MTX exposure. The first group received 50 mg pamapimod once daily (two 25-mg PAMAPIMOD VERSUS MTX IN PATIENTS WITH ACTIVE RA tablets) plus MTX placebo weekly. The second group received 150 mg pamapimod once daily (two 75-mg tablets) plus MTX placebo weekly. The third group received 300 mg pamapimod once daily (two 150-mg tablets) plus MTX placebo weekly. The fourth group received placebo pamapimod once daily (two placebo tablets) plus MTX weekly in increasing doses. Patients randomly assigned to the MTX group received MTX orally once a week, commencing at 7.5 mg (three 2.5-mg tablets) on day 1, which was increased to 15 mg weekly by week 4; a further increase to 20 mg weekly was an option at week 8, depending on whether the patient had painful and swollen joints and did not have signs of significant MTX-related toxicity. Of those in the MTX group who completed 12 weeks of dosing, 71% were taking 15 mg/week and 29% were taking 20 mg/week at the end of the study. The mean cumulative dose of MTX was 156.5 mg. In the 3 pamapimod groups, MTX placebo was escalated to achieve similar nominal mean cumulative doses as in the MTX group, namely, 155.1 mg for the 50-mg group, 152.4 mg for the 150-mg group, and 142.4 mg for the 300-mg group. MTX dosage reductions were permitted for patients who experienced possible MTX-related side effects, and dosage adjustments were performed in the same manner for both active and placebo forms of MTX, due to the blinded nature of the study. In addition to the study drug, patients were to receive folic acid (5 mg weekly) during the study. Oral corticosteroids (ⱕ10 mg/day prednisone or equivalent) and nonsteroidal antiinflammatory drugs were permitted if the dosage was stable for ⱖ4 weeks and ⱖ2 weeks, respectively, prior to baseline and maintained during the study. The following treatments were prohibited during the study: DMARDs, MTX (except as part of protocol-defined study therapy), prednisone or equivalent at a dosage of ⬎10 mg/day, intraarticular or parenteral corticosteroids, biologic agents, cell-depleting therapies, alkylating agents (including cyclophosphamide), any investigational agent other than pamapimod, immunizations, and immunoabsorption columns. Efficacy parameters were assessed at baseline and then routinely throughout the 12 weeks of treatment according to the following schedule. The swollen and tender joint counts and the Stanford Health Assessment Questionnaire disability index (HAQ DI) score (12) were obtained at weeks 2, 4, 8, and 12. An assessment of fatigue using the fatigue subscale of the Functional Assessment of Chronic Illness Therapy questionnaire (FACIT-F) (13) was performed at weeks 4 and 12. All other efficacy assessments (patient’s and physician’s global assessment of disease activity, patient’s assessment of pain, hsCRP level, ESR, and morning stiffness) were performed at weeks 1, 2, 4, 8, and 12. Safety assessments, including EKGs and vital signs, were monitored routinely. Safety laboratory testing (hematology, blood chemistry including CPK level, lipid panel, and urinalysis) was performed at screening and baseline and at weeks 1, 2, 4, 8, and 12. In addition, immunology laboratory testing for rheumatoid factor (RF), anti–cyclic citrullinated peptide (anti-CCP), lymphocyte subtype analysis (CD4⫹, CD3⫹, CD8⫹, and CD19⫹ cells were sorted), anti–doublestranded DNA (anti-dsDNA), antitetanus titer, IgG, IgA, and IgM was performed at baseline and at week 12. All adverse events (AEs) encountered during the study and through 4 weeks following discontinuation of study treat- 337 ment were recorded. AEs were monitored until they stabilized or returned to baseline status. For each AE, the investigator assessed and recorded the intensity (as mild, moderate, or severe), relationship to the study drug (unrelated or remotely, possibly, or probably related), action taken (i.e., whether a treatment was given or a procedure performed and whether the study drug was discontinued or the dose adjusted), and outcome. A serious AE was defined as any experience that resulted in death, was life-threatening, required hospitalization or prolonged an existing hospitalization, resulted in a persistent or significant disability, resulted in a congenital anomaly/ birth defect, or required intervention to prevent one or more of the outcomes listed above. Clinical assessments. The primary efficacy end point was the proportion of patients meeting the ACR 20% improvement criteria (achieving an ACR20 response) at week 12 (14), defined as a 20% improvement from baseline in swollen joint count and tender joint count and in at least 3 of the following 5 variables: HAQ DI score, patient’s global assessment of disease activity, patient’s global assessment of pain, physician’s global assessment of disease activity, and level of an acutephase reactant (primarily CRP; ESR was used only if CRP was missing). Secondary end points included ACR50 and ACR70 responses (50% and 70% improvement, respectively, from baseline in the parameters used to define an ACR20 response), change from baseline in the Disease Activity Score in 28 joints (DAS28) (15), categorical analyses of DAS28/ European League Against Rheumatism response (16), and change from baseline in each parameter of the ACR core set of measures (17) (swollen joint count, tender joint count, patient’s and physician’s global assessment of disease activity, patient’s assessment of pain, HAQ DI score, hsCRP level, and ESR). Additional secondary end points included assessments of the FACIT-F score and the severity and duration of morning stiffness. Statistical analysis. Pamapimod and MTX were expected to have similar efficacy levels, with an assumed ACR20 response rate of 50–70% based on historical data. For a response rate of 70%, a sample size of 50 patients for a given treatment group would provide a 90% confidence interval (90% CI) with a margin of error of ⬃10%. For a response rate of 50%, the same sample size would provide a 90% CI with a margin of error of ⬃12%. Efficacy analyses were based on the intent-to-treat (ITT) population, defined as all patients who received at least 1 dose of double-blind study medication. Patients who prematurely withdrew from the study for any reason, or for whom an assessment was not performed for any reason, were included in the ITT population. Analysis of safety data included all randomized patients who received at least 1 dose of study medication and had at least 1 safety assessment. For the primary efficacy analysis, no formal hypothesis testing was performed. Descriptive statistics, including CI estimates, were used to evaluate differences among treatment groups. Patients with missing ACR20 response values at week 12 (including patients who had withdrawn from the study prior to week 12) were classified as nonresponders for the purpose of the analysis. The primary efficacy variable (ACR20 response at week 12) was analyzed using the Cochran-Mantel-Haenszel test, with region and previous MTX exposure as stratification factors. 338 COHEN ET AL Table 1. Baseline characteristics of the patients* Pamapimod Characteristic Age, years Female, no. (%) Swollen joint count (of 66 joints) Tender joint count (of 68 joints) RF positive, no. (%) RF, IU/ml Anti-CCP positive, no. (%) CRP level, mg/dl ESR, mm/hour Morning stiffness ⱖ45 minutes, no. (%) DAS28 using ESR HAQ DI score FACIT-F score Patient’s global assessment of disease activity, 0–100-mm VAS Physician’s global assessment of disease activity, 0–100-mm VAS Patient’s assessment of pain, 0–100-mm VAS Use of corticosteroids, no. (%) Previous exposure to MTX, no. (%) Previous use of DMARDs, no. (%) Number of previous DMARDs Previous use of anti-TNF agents, no. (%) MTX (n ⫽ 53) 50 mg once daily (n ⫽ 52) 150 mg once daily (n ⫽ 51) 300 mg once daily (n ⫽ 48) 47.3 ⫾ 12.4 44 (83) 15.5 ⫾ 8.4 28.5 ⫾ 14.6 34 (64) 154.4 ⫾ 282.4 32 (60) 1.62 ⫾ 1.91 39.7 ⫾ 22.4 47 (89) 6.40 ⫾ 0.90 1.36 ⫾ 0.65 28.1 ⫾ 11.1 62.4 ⫾ 26.7 51.4 ⫾ 11.5 45 (87) 17.5 ⫾ 9.2 27.0 ⫾ 12.6 37 (71) 308.4 ⫾ 650.3 36 (69) 2.04 ⫾ 2.41 47.8 ⫾ 33.7 46 (88) 6.42 ⫾ 0.97 1.33 ⫾ 0.74 29.9 ⫾ 11.0 55.6 ⫾ 24.2 50.7 ⫾ 12.4 43 (84) 15.7 ⫾ 9.5 27.0 ⫾ 15.2 33 (67) 235.0 ⫾ 411.3 38 (75) 1.95 ⫾ 2.10 44.3 ⫾ 25.1 45 (90) 6.30 ⫾ 1.03 1.37 ⫾ 0.75 30.4 ⫾ 11.3 58.6 ⫾ 24.8 50.2 ⫾ 12.8 40 (83) 19.2 ⫾ 10.9 28.2 ⫾ 14.7 27 (57) 142.6 ⫾ 248.0 33 (69) 2.58 ⫾ 2.95 42.0 ⫾ 22.8 42 (88) 6.61 ⫾ 1.00 1.58 ⫾ 0.74 26.6 ⫾ 11.2 63.6 ⫾ 23.9 58.1 ⫾ 20.8 56.8 ⫾ 19.5 54.4 ⫾ 20.4 59.9 ⫾ 21.2 53.6 ⫾ 25.0 25 (47) 21 (40) 29 (55) 2.4 ⫾ 1.2 4 (8) 49.8 ⫾ 19.5 28 (54) 22 (42) 32 (62) 1.8 ⫾ 0.9 3 (6) 52.2 ⫾ 19.8 21 (41) 16 (31) 28 (55) 2.0 ⫾ 1.2 1 (2) 55.7 ⫾ 24.3 25 (52) 16 (33) 25 (52) 2.2 ⫾ 1.1 3 (6) * Except where indicated otherwise, values are the mean ⫾ SD. MTX ⫽ methotrexate; RF ⫽ rheumatoid factor; anti-CCP ⫽ anti–cyclic citrullinated peptide; CRP ⫽ C-reactive protein; ESR ⫽ erythrocyte sedimentation rate; DAS28 ⫽ Disease Activity Score in 28 joints; HAQ DI ⫽ Health Assessment Questionnaire disability index; FACIT-F ⫽ Functional Assessment of Chronic Illness Therapy–Fatigue; VAS ⫽ visual analog scale; DMARDs ⫽ disease-modifying antirheumatic drugs; anti-TNF ⫽ anti–tumor necrosis factor. For the secondary efficacy analyses, results for categorical parameters were analyzed as described for the primary efficacy analysis. Patients with missing ACR50 and ACR70 response values were considered nonresponders for the week-12 analyses. For all other categorical variables, the observed data were used. Results for continuous parameters were summarized using descriptive statistics. RESULTS Patient characteristics. Study data were collected between February 9, 2006 and June 21, 2007. Of the 271 patients screened, 204 were enrolled; 53 were randomized to receive MTX and 151 to receive pamapimod (52, 51, and 48 patients to the 50-, 150-, and 300-mg groups, respectively). Table 1 summarizes the demographics and baseline characteristics of the 4 treatment groups. The majority of patients were women, and the mean age of the patients ranged from 47.3 years to 51.4 years across the treatment groups. At baseline, the majority of patients tested positive for RF and anti-CCP, 88% of patients reported having morning stiffness of ⱖ45 minutes, and 47% had CRP values that were ⱖ1.0 mg/dl. Data on duration of disease were not captured. Fifty-six percent of patients had reported prior use of DMARDs (mean of 2) for treatment of RA, 37% of patients had prior exposure to MTX, and 2–8% of patients had used an anti-TNF medication. At baseline, approximately half of the patients were using corticosteroids at a dosage equivalent to ⱕ10 mg prednisone once daily. The 4 treatment groups were generally similar at baseline with regard to the ACR core set (17) characteristics of swollen and tender joint counts, HAQ DI score, patientassessed pain, and patient and physician globally assessed disease activity (Table 1). All patients received at least 1 dose of the study drug and were included in both the ITT and safety analysis populations. Overall, 81% of patients completed 12 weeks of treatment; 15.1% of patients in the MTX group and 19.9% of patients in the combined pamapimod groups discontinued study treatment prematurely (Table 2). Reasons for discontinuation were attributed to AEs, lack of efficacy, and refusal of continued treatment (which included noncompliance and withdrawal of consent). The rates of and reasons for discontinuation were similar between the MTX group and the 2 lower- PAMAPIMOD VERSUS MTX IN PATIENTS WITH ACTIVE RA Table 2. Disposition of the patients* Pamapimod 50 mg 150 mg 300 mg MTX once daily once daily once daily (n ⫽ 53) (n ⫽ 52) (n ⫽ 51) (n ⫽ 48) Withdrawn Adverse event 4 (7.5) 3 (5.8) Insufficient therapeutic 3 (5.7) 4 (7.7) response Refused treatment† 1 (1.9) 2 (3.8) Completed 45 (84.9) 43 (82.7) 4 (7.8) 3 (5.9) 10 (20.8) 2 (4.2) 2 (3.9) 42 (82.4) 0 36 (75.0) * Values are the number (%) of patients. MTX ⫽ methotrexate. † Includes patients who withdrew consent and those who were not compliant with the protocol. dose pamapimod groups. The incidence of discontinuation due to an AE was highest in the 300-mg pamapimod group (with a rate of 21%, compared with 6–8% in the other 3 groups). Clinical efficacy. The percentage of patients with an ACR20 response at week 12 was lower in each of the 3 pamapimod groups (23%, 18%, and 31% in the 50-, 150-, and 300-mg groups, respectively) than in the MTX group (45%) (Figure 1A). The percentage with an ACR20 response at week 12 was higher in the 300-mg pamapimod group than in the 2 lower-dose pamapimod 339 groups. Similarly, a greater percentage of patients in the MTX group had an ACR50 response at week 12 compared with the pamapimod groups (23% in the MTX group, versus 12%, 6%, and 13% in the 50-, 150-, and 300-mg pamapimod groups, respectively) (Figure 1B). Few patients (ⱕ8% in each treatment group) had an ACR70 response, and rates were similar in all 4 dosage groups (Figure 1C). A Cochran-Mantel-Haenszel analysis of the ACR20 response at week 12 was used to calculate the weighted difference in proportions, adjusted for stratification factors, between each of the pamapimod groups and the MTX group (pamapimod minus MTX), with a 95% CI for the treatment difference. The results were as follows: ⫺0.22 (95% CI ⫺0.40, ⫺0.05) for the 50-mg group, ⫺0.28 (95% CI ⫺0.45, ⫺0.12) for the 150-mg group, and ⫺0.14 (95% CI ⫺0.32, 0.04) for the 300-mg group. Thus, MTX had superior efficacy to pamapimod, although for the 300-mg dose level, the CI encompassed zero. While the mean DAS28 decreased over time in all groups, by week 12, the MTX group showed the greatest improvements (Figure 1D). For each of the 8 ACR core component criteria, the MTX group showed the greater improvement from baseline compared with the 3 pamapimod groups (Table 3). In the 300-mg pamapimod group, the improvement Figure 1. A–C, Percentages of patients meeting the American College of Rheumatology 20%, 50%, and 70% improvement criteria (achieving ACR20 [A], ACR50 [B], and ACR70 [C] responses, respectively) over the 12-week study period in the intent-to-treat population. D, Changes in the Disease Activity Score in 28 joints (DAS28) over the 12-week study period in the intent-to-treat population. MTX ⫽ methotrexate; QD ⫽ once daily. 340 COHEN ET AL Table 3. Analysis of variance of absolute change from baseline in the American College of Rheumatology core set parameters at week 12* MTX No. of patients Adjusted mean Pamapimod 50 mg once daily No. of patients Adjusted mean Difference 95% CI of difference 150 mg once daily No. of patients Adjusted mean Difference 95% CI of difference 300 mg once daily No. of patients Adjusted mean Difference 95% CI of difference Swollen joint count (of 66 joints) Tender joint count (of 68 joints) Patient’s global assessment of disease activity, 0–100-mm VAS Physician’s global assessment of disease activity, 0–100-mm VAS Patient’s assessment of pain, 0–100-mm VAS CRP level, mg/dl ESR, mm/hour HAQ DI score 47 ⫺7.3 46 ⫺13.3 48 ⫺25.9 46 ⫺27.2 48 ⫺23.4 49 ⫺0.5 47 ⫺12.6 48 ⫺0.5 45 ⫺7.1 0.2 ⫺3.1, 3.5 45 ⫺9.1 4.1 ⫺0.9, 9.2 45 ⫺9.8 16.1 5.5, 26.6 44 ⫺19.1 8.1 ⫺0.7, 16.9 45 ⫺5.1 18.2 7.7, 28.8 45 0.7 1.2 0.4, 2.0 45 ⫺2.6 10.0 2.7, 17.2 45 ⫺0.2 0.2 0.0, 0.5 43 ⫺5.0 2.3 ⫺1.1, 5.6 43 ⫺9.8 3.5 ⫺1.6, 8.6 43 ⫺10.2 15.7 5.0, 26.4 43 ⫺17.9 9.2 0.3, 18.1 43 ⫺9.0 14.4 3.8, 25.0 42 0.5 1.1 0.2, 1.9 43 ⫺0.4 12.2 4.9, 19.6 43 ⫺0.3 0.1 ⫺0.1, 0.4 38 ⫺7.2 0.1 ⫺3.3, 3.6 38 ⫺11.4 1.9 ⫺3.3, 7.2 38 ⫺14.8 11.1 0.1, 22.1 37 ⫺21.3 5.9 ⫺3.3, 15.2 38 ⫺8.9 14.4 3.4, 25.5 37 ⫺0.1 0.5 ⫺0.4, 1.3 38 ⫺5.6 7.0 ⫺0.5, 14.6 38 ⫺0.2 0.3 0.1, 0.6 * 95% CI ⫽ 95% confidence interval (see Table 1 for other definitions). in joint scores was almost as good as with MTX; however, improvements in the patient’s disease activity and pain assessment scores were less than with MTX. CRP levels decreased from baseline for patients in the MTX group and increased for patients in the 50- and 150-mg pamapimod groups; in the 300-mg pamapimod group, there was a modest initial decrease in CRP levels followed by small changes that remained near baseline (Figure 2). FACIT-F scores showed mean increases from baseline over time, suggesting reduced fatigue; these increases were similar across treatment groups (data not shown). The duration of morning stiffness appeared to decline in all groups over the course of the study; while the majority of patients had morning stiffness for ⱖ45 minutes at baseline, by the end of the study the majority of patients experienced morning stiffness for ⱕ45 minutes, with the greatest shift observed in the MTX group (data not shown). Safety. The percentage of patients experiencing at least 1 AE (including AEs experienced during the 4-week followup period) was 58% in the MTX group, 63% in the 50-mg pamapimod group, and 73% in the 150- and 300-mg pamapimod groups (Table 4). Events considered related to study treatment occurred in 42% of MTX-treated patients and in 35–56% of pamapimodtreated patients, showing an increased incidence with dose level. Similarly, the incidence of serious AEs increased with pamapimod dose level, with incidences of 2%, 8%, and 10% in the 50-, 150-, and 300-mg pamapimod groups, respectively, compared with a 4% incidence in the MTX group. Infections were the most frequently Figure 2. Change from baseline in C-reactive protein levels over the 12-week study period. Values are the mean. See Figure 1 for definitions. PAMAPIMOD VERSUS MTX IN PATIENTS WITH ACTIVE RA Table 4. 341 Summary of adverse events* Pamapimod Any adverse event Serious adverse event† Related serious adverse events‡ Adverse event leading to withdrawal Adverse events with ⱖ5% incidence Infections and infestations (system organ class) Skin and subcutaneous tissue disorders (system organ class) Dizziness Hepatic enzyme increased Upper respiratory tract infection Nausea Rheumatoid arthritis Constipation Urinary tract infection Diarrhea Vertigo Headache Pyrexia Fatigue Bronchitis Cough Insomnia MTX (n ⫽ 53) 50 mg once daily (n ⫽ 52) 150 mg once daily (n ⫽ 51) 300 mg once daily (n ⫽ 48) 31 (58) 2 (4) – 4 (8) 33 (63) 1 (2) 1 (2) 3 (6) 37 (73) 4 (8) 1 (2) 4 (8) 35 (73) 5 (10) 2 (4) 10 (21) 13 (25) 12 (23) 14 (27) 17 (35) 2 (4) 6 (12) 4 (8) 9 (19) 3 (6) 2 (4) 3 (6) 6 (11) 4 (8) 1 (2) 3 (6) 1 (2) – 3 (6) 1 (2) 2 (4) 4 (8) 3 (6) 3 (6) 4 (8) – 2 (4) – 2 (4) 1 (2) 2 (4) 2 (4) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 2 (4) – 5 (10) 2 (4) 1 (2) 5 (10) 1 (2) 2 (4) 1 (2) 3 (6) 2 (4) 1 (2) 3 (6) – 1 (2) – 1 (2) 7 (15) 6 (13) 4 (8) – 3 (6) 4 (8) 3 (6) 1 (2) 3 (6) 2 (4) 1 (2) 3 (6) – – – * Values are the number (%) of patients. MTX ⫽ methotrexate. † Defined in Patients and Methods. ‡ Considered by the investigator to be remotely, possibly, or probably related to the study drug. reported serious AEs, occurring in 2%, 2%, and 6% of patients in the 50-, 150-, and 300-mg pamapimod groups, respectively, with 0% in the MTX group. Of these infections, 3 were considered by the investigator to be possibly or probably related to the study drug: sialadenitis (50-mg group), pneumonia (300-mg group), and gastrointestinal (GI) infection (300-mg group). One additional serious AE, GI hemorrhage in the 150-mg pamapimod group, was considered related to the study drug. There were no deaths reported during the 12-week study or 4-week followup period. Withdrawals from the study due to an AE were similar between the MTX group and the 2 lower-dose pamapimod groups (6–8%); in contrast, the rate was substantially higher in the 300-mg pamapimod group (21%). In the MTX group, AEs that led to treatment withdrawal included elevated liver enzyme levels (3 patients) and dizziness (1 patient). In contrast, in the 300-mg pamapimod group, AEs that led to withdrawal included infection (1 patient), skin disorders (2 patients), elevated liver enzyme levels (3 patients), GI disorders (3 patients), and RA flare (1 patient). AEs involving infections or skin disorders as well as dizziness and increased hepatic enzymes occurred with greater frequency in pamapimod-treated patients than in MTX-treated patients (Table 4). Nausea occurred more frequently in the MTX group than in the pamapimod groups (11% of MTX-treated patients, compared with 3% of all pamapimod-treated patients). The proportion of patients experiencing skin disorders trended upward with increasing pamapimod dosage, and while the type of skin disorders varied, the most commonly reported were rash and acne. Skin lesions were predominantly on the face and torso and consisted of maculopapular lesions with or without pustules. The majority of the skin disorders resolved while patients continued pamapimod treatment, and only 2 patients receiving pamapimod discontinued treatment because of the dermatitis. Dizziness appeared to be associated with pamapimod, and the incidence increased with dosage level; only 1 patient (in the 150-mg pamapimod group) withdrew from the study due to dizziness. In the pamapimod groups, dizziness generally occurred soon after treat- 342 ment was started (ranging from 1 day to 12 days) and generally persisted for ⬍2 weeks in the lower-dose groups and for ⬎2 weeks through the duration of the study for patients in the 300-mg group. Several patients had normal values on laboratory measures at baseline but had at least 1 value outside the normal limits following the start of MTX or pamapimod treatment. These laboratory abnormalities included neutrophilia, defined as ⬎9.25 ⫻ 109 cells/liter and a ⬎20% increase from baseline at any postdose time point (13% of patients in the MTX group, 20% in the 50-mg pamapimod group, 12% in the 150-mg pamapimod group, and 25% in the 300-mg pamapimod group), hematuria (8% of patients in the MTX group, 18% in the 50-mg pamapimod group, 10% in the 150-mg pamapimod group, and 15% in the 300-mg pamapimod group), and leukocyturia (9% of patients in the MTX group, 8% in the 50-mg pamapimod group, 6% in the 150-mg pamapimod group, and 13% in the 300-mg pamapimod group). Hematuria and leukocyturia were defined on a scale of 1–4⫹; values ⬎1⫹ and increased by ⬎2⫹ over baseline were recorded as abnormal. Changes in laboratory parameters were observed in all 4 treatment groups. Notably, the MTX group and the 300-mg pamapimod group showed changes from baseline to the end of treatment in levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and CPK. Substantially elevated ALT and/or AST values (i.e., values both ⬎2 times the upper limit of normal [ULN] and ⬎50% over baseline) occurred in 6 MTX-treated patients (11%) and in a total of 12 pamapimod-treated patients (8%) (3 patients in the 150-mg group [6%] and 9 patients in the 300-mg group [19%]). In both MTX- and pamapimod-treated patients, elevated values generally occurred within 2 months after starting the study treatment. Within the MTX group, 3 patients with normal ALT and AST values at baseline discontinued study treatment due to elevations of both ALT (4.2, 14.3, and 4.0 times the ULN) and AST (2.9, 3.3, and 3.8 times the ULN). In all 3 patients, the elevated liver enzyme levels resolved without sequelae within 1 month following discontinuation of MTX. Similarly, within the pamapimod-treated groups, 3 patients, all in the 300-mg group and with normal values at baseline, discontinued the study drug due to elevated liver enzyme levels; all 3 patients had elevated levels of ALT (3.3, 3.5, and 16.4 times the ULN), and 2 of the patients had high levels of AST (5.0 and 7.9 times the ULN). All events were considered possibly or probably related to the study drug. In 2 patients, these AEs resolved without sequelae COHEN ET AL within 1 month following discontinuation of pamapimod; in the third patient, the event was considered ongoing at the time of last contact (30 days after discontinuation of pamapimod). Patients with elevated ALT and AST values had concurrent total bilirubin values that remained within the normal range (0–17 moles/liter); there were no values ⬎15.6 moles/liter. Elevations of CPK levels, ranging from 2.9-fold to 9.4-fold the ULN, were observed in 3 MTX-treated patients (6%) and in 4 pamapimod-treated patients (3%); of the latter patients, 3 in the 150-mg group had values ranging from 2.0-fold to 6.2-fold the ULN, and 1 in the 300-mg group had a value of 2.2-fold the ULN. Two patients, 1 in the MTX group and 1 in the 150-mg pamapimod group, each had 2 reports of elevated CPK values measured ⬃30 days apart; all other patients had only a single episode. There was no trend in time of first onset of elevated CPK levels; the range was 10–58 days following the start of treatment. No patients reported symptoms of muscle pain or weakness, and there were no treatment discontinuations due to elevated CPK levels. There were no changes in RF, anti-CCP, antidsDNA, or immunoglobulin concentrations, lymphocyte subtypes, or antitetanus titer between baseline and 12 weeks in any treatment group. There were no clinically significant changes in EKG findings or vital signs during the study for any treatment group. DISCUSSION This 12-week study demonstrated that treatment with 50-, 150-, and 300-mg once-daily doses of pamapimod, a novel p38 MAPK inhibitor, resulted in lower frequencies of response according to the ACR improvement criteria and the DAS28, compared with MTX started at 7.5 mg/week and increased to 15 mg/week. The 2 lower doses of pamapimod yielded similar frequencies of ACR improvement response, and while the frequency of this response with the 300-mg dose was higher than that with the lower doses, it was well below the frequency of ACR improvement response with MTX. Pamapimod was generally well tolerated, but the overall side effect profile suggested that the 300-mg dose was more toxic than the 2 lower doses or MTX. The results of this study are important, since this is the first large randomized clinical trial evaluating a p38 MAPK inhibitor for which efficacy and safety results have been reported. Several p38 inhibitors have been investigated in the clinic. In terms of efficacy, VX-745 was shown to result in a significantly better ACR20 PAMAPIMOD VERSUS MTX IN PATIENTS WITH ACTIVE RA response than placebo in a phase II study, but development was stopped because of preclinical toxicity findings (18). Doramapimod (BIRB796), showed no efficacy in Crohn’s disease (19). Scio-469 was efficacious in the relief of dental pain (20) and has been tested in phase IIa and IIb trials, but the results are unreported. Why did pamapimod fail to show efficacy? In vitro, pamapimod selectively inhibits p38␣ MAPK and inhibits the production of TNF, IL-1, and IL-6 in both human and rodent cell cultures. Pamapimod also inhibits the in vitro release of constitutively expressed TNF from primary synovial explant cultures, with potency similar to that found in other in vitro assays (10). The drop in CRP levels in the first 2 weeks with pamapimod at the 300-mg dose suggests that at this dosage level, pamapimod may have suppressed proinflammatory cytokine pathways in the patients studied. However, at the lower dosage levels, pamapimod failed to suppress CRP, and the dosage levels were probably too low for this effect to occur. The weakening of CRP inhibition seen after 2 weeks in the 300-mg pamapimod group suggests that inflammatory pathways may have been up-regulated in response to the p38␣ blockade. Candidates for p38␣ bypass might include other p38 isoforms or NF-B, ERK, activator protein 1, and/or JNK pathways. A substantial early decline in CRP levels with subsequent elevation was also seen with BIRB796 in Crohn’s disease (19). There was no decrease in plasma exposures of pamapimod at any dosage level (data not shown), suggesting that metabolism did not account for the lack of effect. The AE profile seen with the 300-mg pamapimod dose (i.e., worse than that with MTX) mitigates against testing higher once-daily dosing levels for long-term treatment of RA. The dominant side effects seen in this trial are consistent with preclinical toxicities and side effects reported with other compounds. Pamapimod caused an increase in infections that were generally mild and primarily of the upper respiratory tract and nasopharynx, skin lesions usually described as folliculitis or acneiform rash, dizziness or vertigo, and GI AEs. Infections are often seen with immunomodulatory compounds. The skin lesions may be infectious in origin, and p38 has been implicated in maintenance of skin barrier function (21). However, skin lesions have been reported with other kinase inhibitors (22), so the precise mechanism is not clear. Skin lesions (23) and dizziness (24) have also been described with other p38 inhibitors. Since p38␣ is found in the cerebellum (25), the maximum concentration–related dizziness may be caused by inhibition of the molecular target. Finally, GI AEs have 343 been reported with other p38 inhibitors in smaller studies and may be a target-related class toxicity perhaps associated with infection or changes in gut flora. Elevation of transaminase levels has occurred with p38 inhibitors as well (19). Preclinical data suggest that p38 is also found in myocardium, developing tissues, skeletal muscle, and hematopoietic cells as well as in the immune system, liver, and central nervous system (26). However, there is no clear correlation between AEs in humans and the first 4 of these tissues. At this point the p38 class side effect profile in RA patients appears to be dizziness, skin lesions, infections, GI AEs, and transaminase elevations. While the results of this study are disappointing, over time the p38 inhibitors have become progressively more potent and selective (27). In addition, studies are ongoing in neuropathic pain, chronic obstructive pulmonary disease, atherosclerosis, and oncologic indications as well as in RA (28). Different selectivity and pharmacokinetic profiles of other p38 inhibitors may offer some advantage over pamapimod. Alternatively, RA may be the wrong indication for p38 inhibitors, as sepsis was for the anti-TNF biologic agents. Additional studies will be needed to define the clinical utility of this new class of immunomodulators. AUTHOR CONTRIBUTIONS Dr. Cohen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Cohen, Caulfield. Acquisition of data. Cheng, Chindalore, Damjanov, Burgos-Vargas, DeLora, Zimany. Analysis and interpretation of data. Cohen, Damjanov, DeLora, Zimany, Travers, Caulfield. Manuscript preparation. Cohen, Chindalore, Damjanov, BurgosVargas, DeLora, Zimany, Travers, Caulfield, D. M. Lidgate (nonauthor; Roche), S. Peng (nonauthor; Roche). Statistical analysis. DeLora, Travers, M. Rabbia (nonauthor; Roche). Medical monitoring. S. Wax (nonauthor; Roche). Programming. J. Chen (nonauthor; Roche), X. Y. Lue (nonauthor; Roche). ROLE OF THE STUDY SPONSOR The study was funded by Hoffman-La Roche (Nutley, NJ) and managed by Roche personnel at several locations (Nutley, NJ; Welwyn Garden City, UK; and Palo Alto, CA). The study was designed by clinicians and scientists at Roche with input from the investigators. Data were collected by the investigators and entered into a database that was maintained by Roche. The data were analyzed by Roche statisticians and programmers. Dr. Caulfield and Dr. Cohen wrote a draft of the manuscript with assistance from a medical writer. All authors reviewed, offered revisions, and approved the content of the manuscript before submission. All authors agreed to submit the final version of the manuscript. 344 COHEN ET AL REFERENCES 1. Goldstein DM, Gabriel T. Pathway to the clinic: inhibition of p38 MAP kinase. A review of ten chemotypes selected for development. Curr Top Med Chem 2005;5:1017–29. 2. Gracie JA, Leung BP, McInnes IB. Novel pathways that regulate tumor necrosis factor-␣ production in rheumatoid arthritis. Curr Opin Rheumatol 2002;14:270–5. 3. Salituro FG, Germann UA, Wilson KP, Bemis GW, Fox T, Su MS. Inhibitors of p38 MAP kinase: therapeutic intervention in cytokine-mediated diseases. Curr Med Chem 1999;6:807–23. 4. Herlaar E, Brown Z. p38 MAPK signalling cascades in inflammatory disease. Mol Med Today 1999;5:439–47. 5. Guan Z, Buckman SY, Pentland AP, Templeton DJ, Morrison AR. Induction of cyclooxygenase-2 by the activated MEKK13 SEK1/MKK43p38 mitogen-activated protein kinase pathway. J Biol Chem 1998;273:12901–8. 6. Lee JC, Kassis S, Kumar S, Badger A, Adams JL. p38 mitogenactivated protein kinase inhibitors—mechanisms and therapeutic potentials. Pharmacol Ther 1999;82:389–97. 7. Hale KK, Trollinger D, Rihanek M, Manthey CL. Differential expression and activation of p38 mitogen-activated protein kinase ␣, ␤, ␥, and ␦ in inflammatory cell lineages. J Immunol 1999;162: 4246–52. 8. Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, et al. Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal– regulated kinase, c-JUN N-terminal kinase, and p38 mitogenactivated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 2000;43:2501–12. 9. Korb A, Tohidast-Akrad M, Cetin E, Axmann R, Smolen J, Schett G. Differential tissue expression and activation of p38 MAPK ␣, ␤, ␥, and ␦ isoforms in rheumatoid arthritis. Arthritis Rheum 2006; 54:2745–56. 10. Hill RJ, Dabbagh K, Phippard D, Li C, Suttmann RT, Welch M, et al. Pamapimod, a novel p38 MAP kinase inhibitor: preclinical analysis of efficacy and selectivity. J Pharmacol Exp Ther 2008. E-pub ahead of print. 11. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. 12. Fries JF, Spitz P, Kraines RG, Holman HR. Measurement of patient outcome in arthritis. Arthritis Rheum 1980;23:137–45. 13. Cella D, Yount S, Sorensen M, Chartash E, Sengupta N, Grober J. Validation of the Functional Assessment of Chronic Illness Therapy Fatigue Scale relative to other instrumentation in patients with rheumatoid arthritis. J Rheumatol 2005;32:811–9. 14. Felson DT, Anderson JJ, Boers M, Bombardier C, Furst D, Goldsmith C, et al. American College of Rheumatology preliminary definition of improvement in rheumatoid arthritis. Arthritis Rheum 1995;38:727–35. 15. Prevoo ML, van ’t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight–joint counts: development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995;38:44–8. 16. Van Gestel AM, Prevoo ML, van ’t Hof MA, van Rijswijk MH, van de Putte LB, van Riel PL. Development and validation of the European League Against Rheumatism response criteria for rheumatoid arthritis: comparison with the preliminary American College of Rheumatology and the World Health Organization/ International League Against Rheumatism criteria. Arthritis Rheum 1996;39:34–40. 17. Felson DT, Anderson JJ, Boers M, Bombardier C, Chernoff M, 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Fried B, et al. The American College of Rheumatology preliminary core set of disease activity measures for rheumatoid arthritis clinical trials. Arthritis Rheum 1993;36:729–40. Weisman M, Furst D, Schiff M, Kauffman R, Merica E, MartinMunley S, et al. A double-blind, placebo-controlled trial of VX745, an oral p38 mitogen activated protein kinase (MAPK) inhibitor, in patients with rheumatoid arthritis (RA) [abstract]. Presented at the 2002 annual European Congress of Rheumatology; 2002 June 12–15; Stockholm, Sweden. Abstract FRI0018. URL: http://www.eular.org. Schreiber S, Feagan B, D’Haens G, Colombel JF, Geboes K, Yurcov M, et al, BIRB 796 Study Group. Oral p38 mitogenactivated protein kinase inhibition with BIRB 796 for active Crohn’s disease: a randomized, double-blind, placebo-controlled trial. Clin Gastroenterol Hepatol 2006;4:325–34. Tong SE, Daniels SE, Nontano T, Chang S, Desjardins P. Scio469, a novel p38␣ MAPK inhibitor, provides efficacy in acute post-surgical dental pain. Clin Pharmacol Ther 2004;75:3. Kobayashi H, Aiba S, Yoshino Y, Tagami H. Acute cutaneous barrier disruption activates epidermal p44/42 and p38 mitogenactivated protein kinases in human and hairless guinea pig skin. Exp Dermatol 2003;12:734–46. Laffitte E, Saurat JH. Kinase inhibitor-induced pustules. Dermatology 2005;211:305–6. Ding C. Drug evaluation: VX-702, a MAP kinase inhibitor for rheumatoid arthritis and acute coronary syndrome. Curr Opin Investig Drugs 2006;7:1020–5. Nikas SN, Drosos AA. SCIO-469 Scios Inc. Curr Opin Investig Drugs 2004;5:1205–12. Lee SH, Park J, Che Y, Han PL, Lee JK. Constitutive activity and differential localization of p38␣ and p38␤ MAPKs in adult mouse brain. J Neurosci Res 2000;60:623–31. Dambach DM. Potential adverse effects associated with inhibition of p38␣/␤ MAP kinases. Curr Top Med Chem 2005;5:929–39. Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, et al. A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008;26:127–32. Clinical Trials.gov. A service of the US National Institutes of Health. URL: http://clinicaltrials.gov. APPENDIX A: INVESTIGATORS Investigators who recruited patients for this study were as follows: Canada: A. Bookman (Toronto), D. Sholter (Edmonton); Croatia: D. Martinovic Kaliterna (Split), S. Novak (Rijeka); Czech Republic: S. Augustinova (Ceske Budejovice), J. Vencovsky (Prague), P. Vitek (Zlin), R. Zahora (Terezin); France: C. Jorgensen (Montpellier), M. Nguyen (Paris); Italy: C. M. Montecucco (Pavia), B. Seriolo (Genoa), G. Valesini (Rome); Mexico: H. Avila-Armengol (Guadalajara), R. Burgos-Vargas (Mexico City), C. Ramos-Remus (Guadalajara); Romania: G. Mirea (Brascov), C. Tanasescu (Bucuresti); Serbia/ Montenegro: N. Damjanov (Belgrade); South Africa: F. Khatib (Johannesburg), D. Nel (Pretoria), B. J. van Rensburg (Bloemfontein); Spain: F. Blanco (La Corunda), S. Marsal (Barcelona), R. Queiro (Oviedo); Taiwan: T.-T. Cheng (Kaohsiung), S. Luo (Taoyuan); United States: J. Anderson (Wichita, KS), R. Capps (Knoxville, TN), V. Chindalore (Anniston, AL), S. B. Cohen (Dallas, TX), K. Colburn (Loma Linda, CA), G. M. Eisenberg (Morton Grove, IL), J. Habros (Scottsdale, AZ), T. Harrington (Danville, PA), H. Holt (Memphis, TN), S. Ishaq (Covington, LA), D. Kirby (Belmont, NC), M. J. Maricic (Tucson, AZ), C. Matejicka (Lancaster, PA), D. Radin (Stamford, CT), G. Roane (Charleston, SC), D. Sandoval (Bend, OR), N. Straniero (South Bend, IN), M. Thurmond Anderle (Amarillo, TX), C. Young (Escondido, CA).