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Efficacy of glucosamine sulfate in knee osteoarthritis : a randomized controlled discontinuation trial Cibere, Jolanda 2004

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EFFICACY OF GLUCOSAMINE SULFATE IN KNEE OSTEOARTHRITIS: A RANDOMIZED CONTROLLED DISCONTINUATION TRIAL by J O L A N D A CIBERE M D , The University Of Saskatchewan, 1993 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Health Care and Epidemiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A April 2004 © Jolanda Cibere, 2004 ABSTRACT Objectives: The primary objective of this study was to determine the clinical efficacy of glucosamine in knee OA in a randomized discontinuation trial. As a secondary objective, the effect of glucosamine on cartilage type II collagen degradation (CII) was evaluated. Methods: A multicenter 24-week randomized double-blind placebo-controlled glucosamine sulfate (GS) discontinuation trial was conducted. Subjects were included if they met the American College of Rheumatology criteria for knee OA, had osteophytes on x-ray, were current users of glucosamine, and had had at least moderate relief of knee pain after starting glucosamine. Subjects received GS at the same dose as prior to the study or placebo (PL) at an equivalent dose. Treatment was continued for 24 weeks or until disease flare. The primary outcome was the proportion of subjects with disease flare in the two groups. Secondary outcomes included time to flare, severity of flare, Western Ontario and McMaster Universities OA index (WOMAC) scores, analgesic medication use and CII degradation markers. Results: The intent-to-treat analysis included 137 subjects (71 GS, 66 PL), aged 40-88 yrs (mean 64) with median baseline W O M A C pain on walking of 13mm (range 0-78mm) and median duration of GS use of 1.5 yrs (range 0 . 1 - 7 yrs). The proportion of subjects who developed a flare in the PL and GS groups was 42% and 45%, respectively (95% confidence interval [CI], -19%, 14%; p=0.76). After adjustment for sex and OA radiographic severity at baseline, the risk of disease flare was similar in the two groups (Cox regression hazard ratio for GS group 0.81; 95% CI 0.47, 1.40; p=0.45). Similarly, no significant differences were seen between treatment groups in the severity of flare, W O M A C scores, analgesic medication use and CII degradation markers. i i Conclusion: In knee OA subjects with moderate to marked subjective improvement with prior glucosamine use, this study provides no evidence of benefit from the continued use of glucosamine sulfate over 6 months. No statistically significant effect of glucosamine sulfate on type II collagen degradation was demonstrated. in TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii ACKNOWLEDGMENTS viii CHAPTER 1 INTRODUCTION 1 1.1 Overview 1 1.2 Study Objectives and Hypotheses 3 1.3 Contributions and Thesis Organization 4 1.4 References 6 CHAPTER 2 BACKGROUND 9 2.1 Osteoarthritis 9 2.2 Alternative Medicine and Glucosamine Use 14 2.3 References 16 CHAPTER 3 GLUCOSAMINE 21 3.1 Articular Cartilage Structure And Bioavailability Of Glucosamine 21 3.2 Efficacy Of Glucosamine For Symptom Modification 23 3.3 Efficacy Of Glucosamine For Disease Modification 26 3.4 References 30 CHAPTER 4 RANDOMIZED DISCONTINUATION TRIAL 36 4.1 Randomized Discontinuation Trial Design Considerations 36 4.2 References 41 CHAPTER 5 CRITICAL ASSESSMENT AND REVIEW OF GLUCOSAMINE LITERATURE 43 5.1 Introduction 43 5.2 Methods 44 5.3 Results 45 5.4 Discussion 48 5.5 References 57 iv CHAPTER 6 RANDOMIZED DOUBLE-BLIND PLACEBO-CONTROLLED GLUCOSAMINE DISCONTINUATION TRIAL IN KNEE OSTEOARTHRITIS 62 6.1 Introduction 62 6.2 Methods 64 6.2.1 Patients 64 6.2.2 Study design 64 6.2.3 Statistical analysis 67 6.3 Results 68 6.4 Discussion 72 6.5 References 86 CHAPTER 7 GLUCOSAMINE SULFATE AND TYPE II COLLAGEN DEGRADATION: RANDOMIZED DISCONTINUATION TRIAL RESULTS ..90 7.1 Introduction 90 7.2 Methods 93 7.2.1 Patients and Study Design 93 7.2.2 Biomarker analysis 94 7.2.3 Study Outcomes 99 7.2.4 Statistical analysis 99 7.3 Results 100 7.4 Discussion 103 7.5 References 113 CHAPTER 8 DISCUSSION AND CONCLUSIONS 118 8.1 Glucosamine and Symptom Modification 118 8.2 Glucosamine and Disease Modification 120 8.3 Strengths and Limitations 122 8.4 Contributions and Recommendations 124 8.5 Conclusions 125 APPENDIX I 126 APPENDIX II 128 LIST OF TABLES Table 5.1: Characteristics of Randomized Controlled Trials of Glucosamine in Knee Osteoarthritis 53 Table 5.2: Summary of Study Population Characteristics for Nine Rigorous Glucosamine Trials 54 Table 5.3: Summary of Study Design Characteristics and Reported Outcomes for Nine Rigorous Glucosamine Trials 55 Table 6.1: Baseline Characteristics of Placebo and Glucosamine Patients 78 Table 6.2: Mean Change in WOMAC and EQ-5D at Final Visit Compared to Baseline and Between-Group Differences in the Intent-To-Treat Population 79 Table 6.3: Multivariate Hazard Ratio of Disease Flare in the Cox Regression Analysis 80 Table 6.4: Mean Change Scores in Non-Flarers and Flarers in the Intent-To-Treat Population and Comparison of Severity of Change in Placebo and Glucosamine Flarers 81 Table 7.1: Baseline Characteristics of Placebo and Glucosamine Groups for Type II Collagen Degradation Markers 108 Table 7.2: Mean Change in Serum and Urine C1,2C/C2C Ratio at Final Visit Compared to Baseline and Between-Group Differences 109 Table 7.3: Mean Change in Serum and Urine C1,2C and C2C at Final Visit Compared to Baseline and Proportion of Undetectable Urine C1,2C and C2C at Final Visit 110 Table 7.4: Multivariable Linear Regression Analysis for the Prediction of Final Serum log C1,2C/C2C Ratio I l l Table 7.5: Mean Change in Serum and Urine C1,2C/C2C Ratio at Final Visit Compared to Baseline in the No-Flare and Flare Subjects and Between-Group Differences 112 LIST OF FIGURES Figure 5.1: Quality Assessment Scores for Glucosamine Trials 56 Figure 6.1: Flow Diagram of Study Enrolment and Conduct 82 Figure 6.2: Proportion of Disease Flare in the Placebo and Glucosamine Treatment Groups in the Intent-To-Treat Population 83 Figure 6.3: Proportion of Analgesic Drug Use at Final Study Vis i t in the Intent-To-Treat Population 84 Figure 6.4: Kaplan-Meier Survival Curves for Time-To-Disease Flare in the Placebo and Glucosamine Groups 85 vii ACKNOWLEDGMENTS My deepest appreciation goes to Dr. John Esdaile and Dr. Jacek Kopec for their guidance and unwavering support of my doctoral studies, for their encouragement of all my research endeavors and for their mentorship which has allowed me to grow and develop as a researcher. Special thanks also go to Dr. Joel Singer and Ms. Anona Thorne for their invaluable support on all aspects of methodology and analysis. Despite their own busy schedules, all members of my supervisory committee have always found the time to read my manuscripts quickly with great attention to detail and provide me with feedback on all aspects of this project. I appreciate their unlimited availability for detailed discussions of my manuscripts and this thesis. Their dedication inspires me to follow the same path. I am grateful to Dr. A. Robin Poole and Ms Mirela Ionescu who gave generously of their time and expertise to conduct the cartilage biomarker assays. I owe thanks to the study co-investigators Dr. Janice Canvin, Dr. David Robinson, Dr. Janet Pope, Dr. Paul Hong and Dr. Eric Grant, who helped with the conduct of this study. I thank all study participants for their interest and contribution to this research. Financial support for this doctoral research was obtained from the Mary Pack Research Fund, the Canadian Institutes of Health Research, and the Michael Smith Foundation for Health Research. Special thanks go to the Arthritis Research Centre of Canada for financial support and for providing me with an environment conducive to research. I appreciate the encouragement of all my colleagues at the Arthritis Research Center. I owe my deepest appreciation to my husband, Joseph, and our children, David, Julia and Samantha. Their encouragement and support have made this work possible, and it is to them that I dedicate this thesis. viii CHAPTER 1 INTRODUCTION 1.1 Overview Osteoarthritis (OA) is the most prevalent joint disease worldwide. Estimates for the prevalence of knee OA vary considerably depending on the population studied and the definition of disease. Above the age of 60 years, radiographic knee OA affects 34%-50% of women and 20%-31% of men.1'2'3'4 Although more prevalent in the elderly, radiographic knee OA already manifests itself in over 10% of the population aged 40 to 54 years of age.1'3'4'5 The prevalence of symptomatic knee OA is estimated to range from 11% to 29% for women and 7% to 14% for men above the age of 65 in population based studies.1'2 More importantly, symptomatic knee OA is a major cause of disability and economic cost.6'7'8 Patients with symptomatic knee OA have significantly greater functional impairment compared to the general population after adjustment for age, sex and other comorbidities.6 Direct and indirect costs are also significantly higher in people with OA compared to non-arthritic persons.7'8 Knee OA is the most common cause for total knee arthroplasty.9 With over 21,000 surgeries performed in 2000 in Canada and a steady increase in the number of procedures performed each year,9 the cost of total knee arthroplasty adds significantly to the overall cost of knee OA. Given the high prevalence of OA in older age groups and the aging of the population in general, the economic burden of knee OA is projected to increase substantially.10 The treatment of knee OA is generally directed at the symptomatic management of the disease. Therapeutic interventions include modification of risk factors, exercise, other non-pharmacological interventions, pharmacological management of pain and joint replacement surgery for advanced disease. No cure for OA exists. However, future prospects 1 for the effective treatment of OA, which may delay the progression of disease, have received increasing attention in recent reviews,11'12 and the search for potentially disease-modifying OA drugs (DMOADs) has intensified. A controversial treatment for knee OA is glucosamine, which has been widely promoted in the media and in books as being able to reduce pain, improve function, delay disease progression, and even rebuild cartilage.13 The promulgation of the benefits of glucosamine has resulted in the widespread use of glucosamine which is available over-the-counter as a nutritional supplement. It has been estimated that 34% to 56% of patients with knee OA are current users of glucosamine.14,15 The public motivation in using glucosamine is not without some foundation, however tenuous. Early in vitro and animal studies have reported chondrometabolic, antireactive and antiarthritic effects of glucosamine.16 In addition, human clinical trials in OA have reported beneficial effects of glucosamine on pain and function. However, based on methodological considerations, the leap into the assumption of efficacy cannot be made from these studies. At the time of initiation of this thesis project, a review of the glucosamine literature yielded 9 full-length articles published in English, only 4 of which were considered to be of adequate scientific rigor.17 In these 4 studies, glucosamine was reported to be efficacious, with 2 trials showing superiority of glucosamine to placebo18'19 and 2 trials showing similar pain relief with glucosamine compared to ibuprofen.20'21 All of these studies were noted to be affiliated with a pharmaceutical producer of glucosamine. At the time of this initial literature review, only one glucosamine trial had been conducted independent of pharmaceutical affiliation.22 The results of this study were available only as an abstract publication, but it had reported no efficacy of glucosamine in the symptomatic treatment of knee OA. 2 2 This negative trial added controversy to the use of glucosamine and raised further questions about its efficacy. The need for additional trials was highlighted by the fact that the US National Institutes of Health awarded substantial funding in 1999 for a major study to evaluate the efficacy of glucosamine.23 The controversy regarding glucosamine was further emphasized by the updated American College of Rheumatology guidelines in 2000, which stated that the recommendation of glucosamine for the symptomatic treatment of knee OA was premature.24 The efficacy of glucosamine as a disease-modifying treatment was not considered in those guidelines, since no such studies had been conducted at that time. It is clear that a state of controversy exists regarding the efficacy of glucosamine and that additional rigorous independent trials would provide further evidence for resolving this controversy. However, the widespread use of glucosamine presents a challenge for the timely conduct of a standard trial. Consequently, a discontinuation trial approach was considered a more feasible and efficient alternative to the evaluation of glucosamine efficacy.25 This thesis presents three papers reporting a recent updated glucosamine literature review, as well as the clinical and biomarker results of a randomized discontinuation trial of glucosamine in knee OA, which provide additional data to the growing body of evidence that will determine the efficacy of glucosamine in the treatment of knee OA. 1.2 Study Objectives and Hypotheses The objectives of this thesis are two-fold. First, the efficacy of glucosamine on symptom modification will be determined relative to placebo in a 6-month multicenter randomized double-blind glucosamine discontinuation trial. Second, the effect of 3 glucosamine on disease-modification will be determined relative to placebo using novel biomarkers that measure the degradation of type II collagen, a component of cartilage. This thesis hypothesizes that discontinuation of glucosamine by patients who have had at least moderate improvement of their OA symptoms coincidental with its use, will result in a flare-up in those OA symptoms. Specifically, this thesis hypothesizes that there will be a difference in the glucosamine and placebo groups with respect to 1) the flare rate, 2) the time to flare, and 3) individual outcomes of pain, stiffness and function. This thesis secondarily hypothesizes that there will be a difference in the glucosamine and placebo groups with respect to type II collagen degradation markers. Because of the discontinuation trial design used, it is expected that type II collagen degradation will increase in the placebo group and remain unchanged in the glucosamine group. 1.3 Contributions and Thesis Organization In general, this thesis contributes to and expands on our current understanding of the efficacy of glucosamine in knee OA with regards to both symptom and disease modification. Specifically, this thesis presents and discusses the results of a unique glucosamine discontinuation trial that provides an alternative perspective on the question of symptomatic efficacy. In addition, this thesis provides a novel contribution in the area of disease modification by reporting the effects of glucosamine on type II collagen degradation. This thesis is comprised of 7 chapters. This first chapter provides a brief introduction to the importance of knee OA, the use of glucosamine in knee OA, as well as the rationale, objectives and unique contributions of this thesis. Chapter 2 reviews the importance of knee 4 OA in terms of prevalence and economic impact and discusses the prevalence of glucosamine use. Chapter 3 discusses the bioavailability of glucosamine, its efficacy as a symptom modifying drug, specifically as it pertains to the state of knowledge prior to the initiation of this thesis, and its efficacy as a disease-modifying drug. Chapter 4 explores considerations related to the randomized discontinuation trial design as well as its advantages and disadvantages. Chapter 5 provides an updated review of the current literature on glucosamine, including trials that were published while this thesis project was ongoing. Chapters 6 and 7 present the discontinuation trial results for symptom and disease modifying effects respectively. The final chapter discusses the interpretation and implications of results, strengths and limitations, as well as the unique contributions of this study. Chapters 5 through 7 are each stand-alone manuscripts, which are either in press, under review or submitted to a major peer-reviewed journal. The work presented in this thesis was conceived, conducted and disseminated by the doctoral candidate. 5 1.4 References 1 ) Lawrence JS, Bremner JM, Bier F. Osteo-arthrosis. Prevalence in the population and relationship between symptoms and x-ray changes. Ann Rheum Dis 1966;25:1-23. 2 ) Felson DT, Naimark A, Anderson J, Kazis, L, Castelli W, Meenan RF. The prevalence of knee osteoarthritis in the elderly. The Framingham Osteoarthritis Study. Arthritis Rheum 1987;30:914-8. 3 ) Van Saase JLCM, Van Romunde LKJ, Cats A, Vandenbroucke JP, Valkenburg HA. Epidemiology of osteoarthritis: Zoetermeer survey. Comparison of radiological osteoarthritis in a Dutch population with that in 10 other populations. Ann Rheum Dis 1989;48:271-80. 4 ) Lethbridge-Cejku M, Scott WW Jr, Reichle R, Ettinger WH, Zonderman A, Costa P, et al. Association of radiographic features of osteoarthritis of the knee with knee pain: Data from the Baltimore Longitudinal Study of Aging. Arthritis Care Res 1995;8:182-8. 5) Sowers M, Lachance L, Hochberg M, Jamadar D. Radiographically defined osteoarthritis of the hand and knee in young and middle-aged African American and Caucasian women. Osteoarthritis Cartil 2000;8:69-77. 6 ) Guccione AA, Felson D, Anderson JJ, Anthony JM, Zhang Y, Wilson PWF et al. The effects of specific medical conditions on the functional limitations of elders in the Framingham study. Am J Publ Health 1994;84:351-8. 7 ) Gabriel SE, Crowson CS, Campion ME, O'Fallon WM. Indirect and nonmedical costs among people with rheumatoid arthritis and osteoarthritis compared with 6 nonarthritic controls. J Rheumatol 1997;24:43-8. 8 ) Gabriel SE, Crowson CS, Campion ME, O'Fallon WM. Direct medical costs unique to people with arthritis. J Rheumatol 1997;24:719-25. 9 ) Health Canada. Arthritis in Canada. An ongoing challenge. Ottawa: Health Canada, 2003. 10) Badley EM, Crotty M. An international comparison of the estimated effect of the aging of the population on the major cause of disablement, musculoskeletal disorders. J Rheumatol 1995;22:1934-40. 11 ) Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum 1998;41:1343-55. 12 ) Dieppe P. Strategies for the prevention of osteoarthritis. Int J Tiss Reac 1993;XV:93-7. 13 ) Theodosakis J, Adderly B, Fox B. The Arthritis Cure. New York: St. Martin's Press; 1997. 14 ) Li L, Maetzel A, Maguire L, Jansz G, Pencharz J, Bombardier C et al. Use of complementary therapies amongst patients with osteoarthritis in Ontario: A telephone survey. J Rheumatol 2001 ;28:1420. 15 ) Penserga EG, Rivera IC, Lucero AL Jr. Treatment of osteoarthritis in the arthritis clinic of the Philippine General Hospital: A preliminary report. Osteoarthritis Cartil 2001;9:S49. 16 ) Setnikar I. Antireactive properties of "chondroprotective" drugs. Int J Tiss Reac 1992;XIV:253-61. 17 ) Cibere J, Esdaile JM. Glucosamine use in osteoarthritis: What is the evidence for its efficacy? J Rheumatol 1999;26:1628. 7 18 ) Noack W, Fischer M, Forster KK, Rovati LC, Setnikar I. Glucosamine sulfate in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:51-9. 19) Reichelt A, Forster KK, Fischer M, Rovati LC, Setnikar I. Efficacy and safety of intramuscular glucosamine sulfate in osteoarthritis of the knee. A randomised, placebo-controlled, double-blind study. Arzneimittelforschung 1994;44:75-80. 20 ) Muller-Fassbender H, Bach GL, Haase W, Rovati LC. Setnikar I. Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:61-9. 21 ) Qiu GX, Gao SN, Giacovelli G, Rovati L, Setnikar I. Efficacy and safety of glucosamine sulfate versus ibuprofen in patients with knee osteoarthritis. Arzneimittelforschung 1998;48:469-74. 22) Houpt JB, McMillan R, Paget-Dellio D, Russell A, Gahunia HK. Effect of glucosamine hydrochloride (GHcl) in the treatment of pain of osteoarthritis of the knee. J Rheumatol 1999;26:2423-30. 23 ) National Institute of Arthritis and Musculoskeletal and Skin Diseases. NIH awards study on glucosamine/chondroitin sulfate for knee osteoarthritis. http://www.niams.nih.gov/ne/press/1999/09_15.htm. 24) American College of Rheumatology Subcommittee on Osteoarthritis Guidelines. Recommendations for the medical management of osteoarthritis of the hip and knee. Arthritis Rheum 2000;43:1905-15. 25 ) Kopec JA, Abrahamowicz M, Esdaile JM. Randomized discontinuation trials: utility and efficiency. J Clin Epidemiol 1993;46:959-71. 8 CHAPTER 2 BACKGROUND 2.1 Osteoarthritis Osteoarthritis (OA) is the most prevalent joint disease worldwide. It is a chronic disease causing deterioration of joint cartilage and development of new bone spurs at the joint margins. OA affects predominantly the hands, knees, hips and spine and less frequently other joints. It is characterized by joint pain, swelling, stiffness, limited motion and difficulty with activities. Estimates of the prevalence of OA vary considerably. In large surveys, self-reported "arthritis" is frequently ascertained. Although OA represents the majority of patients reporting arthritis, such general surveys do not allow for a prevalence estimate of OA itself. The ascertainment of OA by self-report is also fraught with inaccuracy due to the frequent inability of patients to differentiate OA from other musculoskeletal diseases such as rheumatoid arthritis and osteoporosis. Different OA pain questions have variable specificity for OA and hence will result in different prevalence estimates.1 Prevalence estimates from population based studies, which include direct assessments of subjects, are likely more accurate. In addition, such studies often produce joint specific prevalence estimates. However, estimates from such studies can still vary substantially depending on whether OA is defined radiographically or symptomatically. In 2003, Health Canada reported that musculoskeletal conditions affect nearly 4 million Canadians aged 15 years and older, representing 16% of this population. A comparable 15% prevalence of self-reported arthritis was seen in the 1990 US population. A more recent US survey from 2001 found that 33% of US adults aged 18 and above had 9 self-reported arthritis or chronic joint symptoms. This last survey provides a considerably higher estimate, most likely because of a change in case definition, which includes the presence of chronic joint symptoms. The results from these surveys only allow for conclusions of arthritic conditions in general, but not OA specifically. However, the majority of patients with arthritis have OA. 5 Hence these data do provide some insight into the potential magnitude of OA and knee OA. Estimates of the prevalence of knee OA have ranged from 8% to 50% for radiographic knee OA and from 7% to 29% for symptomatic knee OA, depending on the age and gender of the population studied.6'7'8'9'10'11 Since knee OA is more prevalent with 12 increasing age and in women compared to men after the age of 50, a discussion of age and gender specific prevalence estimates is more meaningful. Radiographic knee OA is based on a scoring system to assess the structural degeneration of the knee joint, such as that developed by Kellgren and Lawrence in 1957. The Kellgren and Lawrence radiographic stages are defined as normal (grade 0), questionable (grade 1), mild (grade 2), moderate (grade 3), and severe (grade 4), based 13 predominantly on the size of osteophytes, or bone spurs, at the joint margins. Current studies continue to use this tool frequently for defining radiographic OA, which may have contributed to some consistency for prevalence estimates between studies. Above the age of 60 years, grade 2-4 radiographic knee OA affects 34% to 50% of women and 20% to 31% of men.6'8'9'10 The National Health and Nutrition Examination Survey (NHANES) I reported a considerably lower prevalence of radiographic knee OA with only 18% of women and 8% of men affected above the age of 65 years.7 However, this represents an underestimate of the true prevalence, since a recent rereading of a sample of NHANES I radiographs suggested 10 that >40% of radiographs of middle-aged and elderly participants initially read as normal showed radiographic OA. 3 Although OA is frequently thought of as a disease of the elderly, the disease process is initiated earlier in life and begins to manifest itself in people in their 40's and 50's. Radiographic grade 2-4 knee OA occurs in 12% to 15% of women and 8% to 13% of men aged 40-54 years.6,9'10'11 These data support the notion that radiographic knee OA is extremely common and not confined to elderly persons as previously thought. A more important question relates to whether such radiographic changes are associated with symptoms and hence have an impact on peoples' lives. Studies have consistently reported on the discrepancy between symptoms and radiographic changes of OA. Lachance et al 1 4 recently reported that grade 2-4 radiographic knee OA was associated with knee pain in only 35% of Caucasian women and 51% of African American women 40 to 53 years old. In the Tecumseh Community Health Study, which included 50 to 74 year old subjects, knee pain occurred in 69% of those with grade 2-4 radiographic knee OA. 1 5 Even in subjects with moderately severe (grade 3) knee OA, only 54% of patients had reported knee pain within the previous year in the Baltimore Longitudinal Study of Aging.10 Although this discrepancy has been noted, the severity of radiographic change does correlate with the prevalence of symptoms, such that persons with more severe radiographic knee OA have a higher prevalence of knee pain. 6 , 8 , 1 0 , 1 5' 1 6 As a result of this discrepancy, the prevalence estimates for symptomatic knee OA are lower than those for radiographic knee OA. Symptomatic knee OA is usually defined as radiographic evidence of knee OA and the presence of symptoms in the corresponding joint. The prevalence of symptomatic knee OA has been estimated to range from 11% to 29% for women and 7% to 14% for men above the ft R age of 65 in population based studies. ' 11 These prevalence rates for radiographic and symptomatic knee OA likely represent an underestimate of the true prevalence. In a recent study, grade 1 radiographic changes, previously described as questionable OA, were significantly associated with subsequent development of definite knee OA. 1 7 Similarly, in magnetic resonance imaging (MRI) and arthroscopic knee studies, superficial cartilage lesions on MRI or arthroscopy were undetected on plain radiography in 35%-64% of subjects and deep cartilage lesions were undetected in 18%-21% of subjects.18'19'20 These data suggest that OA prevalence assessed by radiographs represents a mere fraction of the true prevalence. In addition to the high, yet underestimated, prevalence of knee OA, this disease is also a major cause of disability and work disability. Although there is a scarcity of information on OA specific disability in the literature, one can, to some extent, extrapolate from national health surveys, which report on musculoskeletal conditions in general.5 For example, in NHANES I, approximately 75% of individuals with arthritis and rheumatism had physician confirmed OA. 5 In the 2003 Health Canada report, activity limitations were present in over 50% of arthritic persons between 45 and 74 years old and in 66% of those 75 years and above.2 The proportion of people with arthritis who reported activity limitations was twice as high as the proportion among those with other chronic conditions. In a US population survey from 1999, arthritis and rheumatism was identified as the leading cause of 21 22 disability among US adults with 17.5% of all reported disability. Yelin et al reported reduced labor participation rates for persons with musculoskeletal conditions compared to those with other chronic conditions or those with no chronic condition. In OA, population-based studies by Gabriel et al found that work disability was 23 significantly increased among persons with OA compared to non-arthritic controls, as 12 were indirect and nonmedical costs. In knee OA specifically, functional limitations occur frequently. In the NHANES I population, subjects with baseline knee OA were significantly more likely to have difficulty with activities 10 years later compared to those without knee OA, particularly in subjects reporting pain at baseline.25 In Framingham subjects with symptomatic knee OA, functional impairment was significantly increased compared to the remainder of the cohort after adjustment for age, sex and other comorbidities, suggesting that symptomatic knee OA is a major cause of disability in the general population.26 Direct medical costs are also substantial in OA. In a population based study, significantly increased direct medical costs were reported in persons with OA compared to non-arthritic persons.27 In 1995, total joint arthroplasty was estimated to account for an annual cost of $10 billion in the US. 2 8 In Canada, the number of knee replacement surgeries has increased steadily since 1994 with over 21,000 surgeries performed in 2000. Since knee OA is the most common cause for total knee arthroplasty,2 the majority of the cost for arthroplasty surgeries can be attributed to knee OA. Overall, the societal burden of musculoskeletal diseases has been estimated to range from 1.1% to 2.5% of the gross domestic product (GDP) in various countries.29 A more recent US estimate suggested that the costs related to arthritis and other rheumatic conditions were equal to 2.9% of the GDP in 1997.30 Given the high prevalence of knee OA and its association with significant disability, work disability, indirect and direct costs, knee OA is a main contributor to these costs. Furthermore, because of the high prevalence of OA in older age groups and the aging of the population in general, the economic burden of OA 31 is projected to increase substantially. 13 2.2 Alternative Medicine and Glucosamine Use One aspect of OA treatment includes patients' self-management with herbal remedies, nutritional supplements and other complementary therapies. The use of complementary and alternative medicines (CAM) is common in the general population. In a US nationwide survey of the adult population, which was conducted in 1990 and 1997, the prevalence of CAM use had increased from 33.8% to 42.1%.32 In 1997, an estimated 629 million visits were made to alternative medicine practitioners, far exceeding the number of 32 visits made to primary care physicians. In addition, the total out-of-pocket expenses for CAM therapies were estimated at $27 billion, which was comparable with the out-of-pocket expenditures for all physician services.32 In the Canadian National Population Health Survey, the prevalence of CAM use was 22.7% in adults with arthritis and rheumatism, 33 second only to the prevalence in persons with chronic back problems. In OA, CAM therapy use was reported in 47% of patients recruited for a clinical trial and expenditures were estimated at $1127 per year for CAM therapies, compared to $1148 for traditional ambulatory medical care services for OA. 3 4 Glucosamine has been used in North America as a CAM treatment since the early to mid-1990s. It is sold as a nutritional supplement and hence is available over-the-counter. Glucosamine is promoted as an agent that can improve pain and function in OA, as well as slow down OA progression and even rebuild cartilage.35 Promulgation of information on glucosamine in the media and in books35 has resulted in the wide-spread use of glucosamine not only in OA, but also in other rheumatologic conditions. Despite the fact that no trials have been conducted to support the efficacy of glucosamine in rheumatoid arthritis, a recent 36 survey reported that 36% of patients with rheumatoid arthritis were current users. In an 14 Ontario survey of patients with OA aged 55 and older, 34.3% had used glucosamine over the 3-month study period in 1999.37 This study likely underestimates the prevalence of glucosamine use in OA, since the study was limited to patients above age 55, an age group that has been shown to have lower complementary and alternative medicine use than adults aged 35-49.32 In a 1999 Philippine study, 56% of patients with OA from a hospital-based outpatient clinic were current users of glucosamine.38 There is a lack of information on the duration of glucosamine use in the community. Anecdotally, many patients remain on glucosamine treatment for years. Over the past decade, the explosion of public interest in glucosamine has resulted in this supplement being one of the top selling products in health food stores. With a monthly cost ranging anywhere from $15 to $50 for glucosamine, the annual expenditures on this alternative treatment in the population would appear to be substantial. Given the uncertain benefit of glucosamine, as will be discussed in detail in Chapter 3, further investigation of glucosamine will lead to further insights on the efficacy of this costly treatment. 15 2.3 References 1) O'Reilly S, Muir KR, Doherty M. Screening for pain in knee osteoarthritis: which question? Ann Rheum Dis 1996;55:931-3. 2) Health Canada. Arthritis in Canada. An ongoing challenge. Ottawa: Health Canada, 2003. 3 ) Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, Giannini EH, et al. Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum 1998;41:778-99. 4 ) Bolen J, Helmick CG, Sacks JJ, Langmaid G. Prevalence of self-reported arthritis or chronic joint symptoms among adults - United States, 2001. MMWR 2002;51:948-50. 5 ) Badley EM. The effect of osteoarthritis on disability and health care use in Canada. J Rheumatol 1995;(suppl 43)22;19-22. 6 ) Lawrence JS, Bremner JM, Bier F. Osteo-arthrosis. Prevalence in the population and relationship between symptoms and x-ray changes. Ann Rheum Dis 1966;25.T-23. 7 ) Maurer K. Basic data on arthritis knee, hip and sacroiliac joints in adults ages 25-74 years, United States, 1971-1975. Vital Health Stat 11 1979;213. 8 ) Felson DT, Naimark A, Anderson J, Kazis, L, Castelli W, Meenan RF. The prevalence of knee osteoarthritis in the elderly. The Framingham Osteoarthritis Study. Arthritis Rheum 1987;30:914-8. 9 ) Van Saase JLCM, Van Romunde LKJ, Cats A, Vandenbroucke JP, Valkenburg HA. Epidemiology of osteoarthritis: Zoetermeer survey. Comparison of radiological 16 osteoarthritis in a Dutch population with that in 10 other populations. Ann Rheum Dis 1989;48:271-80. 10 ) Lethbridge-Cejku M, Scott WW Jr, Reichle R, Ettinger WH, Zonderman A, Costa P, et al. Association of radiographic features of osteoarthritis of the knee with knee pain: Data from the Baltimore Longitudinal Study of Aging. Arthritis Care Res 1995;8:182-8. 11 ) Sowers M, Lachance L, Hochberg M, Jamadar D. Radiographically defined osteoarthritis of the hand and knee in young and middle-aged African American and Caucasian women. Osteoarthritis Cartil 2000;8:69-77. 12 ) Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum 1998;41:1343-55. 13) Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann Rheum Dis 1957;16:494-502. 14 ) Lachance L, Sowers M, Jamadar D, Jannausch M, Hochberg M, Crutchfield M. The experience of pain and emergent osteoarthritis of the knee. Osteoarthritis Cartil 2001;9:527-32. 15) Carman WJ. Factors associated with pain and osteoarthritis in the Tecumseh Community Health Study. Semin Arthritis Rheum 1989;18(suppl 2): 10-3. 16) Hochberg MC, Lawrence RC, Everett DF, Coraoni-Huntley J. Epidemiologic associations of pain in osteoarthritis of the knee: Data from the National Health and Nutrition Examination Survey and the National Health and Nutrition Examination-I Epidemiologic Follow-up Survey. Semin Arthritis Rheum 1989; 18 (suppl 2):4-9. 17 17 ) Hart DJ, Spector TD. Kellgren & Lawrence grade 1 osteophytes in the knee - doubtful or definite? Osteoarthritis Cartil 2003;11:149-50. 18) Boegard T, Rudling, O, Petersson IF, Jonsson K. Correlation between radiographically diagnosed osteophytes and magnetic resonance detected cartilage defects in the tibiofemoral joint. Ann Rheum Dis 1998;57:401-7. 19) Wada M, Baba H, Imura S, Morita A, Kusaka Y. Relationship between radiographic classification and arthroscopic findings of articular cartilage lesions in osteoarthritis of the knee. Clin Exp Rheumatol 1998;16:15-20. 20 ) Brandt KD, Fife RS, Braunstein EM, Katz B. Radiographic grading of the severity of knee osteoarthritis: Relation of the Kellgren and Lawrence grade to a grade based on joint space narrowing, and correlation with arthroscopic evidence of articular cartilage degeneration. Arthritis Rheum 1991;34:1381-6. 21 ) McNeil JM, Binette J. Prevalence of disabilities and associated health - United States, 1999. MMWR 2001;50:120-5. 22 ) Yelin EH, Katz PP. Labor force participation among persons with musculoskeletal conditions, 1970-87. Arthritis Rheum 1991;34:1361-70. 23 ) Gabriel SE, Crowson CS, O'Fallon WM. Costs of osteoarthritis: Estimates from a geographically defined population. J Rheumatol 1995; (suppl 43)22:23-5. 24 ) Gabriel SE, Crowson CS, Campion ME, O'Fallon WM. Indirect and nonmedical costs among people with rheumatoid arthritis and osteoarthritis compared with nonarthritic controls. J Rheumatol 1997;24:43-8. 25) Davis MA, Ettinger WH, Neuhaus JM, Mallon KP. Knee osteoarthritis and physical functioning: Evidence from the NHANES I Epidemiologic Followup Study. J 18 Rheumatol 1991;18:591-8. 26 ) Guccione AA, Felson D, Anderson JJ, Anthony JM, Zhang Y, Wilson PWF et al. The effects of specific medical conditions on the functional limitations of elders in the Framingham study. Am J Publ Health 1994;84:351-8. 27 ) Gabriel SE, Crowson CS, Campion ME, O'Fallon WM. Direct medical costs unique to people with arthritis. J Rheumatol 1997;24:719-25. 28 ) Lavernia CJ, Drakeford MK, Tsao AK et al. Revision and primary hip and knee arthroplasty. A cost analysis. Clin Orthop Rel Res 1995;311:136-41. 29) March LM, Bachmeier CJM. Economics of osteoarthritis: a global perspective. Clin Rheumatol 1997;11:817-34. 30 ) Yelin E. Cost of musculoskeletal diseases: Impact of work disability and functional decline. J Rheumatol 2003;30(suppl 68):8-l 1. 31) Badley EM, Crotty M. An international comparison of the estimated effect of the aging of the population on the major cause of disablement, musculoskeletal disorders. J Rheumatol 1995;22:1934-40. 32 ) Eisenberg DM, Davis RB, Ettner SL, Appel S, Wilkey S, Van Rompay M et al. Trends in alternative medicine use in the United States, 1990-1997: Results of a follow-up national survey. JAMA 1998;280:1569-75. 33) Fautrel B, Adam V, St-Pierre Y, Joseph L, Clarke AE, Penrod JR. Use of complementary and alternative therapies by patients self-reporting arthritis or rheumatism: Results from a nationwide Canadian survey. J Rheumatol 2002;29:2435-41. 34) Ramsey SD, Spencer AC, Topolski TD, Belza B, Patrick DL. Use of alternative 19 therapies by older adults with osteoarthritis. Arthritis Care Res 2001;45:222-7. 35 ) Theodosakis J, Adderly B, Fox B. The Arthritis Cure. New York: St. Martin's Press; 1997. 36) Li LC, Maetzel A, Pencharz J, Maguire L, Bombardier C, the CHAP team. Use of complementary therapies amongst patients with rheumatoid arthritis. J Rheumatol 2002;29:1571. 37 ) Li L, Maetzel A, Maguire L, Jansz G, Pencharz J, Bombardier C et al. Use of complementary therapies amongst patients with osteoarthritis in Ontario: A telephone survey. J Rheumatol 2001 ;28:1420. 38) Penserga EG, Rivera IC, Lucero AL Jr. Treatment of osteoarthritis in the arthritis clinic of the Philippine General Hospital: A preliminary report. Osteoarthritis Cartil 2001 ;9:S49. 20 CHAPTER 3 GLUCOSAMINE This chapter briefly introduces basic concepts of articular cartilage structure, which is necessary for an understanding of the discussion of possible structure-modifying effects of glucosamine. In addition, issues of pharmacokinetics and bioavailability of glucosamine are introduced. The main focus of this chapter is to review the efficacy of glucosamine as a symptom and disease-modifying treatment in knee OA, a discussion that will highlight the need for further studies. 3.1 Articular Cartilage Structure And Bioavailability Of Glucosamine Glucosamine is an amino sugar that is normally produced in the body and utilized in many tissues. It is a key component of the extracellular matrix of articular cartilage, where it is used by chondrocytes to produce chondroitin sulfate and hyaluronate.1 Chondroitin sulfate and other glycosaminoglycans are attached to a core protein forming aggrecan molecules, which are in turn attached to long hyaluronate strands to produce large proteoglycan molecules.2 These complexes form the basic hydrophilic structure of articular cartilage and are interwoven with type II collagen fibrils, which provide the structural backbone of the extracellular matrix of cartilage. In articular cartilage, turnover of this extracellular matrix occurs normally with a balance in synthesis and degradation. This process is regulated by the metabolic activity of chondrocytes. The catabolism of cartilage matrix is mediated by matrix metalloproteinases (MMPs), which are enzymes that can collectively degrade all components 2 3 of the extracellular matrix. To date, 27 MMPs have been identified. MMP-1, 8 and 13 are thought to play a key role in the degradation of type II collagen,3'4 while MMP-3 is involved 21 in the degradation of aggrecan.2 In OA, this metabolic balance between synthesis and degradation is disrupted, resulting in excess degeneration coupled with insufficient production of the extracellular cartilage matrix. Inflammatory cytokines and mediators such as interleukin-ip, nitric oxide and prostaglandin E2, play a key role in the excess production 2 5 of MMPs by chondrocytes and in the degradation of cartilage. ' Because glucosamine is a key building block for cartilage components, the possibility that supplemental glucosamine may enhance the anabolic process has been entertained.6 Although this concept would allow for a possible explanation of structure modification with glucosamine, the effect on pain and hence symptom modification is less obvious. One theory on pain reduction that has been put forward involves the concept of increased hyaluronate production with supplemental glucosamine.7 Articular injections of hyaluronate are used as a treatment for OA and have been shown to relieve pain.8 Since glucosamine is a building block for hyaluronate, this theory proposes that supplemental glucosamine results in enhanced hyaluronic acid production by synovial cells and therefore pain relief. This theory has not been studied in humans. The concept that supplemental glucosamine provides a substrate to chondrocytes presumes that glucosamine is absorbed and bioavailable. In a review of early pharmacokinetic studies, it was reported that 88.7% of supplemental glucosamine administered orally was absorbed, although only 44% was bioavailable due the extensive hepatic first-pass metabolism.9 A more recent study suggested that first pass metabolism actually occurs in the intestine rather than the liver with a bioavailability of glucosamine of only 19%.10 The elimination half life of radioactivity after single oral dose administration of radiolabeled glucosamine was 58 hours in human pharmacokinetic studies.9 Pharmacological principles dictate that a drug is eliminated from the system after 5 half lives. Hence 22 glucosamine would be eliminated after 290 hours or 12 days. Radiolabeled glucosamine was also shown to be distributed extensively to various tissues including articular cartilage.9 Whether glucosamine reaches the articular cartilage as an intact molecule is not clear from this study, since the radioactivity that was detected in the joints could have resulted equally well from degraded glucosamine.9 However, a more recent study in dogs, using nuclear magnetic resonance spectroscopy, demonstrated the incorporation of radiolabeled glucosamine into proteoglycans, suggesting that glucosamine is indeed bioavailable to chondrocytes." In addition, a recent study showed that glucosamine is the preferred substrate over glucose in in vitro cartilage tissue cultures.12 As a result, if glucosamine is bioavailable and used by chondrocytes for the production of extracellular matrix, then it could be of benefit in the treatment of OA. 3.2 Efficacy Of Glucosamine For Symptom Modification Several glucosamine trials were performed initially in the early 1980's and subsequently in the 1990's.13'14' l5'16'17' ,8'19'20>21'22 All of these studies reported a benefit of 13 14 15 16 17 18 192021 22 glucosamine in 0 A. , J ' , H ' ' J ' , o '" ' , 0 ' , : ' ' z u ' z ' ' z z A critical review of this literature was conducted at the onset of this thesis project in 1998 to determine whether there was any evidence for the efficacy of glucosamine in the treatment of knee OA. 2 3 This evaluation of glucosamine studies revealed suboptimal methodology and/or reporting in most studies, although a definite improvement was noted in study design and reporting in more recent studies. Methodological limitations included open study design,16 difficulties with blinding,13'15'16 13151617 1315 poorly defined disease ' ' ' or study outcome, ' lack of sample size calculations13'14'15'16'17'18'19'22 and lack of intent-to-treat analysis.13'14'15'16'17'22 In addition, the 23 primary outcome was frequently not stated. Three studies evaluated glucosamine in comparison to ibuprofen, a commonly used nonsteroidal anti-inflammatory drug, and reported that glucosamine was as effective as ibuprofen in reducing pain. ' ' However, the lack of a placebo control arm and lack of design as an equivalence trial in these studies 18 21 22 imposes some limitations on the conclusions, ' ' particularly in view of the small sample size of 38 patients used in one study. 1 8 One study applied glucosamine as an intramuscular injection. Glucosamine for injection is not available in North America.19 The study duration in these trials ranged from 3 to 8 wee\[SuMWM*MW& A s a r e s u l t j t h e i o n g . t e r m efficacy of glucosamine had not been evaluated. In addition, a notable finding from our literature review was that all studies had been conducted in affiliation with a pharmaceutical producer of glucosamine. The first study to be conducted independent of pharmaceutical sponsorship was a Canadian study by Houpt et al.2 4 In this 8-week trial, which included 99 patients, no significant difference in the primary outcome of pain reduction was seen between the glucosamine and placebo treated patients as measured by the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index. The study by Houpt et al 2 4 was also the first to evaluate glucosamine hydrochloride, rather than glucosamine sulfate. Although there was no prior indication that glucosamine hydrochloride might be less effective than glucosamine sulfate, the negative trial findings could potentially be explained on that basis. As a result, this negative trial added controversy to the use of glucosamine and raised further questions about its efficacy. The need for further trials was evident. In 1999, the US National Institutes of Health funded a large multicenter trial to evaluate the efficacy of glucosamine. In a meta-analysis by McAlindon et al25 in 2000, which included six glucosamine trials, an effect size of 0.44 was reported with inclusion of all trials. This effect size was 24 considered moderate, according to the scale by Cohen,26 where 0.2 reflects a small effect, 0.5 a moderate effect and 0.8 a large effect. However, the conclusions of the meta-analysis were tempered by the additional result of an effect size of 0.7 for lower quality trials with a quality score below the median, compared to an effect size of 0.3 for higher quality trials with a quality score above the median, and by the finding of a publication bias for positive glucosamine studies. Hence, McAlindon et al 2 5 called for further high quality independent studies of glucosamine. This recommendation was echoed by the updated 2000 American College of Rheumatology guidelines for the treatment of OA, which stated that recommending glucosamine for the treatment of knee OA was premature. From the discussion above and in Chapter 2, it is evident that this thesis was initiated at a time when glucosamine became widely accepted and used in the community despite limited evidence for efficacy. Since then, further glucosamine studies, both positive and negative, have been published. Chapter 5 presents an updated glucosamine literature review. The review not only provides a critical assessment of the current glucosamine literature, but also describes the various glucosamine study populations and study characteristics in an attempt to gain insight into the differences and commonalities amongst negative and positive glucosamine studies. As discussed in Chapter 5, the publication of several additional studies since 1998 on the efficacy of glucosamine as a symptom modifying drug has not reduced the controversy. As a result, the evaluation of glucosamine in a discontinuation trial provides further insight and a unique perspective on the efficacy of glucosamine. 25 3.3 Efficacy Of Glucosamine For Disease Modification Glucosamine has been investigated for its potential disease modifying effects in in vitro ' ' ' ' ' and in animal studies. ' ' Specifically, the effect of glucosamine on different cartilage anabolic and catabolic pathways has been reported, including stimulatory effects on the synthesis of proteoglycan27 and aggrecan,28 as well as inhibition of aggrecan degradation,29 inhibition of MMP-1 (type II collagen degrading enzyme),30 and MMP-3 (aggrecan degrading enzyme),28 and inhibition of pro-inflammatory mediators, such as NF-KB, cyclooxygenase-2, nitric oxide, ' and prostaglanding E 2 . ' Although several of these pathways have been suggested to contribute to the disease-modifying effects of glucosamine, limited human in vivo data are available to support these hypotheses. Nakamura et al 3 0 evaluated 40 OA patients treated with glucosamine and 15 age-matched controls. Serum PGE 2 concentrations and anti-type II collagen antibody titers were reduced to the level of healthy controls after treatment with glucosamine for 3 months. To date, this study has only been reported in abstract form.30 No other human studies have evaluated the biochemical effects of glucosamine on cartilage. In animal studies, a reduction in the development of cartilage lesions was observed in glucosamine treated animals.33'34'35 Altman et al 3 3 evaluated the effect of glucosamine on meniscectomy induced OA in rabbits. Gross and microscopic appearance of cartilage was noted to be more normal in glucosamine treated animals. In addition, a reduction in MMP-1 (type II collagen degrading enzyme) and MMP-3 (aggrecan degrading enzyme) was seen on immunohistochemistry, but not by other analyses.33 In this study, supraphysiologic doses of glucosamine were used.33 In a similar study by Conrozier et al,3 4 using similarly large doses of glucosamine, a reduction in cartilage lesions was reported in a rabbit cruciate ligament 35 transsection model of OA. Oegema et al assessed glucosamine effects in rabbits with 26 enzymatically induced acute joint damage. A positive effect of glucosamine on cartilage glycosaminoglycan (GAG) content was noted in the glucosamine treated rabbits compared to controls. However, this effect was more pronounced in the low dose glucosamine group than the high dose group. In addition, several measurements were not affected by treatment with glucosamine, but rather by the normal response to injury and repair, since the no-glucosamine treated rabbits had similar increases in measurements than the low and high dose glucosamine treated animals.35 Although these animal studies provide further information on the possible disease modifying effects of glucosamine, such effects may be limited to the models studied. That is, acute injury induced cartilage lesions may to some extent be responsive to supplemental glucosamine, which is in increased demand during the acute repair process. The applicability of these findings to human chronic OA is unclear. Given that moderately advanced OA is characterized by excessive cartilage degradation, the biochemical inhibition of such degradation by glucosamine represents a key question that has been incompletely addressed. As noted above, a few glucosamine studies have reported a reduction in MMP-1 ' and MMP-3, ' enzymes involved in the degradation of type II collagen and aggrecan, respectively. Although MMP-1 is thought to be important for the degradation of type II collagen,3'4 it has been suggested recently that MMP-13 may play a more central role in this process than MMP-1.4 Interestingly, MMP-13 was 30 33 evaluated in two of these studies, ' both of which are only available as abstracts, but was found either not to be affected by glucosamine33 or not reported in the results.30 Because the effect of glucosamine on MMP-13 has not been evaluated to any extent, it is not clear whether glucosamine use is associated with an actual reduction in type II collagen degradation. As a result, there is a distinct lack of information of the effect of glucosamine on type II collagen degradation in human in vivo studies. Hence, the need for further evaluations 27 in human clinical trials is evident. As a result, this thesis project includes an evaluation of novel biomarkers to assess type II collagen degradation in OA. The specific biomarkers for type II collagen degradation will be discussed in detail in Chapter 7. Despite the limited human data available for the biochemical effects of glucosamine, studies have evaluated the disease-modifying effects in human trials using alternative methodologies. In the study by Drovanti et al,1 7 cartilage biopsies were taken opportunistically following injury in two glucosamine and two placebo treated patients. Scanning electron microscopy showed slight osteoarthritic changes in the cartilage of glucosamine patients, while severe or definite osteoarthritic changes were noted in the placebo patients. The interpretation of these findings is limited, however, since cartilage sampling may have been selective and was limited to 4 subjects with unknown duration and severity of OA. Other than these preliminary data reported by Drovanti et al , 1 7 the disease-modifying effects of glucosamine had not been evaluated in any human studies at the time of initiation of this thesis project. More recently, two randomized double-blind placebo-controlled trials have evaluated the efficacy of glucosamine on radiographic disease progression in knee OA over 3 years.36'37 Both studies reported significantly reduced joint space narrowing in the glucosamine group compared to placebo after 3 years. A major difficulty with interpretation of these findings has occurred due to the use of weight-bearing full-extension radiographic views of the knee in both studies.38 This radiographic technique uses an unstandardized knee position. Compared to a standardized knee position, the unstandardized one reduces the reproducibility of the 39 measurement for joint space width. Specifically, when the unstandardized weight-bearing full-extension technique is used, the measurement of joint space width was shown to be altered significantly over a 1 to 12 week period simply by a change in joint pain.40 28 Furthermore, the contribution of meniscal extrusion to radiographic joint space narrowing was demonstrated in a magnetic resonance imaging study.41 In this study, all 32 patients with joint space narrowing on plain radiography had meniscal extrusion on MRI.4 1 However, in 17/32 (53%) of these patients no cartilage lesions were seen on MRI. 4 1 This suggests that meniscal extrusion, rather than cartilage degeneration, was responsible for the finding of joint space narrowing on plain radiography. As a result, the progression of joint space narrowing on radiographs may not be due to a progression of cartilage degeneration. Therefore, although these two 3-year studies have been widely touted as evidence for the disease-modifying effects of glucosamine, limited conclusions can be drawn from these studies due to the above considerations. In summary, the evidence for efficacy of glucosamine on pain and symptomatic relief is inconclusive. The recommendation for further methodologically rigorous trials is consistent and echoed by different critical reviews. The disease modifying activity of glucosamine is similarly inconclusive. Although biochemical effects of glucosamine on cartilage tissue have been demonstrated, the extrapolation from in vitro and animal studies to human OA is not possible. Ultimately, the investigation of such effects will be required in human OA trials. Hence, the need for further research is clearly evident. 29 3.4 References 1) de los Reyes GC, Koda RT, Lien EJ. Glucosamine and chondroitin sulfates in the treatment of osteoarthritis: a survey. Progress Drug Res 2000;55:81-103. 2 ) Wollheim FA. Pathogenesis of osteoarthritis, in: Hochberg MC, Silman AJ, Smolen JS, Weinblatt ME, Weisman MH, editors. Rheumatology. 3rd ed. Edinburgh: Elsevier Limited; 2003. p. 1801-15. 3 ) Poole AR, Kobayashi M, Yasuda T, Laverty S, Mwale F, Kojima T, et al. Type II collagen degradation and its regulation in articular cartilage in OA. Ann Rheum Dis 2002;61 (Suppl II):ii78-81. 4 ) Dahlberg L, Billinghurst RC, Manner P, Nelson F, Webb G, Ionescu M et al. Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1). Arthritis Rheum 2000;43:673-82. 5 ) Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage. Arthritis Rheum 2001;44:585-94. 6) Matheson AJ, Perry CM. Glucosamine. A review of its use in the management of osteoarthritis. Drugs Aging 2003;20:1041-60. 7 ) McCarty MF. Enhanced synovial production of hyaluronic acid may explain rapid clinical response to high-dose glucosamine in osteoarthritis. Med Hypotheses 1998;50:507-10. 8 ) Brandt KD, Block JA, Michalski JP, Moreland LW, Caldwell JR, Lavin PT. Efficacy and safety of intraarticular sodium hyaluronate in knee osteoarthritis. 30 ORTHOVISC Study Group. Clin Orthop Rel Res 2001;385:130-43. 9) Setnikar I, Rovati LC. Absorption, distribution, metabolism and excretion of glucosamine sulfate. Arzneimittelforschung 2001;51:699-725. 10) Aghazadeh-Habashi A, Sattari S, Pasutto F, Jamali F. Single dose pharmacokinetics and bioavailability of glucosamine in the rat. J Pharm Pharm Sci 2002;5:181-4. 11 ) Dodge GR, Regatte RR, Hall JO, Borthakur A, Sarma AV, Callaway DA, et al. The fate of oral glucosamine traced by DC-labeling in the dog. Arthritis Rheum 2001;44(suppl):S308. 12 ) Noyszewski EA, Wroblewski K, Dodge GR, Kudchodkar S, Beers J, Sarma AVS, et al. Preferential incorporation of glucosamine into the galactosamine moieties of chondroitin sulfates in articular cartilage explants. Arthritis Rheum 2001 ;44:1089-95. 13) Crolle G, D'Este E. Glucosamine sulphate for the management of arthrosis: a controlled clinical investigation. Curr Med Res Opin 1980;7:104-9. 14) Pujalte JM, Llavore EP, Ylescupidez FR. Double-blind clinical evaluation of oral glucosamine sulphate in the basic treatment of osteoarthrosis. Curr Med Res Opin 1980;7:110-4. 15) D'Ambrosio E, Casa B, Bompani R, Scab G, Scali M. Glucosamine sulphate: a controlled clinical investigation in arthrosis. Pharmatherapeutica 1981;2:504-8. 16) Tapadinhas MJ, Rivera IC, Bignamini AA. Oral glucosamine sulphate in the management of arthrosis: report on a multi-centre open investigation in Portugal. Pharmatherapeutica 1982;3:157-68. 17) Drovanti A, Bignamini AA, Rovati AL. Therapeutic activity of oral glucosamine 31 sulfate in osteoarthrosis: a placebo-controlled double-blind investigation. Clin Ther 1980;3:260-72. 18) Lopez Vaz A. Double-blind clinical evaluation of the relative efficacy of ibuprofen and glucosamine sulphate in the management of osteoarthrosis of the knee on out-patients. Curr Med Res Opin 1982;8:145-9. 19) Reichelt A, Forster KK, Fischer M, Rovati LC, Setnikar I. Efficacy and safety of intramuscular glucosamine sulfate in osteoarthritis of the knee. A randomised, placebo-controlled, double-blind study. Arzneimittelforschung 1994;44:75-80. 20) Noack W, Fischer M, Forster KK, Rovati LC, Setnikar I. Glucosamine sulfate in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:51-9. 21) Miiller-Fassbender H, Bach GL, Haase W, Rovati LC. Setnikar I. Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:61-9. 22) Qiu GX, Gao SN, Giacovelli G, Rovati L, Setnikar I. Efficacy and safety of glucosamine sulfate versus ibuprofen in patients with knee osteoarthritis. Arzneimittelforschung 1998 ;48:469-74. 23 ) Cibere J, Esdaile JM. Glucosamine use in osteoarthritis: What is the evidence for its efficacy? J Rheumatol 1999;26:1628. 24) Houpt JB, McMillan R, Paget-Dellio D, Russell A, Gahunia HK. Effect of glucosamine hydrochloride (GHcl) in the treatment of pain of osteoarthritis of the knee. J Rheumatol 1999;26:2423-30. 25 ) McAlindon TE, LaValley MP, Gulin JP, Felson DT. Glucosamine and chondroitin for treatment of osteoarthritis. A systematic quality assessment and meta-analysis. 32 JAMA 2000;283:1469-75. 26 ) Cohen J. Statistical power analysis for the behavioral sciences. 2nd ed. Hillsdale, NJ: Lawrence Erlbaum Assoc; 1988. 27) Bassleer C, Rovati L, Franchimont P. Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro. Osteoarthritis Cartil 1998;6:427-34. 28 ) Dodge GR, Jimenez SA. Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartil 2003;11:424-32. 29 ) Sandy JD, Gamett D, Thompson V, Verscharen C. Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine. Biochem J 1998;335:59-66. 30 ) Nakamura H, Tanaka M, Masuko-Hongo K, Kato T, et al. Clinical effects and possible mechanisms of glucosamine in the treatment of osteoarthritis. Arthritis Rheum 2001;44 (suppl):S309. 31) Largo R, Alvarez-Soria MA, Diez-Ortego I, Calvo E, Sanchez-Pernaute O, Egido J, et al. Glucosamine inhibits IL-l(3-induced N F K B activation in human osteoarthritic chondrocytes. Osteoarthritis Cartil 2003; 11:290-8. 32) Meininger CJ, Kelly KA, Li H, Haynes TE, Wu G. Glucosamine inhibits inducible nitric oxide synthesis. Biochem Biophys Res Comm 2000;279:234-9. 33 ) Altman RD, Cheung H. Glucosamine sulfate on cartilage: Lapine study. Arthritis Rheum 2001 ;44 (suppl):S308. 34) Conrozier T, Mathieu P, Piperno M, Richard S, Annefeld M, Richard M, et al. 33 Glucosamine sulfate significantly reduced cartilage destruction in a rabbit model of osteoarthritis. Arthritis Rheum 1998;41 (suppl):S147. 35 ) Oegema TR Jr, Deloria LB, Sandy JD, Hart DA. Effect of oral glucosamine on cartilage and meniscus in normal and chymopapain-injected knees of young rabbits. Arthritis Rheum 2002;46:2495-2503. 36 ) Reginster Reginster JY, Deroisy R, Rovati LC, et al. Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial. Lancet 2001;357:251-6. 37) Pavelka K, Gatterova J, Olejarova M, Machacek S, Giacovelli G, Rovati LC. Glucosamine sulfate use and delay of progression of knee osteoarthritis. A 3-year, randomized, placebo-controlled, double-blind study. Arch Intern Med 2002;162:2113-23. 38 ) McAlindon T. Why are clinical trials of glucosamine no longer uniformly positive? Rheum Dis Clin North Am 2003;29:789-801. 39 ) Buckland-Wright JC, Wolfe F, Ward RJ, Flowers N, Hayne C. Substantial superiority of semiflexed (MTP) views in knee osteoarthritis: A comparative radiographic study, without fluoroscopy, of standing extended, semiflexed (MTP), and Schuss views. J Rheumatol 1999;26:2664-74. 40 ) Mazzuca SA, Brandt KD, Lane KA, Katz BP. Knee pain reduces joint space width in conventional standing anteroposterior radiographs of osteoarthritic knees. Arthritis Rheum 2002;46:1223-7. 41 ) Adams JG, McAlindon T, Dimasi M, Carey J, Eustace S. Contribution of meniscal extrusion and cartilage loss to joint space narrowing in osteoarthritis. Clin Radiol 34 1999;54:502-6. 35 CHAPTER 4 RANDOMIZED DISCONTINUATION TRIAL The randomized discontinuation trial (RDT) is a form of randomized controlled trial (RCT).1 Although it has been used infrequently, it can be used as an alternative to the classic RCT for the evaluation of efficacy.1 The RDT design was first described in 1975 as a method to study drug efficacy while minimizing patient exposure to placebo.2 Since then the efficacy of different drug treatments has been demonstrated in various medical conditions using an RDT design.3'4'5'6 In the RDT, basic study design methodologies such as randomization, double-blinding and placebo control require the same considerations as for a standard RCT. 1 However, there are RDT design issues that are distinct from the classic RCT. These will be discussed here, as well as the advantages and disadvantages of the RDT design. 4.1 Randomized Discontinuation Trial Design Considerations One of the main features of an RDT consists of the inclusion of responders into the study. This can be achieved in two ways. Subjects can be entered into phase I of the trial prior to randomization (run-in period) and assessed for response. Alternatively, response to treatment can be assessed retrospectively if a treatment has been available for use in normal clinical practice. This pre-selection of responders for inclusion into an RDT study will tend to increase any difference in outcome compared to that seen in a classic trial.7 In other words, in relation to the source population, which includes all patients with a given condition that might use the study treatment, the RDT design can result in an overestimate of treatment benefit.1 As a result, the quantitative results of an RDT are not generalizable to 36 the source population. However, it has been noted that the qualitative result of an RDT, that is whether a treatment is efficacious or not, is generalizable to the source population. Although this overestimate of benefit can be viewed as a disadvantage, it also confers certain advantages of this study design over the classic RCT. Kopec et al7 demonstrated that the pre-selection of responders into an RDT results in a more efficient trial with reduced sample size requirements. In addition, the exclusion of non-responders from the trial will reduce the likelihood of a type II error, false acceptance of the null hypothesis, compared to the classic RCT. 8 Consequently, a negative trial result from an RDT may lend itself to a more definitive conclusion, assuming that the standard reasons for a negative trial have been considered. The efficiency of an RDT can be significantly improved by increasing the specificity of the criteria for identifying responders.7 Since specificity is related to the number of false responders that are included, more stringent responder criteria will allow for greater efficiency of the study and hence greater ability to show a treatment benefit. This principle was applied in this thesis project by inclusion of only those subjects that had reported at least moderate response to glucosamine. The efficiency of the RDT is further improved by elimination of non-compliers and adverse reactors from the study.7 If a treatment has been used in clinical practice, patients who develop side effects to the treatment will likely stop the treatment and hence be eliminated from the pool of potential study subjects. As a result, one disadvantage of the RDT design is that potential drug toxicity cannot be fully evaluated. In a setting where a drug is widely used in the population, such as was the case with glucosamine, a classic RCT design may present a challenge due to difficulties with recruitment and hence the timely conduct of such a trial. In contrast, such considerations do 37 not represent a concern in the RDT design, since this trial specifically requires the inclusion of subjects who are using the treatment of interest. As a result, the RDT may lend itself particularly well to the evaluation of treatments that are widely used in the community despite inconclusive evidence for efficacy. However, given that subjects have previously been exposed to the study treatment, the risk of unblinding may be greater in an RDT compared to a classic trial.7 An evaluation of the effectiveness of blinding, which is generally recommended for clinical trials, should therefore be considered in the conduct of all RDTs. As will be discussed in Chapter 6, subject blinding was evaluated in this study and was not compromised. A further consideration in the RDT design relates to the outcome measures used. The discontinuation of a treatment will necessitate using a flare or worsening of disease as the outcome. Because RDTs are infrequently used, a flare of disease is not a standard outcome assessment. This difficulty can be overcome by the use of a definition of disease flare that is based on a validated outcome measure, which can be applied equally well in settings of improvement or worsening of symptoms. In OA, the Western Ontario and McMaster Universities (WOMAC) OA index is such an outcome measure. The WOMAC has been validated,9 is extensively used in knee OA, and is recommended as an outcome measure in OA trials.10 Similarly, the physician global assessment has been widely used and is applicable to an evaluation of disease flare.11 The use of these two outcome measures for the definition of disease flare is elaborated in Chapter 6, where the study methodology is discussed in more detail. Since a worsening of symptoms is the usual outcome in an RDT design, the study duration needs to be chosen carefully to allow for observation of such an outcome. Too 38 short a study duration will increase the risk of a type II error of falsely accepting the null hypothesis of no difference. The onset of action of glucosamine is thought to occur within 2 to 12 weeks. As a result, evaluation of flare after discontinuation of glucosamine requires at least 12 weeks of follow-up. This estimate was doubled in the current study with a follow-up of 24 weeks. As discussed in Chapter 3, given that glucosamine appears to be eliminated with a half life of 58 hours after oral administration, a study duration of 24 weeks is reasonable. The outcome of flare also presumes that the treatment being evaluated is not curative. Although glucosamine has been hailed as a cure by the lay press, there is no scientific evidence of a curative effect. Ethical considerations also dictate that subjects who flare should be withdrawn from the study, having reached the end point. This reduces exposure to placebo, which some consider an advantage.2 Because of the risk of flare, the use of concomitant medications, such as analgesics, may be indicated in an RDT and will likely enhance recruitment of potential participants. In addition to the above study design issues, a more important aspect of the difference in RDT to RCT relates to the interpretation of results and the applicability of the findings. In clinical practice it is often not clear how long a patient needs to be maintained on a treatment or whether long-term maintenance is beneficial. This question can be answered using an RDT design. A positive finding from a discontinuation trial not only suggests efficacy of a treatment, but also that continuation of such a treatment confers a benefit. In contrast, a negative RDT result allows for several possible explanations. As mentioned above, a curative treatment does not lead to the development of flare and hence will result in a negative trial. An alternate explanation relates to the possibility that a drug may only be of short-term benefit. However, Kopec et al7 noted that both these explanations 39 seem unlikely, particularly for drug therapies in chronic conditions. A more plausible interpretation of a negative RDT is that the drug has no efficacy. Regardless of the underlying explanation, and assuming that other standard considerations of a negative trial have been excluded, a negative RDT does allow for a conclusion that the long-term maintenance of a treatment is not warranted. From this discussion, it is clear that the RDT study design has some definite advantages over the RCT design, particularly in the setting of a negative trial result. In this thesis, one of the main reasons for employing this study design, was the consideration of feasibility of recruitment given the high prevalence of glucosamine use in the community in patients with knee OA. Additionally, in view of the fact that patients often remain on glucosamine for years, this study was designed as a discontinuation trial to provide novel insight into whether the maintenance of glucosamine treatment was beneficial, a question that had not been answered previously. 40 4.2 References 1 ) Friedman LM, Furberg CD, DeMets DL. Fundamentals of Clinical Trials, 3rd ed. New York: Springer; 1998. 2 ) Amery W, Dony J. A clinical trial design avoiding undue placebo treatment. J Clin Pharmacol 1975;15:674-9. 3 ) The Canadian Hydroxychloroquine Study Group. A randomized study of the effect of withdrawing hydroxychloroquine sulfate in systemic lupus erythematosus. N Engl JMed 1991;324:150-4. 4) Caldwell JR, Furst DE, Smith AL, Clark JA, Bonebrake RA, et al. Flare during drug withdrawal as a method to support efficacy in rheumatoid arthritis: amiprilose hydrochloride as an example in a double blind, randomized study. J Rheumatol 1998;25:30-5. 5 ) Packer M, Gheorghaide M, Young JB, Costantini PJ, Adams KF, et al. Withdrawal of digoxin from patients with chronic heart failure treated with angiotensin-converting enzyme inhibitors. N Engl J Med 1993;329:1-7. 6 ) Stewart JW, Tricamo E, McGrath PJ, Quitkin FM. Prophylactic efficacy of phenelzine and imipramine in chronic atypical depression: likelihood of recurrence on discontinuation after 6 months' remission. Am J Psychiatry 1997;154:31-6. 7 ) Kopec JA, Abrahamowicz M, Esdaile JM. Randomized discontinuation trials: Utility and efficiency. J Clin Epidemiol 1993;46:959-71. 8 ) Quitkin FM, Rabkin JG. Methodological problems in studies of depressive disorder: Utility of the discontinuation design. J Clin Psychopharmacol 1981;1:283-8. 9 ) Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW. Validation study of 41 WOMAC: A health status instrument for measuring clinically important participant relevant outcomes to antirheumatic drug therapy in participants with osteoarthritis of the hip or knee. J Rheumatol 1988;15:1833-40. 10 ) Altman R, Brandt K, Hochberg M, Moskowitz R, Bellamy N, Bloch DA, et al. Design and conduct of clinical trials in patients with osteoarthritis: Recommendations from a task force of the Osteoarthritis Research Society. Osteoarthritis Cartil 1996;4:217-43. 11 ) Ehrich EW, Davies GM, Watson DJ, Bolognese JA, Seidenberg BC, Bellamy N. Minimal perceptible clinical improvement with the Western Ontario and McMaster Universities osteoarthritis index questionnaire and global assessments in patients with osteoarthritis. J Rheumatol 2000;27:2635-41. 42 CHAPTER 5 CRITICAL ASSESSMENT AND REVIEW OF GLUCOSAMINE LITERATURE This manuscript is currently submitted for consideration for publication to Seminars in Arthritis and Rheumatism under the title "Critical assessment and review of glucosamine literature". The candidate is the first author. Drs. John M. Esdaile, Jacek A. Kopec, Anona Thorne, and Joel Singer are co-authors. Dr. Esdaile was involved with the critical review of papers. Drs. Esdaile and Kopec are co-supervisors of the candidate, and Dr. Singer and Ms. Thorne are members of the supervisory committee. The candidate's role in this manuscript involved the development of study methodology, literature search, critical review of papers, all data entry and analysis of results, as well as the writing of the final manuscript. 5.1 Introduction Osteoarthritis (OA) is the most common joint disease worldwide. Symptomatic knee OA is estimated to affect 6% of adults above the age of 30.' OA causes significant disability, work disability and economic cost2'3'4'5'6 and given the aging of the population, the economic burden for OA has been projected to increase substantially. Current treatments are limited to weight loss, exercise and other non-pharmacological interventions, pain management with analgesics or nonsteroidal anti-inflammatory drugs (NSAIDs) and joint replacement surgery for advanced disease. Pharmacological treatment with NSAIDs is frequently limited by side effects, ' while non-pharmacological therapies, such as weight loss or exercise, are not easily implemented in practice or maintained over time. In addition, 43 these treatments are directed at symptom modification and do not alter the progression of disease. Recently, supplemental glucosamine has been promoted in books and in the media as a treatment for OA symptoms and to slow the progression of disease.10 Despite numerous published trials, in 2000, the American College of Rheumatology OA guidelines did not recommend glucosamine as a treatment for OA, stating that such a recommendation was premature.11 In contrast, in Europe, where glucosamine is available as a prescription drug in some countries, the 2000 European League Against Rheumatism recommendations for the management of knee OA included glucosamine, although this recommendation was based on an evaluation of only three glucosamine trials.12 Recent meta-analyses have also reported effect sizes for glucosamine in the treatment of knee OA that range from small to large.13'14'15 However, methodological weaknesses such as difficulties with allocation concealment, lack of standardization of OA diagnosis and outcome assessment, and poor description of blinding and randomization were noted.13'14 Since the publication of these meta-analyses further glucosamine trials, with both positive and negative results, have been published. As a result, the efficacy of glucosamine continues to be controversial. The purpose of this review was to provide a critical assessment and updated review of the published literature on glucosamine for the treatment of knee OA. 5.2 Methods Literature search: An electronic search was conducted in Pre-Medline, Medline, Embase, and the Cochrane Central Register of Controlled Trials from 1966 to February 2003 using the terms osteoarthritis, osteoarthrosis, gonarthrosis, glucosamine and trial as Medical 44 Subject Heading terms and as textwords. In addition, references of journal articles and review articles were systematically reviewed for identification of further publications. Articles were included in this qualitative assessment i f they met the following criteria: 1) randomized controlled trial (RCT) , 2) inclusion of subjects with knee O A , 3) published in English, 4) full-length article. Studies that evaluated glucosamine in combination with chondroitin were excluded. Quality assessment: Studies were scored using the quality assessment scale developed and validated by Jadad et a l . 1 6 This scale assesses study randomization, double blinding and withdrawals/dropouts with a maximum possible score of 5 points. The quality of all studies was assessed independently by two raters (JC, J M E ) . The final quality score was calculated as the mean of the two assessors' scores and converted to a percentage of the maximum attainable score. A rigorous study was arbitrarily defined a priori as one with a score of 60% or more. Studies that qualified as rigorous were then further evaluated for their study populations, study characteristics and reported outcomes. 5.3 Results Our literature search revealed fourteen randomized controlled trials, which met the i t i •. • 17 18 19 20 21 22 "H 24' 'S' ' f i ?7 28 29 30 m i r r . r i inclusion and exclusion criteria. The effect of glucosamine on 17 IK 19 0 ^ 1 2 ^  23 24 S^ 2() ^7 29 symptoms was studied in twelve trials, ' ' •- • a n ( j t w 0 trials reported both structure and symptom modifying effects of glucosamine over three years. 2 8 - 3 0 Study characteristics of these 14 trials are shown in Table 5.1. In general, the earlier studies, which were published in the early 1980's, included smaller patient numbers, used heterogeneous 17 18 19^02! patient populations and were of shorter duration. ' ' ' 45 The mean quality assessment scores for each study are shown in Figure 5.1. Five studies were found to lack methodological rigor and hence were not included in this 17 19 20 21 27 review. ' ' ' ' The remaining nine rigorous studies were evaluated 18 22 23 24 25 26 28 29 30 further. ^ ^ ^ ^ ^ T a b l e 5 2 summarizes the study population characteristics in these nine studies, while Table 5.3 shows study design characteristics and reported findings. The demographic characteristics of the study populations were similar (Table 5.2). Mean age ranged from 54 to 66 years, in keeping with the usual age of subjects with OA. Most studies enrolled a majority of women, while one study enrolled mostly men.24 Mean BMI was reported as 27 and 26 in two studies,28'30 respectively, and mean weight was reported as ranging from 71kg to 82kg in three others. ' ' The remaining four studies did not report 24 28 29 30 either measure. Mean OA duration ranged from 5 to 11 years. ' ' ' Two studies reported that the majority of subjects had an OA duration between 2 and 10 years. ' Houpt et al reported mean symptom duration of 8 years. Radiographic severity of OA was not reported in 3 studies.18'24'25 Of the remaining six studies, four enrolled patients with predominantly mild radiographic changes with 61% to 74% of patients having grade 1 (minimal) or grade 2 (mild) OA. ' ' ' Pavelka et al enrolled similar proportions of mild (grade 2) and moderate (grade 3) radiographic OA. Hughes et al 2 9 enrolled predominantly advanced OA with 60%> of patients having either grade 3 (moderate) or grade 4 (severe) OA. Table 5.3 shows a summary of study design characteristics and study results for the nine rigorous glucosamine trials. With the exception of one study by Houpt et al,2 6 which evaluated glucosamine hydrochloride, all studies used a glucosamine sulfate preparation. In addition, all trials that reported glucosamine to be efficacious, evaluated a glucosamine sulfate compound produced by the Rotta Pharmaceutical Company,18'22'23'24'25'28'30 while Hughes et al, 2 9 who showed no efficacy, evaluated a different glucosamine sulfate product. 46 Analgesic and NSAID use was variable (Table 5.3). Three studies allowed 22 30 26 concomitant use of analgesics - two showed efficacy ' and one did not. Two studies allowed both analgesic and NSAID use. Of these, one showed efficacy28 while the other did not.29 All of these studies allowing concomitant pain medications, except one,22 used patient diaries and reported the use of analgesics and/or NSAIDs to be similar in the two treatment groups. Most studies used an intent-to-treat analysis. Loss to follow-up was minimal in all studies except the 2 three-year studies. Reginster et al 2 3 reported 33% and 36% of patients lost to follow-up in the placebo and glucosamine groups, respectively, while Pavelka et al 2 4 had a loss of follow-up of 46% and 35%, respectively. The most commonly used outcome measures were the Lequesne index, the Western Ontario and McMaster Universities (WOMAC) OA Index, physician global assessment and patient pain assessment, which are recommended outcome measures in OA trials.31 A measurement of radiographic joint space width was used as the outcome in the two studies assessing the structure-modifying effects of glucosamine.28'30 Both studies used an unstandardized weight-bearing extended-view radiographic technique, which was recently deemed insufficient for the evaluation of OA disease progression.32 Seven of 9 studies (78%>) reported efficacy of glucosamine with significant differences in favor of glucosamine 18 22 23 28 30 compared to placebo ' ' ' ' or with no significant difference between glucosamine and 24 25 26 29 ibuprofen treated groups. ' Two studies (22%) found no efficacy of glucosamine. ' Pharmaceutical affiliation was present in all studies that reported glucosamine to be • o 22 23 24 25 28 30 efficacious, whereas the two negative trials were conducted independent of pharmaceutical affiliation.26'29 47 5.4 Discussion This review of rigorous glucosamine trials revealed positive findings in favor of glucosamine for the majority of studies, in keeping with the results of recent meta-analyses.13'14'15 Although the use of a quality assessment scale assists in selecting more rigorous studies for inclusion, the application of such a scale does not guarantee the absence of bias. In addition, the cut off of a 60% quality score for a rigorous study was chosen arbitrarily. However, a change in cut off to a higher level, for example 80%, would have resulted in most positive and all negative studies still being included in this review (Figure 5.1). Similarly a lower cutoff of 50% would have included two additional trials, one with positive and one with negative results. Hence, the overall discussion and conclusions of this qualitative review would not have been altered substantially. The Jadad quality assessment scale evaluates three key trial elements, including randomization, blinding and appropriate description of withdrawals. Only 3/9 rigorous studies in this review achieved a 100% quality score. Thus, in the other six studies, at least one of these important elements was not present, thereby potentially jeopardizing the validity of the study results. In addition to the key areas assessed by Jadad's scale, many other aspects of study design or conduct are not evaluated leaving further room for bias. In this review, most studies achieved full points on being double-blind and describing an appropriate method of blinding. However, blinding was rarely assessed at the end of the study. If unblinding occurs during the study, the results will undoubtedly be biased in favor of glucosamine. Most studies were described as randomized, but the method of randomization was infrequently described. Inappropriate randomization methods are associated with poor allocation concealment and unblinding, resulting in further potential biases. Withdrawals and dropouts were reported by all 9 rigorous studies. High drop-out 48 rates, ranging from 33% to 46%, were seen in the two 3-year studies assessing the effect of glucosamine on joint space narrowing. Although intent-to-treat analyses were performed, the assignment of worst case data to missing values when a greater loss to follow-up is present in the placebo group, will tend to exaggerate the results in favor of glucosamine and 30 hence will result in an overestimate of the beneficial effect of glucosamine. Recent meta-analyses have commented on methodological problems, which may be associated with exaggerated estimates of benefit, including difficulties with allocation concealment, absence of intent-to-treat analysis and lack of a primary efficacy variable stated a priori.n'XA In addition, lack of sample size calculations and lack of standardized OA diagnosis and/or outcome assessment was frequently noted, particularly in earlier studies.14 The importance of standardized techniques for radiographic outcomes has recently been highlighted by Mazzuca et al,33 who reported that a change in knee pain over 1 to 12 weeks was able to significantly alter the measurement of joint space width when a weight-bearing extended-view radiographic technique was employed. Since this radiographic technique was used in the two studies assessing disease progression,28,30 the conclusion of a structure modifying effect of glucosamine may not be valid. The importance of 13 methodological quality on effect size was demonstrated by McAlindon et al, who reported the pooled effect size for glucosamine to be 0.7 (moderate) for trials with a quality score below the median and 0.3 (small) for trials with a quality score above the median. The possibility that different patient subpopulations may respond better to treatment with glucosamine has been raised. In this review, a qualitative comparison of study populations and study design of rigorous trials was undertaken in an attempt to reconcile the results of negative and positive studies. As shown in Tables 5.2 and 5.3, study populations of positive and negative trials were similar with respect to age, gender, degree of obesity 49 and OA duration. Another consideration relates to whether the concomitant use of analgesic and/or NSAIDs can mask a response to glucosamine and hence result in a negative study. As shown in Table 5.3, analgesics and NSAIDs were allowed in both those studies that showed efficacy as well as those that did not. Therefore this does not appear to be an explanation for the difference in findings. Stage of disease may have an effect on whether a drug is found to be efficacious or not. Specifically, subjects with advanced OA may not respond as well to glucosamine compared to those with early OA. Most positive trials have evaluated predominantly mild OA, although some did not report OA severity. Of the two negative trials, one enrolled predominantly patients with mild OA (grade 1 or 2),26 while the other enrolled predominantly advanced OA (grade 3 or 4).29 Whether glucosamine has a differential effect depending on stage of disease has not been investigated. Such an effect cannot be excluded based on these limited data and should receive further consideration in future studies. The question of whether the sulfate component may have a beneficial effect rather than the glucosamine has been raised.34 Since most studies evaluated glucosamine sulfate, this hypothesis cannot be addressed from the available studies, although glucosamine hydrochloride was assessed by one of the negative trials. It is of interest that the studies showing efficacy all used a glucosamine sulfate product manufactured by the Rotta Pharmaceutical Company, while the negative trials used a different glucosamine preparation. The possibility of a product specific response needs to be considered. It is therefore not clear whether the favorable results observed in most trials are generalizable to all glucosamine preparations. In this review, with the exception of the glucosamine product evaluated, the pharmaceutical affiliation in positive studies, and possibly the stage of 50 disease, no other common denominator was found that might explain the dichotomy of positive and negative results in these glucosamine studies. Since this review was conducted, further glucosamine studies in knee OA have been published, including one full-length paper of topical glucosamine,35 an internet-based clinical trial of glucosamine,36 and one abstract of a glucosamine discontinuation trial (current thesis).37 Cohen et al 3 5 reported a significant difference in mean pain reduction between topical glucosamine sulfate and placebo at 8 weeks. However, the active preparation not only included glucosamine sulfate, but also chondroitin sulfate, camphor and peppermint oil, whereas the placebo cream only contained a lesser amount of peppermint oil. Therefore, pain reduction may have been mediated by one of the other ingredients rather than glucosamine. McAlindon et al reported on the feasibility of an internet-based clinical trial with a brief mention of no difference in WOMAC pain scores between glucosamine and placebo treated patients, although detailed glucosamine study results were not discussed. Cibere et al 3 7 reported no efficacy of glucosamine sulfate over 6 months in a randomized controlled glucosamine discontinuation trial, which included subjects who had previously at least moderately improved on glucosamine (current thesis, see Chapter 6). In the study by Cibere et al,3 7 subjects who received glucosamine developed a flare of OA symptoms as frequently, as quickly and as severely as those who received 37 placebo. In summary, the majority of trials to date have reported a beneficial effect of glucosamine. Despite this preponderance of positive results, given the recent negative trials, ' Z , ^' J D ' J ' the methodological issues raised previously ' and the fact that a publication bias exists in the glucosamine literature, the efficacy of glucosamine as a symptom or disease modifying drug cannot be established based on the current evidence. 51 Further high quality trials are needed to evaluate the efficacy of glucosamine and to determine which, i f any, patient subpopulations might respond best to treatment with glucosamine. 52 Table 5.1: Characteristics of Randomized Controlled Trials of Glucosamine in Knee Osteoarthritis Author Year Total number Study duration Disease/Joint studied Comparator drug Setting Crolle et al 1 7 1980 30 3 weeks Osteoarthrosis* Placebo In-patient Pujalte et al1 8 1980 20 6-8 weeks Knee OA Placebo Out-patient Drovanti et al 1 9 1980 80 4 weeks Generalized OA Placebo In-patient D'Ambrosio et al 2 0 1980 30 3 weeks Osteoarthrosis* Placebo In-patient Lopez Vas21 1982 38 8 weeks Knee OA Ibuprofen Out-patient Reichelt et al 2 2 1994 155 6 weeks Knee OA Placebo Out-patient Noack et al2 3 1994 252 4 weeks Knee OA Placebo Out-patient Muller-Fassbender et al 2 4 1994 199 4 weeks Knee OA Ibuprofen In-patient Qiu et al 2 5 1998 178 4 weeks Knee OA Ibuprofen Out-patient Houpt et al 2 6 1999 101 8 weeks Knee OA Placebo Out-patient Rindone et al 2 7 2000 114 8 weeks Knee OA Placebo Out-patient 9R Reginster et al 2001 212 3 years Knee OA Placebo Out-patient Hughes et al 2 9 2002 80 24 weeks Knee OA Placebo Out-patient Pavelka et al 3 0 2002 202 3 years Knee OA Placebo Out-patient OA = osteoarthritis; * joint not specified. 53 Table 5.2: Summary of Study Population Characteristics for Nine Rigorous Glucosamine Trials Mean % BMI or OA duration Kellgren-Lawrence age female weight X- ray grade (years) (kg) C GL C GL C GL C GL C GL Pujalte 65 69 90 80 NR NR NR etal18 Reichelt 57 56 64 66 74 kg 71 kg <2 yrs 28% 23% Grade 1 21% 28% et al 2 2 2-10 yrs 62% 68% Grade 2 50% 33% >10 yrs 10% 9% Grade 3 20% 29% Grade 4 0% 0% U 9% 10% Noack 55 55 62 59 74 kg 75 kg <2 yrs 42% 34% Grade 1 34% 34% et al 2 3 2-10 yrs 47% 51% Grade 2 39% 38% >10 yrs 11% 15% Grade 3 27% 28% Grade 4 0% 0% Muller- 54 54 53 42 NR Mean 5 5 NR Fassben-der et al 2 4 Qiu 56 56 84 73 NR NR NR et al2 5 Houpt 65 64 60 64 82 kg 78 kg Mean 8 8 Grade 1 26% et al 2 6 (symptom Grade 2 48% duration) Grade 3 18% Grade 4 8% Reginster 66 66 78 75 27 27 Mean 8 8 Grade 1 0% 0% et al 2 8 Grade 2 70% 71% Grade 3 30% 29% Grade 4 0% 0% Hughes 62 68 NR Mean 8 Grade 1 9% et al 2 9 Grade 2 31% Grade 3 37% Grade 4 23% Pavelka 64 61 76 79 26 26 Mean 11 10 Grade 1 0% 0% et al 3 0 Grade 2 53% 54% Grade 3 47% 46% Grade 4 0% 0% BMI = body mass index, C = control group, GL = glucosamine group, kg = kilogram, NR = not reported, U=unknown 54 Table 5.3: Summary of Study Design Characteristics and Reported Outcomes for Nine Rigorous Glucosamine Trials Type Concomitant ITT Loss to Outcome Study results Pharma-of Analgesics/ follow- ceutical GL NSAIDs up ( C :%) GL affiliation Pujalte S No/No No 17% 17% Patient pain (1-4), PL -0.24, GL-1.05 Yes etal 1 8 mean change p<0.01 Reichelt S Yes/No Yes 9% 8% Response rate* PL 30%, G L 5 1 % Yes et a l 2 2 p=0.015 Noack S No/No Yes 4% 5% Response rate* PL 37%, GL 52% Yes etal 2 3 p=0.016 Miiller- S No/No Yes 4% 6% Response rate** Ibuprofen 52%, Yes Fassben- GL 4 8 % der et a l 2 4 p=0.67 Qiu S Not No 10% 1% Composite pain score Ibuprofen -4.28, Yes eta l 2 5 reported (0-9), mean change GL -4.82 p>0.05 Houpt HC1 Yes/No Yes 4% 2% WOMAC pain (0- PL 0.8, GL 1.7 No eta l 2 6 20), mean reduction p=0.23 Reginster S Yes/Yes Yes 3 3 % 36% Joint space width PL -0.31,GL -0.06 Yes etal 2 8 (JSW), mean change p=0.043 Hughes S Yes/Y es Yes 8% 5% Patient global pain, PL 1065, GL 1081 No eta l 2 9 mean area under the curve (AUC) p=0.89 Pavelka s Yes/No Yes 4 6 % 35% Joint space width PL -0.19, GL0.04 Yes eta l 3 0 (JSW), mean change p=0.001 C=control group, GL=glucosamine, PL=placebo, S=sulfate, HCl=hydrochloride, NSAIDs=nonsteroidal anti-inflammatory drugs, ITT=Intent-to-treat. *Response rate based on a decrease of >3 points (0-24 scale) in Lequesne index and physician global assessment of moderate or good (worse, unchanged, moderate, good scale). ** Response rate based on a decrease of >2 points (0-34 scale) in Lequesne index if baseline value >12 points or decrease of >1 point in Lequesne index if baseline value <12 points, and physician global assessment of good (poor, moderate, good scale). 55 Figure 5.1: Quality Assessment Scores for Glucosamine Trials Crolle (1980) Pujalte (1980) Drovanti (1980) D'Ambrosio (1981) Lopes Vaz (1982) Reichelt (1994) Noack (1994) Muller-Fassbender (1994) Qiu (1998) Houpt (1999) Rindone (2000) Reginster (2001) Hughes (2002) Pavelka (2002) 190 140 s a - .-" !20 - ISO ISO • /;««;:' ; ' 90 190 I 80 ' ' ,V- " I w - .... = 150 • I70 | . . . • • I I I I I I i i i i i 10 20 30 40 50 60 70 80 Percent of maximum achievable score* 1100 1100 90 100 16 *Quality was assessed using the scale by Jadad et al. A study with a quality score of > 60% was considered rigorous. 56 5.5 References 1) Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum 1998;41:1343-55. 2) Davis MA, Ettinger WH, Neuhaus JM, Mallon KP. Knee osteoarthritis and physical functioning: Evidence from the NHANES I epidemiologic followup study. J Rheumatol 1991;18:591-8. 3) Badley EM. The effect of osteoarthritis on disability and health care use in Canada. J Rheumatol 1995;(suppl 43)22:19-22. 4) Yelin EH, Katz PP. Labor force participation among persons with musculoskeletal conditions, 1970-87. Arthritis Rheum 1991;34:1361-70. 5) Gabriel SE, Crowson CS, O'Fallon WM. Costs of osteoarthritis: Estimates from a geographically defined population. J Rheumatol 1995; (suppl 43)22:23-5. 6) Gabriel SE, Crowson CS, Campoin ME, O'Fallon WM. Indirect and nonmedical costs among people with rheumatoid arthritis and osteoarthritis compared with nonarthritic controls. J Rheumatol 1997;24:43-8. 7) Badley EM, Crotty M. An international comparison of the estimated effect of the aging of the population on the major cause of disablement, musculoskeletal disorders. J Rheumatol 1995;22:1934-40. 8) Gabriel SE, Jaakkimainen L, Bombardier C. Risk for serious gastrointestinal complications related to use of nonsteroidal anti-inflammatory drugs. A Meta-analysis. Ann Intern Med 1991;115:787-96. 9) Smalley WE, Ray WA, Daugherty JR, Griffin MR. Nonsteroidal anti-inflammatory drugs and the incidence of hospitalizations for peptic ulcer disease in elderly 57 persons. A m J Epidemiol 1995;141:539-45. Theodosakis J, Adderly B , Fox B . The arthritis cure. New York: St. Martin's Press; 1997. American College of Rheumatology subcommittee on osteoarthritis guidelines. Recommendations for the medical management of osteoarthritis of the hip and knee. Arthritis Rheum 2000;43:1905-15. Pendleton A , Arden N , Dougados M , Doherty M , Bannwarth B , Bijlsma JWJ, et al. E U L A R recommendations for the management of knee osteoarthritis: report of a task force of the Standing Committee for International Clinical Studies Including Therapeutic Trials (ESCISIT). A n n Rheum Dis 2000;59:936-44. McAl indon T E , LaVal ley M P , Gul in JP, Felson D T . Glucosamine and chondroitin for treatment of osteoarthritis. A systematic quality assessment and meta-analysis. J A M A 2000;283:1469-75. Towheed T E , Anastassiades TP, Shea B , Houpt J, Welch V , Hochberg M C . Glucosamine therapy for treating osteoarthritis (Cochrane review). Cochrane Database Syst Rev 2001; 1:CD002946. Richy F, Bruyere O, Ethgen O, Cucherat M , Henrotin Y , Reginster J Y . Structural and symptomatic efficacy of glucosamine and chondroitin in knee osteoarthritis. A comprehensive meta-analysis. Arch Intern M e d 2003;163:1514-22. Jadad A R , Moore R A , Carroll D , Jenkinson C, Reynolds J M , Gavaghan D J , McQuay H J . Assessing the quality of reports of randomized clinical trials: Is blinding necessary? Controlled C l i n Trials 1996;17:1-12. Crolle G , D'Este E . Glucosamine sulphate for the management of arthrosis: a controlled clinical investigation. Curr M e d Res Opin 1980;7:104-9. 5 8 18) Pujalte JM, Llavore EP, Ylescupidez FR. Double-blind clinical evaluation of oral glucosamine sulphate in the basic treatment of osteoarthrosis. Curr Med Res Opin 1980;7:110-4. 19) Drovanti A , Bignamini A A , Rovati A L . Therapeutic activity of oral glucosamine sulfate in osteoarthrosis: a placebo-controlled double-blind investigation. Clin Ther 1980;3:260-72. 20) D'Ambrosio E, Casa B, Bompani R, Scali G, Scali M . Glucosamine sulphate: a controlled clinical investigation in arthrosis. Pharmatherapeutica 1981;2:504-8. 21) Lopez Vas A . Double-blind clinical evaluation of the relative efficacy of ibuprofen and glucosamine sulphate in the management of osteoarthrosis of the knee on out-patients. Curr Med Res Opin 1982;8:145-9. 22) Reichelt A , Forster K K , Fischer F M , Rovati L C , Setnikar I. Efficacy and safety of intramuscular glucosamine sulfate in osteoarthritis of the knee. A randomised, placebo-controlled, double-blind study. Arzneimittelforschung 1994;44:75-80. 23) Noack W, Fischer M , Forster K K , Rovati L C , Setnikar I. Glucosamine sulfate in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:51-9. 24) Miiller-Fassbender H, Bach GL, Haase W, Rovati L C , Setnikar I. Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:61-9. 25) Qiu G X , Gao SN, Giacovelli G, Rovati L, Setnikar I. Efficacy and safety of glucosamine sulfate versus ibuprofen in patients with knee osteoarthritis. Arzneimittelforschung 1998;48:469-74. 26) Houpt JB, McMillan R, Paget-Dellio D, Russell A , Gahunia H K . Effect of glucosamine hydrochloride (GHcl) in the treatment of pain of osteoarthritis of the knee. J Rheumatol 1999;26:2423-30. Rindone JP, Hiller D, Collacott E, Nordhaugen N, Arriola G. Randomized, controlled trial of glucosamine for treating osteoarthritis of the knee. West J Med 2000;172:91-4. Reginster JY, Deroisy R, Rovati LC, Lee RL, Lejeune E, Bruyere O, Giacovelh G, Henrotin Y, Dacre JE, Gossett C. Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial. Lancet 2001;357:251-6. Hughes R, Carr A. A randomized, double-blind, placebo-controlled trial of glucosamine sulphate as an analgesic in osteoarthritis of the knee. Rheumatology 2002;41:279-84. Pavelka K, Gatterova J, Olejarova M, Machacek S, Giacovelh G, Rovati LC. Glucosamine sulfate use and delay of progression of knee osteoarthritis. Arch Intern Med 2002;162:2113-23. Bellamy N, Kirwan J, Boers M, Brooks P, Strand V, Tugwell P, et al. Recommendations for a core set of outcome measures for future phase III clinical trials in knee, hip, and hand osteoarthritis. Consensus development at OMERACT III. J Rheumatol 1997;24:799-802. Brandt KD, Mazzuca SA, Conrozier T, Dacre JE, Peterfy CG, Provvedini D, et al. Which is the best radiographic protocol for a clinical trial of a structure modifying drug in patients with knee osteoarthritis? J Rheumatol 2002;29:1308-20. Mazzuca SA, Brandt KD, Lane KA, Katz BP. Knee pain reduces joint space width in conventional standing anteroposterior radiographs of osteoarthritic knees. Arthritis Rheum 2002;46:1223-7. 60 Hoffer JL, Kaplan LN, Hamadeh MJ, Grigoriu AC, Baron M. Sulfate could mediate the therapeutic effect of glucosamine sulfate. Metabolism 2001;50:767-70. Cohen M, Wolfe R, Mai T, Lewis D. A randomized, double blind, placebo controlled trial of a topical cream containing glucosamine sulfate, chondroitin sulfate, and camphor for osteoarthritis of the knee. J Rheumatol 2003;30:523-8. McAlindon T, Formica M, Kabbara K, LaValley M, Lehmer M. Conducting clinical trials over the internet: feasibility study. Br Med J 2003;327:484-7. Cibere J, Esdaile JM, Thorne AE, Singer J, Kopec JA, Canvin J, et al. Multicenter randomized double-blind placebo-controlled glucosamine discontinuation trial in osteoarthritis. Arthritis Rheum 2002;45(suppl):S576 61 CHAPTER 6 RANDOMIZED DOUBLE-BLIND PLACEBO-CONTROLLED GLUCOSAMINE DISCONTINUATION TRIAL IN KNEE OSTEOARTHRITIS This manuscript is accepted for publication in Arthritis Care and Research under the title "Randomized double-blind placebo-controlled glucosamine discontinuation trial in knee osteoarthritis". The candidate is first author on this manuscript. Drs. Jacek Kopec, Anona Thorne, Joel Singer, Janice Canvin, David B. Robinson, Janet Pope, Paul Hong, Eric Grant, and John M. Esdaile are co-authors. Study funding was obtained by the candidate from the Mary Pack Research Fund, Vancouver, British Columbia. Drs. Canvin, Robinson, Pope, Hong and Grant evaluated study patients at the other study sites. Drs. Esdaile and Kopec are co-supervisors of the candidate, and Dr. Singer and Ms. Thorne are members of the supervisory committee. The candidate's role in this manuscript involved the development of the study hypothesis and methodology, the clinical evaluation of the majority of the participants, the conduct of the study, all data entry and statistical analysis of results, as well as the writing of the final manuscript. 6.1 Introduction Osteoarthritis (OA) is the most common joint disease worldwide. Symptomatic knee OA occurs in 6% of the population above the age of 301 and increases in prevalence with age. Knee OA causes significant disability, including work disability,2'3'4'5 and is associated 62 with substantial economic costs.6'7 With the aging of the population, the economic burden of OA is projected to increase considerably by the year 2020.8 Current treatments are limited to non-pharmacological interventions, such as weight reduction and exercise, pharmacological management of pain and surgical treatment for severe OA. Therefore, the search for and evaluation of new treatments for OA is an important step toward better management of this disease. Glucosamine has been widely publicized in North America and is currently one of the top selling nutritional supplements. However, the evidence for efficacy of glucosamine is controversial. Glucosamine has been evaluated for the symptomatic treatment of knee OA in a number of studies,9'10'1 '.12,13,14,15,16,17,18,19,20 m Q S t o f w h k h h a v g r e p o r t e d benefit. 9,10,11,12,13,14,15,16,17 H o w e v e i - ; systematic reviews of these studies have commented on methodological issues relating to study design, publication bias and the tendency for pharmaceutical sponsorship to be associated with positive study findings.21'22 Since then, two trials have reported the benefit of glucosamine on the radiographic progression of OA as well as symptomatic improvement over 3 years.2 3'2 4 In contrast, three recent studies have 18 19 20 reported no efficacy of glucosamine in the treatment of symptomatic knee OA. ' ' The differences in these studies has been highlighted in a recent review of the glucosamine literature by McAlindon in an attempt to provide insight into why clinical trials of glucosamine are no longer uniformly positive.2 5 The conclusion from this review was that more research was needed. As a result, despite numerous studies, the evidence for the efficacy of glucosamine in knee OA is inconclusive. This study was initiated to assess the efficacy of glucosamine. Because glucosamine is freely available as an over-the-counter nutritional supplement, its use has become wide-63 spread in the community. As a result, this study was designed as a randomized discontinuation trial (RDT) to evaluate the effect of continuing or withdrawing glucosamine in patients with knee OA. 6.2 Methods 6.2.1 Patients Participants were recruited through newspaper advertising and local posters. Subjects were included if they met the following eligibility criteria: Inclusion criteria - 1) OA of the knee(s) according to the American College of Rheumatology (ACR) diagnostic criteria,27 2) Kellgren-Lawrence grade >2 on anteroposterior radiograph of the knee,28 3) current daily use of glucosamine for at least 1 month, 4) at least moderate improvement in knee pain since starting on glucosamine, measured on a 6-point scale of knee pain worse, unchanged, mildly improved, moderately improved, markedly improved, completely subsided. Subjects were excluded if they met any of the following criteria: 1) chondroitin sulfate use within the previous 2 months, 2) knee injection with hyaluronate in the previous six months or with corticosteroids in the previous 3 months, 3) surgical procedure on either knee in the previous 3 months, 4) narcotic analgesic use, 5) uncontrolled medical condition or planned surgery which could interfere with follow-up, 6) baseline potassium > 5.3mEq/l or baseline creatinine >120mmol/l. 6.2.2 Study design The study was a 6-month randomized, double-blind, placebo-controlled parallel-group glucosamine discontinuation trial performed at 4 centers in Canada. The study was 64 conducted in accordance with the Declaration of Helsinki (1975) and was approved by the institutional review board at each study site. All patients provided written informed consent. Randomization: A central computer-generated randomization code was produced by a researcher not affiliated with the study. Block randomization with a randomly variable block size of 2 to 6 was used. The randomization code was forwarded to the manufacturer of the study medication and was used to label the study medication bottles consecutively from 1 to 160. The randomization codes remained sealed until after the blinded analysis had been carried out. Thus, allocation concealment was maintained and study investigators and patients were blinded throughout the study. Eligible subjects were assigned the next consecutive shady number. Intervention: Glucosamine and placebo tablets were supplied by VitaHealth, Winnipeg. The active drug consisted of glucosamine sulfate formulated as a potassium salt preparation (500mg tablets). The placebo tablets were indistinguishable from the glucosamine tablets and contained excipients only. Patients were randomized to receive either glucosamine sulfate or placebo. The study medication dose was equivalent to the dose of glucosamine taken prior to the study with a maximum of 1500mg per day. Patients who used a dose greater than 1500mg per day prior to the study were treated with 1500mg per day in the study. Compliance with study treatment was evaluated by pill count at each visit, except Week 2. Rescue analgesic medications including acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs) were allowed and recorded by the patient in a daily diary. Other concomitant treatments, including chondroitin sulfate and intra-articular injections with corticosteroids or hyaluronic acid, were not allowed during the study. Patients were followed for 6 months or until disease flare, whichever occurred first. 65 Outcome assessments: Following the screening visit and determination of eligibility, patients were assessed at week 0 (baseline), week 2, 4, 8, 12 and 24 or at any time if a flare occurred. Each study visit included an evaluation of knee symptoms, review of medications and adverse events, an assessment of acetaminophen and NSAID use over the preceding study interval, and a knee examination including warmth, joint effusion, crepitus, joint line tenderness, end-of-range stress pain, and range of motion measured by goniometer. At each visit, patients completed the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index (version VA3.0), which is a validated disease-specific questionnaire frequently used in OA trials assessing pain, stiffness and function,29 as well as the European Quality of Life (EQ-5D) questionnaire, a validated generic health assessment tool, which includes a utility score and a 0-100 visual analog scale (VAS).30 Following all assessments, a physician global assessment was recorded, rating patient status on a scale from 1 to 5 as very poor, poor, fair, good, or excellent.31 A determination of whether disease flare had occurred was made at each visit. If a disease flare was present, the patient was withdrawn from the study. At the final study visit, patient blinding was assessed by asking the patient's opinion whether glucosamine or placebo was received in the study. When all patients had completed the study, a random selection of 11% of glucosamine study drugs (n=8) and a random selection of 2 placebo study drugs were analyzed for glucosamine sulfate content (JR Laboratories Inc., Vancouver, British Columbia). Study outcomes: The primary endpoint was the proportion of patients with disease flare in the glucosamine and placebo groups. Disease flare was defined a priori as either the patient's perception of worsening of symptoms with a concomitant increase in WOMAC pain on walking by at least 20mm, or a significant worsening in the physician global 66 assessment by at least 1 grade (1 to 5 scale). This definition of disease flare was established by consensus amongst two study rheumatologists. A change in W O M A C pain on walking by 20mm was determined to be a clinically important change. A physician global assessment was deemed necessary in the definition of flare, since a change in pain due to flare may not be reflected in the W O M A C pain assessment i f increased use of analgesic medications occurs to control pain. Secondary outcomes included the time to disease flare, the change from baseline to flare visit in W O M A C pain, stiffness, function scores, total W O M A C , and EQ-5D utility and V A S , as well as the proportion of patients using acetaminophen, N S A I D s or both in the two treatment groups at final study visit. Severity of disease flare was assessed in the placebo and glucosamine groups by comparing mean change scores in W O M A C pain, stiffness, function, total W O M A C and EQ-5D utility and V A S scores in patients who flared. 6.2.3 Statistical analysis Baseline characteristics were compared between the placebo and glucosamine treatment groups. The proportion of patients with disease flare was assessed using the C h i -square test. Time to disease flare was evaluated by survival analysis. Kaplan-Meier curves were generated for the placebo and glucosamine treatment groups and the log-rank test was used to test for a statistical difference between the curves. Because of baseline imbalances between treatment groups, a Cox regression analysis was performed with adjustment for imbalanced covariates. The effect of treatment group on the hazard of developing a disease flare was evaluated in an initial univariate model. Gender, study site, and O A radiographic severity were then added to the model to evaluate the effect of treatment on the hazard of developing a disease flare after adjustment for these covariates. In addition, other clinically 67 important covariates were assessed in the Cox regression analysis including age, duration of glucosamine use, glucosamine dosage, duration of OA and analgesic medication use. Because OA radiographic severity was a significant variable in the Cox regression analysis, an interaction between radiographic severity and treatment was evaluated in the Cox regression analysis. The assumptions underlying the proportional hazards model were assessed using residual plots and log-log plots. There was no evidence of violation of any model assumptions. An intent-to-treat (ITT) approach was used. It was decided a priori that patients who were lost to follow-up would be considered to have flared for the purpose of the primary analysis. For the survival analysis, lost to follow-up patients were right censored, and hence they were followed only to their last visit. The sample size calculation was based on assumptions of a 10% flare rate in the glucosamine group and a 30% flare rate in the placebo group. With a power of 80% and an alpha error of 5%, a sample size of 62 patients per group was calculated. Under the assumption of a 10% drop-out rate, the target for recruitment was 138 patients. 6.3 Results A total of 209 patients were screened for the study (Figure 6.1). Of these, 137 met the inclusion and exclusion criteria and were randomized to glucosamine (N=71) or placebo (N=66). One hundred and thirty-four patients completed the study to the predefined outcome of disease flare or 6 months of completed follow-up. Of the remaining three patients, one was lost to follow-up after Week 4, one withdrew from the study at Week 2 due to a cerebrovascular accident, and one withdrew at Week 4 due to a diagnosis of 68 metastatic adenocarcinoma of unknown origin. These three patients had been assigned to the glucosamine treatment group and were considered to have flared for the primary analysis. The baseline characteristics of placebo and glucosamine patients were comparable except for gender and OA radiographic severity (Table 6.1). Seventy percent of patients in the placebo group were female compared to 44% in the glucosamine group. Mild radiographic knee OA (Kellgren-Lawrence grade 2) was present in 64% of placebo and 46% of glucosamine patients. In contrast, moderate OA (grade 3) was present in 33% and 44%, respectively, and severe OA (grade 4) was seen in 3% and 10%, respectively. As a result, glucosamine patients had more severe knee OA based on radiography. However, severity based on WOMAC pain (possible score of 0-500) and function (possible score of 0-1700) was comparable in the two groups with median (range) WOMAC pain scores of 86 (2-279) and 86 (4-301), and median (range) WOMAC function scores of 268 (0-1376) and 294 (2-1240) in the placebo and glucosamine groups, respectively (Table 6.1). Since no analgesic washout was used in this discontinuation trial, these scores, although lower than those reported in standard trials, are consistent with a moderately severely affected patient population as indicated by the range of scores. The majority of patients in both groups used a glucosamine dosage of 1500mg per day prior to the study, with only 3 (5%>) placebo and 5 (7%) glucosamine patients using a larger dosage. The maximum pre-study dose was 2000mg per day. Similarly, the majority of patients had used glucosamine sulfate prior to the study with only 4 (6%) placebo and 3 (4%) glucosamine patients having used pre-study glucosamine hydrochloride. The primary endpoint of a disease flare in the ITT analysis was seen in 28/66 (42%) placebo patients and 32/71 (45%) glucosamine patients (Figure 6.2) The between-group 69 difference of -3% was not statistically significant (95% confidence interval [CI], -19%, 14%; p=0.76). No differences were seen for acetaminophen and NSAID use between the placebo and glucosamine groups. At final study visit, acetaminophen was used by 27% and 21% of placebo and glucosamine patients, respectively (difference 6%; 95% CI, -8%, 20%; p=0.40), NSAIDs were used by 29% and 30%, respectively (difference -1%; 95% CI, -16%, 14%; p=0.92), and both acetaminophen and NSAIDs were used by 20% and 21%, respectively (difference -1%; 95% CI, -15%, 12%; p=0.84) (Figure 6.3). Dosages of acetaminophen and NSAIDs were also not different between the two treatment groups at final study visit (data not shown). Other secondary outcomes of WOMAC pain on walking, pain score, stiffness score, function score and total WOMAC as well as quality of life (EQ-5D utility and VAS) were not significantly different in the placebo and glucosamine groups (Table 6.2). Time to disease flare, assessed by survival analysis, was also similar in the placebo and glucosamine groups. There were no statistically significant differences in Kaplan-Meier survival curves for patients who continued on glucosamine compared to those who were withdrawn from glucosamine (log-rank test, p=0.96) (Figure 6.4). A univariate Cox regression analysis with treatment group as the explanatory variable, revealed a hazard of disease flare of 0.98 (95% CI, 0.58, 1.64; p=0.93) in the glucosamine group compared to placebo (Table 6.3). After adjustment for gender, study site and OA radiographic severity, there was no difference in the risk of disease flare between the placebo and glucosamine patients (hazard ratio 0.81; 95% CI, 0.47, 1.40; p=0.45) (Table 6.3). Age, duration of glucosamine use, glucosamine dosage, duration of OA, and analgesic medication use were not significant in the Cox regression analysis (data not shown) and hence were not included 70 in the final model. However, OA radiographic severity was found to be a significant predictor in the Cox regression analysis with a hazard of disease flare of 2.41 (95% CI, 1.42, 4.09; p=0.001) in the moderate to severe OA group (Kellgren-Lawrence grade 3-4), compared to the mild OA group (Kellgren-Lawrence grade 2). No significant interaction between treatment and OA radiographic severity was seen in the Cox regression analysis. Given that the proportions of patients who met the definition of flare did not differ between treatment groups, it was of interest to determine whether the severity of flare differed between groups. The severity of disease flare was found to be similar in the placebo and glucosamine treated patients who flared (Table 6.4). There were no statistically significant differences in mean change in WOMAC pain on walking, pain, stiffness and function scales, total WOMAC or quality of life between flare patients in the placebo and glucosamine groups. However, mean change scores of flare patients were substantially different from those of non-flare patients. Worsening of WOMAC and EQ-5D was seen in flare patients as indicated by negative change scores, while improvement of these outcomes occurred in non-flare patients as indicated by positive change scores (Table 6.4). Compliance with study drug was excellent. Greater than 80% compliance was seen in 97% of placebo and 97% of glucosamine treated patients. There was no evidence of unblinding at the end of the study. No serious adverse events were reported during the study and there were no differences in adverse events between the glucosamine and placebo groups. Because of the discontinuation design of this study, minimal adverse effects were expected, since all subjects had previously tolerated glucosamine. Mean glucosamine sulfate content was found to be 618 mg per tablet in the glucosamine samples and 0 mg per tablet in the placebo samples. 71 6.4 Discussion Efficacy of glucosamine in knee OA has been reported predominantly in trials with pharmaceutical sponsorship.21 With the recent reporting of negative glucosamine 18 19 20 studies, ' ' the issue of whether glucosamine is efficacious has become more controversial. In this study, we found that knee OA disease flare occurred as frequently, as quickly and as severely in patients who were randomized to continue on glucosamine compared to those who received placebo. As a result, the efficacy of glucosamine as a symptom modifying drug in knee OA is not supported by our study. The randomized discontinuation trial (RDT) approach has been used infrequently to demonstrate efficacy. A key underlying assumption for the RDT study design is that the disease process will worsen or flare when the drug is discontinued. This presumes that the disease under study has not been cured by the drug treatment prior to the trial. Although glucosamine is frequently promoted as a disease modifying drug, there is no evidence that it is curative, and hence an RDT study design is appropriate. A 6-month study design was felt to be sufficient to allow for disease flare to occur. Since pain relief occurs within 2-3 months of treatment with glucosamine, it seems reasonable to expect development of flare within a similar time frame after discontinuation of glucosamine, assuming no curative effect. Because patients in this study were enrolled only if they had subjective improvement on prior glucosamine, the question of whether glucosamine has an initial beneficial effect cannot be answered by this study. Any initial perceived benefit may have been due to a placebo response or natural fluctuation in symptoms over time and hence a null finding would be expected in a discontinuation trial. Alternatively, a temporary initial benefit could 72 be due to glucosamine itself or another component of the nutritional supplement such as sulfate. However, even if an initial benefit had been derived from glucosamine or sulfate, our findings suggest that there is no evidence of benefit with continued use of glucosamine sulfate for the symptomatic treatment of knee OA. Our negative study findings need to be interpreted in the context of the observed confidence interval which indicates, with 95% confidence, that a true difference in proportion of flares is no greater than 14% in favor of glucosamine. Since this study was designed to detect a clinically important difference of 20%, these findings are consistent with our a priori null hypothesis. Furthermore, our negative study findings cannot be explained by a lack of compliance, in view of the fact that 97% of study participants had excellent compliance. Contamination with non-study glucosamine is also an unlikely explanation, since the study procedures were clearly understood by participants, the use of all medications was ascertained at each study visit, and participants understood the importance of rigorously evaluating the efficacy of glucosamine. Similarly, co-interventions were not allowed in the study and no protocol violations occurred. Hence, this is an unlikely explanation for our negative findings. A potential bias towards non-efficacy occurred, because lost-to-follow-up patients were considered to have flared according to our a priori decision. Since all three lost-to-follow-up patients were in the glucosamine group, this resulted in more flares in the glucosamine group and hence introduced a bias toward non-efficacy of glucosamine. However, even with the exclusion of these three patients, there was no difference in the proportion of flares in the glucosamine and placebo groups. In addition, if glucosamine hydrochloride has no or minimal efficacy compared to glucosamine sulfate, the use of glucosamine hydrochloride prior to the study may result in a study finding of no 73 difference. Only 4 placebo and 3 glucosamine patients had used the hydrochloride formulation of glucosamine prior to the study and similar proportions flared (2 patients in each group). Exclusion of these 7 patients did not change the results. Finally, since OA disease activity can fluctuate naturally, a difference between treatment groups may not be appreciable based solely on the primary outcome of disease flare. For this purpose, we evaluated as secondary outcomes the severity of change in WOMAC pain, stiffness and function as well as the time to disease flare in the placebo and glucosamine groups. However, no differences were found in the severity of disease flare or time to disease flare, lending further support to our conclusion of no symptomatic benefit from continued glucosamine use in knee OA. 18 19 20 18 Our findings are in keeping with other recent negative studies. ' ' Houpt et al evaluated a population of knee OA patients with predominantly early radiographic changes where 74% of patients had Kellgren-Lawrence grade 1 or 2 changes.28 They reported no significant difference in WOMAC pain, stiffness or function after 2 months of treatment with glucosamine hydrochloride compared to placebo. In the study by Rindone et al,1 9 where approximately half of the patients had early knee OA (Kellgren-Lawrence grade 1 or 2) they found no differences in pain at rest and pain on walking after 2 months of treatment 20 with glucosamine sulfate or placebo. The most recent study by Hughes et al evaluated patients with more advanced knee OA - 60% of the study population had moderate to severe knee OA (Kellgren-Lawrence grade 3 or 4). In that study, no differences were seen between glucosamine sulfate and placebo groups in pain, use of analgesic medications or in the proportion of responders to treatment over a 6 month study period. These findings suggest that the radiographic stage of OA may not be a factor in non-response to glucosamine 74 treatment. This is further supported by our Cox regression analysis, where no interaction was seen between OA radiographic severity and treatment. Hence, glucosamine was found to be non-efficacious regardless of radiographic severity of OA. Similarly, glucosamine dosage, duration of glucosamine use and analgesic medication use were not significant in the Cox regression analysis and hence did not have an effect on the risk of disease flare in our study. In contrast, it is of interest to note that OA radiographic severity was a significant predictor of flare. Although intuitively one might expect such a finding, this has not been previously reported. There are a number of strengths and limitations of this study. As discussed previously, a disadvantage of the RDT study design is that any initial benefit of glucosamine cannot be evaluated. A further limitation is that the RDT study design has never been used in OA. As a result, no validated definition of disease flare exists. Our definition of disease flare was adopted a priori and was felt to be of clinical importance. Moreover, since the initiation of our study, further publications have lent support to our choice of flare criteria31'32 The minimal clinically perceptible difference (MCPD) in WOMAC pain on walking was reported by Ehrich et al31 to be 11mm on a 100mm VAS. Similarly, the MCPD 31 for physician global assessment was reported to be 0.43 on a 1-5 Likert scale. As a result, the definition for disease flare used in this study, which included a change in pain on walking of 20mm on a 100mm VAS or a change of 1 point on a 1-5 scale on physician global assessment, was adequate to detect a clinically meaningful difference in patient status. In the recent Osteoarthritis Research Society International (OARSI) guidelines on response criteria, the committee recommended that treatment response in an NSAID trial be defined as an absolute decrease in VAS pain by 20mm in association with a relative 75 decrease of pain by 45%.32 An absolute difference of 20mm should therefore be applicable as a flare criterion and hence the OARSI guidelines lend further support to the validity of our choice of flare criteria. Furthermore, the choice of flare criteria are also validated by fact that patients who flared in this study had worsening not only of WOMAC, but also EQ-5D scores, while those who did not flare had improvement over the course of the study in both these outcome measures. The need for rescue medication in a discontinuation trial may be seen as a further limitation due to the potential confounding effect. However, analgesic medication use was included in the secondary analysis of the data and was similar in the placebo and glucosamine groups. In addition, the inclusion of acetaminophen and NSAID use in the Cox regression analysis showed that there was no confounding effect. Furthermore, the use of acetaminophen and NSAIDs may be viewed as a strength of this study, since their use reflects common practice in OA. If a benefit of glucosamine cannot be detected under real world circumstances, then the applicability of glucosamine is limited, even if it is found to be efficacious. There are several advantages to using an RDT study design, including the ability to test the efficacy of a drug which is widely used and minimization of exposure to placebo.26 Furthermore, a particular strength of this study is the selection of patients with at least moderate perceived response to prior glucosamine treatment. The use of such a selective population allows for a more efficient trial, since patients who have previously responded to treatment with glucosamine are more likely to flare on discontinuation of glucosamine than non-responders. Therefore, a treatment difference between glucosamine and placebo, if one exists, can be shown more easily in such a pre-selected population.26 Despite this greater 76 ability to show efficacy, our study results were negative and hence this serves to strengthen our conclusions. In summary, for patients with knee OA with at least moderate subjective improvement with prior glucosamine use, this study provides no evidence of symptomatic benefit from continued use of glucosamine sulfate over and above that found with placebo. 77 Table 6.1: Baseline Characteristics of Placebo and Glucosamine Patients Placebo N=66 Glucosamine N=71 Mean Age (range) [years] 65 (43-88) 64 (40-83) Female gender [%] 70% 44% Mean body mass index (range) [kg/m2] 27 (21-45) 28 (19-49) Median duration of glucosamine use (range) [years] 1.5 (0.1-6.8) 1.7 (0.1-5.4) Pre-study type of glucosamine (%) Glucosamine sulfate 94% 96% Glucosamine hydrochloride 6% 4% Pre-study glucosamine dose (%) >1500 mg per day 5% 7% 1500 mg per day 53% 61% 1000 mg per day 33% 26% 500 mg per day 9% 6% Median duration of physician diagnosed OA (range) [yrs] 3 (0-29) 3 (0-29) Radiographic OA severity [%] Kellgren-Lawrence grade 2 64% 46% Kellgren-Lawrence grade 3 33% 44% Kellgren-Lawrence grade 4 3% 10% Median WOMAC pain on walking (range) [0-100 mm] 12 (0-78) 13 (0-63) Median WOMAC pain (range) [0-500 mm] 86 (2-279) 86 (4-301) Median WOMAC function (range) [0-1700 mm] 268 (0-1376) 294 (2-1240) Median WOMAC total (range) [0-2400 mm] 414(26-1796) 444(10-1671) Analgesic medication use: Acetaminophen only (%) 24% 17% NSAIDs only (%) 35% 35% Both acetaminophen and NSAIDs (%) 14% 16% WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index, NSAIDs nonsteroidal anti-inflammatory drugs 78 Table 6.2: Mean Change in WOMAC and EQ-5D at Final Visit Compared to Baseline and Between-Group Differences in the Intent-To-Treat Population Mean (SD) change from Between-group p-value baseline* difference (95% CI) Placebo Glucosamine (N=66) (N=71) WOMAC Pain on walking (0-100mm) -8 (25) -5 (21) -3 (-11, 4) 0.40 Pain scale (0-500mm) -28 (104) -25 (98) - 3 (-37, 32) 0.88 Stiffness scale (0-200mm) 6(48) 2(42) 4 (-11, 20) 0.57 Function scale (0-1700mm) -63 (318) -58 (270) - 5 (-105, 94) 0.92 Total scale (0-2400mm) -85 (453) -81 (388) -4 (-145, 139) 0.96 EQ-5D Utility score (0-1) -0.04 (0.20) -0.03 (0.16) -0.01 (-0.07,0.05) 0.68 Visual analog scale (0-100) -2 (12) 0.1 (16) -2 (-7, 3) 0.42 *Positive mean change indicates improvement, negative mean change indicates worsening. WOMAC = Western Ontario and McMaster Universities Osteoarthritis Index EQ-5D = European Quality of Life questionnaire SD = standard deviation, CI = confidence interval 79 Table 6.3: Multivariate Hazard Ratio of Disease Flare in the Cox Regression Analysis Variables Hazard ratio (95% CI) p-value Univariate analysis Placebo 1.0 Glucosamine 0.98 (0.58, 1.64) 0.93 Multivariate analysis* Placebo 1.0 " Glucosamine 0.81 (0.47, 1.40) 0.45 *adjusted for gender, study site and radiographic severity of osteoarthritis. CI = confidence interval 80 Table 6.4: Mean Change Scores in Non-Flarers and Flarers in the Intent-To-Treat Population and Comparison of Severity of Change in Placebo and Glucosamine Flarers Mean (SD) change from baseline* Between-group p-value difference (95% CI) for placebo and glucosamine flarers Non-flarers Flarers Placebo Glucos-amine Placebo Glucos-amine (N=38) (N=39) (N=28) (N=32) W O M A C Pain on walking 5 5 -25 -17 -8 (-21,4) 0.20 [0-100] (15) (12) (26) (23) Pain subscale 24 30 -97 -92 -5 (-58, 47) 0.83 [0-500] (75) (47) (98) (103) Stiffness subscale 20 25 -13 -26 13 (-9, 36) 0.24 [0-200] (42) (34) (50) (34) Function subscale 69 96 -243 -246 3 (-138, 143) 0.97 [0-1700] (264) (178) (300) (243) Total scale 114 151 -354 -364 10 (-188, 209) 0.92 [0-2400] (368) (237) (422) (347) EQ-5D Utility score 0.02 0.02 -0.13 -0.10 -0.03 (-0.13,0.06) 0.46 [0-1] (0.18) (0.14) (0.2) (0.16) Visual analog scale 2 6 -6.6 -6.9 0.3 (-8, 8) 0.95 [0-100] (9) (14) (15) (16) *Positive mean change indicates improvement, negative mean change indicates worsening. W O M A C = Western Ontario and McMaster Universities Osteoarthritis Index EQ-5D = European Quality of Life questionnaire SD = standard deviation, CI = confidence interval 81 Figure 6.1: Flow Diagram of Study Enrolment and Conduct Assessed for eligibility (n = 209) Eligible (n= 137) Excluded (n = 72) Did not meet entry criteria (n = 66) Refused to participate (n = 5) Other reasons (n = 1) Analyzed (n = 71) Randomized to glucosamine Randomized to placebo (n =  71) (n = 66) Non-completers (n = 3): Non-completers (n = = 0): Lost to follow-up (n = 1) Discontinued intervention due to concurrent illness (n = 2) Analyzed (n = 66) 82 Figure 6.2: Proportion of Disease Flare in the Placebo and Glucosamine Treatment Groups in the Intent-To-Treat Population 100% — 80% Q> JS 4 -§ 60% a> CO ~ 40% </) | 20% a. 0% Difference -3%; 95% confidence interval -19%, 14% p = 0.76 Placebo Glucosamine 83 Figure 6.3: Proportion of Analgesic Drug Use at Final Study Visit in the Intent-To-Treat Population Acetaminophen use NSAID use 100 90 80 -70 -60 -50 40 ^ 30 20 10 0 Difference 6% 95% Cl -8%, 20% p = 0.40 100 90 80 70 60 50 40 30 20 10 4 Difference -1% 95% Cl -16%, 14% p = 0.92 100 90 80 70 60 50 40 30 20 H 10 0 Acetaminophen and NSAID use Difference -1% 95% Cl -15%, 12% p = 0.84 Placebo Glucosamine Placebo Glucosamine Placebo Glucosamine NSAID = nonsteroidal anti-inflammatory drug, CI = confidence interval 84 Figure 6.4: Kaplan-Meier Survival Curves for Time-To-Disease Flare in the Placebo and Glucosamine Groups Time (days) Solid line indicates glucosamine group; dashed line indicates placebo group. Log-rank test, p=0.96 85 6.5 References 1) Felson DT, Zhang Y. An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum 1998;41:1343-55. 2) Davis MA, Ettinger WH, Neuhaus JM, Mallon KP. Knee osteoarthritis and physical functioning: Evidence from the NHANES I epidemiologic followup study. J Rheumatol 1991;18:591-8. 3) Badley EM. The effect of osteoarthritis on disability and health care use in Canada. J Rheumatol 1995;(suppl 43)22:19-22. 4) Yelin EH, Katz PP. Labor force participation among persons with musculoskeletal conditions, 1970-87. Arthritis Rheum 1991;34:1361-70. 5) Gabriel SE, Crowson CS, O'Fallon WM. Costs of osteoarthritis: Estimates from a geographically defined population. J Rheumatol 1995; (suppl 43)22:23-5. 6) Gabriel SE, Crowson CS, Campoin ME, O'Fallon WM. Indirect and nonmedical costs among people with rheumatoid arthritis and osteoarthritis compared with nonarthritic controls. J Rheumatol 1997;24:43-8. 7) March LM, Bachmeier CJM. Economics of osteoarthritis: a global perspective. Clin Rheumatol 1997;11:817-34. 8) Badley EM, Crotty M. An international comparison of the estimated effect of the aging of the population on the major cause of disablement, musculoskeletal disorders. J Rheumatol 1995;22:1934-40. 9) Crolle G, D'Este E. Glucosamine sulphate for the management of arthrosis: a controlled clinical investigation. Curr Med Res Opin 1980;7:104-9. 10) Pujalte JM, Llavore EP, Ylescupidez FR. Double-blind clinical evaluation of oral 86 glucosamine sulphate in the basic treatment of osteoarthrosis. Curr Med Res Opin 1980;7:110-4. 11) Drovanti A, Bignamini AA, Rovati AL. Therapeutic activity of oral glucosamine sulfate in osteoarthrosis: a placebo-controlled double-blind investigation. Clin Ther 1980;3:260-72. 12) D'Ambrosio E, Casa B, Bompani R, Scali G, Scali M. Glucosamine sulphate: a controlled clinical investigation in arthrosis. Pharmatherapeutica 1981;2:504-8. 13) Lopez Vas A. Double-blind clinical evaluation of the relative efficacy of ibuprofen and glucosamine sulphate in the management of osteoarthrosis of the knee on out-patients. Curr Med Res Opin 1982;8:145-9. 14) Reichelt A, Forster KK, Fischer FM, Rovati LC, Setnikar I. Efficacy and safety of intramuscular glucosamine sulfate in osteoarthritis of the knee. A randomised, placebo-controlled, double-blind study. Arzneimittelforschung 1994;44:75-80. 15) Noack W, Fischer M, Forster KK, Rovati LC, Setnikar I. Glucosamine sulfate in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:51-9. 16) Miiller-Fassbender H, Bach GL, Haase W, Rovati LC, Setnikar I. Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee. Osteoarthritis Cartil 1994;2:61-9. 17) Qiu GX, Gao SN, Giacovelh G, Rovati L, Setnikar I. Efficacy and safety of glucosamine sulfate versus ibuprofen in patients with knee osteoarthritis. Arzneimittelforschung 1998;48:469-74. 18) Houpt JB, McMillan R, Paget-Dellio D, Russell A, Gahunia HK. Effect of glucosamine hydrochloride (GHcl) in the treatment of pain of osteoarthritis of the 87 knee. J Rheumatol 1999;26:2423-30. 19) Rindone JP, Hiller D, Collacott E, Nordhaugen N, Arriola G. Randomized, controlled trial of glucosamine for treating osteoarthritis of the knee. West J Med 2000;172:91-4. 20) Hughes R, Carr A. A randomized, double-blind, placebo-controlled trial of glucosamine sulphate as an analgesic in osteoarthritis of the knee. Rheumatology 2002;41:279-84. 21) McAlindon TE, LaValley MP, Gulin JP, Felson DT. Glucosamine and chondroitin for treatment of osteoarthritis. A systematic quality assessment and meta-analysis. JAMA 2000;283:1469-75. 22) Cibere J, Esdaile JM. Glucosamine use in osteoarthritis: What is the evidence for its efficacy? J Rheumatol 1999;26:1628 (abstract). 23) Reginster JY, Deroisy R, Rovati LC, et al. Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial. Lancet 2001;357:251-6. 24) Pavelka K, Gatterova J, Olejarova M, Machacek S, Giacovelli G, Rovati LC. Glucosamine sulfate use and delay of progression of knee osteoarthritis. A 3-year, randomized, placebo-controlled, double-blind study. Arch Intern Med 2002;162:2113-23. 25) McAlindon T. Why are clinical trials of glucosamine no longer uniformly positive? Rheum Dis Clin North Am 2003;29:789-801. 26) Kopec JA, Abrahamowicz M, Esdaile JM. Randomized discontinuation trials: Utility and efficiency. J Clin Epidemiol 1993;46:959-71. 88 27) Altman RD, Asch E, Bloch D, et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039-49. 28) Kellgren JH, Lawrence JS. Radiological assessment of osteoarthrosis. Ann Rheum Dis 1957;16:494-502. 29) Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW. Validation study of WOMAC: A health status instrument for measuring clinically important participant relevant outcomes to antirheumatic drug therapy in participants with osteoarthritis of the hip or knee. J Rheumatol 1988;15:1833-40. 30) The EuroQol Group. EuroQol - a new facility for the measurement of health-related quality of life. Health Policy 1990;16:199-208. 31) Ehrich EW, Davies GM, Watson DJ, Bolognese JA, Seidenberg BC, Bellamy N. Minimal perceptible clinical improvement with the Western Ontario and McMaster Universities osteoarthritis index questionnaire and global assessments in patients with osteoarthritis. J Rheumatol 2000;27:2635-41. 32 ) Dougados M, LeClaire P, van der Heijde D, Bloch DA, Bellamy N, Altman RD. A report of the Osteoarthritis Research Society International Standing Committee for clinical trials response criteria initiative. Osteoarthritis Cartil 2000;8:395-403. 89 CHAPTER 7 GLUCOSAMINE SULFATE AND TYPE II COLLAGEN DEGRADATION: RANDOMIZED DISCONTINUATION TRIAL RESULTS This manuscript is currently submitted for consideration for publication to the Journal of Rheumatology under the title "Glucosamine sulfate and cartilage type II collagen degradation in patients with knee osteoarthritis: Randomized discontinuation trial results employing biomarkers". The candidate is first author on this manuscript. Drs. Jacek A. Kopec, Anona Thorne, Joel Singer, Janice Canvin, David B. Robinson, Janet Pope, Paul Hong, Eric Grant, Mirela Ionescu, Tatiana Lobanok, A. Robin Poole, and John M. Esdaile are co-authors. Drs. Canvin, Robinson, Pope, Hong and Grant evaluated study patients at the other study sites and contributed to the collection of laboratory samples. Dr. Poole, Ms Ionescu and Ms Lobanok conducted all the biomarker assays. Drs. Esdaile and Kopec are co-supervisors of the candidate, and Dr. Singer and Ms. Thorne are members of the supervisory committee. The candidate's role in this manuscript involved the development of study hypothesis, collection of data and laboratory samples, data entry and statistical analysis of the biomarker results as well as the writing of the final manuscript. 7.1 Introduction Osteoarthritis (OA) is the most common joint disease worldwide. Symptomatic knee OA occurs in 6% of the population above the age of 301 and increases in prevalence with age. Knee OA causes significant disability2'3'4'5 and is associated with substantial economic costs.6'7 Because of the societal impact of OA, the search for disease-modifying treatments 90 has intensified. Recently, the disease-modifying effects of glucosamine were reported in two studies, both of which showed a reduction in radiographic progression of knee OA over 3 8 9 years in glucosamine compared to placebo treated subjects. ' However, because a full-extension unstandardized knee x-ray technique was used in both studies, the evidence for efficacy of glucosamine as a disease modifying treatment is inconclusive. In an attempt to develop outcome measures for OA disease modification that can be assessed over the short-term and that will allow for a direct measurement of cartilage metabolism, the development and evaluation of cartilage biomarkers has been the focus of recent research efforts.10'11 The degradation of cartilage matrix, in particular type II collagen (CII), plays a key role in the pathology of OA. 1 2 In OA, cleavage of each alpha chain of the triple helix of CII is mediated by collagenases, namely matrix metalloproteinase (MMP)-1, MMP-8, and MMP-13. This cleavage results in the generation of a three-quarter length collagen fragment.13 In recent years, antibodies have been developed, which recognize the carboxy-terminal neoepitope of the three-quarter length fragment of CII, including COL2-3/4Cshort (C1,2C) and COL2-3/4Ciong (C2C).13'14 C1,2C measures neoepitope released predominantly from CII, but to a lesser extent from type I collagen as well. In contrast, C2C has 100% specificity for CII.14 The generation of these neoepitopes has been shown to be elevated in OA cartilage compared to controls in in vitro and animal studies.13'15'16'17'18'19'20'21 Recent human studies have reported elevated C2C in OA and inflammatory arthritis patients and an association of elevated C2C with established knee and hip OA. In addition, an elevated ratio of C1,2C/C2C was shown to be predictive of radiographic knee OA progression over 18 months in patients without evidence of generalized OA. 2 4 These studies suggest that C1,2C, C2C and the ratio of these CII degradation products may provide 91 useful information in the evaluation of disease modifying treatments. The inhibitory effects of glucosamine on mediators of cartilage degeneration has been investigated.25'26'27'28'29 A reduction in MMP-1 (CII degrading enzyme)27'29 and MMP-3 (aggrecan degrading enzyme)26'27 activity has been reported in in vitro and animal studies. If glucosamine reduces the activity of MMP-1, which is thought to be important for CII cleavage,12'16 it is reasonable to hypothesize that glucosamine should have an inhibitory effect on CII degradation and hence will have disease modifying effects by inhibition of collagen degradation. However, no studies have evaluated whether reduced MMP-1 activity translates into a reduction in CII degradation. This is an important consideration, since MMP-8 and MMP-13 are also involved in CII degradation. In fact, it has been suggested by Dahlberg et al,1 6 that MMP-13 may play a more central role in CII degradation in OA than MMP-1. Because the effect of glucosamine on MMP-8 and MMP-13 has not been reported, it is not clear whether glucosamine use is associated with an actual reduction in CII degradation. We have recently reported the results from a randomized double-blind, placebo-controlled glucosamine discontinuation trial on symptom modification,30 which showed no reduction in OA disease flares, including time to disease flare, in the glucosamine compared to the placebo group. A secondary objective of this study was to evaluate the effect of glucosamine on CII degradation, measured by C1,2C and C2C assays. 92 7.2 Methods 7.2.1 Patients and Study Design The patients and methods for the glucosamine discontinuation trial have been described previously.30 Briefly, subjects were included if they had knee OA with radiographic evidence of osteophytes (Kellgren-Lawrence grade >2), according to the American College of Rheumatology (ACR) diagnostic criteria,31 were current daily users of glucosamine for at least 1 month, and had at least moderate improvement in OA symptoms on glucosamine. Subjects were excluded if they had used chondroitin sulfate within the previous 2 months, had received a knee injection of hyaluronic acid in the previous six months or corticosteroids in the previous 3 months, had undergone a surgical procedure on either knee in the previous 3 months, were using narcotic analgesic drugs, or had other uncontrolled medical conditions. The glucosamine discontinuation trial was a 6-month randomized, double-blind, placebo-controlled parallel-group study performed at 4 centers in Canada. The study was conducted in accordance with the Declaration of Helsinki (1975) and was approved by the institutional review board at each study site. All patients provided written informed consent. Randomization was done using a central computer-generated block randomization with variable block size. Subjects were randomized to receive either glucosamine sulfate (500mg tablets) or indistinguishable placebo at a dose equivalent to the dose of glucosamine taken prior to the study with a maximum of 1500mg per day. Subjects were followed at regular intervals until disease flare occurred or until six months had elapsed. Regular assessments included an evaluation of knee symptoms, review of 93 medications, knee examination, physician global assessment (1-5 scale), the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index33 and the European Quality of Life (EQ-5D) questionnaire.34 Rescue analgesic medication use, including acetaminophen and nonsteroidal anti-inflammatory drug (NSAID) use, was recorded in a daily diary. At each study visit, a determination of whether disease flare had occurred was made. Disease flare was defined as either the patient's perception of worsening of symptoms with a concomitant increase in WOMAC pain on walking by at least 20mm (0-100mm scale), or a significant worsening in the physician global assessment by at least 1 grade (1-5 scale). If a disease flare was present, the patient was withdrawn from the study. 7.2.2 Biomarker analysis Subjects had serum and urine samples collected at Week 0 (baseline), Week 4, Week 12 and Week 24, or at final study visit if a flare had occurred. Serum and urine samples were stored at -24°C. When all subjects had completed the study, samples were analyzed in triplicate for C1,2C epitope and C2C epitope using commercial ELISA assays. All biomarker analyses were performed blind to treatment and flare status. The C1,2C assay employs a polyclonal rabbit antibody that recognizes the carboxyterminal neoepitope generated by the activity of collagenases in both types I and II collagens.13 In contrast, the C2C assay employs a monoclonal antibody specific for the same cleavage site carboxy terminus in type II collagen.14 C1.2C assay (DELFIA method): The peptide CGPP(OH)GPQG (C1,2C), representing the neoepitope generated by 94 collagenase, was conjugated to ovalbumin and a polyclonal antibody was prepared as described.13 The antibody was purified by Protein A affinity chromatography and biotin-labeled using sulfo-NHS-LC-biotin (Pierce). Sulfo-NHS-LC-Biotin was dissolved in distilled water at a 1 mg/ml concentration and mixed with the purified antibody, previously dialyzed against 0.1M carbonate buffer pH 8.5, at a ratio of 80 pg Biotin per mg antibody. The Biotin-antibody mixture was incubated for 1 hour at room temperature, with stirring, followed by the removal of free Sulfo-NHS-LC-Biotin by dialysis in PBS pH 7.2, without sodium azide. For the quantitative analysis of C1,2C fragments, a DELFIA time-resolved immunofluorometric ELISA assay was used, which greatly reduces the background fluorescence signal. To improve the sensitivity of the immunoassay, the C2C peptide (CGGEGPP(OH)GPQG) is conjugated to keyhole limpet hemocyanin (KLH), using bromoacetic acid-N-hydroxysuccinimide ester (Sigma Chemical Co., St.Louis, MO). White, opaque 96-well plates (Greiner) were coated with C2C peptide-KLH conjugate diluted in PBS pH 7.2 (100 ng/ml, 50 ul/well). After an overnight incubation at 4°C, the plates were washed six times with an automatic plate-washer and blocked with 150 ul/well PBS pH 7.2 containing 1% wt/vol bovine serum albumin (BSA), RIA grade. Standards were prepared by diluting a 1 mg/ml stock solution of C1,2C peptide to 10, 3.33, 1.11, 0.37, 0.12, 0.041 and 0.014 ug/ml in PBS-1% BSA. Standards and samples were added at 50 ul/well, in triplicate, to 96 well U-bottom polypropylene plates (Corning Life Sciences), used as preincubation plates, followed by the addition of 50 pl/well of C1,2C biotinylated antibody, at a dilution in PBS-1% BSA-0.1% Tween 20 (pH 7.2) previously determined by checkerboard analysis. The triplicate 95 nonspecific binding wells consisted of 50 pi PBS-1% BSA and 50 pi PBS-1% BSA-0.1% Tween 20, and the triplicate maximum binding wells contained 50 pi PBS-1% BSA and 50 pi of the C1,2C biotinylated antibody. After an overnight incubation at 4°C, using a multichannel pipette, 50 pi of each preincubated well was transferred to the equivalent well of a Greiner white ELISA plate, coated with C2C peptide-KLH. The ELISA plates were each incubated, with shaking, for 45 minutes at 4°C and then washed three times with PBS-0.1% Tween 20. DELFIA Eu-labeled streptavidin (EG&G WALLAC), diluted 1:1000 in DELFIA assay buffer (EG&G WALLAC), was added to each plate at 50 plAvell and incubated for lh at 37°C. The plates were subsequently washed three times with PBS-0.1% Tween 20 and once with distilled water. Next, 50 ul/well of DELFIA enhancement solution (EG&G WALLAC) were added and the plates were incubated with shaking at room temperature for 30 minutes. Eu-labeled streptavidin is practically non-fluorescent, but when Eu 3 + is dissociated from the conjugate by the acidic chelating detergent DELFIA enhancement solution, it rapidly forms a new highly fluorescent chelate. Fluorescence at 615nm was measured by a EG&G Wallac 1412 plate reader (Turku, Finland). Calibration curve-fitting and interpolation to determine unknown analyte concentrations were performed by the instrument software. Serum and urine values are reported as picomoles of cleavage neoepitope per milliliter (pmol/ml). Urine values are adjusted for creatinine excretion and are expressed per umol creatinine. In cases where no C1,2C epitope was detectable, a value equivalent to half of the lowest standard, or 9.6 pmol/ml, was assigned. C2C assay (DELFIA method): 96 The peptide C G G E G P P ( O H ) G P Q G (C2C), representing the type II collagen specific neoepitope generated by collagenase, was synthesized at a 0.25 mmol scale, using standard Fmoc (9-fluorenylmethoxycarbonyl) chemistry, on a model 433A solid-phase peptide synthesizer (Applied Biosystems Inc., Foster City, CA). The crude peptide was purified by reverse-phase chromatography (Prep-10 Aquapore C8 column, Applied Biosystems Inc.) using an acetonitrile gradient in 0.1% trifluoroacetic acid. The C2C peptide was conjugated to ovalbumin and used to prepare monoclonal antibodies in BALB/c mice, as previously described.35'36 The antibody isotype was determined using a commercial isotype screening kit (Mouse MonoAb ID Kit, Zymed, San Francisco, CA). The antibody was purified from ascitic fluid by Protein A affinity chromatography and biotin-labeled using sulfo-NHS-LC-biotin (Pierce). Sulfo-NHS-LC-biotin was dissolved in distilled water at a 1 mg/ml concentration and mixed with the purified antibody, previously dialyzed against 0.1M carbonate buffer pH 8.5, at a ratio of 80 pg biotin per mg antibody. After an incubation of 1 hour at room temperature, with stirring, the free sulfo-NHS-LC-biotin was removed by dialysis in PBS pH 7.2. An inhibition ELISA for the quantitative analysis of C2C fragments used a DELFIA time-resolved immunofluorometric technique, which greatly reduces the background fluorescence signal. To improve the sensitivity of the immunoassay, the C2C peptide is conjugated to keyhole limpet hemocyanin, KLH, using bromoacetic acid-N-hydroxysuccinimide ester (Sigma Chemical Co., St.Louis, MO). White, opaque 96-well plates (Greiner) were coated with C2C peptide-KLH conjugate diluted in PBS pH 7.2 (100 ng/ml, 50 ul/well). After an overnight incubation at 4°C, the plates were washed six times with an automatic plate-washer and blocked with 150 pl/well PBS pH 7.2 containing 1% 97 wt/vol bovine serum albumin (BSA), RIA grade. Standards were prepared by diluting a lmg/ml stock solution of C2C peptide to 1000, 333.33, 111.11, 37.04, 12.35, 4.12 and 1.37 ng/ml in PBS-1% BSA. Standards and samples were added at 50 ulAvell, in triplicate, to 96 well U-bottom polypropylene plates (Corning Life Sciences), used as preincubation plates, followed by the addition of 50 ulAvell of C2C biotin-labeled antibody, at a dilution in PBS-1% BSA-0.1% Tween 20 (pH 7.2) previously determined by checkerboard analysis. The triplicate nonspecific binding wells consisted of 50 pi PBS-1% BSA and 50 pi PBS-1% BSA-0.1% Tween 20, and the triplicate maximum binding wells contained 50 pi PBS-1% BSA and 50 pi of the C2C biotin-labeled antibody. After an over-night incubation at 4°C, using a multichannel pipette, 50pl of each preincubated well was transferred to the equivalent well of a Greiner white ELISA plate, coated with C2C peptide-KLH. The ELISA plates were each incubated, with shaking, for 45 minutes at 4°C and then washed three times with PBS-Tween. DELFIA Eu-labeled streptavidin (EG&G WALLAC), diluted 1:1000 in DELFIA assay buffer (EG&G WALLAC), was added to each plate at 50 pi /well and incubated for lh at 37°C. The plates were subsequently washed three times with PBS- 0.1% Tween 20 and once with distilled water. Next, 50 pi /well of DELFIA enhancement solution (EG&G WALLAC) were added and the plates were incubated with shaking at room temperature for 30 minutes. Eu-labeled streptavidin is practically non-fluorescent, but when Eu 3 + is dissociated from the conjugate by the acidic chelating detergent DELFIA enhancement solution, it rapidly forms a new highly fluorescent chelate. Fluorescence at 615nm was measured by a EG&G Wallac 1412 plate reader (Turku, Finland). Calibration curve-fitting and interpolation to determine unknown analyte concentrations were performed by the 98 instrument software. Serum and urine values are reported as picomoles of cleavage neoepitope per milliliter (pmol/ml). Urine values are adjusted for creatinine excretion and are expressed per pmol creatinine. In cases where no C2C epitope was detectable, a value equivalent to half of the lowest standard, or 0.72 pmol/ml was assigned. 7.2.3 Study Outcomes The primary outcome was the mean change at final study visit compared to baseline in serum and urine C1,2C/C2C ratio in the glucosamine and placebo groups. Since this study was designed as a discontinuation trial, if glucosamine is effective, one would expect the placebo group to develop an increase in CII degradation markers, while minimal change would be anticipated in the glucosamine group. Secondary outcomes included the change from baseline to final visit in serum and urine C2C and C1,2C, as well as a comparison of the proportion of undetectable urine C2C and C1,2C in the two treatment groups. Secondarily, it was of interest to evaluate whether clinical flare was associated with changes in CII degradation. For this purpose, the mean change in serum and urine C2C, C1,2C and C1,2C/C2C ratio was compared between subjects who flared and those who did not flare. In addition, correlations between serum and urine levels of C2C, C1,2C and C1,2C/C2C ratio were determined. 7.2.4 Statistical analysis Baseline characteristics were compared between the glucosamine and placebo 99 treatment groups. For the primary outcome of biomarker ratio and for the individual biomarker levels, the difference in mean change at final visit compared to baseline in the two treatment groups was analyzed using Student's t-test. The proportion of subjects with undetectable urine C1,2C and C2C was assessed using Chi-square test. Linear regression analyses were performed to evaluate the effect of treatment on the final serum and urine C1,2C/C2C ratio. Because of non-normal distributions, final serum and urine C1,2C/C2C ratios were log transformed. Univariate linear regression analysis was done initially, followed by multivariable linear regression to adjust for imbalanced variables (gender and OA radiographic severity) and potential confounders (baseline C1,2C/C2C ratio, hand OA, menopausal status, OA duration, flare status and baseline WOMAC function). A comparison of the mean change in biomarkers in Flare versus No-flare subjects was performed using Student's t-test. Correlations between serum and urine biomarkers were determined by Spearman's rank correlation coefficient. 7.3 Results Baseline and final visit samples were available in 130 subjects (serum) and 126 subjects (urine). As reported previously,30 glucosamine and placebo groups were balanced at baseline for all clinical variables except gender (placebo 69%, glucosamine 48% females) and OA radiographic severity (placebo 65%, glucosamine 48% Kellgren-Lawrence grade 2) (Table 7.1). With regards to serum and urine C1,2C, C2C and C1,2C/C2C ratio, glucosamine and placebo groups were balanced at baseline (Table 7.1). The primary outcome of serum C1,2C/C2C ratio increased in the placebo group with a mean change (SD) of 0.8 (27.8) and decreased slightly in the glucosamine group with a 100 mean change (SD) of-0.1 (1.8). The between-group difference of 0.9 was not statistically significant (95% confidence interval [CI] -6.0, 7.7; p=0.80) (Table 7.2). Similarly, no statistically significant difference was seen in the mean change of urine C1,2C/C2C ratio in the placebo and glucosamine groups with a mean change (SD) of -0.6 (8.7) and -1.1 (12.4), respectively [difference 0.5; 95% CI -3.3, 4.2; p=0.82] (Table 7.2). Table 7.3 shows the differences in mean change of individual serum and urine CII degradation markers for placebo and glucosamine treated subjects. With the exception of serum C1,2C, there is a trend towards all serum and urine CII degradation products to be reduced in the glucosamine group and unchanged or increased in the placebo group at final visit compared to baseline. Because of large standard deviations, these differences were not statistically significant, although a trend towards significance was seen for serum C2C reduction in the glucosamine group (p=0.12). At final visit, undetectable urine C2C and urine C1,2C was seen in similar proportion of subjects in the placebo and glucosamine groups (Table 7.3). A univariate linear regression analysis with treatment as the explanatory variable, revealed no effect of treatment group on final visit serum C1,2C/C2C ratio. Similarly, after adjustment for potential confounders, treatment was not a significant predictor of final serum C1,2C/C2C ratio. In the final linear regression model, increased baseline serum C1,2C/C2C ratio and increased baseline WOMAC function were significantly associated with an increase in the log of the final serum C1,2C/C2C ratio (Table 7.4). This final linear regression model was able to explain 72% of the variability in final serum log C1,2C/C2C ratio. The use of total WOMAC and WOMAC pain instead of WOMAC function was also significant in this final model (Total WOMAC, p=0.014; WOMAC pain, p=0.048). In uni-101 and multivariable linear regression analysis using final urine log C1,2C/C2C ratio as the outcome, treatment was also not significant. Only baseline urine log C1,2C/C2C ratio was a significant predictor of final urine log C1,2C/C2C ratio (data not shown). Because there was a trend in serum C2C level to be different in the glucosamine and placebo groups, the effect of treatment was investigated further in linear regression analysis as well. After adjustment for confounders, treatment was not significantly associated with final serum log C2C level. Baseline serum log C2C level and baseline WOMAC function were significantly associated with final visit log serum C2C. To evaluate whether CII degradation was associated with a clinical flare, a comparison of the mean change in serum and urine C1,2C/C2C ratio between No-Flare and Flare subjects was undertaken. A trend towards a significant difference was seen for serum C1,2C/C2C ratio with a mean change (SD) from baseline of-1.9 (17.4) and 3.4 (22.1) for the No-Flare and Flare groups, respectively, and a between-group difference of-5.3 (95% CI -12.2, 1.5; p=0.12) (Table 7.5). In addition; although flare was not retained in a backward stepwise multivariable linear regression model, it was borderline significantly associated with final serum log C1,2C/C2C ratio (p=0.16). The mean change in urine C1,2C/C2C ratio was similar between the No-Flare and Flare groups (p=0.92). Similarly, the mean change in individual serum and urine cartilage degradation markers was not significantly different in No-Flare versus Flare subjects (data not shown). Correlations between serum and urine C2C, C1,2C and C1,2C/C2C ratio were found to be low both at baseline and at final visit, ranging from -0.10 to 0.08. 102 7.4 Discussion In recent years, the possibility of disease modification in OA has attracted considerable interest. But, to identify disease modification valid measures of outcome that are sensitive to change are needed. The traditional "hard" outcome in OA, radiographic change, is technically difficult to quantify in a three dimensional joint and requires long-term follow-up. Furthermore, long-term follow-up causes difficulty for maintaining subjects in randomized trials, the study design required to establish efficacy. Thus, alternative intermediate outcomes have been sought. A major alternative approach under consideration for outcome assessment of disease modification in OA involves biochemical measurements. The pathology of OA and the key enzymatic processes involved in the destruction of cartilage have been studied intensively. This improved understanding of OA pathology has led to the recent development of new assays to measure the products of cartilage degradation, such as CII cleavage products, which were assessed in this study in an attempt to confirm the previous suggestion of a disease modifying effect of glucosamine. This study failed to confirm a significant effect of glucosamine sulfate on CII degradation. Had glucosamine affected CII degradation, the CII biomarkers would have been expected to rise in the placebo group and remained more or less stable in the glucosamine treated group. Although the direction of change for the serum C1,2C/C2C was appropriate in the univariate analysis (Table 7.2), treatment was not a significant predictor in the multivariable model after adjustment for potential confounders (Table 7.4). Similarly, there appeared to be an association of treatment with serum C2C in univariate analysis (Table 7.3), which was no longer present after adjustment for confounders. For C1,2C, there 103 was essentially no change (Table 7.3). This might be related to the known cross-reactivity of type I with type II collagen in the C1,2C assay which would reduce the specificity of the assay for CII degradation. Because it is not known what constitutes a clinically important difference in serum C1,2C/C2C ratio or in C2C, it is not clear whether the changes observed in this study from baseline to final visit represent important differences for these biomarkers. The urine biomarker data failed to demonstrate any differences between the glucosamine and placebo treated groups (Tables 7.2 and 7.3). The lack of information on the metabolism of the biomarkers studied and the absence of other studies on urine C2C and C1,2C makes interpretation of the urinary results difficult. A comparison of Flare with No-flare subjects was undertaken to assess whether biomarker outcomes were related to clinical flare. Although not significant, the direction of the difference in the change in serum C1,2C/C2C from baseline to final visit was appropriate (Table 7.3, p=0.12). Similarly, the linear regression analysis demonstrated flare status to be borderline significant (Table 7.4, p=0.16). This suggests that clinical flare may be accompanied by biochemical changes of CII degradation. In addition, worse baseline function and pain on WOMAC were significantly associated with an increased final serum C1,2C/C2C ratio in the linear regression analysis, confirming that CII degradation markers are linked to clinically relevant outcomes. This has not been reported previously for the C1,2C/C2C ratio, although one study did report high correlations of C2C with clinical outcomes in inflammatory arthritis.22 Since functional ability and pain are to some extent related to the severity of OA, these findings are consistent with the notion that biomarkers may be useful as surrogate markers in OA. Further research is required to confirm these 104 findings. CII degradation markers have been developed only recently. As a result, incomplete information exists for these biomarkers as outcome measures in longitudinal studies, which represents a potential limitation of this study. However, some evidence of validity for the use of C1,2C and C2C is available from animal studies15'17'18'19 and recent human 99 9"^  94 15 1V 18 studies. ' ' In studies of spontaneous OA in mice and surgically induced OA in rats, ' C1,2C was detected by immunostaining at the site of cartilage lesions, but not in adjacent normal cartilage, suggesting that OA is associated with generation of the C1,2C neoepitope. Chu et al1 9 reported significant elevations of C2C levels in synovial fluid of dogs that had undergone cranial cruciate ligament rupture compared to controls. This elevation was maintained at 16 weeks following ligament rupture.19 Additional validation for the use of C1,2C, C2C and C1,2C/C2C ratio has come from recent cross-sectional human studies.22'23'24 Fortin et al 2 3 reported that the test-retest correlation was high for C2C (>0.7) and moderate for C1,2C (0.4-0.7). In regression analyses, comparing OA with normal control subjects, elevated C2C was found to be associated with late knee and hip OA, while elevated C1,2C was associated with late hip OA. 2 3 This study suggests that serum C2C is reliable and able to discriminate OA from non-OA. Fraser et al 2 2 measured levels of C2C and other molecular markers in synovial fluid in patients with early and advanced inflammatory arthritis in comparison to patients with OA. C2C levels were elevated to a similar extent in all groups even though significant elevations of MMP-1 were only noted for the inflammatory arthritis groups.22 In the study by Cerejo et al,2 4 which has only been published in abstract form, an elevated baseline serum C1,2C/C2C ratio was associated with radiographic OA disease progression over 18 months in subjects without generalized OA. 105 Longitudinal data on serum C1 ,2C/C2C ratio was not provided in the abstract. It is therefore not clear whether a change in serum C1 ,2C/C2C ratio was also associated with radiographic disease progression in that study and hence whether C1 ,2C/C2C represents an appropriate outcome measure for disease progression. These studies provide preliminary evidence for the validity of CII degradation biomarkers. However, additional research is required and hence our study findings need to be considered in this context. The fact that this study showed an association of WOMAC pain and function as well as a trend towards an association of clinical flare with the serum C1 ,2C/C2C ratio provides further support for the use of these biomarkers. A further limitation of this study includes the possibility of inadequate sample size. Since the primary purpose was to assess symptom modification of glucosamine, the sample size was based on clinical parameters. High intra- and inter-subject variability of biomarker levels was seen in this study. As a result, our sample size may not have been adequate to detect a difference in change score, if one exists. Furthermore, follow-up was limited to 6 months or less, if a flare developed. In the absence of any longitudinal data, it is not clear whether such a time period is sufficient to allow for a detectable change in CII degradation products. Another potential limitation relates to the fact that the presence of OA in other joints contributes to the serum and urine levels of CII degradation markers. Cerejo et al 2 4 noted that C1 ,2C/C2C ratio was only a predictor for disease progression in subjects with non-generalized OA. In our study, information on hand OA was collected and included in the linear regression analyses and no evidence of a confounding effect was noted. However, information on OA in the spine, hips or other joints was not available and hence this represents a potential limitation. 106 In this study, we evaluated the effect of glucosamine on CII degradation only. The possibility of an effect of glucosamine on cartilage biosynthesis or on other degradation enzymes has been raised.26'27 Some studies have reported that glucosamine reduces MMP-3 activity,26'27 which is involved in the degradation of aggrecan and proteoglycans. As a result, the possibility of a disease modifying effect of glucosamine on MMP-3 and proteoglycan degradation pathways cannot be addressed by our study. Similarly, an effect of glucosamine on matrix synthesis cannot be ruled out. However, given that CII degradation plays a key role in the pathogenesis of OA and its progression, and given that this study found no effect of glucosamine on CII degradation, one should equally consider the possibility that glucosamine may have no or minimal disease modifying effects, even if the biosynthetic activity of chondrocytes is enhanced by glucosamine. Further and more comprehensive human research is necessary to elucidate which biopathologic systems, if any, are affected by glucosamine treatment and whether the overall effect results in disease modification. In conclusion, no statistically significant effect of glucosamine sulfate on CII degradation biomarkers was found in patients with knee OA over 6 months. However, because the use of these biomarkers as outcome measures has not been fully validated, our findings need to be interpreted with caution. Further research is needed to validate CII degradation biomarkers and to evaluate possible disease modifying effects of glucosamine using these and other biomarkers. 107 Table 7.1: Baseline Characteristics of Placebo and Glucosamine Groups for Type II Collagen Degradation Markers Placebo Glucosamine N = 65 N = 65 Female gender 69% 48% Radiographic osteoarthritis severity Kellgren-Lawrence grade 2 (%) 65% 48% Kellgren-Lawrence grade 3 (%) 32% 43% Kellgren-Lawrence grade 4 (%) 3% 9% Serum Analysis N = 65 N = 65 Median serum C2C (pmol/ml) [range] 61.1 [1-681] 52.2 [12-446] Median serum C1,2C (pmol/ml) [range] 162 [66-340] 162 [11-390] Median serum C1,2C/C2C ratio [range] 2.8 [0.2-180] 3.5 [0.2-15] Urine Analysis N = 63 N = 63 Median urine C2C (pmol/pmol creatinine) [range] 12.8 [0.2-57] 11.9 [0.1-425] Median urine C1,2C (pmol/pmol creatinine) [range] 14.2 [2-99] 13.2 [2-1154] Median urine C1,2C/C2C ratio [range] 1.4 [0.1-61.8] 1.5 [0.2-63.8] Undetectable urine C2C (%) 14% 13% Undetectable urine C1,2C (%) 25% 19% 108 Table 7.2: Mean Change in Serum and Urine C1,2C/C2C Ratio at Final Visit Compared to Baseline and Between-Group Differences Mean (SD) change from Between-group p-value baseline difference (95% CI) Placebo Glucosamine Serum C1,2C/C2C ratio Urine C1,2C/C2C ratio 0.8(27.8) -0.1(1.8) -0.6(8.7) -1.1(12.4) 0.9 (-6.0, 7.7) 0.5 (-3.3, 4.2) 0.80 0.82 SD = Standard deviation, CI = confidence interval. 109 Table 7.3: Mean Change in Serum and Urine C1,2C and C2C at Final Visit Compared to Baseline and Proportion of Undetectable Urine C1,2C and C2C at Final Visit Mean (SD) change from Between-group P-baseline difference (95% CI) value Placebo Glucosamine Serum C2C (pmol/ml) 3.7 (23.6) -3.5 (28.5) 7.2 (-1.9, 16.3) 0.12 Serum C1,2C (pmol/ml) 9.5 (80.0) 8.5 (64.2) 1.0 (-24.2, 26.2) 0.94 Urine C2C (pmol/ml) -0.6(11.8) -6.9(54.1) 6.3 (-7.6, 20.1) 0.37 Urine C1,2C (pmol/ml) 0.4(17.1) -20.2 (144.9) 20.6 (-15.8, 57.0) 0.27 Undetectable urine C2C (%) 13% 6% 7% (-5%, 18%) 0.36 Undetectable urine C1,2C (%) 24% 16% 8% (-8%, 23%) 0.37 SD = Standard deviation, CI = confidence interval. 110 Table 7.4: Multivariable Linear Regression Analysis for the Prediction of Final Serum log C1,2C/C2C Ratio Variable Reference Parameter 9 5 % confidence p-value group estimate (SE) interval Treatment group Placebo 0.014 (0.082) -0.15, 0.18 0.86 Gender Male 0.043 (0.083) -0.12, 0.21 0.61 OA radiographic severity Mild OA -0.017 (0.085) -0.18, 0.15 0.84 Flare No flare 0.12 (0.083) -0.048, 0.28 0.16 Log baseline serum 0.81 (0.046) 0.72, 0.90 O .001 C1.2C/C2C ratio Baseline WOMAC function 0.034 (0.014) 0.007, 0.062 0.014 per 100 units SE = standard error 111 Table 7 . 5 : Mean Change in Serum and Urine C1,2C/C2C Ratio at Final Visit Compared to Baseline in the No-Flare and Flare Subjects and Between-Group Differences Mean (SD) change from baseline Between-group difference (95% CI) p-value No-Flare Flare Number with serum samples Serum C1,2C/C2C ratio 75 -1.9(17.4) 55 3.4 (22.1) -5.3 (-12.2, 1.5) 0.12 Number with urine samples Urine C1,2C/C2C ratio 74 -0.8(12.3) 52 -1.0 (7.8) 0.2 (-3.6, 4.0) 0.92 SD = Standard deviation, CI = confidence interval 112 7.5 References 1) Felson DT, Zhang Y. 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Arthritis Rheum 2000;43:673-82. 114 17) Stoop R, Buma P, Van Der Kraan PM, Hollander AP, Billinghurst RC, Poole AR, et al. Differences in type II collagen degradation between peripheral and central cartilage of rat stifle joints after cranial cruciate ligament transection. Arthritis Rheum 2000;43:2121-31. 18) Stoop R, Buma P, Van Der Kraan PM, Hollander AP, Billinghurst RC, Meijers THM, et al. Type II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats. Osteoarthritis Cartil 2001;9:308-15. 19) Wu W, Billinghurst RC, Pidoux I, Antoniou J, Zukor D, Tanzer M, Poole AR. Sites of collagenase cleavage and denaturation of type II collagen in ageing and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13. Arthritis Rheum 2002; 46:2087-94. 20) Chu Q, Lopez M, Hayashi K, Ionescu M, Billinghurst RC, Johnson KA, et al. Elevation of a collagenase generated type II collagen neoepitope and proteoglycan epitopes in synovial fluid following induction of joint instability in the dog. Osteoarthritis Cartil 2002;10:662-9. 21) Squires GR, Okouneff S, Ionescu M, Poole AR. The pathobiology of focal lesion development in ageing human articular cartilage and molecular matrix changes characteristic of osteoarthritis. Arthritis Rheum, 2003; 48:1261-70. 22) Fraser A, Fearon U, Billinghurst RC, Ionescu M, Reece R. Barwick T, et al. Turnover of type II collagen and aggrecan in cartilage matrix at the onset of inflammatory arthritis in humans. Relationship to mediators of systemic and local inflammation. Arthritis Rheum 2003;48:3085-95. 115 23) Fortin PR, Abrahamowicz M, Badley E, King LE, du Berger R, Chang E, Poole AR. Discriminatory ability and reliability of serological biomarkers for osteoarthritis. Arthritis Rheum 2003;48(suppl):S684. 24) Cerejo R, Poole AR, Ionescu M, Lobanok T, Song J, Cahue S, et al. The association between cartilage collagenase activity measured in serum and progression of knee osteoarthritis in patients with and without evidence of generalized disease. Arthritis Rheum 2002;46(suppl):S144. 25) Largo R, Alvarez-Soria MA, Diez-Ortego I, Calvo E, Sanchez-Pernaute O, Egido J, et al. Glucosamine inhibits IL-ip-induced NFK(3 activation in human osteoarthritic chondrocytes. Osteoarthritis Cartil 2003;11:290-8. 26) Dodge GR, Jimenez SA. Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartil 2003;11:424-32. 27) Altman RD, Cheung H. Glucosamine sulfate on cartilage: Lapine study. Arthritis Rheum 2001;44(suppl):S308. 28) Sandy JD, Gamett D, Thompson V, Verscharen C. Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine. Biochem J 1998;335:59-66. 29 ) Nakamura H, Tanaka M, Masuko-Hongo K, Kato T, et al. Clinical effects and possible mechanisms of glucosamine in the treatment of osteoarthritis. Arthritis Rheum 2001;44 (suppl):S309. 30) Cibere J, Kopec JA, Thorne AE, Singer J, Canvin J, Robinson DB et al. Randomized double-blind placebo-controlled glucosamine discontinuation trial in knee 116 osteoarthritis. Arthritis Care Res 2004 (in press). 31) Altman R. D, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039-49. 32) Ehrich EW, Davies GM, Watson DJ, Bolognese JA, Seidenberg BC, Bellamy N. Minimal perceptible clinical improvement with the Western Ontario and McMaster Universities Osteoarthritis Index questionnaire and global assessments in patients with osteoarthritis. J Rheumatol 2000;27:2635-41. 33) Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW. Validation Study of WOMAC: A health status instrument for measuring clinically important participant relevant outcomes to antirheumatic drug therapy in participants with osteoarthritis of the hip or knee. J Rheumatol 1988;15:1833-40. 34) The EuroQol Group. EuroQol - a new facility for the measurement of health-related quality of life. Health Policy 1990;16:199-208. 35) Hughes CE, Caterson B, White RJ, Roughlley PJ, Mort JS. Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. J Biol Chem 1992;267:16011-14. 36) Rosenberg, LC, Choi HU, Chang LH, Johnson TL, Pal S, Webber C, et al. Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages. J Biol Chem 1985;260:6304-13. 117 CHAPTER 8 DISCUSSION AND CONCLUSIONS The manuscripts comprising this thesis provide novel insight into the efficacy of glucosamine in knee OA as it relates to both symptom and disease modification. Specifically, this clinical study provides no evidence of symptom modifying effects with maintenance treatment of glucosamine. In addition, no disease modifying effects of glucosamine are evident for type II collagen degradation based on the specific biomarkers used in this study. 8.1 Glucosamine and Symptom Modification With regard to symptom modification, the results from this thesis are in contrast to the majority of published glucosamine literature which has reported positive results, but in keeping with a recent trend towards negative studies by independent researchers. More importantly, this thesis provides a novel perspective on the question of efficacy by virtue of the fact that an RDT design was used. This choice in design was largely driven by considerations of feasibility of study recruitment given the wide-spread use of glucosamine in patients with knee OA. However, the pre-selection of moderate glucosamine responders for inclusion into the study conferred the additional advantage of a greater ability to show a difference between treatment groups, i f one exists. Despite this advantage, the results from this study showed that there was no difference between glucosamine and placebo groups in the proportion of flares, time to flare, severity of flare or analgesic medication use. The interpretation of these results in the context of the RDT study design indicates that, in patients with at least moderate response to prior glucosamine use, the continued use 118 of glucosamine is not beneficial for the symptomatic treatment of knee OA. Although this finding will lead to questions on the efficacy of glucosamine in general, such extrapolation is beyond the conclusion of this study, since the possibility of an initial short term benefit of glucosamine on symptoms cannot be exclude. However, regardless of whether an initial benefit is derived, the conclusion of no symptomatic benefit of glucosamine as a maintenance therapy in knee OA is appropriate. This conclusion has important implications for clinical practice. The concept of maintenance treatment has not been addressed in any prior glucosamine study. As a result, a specific recommendation regarding duration of glucosamine use could not be made previously. This has resulted in patients continuing to take this supplement for prolonged periods of time at substantial personal cost. With the results from this study, the discontinuation of glucosamine for the symptomatic management of knee OA can be supported more confidently. Although a flare may occur on discontinuation of glucosamine, the results from this study provide reassurance that such a flare is not due to glucosamine withdrawal. The differential finding of glucosamine efficacy in the literature was discussed in Chapter 5 and possible reasons for this current dichotomy of positive and negative results were explored. Further research questions are dictated by these considerations. Specifically, the question of whether severity of OA impacts the finding of efficacy has been raised. This issue was evaluated in the Cox regression analysis, but no interaction of treatment with OA radiographic severity was found suggesting that treatment did not have a differential effect depending on OA severity. However, this study was not specifically designed to evaluate such an association. Given that all glucosamine studies to date have included mixed 119 populations in terms of O A severity, this question remains unanswered and requires further exploration. Another consideration relates to the use of the specific glucosamine product. Since most positive studies to date have used glucosamine manufactured by the Rotta Pharmaceutical Company, the possibility of a product-specific response needs to be investigated further. 8.2 Glucosamine and Disease Modification With regards to disease modification, the results from this study demonstrate a lack of a statistically significant effect of glucosamine on type II collagen degradation, as measured by the C2C and C1,2C biomarker assays or their ratio. These results contribute uniquely and in several ways to the current body of biomarker literature, as it pertains to glucosamine. First, no investigations of the effect of glucosamine on type II collagen degradation have been reported, not even in in vitro or animal studies. Secondly, the effect of glucosamine on biomarkers in human subjects has only been evaluated in one study, which measured products of biosynthesis. As a result, this thesis provides novel insight that, in patients with knee OA, glucosamine does not appear to have any disease modifying effects on type II collagen degradation, although the lack of effect is subject to the limitations discussed below. Because the biomarker analysis was secondary in this study, these data should be considered hypothesis generating. The limitations of this analysis, as discussed in chapter 7, provide important considerations for future research. Specifically, large inter-subject variability was demonstrated in our data. This needs to be taken into account in futures studies to ensure adequate sample size. Another important consideration is study duration. 120 Any advantage that biomarkers may have over other modalities for the assessment of disease modification relates to their potential ability to evaluate change over shorter periods of time, which would hopefully lead to substantial reductions in study cost. Whether a 6-month study duration is sufficient to show such change in type II collagen degradation products with glucosamine is not clear. A further key point, which requires evaluation in future studies, is potential confounding due to OA in joints other than the knee. This was incompletely investigated in this study, although our results suggest that the effect of glucosamine on biomarkers is not changed after adjustment for hand OA. Given these limitations, it is clear that additional prospective studies are necessary to evaluate whether glucosamine has any beneficial effects on type II collagen degradation. In addition, further research into the effects of glucosamine on other biomarkers is clearly needed in human trials. Since OA is the result of imbalanced cartilage synthesis and degradation, ultimately, future studies will need to delineate the effect on both metabolic pathways in order to establish whether glucosamine has overall disease modifying effects. An additional finding of the biomarker analysis pertains to the possible association of flare with a change in type II collagen degradation products, a finding that remained borderline significant in multivariable regression analyses after adjustment for confounders. These results suggest that type II collagen degradation products may be of value in predicting progression, at least as defined by clinical flare. Such an association will need to be confirmed in future studies assessing both flare and response in patients with OA. Furthermore, the finding of a significant association of the C1,2C/C2C biomarker with WOMAC pain and function also confirms the potential utility of these biomarkers. To date, three biomarker studies have evaluated C2C, C1,2C and/or C1,2C/C2C ratio in humans. 121 Only one study reported the correlation of biomarkers with function, although predominantly inflammatory arthritis and only C2C were assessed in that study. Whether C1,2C/C2C is superior to the individual biomarkers is unclear due to the paucity of information on each of these biomarkers in general and a lack of direct comparison data specifically. Although the use of the C1,2C/C2C ratio may not be intuitive, it may indeed be superior to the individual markers given that this ratio was predictive of radiographic knee OA progression over 18 months in a previous study. In this thesis, C2C as well as C1,2C/C2C ratio were associated with W O M A C function in patients with knee OA, findings that have not been previously reported. These findings serve to further validate the use of type II collagen degradation in OA research. 8.3 Strengths and Limitations There are several strengths to this clinical trial evaluating the efficacy of glucosamine for symptom and disease modification. A rigorous study methodology, including randomization, double-blinding and placebo-control were implemented. The selection of at least moderate responders to glucosamine into the trial allowed for greater efficiency of this study to show a difference and is a key consideration in an RDT design. Randomization was performed by a person external to the study using a computer-generated randomization list, including block randomization with variable block size. Hence allocation concealment was maintained. Double-blinding was implemented by the use of pre-packaged study medication bottles. An additional strength includes an assessment of patient blinding at final study visit to ensure that no unblinding of patients occurred during the study. The results indicated that blinding was successfully maintained throughout this study. The use of 122 standardized outcome measures, testing of study drug content, and sophisticated statistical analysis are additional strengths of this clinical trial. Furthermore, the evaluation of state-of-the-art cartilage degradation markers adds a further dimension on the efficacy of glucosamine that has not been assessed previously. There are also several limitations to this study, as discussed in detail in Chapters 6 and 7. The use of an RDT design with inclusion of patients who have previously improved on glucosamine does not allow for an assessment of an initial response to glucosamine. Hence efficacy could be present and is not inconsistent with the finding of a negative RDT result, although such efficacy would be short-term at best. The selection of an appropriate definition of flare represents a potential problem in this study design. This challenge was successfully overcome in this study by the use of validated outcome measures and by the use of a clinically relevant change, which was subsequently demonstrated by others to constitute a clinically important difference. Limitations of the biomarker analysis include the paucity of information in the literature in order to select an appropriate biomarker that is sensitive to change. Similarly, a sample size calculation was not possible for the biomarker analysis due to a lack of available data. Hence the negative findings in this study could have occurred as a result of inadequate power to detect a difference between treatment groups. Further limitations of the biomarker analysis include the relatively short study duration of 6 months, which may not be sufficient to detect a change, as well as incomplete knowledge on other important variables that might have an impact on biomarkers. As a result, these biomarker analyses were largely exploratory. 123 8.4 Contributions and Recommendations Despite the limitations noted above, this thesis contributes to and expands on our current understanding of the efficacy of glucosamine in knee O A with regards to both symptom and disease modification. For symptom modification, this thesis provides novel information that glucosamine is not beneficial as a maintenance treatment in knee O A . Hence this study provides a unique perspective on the efficacy of glucosamine that has not been reported previously. The economic implications of this study finding are important, given the considerable cost and high prevalence of glucosamine use in patients with knee O A . In patients who are currently taking glucosamine for symptom modification, consideration should be given to the discontinuation of this supplement. Although patients may be hesitant to stop a treatment, particularly i f it has been perceived as beneficial on pain, the health care practitioner can play an important role in supporting such an attempt and can do so more confidently given these study results. N o recommendation can be made from this study as to whether glucosamine should be initiated and used short-term in the treatment of knee O A . However, given the conflicting evidence for efficacy in the current literature, the possibility that glucosamine has no efficacy should be considered by health care professionals. Patients should be counseled appropriately and fully in this regard, such that they can make an informed decision. With regards to disease modification, the results from this thesis contribute uniquely and in several ways to the current body of biomarker literature, both as it pertains to glucosamine and to the biomarker literature in general. This study is the first to report the effect of glucosamine on type II collagen degradation and suggests that glucosamine does not have any disease-modifying effects on type II collagen, subject to the limitations 124 discussed previously. Another contribution of this thesis is the finding of an association of type II collagen degradation markers with clinically relevant variables including pain and function. In addition, the possible association with flare suggests that these markers may have some validity in the assessment of progression, at least as defined by flare. Because the biomarker analysis was hypothesis generating, no definitive answers are available on the efficacy of glucosamine with regards to disease modification. Further research is necessary to investigate the validity and utility of type II collagen degradation biomarkers and their importance in the overall assessment of disease progression and disease modification. 8.5 Conclusions In summary, this thesis provides definitive evidence that in patients who have previously had a moderately beneficial response to glucosamine, the continued use of glucosamine is not efficacious for the symptomatic treatment of knee OA. Hence, this thesis shows a clear lack of efficacy of glucosamine as a maintenance treatment in knee OA and thus provides a unique contribution to the glucosamine literature. This thesis also provides novel, although preliminary, information that glucosamine has no significant effect on type II collagen degradation. 125 APPENDIX I Declaration of Candidate's Role in Published Manuscripts 126 APPENDIX II Glucosamine Discontinuation Trial Data Collection Forms 128 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm/ yy) Patient Initials: Patient screening evaluation by phone Screening Questions: YES NO Do you have osteoarthritis (wear and tear arthritis) of the knees? (yes) Who diagnosed osteoarthritis? How was osteoarthritis diagnosed? Are you currently taking glucosamine? (yes) Has your knee pain improved on the glucosamine? (yes) Do you have rheumatoid arthritis or another type of arthritis? (no) Are you taking chondroitin? (no) Have you had a knee injection with hyaluronic acid (Synvisc, Orthovisc) in the last 6 months? (no) Have you had a knee injection with steroids (cortisone, Depo-Medrol) in the last 3 months? (no) Have you had any surgical procedures on either knee in the last 3 months? (no) Is any surgical procedure planned on either knee in the next 8 months? (no) If any box above is marked differently than the answer in brackets, the patient is NOT eligible for enrollment in the study. If the patient is eligible, obtain name, address, phone number. Patient's name: Address: Last name First name Initial Telephone: Home ( ) - _ Work ( ) - _ 129 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm/ yy) Patient Initials: Screening visit - Physic ian contact numbers Family Physician: Name: Address: Telephone: Rheumatologist: (if applicable) Name: Address: Telephone: 130 GLUCOSAMINE WITHDRAWAL STUDY D a t e : _ _ / _ _ / (dd/ mm/ yy) Study ID #: Patient Initials: Screening visit - Demographic data Date of birth: _ _ / _ _ /_ (dd/ mm/ yy) Gender: Male Female Ethnicity: Caucasian Chinese Middle Eastern First Nations Marital status: Single Married/common-law Separated Divorced Widowed Black Other Asian, specify Hispanic Other, specify Education level: Highest grade completed: 1 2 3 4 5 6 7 8 9 1 0 1 1 12 (13) University level completed: 1 2 3 4 + Occupation: If retired, last occupation: Employment: Employed, full time _ Employed, part time Student Homemaker Retired Unemployed Disability pension Other, specify 131 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm/ yy) Patient Initials: Screening visit - Patient Selection: Inclusion criteria P a g e Y E S N O Diagnosis of osteoarthritis, based on the following: 6 Knee pain for one month or more 6 Knee pain on most days of the month, when not treated 11 Osteophytes on x-ray of knee 3 Age 18 years or older 6 Patient's knee pain moderately or markedly improved since starting on glucosamine 6, 8 Patient on glucosamine for 1 month or more 8 Patient currently continuing to take glucosamine Patient has reasonable command of the English language Signed written informed consent If any box above is marked "NO", the patient is NOT eligible for enrollment in the study. 132 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm/ yy) Patient Initials: Screening visit - Patient Selection: Exclusion criteria Page YES NO 6,7 Arthritis other than osteoarthritis 8 Knee injection with hyaluronic acid in last six months 8 Knee injection with corticosteroids in last three months 8 Chondroitin in last 2 months 8 Analgesics other than acetaminophen or NSAIDs 7 Surgical procedure on either knee in last three months 7 Planned surgical procedure on either knee in next 8 months 7, 10 Uncontrolled systemic disorder that could interfere with the evaluation 8 Participation in another clinical trial during the study period or one month prior 7 Allergy to any of the ingredients of glucosamine or placebo 10 Hyperkalemia 11 Chondrocalcinosis on x-ray in association with a clinical history of C P P D If any box above is marked "YES", the patient is NOT eligible for enrollment in the study. 133 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Screening visit - Osteoarthritis disease history Osteoarthritis diagnosis date: / (mm/ yy) Have you had knee pain for more than one month? Yes No What makes knee pain worse/better? Do you have morning stiffness? If so, how long does it last? min. Which knee is more painful (either now or prior to taking glucosamine)? Right Left Please note that the more painful knee will be used for assessment during all future visits in this study. Have you ever been told you have arthritis other than osteoarthritis? (for example rheumatoid arthritis, spondylitis, gout, CPPD) No Yes Do you have episodes of sudden painful, swollen, hot knee joints? No Yes Have you been taking glucosamine for more than one month? Yes No Prior to any treatment, how many days in a month would you have knee pain? Every day More than 5 0 % Less than 5 0 % Rarely Has your knee pain changed since starting on glucosamine? Pain is: Worse Unchanged Mildly improved Moderately improved Markedly improved Completely subsided X-rays of knees: D a t e : _ _ / _ _ > 2 years ago: (mm/ yy) Location: 134 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Screening visit - Medical history Medical history List all current medical conditions or illnesses other than osteoarthritis. Condition/illness Date of onset Comments Check here if no current medical conditions/illnesses Have you had a surgical procedure on either knee in the last three months? No Yes Are you planning any surgical procedures on either knee in the next 8 months? No Yes Drug Allergy: No Yes, specify Corn allergy: no yes Note: If the patient is allergic to corn or any of the other ingredients of the glucosamine or placebo tablets, he/she is not eligible for this study. Ingredients of glucosamine/placebo tablets include: Corn starch, microcrystalline cellulose, silicon dioxide, stearic acid, magnesium stearate, dicalcium phosphate dihydrate, potassium salt Glucosamine/placebo tablets do not include the following: Yeast, soy, lactose, sugar, preservatives, artificial colors and flavors 135 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Screening visit - Medications Current medications Type of glucosamine (sulfate, hydrochloride) Dosage Frequency Starting date Acetaminophen NSAID: NSAID: For this study, the patient will take the study medication at a dose identical to the dose of glucosamine listed above. If he/she was taking more than 1500 mg of glucosamine per day, he/she will be allowed a maximum dose of the study medication of 500 mg three times per day. List all current medications used. Include prescription, over-the-counter, herbal medications and vitamins Medication name and dose Starting date Condition used for Have you taken chondroitin in the last 2 months? No Yes Have you had an intraarticular knee injection with -hyaluronic acid in the last 6 months? No Yes -corticosteroids in the last 3 months? No Yes Are you taking part in another clinical trial currently? No Yes 136 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Screening visit - Physical examination of the knees Abnormality Right knee Yes/No Left knee Yes/No Warmth Joint effusion Tender on joint line palpation End-of-range stress pain on passive movement Crepitus Active ROM (record in degrees, eg 4° to 116°) Collateral ligament instability: medial lateral Cruciate ligament instability: anterior posterior Deformity (on standing): varus valgus Study knee: Determination of the study knee will be based on the patient's history of which knee is more painful. If the patient is uncertain as to which knee is more painful, or if both knees are equally painful, the study investigator will choose the study knee based on the physical examination and/or x-ray findings. 137 GLUCOSAMINE WITHDRAWAL STUDY Date: / _ _ / Study ID #: (dd/ mm / yy) Patient Initials: Screening visit - Laboratory tests Laboratory Test Value Check this box if abnormal (put normal values in brackets) C B C : Hgb W B C Platelets Random glucose Creatinine Electrolytes: Na K Cl H C 0 3 138 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Screening visit - X-ray findings of most symptomatic knee X-ray evaluation and grading: Circle the knee that has been identified as more symptomatic and complete the table below only for that knee. Right knee Left knee Chondrocalcinosis (yes/no) Osteophytes (yes/no) Joint space narrowing (yes/no) Subchondral sclerosis (yes/no) Subchondral cysts (yes/no) Grade (0-4) yes = present, no = absent X-ray grading: Grade 0: normal Grade 1: doubtful osteoarthritis Grade 2: minimal osteoarthritis Grade 3: moderate osteoarthritis Grade 4: severe osteoarthritis (based on Kellgren et al, Ann Rheum Dis 1957;16:494-502) 139 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Screening visit - Checklist Checklist: Tasks to be performed at or following the Screening visit Check this box if done Page Personal and demographic data obtained 2,3 Inclusion criteria completed 4 Exclusion criteria completed 5 History and physical examination performed 6-9 Baseline laboratory investigations obtained 10 X-rays of knee(s) ordered or obtained if previously done 11 Ensure that all of the above tasks have been performed! Study continuation: Does the patient meet both the inclusion and exclusion criteria? Yes No If the answer to the above is "YES", the patient will be assigned the next consecutive Study ID number. Study ID #: Mark the Study ID # on all previously completed data sheets! Contact the patient for his/her Week 0 visit within the next 2 weeks! 140 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID#: (dd/ mm / yy) Patient Initials: Week 0 visit - Medications and other treatments List any new medications since last study visit Dose/ Frequency Starting date Condition used for Study medication - - -Check here, if no change in medications has occurred since last study visit Have you used chondroitin since the last study visit? No Yes Have you had any intraarticular knee injection since the last study visit? No Yes Specify: Knee: Right Left Drug: Reason: 141 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 0 visit - Physical examination of knee and physician global assessment Physical examination: Study knee: Right Left Please place check mark to indicate the study knee. See screening visit - physical examination, page 9, for information on which knee is the study knee) Abnormality of knee examination Yes/No Warmth Joint effusion Tender on joint line palpation End-of-range stress pain on passive movement Crepitus Active ROM (record in degrees, eg 4° to 116°) Physician Global Assessment: Patient status is: 1 2 3 4 5 very poor poor fair good excellent Protocol violation: specify: Please note that patients with protocol violations at this point in time will not be able to enter into the study. 142 GLUCOSAMINE WITHDRAWAL STUDY Date: / _ _ / Study ID #: (dd/ mm / yy) Patient Initials: Week 0 visit - Checklist Checklist: Tasks to be performed at Week 0 Check this box if done Page Patient assessments completed 13,14 W O M A C completed EUROQOL completed Analgesic record sheet supplied 16 Study drug supplied for 1 month Instructions supplied to study participant 17 Blood/urine for OA markers obtained Next appointment scheduled in 2 weeks Ensure that all of the above tasks have been performed, if the patient is continuing in the study! Study continuation: Yes No Specify reason: Flare of OA symptoms Protocol violation , specify: Patient wishes to end study: Adverse event Other reason, specify: Please note that patients with protocol violations at this point in time will not be entered into the study. 143 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 0-2 - Analgesic record Day Date Acetaminophen (Tylenol) NSAID Name: Dosage used? How many times taken? Dosage used? How many times taken? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 144 G l u c o s a m i n e w i t h d r a w a l s t u d y Instructions to study participants: 1. The dose of your study medication is one tablet (500mg) times per day. 2. Once you start taking the study medication and for as long as you are in the study, do NOT take any of the glucosamine tablets that you were taking prior to the study. 3. You must bring back your bottle of study medication at each visit, in order to receive the new bottle of study medication. 4. At each study visit you will be given an "Analgesic Record Sheet". We would like you to record every day on this sheet any use of analgesic (pain) medications that you are taking. Analgesic medications include acetaminophen (Tylenol) and anti-inflammatory medications (NSAIDs) such as Ibuprofen (Advil, Motrin), Aspirin (Entrophen), and Indomethacin (Indocid). Please bring the Analgesic Record Sheet with you at each study visit, even if you did not take any pain medications and therefore the sheet is blank. 5. IF Y O U FEEL THAT Y O U HAVE A FLARE OF Y O U R KNEE ARTHRITIS, CALL THE STUDY INVESTIGATORS TO MAKE AN APPOINTMENT. CONTINUE TAKING Y O U R STUDY MEDICATION UNTIL Y O U CAN BE S E E N BY THE STUDY INVESTIGATOR. DO NOT GO BACK ON THE GLUCOSAMINE THAT YOU W E R E TAKING PRIOR TO THE STUDY, UNTIL THE STUDY INVESTIGATOR HAS S E E N Y O U AND TOLD Y O U THAT Y O U HAVE R E A C H E D THE END OF THE STUDY. If you have any questions regarding these instructions, please don't hesitate to call the study investigators at - . 145 GLUCOSAMINE WITHDRAWAL STUDY Date: _ _ / _ _ / _ _ (dd/ mm / yy) Study ID #: Patient Initials: Week 2 visit - Medications/other treatments and adverse events Medications: List any new medications since last study visit Dose/ Frequency Starting date Condition used for Study medication - -Check here, if no change in medications has occurred since last study visit Have you used chondroitin since the last study visit? No Yes Have you had any intraarticular knee injection since the last study visit? N o _ Yes Specify: Knee: Right Drug: Left Reason: Adverse events: None: List adverse events Severity: 1=mild, 2=moderate, 3=severe Additional comments on patient's symptoms and general well-being: 146 GLUCOSAMINE WITHDRAWAL STUDY Date: _ _ / _ _ / _ _ (dd/ mm / yy) Study ID #: Patient Initials: Week 2 visit - Physical examination of knee and physician global assessment Physical examination; Study knee: Right Left Please place check mark to indicate the study knee. See screening visit - physical examination, page 9, for information on which knee is the study knee) Abnormality of knee examination Yes/No Warmth Joint effusion Tender on joint line palpation End-of-range stress pain on passive movement Crepitus Active ROM (record in degrees, eg 4° to 116°) Physician Global Assessment: Patient status is: 1 2 3 4 5 very poor poor fair good excellent Protocol violation: specify: Please note that patients with protocol violations will continue in the study. 147 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 2 visit - Checklist Checklist: Tasks to be performed at Week 2 Check this box if done Page Patient assessments completed 18,19 W O M A C completed EUROQOL completed Previous analgesic record sheet received 16 New analgesic record sheet supplied 21 Next appointment scheduled in 2 weeks Ensure that all of the above tasks have been completed! Study continuation: Yes No Specify reason: Flare of OA symptoms Patient wishes to end study: Adverse event Other reason, specify: If the patient is not continuing in the study, please complete the study termination questions and termination visit check list (pages 39-40). Also ensure that the patient has blood and urine drawn for OA markers. 148 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID#: (dd/ mm / yy) Patient Initials: Week 2-4 - Analgesic record Day Date Acetaminophen (Tylenol) NSAID Name: Dosage used? How many times taken? Dosage used? How many times taken? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 149 GLUCOSAMINE WITHDRAWAL STUDY Date: / _ _ / Study ID #: (dd/ mm / yy) Patient Initials: Week 4 visit - Medications/other treatments and adverse events Medications: List any new medications since last study visit Dose/ Frequency Starting date Condition used for Study medication - -Check here, if no change in medications has occurred since last study visit Have you used chondroitin since the last study visit? No Yes Have you had any intraarticular knee injection since the last study visit? No Yes Specify: Knee: Right Left Drug: Reason: Adverse events: None: List adverse events Severity: 1=mild, 2=moderate, 3=severe Additional comments on patient's symptoms and general well-being: 150 GLUCOSAMINE WITHDRAWAL STUDY Date: _ _ / _ _ / (dd/ mm / yy) Study ID #: Patient Initials: Week 4 visit - Physical examination of knee and physician global assessment Physical examination: Study knee: Right Left Please place check mark to indicate the study knee. See screening visit - physical examination, page 9, for information on which knee is the study knee) Abnormality of knee examination Yes/No Warmth Joint effusion Tender on joint line palpation End-of-range stress pain on passive movement Crepitus Active ROM (record in degrees, eg 4° to 116°) Physician Global Assessment: Patient status is: 1 2 3 4 5 very poor poor fair good excellent Protocol violation: specify: Please note that patients with protocol violations will continue in the study. 151 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 4 visit - Checklist Checklist: Tasks to be performed at Week 4 Check this box if done Page Patient assessments completed 22,23 W O M A C completed EUROQOL completed Previous analgesic record sheet received 21 New analgesic record sheet supplied 25 Previous study drug returned and pill count performed New study drug supplied for one month Blood/urine for OA markers obtained Next appointment scheduled in 4 weeks Ensure that all of the above tasks have been completed! Pill count: Pills remaining in returned bottle = Study continuation: Yes No Specify reason: Flare of OA symptoms Patient wishes to end study: Adverse event Other reason, specify: If the patient is not continuing in the study, please complete the study termination questions and termination visit check list (pages 39-40). Also ensure that the patient has blood and urine drawn for OA markers. 152 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 4-8 - Analgesic record Day Date Acetaminophen (Tylenol) NSAID Name: Dosage used? How many times taken? Dosage used? How many times taken? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 153 GLUCOSAMINE WITHDRAWAL STUDY Date: / _ _ / (dd/ mm / yy) Study ID #: Patient Initials: Week 8 visit - Medications/other treatments and adverse events Medications: List any new medications since last study visit Dose/ Frequency Starting date Condition used for Study medication - -Check here, if no change in medications has occurred since last study visit Have you used chondroitin since the last study visit? No Yes Have you had any intraarticular knee injection since the last study visit? N o _ Yes Specify: Knee: Right Drug: Left Reason: Adverse events: None: List adverse events Severity: 1=mild, 2=moderate, 3=severe Additional comments on patient's symptoms and general well-being: 154 GLUCOSAMINE WITHDRAWAL STUDY Date: / _ _ / _ _ (dd/ mm / yy) Study ID #: Patient Initials: Week 8 visit - Physical examination of knee and physician global assessment Physical examination: Study knee: Right Left Please place check mark to indicate the study knee. See screening visit - physical examination, page 9, for information on which knee is the study knee) Abnormality of knee examination Yes/No Warmth Joint effusion Tender on joint line palpation End-of-range stress pain on passive movement Crepitus Active ROM (record in degrees, eg 4° to 116°) Physician Global Assessment: Patient status is: 1 2 3 4 5 very poor poor fair good excellent Protocol violation: specify: Please note that patients with protocol violations will continue in the study. 155 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 8 visit - Checklist Checklist: Tasks to be performed at Week 8 Check this box if done Page Patient assessments completed 26,27 W O M A C completed EU ROQOL completed Previous analgesic record sheet received 25 New analgesic record sheet supplied 29 Previous study drug returned and pill count performed New study drug supplied for one month Next appointment scheduled in 4 weeks Ensure that all of the above tasks have been completed! Pill count: Pills remaining in returned bottle = Study continuation: Yes No Specify reason: Flare of OA symptoms Patient wishes to end study: Adverse event Other reason, specify: If the patient is not continuing in the study, please complete the study termination questions and termination visit check list (pages 39-40). Also ensure that the patient has blood and urine drawn for OA markers. 156 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 8-12 - Analgesic record Day Date Acetaminophen (Tylenol) NSAID Name: Dosage used? How many times taken? Dosage used? How many times taken? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 157 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 12 visit - Medications/other treatments and adverse events Medications: List any new medications since last study visit Dose/ Frequency Starting date Condition used for Study medication - -Check here, if no change in medications has occurred since last s tudy visit Have you used chondroitin since the last study visit? No Yes Have you had any intraarticular knee injection since the last study visit? No Yes Specify: Knee: Right Left Drug: Reason: Adverse events: None: List adverse events Severity: 1=mild, 2=moderate, 3=severe Additional comments on patient's symptoms and general well-being: 158 GLUCOSAMINE WITHDRAWAL STUDY Date: / / (dd/ mm / yy) Study ID #: Patient Initials: Week 12 visit - Physical examination of knee and physician global assessment Physical examination: Study knee: Right Left Please place check mark to indicate the study knee. See screening visit - physical examination, page 9, for information on which knee is the study knee) Abnormality of knee examination Yes/No Warmth Joint effusion Tender on joint line palpation End-of-range stress pain on passive movement Crepitus Active ROM (record in degrees, eg 4° to 116°) Physician Global Assessment: Patient status is: 1 2 3 4 5 very poor poor fair good excellent Protocol violation: specify: Please note that patients with protocol violations will continue in the study. 159 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 12 visit-Checklist Checklist: Tasks to be performed at Week 1 2 Check this box if done Page Patient assessments completed 30,31 W O M A C completed EUROQOL completed Previous analgesic record sheet received 29 New analgesic record sheet supplied 33-35 Previous study drug returned and pill count performed New study drug supplied for three months Blood/urine for OA markers obtained Next appointment scheduled in 12 weeks Ensure that all of the above tasks have been completed! Pill count: Pills remaining in returned bottle = Study continuation: Yes No Specify reason: Flare of OA symptoms Patient wishes to end study: Adverse event Other reason, specify: If the patient is not continuing in the study, please complete the study termination questions and termination visit check list (pages 39-40). Also ensure that the patient has blood and urine drawn for OA markers. 160 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 12-24 - Analgesic record Day Date Acetaminophen (Tylenol) NSAID Name: Dosage used? How many times taken? Dosage used? How many times taken? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 • 20 21 22 23 24 25 26 27 28 29 30 31 32 161 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID#: (dd/ mm / yy) Patient Initials: Week 12-24 - Analgesic record continued Day Date Acetaminophen (Tylenol) NSAID Name: Dosage used? How many times taken? Dosage used? How many times taken? 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 162 GLUCOSAMINE WITHDRAWAL STUDY Date: _ _ / / Study ID #: (dd/ mm / yy) Patient Initials: Week 12-24 - Analgesic record continued Day Date Acetaminophen (Tylenol) NSAID Name: Dosage used? How many times taken? Dosage used? How many times taken? 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 163 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 24 visit - Medications/other treatments and adverse events Medications: List any new medications since last study visit Dose/ Frequency Starting date Condition used for Study medication - -Check here, if no change in medications has occurred since last study visit Have you used chondroitin since the last study visit? No Yes Have you had any intraarticular knee injection since the last study visit? No Yes Specify: Knee: Right Left Drug: Reason: Adverse events: None: List adverse events Severity: 1=mild, 2=moderate, 3=severe Additional comments on patient's symptoms and general well-being: 164 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 24 visit - Physical examination of knee and physician global assessment Physical examination: Study knee: Right Left Please place check mark to indicate the study knee. See screening visit - physical examination, page 9, for information on which knee is the study knee) Abnormality of knee examination Yes/No Warmth Joint effusion Tender on joint line palpation End-of-range stress pain on passive movement Crepitus Active ROM (record in degrees, eg 4° to 116°) Physician Global Assessment: Patient status is: 1 very poor 2 poor 3 fair 4 good 5 excellent Protocol violation: specify: 165 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Week 24 visit - Checklist Checklist: Tasks to be performed at Week 24 Check this box if done Page Patient assessments completed 36,37 W O M A C completed EUROQOL completed Previous analgesic record sheet received 33-35 Previous study drug returned and pill count performed Study termination questions completed 39 Blood/urine for OA markers obtained Ensure that all of the above tasks have been completed! Pill count: Pills remaining in returned bottle = 166 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Study Termination Questions Questions: 1 . Do you believe that the study medication you were taking was Glucosamine Placebo Unsure A) If the answer above was glucosamine, why do you believe you were taking glucosamine? My knee pain is better compared to before the study My knee pain is the same as before the study My knee pain is worse compared to before the study I had side effects from the study medication Other reason, specify B) If the answer above was placebo, why do you believe you were taking placebo? My knee pain is better compared to before the study My knee pain is the same as before the study My knee pain is worse compared to before the study I had side effects from the study medication Other reason, specify 2 . Are you planning to continue taking glucosamine? Yes No Why, or why not? 167 GLUCOSAMINE WITHDRAWAL STUDY Date: / / Study ID #: (dd/ mm / yy) Patient Initials: Termination visit - Checklist Checklist: Tasks to be performed at Study Termination Check this box if done Page Patient assessments completed W O M A C completed EUROQOL completed Previous analgesic record sheet received Previous study drug returned and pill count performed Study termination questions completed 39 Blood/urine for OA markers obtained Ensure that all of the above tasks have been completed! Pill count: Pills remaining in returned bottle = 168 

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