A Prospective Pilot Study to Evaluate Efficacy and Safety of Euflexxa for the Treatment of Osteoarthritis
Phase: NA
Conditions: Osteo Arthritis Knee
Sponsor: The Cleveland Clinic

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Study Protocol Title: 
A Prospective Pilot Study to Evaluate Efficacy and Safety of Euflexxa for the Treatment of Osteoarthritis 
 
Principal Investigator: Carlos Higuera, MD     Phone: 216-636-1136 
 
Funding Sponsor:  
Ferring Pharmaceuticals, Parsippany, NJ  
 
Project Contact: Peter Shaw 
Senior Director, Medical Affairs – Orthopaedics 
Ferring Pharmaceuticals Inc. 
100 Interpace Parkway, Parsippany, NJ 07054 
Phone: 862-286-5188 Cell: 908-323-3663 
:  
 
Regulatory Sponsor: 
This study is an Investigator Initiated research trial. Each study site will be considered its own regulatory 
sponsor and is responsible for internal data monitoring and any study reporting required by 
ClinicalTrials.gov. 
 
  
 
 

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Introduction   
 
 
Knee osteoarthritis is one of the most common joint disorders which affects a large portion of 
aging population1. One of the treatment options is viscosupplementation, which is intra-articular injection 
of hyaluronic acid IA-HA. Multiple studies have been conducted on the safety and efficacy of IA-HA, but 
none of the studies, to our knowledge, have evaluated the synovial fluid markers. Therefore, it becomes 
essential to investigate the effect of IA-HA on the concentration of various synovial fluid markers.  
 
Background and Significance 
 
The FLEXX trial2 was the first well-controlled, randomized, double-blind, multicenter study 
evaluating the efficacy of Euflexxa (1% sodium hyaluronate) therapy for knee osteoarthritis.  Five-
hundred-and-eighty patients were randomized to receive either Euflexxa or normal saline.  There were 
significant improvements in the Visual analogue scale score Osteoarthritis Research Society International 
responder index, HRQoL, and function at 26-week follow-up2. However, the FLEXX trial did not 
evaluate changes in synovial fluid bio-marker levels. 
The hypothesis of this study is that Euflexxa therapy for knee osteoarthritis will improve levels of 
inflammatory and degenerative synovial fluid bio-markers in patients with knee osteoarthritis.  In 
addition, we also hypothesize that Euflexxa therapy will improve pain, clinical outcomes, and time to total 
knee arthroplasty. 
 
Study Design 
 
Methods 
 A prospective pilot study 
 
Sample 
 
 25 subjects for a pilot study 
 
Inclusion criteria: 
1) OA of the knee by American College of Rheumatology criteria 
2) Those who failed non-pharmacological measures or simple analgesics 
3) moderate to severe pain score of 41 to 90 mm recorded on 100-mm visual analog scale (VAS) 
immediately following a 50-foot walk 
4) bilateral standing anterior-posterior radiograph demonstrating Kellgren and Lawrence grade 2 
or 3 OA of the target knee 
5) ability and willingness to use only acetaminophen as the analgesic (rescue) study medication 
6) unassisted walking 50 feet on a flat surface and going up and down stairs 
7) willingness and ability to complete efficacy and safety questionnaires 
 
Exclusion criteria:  
1. Age less than 18 years 
2. any major injury to the target knee within the prior 12 months 
3. any surgery to the target knee within the prior 12 months  
4. surgery to the contralateral knee or other weight-bearing 
5. inflammatory arthropathies 
6. gout or pseudogout within the previous 6 months 
7. radiographic acute fracture, severe loss of bone density, avascular necrosis, and/or severe bone 
or joint deformity in the target knee 

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8. osteonecrosis of either knee 
9. fibromyalgia, pes anserine bursitis, lumbar radiculopathy, and/or neurogenic or vascular 
claudication 
10. target knee joint infection or skin disorder/ infection within the previous 6 months 
11. symptomatic OA of the hips, spine, or ankle;  known hypersensitivity to acetaminophen, IA-
BioHA, or phosphate-buffered saline solution 
12. Women of childbearing potential who are pregnant, nursing, or planning to become pregnant, 
and those who do not agree to remain on an acceptable method of birth control throughout the 
study 
13. history of immune disorders; vascular insufficiency of lower limbs or peripheral neuropathy 
14. current treatment or treatment of cancer within the previous 2 years (excluding basal cell or 
squamous cell carcinoma of the skin) 
15. active liver or renal disease 
16. any clinically significant abnormal laboratory value [to be defined in detail at a later point] 
17. any intercurrent chronic disease or condition that might interfere with the completion of the 
study 
18. participation in any experimental device study within the prior 6 months or any experimental 
drug study within the prior month 
 
Research Procedures – Define the data to be collected to answer the hypothesis/research 
question(s) 
 
Injection: 
 
Twenty five patients will be assigned to receive Euflexxa (n=10) (1% sodium hyaluronate).  Three 
consecutive injections (2 sets) will be performed with 1 weeks’ time in-between each injection at the 
index visit (Index visit, Week 1±3 days and Week 2±3 days) and at six months (6 month, Week 7, and 
Week 8). 
 
Aspiration (primary outcome) 
 
Knee joint aspiration will be performed prior to the initial injection (at visit 1), and at visit 4 (6 weeks ± 1 
week) and visit 6 (6-months ±2 weeks) as outlined in previous studies which measured synovial fluid bio-
markers.3,4 Following aspiration, the synovial fluid will be placed on ice as soon as possible, and 
transported to the lab. In the lab it will be centrifuged at 1,500 rpm for 10 minutes at a temperature of 
4⁰C. Subsequently, the supernatant fluid will be removed and 1mL of Trizol will be added to the cell 
pellet (sediment). The pellet will be stored at -20⁰C and shipped to Rush University Medical Center for 
future RNA sequencing analyses (cell markers and TLR/Nfk-b Pathway genes). The results of these tests 
(RNA sequencing) will not be shared with the patients, and will not be documented in the medical record. 

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The supernatant fluid will be used for biomarker analysis. Remaining fluid will be frozen and stored for 
future research. The purpose of the aspiration is to measure the following synovial fluid bio-markers3,4: 

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Biomarker 
Measurement Method 
1. Chondroitin 6-sulfate (C6S) 
FACE1 and sizing 
2. Chondroitin 4-sulfate (C4S) 
FACE and sizing 
3. Keratan sulfate (KS) 
FACE and sizing 
4. Hyaluronate (HA) 
FACE and sizing 
5. Matrix metalloproteinase-3 (MMP-3) 
ELISA2 
6. Tissue inhibitor of MMP-1 (TIMP-1) 
ELISA 
7. Tissue Necrosis Factor-alpha (TNF-α) 
ELISA 
8. IL-6 
ELISA 
9. Tumor necrosis factor-stimulated gene 6 (TSG-6) 
ELISA 
10. Inter alpha inhibitor (IαI) 
Western Blot 
 
                                                          
1 FACE- Fluorophore-assisted carbohydrate electrophoresis  
2 ELISA – Enzyme-linked immunosorbent assay 

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Secondary outcome measurements5 
 
 The effect of Euflexxa on pain, using the Visual Analougue Scale (VAS)  
 The effect of Euflexxa on Clinical outcomes using the following standardized scales and 
questionnaires:  
1. Western Ontario and McMaster Universities Arthritis Index (WOMAC)  
2. Knee Society Score (KSS)  
3. Veterans Rand-12 item form (VR-12)  
4. UCLA activity score (UCLA)  
 Time to total knee arthroplasty, which will be collected by asking questions regarding whether or not 
the patient received a total knee arthroplasty will be asked at each visit (Knee surgery?)(Table 1). 
 
Visual analogue scale (VAS), Western Ontario and McMaster Universities Arthritis Index (WOMAC), 
Knee Society Score (KSS), Short Form-12 (SF-12), UCLA activity score (UCLA), and questions 
regarding whether or not the patient received a total knee arthroplasty will be asked at each visit (Table 
1). 
 
 
 
Index 
visit 
/Visit 1 
Visit 2 
(Week 1 
± 3 days) 
Visit 3 
(Week 2 
± 3 days) 
Visit 4 
(Week-6±2 
weeks) (visit 
4) 
Visit 5 
(3-
month±2 
weeks) 
Visit 6 
(6-
month±4 
weeks) 
 
Visit 7 
 (One week 
from visit 6 
± 3 days) 
Visit 8  
(Two weeks 
from visit 6 
± 3 days) 
Visit 9 
(1-year±2 
months)  
Visit 10 
(2-
year±2 
months) 
Aspiration 
X 
 
 
X 
 
X 
 
 
 
 
Injection 
X 
X 
X 
 
 
X 
X 
X 
 
 
VAS 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
WOMAC 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
KSS 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
SF-12 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
UCLA 
X 
X 
X 
X 
X 
X 
 
 
X 
X 
Knee 
Surgery? 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
X 
X 
Adverse 
Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
The subjects will be paid a stipend of 40 dollars for each visit (visit 1 to visit 10). Thus, a total of 
400$ will be paid to the patient if all the visits are completed. Additionally, the subjects will be provided 
free parking for all the visits. 
 
 

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Research Protocol Guidelines (continued) 
 
Data Analysis 
 
Unless otherwise indicated, all testing of statistical significance will be two-sided, and a difference 
resulting in a p-value of less than or equal to 0.05 will be considered statistically significant. Also, after 
each analysis, General Linear Models (GLM) will be used to control for possible confounders, including 
BMI, gender, age and ethnicity. 
 
 
Adverse Events and Data Monitoring Committee(DMC) 
 
Procedural safety will be documented in this study through patient and surgeon reported adverse 
events. AEs will be documented for all cases in this study.  
An Unanticipated Problem Involving Risks to Participants or Others is any event that (1) is 
unforeseen, (2) caused harm or placed a person at increased risk of harm, and (3) is related to the 
research procedures. 
An Adverse Event (AE) is any untoward or unfavorable medical occurrence, including any 
abnormal sign (for example, abnormal physical exam or laboratory finding), symptoms, or disease. 
Adverse events can encompass both physical and psychological harms. 
An Internal Adverse Event (AE) is an untoward medical occurrence, which occurs to participants 
in research conducted by Cleveland Clinic and/or Cleveland Clinic is the IRB of record. 
External Adverse Event (AE) is an untoward medical occurrence experienced by subjects enrolled 
at other institutions for the same study approved at Cleveland Clinic or a different study using the 
same study drug/device. 
A Serious Adverse Event (SAE) is any adverse experience that results in any of the following 
outcomes: 
• death 
• a life-threatening experience 
• inpatient hospitalization or prolongation of existing hospitalization 
• a persistent or significant disability/incapacity 
 a congenital anomaly/birth defect 
• Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon appropriate 
medical judgment, they may jeopardize the patient or subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. 
An Unexpected Adverse Event means any AE not previously known or included in the current 
Investigator’s Brochure, consent form or other risk information. 
Related/Possibly Related means there must be reasonable evidence to suggest the event was 
caused by the drug, device or investigational intervention. 
 
             1. Internal Serious Adverse Events (events that occur to participants enrolled in  
              research being conducted by Cleveland Clinic or when Cleveland Clinic is the IRB of 
              record) must be promptly reported to the IRB using the IRB AE Report Form within 
             10 working days from discovery/awareness which meet any of the following criteria 
             as assessed by the PI/Co-I: 
             a) Serious, Unexpected and Related/Possibly Related. 

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             b) AE’s determined to be occurring at a significantly higher frequency or severity 
             than expected. 
             c) Other Unexpected AE’s, regardless of severity, that changes the risk benefit 
             ratio of the study and results in changes to the Research Protocol or Informed 
             Consent process/document. 
            All Internal SAEs are also reported at continuing review using the AE Summary Log. 
           2. External Serious Adverse Events (events experienced by subjects enrolled at other               
institutions for the same study approved at Cleveland Clinic or a different study using the same study 
device/drug) are reportable to the IRB using the IRB AE Report Form within 10 working days from 
discovery/awareness when: 
a. The External SAE report includes reasonable evidence as assessed by a central monitoring   
entity [Coordinating or Statistical Center, or a Data Safety Monitoring Board (DSMB) or Data 
Monitoring 
Committee (DMC)] that the event is Serious, Unexpected, and Related/Possibly Related AND   
places the subjects or others at a greater risk of physical or psychological harm than was previously 
         known or recognized. This will require a change in the protocol and/or consent document. 
         b. External SAE reports provided by the Sponsor to the investigator indicating the event is 
Serious, Unexpected and Related/Possibly related but without reasonable evidence or DSMB/DMC 
determination  of greater risk are not reportable to the IRB within the 
10 day window. Without Sponsor evidence or assessment the implications of the event cannot be 
determined by the research team and therefore need not be reviewed. These SAE’ shall be placed on 
the AE Summary log to be submitted at the annual continuing renewal. 
3. DEATHS are to be reported to the IRB using the IRB AE Report Form according to 
the following guidelines: 
a) Internal Death 
• Related/possibly related whether expected or unexpected– within 5 working days from 
discovery/awareness 
• not related and expected – at time of continuing review 
• Not related and unexpected – at time of continuing review except cancer studies 
 
b) External Death 
 Related/possibly related and unexpected – within 5 working days from discovery/awareness not 
related whether expected or unexpected – at time of continuing review related/possibly related and 
expected – at time of continuing review 
c) ALL Deaths are also reported at time of continuing review using the AE summary log. 
4. Non-serious Adverse events (Internal and External) that are both Related/Possibly related and 
unexpected are reported on the AE Summary Log at time of continuing review to assess trends. 
5. An IRB staff (a qualified, licensed practitioner assigned to this function by the IRB chair and 
IRB Executive Director) reviews Adverse Event Reports to determine whether they represent 
Unanticipated Problem Involving Risks to Participants or 
Others. Events that are assessed, by either the IRB Staff or Investigator, to place subjects or others 
at a greater risk of harm than was previously known or recognized, or changes the risk/benefit ratio of 
the study, or requires a change in the protocol and/or consent document are referred to Full Board for 
review under Policy #70. 
Events that do not involve risk to Participants or Others or changes to the informed consent or 
protocol do not require further review. Investigators are informed of the determination and the IRB 
file is updated. 
6. The AE Summary Log is reviewed by the IRB at the time of continuing review to identify 
trends in frequency and severity which may impact subject safety. 
 

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This study is an Investigator Initiated research trial. Each study site will be considered its own 
regulatory sponsor and is responsible for internal data monitoring and any study reporting required by 
ClinicalTrials.gov. 
 
Consent 
 
A research personnel will approach the potential subject during the office visit time once 
he/she is identified from the screening process. Then the consenting will be conducted if the 
subject agrees to participate at a private exam room. After the explanation of the study, and 
questions being answered, the subject and the research personnel will both sign the consent form. 

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References 
 
1.  
Zhang Y, Jordan JM. Epidemiology of Osteoarthritis. Clin Geriatr Med. 2010;26(3):355-369. 
doi:10.1016/j.cger.2010.03.001. 
2.  
Altman RD, Rosen JE, Bloch DA, Hatoum HT, Korner P. A double-blind, randomized, saline-
controlled study of the efficacy and safety of EUFLEXXA for treatment of painful osteoarthritis of 
the knee, with an open-label safety extension (the FLEXX trial). Semin Arthritis Rheum. 
2009;39(1):1-9. doi:10.1016/j.semarthrit.2009.04.001. 
 
3. 
Shimizu M, Higuchi H, Takagishi K, Shinozaki T, Kobayashi T. Clinical and biochemical 
characteristics after intra-articular injection for the treatment of osteoarthritis of the knee: 
prospective randomized study of sodium hyaluronate and corticosteroid. Journal of 
Orthopaedic Science. Jan 2010;15(1):51-56. 
4. 
Hasegawa M, Nakoshi Y, Tsujii M, et al. Changes in biochemical markers and prediction of 
effectiveness of intra-articular hyaluronan in patients with knee osteoarthritis. Osteoarthritis 
and Cartilage. Apr 2008;16(4):526-529. 
5. 
Jauregui JJ, Banerjee S, Cherian JJ, Elmallah RD, Mont MA. Rating Systems to Assess the 
Outcomes After Total Knee Arthroplasty. Surgical Technology International. May 
2015;26:289-294.