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1 A MetaAnalysis of the Effectiveness and Safety of Ozone Treatments for Herniated Lumbar Discs Jim Steppan PhD 1 Thomas Meaders BS 1 Kieran Murphy MD FRCPC 2 Mario Muto MD 3. 1 ActiveO, Salt Lake City, Utah www.activeospine.com 2 University of Toronto 3 A. Cardarelli Hospital, Naples, Italy Slide 2 2 Slide 3 3 Slide 4 4 Normal CentralSubarticular Foraminal Extraforaminal Slide 5 5 Slide 6 6 Slide 7 7 Slide 8 8 Slide 9 9 Patient Prepped and Needle Positioned into Herniated Disc Using CT Guidance Needle Penetration in L2 L3 Disc of Patient 027 Slide 10 10 CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial prior to Ozone-Oxygen Gas Injection Contained Intradiscal 3 cc Oxygen/Ozone Gas CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial after Ozone-Oxygen Gas Injection Slide 11 11 The AO-1000 Device Slide 12 12 AO-1000 Design Slide 13 13 MOA Studies Relationship between MOA studies, clinical and animal trials, and previous Italian studies Slide 14 14 Mechanical Analysis of Disc Decompression Slide 15 15 MOA Studies Relationship between MOA studies, clinical and animal trials, and previous Italian studies Slide 16 16 Animal Studies Slide 17 17 Animal Studies Slide 18 18 Histology Slide 19 19 Histology 3 wt% O3 Needle-only control Slide 20 20 Histology 2% O3 Slide 21 21 MOA Studies Relationship between MOA studies, clinical and animal trials, and previous Italian studies Slide 22 22 Disc Anatomy and Composition B). Sketch of Nucleus Pulposus [1] [1] [1] PP Raj, Intervertebral Disc: Anatomy-Physiology-Pathophysiology-Treatment, Pain Practice, 8(1), 18-44, (2008). A). Side View Sketch of Intervertebral Disc Slide 23 23 Disc Anatomy and Composition [2][2] L. Ng., etal., Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy, J. Structural Biology, 1453, 242-257, (2003). Schematic of Aggrecan 2 (PG) IGD: Interglobular domain between G1 and G2 KS: keratan sulfate region cp: core protein GAG: glycosaminoglycan chains CS: chondroitin sulfate brush region N: amine-terminal C: carboxyl-terminal G1, G2, G3: globular domains AFM Image of Fetal Episeal Aggrecan Monomer 2 2 Aggrecan, the major proteoglycan of the disc, is responsible for maintaining tissue hydration through the osmotic pressure provided by its constituent chondroitin and keratan sulfate chains Aggregan consists of a large (over 2000 amino acids) core protein with multiple carbohydrate side chains (glycosaminoglycans (GAGs)) GAG chains of chondroitin sulfate and keratan sulfate covalently bonded to a polypeptide core Slide 24 24 AO-1000 Mechanism of Action (MOA) The AO-1000 creates ozone which subsequently cleaves (directly or indirectly) the proteoglycans (PG) in the nucleus pulposus (NP) It is well known [1],[2] that ozone can react with organic compounds (like PG) in aqueous media both directly via Reaction 1 and indirectly via Reactions 2, 3, and 4. [1][2] [1] [1] Alternative Disinfectants and Oxidants, EPA Guidance Manual, Chapter 3: Ozone, (1999). [2] [2] B Langlais, et al., Fundamental Aspects, Ozone in Water Treatment Application and Engineering, 11-132, (1991). Slide 25 25 ActiveO and Dr. Balagurunathan (U of Utah) characterized the reaction of ozone with GAGs A Chinese hamster ovary (CHO) cell line (CHO was also used in Deweys benchmark work on thermal necrosis) was used to obtain GAGs that were similar in composition to those found in human NP. GAG samples were treated with 2 wt% ozone in oxygen from the AO-1000 device and the samples were then analyzed by high performance liquid chromatography (HPLC) with radioisotope detection. Ozone fragments the GAGs as indicated by the appearance of an elution peak at 13.5 minutes and a decrease in the amount of GAGs eluting at retention time over 40 minutes Exposure of GAGs to an oxygen-only control did not cause any fragmentation. Ozone Reaction with GAGs a. GAGs with no ozone exposureb. GAGs after exposure to ozone MW = 6600 MW = 23,800 Slide 26 26 MOA Studies Relationship between MOA studies, clinical and animal trials, and previous Italian studies Slide 27 27 MOA Studies: Cytokine Testing Left: Image showing puncture and harvest sites Below: Harvested discs transverse aspect Discs frozen in LN 2 @ Lychron Discs shipped to U of U for homogenation Homogenates shipped to Pierce Bio in Mass. for ELISA Slide 28 28 MOA Study: Why Cytokines? IL-1 TNF IL-6 IL-8 Studied 4 unique cytokines Each cytokine measured in triplicate Samples measured neat, e.g. no dilution IL-1 , IL-6, and TNF- are believed to be associated with disc degeneration (IL-1 is neurotoxic) IL-8 linked to enhanced disc healing (IL-8 is neuroprotective) Slide 29 29 MOA Study: Cytokine Analysis Results IL-1B shows increasing trend (w/ 2 wt% being optimal) Slide 30 30 MOA Study: Cytokine Analysis Results IL-6 shows similar increasing trend (w/ 2 wt% being optimal) Observed very elevated levels of IL-6 compared to other cytokines Slide 31 31 MOA Study: Cytokine Analysis Results IL-8 shows similar increasing trend (w/ 2 wt% being optimal) Slide 32 32 2 wt% ozone induces highest response for all cytokines IL-1 and TNF- (pro-inflammatory) cytokine levels found to increase in response to treatment/needle injection IL-8 (anti-inflammatory) found to increase in response to treatment Increase in IL-1 and TNF- 2-days after treatment believed to be due to recent stab incision and needle injection into disc IL-8 produced in response to increased levels of proinflammatory cytokines (IL-1 and TNF- MOA Study: Cytokine Analysis Results Slide 33 33 Ozones Mechanism of Action Primary mechanism of action of the oxygen- ozone mixture injected into the herniated disc is volume reduction (disc decompression) Ozone reacts with the GAGs in the NP which ultimately results in subtle dehydration (volume reduction) of the disc Secondary mechanism of action is related to analgesic/antiinflammatory factors Slide 34 34 Essentially Our model shows that volume removal of 0.28 cc (height reduction of 0.010 inch (0.0254 cm) results in a pressure reduction of 10 psi or dV of 30ul = 1 psi Our model confirms that significant reductions in intradiscal pressure can result from small volume (height) reduction that may be undetectable by CT or MR Slide 35 35 Purpose The purpose of this literature review and analysis was to determine representative outcomes of oxygen/ozone treatment for herniated discs with respect to pain relief, reduction of disability, and risk of complications Slide 36 36 CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial prior to Ozone-Oxygen Gas Injection Contained Intradiscal 3 cc Oxygen/Ozone Gas CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial after Ozone-Oxygen Gas Injection Oxygen/Ozone Treatment Slide 37 37 Methods A random-effects meta-analysis (DerSimonian and Laird Weighted Least Squares) was used to estimate treatment outcomes for oxygen/ozone treatment of herniated discs. A literature search was performed, using search terms associated with ozone treatment of herniated discs, to obtain the relevant studies for the meta-analysis. Slide 38 38 Methods Due to the wide range of study quality, each study was weighted in the meta-analysis by a study-quality score that was based on Type of study randomized, prospective, or retrospective Whether a control arm was used Whether a statistical analysis was performed Whether the study was multi-center or single center Slide 39 39 Methods Separate meta-analyses were performed for each of the pain and function scales most frequently used in the studies: Visual Analog Scale (VAS) Pain assessment (analyzed mean VAS reduction) Oswestry Disability Index (ODI) Pain and function assessment (analyzed mean ODI reduction). Modified MacNab Scale Pain and function assessment (analyzed percentage of patients improving 1 category or more). A meta-analysis for the overall complication rate, based on the reported study complications, was also performed. Slide 40 40 Literature Search Results Twelve (12) articles met the inclusion criteria from our literature search and were included in the meta-analysis. The included articles summarized data from the ozone treatment of over 8000 patients from multiple centers in multiple locations. Slide 41 41 Meta-analyses Results The overall estimated mean improvement: VAS = 3.9 (3.2 4.5, 95% CI) ODI = 25.7 (18.8 32.6, 95% CI) The estimated chance of improvement: Modified MacNab = 79.7% (74.2% 84.2%, 95%CI) The means for the VAS and ODI outcomes are well above both the minimum clinically important difference and the minimum (statistically-significant) detectable change. The estimated chance of complication: = 0.003% (0.000% 0.024%, 95% CI) There were no serious complications reported. Slide 42 Improvement in VAS Scores after Oxygen/Ozone Treatment of Herniated Discs Slide 43 Improvement in ODI Scores after Oxygen/Ozone Treatment of Herniated Discs Slide 44 Improvement in Modified MacNab Scores after Oxygen/Ozone Treatment of Herniated Discs Slide 45 Complication Rate during and/or after Oxygen/Ozone Treatment of Herniated Discs Slide 46 46 Summary of Reported Complications Qing 2005 A few patients demonstrated Hypogastric, lumbar, and lower- extremities pain and distention and difficulty urinating Symptoms resolved without intervention in 24 hours Andreula 2003 Two patients had impaired sensitivity in the lower limb due to periganglionic injection of anesthetic Symptoms resolved without intervention in 2 hours Ying 2005 A few patients had mild pain in the lumbar spine or leg after ozone injection resolved without intervention in several minutes Eight patients had mild respiratory impairment, dyspnea and cornea stimulates due to breathing ozone resolved upon leaving the ozone environment and breathing oxygen Oder 2007 6% of the patients complained of transient aggravation of the symptoms Slide 47 47 Check for Bias Bias - possibility of published articles under- estimating or over-estimating the treatment effect To assess for bias we used a quantitative linear regression test. An unbiased meta-analysis should have good linearity between studies when the precision of each study is plotted against the standardized effect. If the neutral (unbiased) linear regression line (forced through zero on the y-axis) is within the 90% CI of the biased linear regression line, then there is no statistical evidence of bias. Deviation of the biased linear regression line from the neutral (unbiased) line shows the amount of bias between studies. Slide 48 VAS Linear Regression Bias Test Slide 49 ODI Linear Regression Bias Test Slide 50 50 Discussion of Results Pain and function outcomes are similar to the outcomes for lumbar discs treated with surgical discectomy, but the complication rate is much less (