A MetaAnalysis of the Effectiveness and Safety of Ozone Treatments for Herniated Lumbar Discs

Jim Steppan PhD1 Thomas Meaders BS1 Kieran Murphy MD FRCPC2 Mario Muto MD3.
Salt Lake City, Utah www.activeospine.com 2 University of Toronto 3 A. Cardarelli Hospital, Naples, Italy
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1 ActiveO,

Normal

Central

Subarticular

Foraminal

Extraforaminal
4

Patient Prepped and Needle Positioned into Herniated Disc Using CT Guidance

Needle Penetration in L2 L3 Disc of Patient 027

Contained Intradiscal 3 cc Oxygen/Ozone Gas

CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial prior to Ozone-Oxygen Gas Injection

CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial after Ozone-Oxygen Gas Injection

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The AO-1000 Device

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AO-1000 Design

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MOA Studies
Relationship between MOA studies, clinical and animal trials, and previous Italian studies

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Mechanical Analysis of Disc Decompression

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MOA Studies
Relationship between MOA studies, clinical and animal trials, and previous Italian studies

15

Animal Studies

16

Animal Studies

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Histology

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Histology

Needle-only control

3 wt% O3
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Histology
2% O3 2% O3

20

MOA Studies
Relationship between MOA studies, clinical and animal trials, and previous Italian studies

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Disc Anatomy and Composition

A). Side View Sketch of Intervertebral Disc B). Sketch of Nucleus Pulposus[1]

[1]

PP Raj, Intervertebral Disc: Anatomy-Physiology-Pathophysiology-Treatment, Pain Practice, 8(1), 18-44, (2008).

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Disc Anatomy and Composition

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

Schematic of Aggrecan2 (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 Monome

[2] L. Ng., etal., Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy, J. Structural 23 Biology, 1453, 242-257, (2003).

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.

( Rxn 1) O3 PG direct ozone PG oxidation products reaction

decomposition ( Rxn 2) O3 O2 O

( Rxn 3) O H 2 O 2 HO ( Rxn 4) HO PG PG oxidation products ( Rxn 5) H 2 O e electron dissociation H HO impact

[1]

[2]

Alternative Disinfectants and Oxidants, EPA Guidance Manual, Chapter 3: Ozone, (1999). B Langlais, et al., Fundamental Aspects, Ozone in Water Treatment Application and Engineering, 11-132, (1991).

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Ozone Reaction with GAGs

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.
800

800 700 600 500

CPM

0 min-1
700 600 500
CPM

AO-1

MW = 23,800

MW = 6600

400 300 200 100 0 0 20 40 Time (min) 60 80

400 300 200 100 0 0 20 40 Time (min) 60 80

a. GAGs with no ozone exposure

b. GAGs after exposure to ozone

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MOA Studies
Relationship between MOA studies, clinical and animal trials, and previous Italian studies

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MOA Studies: Cytokine Testing

Left: Image showing puncture and harvest sites

Below: Harvested discs transverse aspect

Discs frozen in LN2 @ Lychron Discs shipped to U of U for homogenation Homogenates shipped to Pierce Bio in Mass. for ELISA

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MOA Study: Why Cytokines?

IL-1b, 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)

IL-1

IL-6

IL-8

TNF

Studied 4 unique cytokines Each cytokine measured in triplicate Samples measured neat, e.g. no dilution
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MOA Study: Cytokine Analysis Results

IL-1Beta
1.60

IL-1B shows increasing trend (w/ 2 wt% being optimal)

1.40

1.20

Disc Cytokine Concentration (pg/mL)

1.00

0.80

0.60

0.40

0.20

0.00 0 -0.20 Ozone Exposure (ug O3/g NP) 20 40 60 80 100 120 140 160 180 200

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MOA Study: Cytokine Analysis Results

IL-6
60.00

50.00

Disc Cytokine Concentration (pg/mL)

40.00

30.00

IL-6 shows similar increasing trend (w/ 2 wt% being optimal) Observed very elevated levels of IL-6 compared to other cytokines

20.00

10.00

0.00 0 20 40 60 80 100 120 140 160 180 200 Ozone Exposure (ug O3/g NP)

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MOA Study: Cytokine Analysis Results

IL-8
4.50

IL-8 shows similar increasing trend (w/ 2 wt% being optimal)

4.00

3.50
Disc Cytokine Concentration (pg/mL)

3.00

2.50

2.00

1.50

1.00

0.50

0.00 0 20 40 60 80 100 120 140 160 180 200 Ozone exposure (ug O3/g NP)

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MOA Study: Cytokine Analysis Results

2 wt% ozone induces highest response for all cytokines IL-1b 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-1b 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-1b and TNF-)
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Ozones Mechanism of Action

Primary mechanism of action of the oxygenozone 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

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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
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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

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Oxygen/Ozone Treatment

Contained Intradiscal 3 cc Oxygen/Ozone Gas

CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial prior to Ozone-Oxygen Gas Injection

CT Image of L2-L3 Disc of Patient 027 from the ActiveO Clinical Trial after Ozone-Oxygen Gas Injection

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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.

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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

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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.
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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.

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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.
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Improvement in VAS Scores after Oxygen/Ozone Treatment of Herniated Discs

Ref Lead Author: Study Arm Random Effects Model NA (Comparable Studies) NA Random Effects Model (All Ozone Studies) Year Mean Imp NA NA 3.9 3.5 95% CI Weight 3.21-4.54 NA 2.83-4.18 NA

9
28 20 22 22 23 22 22 22 26 2 28

ActiveO Clinical Trial

Buric: Ozone Arm Muto: All Arms Oder: Bulging Disc Oder: Herniated Disc Buric Oder: Non-Discal Oder: Post Operative Oder: Osteochondrosis

NP
2005 2008 2008 2008 2003 2008 2008 2008

3.7
4.0 4.7 3.5 3.5 3.8 2.7 3.0 2.5 3.6 5.9 4.1

3.02-4.38 0.870
3.03-4.97 1.000 4.65-4.75 0.838 3.21-3.79 0.819 3.19-3.81 0.817 3.36-4.18 0.534 2.31-3.09 0.805 2.48-3.52 0.782 1.91-3.09 0.766 2.29-4.91 0.000 5.56-6.24 0.000 2.40-5.80 0.000

CMS: SpineWand Meta-Analysis 2008 Butterman: Discectomy Arm Buric: Microdiscectomy Arm 2004 2005

0.0

1.0

2.0

Minimum Clinically Important Difference

VAS Minimum Detectable Change

3.0

4.0

5.0

6.0

7.0

Mean Improvement (Before - After) in VAS Score

Comparable Studies Studies Not Meeting AO-1000 (Ref 9) Inclusion/Exclusion Criteria Non-Ozone (Control Arm) Treatments Random Effect Model (All Ozone Studies) Random Effects Model (Comparable Studies)

Improvement in ODI Scores after Oxygen/Ozone Treatment of Herniated Discs

Ref

Minimum Clinically Important Difference

NA NA

NA

21.0

14.14-27.94

NA

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21 28 20 22 22 32 32 22 22 22 27 2 28 21

ActiveO Clinical Trial

Gallucci: Ozone+Steroid Buric: Ozone Arm Muto: All Arms Oder: Bulging Disc Oder: Herniated Disc Leonardi: Long Term Leonardi: Short Term Oder: Non-discal Oder: Post operative Oder: Osteochondrosis Mirzai: SpineWand

NP
2007 2005 2008 2008 2008 2006 2006 2008 2008 2008 2007

28.4
45.8 16.2 30.0 16.5 24.5 10.8 9.8 12.0 5.0 8.0 30.7 36.5 24.2 36.6

22.97-33.83 0.675
42.35-49.17 4.29-28.18 29.55-30.45 14.34-18.66 22.23-26.77 6.54-15.12 4.67-14.83 9.11-14.89 1.2-8.8 3.67-12.33 27.86-33.54 32.88-40.12 7.64-40.83 33.06-40.1 1.000 0.706 0.615 0.609 0.608 0.394 0.388 0.604 0.596 0.591 0.000 0.000 0.000 0.000

Butterman: Discectomy Arm 2004 Buric: Microdiscectomy Arm 2005 Gallucci: Steroid Only 2007

0
Comparable Studies Random Effect Model (All Ozone Studies) Studies Not Meeting AO-1000 (Ref 9) Inclusion/Exclusion Criteria

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ODI Minimum Detectable Change

Lead Author: Study Arm Random Effects Model (Comparable Studies) Random Effects Model (All Ozone Studies)

Year Mean Imp

95% CI

Weight

NA

25.7

18.82-32.61

NA

20

30

40

50
Study Weight

Mean Improvement (Before - After) in ODI Score

Random Effects Model (Comparable Studies) Non-Ozone (Control Arm) Treatments

Improvement in Modified MacNab Scores after Oxygen/Ozone Treatment of Herniated Discs

Percent Chance of Improvement in MacNab Score
50% Ref NA NA Lead Author: Study Arm Random Effects Model (Comparable Studies) Random Effects Model (All Ozone Studies) Year NA NA Imp (%) 79.68 78.17 95% CI 74.22 - 84.24 72.54 - 82.92 Weight NA NA 60% 70% 80% 90% 95% 97.5% 99%

9
36 36 20 35 20 37 34 33 20

ActiveO Clinical Trial

Andreula: Ozone Andreula: Ozone+Steroid Muto: Soft disc herniation Muto: All Muto: Multiple herniations He Ying Qing Muto: FBSS

NP
2003 2003 2008 2004 2008 2003 2005 2005 2008

79.00
70.30 78.30 75.00 75.00 77.00 77.00 93.00 97.00 60.00

65.57 - 88.14 0.463

64.88 - 75.20 1.000 73.28 - 82.60 0.982 73.32 - 76.61 0.644 73.15 - 76.77 0.642 72.30 - 81.11 0.600 71.47 - 81.73 0.389 89.65 - 95.32 0.347 95.29 - 98.10 0.333 53.06 - 66.56 0.586

1.00
Comparable Studies Studies Not Meeting AO-1000 (Ref 9) Inclusion/Exclusion Criteria Random Effect Model (All Ozone Studies)

10.00

100.00
Random Effects Model (Comparable Studies)

Odds of Improvement in MacNab Score

Complication Rate during and/or after Oxygen/Ozone Treatment of Herniated Discs

0% Ref NA Lead Author: Study Arm Random Effects Model (All Studies) Year Complications (%) NA 0.003 95% CI Weight 1% 2% Percent Chance of Complication 3% 4% 5% 6% 7%

0.000 - 0.024 NA

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20 35 36 37 23 21 32 32 28 33 36 34 22

ActiveO Clinical Trial

Muto: All Muto: All Andreula: Ozone+Steroid He Buric Gallucci: Ozone+Steroid Leonardi: Long Term Leonardi: Short Term Buric: Ozone Arm Qing Andreula: Ozone Only Ying Oder: All

NP
2008 2004 2003 2003 2003 2007 2006 2006 2005 2005 2003 2005 2008

0.000
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.499 0.668 2.478 6.120

0.000 - 0.000 0.016

0.000 - 0.000 1.000 0.000 - 0.000 0.987 0.000 - 0.000 0.370 0.000 - 0.000 0.303 0.000 - 0.000 0.065 0.000 - 0.000 0.042 0.000 - 0.000 0.017 0.000 - 0.000 0.009 0.000 - 0.000 0.006 0.000 - 1.062 0.004 0.000 - 1.590 0.001 0.783 - 4.174 0.000 4.235 - 8.005 0.000

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Proportion of Patients with Complication

Summary of Reported Complications

Qing 2005
A few patients demonstrated Hypogastric, lumbar, and lowerextremities 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
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Check for Bias

Bias - possibility of published articles underestimating 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.
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VAS Linear Regression Bias Test

200

VAS Standaridized Effect (Study Treatment Effect / StDev of Study)

150

Bias Linear Regression Line (with 90% CI) y = 4.852x - 6.574 R2 = 0.997 Probability that Regression Line Intersects Y-axis at Zero (p<0.1 Indicates Bias at 90% CI) p = 0.001

20 Muto: All Arms

100

Non-Bias Linear Regression Line 50

9 ActiveO Clinical Trial 23 Buric 22 Oder: Bulging Disc

y = 4.5574x R2 = 0.9872

22 Oder: Herniated Disc 22 Oder: Non-Discal 22 Oder: Post Operative 22 Oder: Osteochondrosis 28 Buric: Ozone Arm

-50 0 5 10 15 20 25 30 35 40 45 Precision (1 / Standard Deviation (StDev) of Study)

ODI Linear Regression Bias Test

160
20 Muto: All Arms

ODI Standaridized Effect (Study Treatment Effect / StDev of Study)

140 Bias Linear Regression Line (with 90% CI) y = 31.31x - 6.83 R2 = 0.97 Probability that Regression Line Intersects Y-axis at Zero (p<0.1 Indicates Bias at 90% CI) p = 0.03

120

100

80 Non-Bias Linear Regression Line 60

22 Oder: Herniated Disc

y = 28.358x R2 = 0.9467

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21 Gallucci: Ozone+Steroid

20

9 ActiveO Clinical Trial

22 Oder: Bulging Disc

22 Oder: Non-discal 32 Leonardi: Long Term 22 Oder: Osteochondrosis 22 Oder: Post operative

-20

32 Leonardi: Short Term 28 Buric: Ozone Arm

0.5

1.5

2.5

3.5

4.5

Precision (1 / Standard Deviation (StDev) of Study)

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 (<0.1%) In addition, the recovery time is significantly shorter for the oxygen/ozone injection than for the discectomy

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Thank You
Kieran.Murphy@uhn.on.ca www.activeospine.com

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