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Dr. Frykberg: Okay. I've been asked to deliver this talk on a recent randomized controlled clinical trial that had been completed and it is in the process of being submitted, and it's on Healing Diabetic Foot Ulcers with Topical Oxygen Therapy. So we'll go over the literature on hyperbaric, as well as topical oxygen therapies, and present a case to you as to why this has been a misunderstood area, and also the basic science and the physiology, and make a case for why it really is an important adjunct to wound care. These are my disclosures. These are the learning objectives as have been published.

I'd like to start out with this graphic which really illustrates the complexities of the diabetic foot, especially since that is what we're going to be talking about today. And it indicates the interrelationships between neuropathy, vascular disease, both microvascular as well as macrovascular disease, motor neuropathy, sensory neuropathy, and autonomic neuropathy, and how these different pathophysiological processes can interact to lead to that high risk foot with an impaired response to infection, and ultimately lead to diabetic foot ulceration. As we know, once a foot is ulcerated, it can become infected, and infection can lead to gangrene, which will lead to amputation. So it's important for us to understand this underlying pathophysiological deficiencies in the diabetic foot. And then, furthermore, we need to recognize the important molecular and cellular deficiencies in any of the chronic wound.

[01:58]

It's a little bit difficult to see all these areas but we deal with impaired angiogenesis, impaired collagen production, excessive protease levels, impaired bacterial killing, et cetera. And as we know for all of these deficits that we find in chronic wounds, the body has a miraculous way to address these with various different types of pathways, and metabolic pathways, and semantic pathways. And we know that oxygen plays a critical role in most of the processes as we'll talk about angiogenesis, redox signaling, growth factor, stimulation and production, collagen production, cellular signaling, cellular metabolism, control of infection through reactive oxygen species production.

So oxygen is a critical component in the wound healing process so we need to be cognizant of that. And therefore, my premise is that oxygen is essential for wound repair and angiogenesis, including ATP, oxidative phosphorylation, which really provides for cellular metabolism, NADPH, oxidase systems, which lead to reactive oxygen species production for bacterial killing, intracellular ROS production or called redox signaling that leads to intracellular signaling for production and synthesis of growth factors and other enzymes. And obviously, oxygen is essential for collagen production as we've seen earlier. So oxygen plays again a critical role in all of these natural processes leading to wound repairs, also angiogenesis.

[03:59]

And we immediately think of hyperbaric oxygen therapy because that's been around for a long time. There's been a lot of study on this and many of you might work in centers that have HBO units. But yet, there has not been a high quality RCT produced. There's been a lot of heterogeneity in the patients, in the wounds, in the standard of care given, the modalities used, the timing. And because of this, in England, a regulatory body called NICE has recommended that HBO not be used as a routine treatment for diabetic foot ulcers or any chronic wound.

Now, that might be in contradistinction to your own centers because this is really not well understood and there has not been a lot of good data, although the physiology sounds perfectly fine. Theoretically, it sounds like it should work on every patient, but it hasn't. Now, topical oxygen therapy has been highly controversial, especially well-opposed by hyperbaric specialists, because it is in fact it's topical. But yet, there is some data that suggest, and we'll show that today, that it does support the role of topical application of pressurized oxygen to wounds to lead to wound repair.

And I'd like to bring out this point when we talk about hyperbaric oxygen. Most centers are now using monoplace chambers where they're putting the patient in, putting them under a little bit above atmospheric pressure breathing 100% oxygen in the chamber. And because of that, they're getting systemic delivery and that's the whole mechanism behind hyperbaric therapy. But what about the topical application of the oxygen, the pressurized oxygen to the wound, while the patient's in the chamber? How do they separate that effect from the other effects that they see?

[06:03]

So there are a lot of certain concerns and questions that heretofore have not been fully addressed, although there had been many, many case reports, perspective, and retrospective series on topical, as well as hyperbaric oxygen for the last 40 or 50 years even. Now, this paper in 2013 was a review by David Margolis out of Philadelphia looking at CMS data for hyperbaric treatment based on a large dataset, and he in fact said, "We could not substantiate any clear benefit of hyperbaric oxygen therapy for the management of diabetic foot wounds or even amputation prevention."

Now, you can imagine that the HBO community wasn't quite very happy about this and there was a lot of concern. But still to this day, there is no clear consistent evidence that hyperbaric therapy, when used in a controlled environment, certainly under controlled study, will clearly lead to benefit for would healing, despite the good theoretical evidence that we have for why it should work. And I'm not saying that it's not a value. It's just that right now, there has not been any clear RCTs and we have to go based on the evidence available.

Now, this a nice graphic from [Chandra Senan 00:07:35] of Ohio State who's done a lot of work on oxygen therapy and it just gives a graphic of the various areas where oxygen plays a role in wound healing. We can see ATP phosphorylation for cellular metabolism. We see ROS production for infection control and production of reactive oxygen species, and notably hydrogen peroxide and superoxide.

[08:05]

We see redox signaling for stimulation synthesis of various growth factors in cytokines. We see its role in collagen production and of course in neovascularization. So it's important to appreciate the varied role that oxygen has in wound repair in this regard.

Now, very importantly as you all know, the center of any chronic wound has very low oxygen tissue levels down to four or six, certainly below 10 millimeters of mercury, as opposed to the periphery of a wound, which would be about 60 millimeters of mercury. This comes from Singer and Clark's paper in 1999 in The New England Journal of Medicine, this graphic. And we also have to recognize that normal tissue PO2 is about 100 millimeters of mercury so that should be pretty much our reference. Normal tissue PO2 is about 100 millimeters of mercury. And as we go from the normal tissue to the edge of the wound, to the center of the wound, there's a very steep gradient drop and oxygen tensions.

I'm going to try to explain this graphic to you, which took me a little while to understand, but we want to reference it to that normal tissue level as we said, which is this green line right here at 100 millimeters of mercury. That's the normal tissue oxygen level. Here, the yellow line is what we would see in the center of a chronic wound, which would be six millimeters of mercury. Now, if we go in reference to the normal tissue level and these graphic lines here, we see that they represent enzymatic activity at various levels of oxygen tensions.

[10:01]

Normal ATP phosphorylation for cellular metabolism occurs almost at 100% if we are at the normal 100 millimeter of mercury, okay? If we look for prolyl hydroxylase collagen production, about 80% at normal tissue oxygen levels, and a little bit less for NADPH oxidase, which is the production of free radicals for managing infection. So these are the levels of enzymatic activity normally in tissue. But if we look at the wounded tissue way down here, we see how reduced those levels of enzymatic activity are because the PO2 levels are so low. So we see a great reduction in the ability to form ATP and the ability to produce collagen in this region here, as well as to produce NADPH oxidase for infection fighting.

Now, if we expanded out to here, a normal oxygen tension, an atmospheric pressure is 760 millimeters of mercury. That's where we go up here. We really see adequate enzymatic activities in normal oxygen levels. This red bar represents our study product, which adds 50 millimeters of mercury or so to that 760 millimeters of oxygen at sea level, leading to about 800 millimeters of partial pressure of oxygen. So we can see how important it is to elevate your PO2 levels if you are to affect normal enzymatic activities within tissues. So I hope I've explained that to you. Just look at how depressed the levels are compared to normal if you really reduce that TcPO2.

[12:06]

And this is really what's happening in the center of a wound, which is why some wounds are so stalled because they just don't have an adequate oxygen tension in them. And I think that's fairly important to understand. Now, this study was produced by Fries also out in his laboratory in Ohio State in 2005, and I like to discuss this because it was using topical oxygen on the backs of dermal wounds in pigs, and they applied topical oxygen to these chronic wounds in pigs. And they applied a wound probe, a dermal probe, two millimeters into the depth of the wound. Why? To measure the tissue oxygen tension.

Now, it's a little bit difficult to see, and I refer you to this paper because it really is good, where it show the positive benefits of topically applied oxygen to the healing of these wounds. And this lower curve, and I'm sorry, it's really hard to see, we see at the start prior to pressurized oxygen being delivered to the wound, the PO2 is about four millimeters of mercury. But after just four minutes of topically-applied oxygen, it went up to 40 millimeters of mercury. This is in the two millimeters deep into the center of these wounds. So in effect, after four minutes, we're seeing almost a tenfold increase in the PO2 levels in these wounds when you're applying topical pressurized oxygen to them.

This graph is just a separation between controlled wounds and the experimental wounds with the oxygen is a clear separation and improved healing rate in neovascularization. I didn't put on another slide which shows the upregulation of VEGF and the more active fibroblastic proliferation, and collagen production as well.

[14:08]

But this evidence really goes to show histologically and physiologically the positive effects that topically-applied oxygen can have on a chronic wound. This was from a recent systematic review of various oxygen therapies including hyperbaric oxygen, including topical oxygen, and even there are topical oxygen producing dressings nowadays. And what they said was there's many suppose – there's many positive outcomes that they found throughout some RCTs, throughout some case studies, throughout some retrospective and prospective studies. So it's called hypothesis-generating information, which was positive in many cases, and you're all aware of positive reports in the literature in all these different types of modalities.

But they all suffer from excessive heterogeneity. Heterogeneity in terms of patient populations, the types of wounds, the types of oxygen treatment, the duration of treatment, the specific dosing, the standard of care, et cetera. So these are common problems throughout the wound care community and they recognize that further randomized clinical studies are warranted. And to achieve this, we needed to have more standardized approach to both methods as well as the subjects. As you could understand, variability is what kills most randomized trials here. So there's good positive evidence. There's good anecdotal evidence but there has not hitherto been a good rigorous control trial in this regard.

Now, internationally as well in the US, there's been growing interest in topical oxygen.

[16:00]

I participated in this review in the journal of wound care in 2017 from the European Wound Management Association. And in this document, we looked at literature on hyperbaric oxygen, on topical oxygen, as well as oxygen producing dressings. And we found that topical oxygen, using a cyclical pressurized system, had a level of evidence rating of 1B, which was fairly good, and it was the only one that was able to provide that. But still, when this was produced, although this was a nice review similar to the [DISMAT] [00:16:42] review we just spoke about, RCT had not yet been completed. So further evidence was necessary.

Now, as you may all well be aware, there are various types of topical oxygen delivery systems. Basically, there's continuous diffusion through cannulas or through dressings, but these are not pressurized, and there is some anecdotal evidence. There's actually some prospective or even clinical trials in this regard, but these clinical trials also suffer from too much variability and somewhat inconsistency.

There's this older generation unit which was a 22 millimeters mercury constant pressure device. We don't see this too much anymore but that only had one ill-conceived randomized trial years ago. And then, there's this study that I'm talking about and the device that I'm talking about, which delivers a cyclical pressurized oxygen in a humidified self-contained chamber that goes from five millibars up to 50 millibars cyclically in this contained system. So you have to recognize there's different types of delivery, different types of units, and different qualities of studies that have been produced in all of these.

[18:07]

I don't think the EPIFLO device is in production any longer. We tested this many years ago. This was through a small cannula dressing over the center of a wound, but the other devices are all on the market, other I think than this device. And I encourage you to visit your exhibitors to understand the difference between these various devices.

Now, recognizing the lack of good evidence in this regard for both hyperbaric, as well as for topical oxygen therapies, CMS had a decision memorandum in 2017 when there was meeting, and they stipulated that there was not good consistent homogeneity of these studies with good high quality evidence. There was too much variability and the types of units, the dosing, the standards of care, et cetera, and the generalizability of the studies that were produced.

And what they – I'm sorry. What they recommended is that they wanted to see a sufficient sample sizes so that the studies – any studies carried out would have sufficient power to adequately detect differences between standard of care and oxygen therapies. They wanted to have consistent standards of care so that we can understand what is the goal standard, apply that goal standard, and compare that to the use of sham or placebo-controlled oxygen devices or hyperbaric oxygen therapy. And make sure that it was also generalizability, which means that that data that you collect in your study isn't just good for the people in your study, but they can be externally validated and generalized to the wider population with a similar type of wounds.

[20:04]

And they also said, "We have to eliminate the heterogeneity of wounds. You can't study venous ulcers mixed with diabetic foot ulcers, mixed with pressure ulcers, mixed with atypical ulcers, because there's too much variability. There's too much noise in those studies, so you have to really restrict it." As you all know, a VLU is certainly different than a diabetic foot ulcer, and it's certainly different from an arterial ulcer, et cetera. So they were just looking for more consistency and more rigorous control of any studies done, and this is really quite important. And they wanted to see the outcome measures of wound healing.

Wound healing, meaning how many – what percentage of your wounds will heal in a certain given time? What is the durability of wound closure? How are the patients functioning? Et cetera. And they wanted to see blinded studies and they did note the study that we're talking about now in their decision memorandum that there was one study that was rigorously controlled with good standard of care, good debridement, good offloading, et cetera. And they wanted to see that kind of study continue on.

And very importantly, intention to treat. Many studies will report on patient completion or per protocol treatments. Per protocol treatment is not intent to treat. Intent to treat means when you randomize a patient to one study group, if they have had one treatment, they are included in that study group from beginning to end, whether they leave the study or not. So you must report your intent to treat data. That's critical and it's standard for any pharmaceutical trial as well. You must look at intent to treat data and that's exactly what CMS wanted. So we conducted this study. This study was in production, if you will, or in process I should say for five or six years, even before we started this study.

[22:01]

Because we wanted to address all the negative concerns that the hyperbaric community would have against this upstart topical oxygen therapy, because I knew that they would bristle at the thought of topical oxygen actually having any effect. But yet, the benchmark to achieve equipoise with the hyperbaric study was very low because they don't have any good study. So we didn't have a high bar to meet so we needed to make it very rigorous. So we conducted this multicenter, multinational study on the use of pressurized cyclical topical oxygen for the management of chronic diabetic foot ulcers.

Our modality looks like this. It's cyclical pressurized with an oxygen generator. These are the disposable bags. The oxygen is cycled between five and 50 millibars so you're getting adequate cyclical compression with humidified 100% oxygen delivered to these patients, usually 90 minutes per day, five days per week. So this is one of the various types of topical oxygen units available but it's the only one using cyclical pressurized topical oxygen.

We had a robust study design. We started with a group sequential design with plans to enroll 220 patients with chronic diabetic foot ulcers, with a two-week standard of care run-in that wasn't rigorous, a controlled standard of care using goal standard offloading, with a boot that has itself undergone randomized control trial for its efficacy. Only ulcers that were not on a proven trajectory to the healing, meaning, they would have had to heal less than 30% within two weeks, were allowed into the study.

[24:04]

And this is fairly standard in most diabetic foot ulcer studies. So if you heal greater than 30% with good offloading standard of care, you cannot be enrolled for this study. And in fact, 23% of our patients failed screening because they did achieve this, so we lost a number of patients for this and other reason. Now, we assigned them to a double line sham-controlled study. Meaning, the investigator nor the patient knew if they were getting active pressurized oxygen because the unit will just make lights and make some sounds. The subjects administered this at home, not in a center but at home, for 90 minutes a day, five days per week. We provided all the study supplies as well.

They just couldn't use any type of an occlusive dressing but we kept the variation to a minimum. Everybody got the same offloading. Everybody got the same wound care supplies, which was just hydrogel and hydrocolloid dressing, weekly wound assessments, weekly wound debridement, and our primary endpoint was the percentage of ulcers in each group that were – achieved 100% healing at 12 weeks, exactly what good robust study should do, and exactly what CMS stipulated that they wanted to see for future trials, okay? And this is pretty routine.

We also did the secondary endpoints of wound recurrence, reduction in wound area, et cetera, et cetera, infections, adverse events, which you would always see. So our results. What I didn't say is the group sequential design specifies that at certain predefined enrolment periods, you're mandated to do an interim analysis, mandated by the study design prospectively.

[26:01]

We had two interim analyses points. The first one was once we completed, 73 patients. When 73 patients have been enrolled and completed their 12-week trial, we are mandated to do an interim analysis. At this first, a priority interim analysis with 73 patients, we stopped the trial. Why was the trial stopped? Because we achieved our primary goal of having a significant difference between the active and the sham group in achieving 100% healing.

So we achieved it, our first A priority group. We were all obviously pleasantly surprised, and we found that at 12 weeks at this interim analysis point, we had almost 42% heal in the active group compared to 13.5% in the sham group, okay? And you can say, "Well, the numbers are low." It doesn't matter. You can have a significant difference with 10 patients in the study if the margin of effect of the treatment is wide enough, and we were mandated to do this analysis. And we're also mandated to stop the trial at this point if we achieve our significance.

So we found significance here and the multivariable analysis show that none of the secondary outcome measures were significant. These are just some images of the various types of study patients. You can see typical type 1 and 2 ulcers. We did allow patients with modest degrees of ischemia down to 0.7 ABI. We did allow modest degrees of renal insufficiency. We allowed A1Cs up to 12. So these are the patients who are usually excluded from trials that we allowed to be enrolled in this trial on purpose because we wanted to treat the more difficult patients. And so these are just some typical images.

So we also found if you look at this separation in this Kaplan-Meier curve, the patients groups were separated right from the beginning.

[28:03]

Look at this significant log-rank test and the separation on the survival curves. These are the active treated people. They separated right from the beginning and I think this picture tells a whole story here. So our patients on the active therapy were four times more likely to heal in 12 weeks than the sham-treated patients, even though we had excluded a number of patients who had failed the run-in. So what this means is these were really difficult to heal ulcers.

We also found in the larger ulcers, let's say over two centimeters in diameter, that the active people had a mean reduction of 64% over 12 weeks, compared to the sham larger ulcers where they actually had an increase in size by 2% over a 12 weeks' time. This was significant as well. So we're seeing positive effects of the topical oxygen in this rigorous trial and we had great compliance. We really had great compliance. It was always a problem in these days. We had about 95% compliance with the active and sham treatment arm, as well as with the offloading boot, because we use the best offloading boot that we could get, and the patients love the boot so they use it, very, very important because we follow this carefully.

If you don't have good patient compliance, your trial is just not good, and we found that that was good. So we used one of the most rigorous protocols ever undertaken for diet, a few trial that was designed to remove the standard of care biases and failures, so we've noted in other trials and other agents, as well as other hyperbaric and topical trials. And we demonstrated that cyclical pressurized topical wound oxygen was significantly healing, was significantly – excuse me, superior in healing recalcitrant DFUs at 12 weeks. And even the larger ones showed a trajectory towards healing.

It was an easy home care therapy with high patient compliance and we believe that because of the results of this trial, this should be considered as a frontline adjunctive, always adjunctive treatment option for non-healing ulcers.

[30:12]

I'm running short on time but I just want to mention this article by William Jeffcoate published this year in diabetes care, looking at all the deficiencies in studies and how we needed to address 21 points in all research studies that are looking to conduct studies for the management of diabetic foot ulcers. This study hit every one of the points. And remember, this just came out this year. Our study was initiated five years ago or so, and certainly formulated five or six years ago. So we hit all of these points. So William Jeffcoate will be very happy of that. And as a result of our study, we were accepted as a late breaking abstract at this year's ADA meeting where we presented this poster and the paper is still in submission.

I want to thank our co-investigators. We can see a number of co-investigators. Some of them are here. I see Matt Garofalo is here. There's been a number of co-investigators. We could not do this without our – the good work of all of our co-investigators here. So I want to thank you very much. I hope that was useful to you. This is a very controversial subject and we did the best job that we could, and this is probably one of the most rigorous types of trial you're going to see in diabetic ulcers.

So with that, I want to thank you for your attention. Hopefully that was useful for you.


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