Intravenous Vitamin C: Pathway to a New Therapy to Save Lives

So, if everybody can settle in, we'll look at getting started. So, welcome everyone. Thank you for um coming out and and being flexible to the change in schedule today. Um I have the pleasure of introducing today's Timothy Johnson Medical Scholar. Please join me in welcoming do Dr. Alpha Barry Fowler III, very distinguished name with a for a distinguished scholar um who is um here at BTC this afternoon.

Um Dr. Fowler currently serves as a director of the Johnson Center for Critical Care and Pulmonary Research at the Virginia Commonwealth University in Richmond and holds the title of full professor at the VCU School of Medicine. He obtained his medical g degree from the medical college of Georgia and then went on to VCU where he completed his internal medicine training followed by a pulmonary and critical care fellowship at the um Colorado I'm sorry University of Colorado um health sciences center in Denver.

Um Dr. Fowler has received numerous honors and awards including the Douglas South Hall Freeman Award from um for volunteer leadership from the American Lung Association of Virginia, an outstanding faculty, distinguished physician and distinguished mentor awards from VC VCU just to name a few. He is the author or co-author on over a hundred peer-review uh publication peer-reviewed publications and currently serves as the PI on several NIH and foundation grants examining novel approaches to treat and improve mortality among patients affected by sepsis and acute respiratory distress syndrome as many of you learned this week. Um and it is on this topic that he will be speaking us today. Um so please let's welcome uh Dr. Feller who will be presenting his seminar introvenous vitamin C pathway to a new therapy to save lives.

So thanks for [Applause] I want to thank Leslie for the invitation. This has been an extraordinary morning. I've talked to a number of individuals. You guys are getting such great training here and the energy that I feel just in this room is amazing. And so I wish you all luck in the future as you pursue your separate careers. So what I'm gonna tell and we can start dropping the lights now. I think we can. Yep. You got Let's Let's take them down even lower because I've got some X-rays here and I just want to be able to project them, right?

Okay. Um, so I had the opportunity to have lunch with a few of you and we were talking one of the question was how the hell did you get into vitamin C? And so what I want to try to do today is to pull you through a pathway of the last 13 years that um has occurred to me and a number of my research colleagues. And I will tell you that the work I'm going to present here today, it is team science and that's the way that this had to go to develop. So I may be up here on the floor, but there are dozens of people at seven different medical centers outside of VCU uh that helped this emerge.

Um, okay. So, uh, the talk I'm going to get out of the way so I can see the slides. Is going to be fundamentally about bacterial sepsis. And a million people just in the United States alone develop sepsis each year. 35 to 40% of those patients will develop some form of acute lung injury. And I might as well go ahead and introduce the term acute respiratory distress syndrome. That is the worst manifestation of acute lung injury where the lungs basically fill up with fluid and a patient drowns. I'll go into that more in a few moments. And the interesting thing here up to this point 35 to 45% 30 to 45% of patients with sepsis induced lung injury will die.

So just focus on this for a moment. A fully loaded 747 can hold a 416 passengers. That is two fully loaded 747s will hold 832 passengers. This is how many people die in the United States every day. So, someone in this country dies of sepsis every 2 minutes and 30 seconds. And if you can think about two 747s crashing every day, that kind of brings it to the forefront.

So, let me show you some pictures. Muhammad Ali had really bad Parkinson's disease and as many patients with Parkinson's disease uh will develop multilobar pneumonia and he developed sepsis from multilobar pneumonia and passed away a couple of years ago but I want to show you this face and I would imagine that most people in the room have never seen this person but yet Paul Allen was the co-founder of Microsoft and Paul had non-hodkkins lymphoma, notice the date here, and died in septic shock and acute respiratory failure in the UCLA Medical Center on the 15th of October.

Globally, let's look at it. sepsis in first and second world countries. An estimated 50.9 million people die and approximately 10% of patients admitted to an in intensive care unit, this is from the so-called lung safe trial, will develop acute lung injury. the acute mortality I just mentioned, patients who survive the long-term morbid morbidity and disability and post-traumatic stress disease that comes after being in an ICU, being on mechanical ventilation for two or three weeks, seeing faces that you don't recognize, being in and out of sedation, in and out of paralysis. Uh the important thing is down here at the bottom 33% of all ICU patient days are accounted for by acute lung injury. 24% of hospital charges occur among ICU patients and ARDS is of the costliest ICU diagnosis.

So, what I'm going to try to do in the next few minutes, um, this is a gentleman we had in critical care a couple of years ago, um, who came in with sudamonus sepsis after bone marrow transplant. And you can see the development here and the change in the chest X-ray and the development of respiratory failure. This is what I'm going to be talking about today. Interesting thing is that up to this point, after about 35 years of continuous research and the expenditure of over a billion dollars between NIH and industry funding, there has not been an effective therapy to save lives for this problem right here.

Um, so look, I know you guys know a lot about anatomy, but I just wanted to sort of bring you down to the level of the alveis. Blood comes out of the right heart, un oxygenated blood circulates through the alvei is oxygenated, returns to the left heart, and is pumped out to the body. And so if you were to make a cross-section of this, we get down to the alvear capillary unit. And here's unoxygenated blood coming by the alvea capillary. It's oxygenated then heads off to the left heart. But also in the process of explaining this, the lung performs a very important barrier function. So nobody in this room is thinking about breathing. It's effortless. It doesn't even occur to you that you're breathing. You don't control your ventilation. Your brain stem controls your ventilation.

So, one of the important things that the lung has is a barrier function between the alvear space and the vascule. And the important thing as time goes by, just the fact that we're all breathing, you're constantly moving moisture down into your lung. And that has to have a way of being cleared. And the way it's cleared is by the water channels. And there are a number of different water channels. They exist on type two and type one alvear epithelium that moves fluid out of the alveis and into the interstatial space which is then carried back into the circulation.

Um, and so I talked with some of you this morning as how this all got started for us. But we were looking back into the literature about vitamin C and some really interesting work was done in the 1990s that showed that in critically ill septic patients that subnormal levels of ascorbic acid or plasma vitamin C was a constant feature in septic patients. Ascorbic levels correlated inversely with the incidence of multiple organ failure. Low levels, higher numbers of organs failed. and directly with survival, low levels, low survival.

So, um, time moves by, we started looking through what was known in the nutritional literature that was looking at all the vitamin levels. And we found as opposed to the normal plasma ascorbic acid level, which is usually between 60 and 80 microar just from the diet or some of our diets. Some of our diets are not good. But uh in the process of looking through the nutritional literature, we find that the levels drop as people get sicker and especially in patients who have had ma massive trauma, burns and sepsis.

So um I talked about this a little bit, but we wanted to model sepsis so that we could control it and treat it. And so we created a model of feces induced peritonitis in the mouse. And the way that was done, we just simply went to the mouse cage, plunked mouse pellets in a 50 ml tube, poured in some normal saline, took it back to the lab, sonicated the hell out of it so that it was a gish, spun it in a centrifuge really hard, and took the cell-free supernent. The supernate went into the fridge overnight and then the next morning we had a number of animals 30 gram wild type mice and each mouse was injected with a tenth of an ml of that solution and it was 100% mortality within 24 hours and then I still remember this and I explained this to some of you here today. Uh, okay. Let's try the vitamin C. Let's try the vitamin C.

And so we did the same experiment. Untreated animals over here, septic animals that were treated here were septic, but at 40 minutes after the septic process, we gave them an intraaritinal injection of 200 micrograms of vitamin C per gram of body weight. Okay. So, the animals received vitamin C parentally and I'll go into why it's not good orally in terms of a treatment regimen. And one of the first things we noticed was in the untreated mice, we had severe lung inflammation, a lot of cellular sequestration in the pulmonary uh walls, in the capillary walls. And you can see here animals were already developing lung injury. Those are proteinacious debris that's in the alvear space. But in the same time frame here were the vitamin C treated animals. We took this a little bit further because we wanted to know one of the one of the findings that's come down over the years is that as sepsis is occurring polymorphuclear neutrfils become activated and do a lot of damage to multiple organs throughout the body especially the lung. And so what we did was we stained mirine lung with a monoconal antibbody to mirine neutrfils. And you can see here sequestered in the lung of these septic animals intense neutrfil sequestration versus no neutrfil sequestration in the vitamin C treated animals. And does this present well?

Um, this is a whole lung preparation at a low power and where all the arrows are are showing microvascular thrombosis in a septic untreated animal. And for those of you in the room, and I know we have two pulmonary people here, for those of you in the room who've even thought about sepsis, sepsis is frequently associated with widespread microvascular thrombosis. And when we treated the animals with vitamin C, the thrombosis was totally eliminated. One thrombus on this particular section.

So uh the question arose at lunch, how did you get into this? How did this develop and evolve? And that's what I wanted you all to hear as we go forward, especially with your question that you asked earlier. So we minced lung and analyzed lung by RTPCR for first of all nuclear factor cappa B activation which drives all of the mirine cytoines and chemocines and then we also use the lung mints to analyze for water channel expression and it looks something like this. So these are septic lungs in animals that are a control animal untreated of feces induced peritonitis series of animals and feces induced peritonitis treated with vitamin C that's the reduced form or the oxidized form dehydrocorbic acid and you can see the dramatic effect on mouse KC that is a cytoine that's equivalent to human interlucanate and over here the LIX cytoine which is a potent inflammatory agent in the mouse the same effect the the paritonitis septic animals a very high expression of licks and then the reduction in the animals that were treated with uh either the reduced or the oxidized form of vitamin C um and here this is very important we looked at one of the water channels expressed in alvear epithelium so-called aquaporin 5. There are 10 aquaporin uh isoypes but we looked at aquaporin five and what you can see the effect of sepsis was to drive the expression of aquaporins down that was responsible for alo fluid clearance and you can see the effect here of the vitamin C on the water channel expression and then over here epithelial sodium channel very similar here they actively are moving vapor out of the lung to keep alvear fluid clearance active and keep alvei dry. And you can see we we saw the same thing here. So we know that vitamin C diminished inflammation as measured by here and that it promoted expression of fluid channels here. The important thing that we were doing all along, we wanted to know were these animals developing lung injury. And so we performed a series of experiments with using what is called lung wet weight dry weight ratios. And the way this is done, an experimental animal has lungs harvested. The lungs are weighed. Then the lung goes into a low temperature oven for three weeks and the lung completely dries down. And then you weigh the lung afterwards. And so you're looking at the wet weight to dry weight ratio. And an elevated wet weight dry weight ratio means the lungs were wet as opposed to the septic lungs as opposed to control. And you can see the impact of vitamin C. the lungs did not lose barrier function in the vitamin C treated mice.

Okay, there's that term again, barrier function. And the other thing we did, we actually performed a study in septic mice to look at alvear fluid clearance. And the way this is done, you've got a mouse mouse that's intubated on a mouse ventilator and you push the endotrachial tube down into the lower lobe and you inject a known concentration of albumin containing water. Pull the endotrachial tube back into the trachea. Mouse continues to ventilate. Then after 30 minutes, you push the endotrachial tube back down to the lower lobe and you aspirate what comes back. And you know the amount of fluid that went in the concentration of the protein and you know the amount of fluid coming out and if there is a lot of water taken out that means alvear fluid clearance is good because the protein concentration is rising. Everybody got that? And what we showed was that vitamin C would promote alvear fluid clearance. So not only were we controlling barrier function, we were increasing the expression of the water channels and we were keeping the lungs dry, the barrier function and promoting alveular fluid clearance.

Along the way, along the way while we were doing all this goes back now about 10 years ago, the concept of neutrfil extracellular trap formation came out. And I want to briefly explain to this after circulating blood neutrfils are exposed to certain levels of live bacteria, cytoines, endotoxins from bacteria or even fungi. the neutrifll, we've known this for years, becomes activated to generate reactive oxygen species and proteasis. And up until, you know, almost 10 or 12 years ago, I've been doing this for a long time, we felt that what was damaging the lungs was protease secretion, reactive oxygen species formation. But what was recognized as time has gone by is after being appropriately stimulated by varying levels of these agents, the DNA in the nucleus begins to unwind and the neutrfil actually disgorgges its genomic DNA. And the thing about this, this is the so-called neutrfil extracellular trap formation. The process is called neptosis. These uh and I know that many people in the room have done with dealt with DNA here and you know that DNA is the consistency of egg white. But you also don't know this is that all of these enzyme systems are active and extracellular are still active and create vascular damage. So the idea between a neutrfil extrc cellular trap formation was that evolutionarily it was probably good for this to occur because when our primitive ancestors became septic this was one of the things that helped to kill organisms because the organisms would become trapped here. And as the work go went on recognizing the existence of nets, neutrfil extracellular traps, it's been shown now in a number of publications that if you go to a patient with sepsis and you just draw a plasma sample and you analyze for cell-free DNA, after it gets to a certain level, it's consistent with at least an 80% mortality.

Okay, so we looked at that in the laboratory. And these are human neutrfils at uh a 20x and a 60x power. And this is what neutrfill nets look like fluorescently when they're stimulated with four ball mirror state acetate to kick out the nets. And then here at a high power, you can see it looks just like a smear, but that's the DNA that's being disorgged from the neutrfll. And what you see here is the effect of vitamin C. It totally shuts down net formation. That'll become relevant in just a few minutes.

Um, so I was speaking to some of you all. Uh, one of my colleagues who's no longer at VCU, Dr. Romesh nrogyn did all the basic molecular work and after we had excuse me after we had been doing this for a couple of years uh I was busy with some clinical work and Romesh walks into my office and he says this has to go into the hospital. We have got to see if this is going to work. And so what we did was we proposed a safety trial to the VCU IRB. This shows you how long ago it was. We proposed to only study patients in the medical respiratory ICU. We proposed randomized double blind placeboc controlled and we wanted to see if there were any adverse events that vitamin C caused. Did it cause the blood pressure to drop? Because we were proposing a similar dose that we had used in the mouse to use in humans. And we wanted to see if it caused high heart rate or nausea. So we studied, excuse me, just a minute.

So what we did was and it was a it was a tiny trial of only 24 patients. We divided up eight placebo eight lowd dose vitamin yeah yeah eight lowd dose vitamin C 50 milligrams per kilogram notice this per day or a high dose like we treated the mouse with 200 milligrams per kilogram per day. what this we chose this dose. I'm going show you why in a second. This is equivalent to 50 milligrams per kilogram infused IV every six hours. Okay. Um, and so the patient would get this total dose, a fourth of the total dose every six hours and the vitamin C was mixed in dextrose 5% in water. And we looked at multiple blood draws over a period of 96 hours. And the protocol was a 4-day infusion. So round the clock for 4 days. The rationale behind going with intravenous as opposed to dropping an NG tube, crushing up vitamin C and giving huge doses orally is that Pettyotti and colleagues working with Mark Lavine from the NIH clearly showed the difference in the plasma levels when you give it introvenously as opposed to even large doses given orally. you only get a plasma level that's basically two to three times the normal dietary level.

So what we proposed to the IRB is that we would use the sequential organ failure assessment score. uh this is used uh moderately frequently in intensive care units and it's a way of looking across these six organ systems and the way it's working looks like this. The higher the number on the sofa score, the more support the patient is having to give to get. This is the oxygenation index here. Uh we looked at the coma score and the higher the lower the coma score the more the patient has coma you get higher numbers. Same thing cardiovascular the more support that a patient is getting from all the way up into norepi norep epinephrine infusion the numbers go up and so on for liver function bone marrow function and platelet and uh renal failure. So the small trial we we screened 31 patients enrolled 24. Here was sort of the breakdown by age and the placebo patients. I mean they were sick. The sofa score was 13. If you're in an intensive care unit and you're rounding on your patient uh who septic a sofa score of eight, you have a sick patient. And so there was a sick population of people here. These were not really that different. and the uh sofa in the high vitamin C was 12. The sofa in the low vitamin C was 11. So these were basically the same.

What we did was we we worked with a medicinal chemist who uh can perform HOCC analysis of the plasma levels. And what we found most interestingly and this had been recorded in the literature septic patients have extremely low plasma vitamin C. A level of 10 microar is scurvy and then you can see the resulting plasma levels in the patients infused with the low dose 300 microar and the high dose 3,000 microar. So what happens to vitamin C? The important thing is during very high oxidative activity and there is a lot of eriththraite licis that's occurring plasmapree iron flows into the circulation and vitamin C is consumed by reducing plasma free iron from feric to ferrus uh by scavenging aquis free radicals and also by the destruction of the oxidized form dehydrocorbic acid and the lower levels permits uncontrolled oxidative activity.

So some of you in the room have read this paper. What we showed in this small trial was that uh putting all of the sofa scores at a level of identification. The highdose vitamin C impacted the sofa score over the four days of infusion. not so much in the green which was the me the low dose but the important thing here you can see what the sofa scores are doing and it's been clearly shown in the literature over the first 72 hours of hospitalization a septic patient who has a rising sofa score has a mortality of 80%. No matter what you're doing fluids antibiotics vasopressors ventilatory support uh we measured an inflammatory peptide C reactive protein. You can see the effects of vitamin C here. We looked at another uh molecule proalcitetonin which is an indicator of sepsis. You can see the effect of the two doses of vitamin C here. Then the last one a little bit of explanation here. Some of you in the room may know this molecule. Thrombomodulin is it decorates the vascular tur and so it hangs in the vascular tur always sort of waving in the circulatory breeze. But what thrombomodulin does is it grabs the protease thromben which occurs during sepsis. It will then hold it adjacent to protein C which activates protein C. And the important thing about activated protein C is that it causes breakdown of the microvascular thrombosis. And what happens is normally thrombomodulin is present on the vascular tur. It's not present in the serum or the plasma. And the way we interpreted the rising thrombolin level was there was vascular injury in the placebo patients. So from this little trial we found that vitamin C was safe. We did not have any adverse events. Mortality was reversed uh was reduced but uh it was a tiny trial. Multiple organ failure went down and these blood markers were attenuated.

Um, so we began looking back through the literature not just the nutrition literature and we found that at uh University of Washington this was a trial that came out of University of Washington uh that was performed here in the year 2002 where patients with surgical critically ill patients were treated with a gram of vitamin C intravenous every eight hours for 28 days and what they showed was in this population patients the incidence of ARDS was lower and multiple organ failure also was lower. Then comes a study that was formed in Japan where Japanese burn surgeons used vitamin C infusing up to 100 grams of vitamin C intravenously in the first 24 hours after a 50% full thickness burn. And they showed that they could cut the amount of resuscitation fluid after a burn of that extent. It's usually uh somewhere in the neighborhood of a 20 liter infusion to keep the blood pressure up because with a burn you're losing so much volume just through the surface of the burn. It lowered the capillary leak and it lowered the amount of resuscitation fluid by 50%. And then there was this trial that was never published where they used antioxidants vitamin C, selenium, an acetylcysteine and vitamin E. And they showed that they could reduce the mortality by 50% but it was never published. That was in 1989.

Um, so let me drop back for a moment. I want you to look at these films. This is a 27year-old mother of three who had widely metastatic ovarian cancer and she was being aggressively treated in the Massie Cancer Center. And on the day that she was admitted to the ICU, her husband came rolling her into the Dalton clinic and the first blood pressure I mean she was sort of delirious was 50 systolic. And I remember this just like yesterday. Nursing took that wheelchair, they roared her up to the medical respiratory ICU and the doors open. We were just finishing rounds. The doors opened and there were these nurses rolling this lady in and we just happened to have a bed and she had a white count of 0.8 uh from her aggressive chemotherapy and had E.coli sepsis from ur urinary tract sepsis. But what I want to show you, you can see how we're transitioning. You can see lung injury developing becoming more widespread. Day three, this woman, I'm going to tell you, she was 27. She was a pistol. There's nobody going to put me on a breathing machine. Uh, you guys may have encountered something like this before, but by day three in the afternoon, her work of breathing was so high, her lungs were flooding with so much fluid, we couldn't keep her oxygen tensions up. So she uh permitted us to be intubated to to intubate her. But the thing I want you to notice is right here. And I want you to notice the level of water in the lung basically did not change. And so what happened is as the septic process was leaving the vascule and getting into the airspaces of the lung, alvear fluid clearance was trashed and she was in the unit. This is day 21. She was actually in the unit for six weeks. Finally at six weeks she went home with a drake and a peg perccutaneous gastrotomy. And the unfortunate thing she died six months later of her unfortunate metastatic cancer. But um I I just want to show you this evolution here in the films in the way the lung becomes wet, the way it impairs oxygenation and ventilation. Um, and it is persistent.

So the story comes back to the alvear capillary unit. What's happening here is neutrfils become activated. They sequester in the microvascular tur. They are causing damage to the alvear epithelium, excuse me, the pulmonary capillary epithelium. And ultimately what happens is the neutrfils complete with get ready for this. The neutrifll nets that I was telling you migrate into the alvear space permeability edema develops as the lung air spaces fill with fluid and then the alvear space just becomes a witch's brew of reactive oxygen species proteases pro-inflammatory lipids cytoines chemocines and cellfree DNA so in my early time on faculty We were talking about this earlier. We were performing bronco alvear lavage to uh here's a patient with ARDS where we were using a broncoscope getting down into the lung and you can see the lavage fluid that's coming back is basically plasma because the lung barrier function has been damaged and the plasma rushes into the air spaces of the lung. But the thing that was important, not only was the cytologic morphology in the airspace of a patient with ARDS neutrfilic, but look here, neutrfil nets were there.

All right. So a just a smidgen about vitamin C physiology. When vitamin C is infused into the vascular space, it comes in contact with sodium vitamin C transporter 2 on the surface of vascular endothelium and vitamin C is literally vacuumed out of the circulation becomes intracellular and then it hits sodium vitamin C transporter one that's on alvear epithelium and the vitamin C moves into the alvear space. We have demonstrated this in our work with septic animals. So the idea that we came up with was if we have this going on by infusing vitamin C it would not only be intravascular but it would also be located intraalvear and that as a process of this that it would sort of save the water channels inside the lung and help promote alvear fluid clearance. And by the preliminary work that we have done decreasing activation of transcription factor nuc nucleifactor capper B increasing water channel expression and ultimately enhancing alvear flprints.

So about that time, the NIH was putting out feeler programs for their so-called um one program. And the title of this program, I'm kidding you not, was novel therapies of lung disease. And this happened to come out right at the time we were finishing this phase one trial. What they wanted was a small multic-enter phase 2 proof of concept. They wanted it. They wanted a real clinical trial. They wanted to because nobody had ever been able to demonstrate a mortality difference in ARDS. They said, "Well, look, let's drop back. Let's just ask them to give us some physiological outcome and some chemical outcome." Um, they wanted data on well-characterized subjects. That was no problem. They wanted a common drug with low toxicity and a drug that had imunol modulating therapy that we proposed for lung disease. All the pre-clinical work we had already done. So we proposed the vitamin C infusion for treatment and sepsis induced acute lung injury, the so-called citrus ALI trial. And what we wanted to do was assess the efficacy of a 4-day infusion, the high dose of vitamin C into patients with septic lung injury. And so I got on the phone and started calling around to some of my colleagues in critical care at other institutions and we enrolled began enrolling patients. We proposed to roll 170 patients with sepsis induced ARDS and 170 patients we enrolled and the trial closed last November and um it was it was good. We had two hospitals the Cleveland Clinic and the Fairview Hospital in Cleveland and then at the medical college of Wisconsin also another hospital Aurora St. looks huge in inner city hospital. Anyway, there were seven enrollment sites.

So, here's the deal. Um, for those of you who are progressing on in clinical medicine, you will come to appreciate that upstream of hospitalization, maybe one, two, or three days, the septic process begins. Um, and like the young woman that I presented to you, she was being aggressively treated for ovarian cancer. All of the badness for her was developing at home as she was becoming sicker and sicker. And during this period of time, what's happening during this risk period is that there is immune cell activation. microvascular thrombosis is occurring and bacteria, endotoxmia and cytoine surges. What happens is all of this goes together to ultimately produce organ injury whether it be kidney, lung, gut, liver patients manifest in different ways. So what we in our first trial whenever a patient hit the door with sepsis before they ever developed acute lung injury they were in the trial. What we proposed to NIH was we had to recognize acute lung injury. And so like the young woman that I presented she went through all of these phases. There is the exudative acute inflammatory. Then there's the lung trying to repair itself and many cell types are proliferating. Finally, the fibiberblast con uh population gets involved and there's the driven to produce collagen in the onset of fibrootic lung disease. And what we proposed to the NIH was if we could jump in and these were patients who were septic and who were intubated on mechanical ventilation that we could prevent this from happening.

So our protocol that we presented was 4-day infusion 200 milligrams per kilogram per day which basically as I said earlier is 50 milligrams per kilogram every six hours. The vitamin C was mixed in investigational pharmacy at the appropriate concentration per body weight. It was then hooded and four 50 ml bags were brought to the ICU, put in a dark ICU refrigerator at minus4 degrees uh at 4° C. And then um the nurse at the time that the study agent, whether it was D5W or whether it was vitamin C, would take the hooded bag out of the uh refrigerator in the ICU call the Pixus, and they would hang it at the bedside, still hooded, and then would infuse it through light protected tubing, which is a blue tubing that is used to prevent any light from coming in contact because vitamin C can be oxidized when it's exposed to light. And what we proposed was to measure the organ failure score over the first four days 0 48 96 hours. And then we were following the plasma scorbate levels as well as a range of biomarkers and I'm going to get into that in a moment.

So we screened over a thousand patients to enroll 170. Out of the 170 we had to eliminate three patients because of the discovery of alvear hemorrhage. And this was in a couple of bone marrow transplant patients and acute eosinaphilic pneumonia. These were exclusion criteria. All patients whether you got treatment or whether you were placebo was ICU standard of care. We enrolled 83 placebo and 84 vitamin C infused patients. This is just briefly a slide to show you the demographics. Uh the gender of male between the two groups was not significant. Okay. The age was not significant. And then looking down through all the races um between the groups, Caucasians and African-Americans, they were not significant. So we had a homogeneous group that we were treating. So things weren't hanging out in the breeze and we were treating somebody we said we weren't treating. The other thing that we wanted was to know that the extent of respiratory failure, the extent of treatment of shock and the sofa scores at the time that the patients came into the trial. None of these were different between the placebo and the vitamin C group.

Here are the plasma ascorbate levels from patients treated with vitamin C. And here again this is 28 microar. This is the level below which patients who were septic with ARDS showed versus the significant increase in the plasma scorbate levels. And we stopped infusion at 96 hours. But even after the following three days out to day seven, the differences were still apparent. So I was speaking with Lesie yesterday before I came here and I told her that the disappointing thing about this as opposed to our phase one trial, we showed that there was no difference in the organ failure scores. And I think my take on this is the patients in the phase one trial we were treating very early. We got to them very early. These patients who we put into this trial, they might have been septic for three or four days before they developed acute lung injury. And I think there was enough organ failure established that our vitamin C treatment was not effective. and we measured C reactive protein and thrombolin and those were not different.

Okay, what we did see interestingly was the plasma cellfree DNA was dramatically reduced in the patients who were treated with vitamin C. Remember the conversation about self-free DNA? We had other important secondary outcomes. Um, if we're giving a therapy that can promote alvear fluid clearance, lessen lung damage, you would think that it would result in more ventilator free days and vitamin C did. But this difference is not significant. What is significant, however, is vitamin C treated patients got out of the ICU significantly earlier than patients who were placebo. And I would just I had mentioned this to some of you in the audience earlier. If you're at the Virginia Commonwealth University and you're sick and you're in the medical respiratory ICU, your typical hospital bill is close to $60,000 a day every 24 hours. because you're paying for nursing, you're paying for medicine, you're paying for ventilator, respiratory care, pharmacy care, drugs, imaging. And so we were very happy to see this because vitamin C was getting patients out of the ICU earlier. And then there's this. I want everybody to look at this. We were able to show that unlike the placebo patients at day 28 and at day 60, the mortality in the placebo patients was 47%. The mortality at day 28 in the vitamin C treated patients was 31%. And at day 60, the mortality had increased slightly to 33%. Here's the important thing that I want you to notice. In the early part of the trial, when the patients are re actively receiving vitamin C, look at the number of patients dying compared to placebo patients. This was a very hard analysis. But what I'm want to show you is what happened to the vitamin C treated patients once vitamin C was stopped. And so the question we're asking going forward, should we have patients while they're in the intensive care unit on infused vitamin C for up to 10 days? That's kind of what we are seeing. The mortality was so steep in the septic untreated patients versus what was happening up here. And then after the infusion was stopped, you can see um

So look, I'm close to the end. What I want to do is share with you a couple of cases. Um uh James Madison University Jr. were with three of her girlfriends and they were spring breaking uh in central Italy and they were staying with a family an Italian family and the oldest son in the family had been vacationing in Morocco. The oldest son got sick and had to be air transported back to uh the town was Milan back to Milan. And this kid came in and he was home. He was obviously sick. He was there for a couple of days while these kids from JMU were there and then he had to be admitted to an area hospital. And so spring break was over. Spring break was over. They jumped on an airplane, flew to uh Charles de Gaulle in Paris and then over the Atlantic. And as the plane was getting closer and closer to continental United States, uh a flight stewart, a flight attendant was walking by and noticed this girl was deeply cyanotic. And so, "Honey, can you can you come back here with me?" And they took her to the back of the plane where the restrooms are. You know, you've been on planes before. And they put her on face mask oxygen. And it's funny, but it's not funny. Is there a doctor on the plane? and a doctor raised his hand who happened to be a psychiatrist and he came to the back of the plane and sat with the patient all the way to Dallas and uh they landed eently landed EMS took her off the plane. They immediately took her to a small area hospital called Fair Oaks up in uh up in Reston near Reston and in the ER she was immediately intubated and mechanically ventilated. the um ICU doctor that came down to take her up to the ICU could not ventilate or oxygenate her and somehow he had the chairman of cardiothoracic surgery on speed dial and he calls Dr. Casarajin down at VCU. They discuss this case. Vig and the ECMO team fly to Fair Oaks and they put this girl on ECMO up there. And then the ambulance brought her back down to VCU by ground. And here is the film that was taken in the emergency room at Fair Oaks. And she was brought down to VCU on ECMO. And this was a Thursday night. My wife and I the next day were heading to the Outer Banks of North Carolina and I was into my second glass of Chardonnay and I'm not kidding. And the phone rings and it's casin. He's a he's kind of a Kurt sort of guy. Barry tells me about this case. I want the girl to get vitamin C. And I'm looking down at this almost finished glass of Chardonnay thinking, well, I have 24 hours to put her into the to the study. Well, Vig, his name is Vig. First name is Vig. I'll come in tomorrow and get this lady in the trial. He said, "No, you don't understand. I don't want her in your trial because she could get a placebo." So he hands the phone off to anesthesia critical care physician that was there. I gave him the dose of vitamin C and what you see here is the evolution of her lung injury. Now the important thing is a bronco alvear lavage was done in her after they got her on ECMO and she came back with an identification of introvirus D68 and that particular virus has been implicated over and over in causing lung injury around the world. So she had inrovirus pneumonia and led to ARDS. Now the important thing is by day seven ECMO was taken off and the patient was extabated to 2 L of oxygen per minute. She was then transferred back to Northern Virginia. She spent one day in the hospital uh same critical care doctor and then he discharged her and he brought her back at my request and took a chest film two weeks later.

So look, the important thing is this. At the time this case came out, uh, and I was writing this up to the World Journal of Critical Care, this paper was published by the University of Illinois. And in that paper, they told this girl, this girl was 21 years old. They told about a 28-year-old woman who had inrovirus D68 infection and ARDS, and she was on mechanical ventilation for 42 days. Drake Peg finally went home on day 55. This girl was home and came back for a doctor's appointment by day 13.

And then a tragic case. This um you're not going to believe it when I tell you this. This is a um 27year-old 25 year old surgical intern and how she ever got into an a surgery residency I'm still never know but she had been living for a number of years with a permanent introvenous line because she was unable to absorb food and she was on continuous hyper for elementation and they took her into a surgical internship. She turns out to be a wonderful sweet woman. But within 30 days of starting her surgical internship, she became dizzy, febrile, uh rigers was taken immediately to the emergency room uh down to the emergency department and they didn't like the way they worked, the way she looked. She was septic in shock. So she was admitted up to the ICU and within 48 hours she was in ARDS. So this particular time I'm with family down in Knoxville, Tennessee and we're looking at the Tennessee River and I'm doing the same thing. One of the pulmonary fellows Amit Barara calls me, "Hey," and I'm saying, "Hey, how you doing?" He said, "What's going on?" I'm saying, "Oh, I'm down at a family reunion." And he told me about her. And so we started treating her on the fourth day of respiratory failure and you can see what happened by day seven. So she recovers from this during the month of August. Her parents come down. She recovers for 30 days and then goes back to surgical internship and it happened again and we treated her again on day four also. um and the outcome was good but subsequently she had to leave surgical internship. I don't know what the follow-up is on her at the present time but um

So I'll stop right here. Um, I guess the thing is this and I was talking to several people here in the room. Changing the practice of medicine is like turning a super tanker. And you all know what I'm talking about. You got the image in your mind. Um, I've got colleagues now uh around the world, New Zealand. I was talking about a colleague in New Zealand who's been studying vitamin C her whole life and she's trying to get the critical care doctors uh in Auckland, New Zealand to use vitamin C and they basically, you know, get out of my face. Um, and so um I was talking to Lesie. Um the manuscript for the phase 2 trial was submitted to JAMAMA and we have tentatively been signaled by JAMAMA that they want to publish the paper. They want to publish the paper as a negative trial but they told me that we can display all of our secondary outcome data. And so hopefully somebody will thumb into the paper and say oh look at this Kaplan Meyer survival curve. vitamin C actually did have an effect.

So, let me stop there. Questions? What kind of questions do you have? Yeah, thank you. Great talk. I was wondering whether vitamin C levels have at all. Yes. the paper that I showed you from the mid 90s from Borelli and colleagues. I haven't kept up with that literature honestly. Uh there's several markers of sepsis that are uh analyzable by protein by Eliza by Luminex technology. But um what Belli showed was that the lower your levels are when you're septic, the higher levels of organ failure occur. The lower your levels, the more chance that you will die. And that's what we showed in the phase one trial. So while vitamin C didn't seem to change the organ level, it seemed really really good at getting water out of the lungs. I was curious if that's ever been used for something like um peripheral edema for secondary to like congestive heart failure or if it could be a therapy for some kind of peripheral edema if it does upregulate these water channels. That's a good question. Um, I'm not sure. I don't know. You know, fioamite is so effective at reducing intravascular volume and getting volume out of

The legs that you're describing in somebody who has congestive heart failure, that, uh, and it's cheap now. I mean, Lasix is nothing. And, uh, you're always when you're taking care of somebody with congestive heart failure, there's always getting the fluid out and lowering the intravascular volume, but you're also giving an agent to lower the, the, uh, systemic vascular resistance so that the heart pumps easier, and all of that sort of a combined effect or can reduce the edema. Yes. Question was pretty upregulating the expression. Yes.

Now, here's the issue. We have a mouse model of lung injury, which is, I mean, it's a model. Uh, you know, there's been years of criticism of animal models and what do they actually show? Um, but in a case of ARDS, by the time I get to the bedside, you've already got all of that neutrophil influx into the alveoli, the witches' brew that I mentioned earlier is damaging all the type one and type two epithelium. The other thing I didn't mention that leads to respiratory failure is all of that neutrophilic oxidative activity will damage your surfactant. And so you have multiple areas of atelectasis associated with the fact that your lung is full of fluid. That's a good question. I but it definitely dramatically upregulated it in septic lung injured animals. I was curious. Well, I mentioned Romesh. Uh, I don't know that he ever did that. Why did you choose a 24-hour continuous infusion as opposed to the regimen that Dr. Merrick had suggested?

Um, we didn't give it by continuous infusion. We gave it every six hours and, uh, we had a little discussion about Dr. Merrick earlier and, um, at one point in the trial, I had to take one of the universities out of the trial, Emory University, but don't tell anybody. Um, and so, uh, the NIH was on my back. Get these patients in the trial. You only have a limited amount of money. And so I called Paul, and this was like February of 2016, and we had published that phase one trial in 2014. And I get him on the phone, "Hey Paul, it's Barry Fowler up the road. Uh, would you be interested in a vitamin C trial that we're doing and coming into, um, as an enrollment site?" And these were his precise words. He said, "I'm sorry, we've lost all equipoise as far as vitamin C is concerned. Ever since you published your paper, it's become our standard of care." They give it intermittently as well. Now, there is a researcher in Brussels who is, who is an an elderly, about to retire anesthesia critical care person, where vitamin C is being given by continuous infusion, which makes sense. Just ask one more question. Could you talk a little bit about the resistance to vitamin C?

The, we really don't have much data for. Well, as I said, it's turning, it's turning a super tanker. That's what it's all about. You know, medicine, rightfully so, is very skeptical of a new therapy, and even after the appropriate trials get done, changing the practice of critical care, the practice of medicine in some way is extraordinarily difficult. Um, but I can tell do this. I get emails and phone calls from family members who have a family member in the ICU in septic shock. "Dr. Fowler, can you please call such and such doctor who's caring for my mother and ask them to use vitamin C?" Um, so I think hopefully when the paper in JAMA is published, even though it was a quote negative study, they will see the Kaplan-Meier curve. Uh, after 40 years of research and multiple trials, a billion dollars spent on trials, there's never been anything that has enhanced survival. And here with 160, 167 patients, we have shown an outcome difference. So, um, JAMA, I got an email from them yesterday. I shared this with Leslie today. They have asked us to state that this trial is negative and that we do not recommend it as a therapy. And I'm going to put a caveat to that until a large phase three trial is done. And so the Merrick protocol, which was giving vitamin C, thiamine, vitamin B6, and hydrocortisone, um, for four days. According to Merrick, it reduced the mortality in septic patients. You guys probably read the Chest paper. Um, which is good. And so now, uh, that's been taken to Emory University, Hopkins, and, and Vanderbilt, and we are part of this trial, the so-called Victus trial, vitamin C, thiamine, and steroids. And so that's about to start. The trial is being funded by a foundation that I never knew about, the Marcus Foundation. Bernard Marcus was the founder of Home Depot. He's now 88 years old. We got to meet him by teleconference when we were down at Emory. I was giving them this stuff. And, uh, he's this relatively healthy, elderly, uh, 88-year-old who had a family member die of sepsis, and he's willing to throw several million dollars behind this trial. So we're part of that trial, and I just came back, but my wife and I just got back from vacation, but just before that, I went to Cleveland, Ohio, and presented to the PEAL network, and PEAL is an acronym, Prevention and Early Treatment of Acute Lung Injury, proposing a thousand-patient trial with vitamin C, and we got the highest number of votes of the six proposals that were submitted. So, we'll see. Um, other questions? Yes, sir. What's the cost of the vitamin?

You would not believe it if I told you. Um, let's put this. You're in sepsis and you're on Meropenem, Zosyn, and Levofloxacin in the ICU. That would probably be $20,000 a day. A vial of vitamin C. And, oh, and I, I should say this also. When we were starting this grant to go into the Citrus trial, uh, we had been using the vitamin C preparation that VCU had been using for two decades. It was made in Ireland, little company in Ireland. And between the time that we had finished the phase one trial and the time that we were start, we got our NIH funding, um, I called the company that had bought this little pharmaceutical firm, and I could never get them to call me back because they were afraid. I think vitamin C, you just give it like you want to give it. They were afraid that the FDA was going to clamp down on them. So again, Romesh walks into my office and says, "Hey, look at this. McGuff Pharmaceuticals out in California produces vitamin C." So we got on the phone, talked to McGuff, the president of the company. He said, "Oh, sure. We'll take care of that." They, they gave the vitamin C free of charge for this trial. And they're also giving, they're also selling it to the Victus trial as well, but the vitamin C is comes in 50 ml vials. It's $80 a vial. Good question.

Rates are actually on the rise, and that was even. Okay. Well, there's the cause of one of them right there because, um, and we were talking earlier. She's an oncologist. People are living longer with diabetes, with all sorts of, um, alcoholic hepatitis, any kind of enteropathy. Uh, patients like my 27-year-old woman being treated aggressively for cancer and becomes immunosuppressed, dramatically immunosuppressed. People are living longer. As people are living longer into their 70s and 80s, here comes acute leukemia. Here comes non-Hodgkin's lymphoma. Here comes multiple myeloma. All of that rolls into aggressive chemotherapy for those kind of illnesses. And, um, many patients in the trial were bone marrow transplant patients that came out down from the bone marrow transplant unit into the medical respiratory ICU because they were septic. So it's older population, multiple organs injured by years, diabetes, ischemic heart disease, uh, all of that feeds into why somebody can become septic, inflammatory bowel disease such as Crohn's disease or ulcerative colitis. There's active translocation of organisms from the lumen of the bowel into the circulation, into the mesenteric circulation. So, I mean, I could just keep going on and on, but that's a good question, and it's what you all are going to have to put up with in the future because the population is getting older, developing gray hair that's falling out along with the teeth. All right. So, uh, but that's, but that's the reason why I was wondering why reduced, reduced, okay. We use the reduced form, okay. Um, there had been some early work with the oxidized dehydroascorbic acid in India, of all places. It wasn't very fruitful research, but Romesh, the genius who used to work with me, wanted to to try DHA, dehydroascorbic acid, and it was also anti-inflammatory, but because the reduced form of ascorbate is so readily available, we didn't pursue that. Good question. What was it about surgery?

Um, young woman, she had a very bizarre, bizarre genetic disease where throughout her GI tract, small benign tumors were growing, and she had gotten through medical school with an IV, an internal jugular line for her whole medical school and was on total parenteral hyperalimentation. And she wasn't in a surgical arena there, but when she got to internship and she was exposed to the nasty D patients with surgical wounds, and she, you know, she was hooking herself up to her hyperal every day when she would get home. That's how it occurred, and it was methicillin-sensitive Staph on both occasions. Good question.

Hey, look, I want to tell you, I've really enjoyed this. This has been so much fun, and, uh, I want to send more people down here. Thank you all very much. Thank you. [Applause]