Public Workshop
Safety of Hemoglobin-Based Oxygen Carriers (HBOCs)
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH, FDA
THE NATIONAL HEART, LUNG, AND BLOOD INSTITUTE, NIH AND
OFFICE OF THE SECRETARY AND OFFICE OF PUBLIC HEALTH AND SCIENCE, DHHS
Natcher Conference Center, Building 45
Main Auditorium, NIH Campus
Bethesda, Maryland
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Tuesday, April 29, 2008
LIST OF PARTICIPANTS
JOSEPH C. FRATANTONI, M.D.
Maxcyte
H. FRANKLIN BUNN, M.D.
Brigham and Women's Hospital,
Harvard Medical School
ABDU I. ALAYASH, Ph.D.
Office of Blood Research and Review, CBER, FDA
ALAN N. SCHECHTER, M.D.
Molecular Medicine Branch, NIDDK, NIH
GEORGE P. BIRO, M.D., Ph.D.
University of Ottawa and
University of Toronto
BARBARA ALVING, M.D.
MACP National Center for Research Resources, NIH
TOBY A. SILVERMAN, M.D.
Office of Blood Research and Review, CBER, FDA
SARA F. GOLDKIND, M.D., M.A.
Office of the Commissioner, FDA
ROBERT M. WINSLOW, M.D.
Sangart, Inc.
STEVEN A. GOULD, M.D.
Northfield Laboratories, Inc.
ABRAHAM ABUCHOWSKI, Ph.D.
Prolong Pharmaceuticals
A. GERSON GREENBURG, M.D., Ph.D.
Biopure Corporation
TIMOTHY N. ESTEP, Ph.D.
Chart Biotech Consulting
JOSEPH DE ANGELO, M.S.
Apex Bioscience, Inc.
SESSION I: WORKSHOP OVERVIEW AND HBOC UPDATE
HBOCS: BIOCHEMICAL AND PHYSIOLOGICAL PERSPECTIVES
NITRIC OXIDE AND NITRITE IONS PHYSIOLOGY, PATHOLOGY AND PHARMACOLOGY
NON-CLINICAL TESTING: STRENGTHS AND LIMITATIONS
QUESTIONS FOR THE FACULTY MEMBERS
SESSION II: CLINICAL EXPERIENCE WITH HBOCS
RISK: BENEFIT CONSIDERATIONS IN CLINICAL TRIALS IN THE CONTEXT OF 21 CFR 50.24 AND 21 CFR 312
CLINICAL DEVELOPMENT OF POLYHEME
HBOCS: CURRENT STATUS AND FUTURE DIRECTIONS
LESSONS LEARNED FROM THE BAXTER EXPERIENCE IN THE DEVELOPMENT OF HBOCS
DEVELOPMENT OF PHP AS AN NO SCAVENGER IN THE TREATMENT OF DISTRIBUTIVE SHOCK
P R O C E E D I N G S
(8:30 a.m.)
MR. GOODMAN: Good morning, everyone. We should get started. I'm Jesse Goodman, Director of the Biologics Center at FDA, and it is really our pleasure to have you here for this scientific meeting. You know, I would really like to thank NHLBI and George Nemo and Simone Glynn for helping sponsor this as well as the Office of the Secretary. And I'm just going to make a few brief introductory comments and then so is Simone.
As you can see from the program, this meeting has really assembled a terrific group of experts to consider the data, including about 40 people from academia, government, and industry. There is over 300 participants signed up from many countries. Now, to preface these concerns about safety of HBOCs in general, have increased over time based on accumulative clinical experience including with newer products.
The purpose of this workshop which FDA began organizing several months ago was to bring forth and have this discussion. As many are aware, on the day before this workshop, a net analysis was published on diverse products. We've already heard considerable commentary on this, methods and perspectives of this analysis, and whatever you think we should certainly consider that review as part of the broad picture in our discussion here today.
Safety concerns about various candidate products are not new. FDA reviewers have identified potential concerns and carefully considered all available data in making their decisions about individual studies. Some of them have been allowed to proceed. Some have not been allowed to proceed. And we have been criticized for both, as being too restrictive, or as being not restrictive enough.
As we review and discuss the data today, we shouldn't lose track that there is tremendous unmet medical need here, whether on the highways of the United States, the battlefields, people who can't be transfused because of failure to immunologically cross match, people who for religious reasons don't want blood. There is a tremendous unmet medical need.
We need you to help us improve and define and advance the science to better predict safety and efficacy of these products. We must better understand the basic and pre-clinical sciences to minimize risk. But even then, nothing, whether a clinical trial or an approved product can be risk free. Without progress, there can be no benefits to those who remain in need every single day.
It is our hope that the presentation and discussion at this workshop will contribute to finding fast forward for further development of these products, but only as appropriate, based on risk benefit analysis of all available relevant data. Scientists both at FDA and NIH working with others will continue to be engaged in helping advance scientific understanding and developing tools for safety and efficacy evaluation of the HBOCs.
So I thank you for coming here today, for your contributions, and for your deliberation and input and also for your consideration of all views on the data and the subject. So with that, I'll turn it over to Dr. Simone Glynn, thank you very much.
(Applause)
MS. GLYNN: Good morning. And it is a pleasure to welcome you on behalf of the National Heart Lung and Blood Institute, at this workshop. The NHLBI is proud to be a co-sponsor of this workshop and as you know, the institute has reported basic research on Oxygen Carrying Red Cell Substitutes for more than 30 years. So I just wanted to remind you or inform you that the institute recently released a strategic plan to serve as a guide for its research and training programs for the next 5 to 10 years.
And the process initially involved a series of thematic, strategic, planning meetings, involving members of both the extramural and the intramural research communities. And one such group concentrated on issues related to global blood safety and availability. And one of the major recommendation from this group was the need to develop alternatives to standard allergenic donor blood, which included the development of safe and effective hemoglobin based oxygen carriers.
So we followed that at the institute by another working group in 2006. And this working group was tasked with formulating research recommendations for basic research studies on -- again, on hemoglobin based oxygen carriers. And a number of recommendations were provided including the need for basic studies to elucidate the mechanisms of adverse reactions primarily the cardiovascular and the cerebral vascular systems, with hemoglobin based oxygen carrier formulations.
The need to conduct studies on the distribution and metabolism of different hemoglobin derivatives, research into the physiology of oxygen delivery, at the level of the microcirculation and the production and distribution of highly purified hemoglobin based oxygen carrier solutions for use by the scientific community. And if you have not seen it, and if you are interested there is a summary of this working group meeting that has been published in this month's issue of Transfusion.
So at the Institute, we are very much interested in the outcome of this workshop, which will review available scientific data and gather informed opinions regarding the safety of the hemoglobin based oxygen carriers in a variety of clinical settings. And the information which emerges from this workshop will serve as a basis, we hope, for further studies to advance the field.
The institute remains very much committed to supporting meritorious research in this area and we certainly look forward to an exciting and productive workshop review over the next couple of days. So thank you.
(Applause)
MR. HOLMBERG: Welcome. I'm Jerry Holmberg. I'm the senior advisor for Blood Policy. And I just want to welcome you on behalf of the secretary and also the Office of Public Health and Science and the Assistant Secretary for Health. I think that as we look at some of the advances that have occurred over the last 30 years, as Simone mentioned as the interest, I think that we have to really reflect on the safety and availability of the products and how are we moving on the various products that are out there.
I think that one of the things that we really have to be concerned about is the safety of any product that we make available to the American public. And so I do greatly appreciate all the support, the research support that is provided by NIH, NHLBI, and also the regulatory review that is undertaken by the Food and Drug Administration.
When Simone mentioned about 30 years of moving ahead and -- and the strategic plan, I just quickly was thinking about well, 30 years, let's say, that would have been back in 1978. And what -- one of my comments that I wanted to make this morning was that I think we have been talking about hemoglobin carriers for probably greater than we have had an energy crisis. And I think that that is a true statement.
And so we really have to be able to analyze the information that is provided today. And without taking too much time, I just want to thank you all for coming here. I really appreciate and look forward to the discussions that take place today. I'm going to turn the meeting over to Dr. Fratantoni.
(Applause)
SECTION I
WORKSHOP OVERVIEW AND HBOC UPDATE
MR. FRATANTONI: Well, good morning. As many of you know I was heading up the review in research aspects of blood substitutes for CBER for a number of years until I left the field in '96 and it was truly an honor to be asked to come back and work with the planning committee and moderate this session at this very important meeting.
I've got a number of general ground rule announcements to make and then I want to talk a little bit about how the meeting is organized. Get the housekeeping out of the way first. Standard comment, we have a very full agenda. Going to have to ask speakers to pay attention to the length of talk and the moderators will work with you on this. We have a warning light system and will try to keep on time as best as we can.
Ask attendees to help in that way also by coming back as soon as possible after breaks and lunch. Would -- after the breaks and -- and if you were outside this morning saw that there is a bell that will announce that the break time is over. Lunch is available on the level above here. The Natcher Cafeteria and it is a -- it is fairly large and we hope we will be able to move people through there in the one hour that has been allotted for lunch.
After the session today, the area above the auditorium, again up at the atrium level, will be available for social gathering, and people can meet there, have discussions, until the building closes at 6:30 p.m.
We would ask that all press questions for FDA be directed to Karen Reilly. And there's Karen Reilly. Want to make finally, something that requires special mention, the organizers want to call attention to the work done by the administrative staff in preparing for this meeting and a special mention to Jennifer Sharpe, Rhonda Dawson and Jim Durum. We'll just give a little hand.
(Applause)
MR. FRATANTONI: Okay, now regarding the meeting itself, can't start without pointing out that the title for the meeting Hemoglobin Based Oxygen Carriers, HBOC, that term was developed at the first FDA NIH Workshop on Safety in 1990. At that time FDA saw that there were some questions that couldn't be answered with data that they had at hand. We pulled together a workshop that led to the first points to consider on safety, and the term HBOCs came out of that meeting.
And this meeting as you can see from your program, there are four sessions. In this first session, we are -- presenting an overview of basic material underlying the HBOCs. In the second session, it is listed as clinical experience and after an introduction by FDA of some of the technical matters and -- and ethical matters there will be presentations from representatives from industry with their clinical data. There will be a discussion after that as there will be after each session.
Session III is divided into two parts. The first will be a series of brief presentations and then panel discussions, moderated by Dr. Klein. And these will be aiming at considering the -- the class effects, these similarities or dissimilarities between the various preparations that have come to be known in recent years.
The second part moderated by Dr. Weiskopf is going to be primarily aimed at discussing safety, primarily at an organ specific manner. And last session, Session IV moderated by Dr. Biro is going to be looking at some way forward looking at biochemical strategic ways of doing things safely and yet learning about the properties and efficacies of these products.
There is the one change in Session IV that I will call to your attention now. The first speaker in Session IV will be Dr. Emanuel. The other speakers will be as -- as listed. Regarding the discussions, written question will be accepted from the audience and will be presented to the Panels at the end of each session. Index cards for writing these questions are in your folders. If you have written the question, please raise you hand, between speakers and there are people who will pick up the cards and bring them forward.
For Session I, I would ask that the questions regard clarification of the factual material that will be presented here, issues of interpretation and analysis will be better served in the later sessions. There are disclosures regarding conflicts of interest. These are provided by the speakers and the list of these are again, in your folder. We encourage speakers to make any information -- any pertinent information available as it is appropriate.
I'm going to call -- I'm going to -- on the first speaker now, the first presentation is on Overview of Oxygen Physiology. It is by Dr. Frank Bunn. He is Professor of Medicine at Harvard Medical School and at the Brigham and Women's Hospital.
MR. BUNN: Thanks, Dr. Fratantoni. I -- it's a pleasure to be here. The -- it is certainly a meeting I've been looking forward to. The -- I have a couple of disclosures. I'm a member of the SAB at Sangart and formally I was a -- had a similar role at Somatogen. When we talk to medical students about oxygen homeostasis, I think that it is almost compulsory to begin with the Fick equation, which says that the oxygen delivery to either the whole organism or to a organ or -- or tissue within the organ is a product of three independent variables. The blood flow, the oxygen carrying capacity of the blood, the hemoglobin concentration, and the unloading of oxygen from -- from the hemoglobin, which is a function of the oxygen-binding curve, so that the -- these three independent variables are controlled in very different ways.
The -- there is complex regulation of blood flow, the erythropoietin is the major hormone that drives red cell production. And the placement of the oxygen binding curve is -- is determined in human red cells by levels of 2,3-DPG and -- and PH.
Now, when we think about hypoxia and adaptation to hypoxia, there are a number of organismal changes that occur acutely with -- and some of these are very obvious. Increased cardiac output, pulmonary basal constriction, systemic vasodilation, one can call -- refer to the third item as hypoxic vasodilation, increased ventilation, and then at a metabolic level, there is a shift to anaerobic glycolysis. And a change in the -- in the position of the oxygen-binding curve, immediately as a function of PH and then soon thereafter changes in -- in red cell 2,3-DPG.
The -- these are events and phenomenon that have been known for a long time. What is a bit more recently appreciated is that accompanying these immediate changes are delayed adaptations to hypoxia that are a result of programming of gene expression. So that there is an induction of genes that will make new blood vessels. Neovascularization, which complements the hypoxic vasodilation. There is Tyrosine hydroxylase, rate-limiting step in dopamine synthesis will increase the carotid body function. Induction of glycolytic enzymes, induction of erythropoietin , these are all mediated by the transcription factor HIF, hypoxia inducible factor.
Now, the increased cardiac output is of course, a direct correlate to what I showed in the previous -- previously in the Fick equation. The induction of erythropoietin as well, and the lowering of oxygen affinity so that there is -- so that all three elements of the Fick equation are encompassed in these changes with -- adaptations to hypoxia.
Now, hypoxic vasodilatation is a topic that I am sure will be visited a number of times during these two days. Because it is -- it is fundamental to understanding how patients or -- who have -- might be in need of oxygen carrying blood substitute, how that their physiology adjusts at an organismal and tissue level. And the mechanism underlying hypoxic vasodilatation has been a subject of great interest and to some -- to some degree controversy.
There are three major mechanisms. These were shown in a slide that I borrowed from the Alabama group who have recently published on this topic. There -- the -- for a number of years one possible mechanism for sensing and signaling hypoxic vasodilation was ATP release from red cells as they profuse hypoxic tissue. The -- and as a result the -- an ATP receptor on endothelial cells would generate nitric oxide for vasodilatation.
Jonathan Stamler and his group at Duke University have promoted the notion that there is a -- a sulphyderal linked nitric oxide, SNO derivative of hemoglobin reactive beta 93 on hemoglobin that altruistically will release nitric oxide as red cells undergo de-oxygenation. And even though a very tiny proportion of hemoglobin would be -- be the SNO derivative, it would suffice to allow for a vasodilation at a point in which it is needed in relation to local hypoxia.
The -- this paper by Isabel et al, reports a transgenic or actually a knock in mouse model, where the beta 93 cysteine in -- in -- is been replaced by an alanine. And they find that there is no change in cardio dynamics. And no evidence that there is any alteration in hypoxic vasodilation with this important mutated hemoglobin circulating in the mouse. So that that provides some fairly strong evidence against the importance of SNO as a regulator and a vasomotor tone in response to hypoxia.
Mike Gladwin and his group at the NIH and collaborators elsewhere propose that nitrite is a source of a nitrogenous compound that would impact on the vasomotor tone. And the -- with the idea that hemoglobin particularly when it is partially saturated with oxygen can function as a nitrite reductase. And I think we will be hearing more about that from -- in Alan Schechter's talk. And -- and as well as others.
Now, getting back to the Fick equation, it's -- it's -- obviously, hypoxic vasodilatation is an important determinant of blood flow to -- to the needy tissue, hypoxic tissue. And that is going to be determined by the oxygen unloading as well as the hemoglobin concentration. Now, the nitrite reduction then would be a way in which hemoglobin can mediate the release of a -- a nitro -- nitrogenous compound to orchestrate and -- and enable as the -- vasodilation to occur.
More relevant to our topic for the next two days, the HBOCs, is that NO can be -- is a substance which can be readily scavenged by free hemoglobin in the circulation. And so there is an issue as to whether or not NO scavenging could impact adversely on vasomotor tone causing vasoconstriction and reduction in blood flow. And this is a topic which I know it will be thoroughly aired during -- during the next two days.
Now, in the terms of designing an optical -- optimal hemoglobin substitute there are a number of important criteria. Prolonged survival in the circulation, physiologically appropriate oxygen affinity, colloid osmotic pressure, slow rate of auto oxidation and minimal NO scavenging. And what -- what I want to talk about are three of these briefly. One would be the prolongation of -- survival in the circulation. I will first talk about that. Then I will talk briefly about NO scavenging. And then finally the issue of what is the appropriate oxygen affinity for optimal delivery with a hemoglobin based blood substitute.
I -- I can't escape going into some ancient history. I -- I first began research with Jim Yantal (phonetic), my mentor at Thorndike Lab at Boston City Hospital, who died last year. And my first research project was on specifically dealing with how free hemoglobin in this -- in the plasma is handled by the kidney. And I actually finished this work when I was in -- I drafted into the Army at Fort Knox, Kentucky. I was in the -- at the Army Research Lab there.
And the studies that we did focused on the mechanism by which hemoglobin is filtered by the kidney. And the hypothesis we worked off of was that the free hemoglobin particularly when it is dilute in the circulation -- disassociates from its tetramer into identical half molecules alpha beta dimers. And that it seemed logical that the filtration of hemoglobin through the glomerulus might be a function of this disassociation process.
It is -- it is clear that albumin with a molecular size similar to hemoglobin tetramer is not filtered through the glomerulus, where hemoglobin readily is. So the -- the thought was then that the mechanism by which you see hemoglobin emerge in the urine with high concentrations in the plasma was related to the extent to which it disassociated into dimers which were more readily filtered.
So to test that hypothesis we used a bi-functional sulphyderal reagent, basically a methyl ether, to crosslink hemoglobin at the beta 93 sulphyderal groups to -- to -- to -- and that would keep -- keep hemoglobin from disassociating. Sandy Simon at New York had -- had -- had shown that this -- this reagent worked quite well to prevent hemoglobin disassociating into dimers. Then as a control used that mono-functional reagent, N - Ethylmaleimide .
And what we showed was that in -- in rats, treated with BM -- BME hemoglobin that the -- on this log scale, you can see that the retention of the hemoglobin in the circulation of the rat was considerably longer than that with either unmodified hemoglobin or not shown here, hemoglobin modified with a mono-functional reagent. So there was a -- the cross-linking then resulted in a marked prolongation of the half-life of the hemoglobin.
And when the rats were nephrectomized there was no difference between normal and BME hemoglobin, indicating that the difference in the survival had to do with renal excretion. Same -- same was observed with dogs, treat -- treated -- hemoglobin treated with BME or normal.
So what the conclusion from -- from this, then was that that the hemoglobin filtered through the glomerulus as an alpha beta dimer and then once it -- it -- it got into the tubule it could be metabolized by the proximal tubule. And till that capacity was overloaded, then you would get free hemoglobin in the urine.
So obviously, it was an important -- important in developing and I -- hemoglobin based blood substitute to prevent this from happening. And in fact, this is a partial list of hemoglobins that have been developed through the years to be tested as oxygen carrying blood substitutes. And all of them are cross-linked so that this transit through the glomerulus is prevented.
Now, I would like to mention -- go on to talk about nitric oxide. It has been -- it has been a -- an assumption and a very reasonable assumption that NO scavenging is a critically important issue in the use and application of hemoglobin based blood substitutes. Obviously, free hemoglobin in the circulation will have access to the endothelium to a greater extent than circulating -- the laminar flow of circulating red blood cells. And therefore, NO that is produced at the local endothelial level could -- could readily be scavenged and that may have deleterious effects on blood flow.
The Somatogen company a number of years ago, developed a -- a cross-linked hemoglobin which was the result of a isopeptide bond created between the two alpha globins and at various lengths of lysine residues were inserted by genetic engineering to make for a di-alpha goblin subunit that -- that would prevent the hemoglobin from disassociating.
And this as expected had -- this di-alpha hemoglobin had a prolonged circulation in the -- compared to free hemoglobin that is -- that -- native hemoglobin that can disassociate. Now, Doug Lemon and John Olson decided to look in depth at -- at the issue of nitric oxide scavenging. And so what they did was to make mutants in the heme pocket which -- significantly reduced the uptake of nitric oxide through the hemoglobin and the -- the conversion of -- of NO to nitrate with oxidation of the heme-iron.
And so what they did was ask whether or not these modified hemoglobins which were -- they showed very -- very elegantly by stop flow analysis did retard NO binding. Whether they might have any effect, physiological effect, on blood flow in the -- in the animal. And so here is a -- a diagram showing the inverse relationship between the ability of the hemoglobin to scavenge nitric oxide and the increase in blood pressure noted.
And you can see that it is clear that unmodified hemoglobin were -- was -- had -- had a -- had a marked pressure response whereas -- genetic modifications that reduced NO uptake, reduced that effect. Now, the question then remains how important this NO scavenging is and what can be -- and if it is important what can be done about it and I believe that we will hear quite a bit more on this issue during this meeting.
A second faith based assumption is that oxygen binding of HBOCs should match that of the red blood cell. I actually had a -- my first grant was from the Army to work on this 40 years ago. And I gave up on it with the cross-linked hemoglobin that I showed you because it had such high oxygen affinity. I thought it will be worthless as a oxygen carrying blood substitute. I had -- I sort of took it as an article of faith that the hemoglobin that circulated in the plasma should match that of the red cell and have a p50 of 26 torr, in order for there to be efficient oxygen unloading to tissues.
But the -- this thinking has been challenged and revisited in a major way by Bob Winslow who has -- postulated that the red blood cells are -- are designed to deliver oxygen in an orderly way to minimize undue vasoconstriction by virtue of facilitated diffusion and a gradient from the red blood cell to the endothelial cell. And when tissue oxygen retention is -- is low, that is in other words, where there is high oxygen consumption, that the -- it -- the oxygen carrier has to be poised in such a way as to not trigger oxygen dependent vasoconstriction.
And -- so that this can be illustrated I think in a couple -- a few illustrative slides here. Just think about a -- a micro vessel whether it is an arterial or a initial capillary, one -- one which is subjected to regulation of vasomotor tone. And subjected to -- therefore, subjected to hypoxic vasodilation. You can see that with -- with the laminar flow of red blood cells there is diffusion of oxygen to the surface of the endothelial -- endothelium and that there is a gradient and so that the oxygen concentration around the red cells can be greater than that, that impaction of the endothelial cell. And this -- this is a -- in a physiologic system, this will allow for a certain maintenance of appropriate vasomotor tone.
Now, if we -- we then flood a hemoglobin based oxygen carrier into the system, and that would be done, say in a patient with severe blood loss, or where the red -- circulating red cells may be marked decreased, you have hemoglobin with a capability of unloading oxygen right at the level of the endothelial cell. And to that extent the oxygen tension at that cite is -- may -- may well is increased particularly if the hemoglobin has an oxygen affinity similar to that of whole blood.
So that the -- the -- so that you are going to get an increase in oxygen retention at the endothelium and what this is going to do if the P50 is close to physiologic, is going to cause vasoconstriction. So you are going to get a narrowing of the lumen of that blood vessel and impairment of blood flow. Now, the -- the -- this problem can be offset by infusion of a hemoglobin that has a high oxygen affinity, where there is less unloading of the oxygen from the oxygen -- oxygen carrying hemoglobin, the HBOC.
And -- and therefore the P02, a level of the blood vessel will be sufficiently low so as not to engage vasoconstriction. So this is a paradigm which I think is one that is worthy of considerable pursuit. And I think I'm going to stop with that and hopefully some of these point will be revisited and -- and better amplified in this -- in future talks. Thank you.
(Applause)
HBOCS: BIOCHEMICAL AND PHYSIOLOGICAL PERSPECTIVES
MR. FRATANTONI: The next presentation will be on the biochemical and physiological perspectives of the HBOCs and this is given by Dr. Abdu Alayash, who is the Chief of the Laboratory of Biochemistry and Vascular Biology at CBER, at FDA. Abdu?
MR. ALAYASH: Thank you, Joe. My presentation will basically focus on as Joe said, the title indicate some biochemical, physiological properties of some HBOCs that we had a chance to work on them. The work is largely done here at CBER. There were such programs that we have been involved with some 18, 19 years ago. Hopefully, some -- some basic aspects will transpire from this -- this presentation. And will be hopefully some use as you deliberate with these important products.
So let me start just with the overall -- I'm sure many of you have seen this slide before. The different approaches that have been used by industry to modify these hemoglobin s. And as you can see, we have basically two classes of product. The fluorocarbon based and the hemoglobin based products. We are not obviously going to talk about the fluorocarbons anymore.
The hemoglobin based -- hemoglobin is basically derived from the red cells, outdated blood, chemically modified and the modifications either takes the form of -- either cross-linking -- cross-linking and the surface of the protein is decorated with some non-protein molecules. Or in some cases the protein is pulverized. In some indication of the -- at least at the research stages now, the hemoglobin is encapsulated with lipid bilayer.
The purpose of modifications is primarily to serve two really basic issues. As Dr. Bunn indicated is obviously to stabilize the tetramer. The tetramer -- the hemoglobin as it released from the red cells when in free form, will break down into dimers. So the idea is to either to stabilize it in the tetrameric form or the polymeric form.
Today, you are going to hear representation of these approaches from Baxter, which is the original (inaudible) hemoglobin , Apex and Sangart. They will be presenting some data on the conjugated hemoglobin, and of course, Northfield and Biopure opted for the pulverized hemoglobin.
Okay. In spite of the bad press, the -- some people in the community, actually believe that there are some promising therapeutic value for these products. But unfortunately as the slide indicate, we are facing a number of issue regarding the toxicity. But if you check the literature these days this is the list that you will come up with, vasoactivity and hypertension GI side effect, pancreatic effect and so on and so forth.
The common thread in all of these reactions is really the -- the -- the -- the issue is either triggered or emanated from the healing prosthetic wound of hemoglobin. And an example of that is -- was vasoactivity. As Bunn had indicated it is very simple reaction between hemoglobin and nitric oxide. But if you really biochemically through some of these events, carefully you can also again, see the role of heme in these reactions.
And anyway you look at it, with the nitric oxide or reactions of hemoglobin without molecules, heme will be oxidized. This is really the main theme of my talk. Regardless, whether it is nitrous oxide or oxidants and so on and so forth. So what drives oxidation? Inside the red cells, and outside the red cells? And as you know, hemoglobin spontaneously oxidizes even within the red cells to a number of species, ferric or the mat, which is non-functional, or even sometimes ferra which is even little bit toxic.
But as you know, in the red cells, we have a very efficient and sematic machinery that reduce the hemoglobin back to its previous functional form. When we have hemoglobin free outside the red cells, of course, you can't control the hemoglobin. Hemoglobin will -- will oxidize spontaneously. And additionally, the hemoglobin will -- the oxidation itself will be actually enhanced by a number of factors, including as you said, the spontaneous oxidation.
If you leave hemoglobin on a bench for 10, 15 hours and you look back at it, it will turn little bit brownish. But it is just the rusting, the oxidation. And of course, the activity with nitrous oxide will also oxidize the hemoglobin to certain extent. And of course, the oxidant, but of course, we have no shortage of oxidants. And incidentally, even the hemoglobin itself when it auto-oxidizes produces oxygen. And can actually if you leave it for long time it will self destruct.
Additionally, in our case, the way you modify the hemoglobin, the manufacturing that goes into producing the hemoglobin, in some cases, can actually enhance the oxidation. In some other cases, may slow it down. And the net result of all of this of course, we can have the effect -- the effectiveness of these hemoglobins. When you accumulate methemoglobin, methemoglobin doesn't carry oxygen. Of course, if it oxidizes fully you break up the hemoglobin. That may actually lead to some issue with the -- with the safety.
I'm going to choose two examples on the manufacturing and on the oxidant. Very briefly. This is the story of hemoglobin that we had to chance to actually look at it. It is human-linked manufactured by Hemosol and they give us this some few years back. And we had some agreement with them. The common scientific name is polymerized hemoglobin. What I am trying to do here is show you how chemical modifications in some cases could actually lead to some undesirable destabilization of the -- of the product.
This cartoon summarized the story. What they tried to do is basically treat the hemoglobin, which is extremely purified form of hemoglobin, A0, with sugar which is a trisaccharide raffinose. And this sugar, before they added to the hemoglobin, they oxidized it to open up the grains, added to the hemoglobin and of course, the sugar will bind to 3-amino acid here in this space which is known as the 2,3-DPGs pocket.
They had done initial work to indicate that the actual cross-linking had occurred. And in fact, if you look at the HBLC in our hands also it looks in a polymeric form. Because the sugar not only goes in the DPG pocket, the sugar actually modifies some of the amino acids on the surface and it produce a polymer.
But if you look at the typical oxygen titration curve here, if you look at typical A0, you get this nice sigmoidal curve. And if you use fresh blood and of course, the curve is shifted. And is again nicely sigmoidal in nature. But if you look at the product which is produced from that addition of that sugar on purified hemoglobin the result is this bizarre form of oxygen. Look at this curve. It is almost linier. Has no sigmoidal nature. Doesn't saturate.
In fact, even if add pure oxygen to it, it would not saturate. So clearly there is something wrong in the chemistry of the hemoglobin. At this rate it would have done something wrong and we tried to sort of get to the bottom this issue, to sort of try to understand what actually went wrong. Two things emerged from an extensive study that we published.
One of them, was the heme itself, if you know the heme is protein and iron, usually sit in the center. Well, we found out that this hemoglobin is actually the -- the heme itself is distorted. The iron instead of being in the middle, it is actually tilted. And that would lead to break up of the heme and iron will be released. And we picked this up with an EPR technique.
The other problem with this product is that their protein as you know, go -- spontaneously they transition from the fully oxygenated to the non-oxygenated. The R and the T form. This hemoglobin is actually locked in the T form, the de-oxy form. And this makes plain -- funny shape polybural curve because you -- you paralyze the hemoglobin in the T4. Remember almost most H factor are to certain extent in the T form. This particular hemoglobin it appears to be actually frozen in one form.
We went down. We broke in the hemoglobin. Now, if they are at the top, we thought if we can take these six fraction, pull them out, look at their properties and if we pull out the bad fraction, maybe if we can put it together we can fix the problem. Couldn't do that. We've broken the hemoglobin into small peptide looking for the reagent.
Where did the reagent go? Remember the reagent was supposed to go here? Unfortunately, we found it bound to (inaudible) amino acid completely different 60-93. And here is the mass fact data to confirm that the masses of the sugar is actually on 60-93, which is way from the area that the reagent was supposed to be here. It was supposed to be here, here, and here.
And here is some calculation of that, the masses to convince ourselves that we are actually looking at the sugars on -- on the wrong side. And here is the close up. The sugar found here, and ironically the pieces of the sugar, not the full sugar, which is called (inaudible) product, we find that on a completely different amino acid.
The point in all of this, the reagent didn't go in the area where it is supposed to go, create some destabilization, pulled some water from the cavity and that may explain the unusual activity. That we unfortunately, didn't have enough of the material to do it. And now, to sort of relate the chemistry to the animal and the story end and there. But the point in here is that when you saw a reagent on your hemoglobin you really need to know what you've done to the protein. From simple experiment, you can actually pull a lot of quite important information.
One more story here, on the -- oxidation. And as I said oxidation can occur by oxidants, by hemoglobin (inaudible) on -- on oxidants. And here is a story that we published recently, which is really very simple story. What we did here and I guess -- remember pure oxide is available physiologically even small amounts can do it.
So we took the hemoglobin, we treated with hydrogen peroxide in 1:1 ratio; very little of hydrogen peroxide. And we found out again, using mass spec, we found out that actually the oxidation of a handful of amino acids is always consistent. Each time you do the experiment we find 60-93 again, is oxidized, irreversibly. And 60-112 and tryptophan and the infamous methionine 55.
Now, when I say reversibly, when you talk to protein chemists, this is unheard of. To actually use little hydrogen peroxide, add it to the protein and you convert sisteic to sisteic acid. Normally you would require a huge amount of hydrogen peroxide and more powerful oxidant. The reason for that is very simple here. What pure oxide did actually radicalize the hemoglobin. And we know that. We have done that. With the pure oxide they create a radical, radical what? Protein and irreversibly damage the protein. The point in all of this, even very little oxidant or actually hemoglobin, can actually radicalize your hemoglobin.
Okay, so the question is we can do chemistry from now, until eternity but obviously there will be a time when you need to ask the question do these simple test-tubes reaction really occur and leave a -- an animal? Does oxidation occur and whether these reactions can actually compromise the ability of hemoglobin to carry oxygen? And more importantly, if it leads to some toxicity?
So we did the following. We've chosen -- and again, depends when you do these experiments, you really need to be careful as far as the choice of amino model, and the extent of your -- your -- your search of the facts. So what we are doing here, we had two identical species. The rat, which we know ahead of time, the rat has the ability to somatically produce ascorbic acids, which is a very powerful reducing agent. While the guinea pig, of course, unfortunately, like humans are -- are not able to produce hydrogen peroxide.
So we infuse these animals with 50 percent exchange transfusion with the same protein which is commercially available oxyglobin. And we looked at the -- the oxidation. But before that you can see in the rats, they maintain normal level of ascorbate, which is very high ascorbate. Nitric oxide guinea pig, after transfusion drop it remain very low. And how about the hemoglobin and circulation? From information of the oxidation that you can see the value almost maintain normal level of -- of hemoglobin, functional hemoglobin, very little oxidation. The -- the guinea pig, almost 50, 60 percent of the hemoglobin turned into met, which is very similar to what actually Bahagas (phonetic) reported years ago. And similar to some clinical data which we published recently in humans.
So what happens with the hemoglobin? The question is out of this oxidation, if you like, we know that hemoglobin will end up with some changes. Can we actually find that in the blood? So we pulled the blood of the animal and we looked at the oxidant modification in the guinea pig. Nothing happens in the rat. And we see here, this is at four hours. This is at 24 hours, and you can see both the alpha and the beta subunits have undergone oxidant modification very similar though we don't have very definitive answer to the oxidation pattern that I showed -- the some of the p-amino acids. So clearly, in the physiology, these things do occur.
We looked for toxicity in the tissue and we've seen very similar to what Baxter and other people have seen. Transient (inaudible) changes and so called, the heart -- cardiac collisions, kidney damage. The rats and the guinea pig are slightly different, so to speak, but we are looking at more sensitive biomarkers to actually relate the chemistry we saw in circulation to the tissue.
The other part of my talk is, do these products deliver oxygen after the oxidation that we have seen in -- in circulation? And we have been looking for a really reliable tissue biomarker. And recently, as Dr. Bunn had indicated, we stumbled on very valuable biomarker. And that is of course, the hypoxia inducible factor, which is -- which is a transitional factor, can control the responses to the hypoxia controlled large number of genes.
Now, they have -- and low oxygen or rather high oxygen is normally degraded through an enzymatic pathway, ultimately by the proteasome. If low oxygen helps binds to the beta subunit, transmit it to the nucleus and bind to the DNA and trigger the activation of a number of key important genes. And these are of course, alpha (inaudible) genes, beta glycolytic genes, (inaudible) genes and so on and so forth. So the point in here, we need something sitting in the cells to tell us whether really oxygen deliver -- being delivered by the hemoglobin. And this is really one thing that we have there, which is an oxygen sensor, you know.
So what happens if the hemoglobin comes with oxygen? Can we see any changes in the genes and the -- and the other responses? Go back to the rat and the guinea pig and here you are looking at the functional ferritin. We pulled the hemoglobin from the circulation and look at the different heme, total heme concentration and other species, but we really concentrating on the ability of hemoglobin as time goes by, clear. And of course, hemoglobin loses the ability to carry oxygen. As you can see they have in the kidney is going up.
And late, in hours you can see a nice coalition between the two. This particular hemoglobin was able to suppress, HIF in the early stages, which means oxygen presumably being delivered. And in the guinea pig, we see -- we see similar thing, but you can see clearly towards the end, hemoglobin turned into absolutely nothing but a cluster of modified hemoglobin and you can see the HIF is extremely high.
The genes, here we are looking at -- at -- at hemoglobin slightly different experiment but we are focused on the rat. Because the rat is basically, cleaner than guinea pig. This is to control oxidation. We didn't want to compound our experiment. Here we comparing the same hemoglobin versus starch, hetastarch. It is 80 percent ET. Which is to chill, exaggerate the hypoxia.
You can see here, with an unoxygen carrying volume expander, the huge increase in the eco-gene and of course, it goes down after some times. This again, the kidney and of you can look at the hemoglobin, there is some suppression early, which is again about 10, 12 hours. Then you can see the EPO rebound to higher level.
Interestingly, few years ago, some in industry thought that this is a new property of hemoglobin, which is induction of erythropoietin but in reality what happens of course, when EPO rebound, it has usually lost the ability to carry oxygen. Hemoglobin has been oxidized and by this time of course, has been cleared. Here, we are looking at the erythropoietin, which basically corresponds with the genes.
Recently, we looked at a rather sensitive organ which is the mitochondria. And here we are looking at cytochrome oxidase, which is -- terminal oxidase in the mitochondria. And it just happens that this protein is also controlled by HIF. Here we are looking at the glycolytic metabolism in case of the -- the same animals, of course. And what happens during normoxic, one subunit of the hemoglobin -- of -- sorry, of the cytochrome oxidase, COX4 1, transformed to COX4 2. Don't confuse it with COX inhibitor. This is cytochrome oxidase.
And what happens, the reason for this because when the mitochondria transfer that, you know, from 4-1 to 4-2, it is to maximize the electron transfer and -- and -- and what you see here again, the -- piece -- piece of HIF starts to fuse around -- increase in the COX4, to very little initially in the case of the oxyglobin. Again, at the mitochondria level where every molecule of oxygen really counts, you can see that the hemoglobin at least, the first 10 hours was able to do what it was supposed to do.
Okay, so clearly, I hope I have convinced you that -- that heme-oxidation is really critical here. And I know that there are a number of people here in the room, and ourselves who started early to think of ways and means to control it. And number of people here in the room, including John Olson and ourselves, and particularly John, started using simple protein type models, which is myoglobin and later on hemoglobin, to reengineer the hemoglobin pocket. The heme pocket, where -- and I presume that these oxidants interact. We, more recently started looking naturally occurring actually, hemoglobin that could have some nice chemistry that we can obviously later on translate it into human.
Recently, we looked at the (inaudible) hemoglobin, which you throw anywhere at it, or oxidant react very, very slowly. The idea is here, that of course, when -- so use that clever chemistry in nature to hopefully do that in human situation. Number of approaches people have tried to put the enzymes from the red cells back to the hemoglobin either cross-linked or not and the whole idea is to control oxygen -- oxygenation or oxidation, rather.
We have recently, we used ascorbate. Of course, as we said it is an important reducing agent, selenium. Even the green tea actually, has some antioxidant property. This is an area that -- and -- or part of the control of oxidation which is heptaglobin, CD 163, which has been really ignored in recent years. This is more recent interest of ours and you are going to hear more from Dominik Schaer who came from Switzerland who will talk about this a little bit more.
But here is a little cartoon which show of course, the conventional thinking that heptaglobin of course, rightfully binds with the dimers and CD 163 also combined with some of these dimers or the tetramers. We actually recently shown that the tetramer even some tetrameric species within the polymerized hemoglobin can be picked up by CD 163 or -- or they heptaglobin. And if we modify the surface of the protein, you can actually enhance either pathway. Or you can enhance both pathways for the clearance. Again, you are going to hear more on that issue tomorrow.
So in summary, almost all HBOC will undergo oxidation. There is no way you can control it and you can actually radicalize the hemoglobin as I have indicated to you because of this transition. If however your HBOC can withstand NON (phonetic) cells, and oxidants (inaudible). All by addition of some of these additives to control slow oxidation, you may actually get away with it. And you can keep the hemoglobin intact and deliver some oxygen.
Finally, the people who actually did the work in my life are listed here. And I would really sincerely like to thank colleagues here with me on the organizing committee for a stunning job and helping us in putting the workshop together. Thank you very much.
(Applause)
NITRIC OXIDE AND NITRITE IONS PHYSIOLOGY, PATHOLOGY AND PHARMACOLOGY
MR. FRATANTONI: Dr. Bunn mentioned that we would be talking about nitric oxide physiology, and now for a more detailed discussion of that, Dr. Alan Schechter. He is the Chief of the Molecular Medicine Branch, of the National Institute of Diabetics, Digestive and Kidney Diseases. Alan?
MR. SCHECHTER: I realize that the time is late and I will try to go through this rapidly. I would like to thank the organizing committee, including myself, for inviting me to present here today. Can we have the lights down please? Thank you. What I will try to do is give a general view of -- for the non-specialists of nitric oxide physiology, pharmacology, and -- and pathology. And just to point of disclosure, that I -- I am a co -- co-inventor of a patent from -- by National Institutes of Health for the use of nitrite salts in the treatment of cardiovascular diseases.
Background slide, most of you probably are familiar with this. The nitric oxide is believed to be the major systemic vasodilators, short-lived free radical which is multiple balanced states, which can undergo reactions with many low and high molecular weight biological compound. For the purpose of this meeting, the fact that nitric oxide can be rapidly destroyed by hemoglobin, a fact that has been known since the very first discovery of -- of nitric oxide by Octivity (phonetic) in the mid-1980s, it was used as an assay for nitric oxide for many years, leads to a paradox about its bioactivity, because of the expectations of the vast amounts of hemoglobin in the body, intracellular and extracellular would destroy virtually all the nitric oxide.
As we have gradually realized over the last 10 or 15 years, the physiological and pharmacological potential of nitric oxide depends upon the balance between destruction and preservation and perhaps transport of nitric oxide through hemoglobin.
The nitric oxide paradigm, which was worked out in the mid-1980s by Furchgott, Ignarro, Murad, and Macata (phonetic), the first three of whom won the Nobel Prize a few years ago, for basically, the ideas described in -- in this cartoon is that either shear stress in the vasculature at the very top of the slides, with certain hormones like ( inaudible ) acting through its receptor can activate a nitric oxide synthase, NOS, in endothelial cells, which converts arginine to citrulline, freeing nitric oxide, much of which diffuses into the smooth muscle below the endothelium and activates guanylate cyclaseto, an active form, which compares GTP and cyclic GMP. And through processes involving calcium fluxes, causes smooth muscle relaxation.
It was also realized but only really studied intensively in the last 10 to 15 years that nitric oxide also diffuses luminally as well as ab luminally into the vascular system and that this process of the NO reactions within the vasculature obviously contributes very greatly to determining the balance of nitric oxide in -- in -- in the body.
The functions of nitric oxide as I indicated are enormous. The regulation of vasodilatone was the first to be described in that initial work that lead to a Nobel Prize but quickly it was realized that there were many other important functions including platelet out-gauge and attachment, changes in circulating selectins and other -- other proteins in activation of super-oxide and the whole complex of reactions involving oxygen radical chemistry.
In addition, it was soon realized that in addition to the NOS in endothelial cells, there were NOS -- there were other NOS enzymes, one in neuronal tissue, the end NOS, which was -- is involved in producing NO in the neuro system which important for neuro transmissions, as well as other processes. And another -- and a third NOS, the iNOS, in macrophage is the inducible NOS, which is important in fibrocytosis and destruction of various pathogens.
And so all in all, nitric oxide I believe that the publication of 75 or a 100,000 papers does indicate some importance is -- is considered a -- a topic of great biomedical interest. My own background for the last 30 years in hemoglobin and sickle cell hemoglobin led -- led to my interest in nitric oxide because of the interactions that were known from long time ago and some recently postulated interactions between hemoglobin and nitric oxide.
Many of these reactions were actually first described in the 19th century, but it was only at the beginning of the 20th century, during the first World War in that -- that the study of oxy- hemoglobin reaction with nitric oxide to lead to methemoglobin nitrate was first studied in -- in detail by Coleman (phonetic) and Rao (phonetic.) And others, for reasons having to do with the use of gases during the First World War.
A second reaction was intensively studied in the '50s and '60s with the advent of EPR, spectroscopy, the reaction of NO with deoxy-hemoglobin to give nitrosoheme hemoglobin with NO hemoglobin with nitrosohemoglobin which I will mention again later. And the third -- a third reaction which as Frank Bunn alluded to was postulated about 12 years ago, primarily by Jonathan Stemlyn, his colleagues at Duke University, who suggested that oxy-hemoglobin could also react with NO to modify the conserved beta 93 cysteine compound which is called S nitrosohemoglobin or SNO hemoglobin and they postulated a -- an important homeostatic function for their -- of great -- and it became the -- the postulate had great theological interest, in that it was -- the idea was that SNO hemoglobin was allosterically controlled and it is dissociation under hypoxic conditions could free NO and lead to increase in -- in blood flow and increased oxygen delivery to compensate for hypoxic conditions.
However, from the very beginning this hypothesis was very controversial but it did serve a function of getting me and many other investigators into the field who were interested in hemoglobin into the field at that time. And in particular, just about then, in '96 and '97, I was fortunate that I initiated a collaboration with Mark Gladwin who had joined the NIH in Critical Care Medicine and Richard Cannon of the National Heart Lung and Blood Institute.
And we began to try to look at the question in humans and virtually all the work I mentioned before about SNO hemoglobin was done in animals and we were not sure how relevant this was to human beings. We decided to -- to investigate the reactions of NO with hemoglobin under physiological conditions and we chose the inhalation methodology of delivering NO, which had been approved by FDA based upon the work of Lawrence A. Paul and others. And it has been approved for use in the treatment of -- of certain pulmonary conditions.
And we were able with the help of Critical Care Medicine nurses, and other staff to have a number of normal volunteers and then later on sickle cell patients and other individuals breath nitric oxide for varying periods and analyze the -- the changes in hemoglobin chemistry that occurred in these individuals. And so you can see under what we considered physiological conditions, what the relevant reactions were.
I will just say, I have long advocated the importance of clinical research of studying phenomenon in humans and I -- I think this is a very good example that -- that things that we found in humans were not necessarily the same as were reported in animals. In any case, the -- the paradigm was have an individual for a couple of hours at rest, we measured nitric oxide derivatives in arterial and venous blood, the individual then breathed nitric oxide at -- at 60 or 80 parts per million for a -- two hours.
We measured the changes in these metabolites and then the inhalation was stopped and then the values went back to -- to zero. And we set up assays for all what we believed were the important NO adducts at this time. And some of the results we got then, and these studies including the use of nitric oxide inhibitors, exercise to -- to test various physiological hypothesis, which I won't go into now, was that we found when individuals breathed, these were normal individual breath nitric oxide for two hours, we got significant increases in nitrosohemoglobin but very interestingly a statistically significant arterial venous difference in all three of these experimental situations.
No increase in SNO hemoglobin which values we got in our assays were much lower than were being reported from further south of here. And no AB differences. But interestingly we found that as you would expect there were marked increases in nitrate. In fact, we have inferred that most of the methemoglobin in the body comes NO reaction. The NO reaction with the oxy-hemoglobin to give methemoglobin nitrate and in addition, how with small increases in nitrate levels with generally significant AP differences both before and after inhalation.
And on this -- on the basis of these studies of normal volunteers we postulated that NO inhalation might lead to NO -- systemic NO increases but that the likely fact is for potential physiological delivery if it occurred, were -- was NO hemoglobin and nitrite. Both of these hypothesis were not warmly accepted. In vitro the half-life of NO hemoglobin is very long, 100 or more hours. And so it did not seem that it could act physiologically and many investigators said nitrate cannot be under physiological condition converted to -- to NO.
But in any case, we believe that data and we wrote a short review and by that time for the New England Journal of Medicine, I think 2003, in which we postulated that the basic overview of nitric oxide pharmacology, physiology, pathology is show in this -- this cartoon that all ordinarily erythrocytes are fairly much immune from interaction with nitric oxide because of unstirred layers, Liao (phonetic) and others, Kim Shapiro, had studied the diffusion and for a variety of hydronamically -- I won't go into this, probably a little diffusion under normal conditions of the NO into the red cell.
With pharmacology such as with nitric oxide inhalation, we postulated a number of reactions the possibility of these anti -- oxygen reductase reduction that (inaudible) has been studying, might contribute to NO formation. But in particular, in terms of red cell, we believed that the major resultant of large scale NO administration as with the inhalation was the formation of nitrosohemoglobin and that could disassociate under -- under physiological conditions to free nitric oxide, perhaps through SNO hemoglobin.
With the idea that we suggested was SNO hemoglobin was an unstable reactive intermediate in nitric oxide metabolism of the red cell. But also that -- that nitrite itself could be formed in the vascular system and nitric oxide could be converted to free NO.
In contrast, we were just beginning to do studies then and from other data that suggest to us that the cell free hemoglobin could be very different, that the NO produced by NO syntheses in the wall of that blood vessel would quickly react with the largely ferrous cell free hemoglobin, convert that to ferric and nitrate and lead to a relative NO deficiency and cause constriction of the vessels and perhaps contribute to some of the pathology of -- of various cardiovascular diseases.
This hypothesis -- this article led to five letters of protest brought by Dr. Stambler and four of his colleagues, five separate letters brought by four of his colleagues saying that none of this could be right. But still five years later, I think what we said here is basically the view we still have. We often initiated studies and this is with Andre Dejam of nitrite levels in humans in plasma and red cells. There was a lot of controversy in the literature. Again, the value bring reported especially for animals, were immensely higher than we were seeing in human normal and individuals and patients.
And we had to work out, largely done by a post doctoral fellow, who is now at the Bingham under Dejam, method both of assaying for nitrite and stabilizing if we felt a stabilization system that we could get stable values of 24 hours. This is -- these data are from a paper in Blood two or three years ago, in which we found that now measurements whole blood nitrite levels in just about a dozen human beings as with regard to 117 animals per liter red blood cells are close to 300 animals per liter suggesting that most of the intravascular nitrite is in the red cells but a significant amount in the plasma and that using these methods, the stabilization solution, one could study nitrites in a systematic way, other than many ideas that it might be a risk factor for various cardiovascular diseases.
We also with our usual propensity to use PowerPoint to draw cartoons did another one of the --this zoom is the interaction of the nitric oxide in the vascular system with the nitric oxide produced by iNOS undergoing reaction with many oxidants reductants to give a whole variety of balance dates which I represent here as N-Oxy and these various balance dates which include proxy nitrite and NO2 and N2O3 and many others can either react to nitrate proteins, tyrosine groups or sulphydral groups or lipids -- could this -- this significant amounts of free NO, as well as oxidation to nitrite which can be reduced to NO by (inaudible) oxidase at low PH or NO and nitrite can go into the red cell and undergo a -- some of the reactions I have been describing.
I don't have time to step through these reactions except to say that the two uncertain issues of importance in this schema is first whether or not there is some nitric oxide being produced within the red cell. Dr. Kelman (phonetic) and his colleagues have published a number of papers over the last few years suggesting that red cell membranes have eNOS activity and convert largely to NO. Other groups have not been able to confirm that but if it is true and it may require certain conditions of substrate, it would be very important to in understanding where the NO -- NO and nitrite in the red cell comes from.
The other aspect is that the -- there is still a question of how the NO can get out of the red cell after any or all these reactions. And this is still controversial. There is an interesting recent suggestion by Kim Shapiro and Gladwin that N2O3 is the active form for efflux in the red cell but I think that that -- those analyses are still ongoing.
But at this point, given our physiological interest in all this, we ask the question is nitrite a vasodilator. We know and this has been known for a long time that vasodilator -- in vasodilators aortic rings have high concentrations. In fact, Dr. Sommerwise (phonetic) did studies like this in 1930 -- well, not with Eric Rings (phonetic), but with -- in animals and people, the studies were done in 1930s at Brigham and in the 1950s by Dr. Brownwald (phonetic) NIH.
The nitrite vasodilates lung profusion models at concentrations of 100 micromolare. I took the byzantine (phonetic) oxidase and the levels but there were papers appearing as recently as three or four years ago, that suggested that there was no vasodilator activity of nitrides.
So we began a collaboration with a group at Loma Linda, Gordon Powers and Chris Hunter and their colleagues and we infused -- they infused nitrite into hypoxic newborn sheep and you can see that blood pressure fell with the infusions. And it fell to a great extent that hypoxic animals than normal animals, suggesting that the hypoxia enhanced this. A very important correlative of this was the measurement of exhaled NO in these sheep and we could when we infused nitrite into an artery, we could see NO being exhaled which was direct proof that nitrite was being converted to NO, was not acting by some other mechanism.
Eventually, a few clinical studies were done with the FDA IND with low levels of nitrite were infused step wise into a few individuals and we found an increase in forearm blood flow which correlated with changes in whole blood nitrite and we could follow the time course of that seeing the effects were almost immediately if seen laterally, but only started occurring after a minute or two in the contra-lateral arm.
And so the conclusion of this work was that nitrite irons caused vasodilatation and physiological concentrations and may contribute to hypoxic vasodilatations. Nitrites probably induced to NO by deoxy heme-proteins and other nitrite reductase mechanisms. And Dr. Gladwin will go into this I think tomorrow in his lecture. Nitrite may be relatively stable tissue and blood source and bioactive NO. And nitrite may be useful for administration by inhalation or infusion as a therapy for various patho-physiological states characterized by a lack of NO.
Now, the time is late and I see the red light so say -- with just to say we have been recently been examining the effect of a ascorbate and the dehydro ascorbate which are in equilibrium on -- on some of these reactions. And we can show, and there is a paper that has just been published in biochemistry by my colleagues (inaudible), that DHA can oxidize HBNO to methemoglobin and presumably free the NO as you can see the time -- the time course of this. And that DHA can increase nitrite levels in erythrocytes and we again, we have worked out a proposed mechanism of this.
Time is late and I won't go into this. You can look at this in your hand out. The only point I would make that I think of general importance for this group, is that other factors like ascorbate or perhaps urate can also affect these reactions and so inferences from studies of pure hemoglobin solutions like the NO hemoglobin solutions are not necessarily valid for what occurs physiologically especially in the red blood cells.
Lastly, it is important that I spend two minutes just mentioning hemolysis. I have worked for 30 years in sickle cell disease and this summarizes the fact that we know that hemolysis is a very important part of sickle cell disease, into vascular hemolysis. And it -- and again, we know that NO, as I said it is a destroyed by hemoglobin on diffusion, limited reaction.
This slide which I made up several years ago, before I was -- got involved in HBOC question, we obviously knew and actually I think it was Bob Winslow who suggested to me in 1993, 15 years ago, that a lot of the problems in the HBOCs was indeed due to nitric oxide. And this was before I'd even paid any attention to nitric oxide.
And this again is just the part of the schemata but what I wanted to -- is that this idea has been taken by Mark Gladwin and his colleagues Martin Steinberg and others into the idea that -- that sickle cell disease may actually have two distinct components. A vasoinclusive components, related to intracellular polymerization. I wouldn't call it erythrocytic sickle, I would call it intercellular hemoglobin mass polymerization.
And the factors of sickle cell disease that are affected by this include the, obviously the hemoglobin level -- include the hemoglobin level for destruction of red cells, the terminal arterials, vasal (inaudible) pain crises, acute chest syndrome and other symptoms. But that was a distinct subset of symptoms including pulmonary hypertension, pryposim, glycolysis and now, there is some data for stroke as well, that is related to the hemolytic component.
Up until now, hemolysis has been largely known as sickle cell disease. Most patients are very well compensated and do not need transfusion. But this -- this approach to it -- to the disease suggests that there are two sets of symptoms which are due to quite different distinct patho-physiological mechanisms.
Lastly, just Frank Bunn alluded to the paper that has impressed, I think may come out this Friday or a month from now, of Nature Medicine from Isabel Patel, in towns at UAB which they made a knock in, replacing beta 93 cysteine with an alanine and you see a change in SNO hemoglobin, but there was no other phenotype in these animals. And in fact, when -- when one does, the classic aortic ring assays with either normal mouse hemoglobin or the without the beta 93 there are in -- in pulmonary arteries of rabbits there are no differences at all.
And the conclusion of this is that under these circumstances there is no evidence that SNO hemoglobin has any physiological importance. And I think all the papers that have come out recently about the important of SNO hemoglobin in blood transfusion, in sickle cell disease and diabetics and pulmonary hypertension have to be thoroughly reevaluated on the basis of this very important finding.
And then just to conclude that nitrite ions cause vasodilatation, physiological concentrations contribute to hypoxic vasodilatation and a relatively stable tissue and blood sources of bioactive NO for endocrine delivery. Nitrite is reduced by deoxyhemoglobin and possibly other heme proteins myoglobin, hemoglobin, et cetera. NO hemoglobin may also be a source of bioactive NO and SNO hemoglobin appears to be an unstable intermediate in NO reactions with hemoglobin.
Cell free hemoglobin and acute and chronic anemias may contribute to pathology by reaction with either NO or nitrite ions. And administration of NO by inhalation of nitrite by inhalation or infusion may compensate for pathology related NO deficiencies. And already there is work in the HBOC field. A recent paper from (inaudible) Paul along these lines. So I think the -- we still have a big problem with NO biochemistry with the blood substitutes. But the -- there is a possibility of how robust it is, I don't know, that the -- there may be a solution. Thank you.
(Applause)
MR. FRATANTONI: I remind people that if you have any questions for these speakers, write them on cards, raise your hands, someone will come down and get the card. The final speaker is going to address the topic of non-clinical studies, the animal models why they do work, why they don't work, a question that's very important one for this conference, is Dr. George Biro, who is the Emeritus Professor at the University of Ottawa, adjunct professor at the University of Toronto. George?
NON-CLINICAL TESTING: STRENGTHS AND LIMITATIONS
MR. BIRO: Good morning, ladies and gentlemen. I'm gratified to have received this invitation to talk and I'm especially gratified to follow the distinguished speakers who have set up an excellent groundwork for what I'm about to say. What I'm going to say has little to do with the molecular and sub-cellular aspects. I'm going to talk about mostly really old-fashioned animal physiology. And what I would like to say is that I left -- retired from the University of Ottawa from which a recent editorial on HBOCs has emanated. I left the University of Ottawa in 1998, I think, worked for Hemosol for about 4 years. Consulted Hemosol for about two more, and went into retirement and been dragged back into the arena.
I set myself three questions for this talk. I wanted to think about the conventional -- the really conventional and old-fashioned ICH and GLP compliant testing for safety into whole animals only. I didn't want to address issues like in vitro testing or the standard issues, only about the things that apply to safety to HBOCs.
Secondly, I wanted to look at the academy laboratory experiments using unique resources that are mostly available only at universities and research institutes and I'm going to use some examples without using actual data. And lastly, I wanted to pose the question of why is it that the safety testing on animals has failed to predict convincingly what has been observed in the clinical trials. So first of all, whole animals only and these were all healthy, normal animals, single dose safety.
The largest problem with single dose safety testing in animals using HBOCs is the fact that there is a very limited applicability to use multiples of the intended clinical dose. The blood volume is limited and there is obviously an issue of interpretation when you're replacing or adding a very large fraction of the blood volume.
There have been two experiments in which practically all of the blood volume has been replaced. One set of experiments by Hemosol replaced 95 percent of the blood volume and studied the animals over the subsequent seven days. All survived in the presence -- in the virtual absence of red cells. Second experiment was done by Baxter and they replaced practically all 98 or 99 percent of the red cells and they also survived. The standard model is to do a standard toxicity panel biochemical histological testing and looking at immediate and short term as well as delayed results.
It is possible using these studies to complete a limited study of the mechanism of effects, but by and large these are small animals and the ability to study mechanisms is quite limited. It is possible to do pathology on these, but it is likely that with a single exposure, a single infusion, there are not likely to be very obvious pathological changes. And there is a possibility to do limited pharmacodynamic and pharmacokinetic studies and these are useful and it is also useful if animal models of relevant disease have been involved; so far, none have.
Repeated dose toxicity is the standard toxicology paradigm. Again, they use whole animals and healthy, normal ones, usually litter mates or at least animals of similar age, size, and of the same species. I would like to offer you an example of one of these, which was conducted by Hemosol, and this was presented at the society for toxicology some years ago as an example for this. Sprague-Dawleys rat were given daily intravenous infusions of Humulin (phonetic) of 10, 20, and 30 milliliters of kilogram. So these represent about 5, 10, and about 20 or 30 percent of the blood volume on 14 consecutive days.
The infusions were either Humulin 10 percent in Ringer's lactate or pentastarch 6 percent in physiological saline. At the time this was done, X10 (phonetic) was not yet on the market. So this was the control, which is obviously not an ideal or appropriate control. But we wanted to use the then available clinical colloid.
The standard panel linked with the hematological clinical resident -- clinical chemistry grows in microscopic examination of all organs, veterinary observations, feed consumption and weight gain, coagulation, special staining by Prussian blue of all the organs for ferric iron, special staining of the testes for spermatogenesis, and quantitative measurement of tissue iron in various target organs.
What I want to emphasize here, is what is the magnitude of the exposure and burden in these animals? At 30 milliliters per kilogram per -- 30 milliliters of Humulin per kilogram, they are exposed to 3 grams of hemoglobin, which represents a cumulative exposure of 14 days or 42 grams which is 5-1/2 -- 5-1/4 times the blood volume. The volume is 30 milliliters and it is 420 milliliters, 800 times the blood volume -- sorry -- this is 5 times the hemoglobin mass.
Iron was given at 11 milligrams which is 20 percent of the total body iron and almost 3 times over the 14 days of the total body iron. Globin was 500 -- 5 times total globin mass and porphyrin was 22 times the normal daily turnover, so these are massive overloads, none of these animals died, all of them survive to the intended time of sacrifice and they were tested and they were a variety of abnormalities, obviously, clinical chemistry was abnormal, liver function tests were abnormal.
Hematology was abnormal, but the interpretation of this changes is difficult because we don't know whether the change in liver function, for example, is due to the disposal of the large amount of globin, or the large amount of porphyrin.
The change in feed consumption may be due to the enormous intravenous protein load, which may play havoc with the hypothalamic signals of satiety and hunger. So it is possible to achieve in these animals a large, manifold multiple of the clinically intended single dose. In larger animals it is possible to make repeated observations and establish a time course for recovery.
There is now out in the public domain one set of experiments in which healthy normal pigs, sheep, as well as dogs and rats have been exposed to Baxter's hemoglobin and subjected to extensive histopathological examination, which revealed widely dispersed small diffused degenerative changes, which were not associated with global changes in either global ventricular function or troponin release.
The perplexing thing is the apparent species specificity. Rats and dogs do not appear to exhibit these lesions, pigs, sheep, and primates do. The mechanism is not understood, but it is possible or probable that there is nitric oxide improvement. And we do not understand the significance of species specificity. So overall, conventional safety testing on large animals -- the procedures are uniform, because there is extensively tested and produced standard operating procedures, the population is homogenized. The rats, and for example, they are often litter mates, same size, same age, and the analysis is mostly done by aggregation.
The variability is minimized because of the aggregation and the large and massive overload may detect common events. The laboratory and the pathology, given long enough exposure for pathology to develop, may help to understand the significance of the clinical chemistry and hematology changes and may allow the determination of reversibility of these changes.
The limitations are that these studies are of limited generalizability. You cannot generalize very easily. They may fail to detect the rare and infrequent events. Unless diseased animal models are used, you cannot identify possible synergistic effect between the disease and the exposure to the agent. So what they may fail and likely do fail is that it fails to identify the specific monitoring requirements for the clinical trials.
Now, my second question was to look at the academic experiments using these unique resources and expertise in university and research institute labs. What I find is this is a wonderland of applied physiology, sophisticated, extremely well-developed methods are used in highly competent and technically excellent health, hence, and they generally are aimed at demonstration of efficacy. They may indicate safety issues, but the major issue in these experiments is to look at the efficacy, and they are comparing with controlled studies.
They use very expensive -- extensive hemodynamic monitoring with additional unique measurements such as oxygen availability in the tissue. Models are reproducing physically relevant conditions such as normovolemic hemodilution, bleeding shock, and delayed resuscitation, animal models such as the spontaneous hypertensive rat, blood flow measurements, both total cardiac output and regional distribution of blood flow, microcirculatory observations using very sophisticated methods and critical oxygen delivery estimates.
The setup and special skills obviously are not available in CROs, and therefore these are not GLP compliant. Unique measurements are available using palladium porphyrin, phosphorescence, platinum microelectrodes and polarographic electrodes to measure tissue PO2 and this distribution within the tissue.
Whole animal hemodynamic and oxygen dynamics can be done in clinically relevant models such as shock and resuscitation and combine, for example, with observations in the conjunctiva microcirculation. Very sophisticated, quantitative microcirculatory observations are made in whole animals through windows mostly looking at accessible parts like skin and the skin pouch.
Global and regional myocardial function can be measured and region can be combined with regional and organ blood flow measurements in whole animals. And these are often combined with tissue oxygen measurements in accessible tissues, such as skeletal muscle. Organ function in other tissues, pancreas, kidney, and in fact animals have been subjected to severe hemorrhagic shock and resuscitation to see if there is amelioration of post ischemic microcirculatory change.
In hearts, in pigs with previously imposed critical coronary stenosis, simulating coronary artery disease has been measured. Again, global and regional myocardial function and blood flow distribution to see what is the effect of an HBOC in the presence of a critical coronary stenosis. But measurements of tissue PO2 have been generally indicative of reasonably well preserved tissue PO2. These have shown that there is reasonable recovery from shock and resuscitation when an HBOC was used.
Quantitative observations have shown differences between the microcirculatory parameters and in the HBOC-resuscitated animals and in controls. Pancreatic organ function has been seen to recover well to post ischemic conditions in the presence of HBOC. And critical coronary stenosis in the presence of HBOC has allowed extreme hemodilution down to a level of a hematocrit of only two percent, whereas animal's hemodilution with albumin would show cardiac failure and die at a hematocrit of about six or seven percent.
In standard hemodynamics and blood flow distribution models, blood flow was measured in all organs, and in the myocardium, blood flow was reasonably well preserved in HBOC hemodiluted animals whereas blood flow has impaired in starch hemodiluted animals. Critical oxygen delivery after similar sequential hemodilution to extremely low hematocrits was shown to have the same critical oxygen delivery level as animals that had been diluted with either/or old or fresh red cells.
Sangart and Intaglietta have shown microcirculatory observations in animals hemodiluted to extremely low hematocrits and compared it with the same degree of hemodilution with decorated albumin. Again, the hemodilution with the HBOC was beneficial. Brain blood flow and oxygenation was measured in the caudate nucleus of rats hemodiluted with an HBOC, and the tissue PO2 in the caudate nucleus was actually increased with maintained blood flow in the presence of an HBOC.
And eventually the HBOC effect of a pharmacological dose of the same dose of an HBOC injected or infused into spontaneous hypertensive rats and the control was the Wistar-Kyoto rat, showed that quantitatively the same dose of Humulin increased the blood pressure bore in a spontaneous hypertensive rat than in the Wistar-Kyoto rat.
So the strength of these sophisticated experiments in academic labs and research institutes are that there are direct indications of efficacy in a really reasonable acceptable criteria. Critical oxygen delivery, critical hematocrit, and there are also indirect indication of safety issues, such as organ or tissue vascular microcirculatory resistance.
Microcirculatory resistance is greatly increased. We have an indication of a possibility of a safety issue. Very powerful tools have been brought to this investigation. Variables of clinical interest have been used in models of clinical relevance and observations were made in highly stressed intervals in contrast to the standard conventional toxicology model.
Pharmacodynamic analysis can be conducted and the investigation is extended to organs of high physiological importance, the heart, the brain, the kidney, pancreas, and the liver. The limitations are actually very specific to the experimental model. There is limited generalizability if the study is conducted in those organs which do not receive a large proportion of the cardiac output and in which the blood flow distribution is not coupled to the metabolic need or other function such as secretory or excretory function.
These studies if they are specific to organs that do not receive the overwhelming majority of the cardiac output, neglect the affected compromise models in morbid conditions, unless they are specifically included and they really have not been.
And focusing a single organ deflects the whole body physiological adjustments that occur in response to any stress. I would remind you of Dr. Bunn's first slide in which the first box is the cardiac output and right under the cardiac output is blood flow distribution. Cardiac output is less important than its distribution.
The striking observation about these is that on balance, they offer strong support for beneficial effects of the HBOC against the controls used. The benefits are seen even in models simulating clinical conditions, for example, hemorrhage and resuscitation, or organ ischemia, or arterial stenosis. In all but two of the studies, actually three -- in all but two of the studies, the control is a non-oxygen transporting colloid. Two of the studies, used either shed red -- shed blood or old and stored red cells and nearly all used healthy normal animals. Only one study that I know of used the spontaneous hypertensive rat.
I was going to say more, but I'm going to cut it short. There has been a failure of the non-clinical testing. The failure has been that by and large these testing showed beneficial effect against the control and failed to predict the adverse clinical outcomes. The published academic experiments have also shown benefits have failed to predict the published outcome. Why? Because there is a great discrepancy between the non-clinical testing and the clinical testing, healthy normal inbred young animals.
In the clinical studies there is a huge heterogeneity of such subjects, age, co-morbid conditions, procedures, et cetera. By and large, the adverse clinical outcomes have been seen more frequently in the aged, in the diabetic, in the atherosclerotic, in the hypertensive, and these adverse clinical outcomes were occurred in this population, which represented a large proportion in these trials than their prevalence in the normal population. I thought that there may be one probable answer to the question of why the animal testing and the human testing diverged.
Endothelial dysfunction not what you thought I was referring to occurs in diabetes, in atherosclerosis, in hypertension, ageing, and others. So the endothelium is an extremely important organ, 20 years ago it first got recognized that the endothelium is very important. Recently there was a review about the endothelial dysfunction in which they quoted, "A 100 years ago textbook of the principles and practice of medicine in which it was said that the age of your arteries defines how long you will live and how well."
The recent review modified this by saying that the health of your endothelium defines how long you will live and how well. Endothelial dysfunction, I am going to ask you to throw away the hardcopy of my slides. I will have a new set tomorrow. Endothelial dysfunction will amplify and exacerbate all the effects that these are supposed to show because they interfere with NO, because they make other vasoactive substances more important.
In conclusion, conventional testing has not been very useful, unconventional testing has failed to predict and the animal experiments should emphasize evaluation of the HBOC effect on important target organs with appropriate attention to physiological adjustments.
Organ blood flow is regulated by an enormously complex interplay of multiple vasodilator mechanisms. These regulate or autoregulate the supply to meet the metabolic demand. The hypertensive effects of HBOCs are also multifactorial and they mediate the vasodilator mechanisms mostly through NO, which are rendered less effective, less bioavailable. And in many highly prevalent human diseases, endothelial dysfunction, which is really manifested in a priori impairment of the NO response. Endothelial dysfunction and HBOCs together exacerbate and make each other worse. Thank you.
(Applause)
QUESTIONS FOR THE FACULTY MEMBERS
MR. FRATANTONI: I have not received any card -- I am receiving a card with question. Okay, I've just got a single question with me. Let me read this one and I'll just give it to one speaker to handle and then we'll go to a break as -- there are more questions coming. Okay, well then can I ask the speakers to join me up here on the panel, we'll just spend a few minutes with these.
Okay, I'm going to hand these questions out to a couple of (off mike) and there is a general question here that we just -- obviously there is one question that asks, will there ever be a substitute as good as your own autologous whole blood; that I think is what this meaning is about. So hopefully we have some approach to that.
Okay, I've got a question for Dr. Bunn. Since HBOC or tetramer injection does not produce systemic vasoconstriction in specialized mouse, you know, the negative imbalance, why is HBOC oxygen delivery vasoconstriction above the (off mike)?
MR. BUNN: I think this is a good point. I don't really have anything to add to what the NOS knockout mouse is an argument I suppose that one could use. I don't think there is any question that vasoconstriction is seen with HBOC administration, and so the question is, you know, how much of a contributor is nitric oxide to this. And I think that's hopefully something we'll gain further insight during the meeting about.
MR. FRATANTONI: For Dr. Schechter, does arginine feeding effect nitric oxide balance?
MR. SCHECHTER: Thank you. I thought -- that is arginine feeding?
SPEAKER: Yeah.
MR. SCHECHTER: Sorry, it is not clear. I suspect not, there have been a number of reports that it does, there is at least one company that supplies arginine candy bars in health food stores and they are apparently widely used. The levels of arginine in the blood are much higher than the KM, the enzyme would indicate as necessary through maximal reduction of NO from the arginine.
People have argued may be within the endothelial cells or other places the levels are lower and by raising the arginine levels one can increase the levels and insert specific tissues where NO is synthesized. There have been a few reports of benefit from arginine administration, but they have not been tested in very large scale controlled studies. I think the verdict is out. I'm a little dubious, but I think we need control trials to establish whether or not it does have value.
MR. FRATANTONI: The question to him -- towards the entire panel, I think it's a question that comes up a lot. Could you explain the difference between vasoactivity and hypertension? Abdul, you want to try that first?
MR. ALAYASH: I tried to explain that before and I was (inaudible) in my simple-minded biochemical mind vasoactivity refers to constrictions of blood vessels as a consequence of that is the hypertension, but to those of us here with a better physiological background could actually explain that. Alan, can you explain it? I mean did I get it right or wrong?
MR. SCHECHTER: Okay. Vasoactivity and hypertension. Well, this is standard physiology. The blood pressure is the result of the interaction between the cardiac output and the peripheral resistance. In almost all studies, the rise in blood pressure is accompanied by the calculated resistance because that's the one method you can measure. You can measure cardiac output, you can measure blood pressure, and you can therefore calculate peripheral resistance which translates into -- this is one of the things I wanted to say -- translates into a constriction of the arterioles principally, unless the blood viscosity changes.
Resistance comprises the constriction of the blood vessels, hindrance and viscosity of the blood. Generally, blood viscosity declines not a great deal from the normal hematocrit of 45. It increases exponentially when you go up the hematocrit at 45. So vasoconstriction accompanies vasoactivity, accompanies the rise in blood pressure in using an HBOC, unless the conditions are such that you really cannot determine the peripheral resistance.
Beyond that, all I can say is that there is not a single factor that determines the blood pressure. There is a host of vasodilators, nitric oxide one, every organ, especially, the ones in which blood flow is coupled to metabolic rate sequence a variety of signals and mediators, the heart, the brain, adenosine is one of the important mediators which is a vasodilator and work synergistically with nitric oxide. In addition, there is a host of vasoconstrictors. The simple adrenal system, endothelin, angiotensin, and there is evidence that HBOCs affect every one of those.
MR. FRATANTONI: Okay, Dr. Bunn you have a question. We'll make this our last one.
MR. BUNN: This is from Dr. Simone, Texas Tech. Do you think that besides the mass the charge on the protein is important in glomerular filtration? Hemoglobin tetramer can also cross the glomerular barrier because it has more -- these more electropositive charge than albumin. This is a very good point. In fact this came up in some detail at Somatogen, where they engineered hemoglobins with different charges with the hypothesis that the more electropositive, the hemoglobin that -- you might get more filtration and it was tested by double-charge mutations to quite rigorously and the result was that that there was not a significant impact of hemoglobin charge on this glomerular filtration.
MR. FRATANTONI: Thank you. We're going to stop there. I'm not getting to all the questions, the time is not going to allow that, so I'll apologize to anyone whose question we did not reach. We're going to -- now it's -- I've got 10:40; we'll reconvene at 5 minutes before 11:00. I want to thank all the speakers for getting this off to a great start.
(Applause)
(Recess)
SESSION II -- CLINICAL EXPERIENCE WITH HBOCS
MS. ALVING: Could you please take your seats now so we can begin the next session?
(Pause)
MS. ALVING: Okay. Our next session is going to be on "Clinical Experience with Hemoglobin Based Oxygen Carriers."
I will be the moderator for the panel this afternoon. I'm Dr. Barbara Alving. I'm the director of the National Center for Research Resources, which is one of the 27 institutes and centers of NIH. In interest of full disclosure, I once worked for the FDA and I am a retired Army Colonel, worked at Walter Reed Army Institute of Research, and thought about the products or potential products that were needed for trauma and for soldiers, and I leave it at that. I'm now a civilian and still very interested in these products and interested in the products for both civilians and soldiers.
And also I think we are very interested as federal -- a federal agency working closely together meaning FDA, NIH, in what is the best way to proceed. And that's going to come out, I think, tomorrow with further discussion. This panel and this session is really going to be about hearing what has been going on in the clinical trials, what has been the experience, and we're -- I think, we're very fortunate to have representatives from several of the companies here to speak about clinical trials.
But first I'd like to introduce Dr. Toby Silverman, and she is going to provide some introduction. She is from the Office of Blood Research and Reviews, CBER, FDA.
Also I'll remind speakers and all of us that we are going to speak very clearly and loudly, and if you cannot hear, please raise your hand in the back to remind the speakers.
(Pause)
MS. SILVERMAN: All right. Okay, can everybody hear me? Fantastic, I'll hold this one here too.
As Dr. Alving said, I'm -- my name is Toby Silverman. I'm the branch chief in the clinical review branch in the Office of Blood Research and Review in CBER, FDA.
My group has evaluated all of the hemoglobin-based oxygen carriers that have come before FDA over the years. I'd like to introduce you to the issues, and I'd like to set the tone here by saying that we will be discussing settings and indications that either have been studied or have been contemplated.
We'll try to set the stage for defining clinical benefits, and after that, endpoints. And then very briefly we'll talk about some unresolved issues. So let's start with settings and indications.
First, how and under what conditions will hemoglobin-based oxygen carriers be used? These are some of the things that people have thought about over the years. It been proposed for initial resuscitation as a bridge to transfusion, as a transfusion alternative, as oxygen therapeutics in various states such as ischemic state stroke, medical anemia.
Some have thought about them as adjuncts, adjunctive therapy, particularly for radiation therapy, and others yet have thought about them of as treatment of pressor-dependant septic shock or SIRS.
There've been a lot of questions about where and by whom such products will be used: Battlefield situations, accident scenes, in transport vehicles, in the hospital, in the emergency room, in surgery, whether elective or urgent, in the ICU, in the oncology ward, in the cath lab on the medical ward.
And some have even thought about using these in physician's office. Other questions that have been raised have been who will control the distribution. Will they be controlled and distributed from the pharmacy, from the blood bank, both, neither?
There had been some questions about medical oversight issues, initial and total dose of product, monitoring of use and utilization review. Certainly there have been issues of clinical laboratory measurements and interference with some perhaps critical laboratory parameters for patient care, and then questions about transfusion or infusion reactions.
Studies that have been conducted in potential indications have included perioperative use, general surgery, orthopedic surgery, GU, GYN cardiac, some with and some without acute normovolemic hemodilution, for the purpose of evaluating these products with transfusion avoidance or reduction in allogeneic transfusion.
Studies in trauma have been conducted and have been proposed for the pre-hospital setting, for the pre-hospital setting into the hospital, and in the hospital. Products have been studied or/and being studied for hemodynamic stabilization, for example, pressor-dependant sepsis and SIRS, in renal failure and in a post-surgical critically ill patients. And these products have been studied in ischemic events, ischemic settings including percutaneous coronary intervention and stroke.
So let's start by trying to define a clinical benefit and the first question I'd like to ask is what's the target?
Well, there are some potential benefits to these products to include, in general, universal compatibility, immediate availability, stability on long-term storage including at room temperature, the fact that these products are pathogen-inactivated or pathogen-reduced, and then in general, an avoidance or reduction of allogeneic red blood cell transfusion.
Potential clinical benefits include oxygen delivery, resuscitation from hemorrhagic shock, treatment of ischemia, radiation sensitization, and again other pharmacologic effects, including taking advantage of the pressor effect of these agents and hemodynamic stabilization.
So let's talk a little bit about endpoints, how do you measure such clinical benefits?
Well, there are -- I work for FDA, so we have to consider some regulatory concepts. We deal with the concept of substantial evidence of effectiveness as defined by the Food Drug and Cosmetic Act and here is the quote, "Evidence consisting of adequate and well-controlled investigations by experts, qualified by scientific training and experience, to evaluate the effectiveness of the drug involved on the basis of which it could be concluded that the drug will have the effect it purports to have under the conditions of use prescribed, recommended or suggested in the labeling."
The Public Health Service Act Section 351 states that licenses for biologics are issued upon showing that the product meets standards designed to ensure continued safety, purity, and potency. And the concept of potency has long been interpreted to include evidence of effectiveness. All hemoglobin-based oxygen carriers are biological drugs. So they're subject not only to the FD&C Act, but also to this provision of the PHSM.
So let's talk about some general endpoint considerations. First, sample sizes must be sufficient to permit adequate assessment of risk versus benefit of use. FDA has said generally separate safety and efficacy data are necessary for each clinical setting for which an indication is sought.
Now what's an indication? And indication is the beneficial effect or effects as determined in clinical investigation or investigations. And the claim should include the setting or settings in which the use of the product is indicated.
General efficacy considerations include an increase in survival, prevention or slowing of disease progression, in other words a decrease in morbidity, or some measurable symptomatic relief. And the real question here is how to apply these general considerations to HBOCs?
In order to do that, CBER has just put out one points to consider in 1990 to which you heard Dr. Fratantoni elude, and then draft guidance in 1997 on the efficacy evaluation of hemoglobin and perfluorocarbon-based oxygen carriers, and then in 2004 draft guidance on criteria for safety and efficacy evaluation of oxygen therapeutics as red blood cell substitutes.
Efficacy and safety considerations are context-specific, and we've talked about some of the contexts, elective surgery and trauma, but the one I haven't talked about yet is blood not available, not appropriate or not acceptable, either due to objections in the use of blood, religious or non-religious, or hemolytic anemias, blood incompatibility, and so forth.
There are other indications that I've alluded to ischemia, as in coronary ischemia and stroke, radiation sensitization, and hemodynamic stabilization are taking advantage of the pressor effect of some of these products.
So how do we measure efficacy? Well, in the various guidance documents, FDA has noted that the population should reflect the clinical population likely to undergo that particular surgery, this is for elective surgery. And the protocols should specify and confirm enrolment of subjects with high transfusion need.
Finally, the hemoglobin based oxygen carrier and the control, which would probably be red blood cells, must be administered for appropriate and evidence-based reasons. Endpoint considerations include reduction and/or avoidance of allogeneic red blood cells, which is a surrogate for risk reduction, including the risk associated with allogeneic red blood cells, which include non cross-matching compatibilities, theoretical immune suppression, transmissible infectious diseases, outcomes related to the age of stored blood, and whatever are known.
Red blood cell transfusion avoidance however does not equate to avoidance of all allogeneic risk. And a delay in allogeneic transfusion without reduction and use of allogeneic red blood cells would not be considered a clinical benefit.
In trauma, some general considerations include -- include the following. Evaluation of clinical outcomes is quite difficult, because of the uncontrolled conditions, variations of the site and extent of injuries, the duration of hypertension, hypoperfusion and hypothermia, and the time interval between injury and access to definitive care.
There are issues related to the difficulties in classifications of trauma severity and the methods for assessing total body oxygen debt to improve evaluation of shock severity, and the success of resuscitation are not currently available.
FDA has said that mortality is an unambiguous endpoint, that's true. And long-term survival, what the good quality of life is the clinical benefit of interest to the patient and the patient's family. But 30-day mortality is not a sensitive measure of the impact of an oxygen therapeutic agent used for early resuscitation.
And present information is insufficient to correlate short-term survival with long-term survival for oxygen therapeutics for a number of reasons. Again, inadequate classifications of trauma severity, the methods for assessing total body oxygen debt to better evaluate shock severity in the success of resuscitation are not currently available, a kind of a circular problem here.
Let's talk a little bit about blood not available, appropriate or acceptable, general considerations. I think it's -- people would agree that it's difficult to devise a single clinical trial that would address all of the situations where blood might not available, or appropriate or acceptable. There is a diversity of clinical situations.
For example, transfusion of avoidance versus other intended uses, when one talks about blood incompatibility in hemolysis, that's not the same as religious objection. The urgency of need is difficult to define and the medical versus the surgical situations would need to be defined.
With these considerations in mind, FDA has suggested that studies in both remote field trauma and elective surgery are needed in order to understand adequately the benefits and risks of oxygen therapeutics in the broadest spectrum of transfusion situations where such products might be used.
However, even that approach does not address the benefit to risk ratio of use in certain settings. For example, there is theme here, ischemia, cardiac, CNS, or other radiations sensitization, or hemodynamic stabilization and taking advantage of the pressor effect.
Studies in both remote field trauma and elective surgery also might not answer fully the question of whether an oxygen therapeutic is as safe as red blood cells in a setting where both are available and the patient is not clinically stable. And the decision whether to use an oxygen therapeutic await the brief time until allogeneic blood is available might actually be quite difficult.
So let's talk a little bit about safety. This is the topic of most of the meeting today. Clinical evaluation of safety, efforts to ensure the quality and completeness of the safety database should be comparable to those made to support efficacy. And this can -- maybe found, this citation may be found in the guidance for industry on pre-marketing risk assessment.
Evaluation of hemoglobin-based oxygen carriers in diverse populations with the wide variety of comorbid conditions -- you heard Dr. Biro has talked this morning, and so the -- this should be fairly self-evident that studying at a variety of comorbid conditions is important.
And the study plans should be designed to capture new or novel adverse events, and changes in the frequency and severity are the mild, moderate, and severe of adverse events of both the background rate or intensity of those events. And there should be pre-specified stopping rules.
In general, there've been a number of toxicities noted for hemoglobin-based oxygen carriers to include, as you've heard earlier, cardiac toxicity with degenerative lesion seen in the left ventricular myocardium in susceptible species such as swine or monkey.
We don't know what the relevance of this is to humans. Myocardial ischemia has been observed clinically. Vasoactivity of the product -- many of these products or most of these products are vasoactive, which probably related, at least in part, to the scavenging of nitric oxide by hemoglobin.
Gastrointestinal effects have been noted to include discomfort, nausea, vomiting, diarrhea, dysphagia, generalized abdominal pain, and there is experimental evidence of enhancement of bacterial translocation across gut epithelium.
These products have proinflammatory activity including procoagulant activity and DIC, and release of procoagulant (inaudible) by simulating leukocytes experimentally. Oxidative stress is a consideration as you heard from Dr. Alayash's talk. Many of these products have been associated with elevations of pancreatic and liver enzymes. And there may be an adverse synergy of free hemoglobin with bacterial endotoxin, and finally neurotoxicity has been raised as a safety concern.
I'm going to show some slides here of eight commercial products. Data are available in the public domain for six.
FDA reviewed these data, which were obtained from peer-reviewed publications, press releases, and testimony presented at the December 2006 Blood Products Advisory Committee meeting. There are a lot of caveats. For each product, data are presented aggregated from all reviewed studies.
This is not by any stretch of the imagination meta-analysis. Controls varied from study to study, and some of the studies I'm going to show you were not controlled. Not all clinical trials conducted with the reviewed products have been published.
Results presented here are not synonymous with line listings of the type that would be reported to FDA in a comprehensive final study report. And this leads to another set of caveats, aggregating information to derive a comprehensive list of adverse events may not give a completely accurate tally of all adverse events that occurred.
Now those of us who did this work made every effort not to count a subject more than once for each category of event which will be represented by a table row. It is possible though that subjects may have been counted more than once because of the reporting methods used in the publications.
In some instances, the number of subjects was back calculated from reported percentages. In these instances, the denominator was assumed to be the number of subjects in each cohort -- that assumption may not be correct. Not all enzyme elevations were captured as adverse events. And the number of subjects with enzyme elevations into clinically significant ranges was not captured uniformly or was not reported at all.
In some instances, only means and standard deviations, not the number of subjects contributing to the data set, were captured. Now let's take a look at some of these.
Here are the eight companies, Apex -- they are in alphabetical order, so nobody is up for particular description -- Apex, Baxter, Biopure, Enzon, Hemosol, Northfield, Sangart and Somatogen.
Two of the companies did not report anything in the public domain. Those are Apex and Enzon. And I believe that you'll be hearing from representatives after this talk. Large studies -- large numbers of subjects are included in the Baxter, Biopure, Hemosol, and Northfield databases.
The number of deaths -- there is an imbalance in the number of deaths, with the exception of Hemosol, in for Baxter, Biopure, Northfield, and Sangart, Sangart reporting two deaths versus zero. Hypertension is a fairly -- is a common feature among these products for those that have reported it, and there is an imbalance for Baxter, Biopure, Hemosol, Sangart, and Somatogen.
Of importance cardiac events, yesterday there was publication to discuss myocardial infarction. There is an imbalance for Baxter, Biopure, Hemosol, Northfield, and Sangart. And then there is a -- there are imbalances in terms of cardiac arrhythmias for the same companies.
So you see that there are some cardiac events of importance and there is an imbalance in deaths. Now we also took a look at pancreas and liver. And as I remind -- I'd like to remind you that not all of the numbers were captured here, Baxter reported a number of cases of frank pancreatitis including hemorrhagic pancreatitis.
There is a small imbalance for Biopure and only one case of pancreatitis was reported in literature for Hemosol. There are excursions in lipase and amylase for these companies, and in some cases these were reported as pancreatic enzyme elevations.
And then a number of these companies have showed changes in the AST or ALT or other liver function tests as you see here. This captures all of the other adverse events to include CNS, respiratory, renal, GI, coagulation, and sepsis and septic shock. What I would like to point out to you is that there is an imbalance in terms of CVA for this company, and a smaller number reported in the literature for Hemosol.
There are imbalances for pneumonia, for respiratory failure, hypoxia and cyanosis, a large imbalance for gastrointestinal events. This category of coagulation defect includes the citation of thrombocytopenia, but also the general category of coagulation defect.
And there is an imbalance again for those that have reported these events. I'd like to bring to your attention, this last one, sepsis, septic shock, multiple organ failure, to show you that there are some imbalances in the literature in terms of this endpoint including Northfield over here, and I'm sure that Dr. Gould will be discussing this later.
So this is a more comprehensive view of the overall safety database for -- that's in the literature. There are some unresolved issues that I'd like to bring to people's attention. We've already eluded to them, the role of public versus proprietary research. There is an urgent need here for better scientific understanding of the chemistry, the redox biology and the pathophysiology of acellular hemoglobins as you heard in the first session today.
Of particular importance is defining a clinical benefit, and once defining a clinical benefit assessing clinically meaningful, readily measurable efficacy endpoints. And I think that there is a critical need for developing predictive surrogate markers of efficacy; we don't have any right now. There is also a critical need to understand clinical safety in terms of dosing and maximum tolerated dose.
We need to define an acceptable benefit to risk profile for each clinical indication based on all of the above, both in studies where subjects are able to provide informed consent, and most particularly in studies where informed consent cannot be obtained.
And finally, I think that there is a critical need for defining a logical, clinical development program for these products. And with that, I'd like to turn it over to Dr. Alving.
(Applause)
MS. ALVING: Thank you, Toby.
Our next speaker is going to be Dr. Sara Goldkind. And she is the senior bioethicist at the FDA and in the GCP program in the Office of the commissioner.
(Pause)
RISK-BENEFIT CONSIDERATIONS IN CLINICAL TRIALS IN THE CONTEXT OF 21 CFR 50.24 AND CFR 312
MS. GOLDKIND: Okay. Good morning.
I'd like to continue to build upon some of the points that Dr. Silverman began to address in her presentation and what I am specifically going to focus on are risk benefit considerations in trials.
And I was asked to focus my remarks on how do we understand risk benefit considerations related to our regulatory dictates 312 and 50.24 which Toby just introduced, and I will go through those further.
But what I would like to do is to bring to your consciousness that while we'll be discussing risk benefit considerations within the context of our regulations, really what we're talking about are ethical concerns. We're talking about ethical considerations for the protection of human subjects, who'll be in these trials and that's really what's captured in the regulations.
And I'm going to present a framework, one of many good frameworks for the discussion of ethical research and the analysis of whether or not research is ethical.
This framework was established by doctors Emanuel, Wendler and Grady. And you have an article in your packets which discusses this in more detail. And Dr. Emanuel will be here tomorrow and will be discussing hemoglobin oxygen carriers more specifically within the context of some of these specific attributes.
I'm going to look at these attributes more generally so that I can give you a framework in which to think about risk benefit across our regulatory spectrum. And I'm not going to discuss all of these attributes. I've highlighted favorable risk benefit ratio, because that's what I'm going to focus on.
But I am going to touch upon what we've described as an unmet need, which is listed as number one, social value is the way Doctors Emanuel, Wendler and Grady referred to it. But it's really the scientific and medical unmet need.
And I'm also going to discuss the interplay between unmet need, scientific validity, and the favor of how we understand risk benefit ratio, and how we see the risks, and what we think are reasonable risks in relation to the benefits within -- touching upon those two first attributes.
Now, Doctors Emanuel, Grady and Wendler added an additional attribute, which they called collaborative partnership, which they described in a different article, and that is not listed here. However, it is pertinent when we look at research that involves the exception from informed consent.
And I'll touch upon that very briefly later. So what are some important caveats to my talk and important messages? One is that before we start to even think about risk benefit ratio, we have to first satisfy conditions of social value and scientific validity.
In other words, we have to convince ourselves if there is a compelling unmet need, and that the protocols that we are designing have int