Question And Answer Session

SESSION CO-CHAIR MATALON: Any questions for Dr. Namboodiri? Michel?

DR. PHILIPPART: I have one comment on the triglycerides. Many years ago, I did an experiment by exposing cultured fibroblasts to various lipids, including triglycerides. You get very good uptake, but EM on these cells shows it's mostly in the form of droplets. So we get it in, and then probably it remains quite inert. So that may not be the whole story.

Of course, we certainly were not using -- I forgot what kind of triglyceride we were using. Definitely it was not Triacetin, as you used, which may have some advantage. But we used probably some medium chain triglyceride. We were pretty sure that's what it was.

And then we got something which sat within the fibroblasts as droplets. And, actually, we could load the whole fibroblast with an enormous amount of droplets, which were fairly inert.

DR. NAMBOODIRI: We measured acetate levels in the brain after Triacetin supplementation, and we found that the levels were increased significantly. So we know it is getting into brain. Our plan is to start early on, soon after birth. In our studies, we find that free acetate is going up in the liver and brain after Triacetin (glyceryl triacetate) administration, so we know that the glyceryl triacetate is not inert.

And the way we are giving it is intra-gastric. So we can do that without too much of a problem and start early on and see what happens. And if we succeed, then we can try to find out if there is a time window where we have to start the administration, beyond which it will not be effective.

All of this can be done in the CD mice the way we are now planning. This acetate source is inexpensive and readily available, and it's, as I said, quite nontoxic. So we are very interested and ready to go ahead with this.

SESSION CO-CHAIR MATALON: Dr. Ledeen?

DR. LEDEEN: Were you surprised that the brain took up as much as the liver of your Triacetin? That seems a little bit surprising.

DR. NAMBOODIRI: We were not really surprised because it is hydrophobic. In half an hour, it should reach every tissue in the body.

DR. LEDEEN: Okay.

DR. NAMBOODIRI: If you give glyceryl triacetate by a gastric route, most of it goes through the liver. So we thought that we would have to saturate the liver before it would get into the brain, but that is not what we are seeing. We see a similar pattern, both in the liver and the brain.

DR. LEDEEN: My next question is, did you check to see whether label from triacetin got into myelin lipids?

DR. NAMBOODIRI: We will look at that.

DR. LEDEEN: That is really a key question, I think.

DR. NAMBOODIRI: The Triacetin experiments were just started, and we haven't checked incorporation into myelin yet. And also so far we have been doing it for very short durations, you know, one hour, two hours. We will need to do a time course.

SESSION CO-CHAIR MATALON: Any question or comment?

PARTICIPANT: The purpose of using Triacetin was to get the acetate across the blood-brain barrier? When you give it orally, does it cross the gastrointestinal tract intact? I would think that most of it would be hydrolyzed in the intestinal lumen. So in order to see if it can enter the brain, you need to see that it can enter the blood intact. Do you have any evidence that the Triacetin gets across the gastrointestinal lumen intact?

DR. NAMBOODIRI: We don't know - when we looked at acetate at two-hours after Triacetin treatment, we do see dramatically increased acetate in the brain.

DR. GANAPATHY: But you are not comparing acetate with the tri-acetate yet. Do you not know the tri-acetate is better than acetate itself?

DR. NAMBOODIRI: We have tried calcium acetate, but you know, we were not getting good uptake in the brain. That is the reason we moved to Triacetin, as opposed to calcium acetate. With calcium acetate, we detected much lower increases in brain acetate levels at very high concentrations, when compared with glyceryl triacetate. So to that extent, Triacetin is more effective.

SESSION CO-CHAIR MATALON: Thank you. Dr. Leone?

DR. LEONE: Yes. I would like to make three points. And perhaps you can comment on it. I think what you are doing is very relevant. And following Dr. Kolodny's suggestion on administering calcium acetate or sodium acetate, depending upon the patients and their baseline physiological problems, we have been following something like, let's see, about 14 patients, looking at brain imaging, looking at clinical development during the years.

You could argue that, of course, you don't know if calcium acetate can deliver as much acetate as required to make myelin, but while we definitely reported just an improvement in ability, especially the young patients, younger than 12 months old, we never saw an increase in myelin looking at the MRIs and T-1 quantitative measures. This is with calcium acetate.

The second point that I have is in the tremor rat where the absolute total deletion of the aspartoacylase we don't even have any nonfunctional enzyme. We do have a little bit of NAA, but in the rat model, we do have myelin; much more than the patients. The patients have, even the four-month-olds have, just a severe total hypomyelination, perhaps one or two percent normal myelination. We can say that in the best patient, we have about two percent of the myelin at the same age the child would have.

And the third point about your hypothesis is that there was a report of a patient with zero NAA. And that patient based on this part, if this was so clear one way straight, then we would have zero myelin. The patient had a mild retardation. And the patient also had hypomyelination, not as severe, not even close to any of the Canavan patients.

So I think your hypothesis and Dr. Ledeen's hypothesis and your group may be very possible, but I think within the context of Canavan disease, we need to be able to look at other potential hypotheses. One of them is that NAA has the active role in toxicity, specifically during development. Dr. Burlina is going to present the case with a low NAA or zero NAA.

DR. NAMBOODIRI: Is the lack of myelin correlated with the severity of the disease?

DR. LEONE: The lack of myelin in our patients? The Canavan patients I follow are a very homogeneous group. They never develop to the point where they can walk, or talk, for example.

You see near zero myelin in the brain of some patients, although you have to know that some of the patients have up to approximately five percent of normal myelin levels in the brain. So perhaps a 5% increase in acetate in the brain should be able to increase myelin in the brain.

DR. BURLINA: We were following a patient, he was supplemented with acetate, and he was not improving at all after one year, we had an opportunity to perform some tests and we found that NAA was increased. So I think we have to be very careful with the acetate supplementation.

SESSION CO-CHAIR MATALON: I think the mouse would be a good idea to check safety of the compound.

Yes. Michel?

DR. PHILIPPART: You mentioned some discrepancy between the size of the enzymes, and I was wondering if you had considered the ratio of the difference between some of this data. Is it possible that one form is an apoenzyme which is then made into the active enzyme in oligodendrocytes

DR. NAMBOODIRI: With our antibody, we don't see a different size. We find the same size, so we don't know why there is the difference between the two findings, whether there is something else going on.

DR. PHILIPPART: Maybe the antibody is already transformed into something else before -

DR. NAMBOODIRI: No. When we do the same experiment with our antibody, with the cytosol, we find 37, but with the myelin fraction, we don't detect a band at all, and we also don't see any activity. So there are some differences between our results and Dr. Ledeen's results.

The problem we have is if it is high molecular weight, small covalent modifications cannot explain that. You may expect a change of 5,000, but not 20,000. So there is something else going on.

SESSION CO-CHAIR MATALON: Brian Ross had a comment.

DR. ROSS: I just have one comment to the question of acetate uptake in the brain, sodium acetate. We can see with radiolabeled acetate that acetate is taken up by the brain, and is metabolized quickly, we believe by the glia. Of course the down side is we can see incorporation into many other molecules in the brain is greater, so that the question is not really will the acetate will get into the bran, but will there be enough around for myelin synthesis after all the other processes it is involved in?

DR. NAMBOODIRI: Yes, that's always a major question because the oxidative path to CO2 is far more significant, and that could cause a problem in terms of acidity and all of that. Those are the questions that we need to look at in terms of toxicity.

SESSION CO-CHAIR MATALON: Ed Kolodny has a comment.

DR. KOLODNY: Perhaps I'll answer the question with regard to acetate in a patient population. When we began to use it years ago, it wasn't clear whether it would do any good, but parents encouraged us because we got very positive feedback with respect to their behavior; their alertness; the children's ability to move.

However, what has I think been a stumbling block in trying to carry out any kind of careful study is that you need clinical markers. In Canavan's disease, it brings them evoked potentials, MR spectroscopy for NAA, head circumference measurements. Perhaps these are some of the markers that could be used, but no study has really been done.

In our experience, the head circumference seems to level off. And the increasing growth of the head seems to be arrested with the use of calcium acetate.

But I would be the first to say that I'm not sure whether it's doing any good. We only have the parents' word. And most parents are looking to any solution as a positive one.

SESSION CO-CHAIR MATALON: I am not sure that the acetate will reduce the NAA level because it doesn't break it down. So that may not be really a good marker. And head size, that's a good one, but I have seen patients with Canavan. Their head does not grow forever.

DR. NAMBOODIRI: Our goal is not to reduce the NAA concentration, but to replace the acetate that was trapped in the NAA, and that was not being liberated.

SESSION CO-CHAIR MATALON: Thank you Dr. Namboodiri. And now we move to Dr. Kolodny's talk.

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