Based on the preclinical efficacy of GCP II inhibitors in models of stroke, ALS and peripheral neuropathy, a potent and selective GCP II inhibitor was chosen to be administered to 77 individuals in three Phase I clinical trials. In the first trial, doses of up to 1500 mg were administered to volunteers. Oral bioavailability of the drug, particularly in the fasted state, was very good. Plasma levels were achieved that were above those needed to produce effects in animal models of diabetic neuropathy and neuropathic pain. The compound was safe and well tolerated at all doses without any CNS effect (EEG, visual tracking, coordination, etc). Gastrointestinal complaints constituted the most common category of adverse event, with dyspepsia being most commonly reported.
In further trials, daily doses of 375 mg and 750 mg were administered for 14 consecutive days to healthy volunteers and to patients with diabetes. In normal volunteers, the safety and tolerability profile was similar to placebo at doses up to 750 mg in the fasted state. In diabetic patients, complaints of mild hypoglycemia were reported at the highest dose, but were not associated with documented laboratory changes. In neither group were there any apparent effects on glutamate sensitive parameters.
GCP II inhibition decreases extracellular excitotoxic glutamate and increases extracellular NAAG, both of which provide neuroprotection. We have demonstrated with our potent and selective GCP II inhibitors efficacy in models of stroke, ALS and neuropathic pain. GCP II inhibition may have significant potential benefits over existing glutamate-based neuroprotection strategies. The upstream mechanism seems selective for excitotoxic induced glutamate release, as GCP II inhibitors in normal animals induced no change in basal glutamate. This suggestion has recently been corroborated by Lieberman and coworkers24 who found that both NAAG release and increase in GCP II activity appear to be induced by electrical stimulation in crayfish nerve fibers and that subsequent NAAG hydrolysis to glutamate contributes, at least in part, to subsequent NMDA receptor activation. Interestingly, even at relatively high doses of compounds, GCP II inhibition did not appear to be associated with learning/memory deficits in animals. Additionally, quantitative neurophysiological testing data and visual analog scales for 'psychedelic effects' in Phase I single dose and repeat dose studies showed GCP II inhibition to be safe and well tolerated by both healthy volunteers and diabetic patients.
GCP II inhibition may represent a novel glutamate regulating strategy devoid of the side effects that have hampered the development of postsynaptic glutamate receptor antagonists.
9. QUESTION AND ANSWER SESSION
DR. MEYERHOFF: Questions? Go ahead.
PARTICIPANT: That was really very interesting stuff, especially with regard to ALS and stroke. I have interest in both areas. I am a clinician.
You administered -- well, first PMPA and then 2-PMPA in stroke and ALS, but in pre-symptomatic stages. So in stroke, I think you gave it before occlusion.
DR. TSUKAMOTO: Right.
PARTICIPANT: And in ALS you gave it at one month, and there really isn't any pathology at that point to that mouse. I think it comes at 90 days. So have you tried further to see if there are effects when the mice become symptomatic or post-occlusion in stroke?
DR. TSUKAMOTO: No, we haven't. One reason is, as you can see, each experiment takes almost a year; it is a very long, intensive study. So what we are interested in is how this drug worked compared to the existing drug. So we used the protocol they used for Rilusol, and we haven't done any modification in that protocol at this point. Yes.
PARTICIPANT: Just now we heard about two different types of GCP, II and III. Then we talked about it had to be knock-out mice, in which GCPII has been knocked out, but there is no symptomatic phenotype. So how specific is your inhibitor for GCPII versus GCPIII?
DR. TSUKAMOTO: I am not familiar with GCPIII, but what I think is -DR. WROBLEWSKA: It appears not GCPIII, we checked 2-PMPA -DR. TSUKAMOTO: Right. What I think is that our inhibitor probably has a good chance to inhibit both GCPII and GCPIII.
DR. WROBLEWSKA: 2-PMPA does it, for sure.
DR. TSUKAMOTO: Right. So in our case, we are working at the glutamate receptors II or III. Probably GCPII inhibitors will shut them both down. Yes?
PARTICIPANT: But you are calling GCPII NAALADase? Does this term refer to GCPII and III?
DR. TSUKAMOTO: Dr. Coyle?
DR. COYLE: Actually, you know, as we got into the 21st Century, I think that term was developed simply as an acronym for the action of the enzyme, and I think GCPII and GCPIII are the appropriate names now, or if you look at the gene, at least for GCPII it is also known as folate hydrolase I, FOH-I.
PARTICIPANT: But if you use this inhibitor, and if it is not specific for II or III, and if it works, what does it do to the folate, because the folate conjugates. It is very critical for the absorption of folic acid in the intestine.
DR. TSUKAMOTO: Yes.
PARTICIPANT: So if you do give this drug to the patients, are you risking the patient's health?
DR. TSUKAMOTO: That is a very good question. We had a collaboration with a researcher at California Davis -- what is his name at University of California?
DR. COYLE: Dr. Halstead.
DR. TSUKAMOTO: Yes, Halstead.
DR. COYLE: Halstead. He was the researcher we worked with.
DR. TSUKAMOTO: Yes. He actually was independently studying this folate hydrase, which turns out to be, as we have been discussing, the same enzyme. So we gave him our inhibitors and see effect of that on the folate and the methionine in metabolism. It didn't show any changes in that study.
DR. COYLE: Actually, the American diet is so fortified with folate, which does not have a polyglutamate tail on it, so that if you eat a normal diet, this would have absolutely no effect on your folate level.
PARTICIPANT: But the individuals who depend on dietary folate, which is predominantly polyglutamate, the inhibitor is going to have an effect.
DR. COYLE: Right. Right.
DR. MADHAVARAO: Would you like to comment on: Since the GCPII knock-out mice did not actually have any abnormalities, is there any actual role for this enzyme there? You know, it assumes that it is the glutamate released from NAAG is actually inducing all this toxicity or mediating the pain. So the pain related effects in the absence of such enzyme in the GCPII knock-out mice, would you not expect that such GCPII mediated pain responses should not be there? So would you like to comment on that?
DR. TSUKAMOTO: I'm not sure if I get your question right. But what Guilford has been focused the last couple of years is really -- we are really focused on reducing glutamate now, but after attending the conference and seeing all the effects that NAAG could have--
I am not really sure that the effect we see in efficacy is something to do with increasing NAAG or decreasing the glutamate. What we care most is that it works.
DR. NAMBOODIRI: I have two questions. Regarding the mechanics of action here, is it acting by preventing the formation of glutamate or is it preventing the release of glutamate by increasing the NAAG level?
DR. TSUKAMOTO: That is again the same type of question. We don't really know yet, but what microdialysis studies tell us is as far as stroke is concerned, we are able to suppress the extra concentration in the glutamate by treating with the GCPII inhibitors.
Now what I cannot prove is that all this glutamate really comes from NAAG hydrolysis. We don't really know that yet, but at least we are able to reduce glutamate, and it correlates with efficacy in our stroke studies. Yes.
DR. NAMBOODIRI: So it is generating glutamate from NAAG?
DR. TSUKAMOTO: Yes, but I cannot say that all the increases you see in stroke are all coming from NAAG hydrolysis by this enzyme? No. We don't have any proof yet.
DR. LIEBERMAN: One is a comment, and it goes to his question. That would be: It would be very interesting to see in the cerebral ligature model, the stroke model, of whether or not the knock-out mouse would give you the same results as the peptidase inhibition.
DR. TSUKAMOTO: Yes. We have done that.
DR. LIEBERMAN: Oh.
DR. TSUKAMOTO: Well, actually, I looked at those, but I am a chemist. Larry, can you comment on this?
DR. WILLIAMS: Using the Hudson knock-out that Barbara Wroblewska described, there is a new part that is smaller -DR. LIEBERMAN: The second question: What is your estimate of why you are getting hydrolysis of NAAG after some sort of an insult, or injury, versus you don't get hydrolysis under resting conditions? Have you infused NAAG into these areas which are showing the damage versus those that have no damage? Is it a substrate activated breakdown or is it an enzyme regulation effect?
DR. TSUKAMOTO: Right. So you are talking about what triggers NAAG peptidase activity in under various conditions? I don't think I am the right person to answer that question, but we have a couple of -DR. WROBLEWSKA: The peptidase works when it is activated by hydrolyzing NAAG, and the actions of NAAG are enhanced when the peptidase is inhibited.
DR. LIEBERMAN: If peptidase action blocks the mGluR by degrading NAAG -- it may be the NAAG that is there is more potent when the enzyme is inhibited.
DR. TSUKAMOTO: And one other factor that we cannot underestimate is the concentration of phosphate, of course, because this enzyme is very sensitive to phosphate concentration, and easily inhibited if you have more than 10 micromolar phosphate. So phosphate might be also another regulator of this enzyme.
DR. COYLE: One perhaps unappreciated fact: When you see that the rise in glutamate gets blocked with the PMPA, it kind of knocks your socks off, and you say, wait a second, what happened to glutamate release?
I should point out that, at least in the rat, the concentration of glutamate is about 3 millimolar, but if ten percent of it -- only ten percent of it is synaptic glutamate, that means it is about 200 micromolar, 300 micromolar -- concentration of NAAG in that region is 200 to 300 micromolar, which means that the NAAG contributes at least equally to the total extracellular burden of glutamate, when it is released and hydrolyzed.
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