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[00:00:51] Alan Whone: Thank you much, Sara. It's a great privilege to talk to you.
[00:00:55] Sara Schaefer: Can you walk me through the actual process of GDNF infusion in [00:01:00] patients? How involved is this process? How often is it generally done in studies? And what are the similarities and differences between this approach and other direct neurosurgical interventions that we may have heard about in the basal ganglia, like grafting?
[00:01:15] Alan Whone: So with trophic factors, what we're talking about are neuro-restorative approaches, the alternative method being cell-based therapies, for people with Parkinson's. And there's good laboratory evidence that pre-clinically in animal models you can achieve this. Although in the five double-blind studies looking with GDNF to date, none have reached their primary outcomes.
Now one of the issues with GDNF is it's a large protein, so it does not cross the blood brain barrier. So you have to take a surgical approach to delivering the protein, and that over time has iterated and been done in several different ways.[00:02:00]
So the first study, which was led by Jane Nutt and colleagues more than 10 years ago now, was an intraventricular approach. And what that showed, if you looked at some of the patients postmortem, was that none of the GDNF penetrated ventricle into the striatum. So then step forward, and there was a next study, which was initially open label and then went to double blind, where they used a intrastriatal approach. So in this situation, a cannula was inserted into each putamen which connected back to pumps in the abdomen. And on that occasion, the infusate, which contained GDNF, did reach the putamen. But it turned out looking at postmortem tissue with histological staining and also at PET scans where there was an [00:03:00] increase in fluorodopa, this was only occurring a few millimeters beyond the catheter tip. The problem was that when you infuse via a very slow rate, because you are giving something continuously otherwise you'll flood the target structure from a pump that is ever running, you can only go at such a slow flow rate that the drug only just diffuses out of the end, where GDNF is very sticky and it binds to heparin binding sites. So it probably wasn't covering the target region of interest. And one of the key things that pharma companies have in drug development, And their first question is are you getting the drug to the target? And the answer was yes, but nowhere near sufficient quantity of spatial distribution to cover the putamen.
And this was also then found to be the same problem with the nurturing studies, which was a GDNF analog that was [00:04:00] not delivered by pumps or catheters, but rather was a viral vector approach. And again, those studies turned out to be negative, and there were two of them. They were both negative again because the drug was given very slowly and probably only diffused out. So the virus was injected, but it didn't get distributed.
So then the next approach, which was our approach, was that rather than having a pump delivering continuously, the patients had a port implanted behind the ear. So this is a bit like the port in the Matrix, and to some extent is the stuff of science fiction.
So they would have a small port behind the ear, and you would penetrate the septum on an every four weeks basis. Externally, that was then connected up to an infusion pump. A bit like the infusion pumps that give chemotherapy, but to deliver much smaller volumes — microliter amounts of fluid. Those ports ran down catheters, where [00:05:00] two were implanted on each side, so two to each putamen. And then on an intermittent basis, you were able to deliver the infuse every four weeks. But because you were going intermittently rather than continuously, and therefore wouldn't flood the structure, you could go a much higher flow rate.
And so we could give 400 microliters into each putamen over about an hour and a half, which is much, much faster. And the physics of that is that you then get something called convection delivery. So rather than diffusion, the infusate moves out through the extracellular space with a convection pressure. And the hypothesis was that this would allow the drug to get to the target region — that all important thing — and it would cover the entire putamen structure.
And in terms of whether or not we achieved that, we did test infusions where we infused gadolinium down into the putamen structure. And what we saw was that the gadolinium was indeed [00:06:00] distributed nearly across the whole putamen volume. so it would seem that we could get this unprecedented coverage of the target structure. But what you don't know is whether GDNF distributes in the same way as gadolinium. But what we did see on the PET scans, and partly that's why they're important, was that there was apparent spreading of the dopamine terminals as evidenced by increased fluorodopa uptake, not just around the catheter tips, but throughout the putamen structure. So that would suggest that you've got target tissue engagement, which is the next thing the pharma companies want. One is to get the drug to the structure of interest, but the other thing is they want to know all about target tissue engagement.
That would suggest you've got target tissue engagement of the GDNF receptor sufficient to have a biological effect, and therefore you had distributed the GDNF. So we think we got the drug there, and we think we engaged the target tissue.
And indeed the [00:07:00] same thing was seen in the study led by Chris Bankovich, which was also reported in Movement Disorders in 2019, which used, again, a viral vector approach, But again, that more of a one-shot approach where you don't have to give it repeatedly cause it's viral vector expression. again, that showed that the virus appeared to have been distributed with the GDNF being given off across the putamen structure, where they injected that virus different from the nurturing study at a flow rate to allow a convection delivery.
So it would seem that both for direct infusion of the protein and viral vector approach, there are means by which you can get the drug to the target tissue with a spatial extent that may then be sufficient to affect a biological outcome.
[00:07:48] Sara Schaefer: Got it. Well, so you already alluded to this FDOPA uptake that you guys looked at in the putamens of these patients after receiving GDNF [00:08:00] infusions. And one interesting thing about this study that you talked about in your introduction is that patients did not improve in terms of off-state UPDRS motor scores, but did increase putamenal FDOPA uptake on scans.
And there's a disconnect there. And you discuss the differential diagnosis, if you will, for this, for why there might be this disconnect. Can you talk a little bit about that?
[00:08:27] Alan Whone: So one of the things to make clear to the audience upfront is that we did not meet any of our secondary outcomes and we didn't meet the primary outcome for the GDNF study that was reported in brain in 2019 where all of those outcomes were clinical benefit over placebo.
And so this was indeed a negative study. And the question is, why was that negative despite that the PET scans were positive? And of course [00:09:00] the important thing is that you improve patients, not scans. And there are a number of potential explanations. So I think that the PET scans are important for the reasons I outlined in my first answer, which is they confirm that the drug reached the target tissue of interest and had a sufficient spatial distribution to fill the putamen volume.
And also that you got target tissue engagement across that volume sufficient to illicit a biological response, which in that case, one could interpret as sprouting of the terminal plexus.
And that potentially is important because there's some potential evidence that in toxic animal models of Parkinson's, GDNF has an effect, possibly not in synuclein animal models. But in fact, this would suggest that in the situation of synuclein, GDNF could have [00:10:00] an effect.
But what you don't know is whether the sprouting of those terminals produced a change in functional pharmacology. So all you are seeing with fluorodopa is that you've got increased binding because you've got increased uptake into the terminal that then binds to the enzyme aromatic amino acid decarboxylase ,where the amount of binding is in part reflective of the density of the terminal plexus.
So, what you might be able to infer is that your terminal plexus is sprouted. But what you cannot infer from the PET scans is that in turn, those sprouted dopamine terminals can lead to restoration of dopamine release.
So in other words, you might have got sprouted terminals, but have no more endogenous dopamine available in the synaptic cleft. And that might therefore explain why the PET scans were positive, but the benefits over placebo [00:11:00] were not seen in the clinical scales.
Other potential explanations may be that the change on PET scans occurs earlier than the changing clinical benefit. And you might consider that possible in the sense that you could get a change at the terminal where the trophic factor was infused to before you get sufficient regeneration the nigrastriatal pathway. So there might be a time disconnect between those two events.
And another potential explanation is the power of scans needed,, in terms of the number of scans needed to see a difference in fluorodopa is probably significantly lower than what you might need to see a clinical benefit, given the inherent noise in test-retest in scales like the UPDRS, for example.
[00:11:50] Sara Schaefer: And this brings us to the current study and the topic of your actual paper, which it seems stemmed from a rather [00:12:00] happenstance observation of your previous study population. Explain how this came about and what you did to explore the issue further.
[00:12:08] Alan Whone: When we did the original study I, as the chief investigator, was kept well away from the study results, which were being done by blinded raters. And this includes that I was kept away from the videos of the participants where the infusions were given every four weeks and videos were recorded every eight weeks. But I didn't see those videos. So although I may have got an impression as to whether individuals were changing over time because I was dealing with them clinically, I didn't know what their practically defined off-state UPDRS appearance was in those videos.
After the study finished, I wanted to help develop less subjective outcome measures. I teamed up with a group of computational scientists at the University of Bristol to develop algorithms for computational modeling to interpret [00:13:00] UPDRS scores with use of the video, which is then less subjective. Of course this isn't unique, although there are some approaches that we are using that may be novel. So this led me to go back to these historic videos and start reviewing them.
And it just so happened that by chance, the first participant video I looked at was the individual whose video is shown in series accompanying the paper and Movement Disorders. And what you see in those videos is that at baseline, the participant in the practically defined off-state looks pretty profoundly off, as you'd imagine. And when in the supramaximal on state, they have the type of peripheral dyskinesias that you see in a supramaximal on. Then over time, the patient is re-videoed. The double blind component of the study ended at week 40. At week 56, the patient begins [00:14:00] demonstrate mild, practically defined off-state dyskinesias.
Now we've got a very large DBS program here in Bristol, one of the biggest in the uk. So I see patients quite often in the practically defined off state, for those who are less familiar, where they've stopped L DOPA overnight and long-acting agents for 24 hours. And of course, in that state, that isn't a sufficient washout period for them to lose all the benefit of L dopa in terms of stiffness and slowness, although they look pretty off. But it certainly is long enough to expect them not to show the type of dyskinetic movement you see peak-dose dyskinesia. That would be very, very unusual.
So, my body jumped up when I saw this lady beginning to show chorea form dyskinesia months of GDNF. I thought, well, that's a bit odd. And then as the video series goes on, they get worse until they reach a plateau point. And at the point of the plateau, the practically defined off-state dyskinesia is [00:15:00] as severe as the super maximal on-state dyskinesia.
Now over the video, the most apparent section where this was seen was when the patient was performing the two point tapping test from the capsid, which is a assessment to look at surgical trials in Parkinson's. And what you see is the patient gets faster tapping between those two points over time. But if you look at our whole study data, you see that there's a very big placebo response on that. So if what you were seeing in the videos was simply that she got faster over time, you couldn't know whether it was a drug effect or a placebo . But that she developed practically defined off-state dyskinesia would be more difficult to put down to a placebo effect, not least because it would unanticipated by the participant.
And you can expect there to be several reasons why people might improve without having received [00:16:00] study drug on tapping between two points — they start competing with themselves, there is the placebo response, and also there is a practice effect and they're more used to being off medication overnight and so on.
But developing dyskinesia in the practically defined off-state seemed unusual. Now, in this particular subject, it so happened they had some further videos after the drug was stopped because we got to the end of the study, which wasn't part of the study protocol. But what happened was the patient, two years later, came back because she'd deteriorated post the end of the study and the cessation of GDNF. She came back for an assessment for DBS where we video all our patients using the same studio studio and video protocol. in that two years you see that she still had practically defined off-state dyskinesia, but they were much more mild. And she didn't go forward for DBS then, but she did a year later. And what you can see in her practically defined, off-drug, off- stimulation state three years after [00:17:00] GDNF, not only does she look much stiffer and much slower than she did at the end of the open phase GDNF study, which was after 80 weeks but the practically defined off-state dyskinesia is resolved.
So this really then looks like a dose response curve. So you see no practically defined off-state dyskinesia for 40 weeks. Then at week 56, it begins to come in. It gets progressively worse until the end of the double blind period, and then it begins to decline, and then stop.
Now of course, dyskinesia is a side effect, not a benefit.
So you can't say this is a benefit of GDNF. But it occurred to me that this might be some tentative evidence, and there are potential other explanations, that the sprouted terminals that we'd seen through the PET scans, at least in some of the patients showed this phenomena were indeed able to produce dopamine and release it. Because they shouldn't have had, to any great extent, of [00:18:00] dopamine around, having stopped exogenous dopamine. So by observing this side effect, you may begin to develop some evidence that you'd restored a functional pharmacology — One of the other key things the pharma companies look for in drug development.
So they look to check the drug gets to the target tissue; they look to check that you've got target tissue engagement; and then they look to check that you've had a functional pharmacological response in addition to looking for clinical benefit. So this is an important gap in the knowledge we've have so far because although trophic factor studies to date have shown improvement in scans and not shown improvement in clinical outcomes against placebo, none of the prior ones have shown evidence that you may be achieving an improvement in functional pharmacology.
And the interesting thing about these patients is that they were 10 years since symptom onset of their Parkinson's. And from the Jeff Kordower of a paper in Brain, there was a question about whether all the terminals would've been gone [00:19:00] by that stage. But this would suggest there's enough there, not only to see sprouting of terminals as evidence by the PET, but also potentially that they could be regenerated to release dopamine. Although with all the caveats, be very cautious about this, because this is a post-hoc, and there are potentially other explanations which we may come to, for why we saw this phenomena.
So having observed that, we then went back and in a blinded way, reviewed all the videos for all the participants, and looked at how often this occurred, and when it occurred, and how it evolved over time.
[00:19:35] Sara Schaefer: And I'll just give a quick summary of my understanding of what you found, which is that there were other patients like this in your sample, and there were also patients in the placebo group who started developing off-state dyskinesia, which, there are a lot of potential explanations for that that you go into in detail in your paper.
I wonder what you think is the most likely [00:20:00] explanation for the off-state dyskinesia that developed in the placebo group.
[00:20:04] Alan Whone: So a couple of things to say: the number of participants very low. It was 41. 21 in the active group and 20 in the placebo group. And what we did see in the GDNF group was that very few of the assessments in the first 40 weeks showed practically defined off-state dyskinesia. But close to under half of them showed practically defined off-state dyskinesia in some of their assessments in the second 40 weeks.
And, Modeling with generalized estimated equation approach suggested that your risk of developing practically defined off-state dyskinesia increased dependent upon the number of infusions you'd had. So there did seem to be some change over time.
In the placebo participants, what we saw was that, again, some of them developed dyskinesias in[00:21:00] the second half of the study — which was an open label extension where the participants had active drug or placebo for the first 40 weeks, and then everyone had active for the second 40 weeks — that in the second 40 weeks where the former placebo patients began to have GDNF, some of them showed this phenomena.
A couple of them had shown this phenomena in the first 40 weeks, which is difficult to immediately understand, given that I've given some lengthy explanation that we tend not to see this in our DBS population. And the potential explanations for that are, one, if you're looking at something like the finger tap test or amongst other motor outcomes, it can be difficult to decide what is a transmitted movement, you know, with your head bombing around as you try and fly your hand between two points. What is a transmitted movement versus what is a dyskinetic movement? So it's possible that at the more mild end, [00:22:00] patients were over called as having practically defined off-state dyskinesia.
But other reasons are, if you cause tissue trauma, you can induce endogenous GDNF release. So it's possible that the placebo patients who were having artificial CSF infused had some tissue disruption, which in itself caused trophic factor release. And that, that explains it. And it's perhaps noteworthy that the GDNF patients who did develop practically defined off-state dyskinesia in the first 40 weeks did in some cases do that before week 36. But none of the placebo patients who developed this — and there are only a couple — did so before the 36 week time point.
And then there are some other explanations we postulated in the paper, one of which was some placebo response, which given the placebo response is felt to be related to anticipated benefit, that seems less likely , because people on the [00:23:00] whole wouldn't anticipate their development and then start mimicking d yskinesia. But nevertheless, it's possible. And the other was that there was an insufficient washout. But these were dyskinesias that appeared very much like peak dose dyskinesia, which have a somewhat different phenomena from biphasic dyskinesia occurring at the end of dose. Maybe that was an explanation, but it would be unusual.
[00:23:25] Sara Schaefer: I personally found the local trauma from infusion causing endogenous GDNF release most compelling. Do you have a favorite among the placebo postulations?
[00:23:40] Alan Whone: Yeah, I guess it's that one. The only thing I would say is that the PET scans in the placebo group did not change between baseline and 40 weeks, whereas there was a very marked change taking what looked like advanced-level [00:24:00] Parkinson's to early-stage Parkinson's PET in the GDNF group. So you'd then have to say, well, if tissue disruption caused GDNF release, why didn't you see benefit in the placebo patients on their PET scans, which we didn't see. We can't rule out, it didn't occur in some of them, because obviously the PET scan outcomes are group effects rather than individual effects.
[00:24:20] Sara Schaefer: Good point. So how did you interpret the results of this analysis with respect to what may be actually going on at the cellular and synaptic level in the brain of these patients? And kind of a related question, what is the scientific understanding of the actual underlying pathophysiology of dyskinesias in Parkinson's disease — dopamine, serotonin, both, other things?
[00:24:46] Alan Whone: Perhaps one thing for the audience to appreciate is that fluorodopa, unlike DAT imaging where the DAT ligund binds to the dopamine, fluorodopa binds to the enzyme AADC, which in itself [00:25:00] present also in adrenergic neurons and serotonergic neuron terminals. So therefore, fluorodopa will, in addition to labeling dopamine terminals, label serotonin terminals. There is relatively sparse innovation of the putamen by serotonin. And we also know that GDNF causes, least in the laboratory, change
in serotonergic neurons. So it is possible that what our PET scans show is not due to regeneration of dopamine nerve endings, but sprouting of serotonergic nerve endings. And you'd have to do experiment again with a DAT transporter to know that it was only the dopamine terminals that were sprouted or not entirely serotonin terminals. So that's one possibility. And if that were the case, it may also be that you then get increased serotonin release.
One hypothesis for why the fetal graft participants [00:26:00] the study reported in the early two thousands by the New York group showed so-called runaway dyskinesia, where those people had to be removed from medication after, and some of them went on, if I recall, to DBS because they had such profound dyskinesia coming from the fetal grafts where what was then found was that the grafted tissue in that American study, contained a significant serotonergic component, whereas I understand it, the Swedish group participants in separate study had a lot less serotonergic cells as part of their grafts implanted, and they didn't see this phenomena. So it may be that the reason we've seen practically defined off state dyskinesia is to do with serotonergic sprouting and serotonin release. But that to my mind seems perhaps an overblown explanation in the sense that the innovation of the putamen by serotonergic neurons is [00:27:00] actually sparse and it's not universally accepted that serotonin is that involved in the evolution of dyskinesia. And I think there is other evidence that there are other reasons why those grafted patients showed runaway dyskinesia — reasons other than the serotonergic component. So, It's something we consider in the paper and it's important and there are many caveats to this paper, and I would say it's tentative evidence of a restoration and functional pharmacology, but perhaps the best, tentative we have. But it has to be taken along with all these caveats that I've described.
[00:27:37] Sara Schaefer: I suppose the naysayers may say that if there's increased serotonin happening because of GDNF causing dyskinesias, and not dopamine, that that may explain why UPDRS motor scores didn't improve in these patients. But , I certainly would leave it to you to tell us what you think is more [00:28:00] likely
[00:28:01] Alan Whone: Well, and I wouldn't be as bold as to use the term naysayers, but certainly this is a field that is frank with contention. And if I recall, when the double-blind study in the early two thousands stopped early by the company that was sponsoring that, which was Amgen. Participants were taking Amgen to the Supreme Court, the US Supreme Court.
So there's a lot of contention in this field. And there are people who think it works and there are people who think it doesn't work and there's perhaps some dogma. I try and have equipose and keep a very open mind. I certainly don't know if it does or not. But I think there is some evidence that it may do. And if you believe restoration is important, it may be worth keeping going, recognizing that many times drug development doesn't occur in one go. Of course can remember the first studies of LDOPA were negative, but that seems to have been [00:29:00] quite a good drug. But it may be worth going and building this to decide whether it's worth taking things forward and accept that it's an iterative process.
[00:29:12] Sara Schaefer: So this nicely segues into my next question, which is: providers and patients alike may look at all of these studies with failed primary and secondary clinical outcomes and be disheartened about the future of the use of GDNF infusion for Parkinson's disease and potential disease modification in these patients.
At this point, what are your thoughts about the future of this procedure and how have your findings in this study impacted your opinion?
[00:29:44] Alan Whone: So in the end, I guess this is the key question. So, so what and what next?
[00:29:48] Sara Schaefer: Right, exactly. So what, and what next?
[00:29:51] Alan Whone: I guess the first question to ask yourself is, is disease modification worth it, given advances in DBS and so on, and better symptom [00:30:00] control? And I think most movement disorder physicians would say it's definitely worth it because we know what happens to people with time and our own DBS patients do great for some years, and then we're back to square one. So disease modification would appear to be worth doing.
So the next question is, it worth all the complexity of restoration where the restorative therapies currently are all neurosurgically applied, given that neuroprotection may be much easier achieved with repurposed drugs? And to my view, the answer is yes because it will appear likely from the regulators that to get a license for neuroprotection, you'd have to see a slowing of approximately 30%, but that does mean those people will still get worse. I think very unlikely the protective agent is going to be halting the progression of Parkinson's. And of course for our patients who are young onset, who appear in clinic thirties and forties while slowing of 30% is [00:31:00] good, it's not gonna be good enough, and they are going to get worse. and it may be to achieve that, you need to be able to give both restorative and protective therapies and in the end it's likely to be a polypharmacy approach if you really want to hold Parkinson's. So I think it probably is still worth looking at restorative therapies despite the problems that it's surgically applied and also despite the problems that it's slightly only to improve motor difficulties at the current time, because you're only targeting the striatum and the rest of the brain's falling apart.
Nevertheless, if you believe it's worth carrying on with restorative therapies, then you've gotta think, is that worth doing and would you go for growth factors or cells? Now the problem with cells are of course, even if you're using IPS you, are still not affecting the innate network. You're putting something in. So it might be that if you could sprout or regenerate your own actual neurons in situ, and their networks, that will be a better way of doing it [00:32:00] themselves. And presently it would appear that trophic factors are way of doing that
[00:32:05] Sara Schaefer: Could you just quickly say what IPS stands for?
[00:32:08] Alan Whone: Induced pluripotent stem cells, so cells that are not taken from fetal tissue, and therefore have less problems with immunogenicity and so on. Both fields are advancing all the time just as symptomatic therapies are advancing. But potentially, trophic factors look like, if it can be achieved, a good way forward.
But it's how on earth do you get it from that laboratory finding to achieving a clinical benefit in patients in clinic? And so I think what the studies to date show are, well, at least you can get the drug there. And it would appear even in advanced Parkinson's, you can get target tissue engagement. And you may be able to get a biological response as evidenced by PET scans. And you may also be able to achieve restoration and functional pharmacology. But given you didn't see [00:33:00] practically defined off-state dyskinesia in the main occurring until after 40 weeks of time, it's very likely that our double-blind study simply wasn't long enough and you probably need to follow people for more than nine months and possibly out to two years. And that's not that surprising when you think about how long it takes for regenerated axons to grow. So in addition to thinking about the science of that, there's a lot to think about clinical trial design and outcome measures to bring these things through. But nevertheless, I think it is worth keeping going.
[00:33:33] Sara Schaefer: So the takeaway I'm getting from you is you maintain optimism that we're getting closer, getting more information, being able to fine tune our studies and our techniques to get better benefit, and ultimately this may prove to be something that's useful in these patients.
[00:33:57] Alan Whone: Yes. And I think it's very important to remain [00:34:00] optimistic. Another question is whether future delivery approaches should be with ports behind the ear, I think is much more debatable. That might just be simply too cumbersome. I think it is important to remain optimistic and not necessarily close everything down. And I guess that comes back to keeping an open mind and just thinking about what's there.
[00:34:20] Sara Schaefer: Well, thank you for this really enlightening conversation about GDNF. I learned a ton from you, and I hope our listeners did too. Thank you for your time.
[00:34:31] Alan Whone: Thank you very much, Sarah.