Chapters Transcript Video Niemann-Pick Disease: Detection Clues and News of a Promising Therapy Hi. And then here's Doctor Visy also to introduce, um, Doctor Hastings and then we can, um, go ahead and get started. Right. That won't take long. Um, I've known Caroline Hastings since she was a resident. I must say there is very few. If she's not the only person I know who can be exceptional in any field. She endeavors from starting, um, NeuroOncology to metabolic diseases, to oncology, to hematology, to fellowship, to becoming the direct that chair, uh, um, the head of the American Society of Pediatric Hematology oncology, running the division, um, in terms of all the scheduling and contracting and, and publishing. Uh, uh, you know, the thing I admire most about Caroline and she has an impressive CV is, but mostly is her emotional and cognitive commitment to make sure every patient, um, can get every, every bit of care, including emotional support that they need. And then she gravitates to those patients who really, um, need that and she goes all out. Uh, um, I must say that, um, she never, uh, uh, exaggerates or, or, or, or doesn't tell the truth except maybe when she, uh, um, wrote an editorial about my accomplishments. Maybe that's the only time I think her interest in metabolic diseases and, and these lysosomal illnesses stemmed from her emotional engagement in families who had such a tragic um course without any opportunity for new, for therapeutic advances and every anyone else would just be engaged and support care. But she took this on and, and, and uh led a um a project to change the disease and intervene with uh therapy leading to an FDA uh studies to alter the disease. Um And which was um done in large part initially here at children's groundbreaking work. Um in contrast to many people who have accomplished as much as she has, she doesn't really talk about it, but she's really a giant at children's. And um I think we're in the institution. You see, and the bear is so lucky to have her. She represents really in my mind, the modeled physician, the scientist, the clinician and the caretaker healer of the patient. Uh So, um II, I am um honored to even be associated with this talk in her. So um I could go on, but I'll stop here. You got the gist of what I think of Caroline. Thank you Elliott. And um good morning to everybody. I really appreciate the opportunity to address this audience this morning. So today I'm gonna tell you a story of how a quest for intervention has put Nieman pick type C or now known as N PC at the forefront of rare disease and and quite frankly, of my academic life. And I can say when this journey started, I really had no idea where it would lead me and to abbreviate the story to fit in this time, I will intentionally be leaving out most of the details. So here are my disclosures relevant to N PC. I serve as a principal investigator for a number of clinical intervention trials and am on several um family foundations and uh scientific advisory boards and also a number of registries. So these are my learning outcomes. And at the conclusion, I hope that you'll be able to describe clinical symptoms and findings that should lead to suspicion for an underlying lysosomal storage disease. Describe an understanding of the mechanism of action of the compound. I'm going to tell you about recognize that benefits as well as toxicities of a drug intervention may be species specific and um may differ due to route dose frequency of administration and then as well to understand future challenges in optimizing intervention. So the story actually begins in 2007 while I was seeing identical toddler twin girls at a satellite clinic for assessment of identically enlarged spleens. And this quickly led to a diagnosis of a rapidly progressive and fatal disease. And shortly after the grim diagnosis, the family asked me to partner with them to learn more about the disease, maybe find some novel interventions and offer their Children really for science, but of course, hoping that they could benefit and quite fortuitously, we heard um about a surprising discovery in a lab at UT Southwestern of a compound that extended life span in N PC mice. So we quickly put together a team to begin to test this compound as a potential repurposed agent for the girls. And as a result of this partnership, we were able to develop the first compound with efficacy in humans. With M PC. We obtained orphan drug designation and our studies and data led to subsequent NIH clinical trials in national and international pharmaceutical trials. And many stories of our partnerships and discoveries have been featured um through the media to increase awareness and this includes on CNN PBS, uh Good Morning America and the Wall Street Journal. Uh It led to the development of an international collaborative effort to bring together a data on our original protocol which we published and this provided supportive data for clinical intervention trials. And then 15 years later, we were able to publish our first phase one study with a second um uh paper currently in submission. So first, I want to back up and talk a little bit about rare disease. Um It's defined as a disease that affects fewer than 200,000 people. And other examples include sickle cell disease and pediatric cancer. DNA H has defined um has identified sorry about 7000 rare diseases. The majority of which are genetic in origin. And the rare, they are hugely impactful with over 30 million Americans being affected, many conditions are serious and life threatening. And due to lack of awareness, challenge exists in research and also in developing therapeutics. Um So in 1983 the Orphan Drug Act was passed uh which gave incentives to researchers and pharmaceuticals in particular, uh which included, for example, tax advantages and exclusivity for a long period of time and also special pricing. And over the past decade, there's been a renewed interest in pursuing therapies um for rare disease. So the li is almost storage diseases are really a prime example of um genetic rare disease. And many are inherited in an autosomal recessive manner. And they're characterized by lysosomal dysfunction which leads to substrate accumulation affecting uh preferentially certain populations. And as you can see, um these include, for example, Neiman pixie acid Stal Mya deficiency gauche and a also the mucoid doses and gangs. So the index cases uh were identical twins and as I mentioned, they were found to have enlarged spleens at approximately 25 months of age, which was detected on a routine physical by their primary care physician. They were otherwise well appearing and they were not thought to have any other findings. I saw them when they were uh 2.5 years of age, a few months later, after having been assessed elsewhere by another pediatric hematologist for possible blood diseases, cancer, infectious ideologies. And that work up was negative. At my visit. They had a Pasco Megale, mild pancytopenia, mild ataxia. Although they were walking and language delay, they didn't engage with toys or with one another, which of course was a major clue that they were developmentally delayed. So when considering a differential diagnosis for splenomegaly, certain findings really should consider us to think about an inborn era of metabolism and some manifestations that are um rather unique to these diseases include bone changes or bone pain, failure to thrive a cherry red spot on ophthalmologic evaluation, unusual facial features. G I symptoms such as chronic diarrhea and very importantly as with our patients developmental regression or neurologic symptoms are a very important clue and should lead to evaluation for a metabolic disease. So, a diagnosis was achieved in our patients. Um by then, what was considered a gold standard uh for pneumonic C, which is a Phillipine test done on a skin biopsy. And this measures the um un aeri cholesterol that is trapped in the lysosome and it is quantified. And our patients then had confirmatory genetic testing which identified two variants on the N PC one gene which and one of which was known at the time to be pathogenic. So a little bit about Neiman Pixie or N PC. It's an autosomal recessive lycos storage disease that was recognized in the 19 nineties as being somewhat unique and distinct from a number of other lycos diseases. It's primarily a cholesterol based disease and it's involved with trafficking or moving cholesterol and lipids um out of the lysosomes, uh which differs from a number of the L SDS which are really related to dysfunctional deficient enzymes. The incidence is approximately 100 and 20,000 to 100 and 50,000 live births. However, this is likely an underestimate because of uh delayed diagnoses and misdiagnoses. Um It, it is uh a disease that affects the function of the N PC proteins. And there's actually two N PC one and N PC two that work in a concerted manner to uh move cholesterol out of cells. So most patients have spleen, Malene at diagnosis and often there is a long um lasting history of splenomegaly and patients may have had an evaluation that was been aborted uh because no ideology could be determined. And interestingly, the spleen doesn't continue to enlarge over their life and may appear to decrease as body size. It increases. The patients do have a number of characteristic neurologic findings um which I'll talk about but may also have psychiatric symptoms. Uh importantly, all cells of the body are affected. Yet the major impact clinically appears to be in the neurologic sys uh system which could be related to the unique susceptibility of cells of the nervous system. So M PC is a debilitating disease often with fatal outcomes in childhood and young adult years. And I I put in often here because I feel this um dialogue has changed as we are now identifying many more adults in um in their fifties, sixties, even seventies that have the disease and and clearly, um they're not gonna die as a result of, of having N PC. So there is no genotype phenotype correlation. Um and interestingly, siblings with the same genotype um often have a a different course of the disease with variability in the age of onset clinical manifestations, the rate of progression as well as the severity. But very importantly, there's no curative therapy for M PC and there are no uh FDA approved disease modifying treatments. So, as mentioned, affected patients typically um inherit this in an autosomal recessive manner and have two pathogenic mutations. 95% of these occur in the N PC one protein, another 4 to 5% on the N PC two protein. And to date, there have been over 500 different mutations reported for N PC. One. Uh there are uh the incident sorry of the disease is similar across the world, as mentioned, it is um pan ethnic. However, um there are some higher gene frequencies due to local founder effects and this um the veteran groups in Israel are particularly interesting because they harbor a number of unusual um mutations. And when inherited in a homozygous manner, a number of them have severe lung disease, which is extremely rare in N PC. And also interestingly, many of these patients are diagnosed at birth and tested due to their surname. So what do we know about the N PC one protein? Um It resides in the late end Zoe um lysosome compartment of the cell and interacts with N PC two protein. It binds um glyco lipids and cholesterol and acts as a shuttle to move them to, to the cell membrane and out into the cytosol where they're um utilized. So it plays a very important role in um the homeostasis of cholesterol and the precise mechanism of how this interacts. Um and the transport is really still being worked out. But we know that dysfunction leads to cellular accumulation of these lipids and early cell death. So, why is this important? Um Well, cholesterol is essential for life. Uh It is required to build and maintain cell membranes. It's used for synthesis and steroid hormones and bile acids. Um Most mammalian cells can make the cholesterol they need. And we have two main sources. One is um uptake of cholesterol carried on plasma like proteins. And the other is from DeNovo cholesterol synthesis from acetyl coa. So, the cholesterol pool is tightly regulated by sensing mechanisms and through these feedback inhibitions when cells sense a cholesterol deficiency synthesis is increased and when they um detect excess synthesis is decreased. So N PC um has variable clinical features in the patients. It's classically a neuro visceral disease, meaning it affects the neurologic system as well as the visceral organs, primarily the liver spleen and then rarely the lungs, it's extremely heterogeneous with respect to age of onset, the constellation of these signs and symptoms, the rate of progression. Um and also the timing of parent onset. And when we do diagnose patients, they can frequently go back 5, 10 years depending on how old the patient is and uh realize that they've been having symptoms for quite a while. So, some of these symptoms like in our patient include uh developmental delay and loss of milestones. Sarria or dysphagia, impaired motor function, particularly affecting ambulation. Um when patients may present with ataxia, as well as fine motor dysfunction and development of dystonia behavioral disturbances, including aggressive behavior, loss of cognition, and um early uh or short term memory loss and difficulty learning, uh seizures. And um interestingly, one of the unique features um in N PC, although it can be seen in some other disorders as well is cataplexy in which patients can appear as a rag doll with sudden complete loss of motor control following some emotional stimulus such as laughing or auditory stimulus such as crackling leaves. Interestingly, our patients uh displayed cataplexy and um which was something that eventually evolved. And when their families would put on these um Disney videos that had some funny scenes right before the funny scenes, the girls knew it was coming, they would start laughing and then they, they would fall over with these Cataplex episodes. So they're really amazing to see another um somewhat unique feature is vertical, vertical supernuclear gaze palsy. It's um sometimes not detected by the patients or families, those family uh as they haven't noticed that the patient has learned to accommodate with moving their head up and down as they cannot move the eyes to look up and down. And then later, they may actually develop um horizontal gaze palsy and acuity is not affected. Hearing loss may also occur in a number of patients. So, as mentioned, um systemic um findings can include prolonged neonatal jaundice, um Cappio magali liver disease, uh splenomegaly and respiratory dysfunction. Psych psychiatric manifestations are more commonly seen in our adult population and can be anxiety, um schizophrenia, bipolar or even frank uh psychosis and in combination, these um clinical manifestations affect the patient in a different way, but all have a significant negative impact on quality of life and length of life and need for lifelong care and support. And so when we see an alignment of several of these different types of signs and symptoms, then we should be thinking about um inborn errors of metabolism. So this is a popular slide amongst uh M PC clinicians given the heterogeneity of the disease. N PC has been categorized by age of onset, starting with the neonatal, early infantile, late infantile juvenile and adult. But it really is a spectrum and most M PC patients will develop a majority of these symptoms. Um But as mentioned, the timing and severity can be different even within family members, the visceral symptoms predominate early in life and in infants with unexplained polys stasis or NBC should be part of that differential. About 10% of patients can uh progress to finate liver failure. And some liver transplants have have even been done prior to the known uh diagnosis. And the remainder of patients, 90% do tend to recover from the liver disease and then go on to develop other um neurologic manifestations. Early dementia is something that's um reported in N PC. And it's often been called the childhood Alzheimer's disease. And in fact, our family made a youtube video that they put out to hopefully increase awareness and talk about this analogy. So um interestingly older patients um may have completed schooling and may have worked even had professions um and families, but in retrospect, they may have a history of clumsiness, forgetfulness um may be needing assistance at school and compared to their siblings, um not as good a student or an athlete, for example. So the mean age of diagnosis of N PC in Children is at 10 years of age and the mean life expectancy is around 16. But we think this has really been changing um since this report came out um due to earlier diagnosis and improved supportive care, and more than 50% of patients will present by 10 years of age, but some won't have symptoms until the sixth or seventh decade of life. And I do believe I likely have the oldest patient um with N PC who's currently 72 years old. So, research and um and um N PC was um really made possible due to our animal models. So, animal models with naturally occurring in PC mutations are in the cat and the mouse. And they have certainly uh led to um an understanding of uh mechanisms of disease and impact of possible new agents. These mice actually resembled the earlier forms of uh onset of disease in humans. So the some of the most severe forms, additionally, mouse models are being made um with unique human mutations. So we can use these to uh test modifying um interventions. Interestingly, there's a cat colony of the lysosomal storage disease in mucopolysaccharidosis at U Penn and I've had the fortune to visit them. And it was really not difficult at all to determine which kid had which disease. Um the physical similarities as well as the neurologic manifestations um compared to affected humans is is really uncanny. So, back to our story in 2007 and the challenges we faced at that time um based on an extensive literature search um on the manifestations, diagnosis and prognosis. Um because I had no prior knowledge or experience of this disease, it was clear that they had early onset um severe form of the disease. Their life expectancy was likely to be 5 to 10 years of age. And um even though labs were conducting research and had a very significant interest in how the N PC proteins were involved with cholesterol and glycolipid lipid transport. Few were actually testing um interventions and labs were also very competitive and un collaborative. And so all of this didn't line up well for us with our timeline to try to make an impact on the girls lives and also to learn from them. So the path to discovery of the important role of hydroxypropyl be the Cyclodextrin or what we now refer to as Cyclodextrin has often been described as serendipitous because of the timing. However, it's really an over oversimplification of the work of innumerable individuals that were involved um in basic science and, and clinical research. So we were fortunate that an interesting finding at UT Southwest by post doc was, was at the same time of our diagnosis and he was replicating an experiment testing a neuro steroid coupled with an excipient, which is what hydroxyl propal beta Cyclodextrin is. And he saw um increased longevity in mice. And when he did a series of experiments, which I'll show you briefly, it was actually the recipient that increased um longevity. And this recipient is a key ingredient in Febreze and it's also in many foods and drugs and it's in high cholesterol and spina, which is I conazole. And this latter detail is actually key to us getting access to a safety profile to support our first IND for drug repurposing. So we now have some basic information on safety, some promising studies in cell culture and mice. And then we needed to figure out how we could quickly um translate this to our two patients and change a research paradigm and move quickly. So first, uh just a little bit of information about Cyclodextrin. This schematic shows the construct of Cyclodextrin which are naturally occurring um cyclic oligosaccharides that derive from um enzymatic conversion of starch. They're composed of a varying uh number of link gluco pyin units. Um and they form this hollow cone like or toid structure. The beta have seven glucose units. So this is a very large hydrophilic molecule that has this um lipophilic um pocket and it's capable of solubilized cholesterol and other hydrophobic molecules. And um it's these properties that really have led to this being used as a delivery vehicle to enhance um bi availability of many uh drugs. And it's also ideal for cholesterol transport and it was used um quite extensively in labs for cholesterol extraction from cells. So, the research in the N PC mice, as I mentioned, was published a few months following the diagnosis of the twins. And this set of experiments uh demonstrated that lifespan was increased in one week old mice that were administered one dose of Cyclodextrin subcutaneously alone or in combination with the neuro which uh confirmed the change was from the Cyclodextrin. So, Lou and colleagues um performed a series of experiments as we can see here, this is the control N PC mouse um which uh has a short life span when treated with aid amide, which from birth, which is a drug that blocks intestinal absorption of cholesterol. They saw improvement of liver function but no change or improvement. Um On longevity when they treated the mice with two different forms of Cyclodextrin that varied by degree of substitution. They actually saw a 40% increase in um the length of life. And then this last set of experiments here when they tested the um neuro steroid rene alone, in combination with Cyclodextrin or Cyclodextrin alone, they saw that identical um increase in um in in lifespan. So another colleague um in the DT lab, Sherina Ramirez, she then um administered subq cyclone to these young mice at weekly intervals and saw a progressive increase um with doubling of lifespan decreased uh neuronal cholesterol and uh glycolipid storage. And she was able to actually quantify that cholesterol content was decreased in every organ and saw a total decrease in in the animal cholesterol pool. So this group as well as others then went on to continue to build on these observations in mice. And then following later in cats, and they tested various doses of Cyclodextrin frequencies, length of exposure routes of administration and the age of animals. And all of this helped us um in our studies. So these labs um discovered that mice had delayed neuro degeneration. However, importantly, it wasn't completely prevented, but they were able to show an increase on um decrease Perini cell loss in the cerebellum, which is actually a Hallmark um pathologic finding in N PC. And this was important because it demonstrated that the Cyclodextrin when given subcutaneously crossed the blood brain barrier in these young mice. And perhaps this was due to this incomplete closure of the blood brain barrier in early development. When they repeated the experiments in um mature mice, which were seven weeks of age, they still demonstrated cholesterol e flux from the organs. But they didn't see the same extent of um lengthened uh life. And maybe there was already um significant tissue damage done or perhaps um you know, more um more drug is needed uh to actually impact that blood brain barrier. So to sum up, we absorbed all of these findings and began our journey to develop in DS. And based on this information, we actually contacted the industrial producer of the lab Cyclodextrin and we ordered a kilo to begin our, our investigations. So we learned a little bit more about Cyclodextrin too. Um We wanted to learn about how much we were going to give. Um and the importance of uh plasma concentrations. We learned at low concentrations that um Cyclodextrin actually served us this cholesterol shuttle between membranes and at high concentrations, it extracted a lot of cholesterol. And because of that um that fine sensing feedback, it drove cholesterol synthesis. So we needed to avoid those high levels and then at very high levels, it was directly neurotoxic. So in 2008, um a year later, we filed our um our first in DS and this was based on the pre clinical data and the NBC mice from the DC lab and also from our formulation of a product for human administration. So we, we formulated a sterile compound, um tested it in a pharmacy and worked with the team at the FDA to develop a clinical protocol with safety measures and some limited outcome measures. And the goal was really to test the safety and efficacy in the affected Children and aim for a similar serum pks in the preclinical model because we of course, hope to see benefit, but we wanted to first do no harm. And we began our infusions in April of 2009, which was about 20 months after they were first seen. And 18 months from diagnosis, we attempted to identify um uh some biomarkers um looking at uh measures of cholesterol synthesis and metabolism and we measured uh drug uh concentrations and clearance. And over the ensuing months and years, we assess clinical manifestations of N PC with neural exams, cognitive assessments, liver function and size, green, size and blood counts. So when the Cyclodextrin infusions were started, um these were given intravenously, the twins were five years of age and they had already progressed rapidly, they had lost speech, they were no longer ambulating independently and they had developed seizures and cataplexy, vertical gaze palsy was also apparent. Um with the initiation of the infusions, we were very surprised to see. Um actually, some improvements with respect to increased alertness, engagement, swallow and ataxia, all of which were neurologic changes. We uh we had assumed um that based on some recent information from the Begley lab at the King's College in the UK, that due to the size of the Cyclodextrin molecule, we would have limited if any crossing across the blood brain barrier, even though we had seen some changes in the animals. And we are clearly seeing some changes in our patients. We um however, we felt that perhaps um with this information as well as our pet scans that showed severe um changes in the in the brain that we needed to better um penetrate the brain tissue. And we look to develop AC NS directed form of treatment as well. So, again, we, we worked on this um with our colleagues at the FDA and we initiated intrathecal treatment um concurrently with intravenous treatments in October of 2010. And this was 18 months following the um initiation of the intravenous um infusions. And again, we were very careful on our dosing so that we did not um exceed what we thought could be um harmful or even toxic levels. And interestingly, at the time that we started this, we had found out that in the cat model, they were going deaf from the um from the CNS directed therapies and our patients um were tested for hearing right before starting this. And, and I have to just say at this time that there was actually little information or little knowledge that patients with N PC developed hearing loss as a result of those disease and we were testing them so that we had a baseline and we could monitor hearing. But what we found is that they actually already had moderate to severe impairment of hearing. And when we started our low dose infusions within weeks, we saw improvement in hearing and this worsened again during a safety clinical hold and then improved again with the restart of infusions. So no doubt it had a, a causal effect on um hearing, which was something we didn't expect at all. Um So with time, we did also note that the patients who already were having seizures had increased seizure frequency for 24 hours following intrathecal infusions. Um This seemed to be some kind of uh neuro irritability that was being uh caused by the by the treatment. Our goal overall was that um we were gonna have stability of the disease and hopefully not worsening and no sustained toxicity. So, our protocol for the IV, as well as the intra delivery was shared on the internet actually by the family, um which meant that it was picked up by innumerable uh patients and clinicians internationally. And suddenly many inquiries were coming in. The protocol was shared. Um We obtained more in DS in the US. We assisted colleagues in Brazil and other parts of the US um who then submitted their own in DS. And then eventually uh four paradigms emerged um with some combination of IV. Um IV, followed by intraco which we uh dubbed sequential um or concurrently or intraco alone. And no doubt that we saw in our patients that they had neurologic benefits with, with the intravenous. But it was unclear if this was due to some penetrability or signaling. And we felt like we needed to explore this a little bit more because we were gaining increasing concern over the toxicity of the uh CNS directed therapy, which was being picked up by the NIH and other investigators. And indeed, they were seeing much more toxicity. So several years following the um in DS, the NIH conducted an early phase trial with CNS directed Cyclodextrin, they used an a Myer reservoir system, they treated three patients, however, they had to abort um due to infections uh which was likely related to the IO access technique. Um A then um open labeled it. Um trial was started with a very large dose range, uh 50 to 1200 mg. I'd like to point out that we hit target concentration at 350. So I was wary about that and, and did not participate in this trial. Um indeed, all patients developed hearing loss and a number of them had this kind of transient increase in seizures as well as post intrathecal fatigue and unsteadiness for um 24 to 72 hours. And as well, a compassionate use um um paper was um was published on three patients that were also treated with intra fecal. Uh They reported patients all developed hearing loss and in both of them, there was some um biomarker changes that appeared uh encouraging as well as some possible um benefits. But overall, I think what we learned from this is that most patients did experience some kind of permanent neurotoxicity such as hearing loss that was very likely dose related. And um and as well these results um um however, we're somewhat encouraging as I mentioned with the biomarker changes. So, following completion of of these studies, a phase 23 trial was started um with intrathecal Cyclodextrin. However, um they did not gain approval from the FDA because of the unfavorable benefit to this balance. So, with all of this in mind, we then really took a very close look at our intro uh Venus data. And in 2019, my colleagues and I published the results of this data acquisition study to evaluate long term outcomes of patients receiving intravenous Cyclodextrin according to our protocol. And um we um performed this as a retrospective review of of these patients. And overall, the long term administration was saved. Individual patients experienced individual benefits. And globally, we saw some improvements in systemic as well as neural cognitive and behavioral symptoms. And interestingly, um when we had a number of patients that received sequential intrathecal, we saw no added benefit of the intrathecal. The only exception being our two patients that had the hearing improvement when receiving low dose. So this review compelled us to reevaluate the role of the CNS directed therapy and focus further on investigation of the IV in a formal clinical trial setting. And this was one of our patients who received compassionate use, who was a rare patient that had lung disease and he had immediate improvement in oxygenation and was able to come off a ventilator following a first infusion of Cyclodextrin and a rapid clearance of uh cholesterol. And he continued on the regular infusions um and had um improve lung function overall, but sadly did succumb to his disease five years later. So based on the results of the compassion that you study and also an effort to confirm similar mechanism of action in humans. As in the preclinical studies, two early phase clinical trials were launched between 2017 and 18. The phase one international study was conducted solely here in Oakland with mild to moderately affected adults and we tested two different dose levels and patients received seven doses over a 14 week period of time. And we had pharmacokinetic and pharmacodynamic end points um as well as assessments of uh cholesterol homeostasis. The trial um was uh completed and overall, we had an excellent uh safety profile. The, the drug was detected. Oops, sorry. And um in the CNS and we were able to determine that it actually had a longer half life in the CS F as compared to the serum. So a companion phase 12 trial was opened in the EU UK and Israel and they tested three different dose levels down to age uh two. And this was a one year uh treatment course and it included clinical outcome measures as well as quality of life measures. So, both of these um early phase trials have been completed. And importantly, we were able to detect Cyclodextrin in the cerebral spinal fluid following intravenous administration. We looked at pharmacodynamics by measuring total Tau protein as well as measurements of 24 a hydroxy cholesterol. So, tau um is a protein that's found primarily in the axons and the CNS axons and it's elevated in neurodegenerative disorders such as Alzheimer's disease and many neurocognitive um uh diseases including N PC. And what we found um we, we don't show this here but what we found with even just a first infusion, a sudden increase in Tau which we think was a release of Tau bolus from neurons, but it was followed by seven weeks later by a reduction in the majority of our patients. And this is a relatively short period of time. Um but we hope but we don't have the data that this may be continued over time. And we think that this suggests that um that Cyclodextrin may be beneficial in reducing the rate of degeneration and death of cells. Serum 24 uh hydroxy cholesterol is a cholesterol metabolite that is produced in the central nervous system and then crosses the blood brain barrier into the circulation where you can measure it in the plasma. We saw peaks that increase following the first infusion and then a lesser peak following the seven infusion. This um is highly suggested that Cyclodextrin crossed the um the blood brain barrier to have this impact. And um we thought this, this, these two findings were very important. So this is a kind of a cartoon pictorial um of what we think is the mechanism in this um left panel we see under normal cir circumstances. The N PC one protein also acting with the N PC two protein moves cholesterol and lipids out to where they're needed. The cytosol when they're defected, the um the cholesterol is trapped. But when we introduce Cyclodextrin, it bypasses these deficient um molecules able to transport this cholesterol out. And the lower um part of this um um the slide shows actually our uh findings in our phase one study that was done here in Oakland. And um this, this is actually a depiction of the Philippine staining impaired liver biopsies. And here on the left side of each pair, we can see that the lighter blue corresponds to stored un aster cholesterol and seven infusions are 14 weeks later. It's much darker showing that there's been a release of this un aster cholesterol content following administration of Cyclodextrin. And we saw significant changes in all of our patients at both those levels. And so we feel that these um that these results of the Phillipine actually correspond to um what we believe to be happening or what was shown to be happening in the uh animal models. And um we also had biochemical markers of cholesterol synthesis and metabolism that correlated with these findings. So in total, our IV clinical trials to date have provided direct and also indirect um and compelling data mechanism of action of with release of accumulated cholesterol from cells in the peripheral organs and also in the central nervous system and restoration of cholesterol homeostasis. And this is very similar to what we're seeing in the animals. The data not shown where changes in the cholesterol metabolites with decreases in some of the serum levels of um uh or precursors such as LAO and also um and also uh markers of um of the feedback mechanism. So we did see as mentioned increased levels of the brain specific cholesterol metabolite 24 hydroxy cholesterol. So this data provided very important um information for the pivotal phase three data. So I just wanna mention as well that um so clinical assessments are becoming now of course, very important and we need to have some mechanism to look at how patients um that are treated with an intervention are actually demonstrating clinical improvement. And we implemented a um a validated clinical severity scoring system. This is uh a sample of this, of 17 domains. It was published by the NIH in 2010 based on a small number of natural history uh patients. And it has become a tool that has been continued to be refined as um we've worked with it um over the years, but it's really not simple to use because of the complexity and everyday practice, the inclusion of some domains that are um affected by supportive care measures, for example, um follow and uh seizures and um also it includes hearing which is um part of the natural progression. But also as we saw can be a direct toxicity from one of the um one of the drugs. So of no clinical stability or maintaining the same score is considered success as we would expect the score to continue to increase over time as patients um gradually worsen. So we've learned many lessons in the last 15 years and we've been able to show a correlation between mechanism of action um between preclinical and human studies. And we've learned some um very important things that route and dose are very important. So is tissue concentration and we really need to be careful that we are um heeding the lessons from, from our um researchers in the lab and not causing harm to our patients. And we, we just want to ensure proper and safe dosing and rout of administration. And um because if we overdo it and it looks like we are causing more harm than we've actually missed the opportunity to treat our patients. So this timeline demonstrates the development. Um that's kind of the story that I've gone through from 2010. Um the earliest um uh accounting in the animal studies and over this 15 year period of time, we've had um more studies done our in DS or Earth and drug designation, which then led to the ability of a sponsor to pick up the studies to, to support our early phase one and phase two trials. And now we are at the uh phase three study. So we've opened a phase three international trial at 23 sites across nine countries. I currently serve as the global uh principal investigator. We hope to enroll 93 patients aged three years and older. Um I'd like to mention as well. There is a substu outside of the United States that was invited by the EN A or European Medicines um agency because they wanted the opportunity to treat um patients from birth as hopefully a preventative uh therapeutic given the um the really encouraging results that have we seen particularly in the younger animals. So we did choose a dose based on um our pharmacokinetic studies and we are using a a composite severity score. Um We are using that 17 domain original NIH score, but additionally applying to other um adapted more hopefully simpler to use scoring systems and have other secondary end points including the Scappy for Ataxia and fine motor and uh swallow and also looking at measures of cognition. So, um we, of course, even though we've learned a lot, we have many challenges and we're always on the search for biomarkers and the correlation with clinical progression and response to intervention. And this has been uh very challenging in this disease. We've certainly learned a lot and um we, we can certainly measure um metabolites of cholesterol um which include bio acids and Oxysterol that are now used for diagnostic methods. But um you know, it's, it doesn't seem to be uh predictive and, and we're still working on correlation with clinical improvement or worsening. So, there's definitely a need for more precise measurement tools both clinically and biochemically. We need to develop um biomarkers that can be of course, easily accessible and reproducible. Um in order for them to be um used. Um uh widespread and natural history studies are very important. We participate in an international registry, but it's becoming more and more difficult um giving um uh that patients have access to clinical trials as well as um compassionate use um drugs. So, we are working um internationally to address some of these challenges at this time. So another area of intense interest is this correlation between genotype and phenotype variability. And as mentioned, even within the same family, you can see um uh some variability in that. So it's not a direct correlation and we know that there are a number of other influences. And this is definitely a hot area of investigation. It's uh newborn screening has um it is something that has been discussed for quite a while and may soon be piloted in some states. We're looking, looking at um onset of clinical symptoms and how this may correlate to uh the genotype. And um we we ultimately feel that we're going to be looking at developing tailored therapy for each patient. And this may be um different depending on this genotypic phenotypic expression of disease. So there's many more research that needs to be done, especially understanding the uh cns penetration of this drug and and really other drugs as well. And we know that probably earlier intervention and may be presymptomatic may be uh key to um to affecting the course of this disease. So I think with Cyclodextrin, we are going on to learn how to better utilize this drug. But we also may need some better transport systems as far as different brain penetrability. And interestingly, we know from the feline model that even with directed cns um infusion of drug that there can be differential concentration in different parts of the brain and likely what we were seeing with hearing is that there was um a very high concentration that surrounded um the cranial nerve that had a direct impact and led to um led to the um deafness that was seen in many patients. So, um we are of course, uh looking at many other types of therapies. Um Cyclodextrin intravenously is the only clinical trial that's open right now. Um Internationally, there has been a recent study looking at a heat shock protein called AOL and it the study was completed and it was um somewhat favorable and it is undergoing resubmission at this time to the FDA, an amino acid acetyl leine which has been tested in various forms of ataxia has also been um tested in Neiman pick and uh specifically looking to ameliorate neurologic symptoms and that looks encouraging as well and the trial has been completed. And um we're waiting to hear about uh that if it becomes an indication as well, we do feel that combination approaches are gonna be really important because um one drug Milos, which is a substrate reduction therapy that is approved for use in F in Gauthier disease has been used off label in N PC. And there are studies suggesting highly that it's used long term will uh lessen the a rate of progression of neurologic symptoms. And so many patients came into these studies already on Nilesat and we saw that those patients had increased benefit when used with um with Cyclodextrin and also when used with a. So there are other um types of, of uh mechanisms that are being explored. And gene therapy is also a consideration that's, that's very early in investigation in animal models. So patients with N PC like chronic or or other progressive diseases benefit from many supportive care measures. And as you may imagine, a multidisciplinary approach is really important and critically, these patients need a medical home and this is likely the biggest challenge patients with chronic disease face. Um I've become interestingly a medical home for a number of these patients because they're turned away frequently by their primary care doctors or other subspecialists. And I think that all of us really do need to get involved in taking care of kids with rare disease and and prevent this from happening. So, early diagnosis of rare disease is critical. I'm not gonna really go through these, but just know that we really should have a heightened suspicion if we have a combination of signs and symptoms that don't fully make sense or we don't have an answer. And certainly the combination of splenomegaly cognitive decline, um psychiatric history and um a movement disorder such as ataxia, all should really alert you to the possibility of an important era of metabolism. And then some of the unique features, for example, cataplexy and vertical gaze palsy should also um lead you to suspect a lysosomal disease. So uh the laboratory diagnostic algorithm algorithm is really not as complex as this will look. Um the skin biopsy for Phillipine staining that we did in our patients is now really no longer done first line as we have uh biomarker and genetic uh gene panels that are available. However, um with um with uncertainty or or for confirmatory testing, uh Phillipine staining is still used. So for our patients, our story is continuing. Um many more people that are listed here were actually involved along the way. It was really an international effort involving patients. A lot of motivated families who are willing to risk everything, scientists, clinicians um and media to improve the awareness of this disease. And um so this has been a fun and at times risky path to take. Um but very exciting. And then finally, I wanted to conclude with the twins, they received um Cyclodextrin infusions for over 10 years and they continued to enjoy a comfortable quality of life. And at age 15 years, having really outlived all expectations for life expectancy, they passed away 30 minutes apart in their birth order um after a short bout of parainfluenza in July of 2019. Um but their parents remain grateful for all that was done and they really moved um the bar on this and continue to be involved with the parent community and forging ahead with scientists and also pharmaceutical companies to work to um create a hopefully a better life with patients with N PC. So Thank you to all of you for participating and I hope that, um, I was able to excite you about um, being involved in rare disease. Thank you so much. That was um, a really interesting talk on a topic that we don't, um, learn about very much in other settings. So, um, there are a couple of questions um, that Dr Viki actually had that I'm gonna ask, um, then we'll see if we have time to get to the other ones. Um Doctor Bins was wondering if um the fetal ultrasound shows um changes um like organ amygdala or placental enlargement in these patients. I have never heard that um patients have been born with massive hepatomegaly and splenomegaly. So presumably that could have been picked up in utero, but I don't know what, what timing. Yeah. Thank you. And then another one was focused on um any trials with um neuros stem cells um and implantation or any like talk of doing trials um with intracerebral um stem cells. So, um no, is the answer uh to that. I know that there are a number of labs uh particularly in the UK at um Oxford that are looking at various uh gene gene therapies and it's a very complicated disease to do gene therapy in. But I've not heard about utilizing um stem cells at, at this point or in in utero um method. Of course, we'd have to have really early diagnosis which would mean that um a prior family member or sibling probably would have been um diagnosed and then, then we can do prenatal diagnosis certainly in the patients, but I'm not aware of any, any therapies that early on. Thank you. Um And then let's see there was another one from him as well. Um Well, there's a comment um from Cecilia fairly saying that this is an amazing, um This is amazing progress for N PC. Um And that the progress is very exciting and thank you for sharing with us. Um There is a request to show the slide with Tau again and um speak to kind of the um changes that were noted there. Um I don't know if you can go back to that one. Let me see. Um Well, all of our patients had all, well, there were, there were a couple that were low but 1000 is is very elevated. So the majority of our patients in the phase one study and um and all of our patients in our phase 12 study had extremely elevated levels of Tau that correlate with neuro degeneration. And we don't know how long it would take for Tau to change or we weren't even sure we would see that. Um I we, we really can't explain this one that goes up, but what we saw was a general trend in the majority of our patients over and this is only a three month period of time. Um And you know, since this is CS F, we didn't really have the ability to do lumbar punctures a year later and see where that might be. But we think that there may be, um there's at least some impact on the TAU protein which we would not expect to decrease over time. We would expect it to be stable or to continue to increase. Ok. Great. And then there at the beginning of that top graph, it looks like you can see the baseline measures, which is what um another part of that question was um the base line here. All right. And then let's see. Um So I think that's about it for today. Thank you so much for joining us and thanks to everybody um in the audience for joining as well. Um Yeah, thank you, Doctor Hastings and thank you everybody else. Created by
