Hi, my name is Riccardo Perfetti. I'm the chief medical officer at Applied Therapeutics. I'd like to welcome you at the galactosemia webinar at ACMG. The title of our webinar today is Progressive Worsening of Central Nervous System Phenotype in Children with Classic Galactosemia, a cross-sectional analysis. We will be discussing disease manifestation in children with galactosemia with a specific focus on the CNS phenotype. We will review recent evidence demonstrating that galactosemia is a progressive disease, significantly worsening with age. We also will review the current knowledge on disease pathogenesis and potential opportunities for intervention through clinical trials. This is our disclosure slide. Myself and Dr. Lawson will present to you data on galactosemia today. Galactosemia is a rare, slowly progressive metabolic disease caused by a genetic inability to break down galactose. Galactose is not only found in food, but it's also synthesized by cells in the body. There are approximately 3,000 people affected by galactosemia in the United States, and 80 new births per year have the diagnosis of galactosemia. In the United States and in Europe, the newborn screening allows for early detection of people with galactosemia, for children with galactosemia, and these children are placed immediately on a galactose-free diet. Unfortunately, that does not prevent endogenous production of galactose and does not prevent the long-term consequences of the disease, which are typically characterized by relevant, important CNS phenotypes that impair cognition, memory, and also has important behavioral components. The enzyme aldose retutase is the enzyme that converts galactose into galactitol. Galactitol is an aberrant, toxic metabolite that is not produced in healthy individuals, but in people with galactosemia, builds up in tissues and organs and causes those long-term disease complications I alluded to. AP007 is a novel CNS penetrant aldose retutase inhibitor, which prevents galactitol formation and the accumulation in adult galactosemia patients, as we have recently demonstrated, and we are investigating this, the ability of AP007 to block the production of galactitol in the pediatric population. The slide that you see projected depicts the biochemistry of galactose. Galactose is a substrate for energy production, is metabolized through different steps. The genetic defects on this pathway leads to different forms of the disease. The most common is the GAL-T gene deficiency, also called plastic galactosemia. Individuals with a genetic defect on the GAL-T gene accumulates GAL-1T and galactose, and galactose, when accumulating, could be transformed by the enzyme aldose retutase in something that in healthy individuals is never produced, is galactitol. Galactitol accumulates in various tissues and is toxic to those tissues. AP007 blocks the making of galactitol, and therefore galactose could be redirected over energy-efficient pathway or eliminated as part of the catabolic pathway of the molecule itself. To test the hypothesis that inhibiting aldose retutase is relevant in correcting the clinical phenotype, we use an animal model of galactosemia, an animal model, a rat model, that is deficient for the GAL-T enzyme. Animals that are deficient for the GAL-T enzyme accumulate high level of galactitol. As you can see on the left part of this slide, three different tissues that were investigated, liver, brain, and plasma, have very high level of galactitol as described by the green, the gray bar. The gray bar, the animal untreated, have very high level of this metabolite. If they are treated with an inhibitor of aldose retutase, the production of this apparent metabolite is inhibited. This happens without creating a compensatory increase in precursor substrate like galactose or GAL-1P. So inhibiting galactitol is not associated with a compensatory increase in precursor metabolite. This is the biochemistry. What is obviously relevant is to see whether by correcting the biochemistry translate in improvement in the phenotype. Animals' rat model of GAL-T deficient have some relevant cognitive behavioral organ-specific abnormality that very closely resemble galactosemia in humans. Cognition and memory could be tested through different modality in animal model. One of the tests that I'm describing in this slide is the water maze. Rats are placed in a maze that is immersed in water and they need to be able to navigate the maze and eventually be able to get out of the maze. The time that takes them to get out of that maze is one of the elements that are measured. The second time around you put a rat in a maze, a normal rat would have learned how to get out of the maze, would do that much faster than the first time. If you focus on the panel on the left of your slide, in blue, you see the wild-type animals. So the first time around takes them some time to figure the way out, but then if you put them back in the maze they can get out of the maze much faster. This is not the case for the GAL-T deficient rats. It takes them much longer time. So they have a cognitive memory impairment. If they are treated with AT007, in the green bar that you see in this graph, you see that they behave very similar to the wild-type control, so they have prevented that memory and cognitive deficiency. In patients with galactosemia, another important element of the phenotype of the disease is the development of cataract. In the center of this slide, you see a picture of the eyes of the rats, and the picture in the middle is the picture of animals that are carrying the gene defect that are not treated. You can clearly see the cataract in the eye of this rat. However, on top of that, you see the wild-type, which obviously does not have cataract, and at the bottom you see the animals that have been treated with AT007, where you have the prevention of formation of cataract. You can even build a dose-response curve to compare the degree of AT007 exposure versus the ability of preventing the cataract, which is shown by the graph on the screen right of the slide. So, how do we know that galactitol is responsible for the long-term CNS complication of the disease? So, the rat model, which as I mentioned is an animal model that express the enzyme aldose regulates to level very similar to human, the reduction of galactitol results in a normalization of the CNS outcome of the disease. So, from many different points of view, learning, cognition, memory, motor skill, and those are measured by different tools. The mouse model, which has a level of the aldose regulates that is very different from human, has a barely detectable level of aldose regulates activity, doesn't produce galactitol field. Even if you delete the GALT gene, you're able to produce a biochemical genetic defect that doesn't translate in CNS outcome. These animals do not have an elevated level of galactitol. In humans, the GALK deficiency patients are missing the GALK enzyme and they do not produce galactose metabolites such as GAL1P, but they also have an elevated level of galactitol. So, that elevated level of galactitol is sufficient to produce a CNS phenotype, which is similar to the one that we more commonly see in GALT deficient patients. There is a small number of biochemical variants of classic galactosemia. This is characterized by a patient with a residual GALT activity, this patient low level of galactitol when compared to classic galactosemia. There are some residual functions that provide a less severe outcome. All of this data supported the development of the clinical program that has two major components. One in healthy volunteers exploring safety pharmacokinetic, but also investigating the ability of AT007 to be CNS penetrant. And another component of the study in the adult patients with galactosemia to again validate the safety tolerability, characterize the pharmacokinetic, but also investigate pharmacodynamic with the reduction of galactitol. For the adult galactosemia part of the study, three different doses were investigated, 5, 20, and 40 milligrams per kilogram per day, and obviously there was a placebo comparative group. This study is now finished and we have the result of both the healthy volunteers and the adult study in patients with galactosemia. The study in healthy volunteers enrolled approximately 80 patients that demonstrated that AT007 was safe and well tolerated. We also demonstrated that AT007 is brain penetrant in a dose-dependent manner. From a pharmacokinetic, we were able to observe a classical dose-dependent accumulation of AT007 in the bloodstream, suggestive of a once-daily potential dose of administration in the clinic. In the study in adult patients with galactosemia, in addition to verify once again safety, tolerability, and pharmacokinetic, we're also able to investigate the pharmacodynamic, whether the doses that we had tested were able to produce a meaningful reduction of Galactitol. And we observed that the doses of 20 and 40 milligram produced a statistically significant reduction of Galactitol that was rapid and sustained over time. All patients exposed to 8007 responded to the intervention with a reduction of Galactitol. This reduction was statistically significant to all patients that were exposed to 20 and to 40 milligram per kilogram per day. So in summary, the clinical data so far produce clear evidence of the reduction of Galactitol. They also clearly show that the drug is safe and well-tolerated from a pharmacodynamic and pharmacokinetic relationship support. Once daily dosing, they indicate that the suppression of Galactitol is rapid and sustained over time. And an exploratory investigation of the MR spectroscopy of the presence of Galactitol in the brain also suggests a reduction of Galactitol in the brain. And with this summary, I would like now to introduce you to Dr. Lawson who will present the pediatric study. Hello, my name is Francesca Lawson and I'm the head of development applied therapeutics. And today I will give you an update on our galactosemia pediatric program. Galactosemia is a progressive and debilitating disease. Before the implementation of the mandatory genetic newborn screening, the disease was often fatal. And the cause of death often was liver failure, renal failure or septicemia. Galactosemia like many rare diseases is very heterogeneous but there are common patterns of signs and symptoms in the various age groups. For example, in infant and toddlers, the most common and important impairment is often a speech and language delay. Toddlers also manifest coordination problems, developmental delays, growth delays. In school-aged children, we start observing learning delays and these young children continue to present an impairment on fine and gross motor skills. They have growth problem, they have speech and language problems. Then in the adolescent years, the majority of the females have fertility problems including primary ovarian insufficiency. These older children continue to have growth delays, learning delays, and some start manifesting mental health problems such as anxiety and socialization problems. And some start exhibiting some tremor. The adult phenotype is often characterized by worsening tremor. And many adults also have a history of seizure. Some suffer psychiatric diseases such as severe anxiety, major depression, ADD, and some exhibit early onset cataract. The Action Galactosemia Kids Study is a placebo-controlled, double-blind, randomized trial in children with galactosemia. The study consists of an initial intrapatient dose escalation that is designed to identify the optimal dose in each of the three age groups that we are evaluating, the two to six year age old, the seven to 12 years old, and the 13 to 17 years old. Upon completion of the dose finding study, once we have identified the optimal dose, the children will transition without any treatment interruption to the three month assessment of the biomarker Galactitol. And the result of this part will form the basis for the NDA submission and their accelerated approval. Upon completion of the biomarker component, then the children still without any treatment interruption will move to the long-term safety and clinical outcomes part. In this part, the children will be evaluated every six months for their clinical outcomes, and this will be evaluated by an independent committee, a data monitoring committee. I mentioned earlier that the study is placebo-controlled. Therefore, at the end of the trial, all the children that were randomized to placebo will cross over to an open-label active treatment extension. Now, the long-term clinical outcome assessment encompass four quadrants, speech, cognition, behavior, and motor skills. And each of these domains include specific subdomain. So the speech includes the subdomain of the expressive language and the receptive language that we will evaluate using the oral and written language skills two, the OWLs two. The cognition and learning include the assessment of cognitive and intellectual development, attention, executive function, memory, and processing speed. And this will all be performed using the NIH toolbox cognition battery. The behavioral psychosocial domain include assessment of any atypical behavior, mental health, particularly anxiety and depression, adaptability, social skills, activities of daily living, and communication. And to assess this, we perform two tests. One is the behavior assessment system for children, the BASC, and the other one is the Vineland adaptive behavior scales. Now, for the motor function domain, we assess the dexterity and balance through the NIH toolbox motor battery. We assess the gait coordination through the SARA, that is a scale for assessment of ataxia. And then we evaluate tremor using the spiral joint test. How do we know that these are relevant outcomes? They worsen over time? Well, the galactosemia population has not yet been studied prospectively in a longitudinal study where the same children fall over time. However, individual patients have been evaluated over time and have exhibited progressively worsening symptoms. We have conducted a cross-sectional analysis of the first 19 pediatric patients at baseline. These children range between 3 and 15 years of age and were evaluated across all four quadrants, speech, cognition, behavior, and motor skills. And this analysis showed that disease severity increased with age, confirming the galactosemia progressively worsens over time. These 19 participants, as I mentioned, ranged in age between 3 and 15. 11 were females, 8 were males. We had 7 patients in the youngest group of 2 to 6 years old, although we didn't have anybody younger than 3. 9 patients were in the intermediate group, so 7 to 12, and 3 patients were in the older group, 13 to 17, but we didn't have anybody older than 15. In terms of genotype, 9 patients were homozygous for the Q188R mutation, 1 patient was homozygous for the K285M mutation, and 9 patients carried two different mutations on each of the alleles, and one of these 9 heterozygous children carried a biochemical variant. The table below shows that the percent of patients with severe impairment defined as children with a score that is at least a two standard deviation below the norm. And the table shows that these percentages for the three age groups and for the four domain, speech, cognition, adaptive behavior, and motor function. As you can see, the youngest kids, the 2 to 6 year old, we had zero patients with severe impairment. In the intermediate age group, the 7 to 12, we had one third of the children had a severe speech impairment, half of the kids had a severe motor function impairment, three quarter of the kids had a severe cognition impairment, and no kids start showing a severe adaptive behavior issues at that age. Then in the older age group, we had two third of the kids who had both a severe speech impairment and a severe motor impairment, and all the kids had a severe cognition impairment. And then we start observing that one third of the kids start showing severe impairment of their adaptive behavior. This slide shows the individual characteristics of the 19 participants. So in terms of their age, gender, their baseline plasma level of galactitol, their gout enzyme activity, and their gene mutation. So this slide just shows the result of the cross-sectional analysis, and each graph obviously shows each domain, speech, cognition, adaptive behavior, and motor skills. So the horizontal axis shows the age, and the vertical axis on the left shows the scale. The light green color on the top shows the score in the average range. The intermediate yellow color shows score below average. They have one standard deviation below the norm. And at the bottom, the red color identifies a severe impairment, so score that had at least two standard deviations below the norm. The solid green line represent the mean scores of all the children, and the gray shadow area represented the standard deviation. So you can see that in the younger age group in each domain, the majority of the kids have either an average range score, or some have maybe a below average score. In the intermediate age group, the majority of kids have below average score, and then some have already a severely impaired score. And then the adolescent, so the older age group, the majority have a significant deficit in most of the domains. So, and the slope you see is really similar in the various domains, and maybe is even steeper for the cognition domain. This slide shows the results of the sensitivity analysis that does not include the biochemical variant patient. And as you can see, the results are very similar to the primary analysis results. In conclusion, galactosemia is a slowly progressive rare disease which worsens over time. The complications of the disease are caused by accumulation of the toxic aberrant metabolite galactitol that is naturally produced by the human body, even in patients following a galactose-free diet. We have demonstrated that the reduction of galactitol induced by AT007 was able to prevent CNS symptoms in a rat model of galactosemia. We have also demonstrated that AT007 treatment in adults with galactosemia significantly reduced galactitol levels by approximately 50%, and a similar degree of reduction was shown to be effective in the preclinical model. We are currently performing a pediatric biomarker study, and all patients will seamlessly transition to the long-term part of the study, which will evaluate the long-term clinical outcomes. We hope that this presentation has highlighted the importance of finding a therapy for this progressive and debilitating disease. For all of you who have supported our clinical development program, we want to sincerely thank you for your continuous involvement. For any additional information, please visit our website, or feel free to reach out directly to me or Dr. Perfetti. Thank you very much.

galactosemia

metabolic disease

central nervous system phenotype

cognition

galactitol

clinical outcomes