Retrotope, Inc. A Clinical Stage Pharmaceutical Company

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1 Reinforcing the molecules of life Retrotope, Inc. A Clinical Stage Pharmaceutical Company Retrotope has created a proprietary drug platform to preserve and restore cellular health in degenerative diseases. The unique mechanism of Retrotope s platform prevents cellular damage and recovers cellular function that has been damaged by oxidative stress and lipid peroxidation. Retrotope has a rapid route to high value drug proof of concept. The first product candidate, RT001, is being tested in Friedreich s ataxia (FRDA), a high value orphan disease with no current treatment. Orphan drug development takes on average 3-5 years and <$50MM; this is significantly faster and more cost effective compared to larger indications. The FRDA orphan indication market is estimated to be ~$1B/year. Should Retrotope s clinical plan for RT001 in FRDA be realized, an NDA would be submitted in Retrotope s transformational platform technology of stabilized lipid drugs, designed to halt lipid peroxidation, has the potential to treat a wide variety of other important diseases such as Parkinson s, Alzheimer s, diabetic retinopathy, and mitochondrial diseases. These diseases affect tens of millions of people, incurring billions of dollars in annual healthcare costs. Drug Platform Rationale Typical drugs target enzymes, proteins, or gene pathways. However, many biochemical processes are not controlled by enzymes (Keller, 2015). These processes are not often addressed therapeutically, in part, because modern drug discovery is usually based on biochemical pathway mapping informed by genomic analysis, and such approaches are blind to non-genetically encoded events. Non-enzymatic in vivo processes include a large group of oxidation reactions. The resulting oxidative damage is detrimental and, in diseased cells, cannot be controlled by antioxidants. The reasons for this inefficiency include the stochastic nature of damage: whenever a reactive oxygen species (ROS) appears as a result of a metabolic process or an environmental insult, it oxidizes a nearby molecule so quickly that there is no time for nearby antioxidant molecules to diffuse close enough to neutralize it. Antioxidants are typically present in cells at levels close to saturation, through enzymatically controlled active transport, and their concentrations cannot be easily be further increased. In addition, excessive levels of antioxidants may interfere with required redox processes, and result in a net detrimental effect. This explains why clinical trials of antioxidants in humans often result in no positive or negative effects, even though the disease aetiology is oxidative in nature. This leads to the paradigm Retrotope set out to solve: 1

2 ! Most organisms need a constant supply of oxygen for life, but oxygen causes damage which leads to illness and death! All tissues require oxygen for metabolism to produce ATP in a complex bioenergetic electron transfer chain, which also produces physiological amounts of reactive oxygen species.! Up to 2% of all inhaled oxygen is converted into damaging Reactive Oxygen Species (ROS)! ROS reactions in a cell break apart molecules and result in cell damage; polyunsaturated fatty acids (PUFAs) are very susceptible to ROS; and certain specific bonds in the PUFAs are damaged.! Poly unsaturated fatty acids (PUFAs) are essential nutrients. They have to be ingested because animals cannot make them, the body naturally distributes them throughout the tissues, and they are a required part of the membranes of all cells.! The most malicious type of oxidative damage occurs as lipid membrane peroxidation in the phospholipids comprising the membranes. PUFAs are essential for the active function of membranes that rely on rapid interactions between membrane-bound proteins, such as mitochondrial and neuronal membranes. PUFAs, which are liquid fats, enable these interactions by imparting fluidity to membranes. This property is linked to the number of double bonds in a PUFA molecule. Moreover, biologically relevant PUFAs contain double bonds separated by methylene groups (-CH2-), known as bisallylic methylene groups. These C-H bonds are a weak link in the fatty acid chain, and are 2

3 known to be cleaved by a variety of ROS species. Alkyl peroxyl radicals (a prevalent type of ROS) are formed by a variety of ROS stresses, and abstract a hydrogen atom from the susceptible bis-allylic methylene groups. This results in a formation of a damaging, nonradical alkyl peroxide (also a type of ROS) and a new radical which reacts further with plentiful oxygen molecules abundant in the lipid membrane, yielding another alkyl peroxyl radical in a cyclic chain reaction, each step of which permanently damages the membrane fats. This peroxidation chain reaction is an insidious type of non-enzymatic damage process, because the cyclic chain multiplies the damage originating from a single initiating ROS species many times before it is quenched by an antioxidant or two radicals combining, both rare events. Hence, membrane lipid peroxidation, unlike all other types of stoichiometric (one ROS damages one biomolecule) oxidation, is responsible for massively amplifying biological damage at levels that can lead to disease and cell death at multiple levels (molecular, membrane, organelle, cell, and organ). The only current remedy against lipid oxidation chain reactions is to intercept the ROS species by an antioxidant. However, the saturating levels of the major chain-breaking antioxidant molecules are one in a thousand to two thousand lipid molecules in a membrane [Packer 1993]. Supplying more would not help, as the membrane cannot incorporate more than a saturating amount. Doing so would lead to changes in the membrane physico-chemical properties, and be incompatible with membrane function (i.e. toxic). There are other processes that can terminate the chain: two radicals can recombine; or a chain propagating lipid radical can oxidize a membrane protein, but these are rare, and it is highly likely that the species next to a PUFA radical will be another susceptible PUFA, thus sustaining the chain. Estimates suggest that hundreds to thousands of PUFA residues get oxidized before the chain terminates. This damage decreases membrane fluidity and compromises the ability of the lipid membranes to participate as needed in the mitochondrial electron transport chain. Moreover, peroxidized PUFA residues decompose further, generating numerous types of highly reactive (toxic) carbonyl compounds, which broadcast damage, cross-linking biomolecules, damaging proteins and causing DNA mutations, particularly in mitochondria. Evidence of the Role of Lipid Peroxidation in Neurodegenerative Disease 3

4 There is a broad body of literature supporting the role of lipid peroxidation in degenerative diseases.! Neurodegenerative Diseases Cobb et al, 2015 " This review proposes a significant role for oxidative stress/nitrite stress and more specifically, lipid peroxidation (LPO) and other lipid modifications, by triggering microglial activation to elicit a neuroinflammatory state potentiated by diabetes or abnormal dopamine metabolism. Subsequently, sustained distress in the neuro-inflammatory state overwhelms cellular defenses and prompts neurotoxicity resulting in the onset or amplification of brain damage.! Parkinson s Ruipérez et al, 2010 " We propose that association of α-synuclein with oxidized lipid metabolites can lead to mitochondrial dysfunction in turn leading to dopaminergic neuron death and thus to Parkinson s disease.! Alzheimer s Butterfield et al, 2013 " Oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. The tissues with the highest oxidative metabolic requirements: brain, retina, cardiac muscle and skeletal muscle are the most sensitive to the toxic effects of excessive oxidative stress. Many diseases of lipid peroxidation involve multiple organs/tissues resulting in a great variety of clinical syndromes with phenotypic characteristics and multiple genotypes. Neurodegenerative diseases constitute a major group of inherited pathologies related to oxidative stress. Lipid peroxidation, abnormal proteins and toxic metabolites interfere with a number of metabolic pathways. Since PUFA oxidation products are known to be involved in a host of degenerative diseases, what can be done to stop this, given that antioxidants are inefficient? Retrotope s Mechanism of Action The lipid peroxidation chain reaction is the target of Retrotope s drug platform. This chain reaction results in cell damage, death and disease. To halt this damage process Retrotope s drug platform targets the root cause of disease, the amplification of the original disease trigger by lipid peroxidation, and stops it from ever starting. Since PUFAs also turn over in diseased as well as normal cells, Retrotope s drug can both maintain and restore health and function to them. 4

5 The initiation event of the lipid peroxidation chain reaction is caused by ROS ripping a hydrogen off a bis-allylic (between the double bonds) methylene carbon in the lipid- this is the rate determining step of the chain reaction in lipid peroxidation. If one could slow down the initiation rate, it would have a large effect down-regulating PUFA oxidation by eliminating all of the downstream multiplying cycles of damage from each abstraction. Retrotope reduces the initial abstraction rate by replacing hydrogen atoms at bis-allylic methylene sites with deuterium atoms. Deuterium is naturally present and is recognized by living systems as a normal variation of hydrogen (typically hydrogen in all natural substances consists of ~ 1 deuterium per 7000 hydrogens). Deuterium is also responsible for a wellknown isotope effect (IE): reactions involving cleavage of a C-H bond are slowed down substantially when H is replaced with D. This substitution reduces that ability of the C-H bond to be broken. 5

6 Novel Target Retrotope s first clinical candidate: RT001 RT001: is deuterated linoleic acid, a stabilized lipid mimetic of Omega 6 fatty acid! Deuterium placed at specific bisallylic position! Resulting in a lipid stabilized fold by isotopic effect! RT001 halts lipid peroxidation!...without being used up, a nonstoichiometric effect o Linoleic acid (susceptible fat) RT001 = Stabilized fat 7 PUFAs specifically substituted with deuterium at bis-allylic positions can be made in large quantities and reasonably cheaply using well-optimized synthetic methods. This modification is both natural (deuterium exists in nature) and game-changing. Whereas the lipid peroxidation process is autocatalytic the stabilization of the initiating step is anti catalytic, causing at each step a multiplicative > 10-fold isotope reduction, essentially shutting down the chain process quickly. Hence, the susceptible target bonds of the chain reaction are fireproofed against the damage of ROS. Importantly, enzymatic processes involving PUFAs, such as β-oxidation, transformations involving other enzymes (all stoichiometric 1:1 enzyme: substrate reactions) are largely unaffected. 6

7 In addition, Retrotope has discovered this fireproofing process requires only a fraction (15-20%) of the total PUFAs need to be deuterated for the chain reaction of lipid peroxidation to be effectively shut down (Hill 2012). Co-enzyme Q deficient yeast cells (bottom row in each panel) recover full viability in presence of only ~20% deuterated lipids and 80% non-deuterated lipids Deuterated PUFAs thus represent a novel type of sensitive and specific drug which is identical to natural PUFAs, EXCEPT that they prevent damaging, non-enzymatic oxidation processes without interfering substantially with biologically necessary enzymatic transformations. Because PUFAs in membranes turn over rapidly even when the cells do not (and especially in high energy tissues such as brain and retina), deuterated PUFAs rapidly replace the original hydrogen containing molecules in all compartments in all tissues. All of the active transport used to transfer normal PUFAs from orally ingested foods work the same for deuterated PUFAs, and transport them wherever they are needed. As a result, D-PUFAs rapidly incorporate into brain, retina, and other difficult to treat tissues. Preclinical Data Retrotope s drug platform, deuterated PUFAs, and its first drug candidate RT001 (11,11-dideutero-linoleic acid ethyl ester) are unique in drug discovery, a foodstuff that has no pharmacological effect that in the deuterated form is a sensitive and specific drug. It also, due to the underlying role of radical lipid peroxidation in many diseases of degeneration, works across many indications without any observable side effects. The mechanism targets a 7

8 fundamental degeneration pathway that is both in common to many diseases, and is required for the phenotype to develop. Retrotope currently has data in multiple models and across multiple diseases all with similar, but not identical, drugs, targeting the same mechanism of action: lipid peroxidation. Data has been generated using the two lead product candidates, RT001 (an omega 6 polyunsaturated fatty acid) and RT002 (11,11-,14,14 tetra-deutero-linolenic acid ethyl ester, an omega 3 polyunsaturated fatty acid). The diseases studied include: Friedreich s ataxia (primary patient cells, murine knock in, and yeast cells); Alzheimer s neurons (HNE, Fe toxicity and lactase models); Retinal (retinal ganglia, RPE, and MVE cells and AMD and diabetic retinopathy mice); Muscle H9C2 myoblasts (mitochondrial toxicity with Fe, tbu00h); Parkinson s (synuclein toxicity & MPTP mouse model); NCL (Neuronal Ceroid Lipofuscinosis); Huntington s (UCLA); Smith-Lemli-Opitz Syndrome (Vanderbilt). Below are a summary of a few of these experimental results. In the Sea Horse instrument, H9C2Myoblasts were treated with 25µM RT h prior to assay of mitochondrial respiration. Oxidative damage was induced by treatment with 1 mm FeSO4 for 2 hours. The results showed RT001 inhibition of lipid peroxidation restores full mitochondrial function. Friedreich s ataxia: Lipid peroxidation resulting from oxidative stress plays a pivotal role in key processes associated with FRDA. RT001 s mechanism of action is not that of an antioxidant, but instead functions by blocking the autocatalytic ROS-induced chemical reactions that severely damage membrane lipids. Since the PUFAs in these membranes are regularly replaced with PUFAs from dietary sources of essential fatty acids, deuteriumstabilized linoleic acid and its derived PUFAs are incorporated into the membranes and restore membrane function and cellular health. We have tested our approach in in vitro 8

9 FRDA models and demonstrated that RT001 and other D-PUFAs prevent cell death and restore stressed cells to health. The human cell FRDA model is a primary human FRDA fibroblast cell line (Coriell designation GM3665) with GAA expansions of 790 and 1357 repeats in the first intron of the FRDA disease gene. The FRDA patient cells are presented with a toxic challenge of iron and BSO (A glutathione synthesis inhibitor) creating an environment similar to that of a FRDA patient cell. The vector control and cells exposed to normal fatty acids show high levels of lipid peroxidation (LPO) in membranes as seen with the BODIPY dye. RT001, RT002 return LPO to baseline and show low levels of lipid peroxidation in membranes. RT001 and RT002 Protect Against Peroxidation in Friedreich s ataxia Patient Cells: Wilson lab, UPENN, USA Fe + BSO Parkinson s disease: There is a substantial body of evidence linking lipid peroxidation to neurological disease, prompting mitochondrial loss of functions, and neurotoxicity that results in the onset or amplification of brain cell damage. An important mechanistic study showed that oligomeric α-synuclein, while toxic to a co-culture of neurons and astrocytes via an increase in lipid peroxidation in this in vitro model of Parkinson s disease, is non-toxic to those same cells in the presence of RT001. This establishes the role of RT001 in neutralizing damage from toxic triggers of ROS generation. MPTP is a neurotoxin precursor to MPP+, which causes permanent symptoms of Parkinson's disease by destroying dopaminergic neurons in the substantia nigra of the brain. It has been used to study disease models in various animal studies. In an in vivo orally dosed animal model of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced Parkinson s disease, RT001 statistically increased dopamine in the striatal nigra after 12 days dosing. 9

10 The MPTP mice were dosed for six days on diets with either 0.3 wt. % RT001/002 D-PUFAs (treated) or H-PUFAs (control) in diet. The mice then received either a sham saline injection or a single bolus MPTP injection, and followed for another six days on differential diets. After 12 days, the mice were sacrificed and striatal damage was assessed. Compared to the baseline, the RT001 treated mice had brain deuterium level increased by 14 fold indicating presence of RT001 in the brain. These mice had dopamine levels increased by more than 2.5 fold, a statistically significant increase that demonstrated RT001 protects against MPTPinduced striatal damage in this Parkinson s mouse model. (Toxicol Lett. 2011) An in-vitro cell study in Parkinson s disease (PD) looked at the effect of oligomeric a- synuclein (α-syn) in primary co-cultures of neurons and astrocytes (Angelova et al, 2015). In this important mechanistic study, data showed that oligomeric α-syn, while toxic to striatal nigra cells via increased lipid peroxidation, was non-toxic to those same cells in the presence of RT001, demonstrating that inhibition of lipid peroxidation with D-PUFAs protected cells from cell death induced by oligomeric α-syn. Thus, lipid peroxidation induced by misfolding of α-syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson s disease. Alzheimer s Disease: In yet another in vivo animal model, the laboratory of Mark Mattson, Chief of Neurosciences of the National Institute of Aging, did an extensive and well powered triple transgenic Alzheimer s mouse (3xTG mouse) model. After 4 month s dosing with either standard amounts of normal PUFA diets versus equivalent amounts of stabilized PUFA (RT001 and RT002) diets, extensive biochemical analysis of brain tissues was performed. The data shows that the toxic amyloid beta fragments 38, 40, and 42 characteristic of Alzheimer s disease, and oxidation damage markers consisting of isoprostanes and neuroprostanes were reduced. The data showed a consistent and statistically significant lowering of amyloid beta 38 and 40, and strong trends for lower amyloid beta 42 and the ratio of abeta 40/42. This correlated with lowered oxidation damage markers. 10

11 Disease Modifying D-PUFAs reduce amyloid beta in the hippocampus of 3xTG mouse model AB 38 * AB 40 * H-PUFA D-PUFA 0 H-PUFA D-PUFA Mattson lab, NIA, NIH 14 And Iso- and Neuro-prostanes (Ox stress markers) are also down-regulated Mattson lab, NIA, NIH, Milne lab, Vanderbilt 15 In addition IND-enabling toxicology was completed with the lead candidate, RT001, and no test article-related effects were noted in any parameter examined. The Maximum Tolerated Dose in Rats was 502 mg/kg/day after oral daily dosing for 26 weeks. Note that FDA considers Linoleic acid (non-deuterated) as Generally Recognized As Safe (GRAS) and has no upper limit under this designation. The FDA approved drug Intralipid (60% linoleic acid) is routinely dosed at levels greater than 10-fold the RT001 max g/day dose in adult. While RT001 is considered a novel chemical entity drug by FDA, it benefits from the strong safety profile of the non-deuterated parent fatty acid as both a nutrient and an approved IV drug. 11

12 Patent Protection Retrotope is the exclusive IP & leader in the field for isotopically modified fatty acids, lipid drugs, with over 20 patents filed and 6 issued or allowed. The summary of Retrotope s patent families is below. Summary Retrotope s transformational platform technology of stabilized lipid drugs, designed to halt lipid peroxidation, has the potential to treat a wide variety of other important diseases such as Parkinson s, Alzheimer s, diabetic retinopathy, and mitochondrial diseases. These diseases affect tens of millions of people, incurring billions of dollars in annual healthcare costs. Early clinical data is available under CDA from a first-in-human trial of RT001 in Friedreich s ataxia patients. 12

13 REFERENCES: Angelova et al. Journal of Neurochemistry 2015: Butterfield et al. Free Radical Biology and Medicine 2013 Cobb et al. Neurobiology of Disease Vol. 84, 2015:4 21 Hill S. et al. Free Rad Biol Med 2012 Keller MA, Piedrafita G, Ralser M. The widespread role of non-enzymatic reactions in cellular metabolism. Current Opinion Biotech. 2015:34; Packer L., Fuchs J. Vitamin E in health and disease. Marcel Decker, 1993 Ruipérez et al. Progress in Lipid Research Vol. 49, 2010: Toxicol Lett Nov 30;207(2): For more information, please contact: Dr. Robert Molinari, CEO bob@retrotope.com Retrotope, Inc El Camino Real, Suite 201 Los Altos, CA, USA 13