Small-Cap Research - Semantic Scholar

May 22, 2013 Jason Napodano, CFA 312-265-9421 [email protected]

Small-Cap Research scr.zacks.com

111 North Canal Street, Chicago, IL 60606

Amarantus BioScience Holdings, Inc. AMBS: MANF and LymPro Present Massive Upside To the Amarantus Story Current Recommendation Prior Recommendation Date of Last Change Current Price (05/22/13) Target Price

Neutral N/A 02/01/2013 $0.04 $0.25

(AMBS-OTC)

INITIATION We are initiating coverage of Amarantus Bioscience with a Neural rating and $0.25 target. First among the key points of our recommendation is noting the risk inherent in investing in any stock trading this low in price hence the Neutral rating. That said, we do not take target prices lightly, and our $0.25 target clearly points to massive upside in the shares. It is clear to us after a thorough review of the science around both MANF and LymPro that there is significant opportunity in the shares, and the current price of $0.04 a market capitalization of around $16 million vastly under-appreciates this potential. We originally became attracted to the story because of MANF for Parkinson s disease, but now believe orphan indications around ischemic brain damage present meaningful upside to the story. Likewise, LymPro could be a game-changing in the Alzheimer s diagnostic market.

SUMMARY DATA 52-Week High 52-Week Low One-Year Return (%) Beta Average Daily Volume (sh)

$0.17 $0.01 -65 -0.05 2,605,962

Risk Level Type of Stock Industry

Ultra High / Speculative Small-Growth Biotech

ZACKS ESTIMATES Shares Outstanding (mil) Market Capitalization ($mil) Short Interest Ratio (days) Institutional Ownership (%) Insider Ownership (%) Annual Cash Dividend Dividend Yield (%)

384 $16 N/A 0 5 $0.00 0.00

5-Yr. Historical Growth Rates Sales (%) Earnings Per Share (%) Dividend (%)

N/A N/A N/A

P/E using TTM EPS

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P/E using 2013 Estimate P/E using 2014 Estimate

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Revenue (In millions of $)

Q1 (Mar)

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Year (Dec)

2012

0A

0A

0A

0E

0E

2013

0A

0E

0E

0E

0E

2014

0E

2015

0E

Earnings per Share (EPS is operating earnings before non-recurring items)

2012 2013 2014 2015

Q1 (Mar)

Q2 (Jun)

Q3 (Sep)

Q4 (Dec)

Year (Dec)

-$0.02 A -$0.01 A

-$0.01 A -$0.00 E

-$0.00 A -$0.00 E

-$0.00 E -$0.00 E

-$0.03 E -$0.01 E -$0.01 E -$0.01 E

© Copyright 2013, Zacks Investment Research. All Rights Reserved.

Amarantus BioScience Holdings, Inc. is a holding company for two business unit organization that includes Amarantus Therapeutics, a focused biotechnology company with a preclinical therapeutic candidate for the treatment of Parkinson s and ischemic diseases, and Amarantus Diagnostics, a diagnostic company developing a blood test for Alzheimer s and Parkinson s disease. Below we discuss each division and why we believe the sum of parts is an attractive investment opportunity despite the low stock price.

An Introduction To Amarantus Therapeutics Amarantus Therapeutics lead development program is a Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) currently in preclinical testing for neurodegenerative diseases, specifically Parkinson s disease, and in reducing ischemic damage and promoting behavioral recovery post stroke or a traumatic brain injury. MANF is a 20 kDa, recombinant, secreted human protein with specific neuroprotective selectively for dopaminergic neurons, but not for gabaergic or serotonergic neurons. Research shows that MANF supports dopaminergic neuron survival greater than glial cell line-derived neurotrophic factor (GDNF), the current gold standard and most extensively studied neurotrophic factor for neuroprotection, in the ventral midbrain. Scientific research shows that MANF selectively promotes survival of dopaminergic neurons and is neuroprotective in transient focal ischemia through inhibition of cell apoptosis and necrosis. MANF is upregulated in unfolded protein response (UPR) and reduces endoplasmic reticulum (ER) stress-induced cell death. In fact, data suggest MANF is superior to Tau in preventing cell apoptosis in UPR. In both Drosophila and rodent models of Parkinson s disease, data is suggestive of a neuroprotective and neurorestorative effects. Specifically, Drosophila mutants with complete loss of MANF demonstrate a total loss of dopaminergic neurons and eventual cell death, as well as membrane traffic alterations. Studies show human MANF can be added to rescue mutant Drosophila, confirming a conserved evolutional role for the molecule. This is of particular interest for those in the development of therapeutic agents for the treatment of Parkinson s disease, which is characterized by widespread neuroregeneration, particularly of dopaminergic neurons that arise in the substantia nigra (SN) region of the brainstem and innervate the striatum. Parkinson s disease (PD) is a neurodegenerative brain disorder that results from the death of dopamine-generating cells in the substantia nigra (SN) region of the midbrain. PD is also characterized by the accumulation of a protein called alpha-synuclein into inclusions called Lewy bodies in neurons. The cause of PD is generally idiopathic, although some atypical cases have a genetic origin. PD patients often exhibit marked reduction in motor control and an increase in tremors, hypokinesia, rigidity, bradykinesia, and postural instability (parkinsonism). However, as the disease progresses, patients often exhibit non-motor symptoms that include autonomic dysfunction, neuropsychiatric problems (mood, cognition, behavior or thought alterations, psychosis), and sensory and sleep difficulties. In humans and animals, dopamine is a neurotransmitter; a chemical released by dopaminergic neurons to send signals to other neurons. Dopaminergic neurons are the cells responsible for releasing dopamine, and degenerate in the substantia nigra of Parkinson s patients. This degeneration causes nerve terminals from the striatum to retract towards their cells bodies in the substantia nigra. The loss of dopaminergic nerve terminals in the striatum leads to reduced dopamine levels in the striatum and a worsening of the disease. There is no cure for PD. Instead, physicians attempt to manage the symptoms of the disease through a multidisciplinary approach that may include pharmacological, social, and surgical options. The most common pharmaceutical treatment options are those with look to increase the level of dopamine in the brain. These include dopamine replacement therapies (DRT) combined with dopa-decarboxylase inhibitors, dopamine agonists, and MAO-B inhibitors. The treatment option is often tailored specifically for the patient based on the stage and severity of the disease and the balance between good symptom control and side-effects resulting from enhancement of dopaminergic function. Amarantus MANF, a neurotrophic factor under development for the treatment of Parkinson s disease, offers and unique, non-DRT approach, with the promise of safely and effectively protecting dopamine producing neurons from death and rejuvenating dying cells to ultimately stop the progress of Parkinson s disease and restore normal function to patients.

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Review Of Scientific Literature Suggests MANF Has Meaningful Potential We have reviewed twenty scientific papers in an attempt to better understand the mechanism of action, preclinical data, a commercial potential for Amarantus Therapeutics Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF). Below is a review of those papers, in chronological order from the date they were first published. Paper # 1 Petrova PS, Raibekas A, Pevsner J, Vigo N, Anafi M, Moore MK, Peaire AE, Shridhar V, Smith DI, Kelly J, Durocher Y, Commissiong JW. (April 2003) MANF: A New Mesencephalic, Astrocyte-Derived Neurotrophic Factor with Selectivity for Dopaminergic Neurons. Journal of Molecular Neuroscience, Vol.20/2:173-187. The Petrova et al. paper above was the first characterization we found on MANF. Petrova et al. initially derived the homologous native molecule from a rat mesencephalic type-1 astrocyte cell line and recombinant MANF subcloned from a cDNA encoding human arginine-rich protein. The group discovered MANF selectively protects nigral dopaminergic neurons, versus GABAergic or serotonergic neurons. In fact, the MANF-mediated survival was quite pronounced. For example, when compared to glial cell line-derived neurotrophic factor (GDNF), at the time the gold standard for dopaminergic neuroprotection, and brain-derived neurotrophic factor (BDNF), Petrova et al. found that MANF was more selective in the protection (i.e. percentage survival) of dopaminergic neurons at lower (0.05 0.25 ng/mL) and middle (0.5 2.5 ng/mL) concentrations (MANF>GDNF>BDNF). GDNF was more selective then MANF at higher concentrations (25 50 ng/ml). The paper concludes that the selective, neuroprotective effect of MANF for dopaminergic neurons is suggestive of utility in the treatment of Parkinson s disease.

Paper # 2 Petrova PS, Raibekas A, Pevsner J, Vigo N, Anafi M, Moore MK, Peaire A, Shridhar V, Smith DI, Kelly J, Durocher Y, Commissiong JW. (2004) Discovering Novel Phenotype-Selective Neurotrophic Factors to Treat Neurodegenerative Diseases. Progress in Brain Research, Vol.146: 168-183. Petrova et al. came back in 2004 with their second paper on MANF. The group identifies the challenges of developing a therapeutic candidate for Parkinson s disease, noting there has yet to be a dramatic breakthrough in treatment options because of a lack of fundamental understanding for the cause of the disease. The paper points to two essential requirements for an effective treatment: arresting the further neuronal death after diagnosis and protecting the remaining dopaminergic neurons over the long-term. Results of the research presented in the paper imply that the death of dopaminergic neurons in the substantia nigra is a discrete specific event in Parkinson s patients. A therapeutic candidate, such as MANF, which has been shown to offer selective neuroprotection of dopaminergic neurons, could be an effective treatment option. The authors even predict that the concept of homotypic, astrocyte-derived neurotrophic factors from the mesencephalon will likely prove to be important in the future treatment of the disease. However, the paper notes the future challenges of transferring this cellular and molecular mechanism to an in vivo clinical setting, and cautions that patients with long-standing Parkinson s disease, who have lost 100% of their dopaminergic neurons, will likely not benefit from such a treatment option.


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Paper # 3 Zhou C, Xiao C, Commissiong JW, Krnjevic K, Ye JH. (December 2005). Mesencephalic AstrocyteDerived Neurotrophic Factor Enhances Nigral [gamma]-Aminobutyric Acid Release. NeuroReport Vol.17:293-297. Zhou et al. provide their findings on the effects of MANF on -aminobutyric acid type A (GABAA) receptor-mediated inhibitory postsynaptic currents. The paper provides evidence that MANF modulates GABAergic transmission to the dopaminergic neurons of the substantia nigra. The group suggests the mechanism of action is presynaptic, with a metabolic process within the nerve terminals as evidenced by the slow onset and offset of action. It is noted that other neurotrophic factors, such as GDNF and BDNF through distinct mechanisms, also modulate synaptic transmission by increasing transmitter release. Zhou et al. concludes that MANF is much more potent as an enhancer of GABAergic synapses on nigral dopaminergic neurons than other neurotrophins that also act presynaptically, and that this GABAmediated inhibition of dopaminergic neurons cell firing may contribute to its protective mechanism of action. Paper # 4 Dauer, William (November 2007). Neurotrophic Factors and Parkinson s Disease: The Emergence of a New Player? Science STKE Vol.411, pe60. Dauer focuses his paper on conserved dopamine neurotrophic factor (CDNF), a homolog of MANF, and concludes that CDNF offers similar neuroprotection to GDNF, the gold standard and most extensively studied neurotrophic factor for neuroprotection. The paper goes into a brief background on GDNF and BDNF. BDNF was the first protein identified to directly support the survival of dopamine neurons in vitro, but the effects did not transfer to in vivo studies. GDNF demonstrates highly effective protection of dopaminergic neurons both in vitro and in vivo, but has two key issues that limit the commercial viability of the agent. Firstly, the mode of delivery remains unclear because the required continuous high doses may lead to receptor down-regulation. And secondly, some patients may develop neutralizing antibodies that could limit the utility and conceivably lead to serious side effects. Dauer notes, after conducting a simple study inducing midbrain dopaminergic neuron cell death in rodents through injection of a neurotoxic agent, 6-hydroxydopamine (6-OHDA) into the striatum, that CDNF does confer neuroprotection similar to GDNF. However, the author concludes that there is little evidence at this stage to suggest CDNF will have superior commercial potential than GDNF until additional studies are conducted. Paper # 5 Apostolou A, Shen Y, Liang Y, Lou Y, Fang S. (May 2008). Armet, a UPR-Upregulated Protein, Inhibits Cell Proliferation and ER Stress-Induced Cell Death. Experimental Cell Research Vol.14:2454-2467. Apostolou et al. begin their detailed paper with an overview of the endoplasmic reticulum (ER), an organelle responsible for folding and modification of proteins destined for the secretory pathway. Previous research concludes that the accumulation of misfolded proteins in the ER causes stress that initiates the unfolded protein response (UPR). This response, through both adaptive and apoptotic mechanisms, contributes to disease pathogenesis, specifically in Parkinson s disease. The authors found that Armet (arginine-rich, mutated in early stage tumors), the previous name for MANF before it had been better characterized, is up-regulated in UPR, and may have a protective role against 6-OHDA in a Parkinson s disease rodent model. The authors point to the previous work done by Lindholm et, published in Nature Vol. 448 (2007) noting the protective role of CDNF (Armet-L1), and the similarities between CDNF and MANF. The group also speculates that MANF may play a role in facilitating the removal of misfolded proteins from the ER by enhancing protein folding and/or degradation. Paper # 6 Lindholm P, Andressoo J-O, Kalkkinen N, Kokaia Z, Lindvall O, Timmusk T, Peranen J, Saarma M (July 2008). MANF is Widely Expressed in Mammalian Tissues and Differently Regulated After Ischemic and Epileptic Insults in Rodent Brain. Molecular and Cellular Neuroscience. Vol.39:356-371. The Lindholm et al. manuscript is a detailed characterization through situ hybridization and immuno-histochemistry of MANF expression. Relatively high levels of MANF were detected in the cerebral cortex, hippocampus and cerebellar Purkinje cells of both developing and adult mice. The group found that MANF expression was widespread in the hippocampal formation and more restricted in cerebral cortex following global forebrain ischemia. Thus, the widespread expression of MANF following ischemic and epileptic brain insults (associated with neuronal death) suggest that it has important functions both under normal and pathological conditions in many tissue types. The paper also elaborates on a Mizobuchi et al. paper from 2007 that characterizes MANF as an ER resident protein. Lindholm et al. widen the reach of MANF, noting that the protein was detected in the culture media and transiently transfected cells during their studies, showing that MANF does not only reside in the ER, but that it is also secreted from the cells. The group also found MANF to be present in trigeminal and DRG ganglia of embryonic mice, suggesting that it has a function in the development of these ganglia in vitro. The paper concludes by suggesting MANF has a larger role than previously believed as just a neurotrophic factor.


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Paper # 7 Tadimalla A, Belmont PJ, Thuerauf DJ, Glassy MS, Martindale JJ, Gude N, Sussman MA, Glembotski CC (October 2008). Mesencephalic Astrocyte-Derived Neurotrophic Factor Is an Ischemia-Inducible Secreted Endoplasmic Reticulum Stress Response Protein in the Heart. Circulation Research Vol.103:1249-1258. The Tadimalla et al. paper published in Circulation was the first to conclude potential utility of MANF in protecting cardiac myocytes following a myocardial ischemia. The group studied the effects of MANF on preventing or repairing ischemic damage through induced myocardial ischemia / reperfusion. Myocardial ischemia activates an endoplasmic reticulum stress response, which in turn activates a transcription factor, ATF6, which induces expression of MANF. Results show the activated MANF, induced by external ATF6 activation, protects myocytes from apoptosis. Extracellular recombinant MANF (r-MANF) has a similar cardioprotective effect. Furthermore, knockdown of endogenous MANF with micro-RNA increased cell damage and / or death, confirming MANF s role in cardioprotection. The group concludes that MANF functions in an autocrine / paracrine manner to modulate myocardial damage from ER stress, including ischemia; and that the role of MANF in the heart should be further explored.

Paper # 8 Palgi M, Lindstrom R, Peranen J, T. Piepponen P, Saarma M, Tapio I. Heino TI (December 2008). Evidence that DmMANF is an Invertebrate Neurotrophic Factor Supporting Dopaminergic Neurons. Proceedings of the National Academy of Sciences. Vol.106: 2429 2434. The Palgia et al. paper is the first we reviewed that supports evidence of MANF in invertebrates, specifically in Drosophila ( fruit flies ). The group demonstrates that DmMANF is required in Drosophila embryogenesis for the maturation of the nervous system. Furthermore, DmMANF null mutants revealed a total loss of dopaminergic neuritis and drastic reduction in dopamine levels, followed by subsequent nonapoptotic cell death. Palgia et al. then successfully rescued these mutant DmMANF null flies by the addition of human MANF, finding that the ability of human MANF to replace the function of DmMANF in Drosophila suggest a conserved evolutional role for NTFs, and that human MANF is the ortholog of the Drosophila DmMANF gene. Thus, this makes the Drosophila model very attractive to study the MANF signaling pathway for the treatment of Parkinson s disease. Paper # 9 Parkash V, Lindholm P, Peranen J, Kalkkinen N, Oksanen E, Saarma M, Leppanen VM, Goldman A (March 2009). The Structure of the Conserved Neurotrophic Factors MANF and CDNF Explains Why They Are Bifunctional. Protein Engineering, Design & Selection. Vol.22: 233-24. Parkash et al. report on the structures of both MANF and CDNF through crystallography, finding that neither protein structure resembles any known growth factor. The group finds that the N-terminal domain is a saposin-like lipidbinding domain responsible for binding lipids or membranes (allowing membrane bonding). The natively unfolded MANF C-terminus contains a CKGC disulphide bridge, such as reductases and disulphide isomerases, consistent with a role in ER stress response. The authors conclude that this unique structure explains the bifunctional role of MANF and CDNF, ascribing the neuroprotective function to the N-terminal SAPLIP domain, and cytoprotection and involvement in the ER stress response to the natively unfolded C-terminal CXXC domain. Paper # 10 Airavaara M, Shen H, Kuo C-C, Peränen J, Saarma M, Hoffer B, Wang Y (2009). Mesencephalic Astrocyte-Derived Neurotrophic Factor Reduces Ischemic Brain Injury and Promotes Behavioral Recovery in Rats. The Journal of Comparative Neurology. Vol.515: 116-124. The Airavaara et al. paper was perhaps the best proof-of-concept animal data generated with MANF to date. The author examined the protective effects of various concentrations of MANF (0, 1, 6, 12, 24 g in phosphate buffered saline vehicle) in rats following an induced ischemia brain injury via middle cerebral artery occlusion (MCAo). We provide a summary of the findings below:


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Cerebral infarction volume was significantly reduced in rats pretreated with MANF (6 g was best and highly statistically significant vs. phosphate buffered saline control). Similarly, the area of largest infarction was significantly reduced by MANF at a dose of 6 g. Pretreatment with MANF reduced TUNEL labeling, suggesting that MANF may have antiapoptotic activity close to the site of injection. The authors theorize that the protective effect of MANF is mediated through inhibition of NDA fragmentation. MANF promoted behavioral recovery after MCAo induced ischemia on days 2, 7, and 14. MANF did not alter cortical blood flow or systemic blood pressure, blood gases, electrolytes, body or brain temperature, suggestive of good tolerability at an efficacious dose. Airavaara et al. conclude that MANF has neuroprotective effects against CNS injury in a transient focal ischemia model. The protective response of MANF may involve the inhibition of apoptosis / necrosis. The effectiveness of MANF pretreatment in stroke may be clinically useful for patients susceptible to ischemic events, for example, those suffering from transient ischemic attacks. Furthermore, because MANF expression is enhanced after brain ischemia, and overexpression of MANF reduces ER stress, it is possible that MANF is involved in endogenous protection against ischemia in patients.

Paper # 11 Yong-Qiang Yu, Lian-Cheng Liu, Fa-Cai Wang, Yan Liang, Da-Qin Cha, Jing-Jing Zhang, Yu-Jun Shen, Hai-Ping Wang, Shengyun Fang and Yu-Xian Shen (September 2009). Induction Profile of MANF/ARMET by Cerebral Ischemia and its Implication for Neuron Protection. Journal of Cerebral Blood Flow & Metabolism. September 23:1 13. The authors of this paper conducted a similar experiment and analysis to the Airavaara et al. paper discussed above. Yong-Qiang et al. induced cerebral ischemia in rodents via middle cerebral artery occlusion (MCAo). The group discovered that MANF/ARMET is extensively upregulated in ischemic regions in response to the ischemic insult, and that expression is earlier and more sensitive to ischemic stimulation than C/EBP homologous protein (CHOP). The authors also found that ARMET promoted neuron proliferation and prevented neuron apoptosis induced by tunicamycin. Finally, Yong-Qiang et al. studied the ability of MANF/ARMET to prevent ER stressinduced apoptosis through TUNEL staining (a method for detecting DNA fragmentation). The authors discovered statistically less TUNEL-positive cells in the ARMET-treated neurons group vs. the negative control (bovine serum albumin) and an active comparator in Tau. Tau is widely believed to modulate the stability of axonal microtubules, but the authors show that MANF/ARMET is far superior to Tau in preventing cell apoptosis.


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Paper # 12 Voutilainen M, Back S, Porsti E., Toppinen L, Lindgren L, Lindholm P, Peranen J, Saarma M and Tuominen R (July 2009). Mesencephalic Astrocyte-derived Neurotrophic Factor is Neurorestorative in a Rat Model of Parkinson s Disease. Journal of Neuroscience, Vol.29(30):9651 9659. Voutilainen et al. examined whether MANF had neuroprotective and neurorestorative effects in an experimental model of Parkinson s disease in rats. The authors also studied the distribution and transportation of intrastriatally injected MANF in the brain and compared it with glial cell line-derived neurotrophic factor (GDNF). The paper shows that intrastriatally injected MANF protected nigrostriatal dopaminergic nerves from 6-OHDA-induced degeneration as evaluated by counting tyrosine hydroxylase (TH)-positive cell bodies in the substantia nigra (SN) and TH-positive fibers in the striatum.

More importantly, the authors found that MANF also restored the function of the nigrostriatal dopaminergic system when administered either 6 hours before or 4 weeks after 6-OHDA administration in the striatum. Plus, MANF was distributed throughout the striatum more readily than GDNF. The mechanism of MANF action differs from that of GDNF because intrastriatally injected MANF was transported to the frontal cortex, whereas GDNF was transported to the SN. Thus, the authors hypothesize that superior protection may be achieved with various neurotrophic factors. The conclusion of the paper is that MANF is readily distributed throughout the striatum and has significant therapeutic potential for the treatment of Parkinson s disease.

Paper # 13 Lindholm P, Saarma M (September 2009). Novel CDNF/MANF Family of Neurotrophic Factors. Developmental Neurobiology. Vol.70:360-371. In this paper, Lindholm summarizes the recent research on MANF. The author comments on the structural work conducted by Parkash et al., noting the crystal structure analysis revealed that CDNF and MANF consist of two domains; an amino-terminal saposin-like domain that may interact with lipids or membranes, and a presumably unfolded carboxy-terminal domain that may protect cells against endoplasmic reticulum stress. Lindholm notes work done by Airavaara et al. showing CDNF and MANF protect midbrain dopaminergic neurons and restore motor function in 6-OHDA rat model of Parkinson s disease. The paper summarizes work done by Voutilainen et al. showing the greater repair efficiency of MANF vs. GDNF based on diffusion, and hypothesizes that CDNF may offer similar potential if tested. Lindholm believes the efficient diffusion of MANF may be of critical importance for its possible clinical use, since growth factors delivered to human brain have to diffuse significant distances to reach all target cells.


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MANF Offers Improved Diffusion Volume vs. GDNF

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Paper # 14 Airavaara M, Chiocco MJ, Howard DB, Zuchowski KL, Peränen J, Liu C, Fang S, Hoffer BJ, Wang Y, Harvey CA (June 2010). Widespread Cortical Expression of MANF by AAV Serotype 7: Localization and Protection Against Ischemic Brain Injury. Experimental Neurology. Vol.225:104-113. This article by Airavaara et al. is interesting because it discusses the potential for yet another indication for MANF, this time in stroke. The authors infect adult rats by injecting adeno-associated virus (AAV) serotype 7 encoding MANF into the cerebral cortex, then induce ischemia by right middle cerebral artery (MCA) ligation a week later. The group conducted behavioral monitoring by using body asymmetry analysis, neurological testing, and locomotor activity. Data showed that AAV-induced MANF expression is redistributed in neurons and glia in cerebral cortex after ischemia, and that pretreatment with AAV-MANF reduced the volume of cerebral infarction and facilitated behavioral recovery. Airavaara et al. conclude that intracortical delivery of AAV-MANF increases MANF protein production, which reduces ischemic brain injury and promotes behavioral recovery in rats.

Paper # 15 Glembotski CC (October 2010). Functions for the Cardiomyokine, MANF, in Cardioprotection, Hypertrophy and Heart Failure. Journal of Molecular and Cellular Cardiology. Vol.51(4):512-517. Glembotski defines cardiomyokines as heart-derived secreted proteins that affects cardiovascular function autocrine, paracrine and/or endocrine mechanisms. Previous research demonstrates that expression of MANF may play an important role in facilitating myocardial survival from ischemic injury, as well as modulating cardiac hypertrophy and heart failure. Thus, the author considers MANF to be a cardiomyokine. This novel function of MANF suggests that it may have important roles in the ER stressed ischemic heart. Additional work shows this protection has been seen in multiple cell and tissue types, including the ischemic brain, as well as in animal models of neurodegenerative disorders such as Parkinson s disease. Finally, Glembotski also notes that MANF overexpression decreases proliferation of cultured cancer cells, and decreases cardiomyocyte hypertrophy. As such, a MANF-based cancer therapy is conceivable. Paper # 16 Voutilainen MH, Bäck S, Peränen J, Lindholm P, Raasmaja A, Männistö PT, Saarma M, Tuominen RK. (December 2010). Chronic Infusion of CDNF Prevents 6-OHDA-Induced Deficits in a Rat Model of Parkinson s Disease. Experimental Neurology. Vol.228:99-108. In previous work on the subject, the author notes MANF is distributed throughout the striatum more readily than GDNF, and hypothesizes that CDNF offers a similar potential. To test this hypothesis, Voutilainen et al. study the effects of two-week infusions of CDNF, MANF and GDNF in a rat 6-OHDA Parkinson model. The authors found that both MANF and CDNF have potential as a neuroprotective or even neurorestorative therapies Parkinson s. Paper # 17 Hellman U, Arumäe U, Yu L, Lindholm P, Peränen J, Saarma M, Permi P (November 2010). Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) Has a Unique Mechanism to Rescue Apoptotic Neurons. The Journal of Biological Chemistry. Vol.286:2675-2680. The team behind this paper used NMR spectroscopy to solve a three-dimensional solution of full-length MANF as well as its isolated C-terminal domain, dubbed C-MANF. The group discovered that individual N- and C-terminal domains are well defined, with the N-terminal domain homologous (both share closed left fold) to the lipid and membrane binding protein superfamily, saposins. Hellman et al. found that the C-terminal has an independent functional role similar to that of Ku70, which has been shown to inhibit the proapoptotic Bax and prevent mitochondrial cell death signaling.


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MANF Structure

The paper published by Hellman et al. supports the intracellular mode of action of MANF. The team concludes that MANF is an exceptional neurotrophic factor that can protect the cells both intracellularly and in vivo extracellularly. Paper # 18 Palgi M, Greco D, Lindström, R, Auvinen P, Heino TI, (April 2012). Gene Expression Analysis of Drosophilaa MANF Mutants Reveals Perturbations in Membrane Traffic and Major Metabolic Changes. BMC Genomics. Vol.13:134-170. Palgi et al. attempt to clarify the role of MANF in multicellular organism development by conducting microarraybased analysis of the transcriptional changes induced by the loss and overexpression in Drosophila (fruit flies). The group notes the most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal / zygotic and only zygotic MANF mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. Palgi el al. conclude that besides a neurotrophic factor, MANF is an important cellular survival factor needed to overcome the unfolded protein response (UPR), especially in tissues with high secretory function. In the absence of MANF, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as Parkinson s disease, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD, and this paper provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons. Paper # 19 Oh-Hashi K, Tanaka K, Koga H, Hirata Y, Kiuchi K (November 2011). Intracellular Trafficking and Secretion of Mouse Mesencephalic Astrocyte-derived Neurotrophic Factor. Molecular and Cellular Biochemistry.Vol.363: 35-41. Oh-Haski et al. identified two variants of MANF mRNA, a wild type (wt-MANF) that contains exon-1 and one presumably not secreted ( N-MANF) in neuro2a-cells that lacks exon-1. The paper shows that the amount of intracellular -MANF was much lower than that of wt-MANF. Furthermore, -MANF was not detected in the culture medium after its transient transfection into Neuro2a cells. The authors conclude that the secretion of MANF is regulated via COPII-mediated transport and that its C-terminus could be responsible for its retention in the endoplasmic reticulum (ER) through GRP78. The alternate variant, -MANF, may be less stable in cells than wtMANF and may not be secreted extracellularly. This has potential implications in the potential onset and progression of cell death, and how a commercial therapeutic could be developed from recombinant MANF. Paper # 20 Shen Y, Sun A, Wang Y, Cha D, Wang, H, Wang F, Feng L, Fang S, Shen Y. (November 2012). Upregulation of Mesencephalic Astrocyte-derived Neurotrophic Factor in Glial Cell is Associated with IschemiaInduced Glial Activation. Journal of Neuroinflammation. Vol.9:254-275. The Shen et al. paper is the first we ve seen to characterize MANF expression in the different types of glial cells. The results show that, unlike its name 'mesencephalic astrocyte-derived neurotrophic factor' suggests, the astrocytes were not the major source of MANF in the brain tissue, and that MANF-positive cells were neurons, not astrocytes. That being said, the authors discovered that severe cerebral ischemia could induce MANF expression in astrocytes and oligodendrocytes. This is important because astrocyte activation is one of the key components of cellular responses to brain injuries and neurodegeneration. The authors also note that MANF expression was easily induced by ER stress inducers and nutrition deprivation, confirming work done by others suggesting that expression of MANF in glial cells is stress inducible. The group s previous work shows that recombinant human MANF was protective to neurons, suggesting that induction of MANF is probably protective to neural cells. The paper concludes that characteristics of MANF expression in different types of glial cells suggest that upregulated MANF expression is associated with activated glial cells, which will helps to understand the function in ischemia-induced neural injury.


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Amarantus Shows MANF is Superior to GDNF In Parkinson s Model The Petrova et al. (April 2003) paper noted above was the first to characterize mesencephalic astrocyte-derived neurotrophic factor (MANF) in comparison to glial cell-derived neurotrophic factor (GDNF). Previous work on GDNF yielded encouraging results. GDNF has shown therapeutic potential in rodent (Hoffer et al.,1994, Tomac et al.,1995, Kearns and Gash,1995), and primate (Gash et al.,1996, Zhang et al.,1997) models of PD. Unfortunately, work with GDNF never progressive to pivotal human studies due to the intracerebral delivery method and the fact that the drug was poorly distributed in the brain, specifically at the substantia nigra (SN) where dopaminergic neurons begin to die in patients with Parkinson s disease. As a result of the failure to adequately deliver the drug, companies like Amgen, Biovail, and organizations like the Michael J. Fox Foundation (MJFF) have taken a step-back from GDNF. Privately-held Canadian MedGenesis acquired the exclusive rights to GDNF from Amgen in 2010. MedGenesis was also previously working with Biovail and MJFF on GDNF development, but the collaboration ended in 2010 following the failure of a phase 2 (n=34) study in PD patients, most likely the result of poor delivery of the drug to the target tissue in the putamen and limited technology available at that time to provide for accurate, precise and verified distribution of the therapeutic agent across a therapeutically relevant fraction of the target structure within the brain. We note previous open-label work with GDNF in phase 1 (n=15) yielded encouraging results. MedGenesis is refocusing its efforts with GDNF with a convection enhanced delivery (CED) platform. CED is designed to expose the brain parenchymal target tissues with GDNF by facilitating a wider, more consistent, more homogenous drug distribution, and longer drug exposure across the blood-brain barrier. MedGenesis raised approximately $5 million in January 2012, also recently secured a grant (undisclosed amount) from the Deutsche Parkinson Vereinigung e.V. The company is reconducting the phase 2 study with data expected later in 2013. We find it highly encouraging from Amarantus standpoint that MedGenesis can secure $5 million in investorfunding and a grant from the Deutsche Parkinson Vereinigung e.V for GDNF considering the previous failure in human studies. It is a testament to need for treatment options and the desire of investors and institutions to develop a cure using neurotrophic factors. We are particularly interested in the outcome of the second phase 2 study with GDNF via CED. We think success could spark a major re-valuation of MANF in the eyes of investors. Amarantus has done considerable work over the past few quarters demonstrating the superiority of MANF to GDNF. Specifically, on January 9, 2013, Amarantus presented positive preclinical efficacy data for MANF in a neurorestoration 6-hydroxydopamine (6-OHDA) rat model of Parkinson s disease. The data demonstrate that unlike GDNF, MANF significantly reduces behavioral deficits, increases dopaminergic nerve terminal reinnervation of the striatum, and increases dopamine concentrations in the striatum when MANF is administered directly to the substantia nigra (SN). This research is in contrast to work done by Voutilainen et al. in 2010 suggesting that only CDNF shows a meaningful benefit in the 6-OHDA rat model of PD (side note: we believe Voutilainen et al. have ulterior motives with respect to highlighting the potential of CDNF vs. other neurotrophic factors). Background In humans and animals, dopamine is a chemical released by dopaminergic neurons to send signals to other neurons, and is known as a neurotransmitter. Dopaminergic neurons are the cells responsible for releasing dopamine, and degenerate in the SN of Parkinson s disease patients. This degeneration causes dopaminergic nerve terminals from the striatum to retract towards their cells bodies in the SN. The loss of these nerve terminals in the striatum leads to reduced dopamine levels in the striatum. Therefore, drug treatment to the SN that increases innervation of the striatum is critical as a basis for functional recovery in Parkinson s disease. In the study, rodents were lesioned with 6-OHDA on one side of their brain (t = 0). Behavior was tested for baseline (t =1 week) and vehicle, MANF (3 g, 10 g or 36 g) and GDNF (10 ug) were injected in different groups of animals at t = 2 weeks. Behavior was tested for drug effect at t = 4 weeks (2 weeks post-treatment) and at t = 6 weeks (4 weeks post-treatment).


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The data produced comparing optimal doses to the SN of MANF = 10 g and GDNF = 10 g in this model indicate the following at four weeks post-treatment: MANF reduced behavioral deficits by 53%, whereas behavioral deficits with GDNF increased by 20%; MANF produced a 14.4% reinnervation of the striatum, whereas striatum innervation with GDNF was reduced by 9.9%; MANF increased dopamine concentrations in the striatum, whereas striatum dopamine concentrations with GDNF did not increase.

The behavioral data achieved statistical significance (p