ZyStor’s lead product candidate, ZC-701, is an enzyme replacement therapy for the treatment of Pompe disease. ZC-701 is a chimeric human recombinant protein containing a signal peptide derived from human IGF-II, a GILT tag derived from human IGF-II, a three amino acid spacer and residues 70-952 of human GAA. IGF-II has the ability to bind with high affinity to the CI-MPR receptor, which is present on the surface of many mammalian cell types.1,2  As a result, a protein tagged with IGF-II targets the identical receptor targeted by M6P, thereby sharing the identical endocytic pathway for lysosomal targeting with M6P containing proteins.

Based on pre-clinical animal studies, ZC-701 has demonstrated the ability to clear glycogen effectively from a range of skeletal muscle tissues and greater potency than recombinant human GAA (“rhGAA” or “wtGAA”). Based on these and other studies, the Company believes ZC-701 can be administered at lower doses and will be shown to be more effective than Myozyme in the treatment of Pompe disease.

Pre-clinical Studies

ZyStor has evaluated the uptake kinetics into myoblasts of ZC-701 as compared to both Myozyme and wtGAA in animals. In these in vitro studies, the Company found the uptake kinetics of wtGAA to be almost identical to that of Myozyme. In contrast, ZC-701 was found to be taken up by myoblasts, a precursor type of muscle cells, almost 30 times more efficiently than Myozyme. The results of these studies are shown in the figure below.

In collaboration with Dr. Byrne, the Company conducted a multi-injection, two-dose, controlled animal study to evaluate the potential efficacy of ZC-701 in Pompe disease. The study compared 5 mg/kg and 20 mg/kg doses of ZC-701 with wtGAA of the same doses as well as a phosphate buffered saline (“PBS”) control. Four injections at weekly intervals were administered to nine-week old Pompe mice. Five mice per group were used in the study, representing 25 mice in total. The mice were sacrificed one week after the final injection. Tissues assayed included heart, diaphragm, quadriceps, soleus, TA, gastrocnemius, tongue and EDL. Soleus, TA, gastrocnemius and EDL are muscles in the calf. The assays measured the amount of glycogen in the tissue, and the results are shown in the figure below. In each tissue, ZC-701 was found to reduce glycogen more effectively than wtGAA, which in certain cases such as in the quadriceps, TA and EDL did not perform better than the PBS control. ZC-701 also demonstrated a dose response with the 20 mg/kg dose reducing glycogen by a larger amount than the 5 mg/kg dose in most of the tissues assayed. 5 mg/kg ZC-701 cleared more glycogen than did 20 mg/kg wtGAA in all tissues indicating at least four-fold greater potency. Data published by Zhu et al., Raben et al., and in an FDA filing suggest that Myozyme, which is currently approved at a 20 mg/kg dose, would require a dose of up to 100 mg/kg to reach the same level of efficacy that has been demonstrated by ZC-701 in this animal study.3,4,5  Moreover, 5 mg/kg of ZC-701 was shown to be more efficacious than 20 mg/kg of recombinant human GAA in all tissues.

ZyStor believes the results from this study demonstrate that ZC-701 clears glycogen effectively from a wide range of muscular tissues, is significantly more potent than wtGAA and is likely to be significantly more effective than Myozyme in treating Pompe disease patients.

Proof-of-Concept in MPS VII Murine Model

The ability of the GILT tag to serve as a lysosomal targeting agent for enzyme replacement therapy has been demonstrated in a study conducted by Jonathan H. LeBowitz, Ph.D. et al. using a murine model of MPS VII, a type of LSD. In the murine model, the MPS VII mice lack the lysosomal enzyme, murine â-glucuronidase (“mGUS”), and display a disease progression similar to that observed in human patients lacking human â-glucuronidase (“hGUS”). Multiple infusions of mGUS have been shown to be effective in improving many of the symptoms and their underlying causes in the MPS VII mice.6,7  The results of Dr. LeBowitz’s study were published in the Proceedings of the National Academy of Sciences in March 2004.8  This manuscript can be found at http://www.pnas.org/cgi/content/full/101/9/3083 
  
In the study, MPS VII mice were injected with 1 mg/kg of hGUS, hGUS-GILT or hGUS-GILT-F1 (where F1 is endoglycosidase F1 which removes most of the oligosaccharides, including all of the M6P) in 125 ìl of PBS control solution. hGUS and hGUS-GILT-F1 were each administered in six animals, while hGUS-GILT was given to seven animals. Additionally, six control animals received PBS alone. The animals were sacrificed 24 hours after injection, and the liver, spleen, kidney, heart, and lung were removed for biochemical and histochemical analysis. Also, to determine the effectiveness of hGUS and hGUS-GILT at reversing lysosomal storage pathology, three adult animals in each group were administered three weekly doses of 1 mg/kg of either hGUS, hGUS-GILT or PBS. These animals were sacrificed one week after the third injection, and the organs were removed for histopathology analysis with light or electron microscopy.

The study demonstrated that in the animals sacrificed after a single injection, hGUS-GILT was more effective at reaching kidney, heart and lung tissue than native hGUS and that hGUS-GILT-F1, which no longer showed any M6P-mediated uptake by fibroblasts, was delivered as effectively as native hGUS to the same tissues. The study also revealed that the liver tissue received comparable amounts of the three enzymes while the spleen received less of hGUS-GILT and even less of hGUS-GILT-F1 than native hGUS. This suggests that the removal of M6P diverts much of the enzyme from the reticuloendothelial cells. These findings are summarized in the figure below.

In the animals that received three weekly injections, the study examined a wide range of tissues, representing the same tissues that were examined in prior studies of enzyme replacement therapies in MPS VII mice, one week after the last dose. The study compared the tissues of the mice that received native hGUS versus hGUS-GILT and showed that both enzymes completely cleared storage material in several tissues. However, there were two notable differences in which the hGUS-GILT appeared to be more effective in clearing the storage material. First, in the kidney tissue, the glomerular visceral epithelial cells and the renal tubular cells had considerably less storage in the mice treated with hGUS-GILT. Second, in bone tissue, the osteoblasts were cleared almost completely in the mice treated with hGUS-GILT, while the mice treated with native hGUS showed only minimal or moderate reductions in storage material in the osteoblasts. The results of this examination are summarized in the figure below.

1  Morgan, D.O., Edman, J.C., Standring, D.N., Fried, V.A., Smith M.C., Roth, R.A. & Rutter, W.J. Nature. 1987. 329: 301-307.
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3  Zhu Y, Jiang JL, Gumlaw NK, Zhang J, Bercury SD, Ziegler RJ, Lee K, Kudo M, Canfield WM, Edmunds T, Jiang C, Mattaliano RJ, Cheng SH.  Glycoengineered Acid Alpha-Glucosidase with Improved Efficacy at Correcting the Metabolic Aberrations and Motor Function Deficits in a Mouse Model of Pompe Disease. Mol Ther. 2009 Jun;17(6):954-63.
4  Raben N, Danon M, Gilbert AL, Dwivedi S, Collins B, Thurberg BL, Mattaliano RJ, Nagaraju K, Plotz PH.  Enzyme Replacement Therapy in the Mouse Model of Pompe Disease. Mol Genet Metab. 2003 Sep-Oct;80(1-2):159-69.
5  Myozyme® Summary Basis for Approval. Pharmacology Reviews.  Available at: http://www.accessdata.fda.gov/drugsatfda_docs/nda/2006/125141s000_MyozymeTOC.cfm)..
6  Sands, M.S., Vogler, C., Kyle, J.W., Grubb, J.H., Levy, B., Galvin, N., Sly, W.S. & Birkenmeier, E.H. J. Clin. Invest. 2004. 93: 2324-2331.
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8  LeBowitz, J.H., Grubb, J.H., Maga, J.A., Schmiel, D.H., Vogler, C., Sly, W.S. PNAS. 2004. 101,9: 3083-3088.