US007.404951B2 (12) United States Patent (10) Patent No.: US 7.404,951 B2 Teichberg (45) Date of Patent: Jul. 29, 2008 (54) METHOD AND COMPOSITION FOR DiGiorgio et al. "Gabaergic Systems in Brain Regions of Glutamate PROTECTING NEURONAL TISSUE FROM Lesioned Rats', Italien Journal of Biochemistry, 34(1): 19-28, 1985. DAMAGE INDUCED BY ELEVATED p. 19, Line 15. GLUTAMATE LEVELS Engelhardt etal. “The Diagnostic Value of Enzyme Determination in Cerebrospinal Fluid'. Medizinische Klinik, München, 71 (17): 699 (75) Inventor: Vivian I. Teichberg, Savyon (IL) 702, 1976. p. 701. Andrae etal. “Pyruvate and Related O-Ketoacids ProtectMammalian (73) Assignee: Yeda Research And Development Co. Cells in Culture Against Hydrogen Peroxide-Induced Cytotoxicity'. Ltd., Rechovot (IL) Toxicology Letters, 28:93-98, 1985. Avramis et al. "A Randomized Comparison of Native Escherichia (*) Notice: Subject to any disclaimer, the term of this coli Asparaginase and Polyethelyne Glycol Conjugated patent is extended or adjusted under 35 Asparaginase for Treatment of Children With Newly Diagnosed U.S.C. 154(b) by 95 days. Standard-RiskAcute Lymphoblastic Leukemia: A Children's Cancer Group Study”. Blood,99(6): 1986-1994, 2002. (21) Appl. No.: 10/522,415 Cavallini et al. “The Protective Action of Pyruvate on Recovery of Ischemic Rat Heart: Comparison With Other Oxidizable Substrates'. (22) PCT Filed: Jul. 31, 2003 Journal of Molecular Cell Cardiology, 22: 143-154, 1990. Desagher et al. “Pyruvate Protects Neurons Against Hydrogen Per (86). PCT No.: PCT/LO3/OO634 oxide-Induced Toxicity'. The Journal of Neuroscience, 17(23): 9060-9067, 1997. S371 (c)(1), Gramsbergen et al. “Pyruvate Protects Against 3-Nitropropionic (2), (4) Date: Jan. 26, 2005 Acid Neurotoxicity in Corticostriatal Slice Cultures'. Neuropharmacology, 11(21): 2743-2747, 2000. (87) PCT Pub. No.: WO2004/012762 Hosoya et al. “Blood-Brain Barrier Produces Significant Efflux of L-Aspartic Acid But Not D-Aspartic Acid: In Vivo Evidence Using PCT Pub. Date: Feb. 12, 2004 the Brain Efflux Index Method”. Journal of Neurochemistry, 73: 1206-1211, 1999. (65) Prior Publication Data Lee et al. “Protection by Pyruvate Against Transient Forebrain US 2006/0024284 A1 Feb. 2, 2006 Ischemia in Rats'. The Journal of Neuroscience, 21(RC171): 1-6, 2001. Related U.S. Application Data Liu et al. “P-Glycoprotein Regulated Transport of Glutamate at Blood-Brain Barrier'. Acta Pharmacol Sin, 22(2): 111-116, 2001. (60) Provisional application No. 60/399,708, filed on Aug. Matsumoto et al. “Role of Pyruvate in Ischemia-Like Conditions on 1, 2002, provisional application No. 60/430,689, filed Cultured Neurons'. Neurological Research, 16: 460-464, 1994. on Dec. 4, 2002. 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WO WO99,21565 6, 1999 Monganetal. “Intravenous Pyruvate Prolongs Survival During Hem orrhagic Shock in Swine', AJP Heart and Circulatory Physiology, OTHER PUBLICATIONS 277(46): H2253-H2263, 1999. Rumigny et al. Biochemical Pharmacology, vol. 30, pp. 305-312, (Continued) 1981.* Geng et al. Journal of Neurochemistry, vol. 68.No. 6, 1997, p. 2500 Primary Examiner Ruth A. Davis O6.* Assistant Examiner Tiffany M Gough BL4820 Biochemistry Techiniques ttp://www.bio.mtu.edu/ campbell/b14820/lectures/lec4/gotw41.htm, 1996.* (57) ABSTRACT EC2.6.1.1 to EC2.6.1.50) http://www.chem.qmul.ac.uk/iubmb? enzyme/EC2/0601a.html, 1976.* Haring et al. Protein Engineering, vol. 15, 2002, p. 603-610.* A method of reducing extracellular brain glutamate levels. 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Experimental Neurology, 183: 700-704, 2003. * cited by examiner U.S. Patent Jul. 29, 2008 Sheet 1 of 22 US 7.404,951 B2 200 !=<= N,LOCNON LOO 20 40 60 8O 100 Min Fig. 1b U.S. Patent Jul. 29, 2008 Sheet 2 of 22 US 7.404.951 B2 140 120 100 8 O i 6 O 1 2 O 1 O O 8 O 6 O 4 O 2 O O Mi Fig.2b U.S. Patent Jul. 29, 2008 Sheet 3 of 22 US 7.404,951 B2 Pyr/OxalmM) Fig. 3 U.S. Patent Jul. 29, 2008 Sheet 4 of 22 US 7.404.951 B2 100 6 O O.O 0.2 0.4 0.6 Lipoamide mM) Fig. 4a O 5 10 Thiaminepyrophosphate uM) Fig. 4b U.S. Patent Jul. 29, 2008 Sheet 5 of 22 US 7.404,951 B2 O 20 40 60 80 150 10 O 5 O O 20 40 60 80 U.S. Patent Jul. 29, 2008 Sheet 6 of 22 US 7.404,951 B2 140 120 8 O S. 60 4. O 2 O O 15 3O 45 60 75 90 105 Min Fig. 7 U.S. Patent Jul. 29, 2008 Sheet 7 of 22 US 7.404,951 B2 500 120 400- 1OO Pyr/Oxal (uM/I) 80 % of basal 3OO Glu 60 200 40 100- 20 O O Fig. 8 20 40 60 80 100 Min 250 200 2 O 150 (5 O c O 100 50 O 50 100 150 200 250 Fig. 9 Min U.S. Patent Jul. 29, 2008 Sheet 8 of 22 US 7.404,951 B2 O 20 40 60 80 100 MIN Fig. 10 U.S. Patent Jul. 29, 2008 Sheet 9 of 22 US 7.404.951 B2 140 120 S 100 (f) CD 80 O 3 60 S 40 P CP 20 O 20 40 60 80 100 120 140 160 Time (min) Fig.11 U.S. Patent Jul. 29, 2008 Sheet 10 of 22 US 7.404,951 B2 50 4. O 23 OO 1 O O 500 1000 Glu uM Fig. 12 80 4.6 OO 2 O O 500 1 OOO Glulu Molar Fig. 13 U.S. Patent Jul. 29, 2008 Sheet 11 of 22 US 7.404,951 B2 140 120 100 4.68 OOO 2 O 0 15 30 45 60 75 90 105 120 135 150 Time Fig. 14 U.S. Patent Jul. 29, 2008 Sheet 12 of 22 US 7.404,951 B2 O 15 30 45 6.O 75 90 105 120 135 150 Time Fig. 15 120 110 1 O O 9 O 8 O 5 O O 20 40 60 80 100 120 140 160 Time Fig. 16 U.S. Patent Jul. 29, 2008 Sheet 13 of 22 US 7.404,951 B2 120 100 60 40 O 5 10 15 ml of blood Substituted with 6% hetastarch Fig. 17 112233500 O5O5O OOOOO OOOOO 500 O 1000 2000 3OOO 4000 Time (seconds) Fig. 18 U.S. Patent Jul. 29, 2008 Sheet 14 of 22 US 7.404,951 B2 1OOOOO E E Ol Sl 2 CD 10000 O O ? O) O - 1000 O 250 5OO 750 1OOO 1250 Fig. 19 200 as 150 SO 2 r CD s: X is 100- S as S SS o 50 3. O 50 100 150 200 Min Fig. 20 U.S. Patent Jul. 29, 2008 Sheet 15 of 22 US 7.404,951 B2 12000 S 11 OOO T S 1 OOOO O Sl c o 9000 5, 8000 (S 7000 6000 O 50 100 150 200 Time Fig. 21 Saline Saline + Saline Pyruvate/ Oxaloacetate Fig. 22 U.S. Patent Jul. 29, 2008 Sheet 16 of 22 US 7.404,951 B2 70 60 0.2 uM "" 0.2 uMH) Glu + 250 uM Glu 50 40 Time O 50 100 150 Fig. 24 Min U.S. Patent Jul. 29, 2008 Sheet 17 of 22 US 7.404,951 B2 120 100 8O 60 40 20 Pyrloxal O 50 100 150 Time Fig. 25 ISF GLU 1 uM GLN/Na Na/KATPase GLU/NA see to e -Nasi GLUss 40AM GLNHo GLU + NH3 GLN GPT plasma GLU GLU + Pyruvate --> o-KG + Ala 40 uM GLU + Oxaloacetate -->o-KG + Asp GOT Fig.