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`Apotex Ex. 1012
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`Apotex v. Auspex
`IPR2021-01507
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`SHORT COMMUNICATIONS 567 volumes (v/w) of 1.15 % KC1 and was centrifuged at IOOOO × g for IO rain. The super- natant fraction was centrifuged in a Spinco ultracentrifuge at a maximum speed using a No. 3o head for 3o min to sediment the microsomes. The postmicrosomal super- natant fraction was kept as a source of glucose-6-phosphate dehydrogenase. The microsomal fraction was washed once by recentrifuging in 1.15 % KC1 and was sus- pended in one volume of KC1 for every g of the initial liver weight. o-Nitrophenol was assayed spectrophotometrically ~. Formaldehyde was assayed colorimetrically after distillation 4. Hexamethylenetetramine was used as a standard for formaldehyde. As shown in Table I, substitution of deuterium for hydrogen in the methyl group resulted in approx. 50 To reduction in the rate of 0-demethylation as evidenced by the ratio of the velocity constants, kn/k2H, of almost 2 regardless of which product was measured. Determination of the Michaelis constants by the LINEWEAVER-BURK plot 5 showed an average Km*H/Km H of 0.4 (9.6" lO -5 M/2.5" lO -4 M) in three separate de- terminations indicating, if any, a stronger binding of the deuterated o-nitroanisole to the O-demethylating enzyme. Thus the difference in the rate of metabolism between unlabeled and deuterated o-nitroanisole must be due to differences in the rates of C-H and C-2H bond breaking. It is of interest that we observed no kinetic isotope effect in a somewhat analogous reaction, the oxidation of [C2H~tolbutamide to the hydroxy metabolite of tolbutamide by the microsomal system e. On the other hand, the in vitro rate of oxidation of [3'-2H]5-butyl-5-ethylbarbituric acid (Neonal) to 5-ethyl-5-(3'-hydroxybutyl)-barbituric acid was slower than the unlabeled Neonal and the deuterated Neonal accordingly prolonged the sleeping time of mice v. Deuterium kinetic isotope effects were also reported for the oxidation of [~,c¢-2H2~tyramine s catalyzed by monoamine oxidase and of [C2H3]morphine 9, an N-demethylation re- action catalyzed by liver microsomes. This investigation was supported in part by a U. S. Public Health Service Grant AM o6629 from the National Institute of Arthritis and Metabolic Diseases. Departments of Biomedical Research and Pharmaceutical Chemistry, Stanjord Research Institute, Menlo Park, Calif. (U.S.A.) C. MITOMA D. M. YASUDA J. TAGG M. TANABE I A. ][.VOGEL, A Textbook of Practical Organic Chemistry, 3rd ed., John Wiley and Sons, New York, 1956, p. 67o. 2 H. I. HEILBRON AND H. M. BUNBURY, Dictionary of Organic Compounds, Vol. 3, Oxford Uni- versity Press, New York, 1953, p. 632. 3 K. J. NETTER, Arch. Exptl. Pathol. Pharmakol., 238 (196o) 292. 4 M. J. BOYD AND M. A. LOGAN, J. Biol. Chem., 146 (1942) 279. 5 H. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. 6 J. TAGG, D. M. YASUDA, M. TANABE AND C. MITOMA, Biochem. Pharmacol., 16 (1967) 163. 7 J. SOBOREN, D. M. YASUDA, iVY. TANABE AND C. MITOMA, Federation Proc., 24 (1965) 427 . 8 B. BELLEAU, J. BURBA, M. PINDELL AND J. REIFENSTEIN, Science, 133 (1961) lO2. 9 C. ELISON, I-I. RAPOPORT, R. LAURSEN AND H. W. ELLIOTT, Science, 134 (1961) lO78. Received December 9th, I966 Biochim. Biophys. Acta, I36 (I967) 566-567
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`Apotex Ex. 1012
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