Document 9Lq9x4kr7e15JeKz7O0OQjMVD

FJ r..K4*< Vot Ifl pp tv to 629. 1911 Primed m Great Britain All rightt revrved <\ jr A Of 00IS-4IM It OJOm-OJBloDO Copjfuhl o ! I Ptn. Lid <-* ACTION OF N.N-DIETHYLACETAMIDE ON HEPATIC MICROSOMAL DRUG-METABOLIZING ENZYMES F. E. Beyhl and E. Lindner Hoechst AC, D-6230 Frankfurt am Main HO, Bundesrepublik Deutschland PLAINTIFF'S EXHIBIT (Received 29 December 1980) DOW-1429 Summary--The action of N.N-diethylacetamide on liver microsomal drug-metabolizing studied in vitro using rat-liver homogenates, and in into in mice. In vitro, N^i-diethylacetamide had no effect on a series of mixed-function oxidases or on cytochrome c reductase but in vivo, these enzymes were inhibited. 45 min after a single, ip dose of 150 mg of the subslance/kg body weight and there was also an increase in hexobarbiial sleeping lime. This discrepancy between the in vitro and the in vivo results may be due to oxidative N-dealkylation of diethylacetamide occurring in vivo leading to enzyme inhibition. Introduction In pharmacological and toxicological studies and in some other uses, water-insoluble compounds are often administered in water-miscible organic solvents such as dimethylsulphoxide, glycerol, propylene glycol, polyethylene glycol, and Af.JV-diethylacetamide. It has been shown that these vehicle compounds are not as biologically inert as they were originally thought to be (Budden. Kiihl A Bahlsen, 1979; Budden, Kiihl A Buschmann, I978a.b,c; Gergcly, 1970). Budden er al. (1978a.b,c) showed that amongst other effects, they can prolong hexobarbital sleeping time. Thus these substances can interfere with the biotransformation and the pharmacokinetics of compounds to be tested in pharmacological or toxicological studies. We studied the effect of N.N-diethylacetamide on oxidative drug metabolism in tnoo and in vitro because this solvent has structural similarities to the aliphatic amide, 2-hydroxy-2-ethylbutyryl NJi-diethylamide (HOE 17,879). a compound which has been shown to prolong drug action times (Beyhl A Lindner. 1976; Lindner. 1960) and to inhibit hepatic microsomal drug-metabolizing mixed-function oxidases, both in vivo (Beyhl A Lindner, 1973 A 1976) and in vitro (Beyhl 1980a). but to have no effect on hepatic micro somal NADPH-dependent cytochrome c reductase in nco (Beyhl A Lindner. 1976) or in vitro (Beyhl. 1980a) Experimental Materials. The following chemicals were used: saccharose, inorganic salts, and buffer substances obtained from Merck (Darmstadt) and RiedelDeHaen (Seelzel biochemicals obtained from Boehnnger (Mannheim). iV.iV-dicthylacetamide and coumartn obtained from Merck, aminopyrme obtained from Hoechst (Frankfurt), sodium hexobar bital obtained from Bayer (Leverkusen), and 4-methylumbelliferone obtained from EGA-Chetme (Stemheiml. 4-Methoxybiphenyl and 7-ethoxycoumarin were the gifts of Dr Sinharay (Hoechst). in vivo study Treatment of animals. Male mice of the NMRI strain obtained from our breeding station, were divided at random into two groups of 16 animals. The mice of one group were injected ip with an aqueous solution containing I SO mg Af^f-diethylacetamide/kg body weight and those of the other (control) group were injected with the same amount of water. After 45 min six animals from each group were injected iv with 100 mg sodium hexobarbital/kg body weight for the measurement of hexobarbital sleeping time according to Lindner (I960). The remaining animals were killed by a blow on their heads and bleeding; the livers were removed, weighed, and frozen immediately by immersion into liquid nitrogen. The livers were stored at - 20C until further handling Activities of drug-metabolizing enzymes were not deteriorated by this procedure (F. E Beyhl unpublished data 1980). Biochemical measurements. Alter thawing the livers were homogenized in ice-cold isotonic saccharose sol ution with a glass/polytetraffuoroethylene homogentzer of the Potter-Elvehjem (1936) system driven by compressed-air motor (Press!uft-Gdtz, Mannheim). From, these crude homogenates, microsomes were prepared by the calcium chloride precipitation method as died previously (Beyhl A Mayer, 1980) The activities of certain enzymes in these microsomes were assayed as described below. Methoxybiphenyl O-demethylase was assayed according to Beyhl (1980b; d. Bridges. Creaven A Williams. 1965: Crea ven. Davies A Williams, 1967; Davies A Creaven. 1964) Aminopyrme Af-demethyiase was determined following Leber. Degkwitz A Staudinger (1969; cf. Volz A Kellner, 1980) Coumarin 7-hydroxylase was assayed by our own method (F. E Beyhl unpublished data. 1981: cf. Creaven, Parke A Williams. 1965: Fink A von Kerekjarto. 1966; von Kerekjarto. 1966: von Kerekjarto. Kratz A Staudinger. 1964) with 4-methylumbeliilerone as the fluorescence standard, and microsomal NADPH-dependent cytochrome c reduc tase was measured according to Geveland A Smuckler (1965) All the resulting values were tested for statistical significance using Student's r test (Ther. 1965) . In vitro studies For the in citro studies, the livers of untreated Wistar 9210U1 rtowV 1 628 F. E. Beyhl and E. Undnek rats, of either sex, obtained from our breeding station were homogenized either in isotonic saccharose sol ution for the preparation of calcium chloride-prectpitated mtcrosomes as described above or in isotonic potassium chloride solution for the preparation of 13.000 g supernatants (Beyhl & Mayer, 1980). In the 13.000k supernatants, the activities of aminopyrine N-demethylase and cytochrome c reductase were de termined as outlined above: in previous studies it had been shown that these microsomal enzymes could be measured without disturbance of the cytosolic com ponents present in these supernatants (F. E. Beyhl. unpublished data, 1980). In the microsomal prep aration. the activities of methoxybiphenyl O-demethylase and coumarin 7-hydroxylase were measured by the methods cited above, and that of 7-ethoxycoumarin O-deethyUse was determined by our own method (F. E. Beyhl unpublished data. 19^1; cf. Aitio. 1978; Ullrich & Weber. 1971). All the enzyme activi ties were assayed in the absence and in the presence of the N.N-diethylacetamide at concentrations of 01. 1-0 and 10 rim. in the assay medium (cf. Beyhl 1980a). Again, the resulting values were tested for statistical significance using Student's r test. Results The results are listed in Tables 1 and Z All the enzyme activities measured in the in riro experiment were significantly decreased and hexobarbital sleeping time was doubled. In the in rirro experiment N.Ndiethylacetamide did not have a significant effect on the activity of any of the enzymes. either mixed-function oxidases or cytochrome c reductase. Other microsomal enzyme inhibitors either inhibit the mixed-function oxidases, as a result of in teraction with cytochrome P-4S0, the terminal oxi dase of the microsomal redox chain (Beyhl 1980a,c; Cooper. Axelrod & Brodie, 19S4) or inhibit both the mixed-function oxidases and cytochrome c reductase (Beyhl 1981). Thus Af.Af-diethylaceumide would not be expected to affect drug-metabolizing enzymes or hexobarbital sleeping time in vivo. However our results show that Af.Af-diethylacetamide reduces both mixed-function oxidase and cytochrome c reductase activities in vivo and. as a consequence of the thus impaired drug biotransformation. prolongs hexobar bital sleeping time. The increased hexobarbital sleep ing time was also observed by Budden et al. (1978a). In view of the in vitro results this effect on drugmetabolizing enzymes cannot be explained as result ing from enzyme inhibition by Af.Af-diethylacetamide itself. We would like to suggest that in rieo, N.Ndiethylacetamide is Af-dealkylated to form a reactive metabolite which blocks both drug metabolism by mixed-function oxidases and microsomal electron transport by cytochrome r reductase. A similar mech anism for inhibition of drug-metabolizing enzymes is being discussed for diethylamino-ethy! Z2-diphenylvalerate hydrochloride (SKF 525-A; Proadifen), a well-known inhibitor of mixed-function oxidases (Barber St Wilson. 1980: Schenkman. Wilson & Cintl 19721 Studies on this metabolic activation of Af,Afdiethylacetamide are in progress. DiscussiM The lack of enzyme inhibition observed in vitro shows that Af.Af-diethylacetamide does not inhibit Acknowiedgements--Tbe authors are grateful (o Mrs Fey. Mrs Meier. Mrs Noll and Mr Weinrauch for their assist ance and to Dr Sinharay for synthesizing 4-methoxybiphenyl and 7-ethoxycoumaria. Table 1. Action of MN-Jierhyiacrtamide on hepatic parameters in mice in vivo Croup Control Treated Hexobarbital sleeping time (mini 50 11 101 17 (202* .) Aminopyrine Af-demethylase (U g liver) 0*52 0031 0315 0047 (69-7*.) Methoxybiphenyl O-demcthylaae (U/g liver) 0179 001 0127 002 (709*J Coumarin. 7-hydroxylase (mU/g liver) 506 093 2-72 107 (5>8*J V"t -- -H +l!o Cytochrome e reductase (U/g liver) Vaiuea an means 1SD for groups of ten animals lexcept hexobarbital sleeping ume. six animals! Values in parentheses arc percentages of the corresponding control value. All values for the treated group differ significantly (Student's r test) from those of the corresponding control group (P < 00005! Table 2 Effect of N.N-dirrhr/arrramidr on rat-ilrer encyme activities in vitro Enzyme activities (*. of controll .V.jV-Diethylacetatnide concn (him) 01control) Ol 10 10 Aminopyrine Af-demethylase 100 3-9 105-2 11-2 102 1 228 804 13 2 Methoxy biphenyl O-demcihylase 100 15 99-2 19 103-9 2-7 106-2 24 Ethoxycoumann O-deethylaie 100 8 5 1007 9-2 1004 6-5 96-5 5-3 Coumarin 7-hydroxyiase 991000820 04 37-91 82-8 7-4 Cytochrome c reductase 100 26 99-8 28 111 8 2 7 111 5 50 Values are means I SO. None of the values differs significantly (Student's t test) from that of the corresponding control group. a-. . - 921001 RowVerK i hrome c on either -tilt of inntnal oxi- I980a.c; both the reductase ould not 'mes or er our ;es both reductase he thus exobar>1 sleep11978a). n drugas resultacetamide rro. N.N- 2 reactive oiism by electron tr mechrymes is iiphenyl-tdifenl a oxidases i A Cinti. i of N.N- i % i i 4 1 > Mrs Fey. ietr assisltoxybiphe- I i -irome ::ase rr) 35 JI .) entheses nt's r (estl i N.N-Diethylacetamidc and microsomal enzymes 629 REFERENCES Aitio. A. (19781 A simple and sensitive assay of 7-ethoxycoumann dcethylation. Analvt. Biochem. 85. 488. Barber. P. J. St Wilson. B. J. (1980V Microsomal conver sion of SKF 525-A and SKF 8742-A Biochem. Pharmac 29.2577. ' Beyhl. F. E. (1980aV Interaction of the enzyme inducer. HOE 17,879. with microsomal enzyme systems. In Microtomes, Drug Oxidations, and Chemical Carcinoge nesis. Edited by M. J. Coon. A. H. Conney. R. W. Estabrook. 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