Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Biotransformation of tri-substituted methoxyamphetamines by Cunninghamella echinulata B. C. Foster, J. McLeish, D. L. Wilson, L. W. Whitehouse, J. Zamecnik & B. A. Lodge To cite this article: B. C. Foster, J. McLeish, D. L. Wilson, L. W. Whitehouse, J. Zamecnik & B. A. Lodge (1992) Biotransformation of tri-substituted methoxyamphetamines by Cunninghamella echinulata, Xenobiotica, 22:12, 1383-1394, DOI: 10.3109/00498259209056689 To link to this article: http://dx.doi.org/10.3109/00498259209056689
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Date: 25 April 2016, At: 09:50
XENOBIOTICA,
1992, VOL. 22,
NO.
12, 1383-1394
Biotransformation of tri-substituted methoxyamphetamines
by Cunninghamella echin ula fa B. C. FOSTER?, J. McLEISH, D. L. WILSON, L. W. WHITEHOUSE, J. ZAMECNIK and B. A. LODGE Bureau of Drug Research, Sir Frederick Banting Research Centre, Health Protection Branch, Ottawa, Ontario, Canada K1 A OL2
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Received 2 January 1992; accepted 27 June 1992 1. Four trimethoxyamphetarnine analogues were incubated with the filamentous fungus Cunninghamella echinulata. 2. 2,4,5-Trimethoxyamphetamine and 2,5-dimethoxy-4-ethoxyamphetamine were poorly metabolized by C. echinulata ATCC 9244and C. echinulata var. elegans ATCC 9245. 2,5-Dimethoxy-4-(n)-propoxyamphetamine was mainly metabolized through Nacetylation and O-dealkylation with minor amounts of several aliphatic hydroxylation metabolites formed. 2,5-Dimethoxy-4-methylthioamphetamine was extensively metabolized to the corresponding sulphoxide.
3. 2,5-Dimethoxy-4-methylthioamphetaminemetabolism was inhibited by ethanol and quinidine. Sparteine did not inhibit the formation of the sulphoxide and may have shunted the substrate through alternate metabolic pathways.
4. Incubation conditions can affect the rate and extent of fungal biotransformation of 2,5-dimethoxy-4-methylthioamphetamine, and influence dextrose utilization, ammonia formation and pH.
Introduction In this era of growing social awareness and concern over the treatment of animals in scientific studies, microbial models have provided an alternative method for the study of drug metabolism. T h e two filamentous fungal strains, Cunninghamella echinulata ATCC 9244 and C . echinulata var. elegans ATCC 9245 examined in this study had been used by numerous investigators to validate the concept of microbial models of mammalian metabolism (Clarke et al. 1985, Foster et al. 1989b, 1990a, Rosazza and Duffel 1986, Smith and Rosazza 1975). Since amphetamines are metabolized in mammalian systems by several pathways, including aromatic hydroxylation, aliphatic hydroxylation, N-dealkylation, oxidative deamination, Noxidation and conjugation (Caldwell 1976) they are an excellent class of compounds for validating metabolic models. As a continuation of earlier studies to examine the effect of ring substitution on the fungal biotransformation of aryl alkyl amphetamines (Foster et al. 1989b), 4-monosubstituted amphetamines (Foster et al. 1989b), 4monosubstituted amphetamines (Foster et al. 1990a), and 2-, 3-, and 4-methoxyamphetamines (Foster et al. 1991a) we now report the C . echinulata-mediated biotransformation of four 2,4,5-tri-substituted amphetamines analogues, namely, 2,4,5-trimethoxyamphetamine(TMA-2, l a , figure 1; Sargent et al. 1976), 2,sdimethoxy-4-ethoxyamphetamine (lb), 4-(n)-propoxy-2,5-dimethoxyamphetamine (Ic) and 2,5-dimethoxy-4-methylthioamphetamine( I d , Nichols and Shulgin 1976).
t T o whom correspondence should be sent. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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R1 G
C : 2
OIWI-$
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CH30
la
CH30
lb
CHjCH20
lc Id
CH3CH2CH20
10
H
CH3S
H H H H H
If
COCH3
lg
COCH3
lh
COCHS
li
COCH3
li
COCH3
lk
H
11
H H
lm In
HO-CH2CH2CH20
lo
CH3CH(OH)CH20
H
1P
HO-CH2CH2CH20
COCHj
lq
CH~CH(OH)CH~O
COCH3
H
Figure 1. Structures of substrates and metabolites
Materials and methods Chemicals, substrates and reference compounds 2,4,5-Trimethoxyamphetamine hydrochloride (la) m.p. 98-99°C; 2.5-dimethoxy-4-ethoxyamphetamine hydrochloride (lb) m.p. 13Cb131"C; 2,5-dimethoxy-4-(n)-propylamphetaminehydrochloride (lc) m.p. 134-135°C; 2,5-dimethoxy-4-methylthioamphetatninehydrochloride (Id) m.p. 136138°C; and 2,s-dimethoxyamphetamine hydrochloride (le) m.p. 74-75°C; were synthesized for and obtained from the Bureau of Drug Research collection of reference standards. Crude /Pglucuronidase containing aryl-sulphatase extracted from molluscs (Type H-2) and sparteine sulphate were obtained from Sigma (St. Louis, MO, USA). Quinidine sulphate dihydrate was obtained from Aldrich Chemicals (Milwaukee, WI, USA). All other chemicals were of analytical grade and obtained from regular commercial sources. The sulphoxide of 2,5-dimethoxy-4-methylthioamphetamine(1k) was prepared from 300 mg (1.1 mmol) 2,5-dimethoxy-4-methylthioamphetamine hydrochloride by reaction with 230 mg (1.1 mmol) 3-chloroperoxybenzoic acid (85%) dissolved in 20 ml chloroform at room temperature for 96 h. T h e organic layer was washed with 5% K,CO, in saturated brine and dried over anhydrous MgSO,. T h e solution was filtered and evaporated to dryness. T h e crude product, containing 20-25% sulphone, was dissolved in isopropanol and triturated with concentrated HCI. T h e sulphoxide was recrystallized from ethanol/diethyl ether to give white crystals, yield 48%. T h e 'H-n.m.r. (400MHz) in D,O spectrum for sulphoxide hydrochloride showed signals at S,7.19 ( l H , s); 6.99 (1 H, s); OCH, 3-83(3H, s);OCH3 3.82 (3H,s); C H 3-63 (lH,m); CH, 2.95 (2H,m); CH,SO 2.83 (3H,s); C-CH, 1.24 (3H,d). T h e 'H-n.m.r. spectrum for 2,5-dimethoxy-4-methylthioamphetaminehydrochloride showed signals at 6,671 (2H, s); OCH, 3.68 (6H, s); C H 3.48 ( l H , m); CH, 2.73 (2H, m); SCH, 2.30 (3H, s) and C-CH, 1.14 (3H, d). Incubation Procedure Stage 1 fungal cultures were grown in 50 ml of complex medium containing 3.0 g/lOO ml trypticase soy broth (BBL, Becton Dickinson and Co., Cockeysville, M D , USA) and 0.72 g/100ml yeast extract (Difco). This medium was inoculated by loop with spores from cultures of C . echinulata ATCC 9244 or C .
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echinulata var. elegans ATCC 9245. After 72 h, the spent medium was decanted and replaced with 50 ml of sterile deionized water. T h e cultures were homogenized with a Waring blender and 2ml of the homogenate was added to stage 2 flasks of 25 ml dilute (20%) complex medium containing 1 ml of dextrose (20g/100ml)and 1 ml of filter sterilized substrate (2 mg/ml). In an interaction study, substrate was added alone and in combination with either ethanol, sparteine, or quinidine 48h after the start of Stage 2 cultures. All cultures were protected from light and incubated at 28°C on a New Brunswick model G-2 flatbed shaker at 250 rpm in 250 ml (stage 1) and 125 ml (stage 2) long-necked, metal-capped Erlenmeyer flasks. Culture controls were prepared by incubating the fungus in the absence of substrate. Substrate controls consisted of sterile medium containing the drug substrate, but without inoculum. Sample preparation Duplicate 1 ml samples were withdrawn up to 7 days after addition of substrate from all incubation and control broths and stored frozen (-20°C) until required. After thawing, samples were mixed with 1ml of an aqueous solution of 17.3 pg/ml2,5-dimethoxyamphetaminehydrochloride (le, internal standard).The samples were extracted twice by vortexing with 3 ml ethyl acetate for 1 min, then the organic layer was filtered through a Pasteur pipettecontainingglass wool and anhydrous Na,SO, (method A). In method B, samples were basified using solid K,CO, and extracted as outlined in method A. Method C was identical to method A, with the exception that 50p1 of acetic anhydride was added to the aqueous mixture prior to each extraction. T h e extracts obtained from methods A-C were reduced to approximately loop1 under a stream of dry nitrogen at 35°C and analysed by injecting 1 pl into the g.1.c. and g.1.c.-mass spectrometry apparatus. Instrumentation and analyses G.1.c. analyses were conducted on a Hewlett-Packard model 5890A gas chromatograph equipped with a ChemStation and flame ionization detector using a 15 m DB-1 megabore column with a film thickness of 1.5 pm and a 0.53 pm diameter (J&W Scientific, Inc., Folsom, CA, USA). Standard operating conditions were: splitless mode: injection port temperature, 250°C; detector temperature, 225°C; He, carrier gas 15 ml/min; make-up gas 30ml/min; H, 60ml/min; air, 320ml/min and oven temperature, 185°C. G.1.c. data are given in table 1. Electron-impact mass spectrometric analysis was performed on a Finnigan-MAT 4610B gas chromatograph-mass spectrometer. T h e g.1.c. conditions were as follows: Initial oven temperature of 70°C was held for 1 min, then increased at a rate of 25.O0C/minfor 2.7 min, and then increased at a rate of 8WClmin for 15.0min to a final oven temperature of 260" which was maintained for 10.0min; injection port temperature, 250°C; splitless mode equipped with a DB-5 15 m narrowbore (0.25 mm) column with a stationary phase film thickness of 0.25 pm (J&W Scientific Inc.). Operating conditions for the mass spectrometer were: separator temperature, 260°C; source temperature, 150°C; emission ion current, 320pA; and electron energy, 70eV. Mass spectrometric data are given in table 1. Proton n.m.r. spectra were recorded on a Bruker AM 400 spectrometer equipped with an Aspect 3000 computer and process controller. Spectra were recorded with a sweep width of about 5000 Hz, with 32 K data points, giving a final resolution of about 0.3 Hz/point. The chemical shifts ( b H were ) reported in ppm relative to tetramethylsilane as internal reference. Ammonia and dextrose were determined using an Abbott VP clinical chemistry analyser. T h e pH of the incubation broth was determined using a Fisher p H meter, model 91 5. Data analysis Group data are expressed in terms of the free base and presented as means and standard deviations unless otherwise indicated. T h e Student's t-test was used to identify significant differences between groups, with values of p < 0.05 being considered significant.
Results Metabolism of 2,4,5-trimethoxy- and 2,5-dimethoxy-4-ethoxyamphetamine G.1.c. analysis of day 7 extracts (method B) revealed that 2,4,5-trimethoxyamphetamine (1a) and 2,5-dimethoxy-4-ethoxyamphetamine(1b) were poorly metabolized by both C. echinulata ATCC 9244 (89 and 88% recovery of unchanged substrate, respectively) and C. echinulata var. elegans ATCC 9245 (108 and 91% recovered, respectively; table 2). T h e corresponding N-acetyl metabolites (1f and g) of these substrates were not detected. No other metabolites were detected with either of these substrates (methods A-C,see Sample preparation).
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Table 1. Gas-liquid chromatography retention times ( t R )and electron-impact mass spectrometric data for substrates and metabolites.
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Compoundt
Mass spectrometric data m/z (percentage relative abundance)
tRf
la lb lc Id le If 1g lh
3.48 400 5.30 6.49 2.07 1051 12.19 16.23
li 1j lk 11 lm In lo 1P
19.96 5.99 12.97 1468 7.12* 1 0 51* 11.53* 14.12*
1q
15.15'
lr
13.04*
225(2) 182(87) 167(20) 151(8) 139(5) 121(1) 44(100) 239(4) 196(96) 181(7) 167(17) 153(20) 137(10) 122(3) 44(100) 253(9) 210(100) 168(59) 167(49) 153(61) 137(28) 122(12) 44(98) 241(3) 198(63) 183(13) 167(5) 152(4) 137(3) 121(2) 44(100) 195(23) 180(8) 164(7) 152(74) 137(41) 121(19) 91(26) 77(25) 44(100) 267(21) 208(100) 193(9) 181(72) 167(6) 151(19) 136(6) 86(5) 44(56) 281(21) 222(100) 195(56) 167(55) 153(7) 137(16) 86(5) 44(56) 295(61) 236(100) 210(24) 209(59) 194(47) 179(30) 167(94) 153(24) 137(44) 122(23) 86(22) 44(49) 283(30) 224(100) 197(42) 183(10) 167(12) 152(7) 137(3) 86(12) 44(79) 237(51) 194(5) 178(100) 121(40) 86(61) 44(93) 257(1) 214(34) 199(22) 197(100) 167(12) 44(46) 273(2) 230(1) 197(1) 167(1) 44(100) 211(2) 168(54) 153(22) 13718) 12216) 44(100) 269(2) 226(61) 168(57) 167(44) 153(49) 137(28) 122(12) 59(10)45( 19)44( 100) 269(3) 226(78) 168(28) 167(25) 153(28) 137(12) 122(6) 44(100) 311(42) 252(96) 225(42) 194(60) 179(21) 168(30) 167(100) 153(16) 137(44) 122(16) 86(21) 59(11) 44(84) 311(17) 252(98) 225(40) 194(21) 179(9) 168(14) 167(100) 153(10) 137(22) 122(9) 86(10) 57(13) 44(90) 43(23) 268(15) 226(4) 194(1) 167(5) 137(5) 122(2) lOl(79) 73(8) 44(100)
t Identities of the numbered compounds are shown in figure 1. $ T h e g.1.c. conditions are described in Materials and methods. * Retention times are obtained from g.1.c.-mass spectrometry analysis. Table 2.
Recovery of 2-, 4-, 5-trisubstituted amphetamines unchanged from incubations with Cunninghamella echinulata ATCC 9244 and C. echinulata var. elegans ATCC 9245. ~
ATCC 9244 Substrate la lb lc Id
Day 3
Day 7
50.3 2.0 (84)t 55.4 2.4 (93) 27.5 f4.5 (46) 21.4 f 4 3 (36)
53.4 f4.2 (89) 52.6 & 9.6 (88) 23.0& 3.0 (38) 9.4 f3.1 (16)
+
~
ATCC 9245 Day 4
Day 7
Values are in pg/ml (percentage recovery) and are expressed as means + S D (n=3). 2,4,5Trimethoxyamphetamine (la); 2,5-dimethoxy-4-ethoxyamphetamine(1b); 2,5-dimethoxy-4-(n)propylamphetamine (lc); 2,5-dimethoxy-4-methylthioamphetamine(Id) were the substrates used. t Percentage concentration of unmetabolized substrate relative to the concentration of the parent compound recovered from substrate controls.
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Metabolism of 2,5-dimethoxy-4-(n)-propoxyamphetamine In addition to 2,5-dimethoxy-4-(n)-propoxyamphetamine(1c) which accounted for 38% of the added dose, seven new g.1.c. peaks were detected in extracts (method B) from C. echinulata ATCC 9244 incubation broths (table 2). Recovery of 2,S-dimethoxy-4-(n)-propoxyamphetamine from extracts of C. echinulata var. elegans ATCC 9245 was > 80%. T h e N-acetyl derivative of 2,5-dimethoxy-4-(n)propoxyamphetamine, namely, N-acetyl-2 ,5-dimethoxy-4-(n)-propoxyamphetamine (1h) was determined unequivocally by comparison with an authentic standard (table 1) to be the major metabolite formed by both organisms. Five of the g.1.c. peaks (lm-q; figure 1) were isolated and identified from incubation broth extracts (method B) of 2,5-dimethoxy-4-(n)-propoxyamphetamine and C . echinulata ATCC 9244 (table 2) on the basis of their g.1.c.-mass spectrometry properties. 2,5-Dimethoxy-4-hydroxyamphetamine ( l m ) was identified on the basis of the ions at m/z211 (M'), m/z 168 (C,H,,O;, indicative o f p a r a 0-dealkylation) and m/z44 (CH, - C H = N + H 2 ,intact side-chain). Four metabolites (In-q) were found to have similar properties with ions at m/z 44, 167, 168, and either m/z 225 or 226. These ions are consistent with those expected for a compound having one oxygen atom added to the 4-substituent side-chain. Two of the metabolites ( I n and 0) had an apparent molecular ion at m/z 269 and a base peak of m/z 44 (unsubstituted amine). The presence of m/z 59 (C,H,O+) in I n was characteristic of either C, or C, aliphatic hydroxylation. The C, product could readily rearrange to l m and this compound has been tentatively identified as 2,5-dimethoxy-4-(3-hydroxyprop0xy)amphetamine (In). T h e Aetabolite l o differs from I n by the absence of m/z 59. On the basis of g.1.c. and mass-spectral characteristics (lo was tentatively assigned the structure of 2,5-dimethoxy-4-(2-hydroxypropoxy)amphetamine. Two of the metabolites ( l p and q) had additional ions at m/z86 (CH,-CH = N + H C O C H , ) and m/z311 (M' of 269+42). T h e presence of m/z59 in l p suggested that this product was the corresponding N-acetyl derivative of In. T h e presence of m/z 43 ( C 2 H 3 0 + ) ,characteristic of secondary alcohol mass-spectral cleavage in l q would support the identification of this compound as the corresponding N-acetyl metabolite of lo. T h e seventh metabolite (lr), had ions consistent with that expected for a single oxidation product, but there were insufficient data to assign a definitive structure for this metabolite.
Metabolism of 2,5-dimethoxy-4-methylthioamphetamine In addition to the unchanged substrate (1d ) a single peak (1k) which accounted for most of the metabolized substrate was found in extracts (method B) from C. echinulata ATCC 9244 and C. echinulata var. elegans ATCC 9245 incubation broths containing 2,5-dimethoxy-4-methylthioamphetamine (1d; table 2). Analysis of this peak by g.1.c.-mass spectrometry characteristic ions at m/z 257 and 44 (M+ and CH,CH = N + H 2 )respectively, which were consistent with a single oxidation product associated with the aromatic ring (table 1). Other diagnostic ions at m/z 214 (M-43) and m/z 197 (m/z 214-OH) indicative of addition of a single oxygen to the sulphur. Comparison with spectra obtained from an authentic standard confirmed the identity of this metabolite as 2,5-dimethoxy-4-methylthioamphetaminesulphoxide (lk). T h e sulphone (11) was not detected in these extracts. Total recovery of 2,5-dimethoxy-4-methylthioamphetamineand the sulphoxide from day 7 incubation broths was not increased by homogenization of the cultures.
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2,5-Dimethoxy-4-methylthioamphetaminesubstrate controls without fungus were incubated at pH4.23, 7.23, and 9.30 for 7 days to determine whether the sulphoxide was a chemical breakdown product of the substrate rather than a fungal metabolite. G.1.c. analysis of the extracts (method A) from p H 7.23 and 9.30 samples revealed trace amounts ( < 0.1%) of a product having the retention characteristics of the sulphoxide metabolite. No sulphoxide was detected in the pH4.23 chromatograms. Enzyme hydrolysis using crude p-glucuronidase, of aliquots from all substrates neither increased recovery of the parent compounds nor their metabolites, nor did it yield additional metabolites (methods A and B). Since there was no evidence for the formation of the alcohol or ketone deamination products from these substrates, the incubation broths were not examined for acidic metabolites.
Effect of incubation conditions on metabolism and growth Based on the above results that 2,5-dimethoxy-4-methylthioamphetamine(1d) was the most extensively metabolized substrate with C. echinulata ATCC 9244, this compound and fungal strain were chosen for additional studies. T o determine whether a change in the incubation environment would affect the transformation of 2,5-dimethoxy-4-methylthioamphetamine, the procedure was altered to include flasks incubated on 30" angle brackets in addition to flasks incubated in the usual flat position. T h e fungus grew as numerous micropellets ( < 4 mm diameter) rather than as the normal single macropellet found in the flat incubation broths. Also, there was no surface wall growth in the cultures incubated at 30". T h e disappearance of substrate in both the flat (day 0-7) and 30" (day 0-4) incubations was linear ( r 2 = 0.9036 and 0.9790, respectively) with slopes of the regression lines being - 8-2pg/day and - 14-4pglday, respectively. No substrate was found in the incubation broth of the 30" cultures at day 7 (table 3). T h e formation of the
Table 3. Metabolism of 2,5-dimethoxy-4-methylthioamphetamineby cultures of Cunninghamella echinulata ATCC 9244 incubated either on flat or 30" angle brackets. Method of culture Flat Substrate/ metabolite Day 2 Day 4 Day 7
30" Angle
Id
lk
Id
lk
55.1 k8.5 ( 9 2 ~ 19.8 2.2 (33) 7.67 k 2.6 (13)
ND
24.1 &107* (40) 2-6*03* (4) ND*
32.0&4.3* (49H 59.1 _f9.2* (91) 62.2 k 10.0 (96)
+
28.7 & 3.2 (45) 51.4k 5.5 (80)
Values are in pg/ml (percentage recovery) and expressed as means & S D (n=5). 2,5-Dimethoxy-4methylthioamphetamine (Id); 2,5-dimethoxy-4-methylthioamphetaminesulphoxide ( l k ) were the substrates. ND, not detected. t Percentage concentration relative to the concentration of the parent compound recovered from substrate controls. 1T h e percentage recovery of the sulphoxide was based on the concentration of the sulphoxide relative to the concentration of the original thioamphetamine substrate (Id), corrected for molarity (% x [wt l d base/wt lk]). Denotes statistically different from the corresponding value obtained on the flat brackets.
1389
I
Q
4)
6-1
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70 60 50 40 30 20
r
0
10
0
0
1
2
3
4
5
6
7
Time of incubation (days) Figure 2. Analysis of Cunninghamella echinulata ATCC 9244 culture broths incubated under different conditions in the presence of the substrate 2,5-dimethoxy-4-methylthioamphetamine. Incubations were carried out on flat (B)or 30" angle (A)brackets, substrate controls ( 0 ) .(A) broth pH; (B) dextrose utilization (closed symbols) and ammonia formation (open symbols); and (C) substrate metabolism (closed symbols) and formation of the metabolite 2,5-dimethoxy-4methylthioamphetamine sulphoxide (open symbols) (n= 5).
sulphoxide metabolite was also linear for the flat samples between days 2 and 7 ( r 2= 0.9671, slope 10-1pg/day) whereas its formation in 30" cultures was linear up to day 4 ( r 2=0*9977, slope 14.8 pg/day). Examination of aliquots of the incubation broths analysed for pH, dextrose and ammonia (figure 2) during this comparative study revealed a relationship between biotransformation, dextrose utilization, ammonia formation and pH. T h e rate of dextrose utilization and the rate of ammonia formation mirrored in the p H as an earlier alkaline p H shift was directly related to the faster biotransformation of 2,5-dimethoxy-4-methylthioamphetamine. InfEuence of drug concentration Mammalian drug metabolism is often concentration dependent. T o determine whether this also occurs in fungal metabolism, the concentrations of 2,s-dimethoxy4-methylthioamphetamine were varied from 0.4 to 2.0 mg/flask and 10.0mg/flask in cultures of C. echinulata ATCC 9244 (figure 3). It is evident that the slopes for the
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Time o f incubation (days)
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Figure
3. Effect of substrate concentration on the metabolism of 2,5-dimethoxy-4methylthioamphetamine and formation of 2,5-dimethoxy-4-methylthioamphetaminesulphoxide by Cunninghamella echinulata ATCC 9244. Substrate concentration: 0.4 ( 0 ) ;2.0 (A);and 10mg/flask (m). Unchanged substrate (closed symbols) and the metabolite 2,5-dimethoxy-4-methylthioamphetamine sulphoxide (open symbols) (n= 5 ) .
Table 4. Metabolism of 2,5-dimethoxy-4-methylthioamphetamineCunninghamella echinulata ATCC 9244 in the presence of equimolar (lx) or fivefold (5x) higher concentrations of quinidine and sparteine. Day 4 Substrate/ metabolite Control Quinidine l x 5x Sparteine l x 5x
Day 7
Id
lk
25.7 2.3 (43)t 33.6 7.4* (56) 57.0&11.2* (95) 23.1 5.0 (39) 21.1 58.9 (35)
19.6f6.5 (3 1)$ 21.4 & 4.0 (33) 7.0 k 0.1 * (11) 21.6 f6.8 (34) 27.4 5.3* (44)
Id
lk
33.8 f11.8 (53)
Values are in pg/ml (percentage recovery) and expressed as means & S D ( n = 5 ) . 2,5-Dimethoxy-4methylthioamphetamine (1 d); 2,5-dirnethoxy-4-methylthioamphetamine sulphoxide (1k). t Percentage concentration relative to the concentration of the parent compound recovered from substrate controls. $ T h e percentage recovery of the sulphoxide was based on the percentage ratio of the sulphoxide concentration to the concentration of ( 1 d) free base, corrected for molarity (% x [wt Id base/wt lk]). * Denotes statistical significant difference, p < 0.05 control value.
disappearance of substrate and formation of the sulphoxide at the higher dose were greater than those for the two lower drug concentrations. For direct comparison of the three substrate concentrations the data was transformed to percent recovery. Disappearance of 04-10.0 mg/flask 2,5-dimethoxy-4-methylthioamphetaminewas linear for all concentrations, r 2 =0.9926 (slope - 12*2%/day),0.9844 (slope - 12.3% /day) and 0.8648 (slope - 14*3%/day),respectively. T h e formation of the sulphoxide metabolite was also linear with r2 values for 2.0 and lO.Opg/ml being 0.9835 (slope 12-3%/day)and 0.9921 (slope 164%/day), respectively. A minor new peak with a
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longer retention time was detected in extracts (method B) of the lO.Omg/flask samples. G.1.c.-mass spectrometry analysis identified this peak as the 2,sdimethoxy-4-methylthioamphetaminesulphone (1 1).
Metabolic probes Quinidine and sparteine can also provide information on the metabolic mechanisms involved in the fungal biotransformation (Foster et al. 1989a, c, 1990b). After a 7-day incubation, quinidine had a concentration-dependant effect decreasing the metabolism of 2,5-dimethoxy-4-methylthioamphetamine(Id) to the sulphoxide (1k; table 4). Incubation in the presence of sparteine had a limited effect in decreasing the amount of sulphoxide formed at day 7. Total recovery from incubations with sparteine ranged from 57 to 79%, compared with 89-103% recovery in controls and cultures containing quinidine, thus indicating that in the presence of sparteine other metabolite pathways were involved in the metabolism of this substrate (table 4). No metabolites other than the sulphoxide were detected in incubations with either quinidine or sparteine. In a concomitant study with ethanol it was observed that the sulphoxide accounted for 19% of the total recovered substrate (85%).
Discussion Comparative biotransformation Hydroxylation at the cr-carbon and (w-1)-position was responsible for the formation of major metabolites in the C . elegans biotransformation of a homologous series of O-alkyl paracetamol ethers (Reddy et al. 1990). T h e rank order of 0dealkylation for these O-alkyl substrates was ethyl > isopropyl > n-propyl > nbutyl >methyl. Two of the four amphetamine analogues, 2,4,5-trimethoxyamphetamine and 2,5-dimethoxy-4-ethoxyamphetamine,were poorly metabolized by both C . echinulata ATCC 9244 and C. echinulata var. elegans ATCC 9245. 2,5-Dimethoxy-4-(n)-propoxyamphetaminewas metabolized by cultures but to a lesser extent in C. echinulata var. elegans ATCC 9245. C. echinulata ATCC 9244 through at least three different biotransformation pathways (in order of importance): Nacetylation (1h), O-dealkylation ( l m ) and aliphatic hydroxylation ( I n and o ) . Aliphatic hydroxylation occurred at the terminal (0) and adjacent (w-1)-position. These metabolites were then substrates for N-acetylation. These results were surprising in that the corresponding methyl and ethyl 4-mono-substituted derivatives were readily metabolized by C . echinulata ATCC 9244 (Foster et al. 1990a) through O-dealkylation and N-acetylation. O-Dealkylation was the predominant pathway observed with 4-propoxyamphetamine (Foster et al. 1990a). T h e addition of methoxy substituents at the 2- and 5-positions has significantly altered the expected metabolic profile of these compounds. Steric hindrance may be important in limiting O-dealkylation but the relative importance of this pathway with 2,sdimethoxy-4-(n)-propoxyamphetamine would indicated that other factors such as electronic effects and lipophilicity may also be important. 2,5-Dimethoxy-4-methylthioamphetamine was extensively metabolized by both cultures to the sulphoxide (lk) and under high substrate concentrations, to the sulphone metabolite (11). Sulphoxide formation is common in fungi, and both sulphoxide and sulphone formation have been previously reported for C. echinulata (Smith et al. 1983). A neutral or basic environment caused chemical breakdown of 2,5-dimethoxy-4-methylthioamphetamineto the sulphoxide, to a minor degree. The amount of sulphoxide found after incubation in the presence of the fungus,
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however, was higher than that found in media controls and it can be concluded that the vast majority of the sulphoxide was a product of fungal metabolism. N-Acetylation of the primary amine was an important fungal metabolic pathway in all of the amphetamines previously studied (Coutts et al. 1979, Foster et al. 1989b, c). In a homologous series of 4-substituted 0-alkyl substrates the rank order for N-acetylation significantly decreased with increasing chain length and was not detected with the n-propyl and n-butyl substrates (Foster et al. 1990a). T h e 4methoxy-, 4-ethoxy-, and 4-methylthio-2,5-dimethoxyamphetaminesdo not appear to be good substrates for the fungal N-acetyltransferase. Only 2,5-dimethoxy-4and adjacent (w-1) aliphatic hydroxylation (n)-propoxyamphetamine and the (0) products were substrates for the acetyltransferase. Although oxidative deamination was a major biotransformation process associated with the mammalian metabolism of amphetamines to the corresponding ketones and acids (Caldwell 1976), ketones were not detected with any of the substrates employed in this study.
Effect of incubation conditions on metabolism Incubation of C . echinulata ATCC 9244 cultures on the 30"angle brackets had a marked effect on morphology. T h e fungus grew as numerous micropellets rather than the normal single macropellet found in the flat incubation broths. All pellets were < 4 mm in diameter and most were < 2 mm in diameter. Not only was the rate and extent of 2,.5-dimethoxy-4-methylthioamphetamine metabolism to the sulphoxide increased, compared with that observed with the flat incubation cultures, but there was an increased rate of dextrose utilization, and ammonia formation and subsequent alkaline p H shift. This increase in metabolism could have been a result of enhanced diffusion of nutrients such as dextrose and oxygen due to the enlarged surface area of the micropellets (Righelato 197.5) relative to the macropellets. Nutrient concentrations (possibly due to catabolite repression) and substrate diffusion are probably the rate-limiting steps within the macropellets found under the flat incubation conditions. These results are consistent with those found with tranylcypromine (Foster et al. 1991b). Influence of drug concentration T h e concentration study showed that under these conditions drug concentration had an effect on biotransformation. At the highest substrate concentration, the sulphoxide was further oxidized to the corresponding sulphone and the slopes for the biotransformation of 2,5-dimethoxy-4-methylthioamphetamine and for the formation of the sulphoxide were higher than those for the two lower concentrations. This may indicate that there is an enzyme or second pathway to the sulphoxide at the higher concentration. Effect of metabolic probes Acute doses of ethanol are known to inhibit drug metabolism whereas chronic consumption results in induction of cytochrome P450 (Lieber 1990). In the mouse (Iverson et al. 1975) and rat (Creaven and Barbee 1969), ethanol pretreatment increased the recovery of amphetamine with a concomitant decrease in the formation of the aromatic hydroxylation product. Moody et al. (1990) reported that there was a statistically insignificant decrease in aromatic hydroxylation. Ethanol had an inhibitory effect on the fungal metabolism of 2,.5-dimethoxy-4-methylthioamphetamine to the sulphoxide, which was consistent with earlier results reported
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for nifedipine (Foster et al. 1990b) and phenazopyridine (Foster et al. 1 9 9 1 ~ ) . Incubation of stage 2 cultures with ethanol may then provide a model for examining the effect of acute ethanol administration. Whether the effect of ethanol on Soxidation was specific or was a general effect on fungal physiology was not known. In humans, genetic defects had been observed for several oxidative reactions with different substrates, including the prototype sparteine (Eichelbaum et al. 1979). Quinidine specifically binds to the sparteine/debrisoquine cytochrome P450 isozyme termed P450IID6 in the human (Gonzalez et al. 1988), thereby inhibiting its activity (Otton et al. 1988). In male Lewis rats quinidine significantly decreased amphetamine metabolism to p-hydroxyamphetamine (Moody et al. 1990). Quinidine and sparteine decreased the metabolism of propranolol (Foster et al. 1989a) and methoxyphenamine (Foster et al. 1989c) by C . echinulata. If the C . echinulata orthologue of P450IID6 is involved in S-oxidation of the tri-substituted amphetamine, then concomitant addition of either quinidine or sparteine should inhibit this reaction. Sparetine significantly enhanced the fungal metabolism of 2,sdimethoxy-4-methylthioamphetamineby day 7. T h e combined recovery of unchanged substrate and the sulphoxide is less than that of the controls. This may indicate that metabolism was shunted through alternative enzymic pathways to the sulphoxide and into metabolic pathway(s) which resulted in the formation of undetected product(s). However, the concentration-dependent inhibitory effect of quinidine on 2,5-dimethoxy-4-methylthioamphetaminemetabolism indicates that the fungal equivalent of P450IID6 may be involved in the formation of the sulphoxide. This observation, although apparently different to that obtained with sparteine has been observed previously with the fungal metabolism of nifedipine to dehydronifedipine (Foster et al. 1990b). T h e results from the above studies indicate that microbial models of mammalian metabolism may be sensitive to the same probe compounds used in mammalian studies. T h e incubation time frame for microbial systems, however, may be longer than those used in mammalian studies. Extrapolation of results from microbial inhibitor studies must also take into account possible effects of the probe compounds on other enzymes and transport systems which may be involved in intact mammalian systems.
Acknowledgements We gratefully acknowledge the technical assistance of R. Duhaime and D. L. Litster. We thank D r B. A. Dawson and Mr J. C. Ethier for the determination of n.m.r. and M S data, respectively.
References CALDWELL, J., 1976, The metabolism of amphetamines in mammals. Drug Metabolism Reviews, 5,219280. CLARKE, A. M., MCCHESNEY, J . D., and HUFFORD, C. D., 1985, The use of microorganisms for the study of drug metabolism. Medicinal Research Reviews, 5 , 231-253. COUTTS, R. T.,FOSTER, B. C., JONES, G. R., JONES, G . R . , and MYERS, G. E., 1979, Metabolism of ( ) - N (n-propy1)amphetamine by Cunninghamella echinulata. Applied and Environmental Microbiology, 37,429432. CREAVEN, P. J., and BARBEE, T., 1969, The effect of ethanol on the metabolism of amphetamine by the rat. Journal of Pharmacy and Pharmacology, 21, 859-860. EICHELBAUM, M . , SPANNBRUCKER, N., STEINKE, B., and DENGLER, H. J., 1979, Defective N-oxidation of sparteine in man: a new pharmacogenetic defect. European Journal of Clinical Pharmacology, 16, 183-187.
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FOSTER, B. C., BUTTAR, H. S., QURESHI, S. A., and MCGILVERAY, I. J., 1989a, Propranolol metabolism by Cunninghamella bainieri. Xenobiotica, 19, 539-546. F. M., 1989b, Biotransformation of aryl alkylamines by FOSTER,B. C., COUTTS,R. T., and PASUTTO, Cunninghamella bainieri. Xenobiotica, 19, 531-538. FOSTER,B. C., LITSTER,D. L., and LODGE,B. A,, 1991a, Biotransformation of 2-, 3-, and 4methoxyamphetamines by Cunninghamella echinulata. Xenobiotica, 21, 1337-1 346. J., and COUTTS,R. T . , 1991b. The biotransformation of FOSTER,B. C., LITSTER,D. L., ZAMECNIK, tranylcypromine by Cunninghamella echinulata. Canadian Journal of Microbiology, 37, 791-795. J., and LODGE,B. A,, 1990a, FOSTER, B. C., NANTAIS, L. M., WILSON,D. L., BY, A. W., ZAMECNIK, Fungal metabolism of 4-substituted amphetamines. Xenobiotica, 20, 583-590. B. H., ZAMECNIK, J., DAWSON,B. A,, WILSON,D . L., DUHAIME, R., FOSTER,B. C., THOMAS, SOLOMONRAJ, G., MCGILVERAY, I. J., and LODGE,B. A,, 1991c, Aromatic hydroxylation and sulfation of phenazopyridine by Cunninghamella echinulata. Canadian Journal of Microbiology, 37, 504-508. FOSTER, B. C., WILSON,D. L., and MCGILVERAY, I. J., 1989c, Effect of sparteine and quindine on the metabolism of methoxyamphetamine by Cunninghamella bainieri. Xenobiotica, 19, 445-452. I. J., 1990b, Interaction or ethanol, quinidine, and FOSTER, B. C., WILSON,D. L., and MCGILVERAY, sparteine with the metabolism of nifedipine by Cunninghamella echinulata. Biopharmaceutics and Drug Disposition, 11, 735-738. GONZALEZ, F. J., SKODA, R. C., KIMURA, S., UNENO,M., ZANGLER, U. M., NEBERT, D. W., GELBOIN, H. V., HARDWICK, J. P., and MEYER,U. A , , 1988, Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature, 331, 442-446. F., COLDWELL, B. B., DOWNIE, R. H., and WHITEHOUSE, L. W., 1975, Effect of ethanol on the IVERSON, toxicity and metabolism of amphetamine in the mouse. Experientia, 31, 679-680. LIEBER,C. S., 1990, Interaction of alcohol with other drugs and nutrients: implication for the therapy of alcoholic liver disease. Drugs, 40 (Suppl. 3), 23-44. MOODY, D. E., RUANGYUTTIKARN, W., and LAW,M. Y., 1990, Quinidine inhibits in oiwo metabolism of amphetamine in rats: impact upon correlation between GC/MS and immunoassay findings in rat urine. Journal of Analytical Toxicology, 14, 3 11-31 7. NICHOLS,D. E., and SHULGIN, A. T., 1976, Sulfur analogs of psychotomimetic amines. Journal of Pharmaceutical Sciences, 65, 1554-1 555. OTTON,S. V., BRINN,R. U., and GRAM, L. F., 1988, In witro evidence against the oxidation of quinidine by the sparteine/debrisoquine monooxygenase of human liver. Drug Metabolism and Disposition, 16, 15-17. REDDY,C. S. G., ACOSTA,D., and DAVIS,P. J., 1990, Microbial models of mammalian metabolism: biotransformations of phenacetin and its 0-alkyl homologues with Cunninghamella species. Xenobiotica, 20, 1281-1297. RIGHELATO, R. C., 1975, Growth kinetics of mycelial fungi. In The Filamentous Fungi, Vol. 1, edited by J. E. Smith and D. R. Berry. (New York, J . Wiley and Sons), pp. 79-103. J. P. N., and DUFFEL,M. W., 1986, Metabolic transformations of alkaloids. Alkaloids, 27,323ROSAZZA, 405. SARGENT, T., SHULGIN, A. T., and KUSUBOV, N., 1976, Quantitative measurement of demethylation of '4C-methoxyl-labelled DMPEA and 2,4,5-trimethoxyamphetaminein rats. Psychopharmacology Communications, 2, 199-206. SMITH, R. V., DAVIS,P. J., and KERR,K. M., 1983, Microbial transformations of pergolide to pergolide sulphoxide and pergolide sulphone. Journal of Pharmaceutical Sciences, 72, 733-736. J. P., 1975, Microbial models of mammalian metabolism. Journal of SMITH,R. V., and ROSAZZA, Pharmaceutical Sciences, 64, 1737-1 759.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Biotransformation of tri-substituted methoxyamphetamines by Cunninghamella echinulata B. C. Foster, J. McLeish, D. L. Wilson, L. W. Whitehouse, J. Zamecnik & B. A. Lodge To cite this article: B. C. Foster, J. McLeish, D. L. Wilson, L. W. Whitehouse, J. Zamecnik & B. A. Lodge (1992) Biotransformation of tri-substituted methoxyamphetamines by Cunninghamella echinulata, Xenobiotica, 22:12, 1383-1394, DOI: 10.3109/00498259209056689 To link to this article: http://dx.doi.org/10.3109/00498259209056689
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Date: 25 April 2016, At: 09:50
XENOBIOTICA,
1992, VOL. 22,
NO.
12, 1383-1394
Biotransformation of tri-substituted methoxyamphetamines
by Cunninghamella echin ula fa B. C. FOSTER?, J. McLEISH, D. L. WILSON, L. W. WHITEHOUSE, J. ZAMECNIK and B. A. LODGE Bureau of Drug Research, Sir Frederick Banting Research Centre, Health Protection Branch, Ottawa, Ontario, Canada K1 A OL2
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Received 2 January 1992; accepted 27 June 1992 1. Four trimethoxyamphetarnine analogues were incubated with the filamentous fungus Cunninghamella echinulata. 2. 2,4,5-Trimethoxyamphetamine and 2,5-dimethoxy-4-ethoxyamphetamine were poorly metabolized by C. echinulata ATCC 9244and C. echinulata var. elegans ATCC 9245. 2,5-Dimethoxy-4-(n)-propoxyamphetamine was mainly metabolized through Nacetylation and O-dealkylation with minor amounts of several aliphatic hydroxylation metabolites formed. 2,5-Dimethoxy-4-methylthioamphetamine was extensively metabolized to the corresponding sulphoxide.
3. 2,5-Dimethoxy-4-methylthioamphetaminemetabolism was inhibited by ethanol and quinidine. Sparteine did not inhibit the formation of the sulphoxide and may have shunted the substrate through alternate metabolic pathways.
4. Incubation conditions can affect the rate and extent of fungal biotransformation of 2,5-dimethoxy-4-methylthioamphetamine, and influence dextrose utilization, ammonia formation and pH.
Introduction In this era of growing social awareness and concern over the treatment of animals in scientific studies, microbial models have provided an alternative method for the study of drug metabolism. T h e two filamentous fungal strains, Cunninghamella echinulata ATCC 9244 and C . echinulata var. elegans ATCC 9245 examined in this study had been used by numerous investigators to validate the concept of microbial models of mammalian metabolism (Clarke et al. 1985, Foster et al. 1989b, 1990a, Rosazza and Duffel 1986, Smith and Rosazza 1975). Since amphetamines are metabolized in mammalian systems by several pathways, including aromatic hydroxylation, aliphatic hydroxylation, N-dealkylation, oxidative deamination, Noxidation and conjugation (Caldwell 1976) they are an excellent class of compounds for validating metabolic models. As a continuation of earlier studies to examine the effect of ring substitution on the fungal biotransformation of aryl alkyl amphetamines (Foster et al. 1989b), 4-monosubstituted amphetamines (Foster et al. 1989b), 4monosubstituted amphetamines (Foster et al. 1990a), and 2-, 3-, and 4-methoxyamphetamines (Foster et al. 1991a) we now report the C . echinulata-mediated biotransformation of four 2,4,5-tri-substituted amphetamines analogues, namely, 2,4,5-trimethoxyamphetamine(TMA-2, l a , figure 1; Sargent et al. 1976), 2,sdimethoxy-4-ethoxyamphetamine (lb), 4-(n)-propoxy-2,5-dimethoxyamphetamine (Ic) and 2,5-dimethoxy-4-methylthioamphetamine( I d , Nichols and Shulgin 1976).
t T o whom correspondence should be sent. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
R. C . Foster et al.
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R1 G
C : 2
OIWI-$
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CH30
la
CH30
lb
CHjCH20
lc Id
CH3CH2CH20
10
H
CH3S
H H H H H
If
COCH3
lg
COCH3
lh
COCHS
li
COCH3
li
COCH3
lk
H
11
H H
lm In
HO-CH2CH2CH20
lo
CH3CH(OH)CH20
H
1P
HO-CH2CH2CH20
COCHj
lq
CH~CH(OH)CH~O
COCH3
H
Figure 1. Structures of substrates and metabolites
Materials and methods Chemicals, substrates and reference compounds 2,4,5-Trimethoxyamphetamine hydrochloride (la) m.p. 98-99°C; 2.5-dimethoxy-4-ethoxyamphetamine hydrochloride (lb) m.p. 13Cb131"C; 2,5-dimethoxy-4-(n)-propylamphetaminehydrochloride (lc) m.p. 134-135°C; 2,5-dimethoxy-4-methylthioamphetatninehydrochloride (Id) m.p. 136138°C; and 2,s-dimethoxyamphetamine hydrochloride (le) m.p. 74-75°C; were synthesized for and obtained from the Bureau of Drug Research collection of reference standards. Crude /Pglucuronidase containing aryl-sulphatase extracted from molluscs (Type H-2) and sparteine sulphate were obtained from Sigma (St. Louis, MO, USA). Quinidine sulphate dihydrate was obtained from Aldrich Chemicals (Milwaukee, WI, USA). All other chemicals were of analytical grade and obtained from regular commercial sources. The sulphoxide of 2,5-dimethoxy-4-methylthioamphetamine(1k) was prepared from 300 mg (1.1 mmol) 2,5-dimethoxy-4-methylthioamphetamine hydrochloride by reaction with 230 mg (1.1 mmol) 3-chloroperoxybenzoic acid (85%) dissolved in 20 ml chloroform at room temperature for 96 h. T h e organic layer was washed with 5% K,CO, in saturated brine and dried over anhydrous MgSO,. T h e solution was filtered and evaporated to dryness. T h e crude product, containing 20-25% sulphone, was dissolved in isopropanol and triturated with concentrated HCI. T h e sulphoxide was recrystallized from ethanol/diethyl ether to give white crystals, yield 48%. T h e 'H-n.m.r. (400MHz) in D,O spectrum for sulphoxide hydrochloride showed signals at S,7.19 ( l H , s); 6.99 (1 H, s); OCH, 3-83(3H, s);OCH3 3.82 (3H,s); C H 3-63 (lH,m); CH, 2.95 (2H,m); CH,SO 2.83 (3H,s); C-CH, 1.24 (3H,d). T h e 'H-n.m.r. spectrum for 2,5-dimethoxy-4-methylthioamphetaminehydrochloride showed signals at 6,671 (2H, s); OCH, 3.68 (6H, s); C H 3.48 ( l H , m); CH, 2.73 (2H, m); SCH, 2.30 (3H, s) and C-CH, 1.14 (3H, d). Incubation Procedure Stage 1 fungal cultures were grown in 50 ml of complex medium containing 3.0 g/lOO ml trypticase soy broth (BBL, Becton Dickinson and Co., Cockeysville, M D , USA) and 0.72 g/100ml yeast extract (Difco). This medium was inoculated by loop with spores from cultures of C . echinulata ATCC 9244 or C .
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echinulata var. elegans ATCC 9245. After 72 h, the spent medium was decanted and replaced with 50 ml of sterile deionized water. T h e cultures were homogenized with a Waring blender and 2ml of the homogenate was added to stage 2 flasks of 25 ml dilute (20%) complex medium containing 1 ml of dextrose (20g/100ml)and 1 ml of filter sterilized substrate (2 mg/ml). In an interaction study, substrate was added alone and in combination with either ethanol, sparteine, or quinidine 48h after the start of Stage 2 cultures. All cultures were protected from light and incubated at 28°C on a New Brunswick model G-2 flatbed shaker at 250 rpm in 250 ml (stage 1) and 125 ml (stage 2) long-necked, metal-capped Erlenmeyer flasks. Culture controls were prepared by incubating the fungus in the absence of substrate. Substrate controls consisted of sterile medium containing the drug substrate, but without inoculum. Sample preparation Duplicate 1 ml samples were withdrawn up to 7 days after addition of substrate from all incubation and control broths and stored frozen (-20°C) until required. After thawing, samples were mixed with 1ml of an aqueous solution of 17.3 pg/ml2,5-dimethoxyamphetaminehydrochloride (le, internal standard).The samples were extracted twice by vortexing with 3 ml ethyl acetate for 1 min, then the organic layer was filtered through a Pasteur pipettecontainingglass wool and anhydrous Na,SO, (method A). In method B, samples were basified using solid K,CO, and extracted as outlined in method A. Method C was identical to method A, with the exception that 50p1 of acetic anhydride was added to the aqueous mixture prior to each extraction. T h e extracts obtained from methods A-C were reduced to approximately loop1 under a stream of dry nitrogen at 35°C and analysed by injecting 1 pl into the g.1.c. and g.1.c.-mass spectrometry apparatus. Instrumentation and analyses G.1.c. analyses were conducted on a Hewlett-Packard model 5890A gas chromatograph equipped with a ChemStation and flame ionization detector using a 15 m DB-1 megabore column with a film thickness of 1.5 pm and a 0.53 pm diameter (J&W Scientific, Inc., Folsom, CA, USA). Standard operating conditions were: splitless mode: injection port temperature, 250°C; detector temperature, 225°C; He, carrier gas 15 ml/min; make-up gas 30ml/min; H, 60ml/min; air, 320ml/min and oven temperature, 185°C. G.1.c. data are given in table 1. Electron-impact mass spectrometric analysis was performed on a Finnigan-MAT 4610B gas chromatograph-mass spectrometer. T h e g.1.c. conditions were as follows: Initial oven temperature of 70°C was held for 1 min, then increased at a rate of 25.O0C/minfor 2.7 min, and then increased at a rate of 8WClmin for 15.0min to a final oven temperature of 260" which was maintained for 10.0min; injection port temperature, 250°C; splitless mode equipped with a DB-5 15 m narrowbore (0.25 mm) column with a stationary phase film thickness of 0.25 pm (J&W Scientific Inc.). Operating conditions for the mass spectrometer were: separator temperature, 260°C; source temperature, 150°C; emission ion current, 320pA; and electron energy, 70eV. Mass spectrometric data are given in table 1. Proton n.m.r. spectra were recorded on a Bruker AM 400 spectrometer equipped with an Aspect 3000 computer and process controller. Spectra were recorded with a sweep width of about 5000 Hz, with 32 K data points, giving a final resolution of about 0.3 Hz/point. The chemical shifts ( b H were ) reported in ppm relative to tetramethylsilane as internal reference. Ammonia and dextrose were determined using an Abbott VP clinical chemistry analyser. T h e pH of the incubation broth was determined using a Fisher p H meter, model 91 5. Data analysis Group data are expressed in terms of the free base and presented as means and standard deviations unless otherwise indicated. T h e Student's t-test was used to identify significant differences between groups, with values of p < 0.05 being considered significant.
Results Metabolism of 2,4,5-trimethoxy- and 2,5-dimethoxy-4-ethoxyamphetamine G.1.c. analysis of day 7 extracts (method B) revealed that 2,4,5-trimethoxyamphetamine (1a) and 2,5-dimethoxy-4-ethoxyamphetamine(1b) were poorly metabolized by both C. echinulata ATCC 9244 (89 and 88% recovery of unchanged substrate, respectively) and C. echinulata var. elegans ATCC 9245 (108 and 91% recovered, respectively; table 2). T h e corresponding N-acetyl metabolites (1f and g) of these substrates were not detected. No other metabolites were detected with either of these substrates (methods A-C,see Sample preparation).
B. C . Foster
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Table 1. Gas-liquid chromatography retention times ( t R )and electron-impact mass spectrometric data for substrates and metabolites.
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Compoundt
Mass spectrometric data m/z (percentage relative abundance)
tRf
la lb lc Id le If 1g lh
3.48 400 5.30 6.49 2.07 1051 12.19 16.23
li 1j lk 11 lm In lo 1P
19.96 5.99 12.97 1468 7.12* 1 0 51* 11.53* 14.12*
1q
15.15'
lr
13.04*
225(2) 182(87) 167(20) 151(8) 139(5) 121(1) 44(100) 239(4) 196(96) 181(7) 167(17) 153(20) 137(10) 122(3) 44(100) 253(9) 210(100) 168(59) 167(49) 153(61) 137(28) 122(12) 44(98) 241(3) 198(63) 183(13) 167(5) 152(4) 137(3) 121(2) 44(100) 195(23) 180(8) 164(7) 152(74) 137(41) 121(19) 91(26) 77(25) 44(100) 267(21) 208(100) 193(9) 181(72) 167(6) 151(19) 136(6) 86(5) 44(56) 281(21) 222(100) 195(56) 167(55) 153(7) 137(16) 86(5) 44(56) 295(61) 236(100) 210(24) 209(59) 194(47) 179(30) 167(94) 153(24) 137(44) 122(23) 86(22) 44(49) 283(30) 224(100) 197(42) 183(10) 167(12) 152(7) 137(3) 86(12) 44(79) 237(51) 194(5) 178(100) 121(40) 86(61) 44(93) 257(1) 214(34) 199(22) 197(100) 167(12) 44(46) 273(2) 230(1) 197(1) 167(1) 44(100) 211(2) 168(54) 153(22) 13718) 12216) 44(100) 269(2) 226(61) 168(57) 167(44) 153(49) 137(28) 122(12) 59(10)45( 19)44( 100) 269(3) 226(78) 168(28) 167(25) 153(28) 137(12) 122(6) 44(100) 311(42) 252(96) 225(42) 194(60) 179(21) 168(30) 167(100) 153(16) 137(44) 122(16) 86(21) 59(11) 44(84) 311(17) 252(98) 225(40) 194(21) 179(9) 168(14) 167(100) 153(10) 137(22) 122(9) 86(10) 57(13) 44(90) 43(23) 268(15) 226(4) 194(1) 167(5) 137(5) 122(2) lOl(79) 73(8) 44(100)
t Identities of the numbered compounds are shown in figure 1. $ T h e g.1.c. conditions are described in Materials and methods. * Retention times are obtained from g.1.c.-mass spectrometry analysis. Table 2.
Recovery of 2-, 4-, 5-trisubstituted amphetamines unchanged from incubations with Cunninghamella echinulata ATCC 9244 and C. echinulata var. elegans ATCC 9245. ~
ATCC 9244 Substrate la lb lc Id
Day 3
Day 7
50.3 2.0 (84)t 55.4 2.4 (93) 27.5 f4.5 (46) 21.4 f 4 3 (36)
53.4 f4.2 (89) 52.6 & 9.6 (88) 23.0& 3.0 (38) 9.4 f3.1 (16)
+
~
ATCC 9245 Day 4
Day 7
Values are in pg/ml (percentage recovery) and are expressed as means + S D (n=3). 2,4,5Trimethoxyamphetamine (la); 2,5-dimethoxy-4-ethoxyamphetamine(1b); 2,5-dimethoxy-4-(n)propylamphetamine (lc); 2,5-dimethoxy-4-methylthioamphetamine(Id) were the substrates used. t Percentage concentration of unmetabolized substrate relative to the concentration of the parent compound recovered from substrate controls.
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Metabolism of 2,5-dimethoxy-4-(n)-propoxyamphetamine In addition to 2,5-dimethoxy-4-(n)-propoxyamphetamine(1c) which accounted for 38% of the added dose, seven new g.1.c. peaks were detected in extracts (method B) from C. echinulata ATCC 9244 incubation broths (table 2). Recovery of 2,S-dimethoxy-4-(n)-propoxyamphetamine from extracts of C. echinulata var. elegans ATCC 9245 was > 80%. T h e N-acetyl derivative of 2,5-dimethoxy-4-(n)propoxyamphetamine, namely, N-acetyl-2 ,5-dimethoxy-4-(n)-propoxyamphetamine (1h) was determined unequivocally by comparison with an authentic standard (table 1) to be the major metabolite formed by both organisms. Five of the g.1.c. peaks (lm-q; figure 1) were isolated and identified from incubation broth extracts (method B) of 2,5-dimethoxy-4-(n)-propoxyamphetamine and C . echinulata ATCC 9244 (table 2) on the basis of their g.1.c.-mass spectrometry properties. 2,5-Dimethoxy-4-hydroxyamphetamine ( l m ) was identified on the basis of the ions at m/z211 (M'), m/z 168 (C,H,,O;, indicative o f p a r a 0-dealkylation) and m/z44 (CH, - C H = N + H 2 ,intact side-chain). Four metabolites (In-q) were found to have similar properties with ions at m/z 44, 167, 168, and either m/z 225 or 226. These ions are consistent with those expected for a compound having one oxygen atom added to the 4-substituent side-chain. Two of the metabolites ( I n and 0) had an apparent molecular ion at m/z 269 and a base peak of m/z 44 (unsubstituted amine). The presence of m/z 59 (C,H,O+) in I n was characteristic of either C, or C, aliphatic hydroxylation. The C, product could readily rearrange to l m and this compound has been tentatively identified as 2,5-dimethoxy-4-(3-hydroxyprop0xy)amphetamine (In). T h e Aetabolite l o differs from I n by the absence of m/z 59. On the basis of g.1.c. and mass-spectral characteristics (lo was tentatively assigned the structure of 2,5-dimethoxy-4-(2-hydroxypropoxy)amphetamine. Two of the metabolites ( l p and q) had additional ions at m/z86 (CH,-CH = N + H C O C H , ) and m/z311 (M' of 269+42). T h e presence of m/z59 in l p suggested that this product was the corresponding N-acetyl derivative of In. T h e presence of m/z 43 ( C 2 H 3 0 + ) ,characteristic of secondary alcohol mass-spectral cleavage in l q would support the identification of this compound as the corresponding N-acetyl metabolite of lo. T h e seventh metabolite (lr), had ions consistent with that expected for a single oxidation product, but there were insufficient data to assign a definitive structure for this metabolite.
Metabolism of 2,5-dimethoxy-4-methylthioamphetamine In addition to the unchanged substrate (1d ) a single peak (1k) which accounted for most of the metabolized substrate was found in extracts (method B) from C. echinulata ATCC 9244 and C. echinulata var. elegans ATCC 9245 incubation broths containing 2,5-dimethoxy-4-methylthioamphetamine (1d; table 2). Analysis of this peak by g.1.c.-mass spectrometry characteristic ions at m/z 257 and 44 (M+ and CH,CH = N + H 2 )respectively, which were consistent with a single oxidation product associated with the aromatic ring (table 1). Other diagnostic ions at m/z 214 (M-43) and m/z 197 (m/z 214-OH) indicative of addition of a single oxygen to the sulphur. Comparison with spectra obtained from an authentic standard confirmed the identity of this metabolite as 2,5-dimethoxy-4-methylthioamphetaminesulphoxide (lk). T h e sulphone (11) was not detected in these extracts. Total recovery of 2,5-dimethoxy-4-methylthioamphetamineand the sulphoxide from day 7 incubation broths was not increased by homogenization of the cultures.
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2,5-Dimethoxy-4-methylthioamphetaminesubstrate controls without fungus were incubated at pH4.23, 7.23, and 9.30 for 7 days to determine whether the sulphoxide was a chemical breakdown product of the substrate rather than a fungal metabolite. G.1.c. analysis of the extracts (method A) from p H 7.23 and 9.30 samples revealed trace amounts ( < 0.1%) of a product having the retention characteristics of the sulphoxide metabolite. No sulphoxide was detected in the pH4.23 chromatograms. Enzyme hydrolysis using crude p-glucuronidase, of aliquots from all substrates neither increased recovery of the parent compounds nor their metabolites, nor did it yield additional metabolites (methods A and B). Since there was no evidence for the formation of the alcohol or ketone deamination products from these substrates, the incubation broths were not examined for acidic metabolites.
Effect of incubation conditions on metabolism and growth Based on the above results that 2,5-dimethoxy-4-methylthioamphetamine(1d) was the most extensively metabolized substrate with C. echinulata ATCC 9244, this compound and fungal strain were chosen for additional studies. T o determine whether a change in the incubation environment would affect the transformation of 2,5-dimethoxy-4-methylthioamphetamine, the procedure was altered to include flasks incubated on 30" angle brackets in addition to flasks incubated in the usual flat position. T h e fungus grew as numerous micropellets ( < 4 mm diameter) rather than as the normal single macropellet found in the flat incubation broths. Also, there was no surface wall growth in the cultures incubated at 30". T h e disappearance of substrate in both the flat (day 0-7) and 30" (day 0-4) incubations was linear ( r 2 = 0.9036 and 0.9790, respectively) with slopes of the regression lines being - 8-2pg/day and - 14-4pglday, respectively. No substrate was found in the incubation broth of the 30" cultures at day 7 (table 3). T h e formation of the
Table 3. Metabolism of 2,5-dimethoxy-4-methylthioamphetamineby cultures of Cunninghamella echinulata ATCC 9244 incubated either on flat or 30" angle brackets. Method of culture Flat Substrate/ metabolite Day 2 Day 4 Day 7
30" Angle
Id
lk
Id
lk
55.1 k8.5 ( 9 2 ~ 19.8 2.2 (33) 7.67 k 2.6 (13)
ND
24.1 &107* (40) 2-6*03* (4) ND*
32.0&4.3* (49H 59.1 _f9.2* (91) 62.2 k 10.0 (96)
+
28.7 & 3.2 (45) 51.4k 5.5 (80)
Values are in pg/ml (percentage recovery) and expressed as means & S D (n=5). 2,5-Dimethoxy-4methylthioamphetamine (Id); 2,5-dimethoxy-4-methylthioamphetaminesulphoxide ( l k ) were the substrates. ND, not detected. t Percentage concentration relative to the concentration of the parent compound recovered from substrate controls. 1T h e percentage recovery of the sulphoxide was based on the concentration of the sulphoxide relative to the concentration of the original thioamphetamine substrate (Id), corrected for molarity (% x [wt l d base/wt lk]). Denotes statistically different from the corresponding value obtained on the flat brackets.
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I
Q
4)
6-1
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70 60 50 40 30 20
r
0
10
0
0
1
2
3
4
5
6
7
Time of incubation (days) Figure 2. Analysis of Cunninghamella echinulata ATCC 9244 culture broths incubated under different conditions in the presence of the substrate 2,5-dimethoxy-4-methylthioamphetamine. Incubations were carried out on flat (B)or 30" angle (A)brackets, substrate controls ( 0 ) .(A) broth pH; (B) dextrose utilization (closed symbols) and ammonia formation (open symbols); and (C) substrate metabolism (closed symbols) and formation of the metabolite 2,5-dimethoxy-4methylthioamphetamine sulphoxide (open symbols) (n= 5).
sulphoxide metabolite was also linear for the flat samples between days 2 and 7 ( r 2= 0.9671, slope 10-1pg/day) whereas its formation in 30" cultures was linear up to day 4 ( r 2=0*9977, slope 14.8 pg/day). Examination of aliquots of the incubation broths analysed for pH, dextrose and ammonia (figure 2) during this comparative study revealed a relationship between biotransformation, dextrose utilization, ammonia formation and pH. T h e rate of dextrose utilization and the rate of ammonia formation mirrored in the p H as an earlier alkaline p H shift was directly related to the faster biotransformation of 2,5-dimethoxy-4-methylthioamphetamine. InfEuence of drug concentration Mammalian drug metabolism is often concentration dependent. T o determine whether this also occurs in fungal metabolism, the concentrations of 2,s-dimethoxy4-methylthioamphetamine were varied from 0.4 to 2.0 mg/flask and 10.0mg/flask in cultures of C. echinulata ATCC 9244 (figure 3). It is evident that the slopes for the
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Time o f incubation (days)
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Figure
3. Effect of substrate concentration on the metabolism of 2,5-dimethoxy-4methylthioamphetamine and formation of 2,5-dimethoxy-4-methylthioamphetaminesulphoxide by Cunninghamella echinulata ATCC 9244. Substrate concentration: 0.4 ( 0 ) ;2.0 (A);and 10mg/flask (m). Unchanged substrate (closed symbols) and the metabolite 2,5-dimethoxy-4-methylthioamphetamine sulphoxide (open symbols) (n= 5 ) .
Table 4. Metabolism of 2,5-dimethoxy-4-methylthioamphetamineCunninghamella echinulata ATCC 9244 in the presence of equimolar (lx) or fivefold (5x) higher concentrations of quinidine and sparteine. Day 4 Substrate/ metabolite Control Quinidine l x 5x Sparteine l x 5x
Day 7
Id
lk
25.7 2.3 (43)t 33.6 7.4* (56) 57.0&11.2* (95) 23.1 5.0 (39) 21.1 58.9 (35)
19.6f6.5 (3 1)$ 21.4 & 4.0 (33) 7.0 k 0.1 * (11) 21.6 f6.8 (34) 27.4 5.3* (44)
Id
lk
33.8 f11.8 (53)
Values are in pg/ml (percentage recovery) and expressed as means & S D ( n = 5 ) . 2,5-Dimethoxy-4methylthioamphetamine (1 d); 2,5-dirnethoxy-4-methylthioamphetamine sulphoxide (1k). t Percentage concentration relative to the concentration of the parent compound recovered from substrate controls. $ T h e percentage recovery of the sulphoxide was based on the percentage ratio of the sulphoxide concentration to the concentration of ( 1 d) free base, corrected for molarity (% x [wt Id base/wt lk]). * Denotes statistical significant difference, p < 0.05 control value.
disappearance of substrate and formation of the sulphoxide at the higher dose were greater than those for the two lower drug concentrations. For direct comparison of the three substrate concentrations the data was transformed to percent recovery. Disappearance of 04-10.0 mg/flask 2,5-dimethoxy-4-methylthioamphetaminewas linear for all concentrations, r 2 =0.9926 (slope - 12*2%/day),0.9844 (slope - 12.3% /day) and 0.8648 (slope - 14*3%/day),respectively. T h e formation of the sulphoxide metabolite was also linear with r2 values for 2.0 and lO.Opg/ml being 0.9835 (slope 12-3%/day)and 0.9921 (slope 164%/day), respectively. A minor new peak with a
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longer retention time was detected in extracts (method B) of the lO.Omg/flask samples. G.1.c.-mass spectrometry analysis identified this peak as the 2,sdimethoxy-4-methylthioamphetaminesulphone (1 1).
Metabolic probes Quinidine and sparteine can also provide information on the metabolic mechanisms involved in the fungal biotransformation (Foster et al. 1989a, c, 1990b). After a 7-day incubation, quinidine had a concentration-dependant effect decreasing the metabolism of 2,5-dimethoxy-4-methylthioamphetamine(Id) to the sulphoxide (1k; table 4). Incubation in the presence of sparteine had a limited effect in decreasing the amount of sulphoxide formed at day 7. Total recovery from incubations with sparteine ranged from 57 to 79%, compared with 89-103% recovery in controls and cultures containing quinidine, thus indicating that in the presence of sparteine other metabolite pathways were involved in the metabolism of this substrate (table 4). No metabolites other than the sulphoxide were detected in incubations with either quinidine or sparteine. In a concomitant study with ethanol it was observed that the sulphoxide accounted for 19% of the total recovered substrate (85%).
Discussion Comparative biotransformation Hydroxylation at the cr-carbon and (w-1)-position was responsible for the formation of major metabolites in the C . elegans biotransformation of a homologous series of O-alkyl paracetamol ethers (Reddy et al. 1990). T h e rank order of 0dealkylation for these O-alkyl substrates was ethyl > isopropyl > n-propyl > nbutyl >methyl. Two of the four amphetamine analogues, 2,4,5-trimethoxyamphetamine and 2,5-dimethoxy-4-ethoxyamphetamine,were poorly metabolized by both C . echinulata ATCC 9244 and C. echinulata var. elegans ATCC 9245. 2,5-Dimethoxy-4-(n)-propoxyamphetaminewas metabolized by cultures but to a lesser extent in C. echinulata var. elegans ATCC 9245. C. echinulata ATCC 9244 through at least three different biotransformation pathways (in order of importance): Nacetylation (1h), O-dealkylation ( l m ) and aliphatic hydroxylation ( I n and o ) . Aliphatic hydroxylation occurred at the terminal (0) and adjacent (w-1)-position. These metabolites were then substrates for N-acetylation. These results were surprising in that the corresponding methyl and ethyl 4-mono-substituted derivatives were readily metabolized by C . echinulata ATCC 9244 (Foster et al. 1990a) through O-dealkylation and N-acetylation. O-Dealkylation was the predominant pathway observed with 4-propoxyamphetamine (Foster et al. 1990a). T h e addition of methoxy substituents at the 2- and 5-positions has significantly altered the expected metabolic profile of these compounds. Steric hindrance may be important in limiting O-dealkylation but the relative importance of this pathway with 2,sdimethoxy-4-(n)-propoxyamphetamine would indicated that other factors such as electronic effects and lipophilicity may also be important. 2,5-Dimethoxy-4-methylthioamphetamine was extensively metabolized by both cultures to the sulphoxide (lk) and under high substrate concentrations, to the sulphone metabolite (11). Sulphoxide formation is common in fungi, and both sulphoxide and sulphone formation have been previously reported for C. echinulata (Smith et al. 1983). A neutral or basic environment caused chemical breakdown of 2,5-dimethoxy-4-methylthioamphetamineto the sulphoxide, to a minor degree. The amount of sulphoxide found after incubation in the presence of the fungus,
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however, was higher than that found in media controls and it can be concluded that the vast majority of the sulphoxide was a product of fungal metabolism. N-Acetylation of the primary amine was an important fungal metabolic pathway in all of the amphetamines previously studied (Coutts et al. 1979, Foster et al. 1989b, c). In a homologous series of 4-substituted 0-alkyl substrates the rank order for N-acetylation significantly decreased with increasing chain length and was not detected with the n-propyl and n-butyl substrates (Foster et al. 1990a). T h e 4methoxy-, 4-ethoxy-, and 4-methylthio-2,5-dimethoxyamphetaminesdo not appear to be good substrates for the fungal N-acetyltransferase. Only 2,5-dimethoxy-4and adjacent (w-1) aliphatic hydroxylation (n)-propoxyamphetamine and the (0) products were substrates for the acetyltransferase. Although oxidative deamination was a major biotransformation process associated with the mammalian metabolism of amphetamines to the corresponding ketones and acids (Caldwell 1976), ketones were not detected with any of the substrates employed in this study.
Effect of incubation conditions on metabolism Incubation of C . echinulata ATCC 9244 cultures on the 30"angle brackets had a marked effect on morphology. T h e fungus grew as numerous micropellets rather than the normal single macropellet found in the flat incubation broths. All pellets were < 4 mm in diameter and most were < 2 mm in diameter. Not only was the rate and extent of 2,.5-dimethoxy-4-methylthioamphetamine metabolism to the sulphoxide increased, compared with that observed with the flat incubation cultures, but there was an increased rate of dextrose utilization, and ammonia formation and subsequent alkaline p H shift. This increase in metabolism could have been a result of enhanced diffusion of nutrients such as dextrose and oxygen due to the enlarged surface area of the micropellets (Righelato 197.5) relative to the macropellets. Nutrient concentrations (possibly due to catabolite repression) and substrate diffusion are probably the rate-limiting steps within the macropellets found under the flat incubation conditions. These results are consistent with those found with tranylcypromine (Foster et al. 1991b). Influence of drug concentration T h e concentration study showed that under these conditions drug concentration had an effect on biotransformation. At the highest substrate concentration, the sulphoxide was further oxidized to the corresponding sulphone and the slopes for the biotransformation of 2,5-dimethoxy-4-methylthioamphetamine and for the formation of the sulphoxide were higher than those for the two lower concentrations. This may indicate that there is an enzyme or second pathway to the sulphoxide at the higher concentration. Effect of metabolic probes Acute doses of ethanol are known to inhibit drug metabolism whereas chronic consumption results in induction of cytochrome P450 (Lieber 1990). In the mouse (Iverson et al. 1975) and rat (Creaven and Barbee 1969), ethanol pretreatment increased the recovery of amphetamine with a concomitant decrease in the formation of the aromatic hydroxylation product. Moody et al. (1990) reported that there was a statistically insignificant decrease in aromatic hydroxylation. Ethanol had an inhibitory effect on the fungal metabolism of 2,.5-dimethoxy-4-methylthioamphetamine to the sulphoxide, which was consistent with earlier results reported
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for nifedipine (Foster et al. 1990b) and phenazopyridine (Foster et al. 1 9 9 1 ~ ) . Incubation of stage 2 cultures with ethanol may then provide a model for examining the effect of acute ethanol administration. Whether the effect of ethanol on Soxidation was specific or was a general effect on fungal physiology was not known. In humans, genetic defects had been observed for several oxidative reactions with different substrates, including the prototype sparteine (Eichelbaum et al. 1979). Quinidine specifically binds to the sparteine/debrisoquine cytochrome P450 isozyme termed P450IID6 in the human (Gonzalez et al. 1988), thereby inhibiting its activity (Otton et al. 1988). In male Lewis rats quinidine significantly decreased amphetamine metabolism to p-hydroxyamphetamine (Moody et al. 1990). Quinidine and sparteine decreased the metabolism of propranolol (Foster et al. 1989a) and methoxyphenamine (Foster et al. 1989c) by C . echinulata. If the C . echinulata orthologue of P450IID6 is involved in S-oxidation of the tri-substituted amphetamine, then concomitant addition of either quinidine or sparteine should inhibit this reaction. Sparetine significantly enhanced the fungal metabolism of 2,sdimethoxy-4-methylthioamphetamineby day 7. T h e combined recovery of unchanged substrate and the sulphoxide is less than that of the controls. This may indicate that metabolism was shunted through alternative enzymic pathways to the sulphoxide and into metabolic pathway(s) which resulted in the formation of undetected product(s). However, the concentration-dependent inhibitory effect of quinidine on 2,5-dimethoxy-4-methylthioamphetaminemetabolism indicates that the fungal equivalent of P450IID6 may be involved in the formation of the sulphoxide. This observation, although apparently different to that obtained with sparteine has been observed previously with the fungal metabolism of nifedipine to dehydronifedipine (Foster et al. 1990b). T h e results from the above studies indicate that microbial models of mammalian metabolism may be sensitive to the same probe compounds used in mammalian studies. T h e incubation time frame for microbial systems, however, may be longer than those used in mammalian studies. Extrapolation of results from microbial inhibitor studies must also take into account possible effects of the probe compounds on other enzymes and transport systems which may be involved in intact mammalian systems.
Acknowledgements We gratefully acknowledge the technical assistance of R. Duhaime and D. L. Litster. We thank D r B. A. Dawson and Mr J. C. Ethier for the determination of n.m.r. and M S data, respectively.
References CALDWELL, J., 1976, The metabolism of amphetamines in mammals. Drug Metabolism Reviews, 5,219280. CLARKE, A. M., MCCHESNEY, J . D., and HUFFORD, C. D., 1985, The use of microorganisms for the study of drug metabolism. Medicinal Research Reviews, 5 , 231-253. COUTTS, R. T.,FOSTER, B. C., JONES, G. R., JONES, G . R . , and MYERS, G. E., 1979, Metabolism of ( ) - N (n-propy1)amphetamine by Cunninghamella echinulata. Applied and Environmental Microbiology, 37,429432. CREAVEN, P. J., and BARBEE, T., 1969, The effect of ethanol on the metabolism of amphetamine by the rat. Journal of Pharmacy and Pharmacology, 21, 859-860. EICHELBAUM, M . , SPANNBRUCKER, N., STEINKE, B., and DENGLER, H. J., 1979, Defective N-oxidation of sparteine in man: a new pharmacogenetic defect. European Journal of Clinical Pharmacology, 16, 183-187.
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FOSTER, B. C., BUTTAR, H. S., QURESHI, S. A., and MCGILVERAY, I. J., 1989a, Propranolol metabolism by Cunninghamella bainieri. Xenobiotica, 19, 539-546. F. M., 1989b, Biotransformation of aryl alkylamines by FOSTER,B. C., COUTTS,R. T., and PASUTTO, Cunninghamella bainieri. Xenobiotica, 19, 531-538. FOSTER,B. C., LITSTER,D. L., and LODGE,B. A,, 1991a, Biotransformation of 2-, 3-, and 4methoxyamphetamines by Cunninghamella echinulata. Xenobiotica, 21, 1337-1 346. J., and COUTTS,R. T . , 1991b. The biotransformation of FOSTER,B. C., LITSTER,D. L., ZAMECNIK, tranylcypromine by Cunninghamella echinulata. Canadian Journal of Microbiology, 37, 791-795. J., and LODGE,B. A,, 1990a, FOSTER, B. C., NANTAIS, L. M., WILSON,D. L., BY, A. W., ZAMECNIK, Fungal metabolism of 4-substituted amphetamines. Xenobiotica, 20, 583-590. B. H., ZAMECNIK, J., DAWSON,B. A,, WILSON,D . L., DUHAIME, R., FOSTER,B. C., THOMAS, SOLOMONRAJ, G., MCGILVERAY, I. J., and LODGE,B. A,, 1991c, Aromatic hydroxylation and sulfation of phenazopyridine by Cunninghamella echinulata. Canadian Journal of Microbiology, 37, 504-508. FOSTER, B. C., WILSON,D. L., and MCGILVERAY, I. J., 1989c, Effect of sparteine and quindine on the metabolism of methoxyamphetamine by Cunninghamella bainieri. Xenobiotica, 19, 445-452. I. J., 1990b, Interaction or ethanol, quinidine, and FOSTER, B. C., WILSON,D. L., and MCGILVERAY, sparteine with the metabolism of nifedipine by Cunninghamella echinulata. Biopharmaceutics and Drug Disposition, 11, 735-738. GONZALEZ, F. J., SKODA, R. C., KIMURA, S., UNENO,M., ZANGLER, U. M., NEBERT, D. W., GELBOIN, H. V., HARDWICK, J. P., and MEYER,U. A , , 1988, Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature, 331, 442-446. F., COLDWELL, B. B., DOWNIE, R. H., and WHITEHOUSE, L. W., 1975, Effect of ethanol on the IVERSON, toxicity and metabolism of amphetamine in the mouse. Experientia, 31, 679-680. LIEBER,C. S., 1990, Interaction of alcohol with other drugs and nutrients: implication for the therapy of alcoholic liver disease. Drugs, 40 (Suppl. 3), 23-44. MOODY, D. E., RUANGYUTTIKARN, W., and LAW,M. Y., 1990, Quinidine inhibits in oiwo metabolism of amphetamine in rats: impact upon correlation between GC/MS and immunoassay findings in rat urine. Journal of Analytical Toxicology, 14, 3 11-31 7. NICHOLS,D. E., and SHULGIN, A. T., 1976, Sulfur analogs of psychotomimetic amines. Journal of Pharmaceutical Sciences, 65, 1554-1 555. OTTON,S. V., BRINN,R. U., and GRAM, L. F., 1988, In witro evidence against the oxidation of quinidine by the sparteine/debrisoquine monooxygenase of human liver. Drug Metabolism and Disposition, 16, 15-17. REDDY,C. S. G., ACOSTA,D., and DAVIS,P. J., 1990, Microbial models of mammalian metabolism: biotransformations of phenacetin and its 0-alkyl homologues with Cunninghamella species. Xenobiotica, 20, 1281-1297. RIGHELATO, R. C., 1975, Growth kinetics of mycelial fungi. In The Filamentous Fungi, Vol. 1, edited by J. E. Smith and D. R. Berry. (New York, J . Wiley and Sons), pp. 79-103. J. P. N., and DUFFEL,M. W., 1986, Metabolic transformations of alkaloids. Alkaloids, 27,323ROSAZZA, 405. SARGENT, T., SHULGIN, A. T., and KUSUBOV, N., 1976, Quantitative measurement of demethylation of '4C-methoxyl-labelled DMPEA and 2,4,5-trimethoxyamphetaminein rats. Psychopharmacology Communications, 2, 199-206. SMITH, R. V., DAVIS,P. J., and KERR,K. M., 1983, Microbial transformations of pergolide to pergolide sulphoxide and pergolide sulphone. Journal of Pharmaceutical Sciences, 72, 733-736. J. P., 1975, Microbial models of mammalian metabolism. Journal of SMITH,R. V., and ROSAZZA, Pharmaceutical Sciences, 64, 1737-1 759.
