XENOBIOTICA,
1991, VOL. 21,
NO.
7, 847-857
Regioselectivity and stereoselectivity of the metabolism of the chiral quinolizidine alkaloids sparteine and pachycarpine in the rat T. EBNERt, C. 0. MEESE and M. EICHELBAUM
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Dr Margarete Fischer-Bosch-Institut fur Klinische Pharmakologie, Auerbachstr. 112, D-7000 Stuttgart 50, Germany
Received 25 Nowember 1990; accepted 21 December 1990 1. The metabolism of (-))-sparteine and (+)-sparteine (pachycarpine) was investigated in male Sprague-Dawley rats by g.1.c.-mass spectrometry, and 13C- and 'H-n.m.r. spectroscopy. The structure of the major metabolite of (-)-sparteine was confirmed to be 2,3-didehydrosparteine by g.1.c.-mass spectrometry after alkaline sample work-up. 'H-n.m.r. spectroscopy showed that this metabolite exhibits the structure of the carbinolamine (2S)-hydroxysparteine in aqueous solution of neutral pH. No other metabolites with an enamine structure were observed by g.1.c.-mass spectrometry and 13C-n.m.r. spectroscopy. 2. Pachycarpine is metabolized in wiwo and in witro stereoselectively to the aliphatic alcohol (4s)-hydroxypachycarpine as the main metabolite. 3. The formation of the 2.3-didehydrosparteine proceeds via stereospecific abstraction of the axial 28 hydrogen atom. Inhibition in witro studied with purified rat liver microsomes demonstrated that both sparteine enantiomers are metabolized by the same cytochrome P450 isozyme. Therefore this enzyme exhibits marked substrate and product stereoselectivity for the metabolism of the two enantiomeric quinolizidine aikaloids.
Introduction The metabolism of the quinolizidine alkaloid (-)-sparteine (SP) has been studied intensively both in humans and laboratory animals. Interest in its metabolism has been stimulated by the observation that the formation of its two major metabolites in man, wiz. 2,3- and 5,6-didehydrosparteine, exhibits a genetic polymorphism (Eichelbaum et al. 1979, 1986). About 7-10% of the European population exhibit impaired formation of sparteine metabolites and are therefore defined as poor metabolizers (PM), with the remainder of the population being defined as extensive metabolizers (EM). PMs are characterized by a severe impaired capacity to form sparteine metabolites and almost exclusively unchanged sparteine is found in the urine. This is due to the absence of the cytochrome P-450 IID6 isozyme which catalyses the biotransformation of SP, debrisoquine and about 30 other drugs. The metabolism of debrisoquine (Kobayashi et al. 1989) and metoprolol (Lancaster et al. 1990) in rat is catalysed by cytochrome P-450 I I D l . This isozyme belongs to the same subgroup of cytochrome P450 enzymes as the human cytochrome P-450 IID6 which catalyses the biotransformation of the drugs undergoing the sparteine/debrisoquine polymorphism (Nebert et al. 1989). However, in contrast to humans, rats seem to excrete no 5,6-DDHSP and only
t To whom correspondence should be addressed. 0049-8254/91 $ 3 9 0
0 1991 Taylor & Francis Ltd.
T . Ebner et al.
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848
2,3-DDHSP was identified in previous studies (Eichelbaum et al. 1986). T h e structure of this metabolite was assigned indirectly by g.1.c.-mass spectrometry (figure 1). The metabolism of the (+ )-enantiomer of sparteine, pachycarpine (PA), has not been investigated as yet. This alkaloid which is much less frequently occurring in plants than (-)-sparteine, was used as a ganglion-blocking agent for the treatment of supraventricular tachycardia (Erina 1960). Preliminary studies with this drug showed that PA also is metabolized extensively in Sprague-Dawley rats. T h e oxidative metabolism leads to the predominant formation of a hydroxy metabolite whose structure was unequivocally confirmed by high-resolution and twodimensional ‘H- and I3C-n.m.r. techniques as ( +)-(423)-hydroxypachycarpine ((4S)-hydroxy-PA) with the hydroxyl-group exclusively in the axial position (Ebner et al. 1989a) (figure 1). We here report in vivo and in vitro studies concerning the formation of metabolites of both sparteine enantiomers in rat. It was the aim of our studies to examine whether both S P enantiomers are metabolized by the same cytochrome P450 isozyme. In addition, results concerning regioselectivity and stereoselectivity in the metabolism of the two enantiomeric alkaloids could provide some information about possible reaction mechanisms leading to the formation of the metabolites.
Materials and methods Chemicals [2S-*H]- and [2R-’H]-SP were synthesized as described (Ebner et al. 1989 b), isotopic purity 3 9 8 % ’H,. PA and (+)-(4S)-hydroxy-PA was isolated as described (Ebner et al. 1989a). ( +)-2,3-DDHSP, and [2-’H]-2,3-DDHSP (isotopic purity>98Yo ’H,) (Ebner et al. 1989c), 5.6-DDHSP (Leonard et al. 1955), perchlorates of 11,12-didehydrosparteine(Leonard et al. 1955) and 17-hydroxysparteine (Rink and Grabowski 1956), 17-hydroxylupanine (Wiewiorowski et al. 1967), 17-ethylsparteine (Rink and Crabowski 1956), 2- (Bellet 1950), 15- (Golebiewski and Spenser 1985),
Sparteine
Pachycarpine
M e t a b o l i t e s m o n i t o r e d by g l c - m a s s s p e c t r o m e t r y
A
B
Sporteine m e t a b o l i t e s
C
D
E
Pochycorpine m e t a b o l i t e s
Figure 1. Structure of sparteine, pachycarpine and its metabolites identified in the urine of rats by g.1.c.-mass spectrometry after alkaline sample work-up. A = 2,3-DDHSP B=lupanine; C=(4S)-hydroxy-PA; D = 2,3didehydro-parchycarpine;E= 5,6didehydro-pachycarpine.
Chiral quinolizidine alkaloids in the rat
849
and 17-oxosparteine (Rink and Grabowski 1956) were prepared according to the published methods. SP and PA monohydrogen sulphates pentahydrates were prepared from the distilled bases (Meese and Ebner 1988). Quinidine was purchased from Aldrich (Steinheim, Germany) as well as other chemicals. NADPH tetrasodium salt was obtained from Sigma (Deisenhofen, Germany), K13CN was obtained from Promochem (Wesel, Germany). (+)-Propafenone hydrochloride was a generous gift of Knoll AG (Ludwigshafen, Germany).
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Animals Male Sprague-Dawley rats (about 200 g bodyweight) were obtained from the Zentralinstitut fur Versuchstierzucht (Hannover, Germany), animals were kept in a 12 h dark-light cycle and had free access to standard diet (Standarddiat RattenlMause, Altromin, Lage, Germany) and water. Microsomes Rats were killed by decapitation, livers were removed, washed with ice-cold 0.1 M phosphate buffer (pH7.4) and were stored minced at -80°C until work-up. Purified rat liver microsomes were prepared (Osikowska-Evers and Eichelbaum 1986, Dayer et al. 1984), protein concentration and content of cytochrome P450 was determined as described (Lowry et al. 1951, Omura and Sat0 1964). Prepared microsomes containing 2mg/ml of microsomal protein in 0.1 M phosphate buffer (pH 7.4) were stored at -80°C. Incubations with rat liver microsomes were performed as described (Osikowska-Evers and Eichelbaum 1986). Each incubation mixture contained 50 pg of microsomal protein, substrate concentrations were 0.5,0.75, 1,2,5,10,20,40,80, 160, 320,640, 1280 and 2 5 6 0 ~The ~ . final volume of the incubation mixtures was 250~1,and incubation time was 5 min. Linearity of the rate of metabolite formation with respect to concentration of microsomal protein (50-1 50pglml) and incubation time (5-20 min) was observed. Inhibition studies were started by addition of substrate and NADPH after a preincubation period with the respective inhibitors of 10min. Incubations were stopped by addition of lop1 of 30% (w/v) perchloric acid. All incubations were made as duplicates. In vivo studies Sulphates of SP or PA (0.094mmol/kg body wt) and synthetic monoperchlorate of (+)-2,3-DDHSP (0.04 mmol/kg body wt) were dissolved in water and administered orally. Rats were housed in metabolic cages for 16 h and urine was collected over this period. Several experiments were also performed with an additional 16-24 h collection period. Urine samples were stored at -30°C until analysed. Analytical methods Gas chromatography. Rat urines were analysed by a capillary g.1.c. system (Ebner et al. 1989 a) with a modified temp. programme: injection temp. (on-column) 3 5 T , heating rate 30"C/min, final temp. 210"C, final time 11.5min. For the detection of lupanine, the final temp. of the g.1.c. was raised to 270°C for 15 min. The following retention times were observed (min): S P 13.8; 5,6-DDHSP 15.1; 2.3-DDHSP 15.5; 17-ethylsparteine 17.1; (4S)-hydroxy-PA 19.0; 17-oxosparteine 19.7; 2-oxosparteine (lupanine) 21.9; 15-oxosparteine 22.5. Standard curves were determined by the use of the synthetic reference compounds, using 17-ethylsparteine as internal standard. The dichloromethane extracts of the alkalized incubation mixtures were analysed by capillary g.1.c. as described (Osikowska-Evers and Eichelbaum 1986). For determination of the standard curves, synthetic reference compounds and substrate (40 PM) were added to the incubation mixtures, which contained heat-denatured microsomes. The standard mixtures were incubated simultaneously with the incubation experiments. Standard curves were obtained using the optically pure synthetic metabolites. The linearity of the analytical assay was proven over a concentration range of 10-500 pmol/incubation mixture. Mass spectra. Rat urines and incubation mixtures were analysed using a H P 5790 M S D mass spectrometer (EI ionization, 70eV) coupled to g.1.c. to confirm the identity of the formed metabolites. Isotopic contents of synthetic deuterated compounds and excreted metabolites were determined by selected ion monitoring in coupled g.1.c.-mass spectrometry using a Hewlett-Packard HP 5985 A mass spectrometer, PI/CI ionization (NH,, 72 eV). Isotopic composition of [2R-'H]-SP and [2S-'H]-SP was determined from the molecular ion [ M + l ] + m/z=236 (Ebner et al. 1989b), of synthetic (Ebner et al. 1989 c) and urinary excreted [2-'H]-2,3-DDHSP from the respective molecular ion [M 11 m / z = 234.
+
+
N.m.r. spectra. N.m.r. measurements were performed on a Bruker WP 80 (13C-n.m.r.) or CPX 300 ('H-n.m.r.). For I3C-n.m.r. measurements of synthetic iminium perchlorates (40pmol), 2 ml of 0.1 M phosphate buffer (pH 7.4) was used as solvent with dioxane as internal standard (6 =69.26 ppm). After addition of 4Opmol of K13CN the pH was adjusted to 7.4 by 0.1 M HCl. Samples of rat urine (5 ml) were lyophilized, redissolved in 2 ml of phosphate buffer and centrifuged. After addition of dioxane and K"CN the pH was adjusted to 7.4. 'H-n.m.r. of rat urine samples was performed with 5ml of lyophilized urine dissolved in 2ml of deuterium depleted water. C2H3CN was added as internal
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standard (6 = 2.078 ppm). The resonance of [2R-2H]-(2S)-hydroxysparteine (Ebner et 01. 1990a) showed a chemical shift of 6 =4.56 ppm.
Analysis of results Enzyme kinetics were calculated using a computerized nonlinear least square iterative procedure (Wilkinson 1986).
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Results After administration of SP and PA to male Sprague-Dawley rats (n= 5) marked differences in the metabolic pattern of the excreted metabolites were observed by g.1.c. (figure 2). For SP, 2,3-DDHSP was found as the main metabolite, the structure of which was confirmed by direct comparison with the synthetic reference compound. T h e mass spectra as well as the retention times of both the synthetic and the metabolic product were identical. After administration of stereospecifically labelled [2S-*H]-SP, the excreted 2,3-DDHSP exhibited the same mass spectrum as the unlabelled metabolite. In contrast, the main metabolite which was excreted after administration of [ZR-'H]-SP retained more than 95% of the label. With temperatures in the g.1.c. temperature programme (see Experimental section) for the analysis of urine samples, a metabolite with a considerable longer retention time (21.9min) was observed (figure 2). T h e mass spectrum of this metabolite exhibits a molecular ion m / z = 248, which is compatible with an 0x0 derivative of SP. Comparison with the mass spectra of synthetic 15-oxosparteine ( m / z (rel. abundance, yo):248 (30.0, M'), 137 (25-3), 98 (100)) or 17-oxosparteine ( m / z(rel. abundance, %): 248 (45-8, M'), 110 (72.0), 97 (loo)), showed no identity with either compound. In contrast, full accordance with 2-oxosparteine (lupanine) was found ( m / z (rel. abundance, yo):248 (39.6, M'), 137 (100)). When synthetic
4
!
3
.I
-----I8
Time [ m i n ]
I
14
WCLL
1
17
I
20
I
23
Figure 2. G.1.c. separation of SP and metabolites excreted in rat urine. 1: Unchanged SP; 2: 2,3-DDHSP; 3: 17-ethylsparteine (internal standard); 4: lupanine; the arrow indicates retention time of 5,6-DDHSP.
Chiral quinolizidine alkaloids in the rat
851
Table 1. Cumulative urinary excretion of unchanged sparteine enantiomers and metabolites after oral administration of sparteine (SP) and pachycarpine (PA) sulphates to rats. Pachycarpine ~
~~
Unchanged PA 2,451.0
~
(4S)-Hydroxy-PA
5,6-DDHPA
2,3-DDHPA
Total
58.6 & 6.2
0.3f0.1
0.3 f0.05
61.6f 6.2
2,3-DDHSP
Lupanine
Total
16.9f6.0
6.3 f 0.8
25.2k6.5
Sparteine Unchanged SP
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2.1 f O . 5
Values are meansfSD for @-16 h excretion, expressed as percentage of dose. The doses were five rats. 5,6-DDHPA, 2,3-DDHPA=5,6-, and 94pnol/kg given to each of 2,3-didehydro-pachycarpine.
(+)-2,3-DDHSP was given to rats (n=2), 13.070 and 10.4% of the dose was recovered unchanged, and 16.4% and 16.1Yo of the dose was excreted as lupanine. Other didehydro metabolites such as 5,6-DDHSP or 11,12-didehydrosparteine were not observed during the in wiwo studies by g.1.c.-mass spectrometry. Following administration of PA, the main metabolite (4S)-hydroxy-PA was formed, and only traces of 5,6- and 2,3-didehdropachycarpine could be quantified by g.1.c. using the synthetic dextrorotary analogues as reference compounds. T h e urinary recoveries of SP and PA and its metabolites are listed in table 1. When K13CN was added to buffered (pH 7.4) aqueous solutions of iminiumstructured synthetic sparteine derivatives, the formation of different cyanosparteines was observed. T h e 13C-n.m.r. spectra of such samples exhibit the signal of the 13CN-group of the different cyanosparteines (table 2), whereas the natural abundance of the 13C resonances of the other 15 carbon atoms are not detected. If excess K12CN is added to the samples, a marked decrease of the 13C nitrile resonance, due to a rapid exchange of the cyano group, was found. After addition of K13CN to samples of rat urine, which was excreted after administration of SP, the resonance of the (%a-cyanosparteinewas observed and no respective signal of other compounds listed in table 2 was found. Resonances at 124.2, 123-0, 122.4, and 122.3ppm were also present, but these may also be obtained with blank urine samples and are therefore caused by reaction products of physiological components of the urine samples with the labelled cyanide.
Table 2.
13C-n.m.r. chemical shifts of the 13CNgroup of different cyanosparteines in aqueous buffered solution (pH 7.4). Compound
Chemical shift (p.p.m.)
6-' 3CN-sparteine 1l-'3CN-sparteine 6- "CN- 17-oxosparteine ( 2 3 - l 3CN-sparteine (17S)-'3CN-2-oxosparteine (17S)-13CN-sparteine *H,-dioxane: 6=69.26p.p.m., "CN-: 6= 116.79p.p.m.
119.79 119.86 120.13 121.31 124.01 126.92
T . Ebner et al.
852 Table 3.
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Liver sample no.
Enzyme kinetic parameters for hepatic microsomal oxidation of pachycarpine (PA). KnI
Vmex
ViUSJKSl
Metabolite
(PM)
(pmol/min per mg)
(min- ’)
1
(4S)-Hydroxy-PA 5,6-DDHPA
3.1 3.9
771 294
249 75
2
(4S)-Hydroxy-PA 5,6-DDHPA
3.9 5.9
498
128 29
1-71
Only one deuterium resonance was recorded by ’H-n.m.r. apart from the signal of C2H3CN (C2H3:6=2.078ppm) which was used as internal standard with rat urine samples after administration of [2R-’H]-SP. This resonance can be assigned to the axial methine deuterium atom in position 2 of the carbinolamine [2R-’H](2s)-hydroxysparteine (Ebner et al. 1990a). This carbinolamine derivative of sparteine is spontaneously formed when the synthetic perchlorate of [2-2H]-2,3DDHSP is added to blank urine or to buffered aqueous solutions ( p H 4-8). Biphasic Michaelis-Menten kinetics were calculated for the formation of 2,3DDHSP in the course of in vitro experiments with purified liver microsomes of male Sprague-Dawley rats. Enzyme kinetic parameters were calculated ( n = 2) for the high-affinity ( K , 2.0 f0.4 p ~ V,,,l294 , +_ 97 pmol/min per mg) and the lowaffinity component ( K , 1235 & 40 p ~ V,,, , 6346 & 884 pmol/min per mg). High substrate concentrations (concn of SP> 1 mM) resulted in the formation of very small quantities of two didehydrosparteines, identified by g.1.c.-mass spectrometry after alkaline work-up as 5,6-DDHSP and 11,12-didehydrosparteine(less than 50pmol/ml incubation mixture). These products were also found when SP was incubated with heat-denatured (30 min, 90°C) microsomes. No lupanine was formed during the in vitro experiments with rat liver microsomes and rat liver homogenates. The formation of (4S)-hydroxy-PA and 5,6-didehydropachycarpine exhibited monophasic enzyme kinetics. I n contrast to the formation of the main metabolite of S P no low-affinity/high-activity component was observed. Results of the in vitro experiments are listed in table 3 . As a measure for the enzymic activity at low, non-saturating concentrations the intrinsic clearance (CL,) was calculated from the Vmax/Km term. T h e influence of different inhibitors on the metabolism of both sparteine enantiomers was examined using the in vitro system mentioned above. (&)-Propafenone (Siddoway et al. 1987) and quinidine (Otton et al. 1988, Spiers et al. 1986) were used as inhibitors since these drugs are known to be potent competitive inhibitors of the human sparteine metabolism in vivo and in vitro. Both compounds showed marked competitive inhibition for the formation of 2,3-DDHSP from S P and (4S)-hydroxy-PA from PA. Dixon plots for (+_)-propafenoneare shown in figure 3. T h e estimated Ki values of (+-)-propafenone were 180nM for S P and 260nM for PA. T h e respective values of quinidine were 2 7 0 n ~(SP) and l 0 O n ~ (PA). When SP was added to incubations with PA (substrate concentration= 8 0 ~ ~ ) the formation of (4S)-hydroxy-PA was also inhibited. SP concentrations of 5 p ~ , ~ O P Mand , 8 O p revealed ~ a 43%, 70%, and 89%, respectively, inhibition of the formation of the main metabolite of PA.
853
Chiral quinolizidine alkaloids in the rat
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-1
0
1
2
3
2
3
Propafenone [ wMI
0.008 0.006 0.004 0.002
?
0.000 -1
0
1
r
Propafenone [ pM1 Figure 3. Competitive inhibition of 2,3-DDHSP formation from sparteine (SP) and (4S)-hydroxy-PA formation from pachycarpine (PA) by ( f)-propafenone. Each point is the mean of two determinations; substrate concentrations were 2 , s and 1 5 , 10 and 2 0 p M (PA).
0 (SP); ~ ~
Discussion T h e structure of the main metabolite of sparteine in the rat was confirmed by direct comparison with the synthetic compound using analytical and spectroscopic methods. T h e enamine base 2,3-DDHSP was identified by g.1.c. and g.1.c.-mass spectrometry, the structure of which had previously been assigned indirectly. Previous studies of other substrates have demonstrated that biodegradation products or intermediate metabolites with electrophilic properties such as iminium ions react very easily with cyanide (Murphy 1973, Ho and Castagnoli 1980). Therefore, radiolabelled 14C-cyanide has been used in in oitro studies for the trapping of iminium structured metabolites (Whittlesea et at. 1991). Synthetic cyanosparteines can be prepared by reaction of the corresponding iminium derivatives of S P with cyanide (Leonard et al. 1955, Ebner et al. 1 9 8 9 ~ )T. h e use of the 14C radiolabel, however, provides no information about the chemical structure of the products. I n contrast, the use of 13C-labelled drugs in conjunction with 13C-n.m.r. spectroscopy has been shown to be superior for monitoring drug
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[ 2 R -2Hl-Sparteine
I
2i
*
Oxidative metabolism
c-^t'_zi?
Excreted i n urine, pH 4-8 (Monitored by 2H-nmr)
"0
[ 2 R -'H]-(2S)-hydroxysparteine
'"2"
/
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Alkaline work-up
[2-'H1-2,3-didehydrosparteine (Measured by glc
Figure 4.
[2R -2Hl-(2S -''CN)-sparteine
- mass spectrometry)
(Monitored by ISC-nmr)
Formation, detection, and chemical transformations of the main metabolite of [2R-*HI-sparteine.
At pH 4 3 the 1,2-didehydrosparteinium ion is formed by acid-catalysed, non-enzymic dehydration.
metabolism (Meese et al. 1990, Meese and Fischer, 1990). Therefore, stable labelled I3C-cyanide and '3C-n.m.r.-spectroscopy was used for the screening of sparteine metabolites in rat urine. This method offers additional information on the structure of the metabolically formed products. T h e observation of only the one 3C-n.m.r. resonance signal corresponding to authentic (2s)-cyanosparteine provides further evidence for the structure of the main urinary excreted metabolite of SP in the rat. In contrast, urine of human extensive metabolizers of SP exhibited two resonances, according to two '3C-cyanosparteines, viz. (2s)- and 6-cyanosparteine, which were formed from the two human metabolites of SP (data not shown). In full accordance with our results obtained with the human major metabolite of SP (Ebner et al. 1990a), the main metabolite of SP in the rat exhibits the structure of (2s)-hydroxysparteine in neutral aqueous solution. T h e free enamine base 2,3DDHSP which is detected by g.1.c.-mass spectrometry has to be considered as an artefact formed during the strongly alkaline analytical work-up. No other metabolite of S P with enamine structure was found in the urine of rats by g.1.c.-mass spectrometry, or I3C- and 2H-n.m.r. spectroscopy. However, lupanine (2-oxosparteine) is a secondary metabolite of SP, which is probably formed by oxidation of the carbinolamine derivative of 2,3-DDHSP, (2s)hydroxysparteine. Lupanine was recently reported to be a second metabolite of SP in rat (Chaudhuri and Keller 1990). Similar lactame-structured metabolites were reported for other drugs (Ruenitz et al. 1979, Schwartz and Kolis 1973), the formation of which is catalysed by various cytosolic (Brandange and Lindblom 1979) or microsomal (Obach and Vunakis 1990) iminium oxidases. 5,6-DDHSP was completely absent from samples of rat urine and only traces of this compound beside other oxidation products were formed in vitro, by non-enzymic oxidation.
'
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Chiral quinolizidine alkaloids in the rat
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Thus, the assumed formation of 5,6-DDHSP by rat liver microsomes, as reported in the literature (Guengerich 1984), is not consistent with our results. PA is metabolized in the rat to a stable aliphatic alcohol, o i z . (4S)-hydroxy-PA by the same enzyme which catalyses the formation of the main metabolite of SP, excreted in the urine as (2S)-hydroxy-SP. This carbinolamine dehydrates very readily and only the enamine 2,3-DDHSP can be extracted into non-polar solvents. Since exchange of the hydroxyl group may also take place in aqueous solution, it is not possible to find out whether the hydroxyl group of (2S)-hydroxy-SP is transferred to the sparteine molecule by the enzyme directly, as it is obviously the case for the (4S)-hydroxy-PA. However, our results with the stereospecifically labelled analogues [2R-’H]- and [2S-’H]-SP (Ebner et al. 1990a, b, Ebner and Meese 1988) clearly indicate a high degree of stereoselectivity of the metabolizing cytochrome P450 isozyme for the abstraction of the axial /3-hydrogen. This is closely related to the stereoselective hydroxylation of PA to (4S)-hydroxy-PA. The axial b-hydrogens in positions 2 or 4 are abstracted exclusiveIy during the formation of the main metabolites of the respective enantiomers. In the course of the formation of the 5,6-DDHSP, the human minor metabolite, and also the 5,6didehydropachycarpine in the rat, the hydrogen atom at position 6, which is also located on the /hide of the SP molecule, is also abstracted. Thus, the enzymic attack seems to occur from the sterically less-hindered /%side. A reaction mechanism similar to the formation of the aliphatic alcohol (4S)-hydroxy-PA, i.e. direct C-hydroxylation of the carbon atom C-2 by the enzyme to yield (2S)-hydroxy-SP is therefore also probable for the formation of the main metabolite of SP. The cytochrome P450 isozyme also shows a high degree of regioselectivity, as only positions of ring A of the tetracyclic sparteine molecule are involved in metabolic reactions in mammals. The binding of the molecule is mediated by the second nitrogen (N-16) as was proposed by different authors (Meyer et al. 1986, Wolff et al. 1985). Small changes in orientation between the SP and PA molecules bound to the enzyme may cause alteration in product formation of the two sparteine enantiomers. Variations of the structure of the binding sites of the human cytochrome P-450 IID6 and rat cytochrome P-450 I I D l could explain the divergencies in the metabolic pattern for the metabolism of SP between the two species. Results about marked differences in the affinity for different competitive inhibitors of the human and rat isozyme reported for the metabolism of debrisoquine (Kobayashi et al. 1989) and metoprolol (Lancaster et al. 1990) are also consistent with differences in the geometry of the binding sites of the two enzymes. Our investigations on the metabolism of PA are the first reported case of a metabolic reaction at an aliphatic CH, group of the sparteine molecule not adjacent to nitrogen. In addition, ’H-n.m.r. spectroscopy and the use of l3C-labe1led reagents, such as cyanide, together with 3C-n.m.r. spectroscopy, is another example for the use of n.m.r. methods for the identification of metabolic products. Finally, these results indicate that both enantiomers of this quinolizidine alkaloid are metabolized by the same cytochrome P450 isozyme which has a remarkable regioselectivity and stereoselectivity for the two enantiomers.
Acknowledgements This work was supported by the Robert-Bosch Foundation, Stuttgart. Excellent technical assistance of Ms S. Hod1 and Mr J. Rebel1 is gratefully acknowledged.
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References BELLET,P. M., 1950, Contribution a I’etude de la lupanine et ses derrivkes. Annales Pharmaceutiques Francaises, 8, 551. BRANDANGE, S., and LINDBLOM, L., 1979, T h e enzyme ‘aldehyde oxidase’ is an iminium oxidase. Reaction with nicotine A’(5’iminiumion. Biochemical and Biophysical Research Communications, 91, 991. CHAIJDHURI, A,, and KELLER, W. J., 1990, New mammalian metabolites of sparteine. Life Sciences, 47, 319. DAYER, P., GASSER, R., GUT,J., KRONBACH, T., ROBERTZ, G . M., EICHELBAUM, M., and MEYER, U. A,, 1984, Characterisation of a common genetic defect of cytochrome P-450 function (Debrisoquinesparteine type polymorphism). Biochemical and Biophysical Research Communications, 125, 374. EBNER,T . , and MEESE,C. O., 1988, The use of deuterated analogues to study the mechanism of sparteine oxidation in man. Naunyn-Schmiedeberg’s Archives of Pharmacology, Supplement, 337, R 121, 482. EBNER,T., EICHELBAUM, M.. FISCHER, P . , and MBESE,C. O., 1989a, u b e r die stereospezifische Hydroxylierung von ( +)-Spartein (Pachycarpin) in der Ratte. Archiv der Pharmazie, 332, 399. EBNER, T., REDELL, J., FISCHER, P., and MEESE,C. O., 1989 b, Synthesis and spectroscopic stereospecificity assay of the deuterated quinolizidine alkaloids (2S)-[’H]- and (2R)-[’H]-sparteine. Journal of Labelled Compounds and Radiopharmaceuticals, 27, 485. H., REMBERG, G., and MEESE,C. 0.. 1989c, 2,3-Didehydrospartein. Liebigs EBNER,T., LACKNER, Annalen der Chemie, 197. EBNER, T., MEESE,C. O., FISCHER, P., and EICHELBAUM, M., 1990a. T h e use of stable label isotope-high resolution NMR technique for metabolic studies: The metabolism of sparteine. European Journal of Pharmacology, 183, 1640. EBNER, T., MEESE,C. O., and EICHELBAUM, M., 1990b, Stable isotope studies on the stereochemistry of the polymorphic oxidation of sparteine and debrisoquine in man. Chirality and Biological Activity, edited by B. Holmstedt, H. Frank, B. Testa, (New York: Alan R. Liss), p. 191. EICHELUAUM. M., SPANNBRUCKER, N., STEINCKE, B., and DENGLER, 11. J., 1979, Defective N-oxidation of sparteine in man: a new pharmacogenetic aspect. European Journal of Clinical Pharmacology, 16, 183. EICHEI.nAUM, M., REETZ,K. P., SCHMIDT, E. K., and ZEKORN, C., 1986, T h e genetic polymorphism of sparteine metabolism. Xenobiotica, 16, 465. ERINA, E. V., 1960, Pharmacology of ganglionic transmission. Handbook of Experimental Pharmacology, edited by D. A. Kharkevitch (Berlin, Heidelberg, New York: Springer Verlag), pp. 425-438. W. M., and SPENSER, 1. D., 1985, Lactams of sparteine. Canadian Journal of Chemistry, GOLEBIEWSKI, 63, 716. GUENGERICH, F. P., 1984, Oxidation of sparteines. Evidence against the formation of N-oxides. Journal of Medicinal Chemistry, 27, 1101. Ho, B., and CASTAGNOLI, N ., 1980, Trapping of metabolically generated electrophilic species with cyanide ion: metabolism of 1-benzylpyrrolidine. Journal of Medicinal Chemistry, 23, 133. KOBAYASHI, S., MURRAY, S.,WATSON,D., SESARDIC, D., DAVIES, D. S. and BOOBIS,A. R., 1989, T h e specificity of inhibition of debrisoquine 4-hydroxylase activity by quinidine and quinine in the rat is inverse of that in man. Biochemical Pharmacology, 38, 2795. LANCASTER, D. L., ADIO,R. A,, TAI,K. K., SIMOOYA, 0. O., BROADHEAD, G. D., TUCKER, G. T., and LENNARD, M . S., 1990, Inhibition of metroprolol metabolism by chloroquine and other antimalarial drugs. Journal of Pharmacy and Pharmacology, 42, 267. LEONARD, N. D., THOMAS, P. D., and GASH, V. W., 1955, Structures and reactions of the dehydrosparteines and their salts. Journal of the American Chemical Society, 77, 1552. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., 1951, Protein measurement with Folin-phenol-reagent. Journal of Biological Chemistry, 193, 265. MEESE,C. O., and EBNER, T., 1988, Synthesis of 5-, 6-, and 7-deutero sparteines. Journal of Labelled Compounds and Radiopharmaceuticals, 25, 329. P., 1990, Tracing the human metabolism of stable isotope-labelled drugs by MEESE,C. O., and FISCHER, ex wivo NMR spectroscopy. A revision of S-carboxymethyl-L-cysteine biotransformation. Zeitschrz’ftfur Naturforschung, 45 c , 139~-143. D., and FISCHER, P., 1990, Revision of S-carboxymethyl-I,-cysteine metabolism MEESE,C. O., SPECHT, by ”C-NMR spectroscopy. Fresenius Journal of Analytical Chemistry, 331, 130. MEYER,U. A., GUT,J., KRONBACH, T . , SKODA, C., MEIER,U. T., and CATIN,T., 1986, T h e molecular mechanisms of two common polymorphisms of drug oxidation-evidence for functional changes in cytochrome P-450 isoenzymes catalyzing bufuralol and mephenytion oxidation. Xenobiotica, 16, 449. P. J., 1973, Enzymatic oxidation of nicotine to nicotine A1(s’iminiumion. Journal of Biological MURPHY, Chemistry, 248, 2796. D. W., NELSON,D. R., ADESNIK,M., COON,M . J., ESTABROOK, R. W., GONZALEZ, F. J . . NEBERT, GUENGERICH, F. P., GUNSALUS, I. C., JOHNSON, E. F., KEMPER, B., LEVIN,W., PHILLIPS, I. R.,
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SATO,R., and WATERMAN, M. R., 1989, The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci. D N A , 8 , 1. H. V., 1990, Nicotinamide adenine dinucleotide (NAD)-dependent OBACH,R. S., and VUNAKIS, oxidation of nicotine A*%ninium ion to cotinine by rabbit liver microsomes. Biochemical Pharmacology, 39, R1. OMURA, T., and SATO,R., 1964, The carbon monoxide binding pigment of liver microsomes. Evidence for its haemoprotein nature. Journal of Biological Chemistry, 239, 2370. OSIKOWSKA-EVERS, B. A., and EICHELBAUM, M., 1986, A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver. Life Sciences, 38, 1775. OTTON,S. V., CREWE, H. K., LENNARD, M. S., TUCKER, M. S., and WOODS,H. F., 1988, Use of quinidine inhibition to define the role of sparteine/debrisoquine cytochrome P-450 in metoprolol oxidation by human liver microsomes. Journal of Pharmacological and Experimental Therapeutics, 247, 242. RINK,M., and GRABOWSKI, K., 1956, Zur Kenntnis des 17-Hydroxysparteins. Archie, der Pharmazie, 289, 695. RUENITZ, P. C., MCLENNON, T. P., and STERNSON, L. A., 1979, Biotransformation of azapetine to oxazapetine in rat and liver fractions. Drug Metabolism and Disposition, 7 , 204. SCHWARTZ, M. A., and KOLIS,S. J., 1973, Oxidation of an amino carbinol by an NAP+-dependent microsomal dehydrogenase. Drug Metabolism and Disposition, 1, 322. SIDDOWAY, L. A,, THOMPSON, K. A., MCALLISTER, C. B., WANG,T., WILKINSON, G. R., and RODEN, D . M., 1987, Polymorphism of propafenone metabolism and disposition in man: clinical and pharmacokinetic consequences. Circulation, 7 5 , 785. SPIERS, C. J., MURRAY, S., BOOBIS, A. R., SEDDON, C. E., and DAVIES, D . S., 1986, Quinidine and the identification of drugs whose elimination is impaired in subjects classified as poor metabolisers of debrisoquine. British Journal of Clinical Pharmacology, 22, 739. WHITTLESEA, C. M. C., LAM,L., and GORROD, J. W., 1991, In vitro metabolism of some therapeutic alicyclic amine drugs by rabbit hepatic microsomes in the presence of 14C sodium cyanide. Presented at the 4th international symposium on the biological oxidation of nitrogen in organic molecules, Munich 1989. Progress in Pharmacology and Clinical Pharmacology (in press). WIEWIOROWSKI, M., EDWARDS, 0. E., and BRATEK-WIEWIOROWSKA, M. D., 1967, Conformation of the C , , lupine alkaloids. Canadian Journal of Chemistry, 45, 1447. WILKINSON, L., 1986, Systat: the Systems for Statistics (Evanston, IL: Systat). WOLFF,T., DISTELRATH, L. M., WORTHINGTON, M. T., GROOPMAN, J. D . , HAMMONS, G . J., KADLUBAR, F. F., PROUG, R. A., MARTIN, M. V., and GUENGERICH, F. P., 1985, Substrate specifity of human liver cytochrome P-450 debrisoquine 4-hydroxylase probed using immunochemical inhibition and chemical modeling. Cancer Research, 45, 21 16.
1991, VOL. 21,
NO.
7, 847-857
Regioselectivity and stereoselectivity of the metabolism of the chiral quinolizidine alkaloids sparteine and pachycarpine in the rat T. EBNERt, C. 0. MEESE and M. EICHELBAUM
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Dr Margarete Fischer-Bosch-Institut fur Klinische Pharmakologie, Auerbachstr. 112, D-7000 Stuttgart 50, Germany
Received 25 Nowember 1990; accepted 21 December 1990 1. The metabolism of (-))-sparteine and (+)-sparteine (pachycarpine) was investigated in male Sprague-Dawley rats by g.1.c.-mass spectrometry, and 13C- and 'H-n.m.r. spectroscopy. The structure of the major metabolite of (-)-sparteine was confirmed to be 2,3-didehydrosparteine by g.1.c.-mass spectrometry after alkaline sample work-up. 'H-n.m.r. spectroscopy showed that this metabolite exhibits the structure of the carbinolamine (2S)-hydroxysparteine in aqueous solution of neutral pH. No other metabolites with an enamine structure were observed by g.1.c.-mass spectrometry and 13C-n.m.r. spectroscopy. 2. Pachycarpine is metabolized in wiwo and in witro stereoselectively to the aliphatic alcohol (4s)-hydroxypachycarpine as the main metabolite. 3. The formation of the 2.3-didehydrosparteine proceeds via stereospecific abstraction of the axial 28 hydrogen atom. Inhibition in witro studied with purified rat liver microsomes demonstrated that both sparteine enantiomers are metabolized by the same cytochrome P450 isozyme. Therefore this enzyme exhibits marked substrate and product stereoselectivity for the metabolism of the two enantiomeric quinolizidine aikaloids.
Introduction The metabolism of the quinolizidine alkaloid (-)-sparteine (SP) has been studied intensively both in humans and laboratory animals. Interest in its metabolism has been stimulated by the observation that the formation of its two major metabolites in man, wiz. 2,3- and 5,6-didehydrosparteine, exhibits a genetic polymorphism (Eichelbaum et al. 1979, 1986). About 7-10% of the European population exhibit impaired formation of sparteine metabolites and are therefore defined as poor metabolizers (PM), with the remainder of the population being defined as extensive metabolizers (EM). PMs are characterized by a severe impaired capacity to form sparteine metabolites and almost exclusively unchanged sparteine is found in the urine. This is due to the absence of the cytochrome P-450 IID6 isozyme which catalyses the biotransformation of SP, debrisoquine and about 30 other drugs. The metabolism of debrisoquine (Kobayashi et al. 1989) and metoprolol (Lancaster et al. 1990) in rat is catalysed by cytochrome P-450 I I D l . This isozyme belongs to the same subgroup of cytochrome P450 enzymes as the human cytochrome P-450 IID6 which catalyses the biotransformation of the drugs undergoing the sparteine/debrisoquine polymorphism (Nebert et al. 1989). However, in contrast to humans, rats seem to excrete no 5,6-DDHSP and only
t To whom correspondence should be addressed. 0049-8254/91 $ 3 9 0
0 1991 Taylor & Francis Ltd.
T . Ebner et al.
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848
2,3-DDHSP was identified in previous studies (Eichelbaum et al. 1986). T h e structure of this metabolite was assigned indirectly by g.1.c.-mass spectrometry (figure 1). The metabolism of the (+ )-enantiomer of sparteine, pachycarpine (PA), has not been investigated as yet. This alkaloid which is much less frequently occurring in plants than (-)-sparteine, was used as a ganglion-blocking agent for the treatment of supraventricular tachycardia (Erina 1960). Preliminary studies with this drug showed that PA also is metabolized extensively in Sprague-Dawley rats. T h e oxidative metabolism leads to the predominant formation of a hydroxy metabolite whose structure was unequivocally confirmed by high-resolution and twodimensional ‘H- and I3C-n.m.r. techniques as ( +)-(423)-hydroxypachycarpine ((4S)-hydroxy-PA) with the hydroxyl-group exclusively in the axial position (Ebner et al. 1989a) (figure 1). We here report in vivo and in vitro studies concerning the formation of metabolites of both sparteine enantiomers in rat. It was the aim of our studies to examine whether both S P enantiomers are metabolized by the same cytochrome P450 isozyme. In addition, results concerning regioselectivity and stereoselectivity in the metabolism of the two enantiomeric alkaloids could provide some information about possible reaction mechanisms leading to the formation of the metabolites.
Materials and methods Chemicals [2S-*H]- and [2R-’H]-SP were synthesized as described (Ebner et al. 1989 b), isotopic purity 3 9 8 % ’H,. PA and (+)-(4S)-hydroxy-PA was isolated as described (Ebner et al. 1989a). ( +)-2,3-DDHSP, and [2-’H]-2,3-DDHSP (isotopic purity>98Yo ’H,) (Ebner et al. 1989c), 5.6-DDHSP (Leonard et al. 1955), perchlorates of 11,12-didehydrosparteine(Leonard et al. 1955) and 17-hydroxysparteine (Rink and Grabowski 1956), 17-hydroxylupanine (Wiewiorowski et al. 1967), 17-ethylsparteine (Rink and Crabowski 1956), 2- (Bellet 1950), 15- (Golebiewski and Spenser 1985),
Sparteine
Pachycarpine
M e t a b o l i t e s m o n i t o r e d by g l c - m a s s s p e c t r o m e t r y
A
B
Sporteine m e t a b o l i t e s
C
D
E
Pochycorpine m e t a b o l i t e s
Figure 1. Structure of sparteine, pachycarpine and its metabolites identified in the urine of rats by g.1.c.-mass spectrometry after alkaline sample work-up. A = 2,3-DDHSP B=lupanine; C=(4S)-hydroxy-PA; D = 2,3didehydro-parchycarpine;E= 5,6didehydro-pachycarpine.
Chiral quinolizidine alkaloids in the rat
849
and 17-oxosparteine (Rink and Grabowski 1956) were prepared according to the published methods. SP and PA monohydrogen sulphates pentahydrates were prepared from the distilled bases (Meese and Ebner 1988). Quinidine was purchased from Aldrich (Steinheim, Germany) as well as other chemicals. NADPH tetrasodium salt was obtained from Sigma (Deisenhofen, Germany), K13CN was obtained from Promochem (Wesel, Germany). (+)-Propafenone hydrochloride was a generous gift of Knoll AG (Ludwigshafen, Germany).
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Animals Male Sprague-Dawley rats (about 200 g bodyweight) were obtained from the Zentralinstitut fur Versuchstierzucht (Hannover, Germany), animals were kept in a 12 h dark-light cycle and had free access to standard diet (Standarddiat RattenlMause, Altromin, Lage, Germany) and water. Microsomes Rats were killed by decapitation, livers were removed, washed with ice-cold 0.1 M phosphate buffer (pH7.4) and were stored minced at -80°C until work-up. Purified rat liver microsomes were prepared (Osikowska-Evers and Eichelbaum 1986, Dayer et al. 1984), protein concentration and content of cytochrome P450 was determined as described (Lowry et al. 1951, Omura and Sat0 1964). Prepared microsomes containing 2mg/ml of microsomal protein in 0.1 M phosphate buffer (pH 7.4) were stored at -80°C. Incubations with rat liver microsomes were performed as described (Osikowska-Evers and Eichelbaum 1986). Each incubation mixture contained 50 pg of microsomal protein, substrate concentrations were 0.5,0.75, 1,2,5,10,20,40,80, 160, 320,640, 1280 and 2 5 6 0 ~The ~ . final volume of the incubation mixtures was 250~1,and incubation time was 5 min. Linearity of the rate of metabolite formation with respect to concentration of microsomal protein (50-1 50pglml) and incubation time (5-20 min) was observed. Inhibition studies were started by addition of substrate and NADPH after a preincubation period with the respective inhibitors of 10min. Incubations were stopped by addition of lop1 of 30% (w/v) perchloric acid. All incubations were made as duplicates. In vivo studies Sulphates of SP or PA (0.094mmol/kg body wt) and synthetic monoperchlorate of (+)-2,3-DDHSP (0.04 mmol/kg body wt) were dissolved in water and administered orally. Rats were housed in metabolic cages for 16 h and urine was collected over this period. Several experiments were also performed with an additional 16-24 h collection period. Urine samples were stored at -30°C until analysed. Analytical methods Gas chromatography. Rat urines were analysed by a capillary g.1.c. system (Ebner et al. 1989 a) with a modified temp. programme: injection temp. (on-column) 3 5 T , heating rate 30"C/min, final temp. 210"C, final time 11.5min. For the detection of lupanine, the final temp. of the g.1.c. was raised to 270°C for 15 min. The following retention times were observed (min): S P 13.8; 5,6-DDHSP 15.1; 2.3-DDHSP 15.5; 17-ethylsparteine 17.1; (4S)-hydroxy-PA 19.0; 17-oxosparteine 19.7; 2-oxosparteine (lupanine) 21.9; 15-oxosparteine 22.5. Standard curves were determined by the use of the synthetic reference compounds, using 17-ethylsparteine as internal standard. The dichloromethane extracts of the alkalized incubation mixtures were analysed by capillary g.1.c. as described (Osikowska-Evers and Eichelbaum 1986). For determination of the standard curves, synthetic reference compounds and substrate (40 PM) were added to the incubation mixtures, which contained heat-denatured microsomes. The standard mixtures were incubated simultaneously with the incubation experiments. Standard curves were obtained using the optically pure synthetic metabolites. The linearity of the analytical assay was proven over a concentration range of 10-500 pmol/incubation mixture. Mass spectra. Rat urines and incubation mixtures were analysed using a H P 5790 M S D mass spectrometer (EI ionization, 70eV) coupled to g.1.c. to confirm the identity of the formed metabolites. Isotopic contents of synthetic deuterated compounds and excreted metabolites were determined by selected ion monitoring in coupled g.1.c.-mass spectrometry using a Hewlett-Packard HP 5985 A mass spectrometer, PI/CI ionization (NH,, 72 eV). Isotopic composition of [2R-'H]-SP and [2S-'H]-SP was determined from the molecular ion [ M + l ] + m/z=236 (Ebner et al. 1989b), of synthetic (Ebner et al. 1989 c) and urinary excreted [2-'H]-2,3-DDHSP from the respective molecular ion [M 11 m / z = 234.
+
+
N.m.r. spectra. N.m.r. measurements were performed on a Bruker WP 80 (13C-n.m.r.) or CPX 300 ('H-n.m.r.). For I3C-n.m.r. measurements of synthetic iminium perchlorates (40pmol), 2 ml of 0.1 M phosphate buffer (pH 7.4) was used as solvent with dioxane as internal standard (6 =69.26 ppm). After addition of 4Opmol of K13CN the pH was adjusted to 7.4 by 0.1 M HCl. Samples of rat urine (5 ml) were lyophilized, redissolved in 2 ml of phosphate buffer and centrifuged. After addition of dioxane and K"CN the pH was adjusted to 7.4. 'H-n.m.r. of rat urine samples was performed with 5ml of lyophilized urine dissolved in 2ml of deuterium depleted water. C2H3CN was added as internal
T.Ebner et al.
850
standard (6 = 2.078 ppm). The resonance of [2R-2H]-(2S)-hydroxysparteine (Ebner et 01. 1990a) showed a chemical shift of 6 =4.56 ppm.
Analysis of results Enzyme kinetics were calculated using a computerized nonlinear least square iterative procedure (Wilkinson 1986).
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Results After administration of SP and PA to male Sprague-Dawley rats (n= 5) marked differences in the metabolic pattern of the excreted metabolites were observed by g.1.c. (figure 2). For SP, 2,3-DDHSP was found as the main metabolite, the structure of which was confirmed by direct comparison with the synthetic reference compound. T h e mass spectra as well as the retention times of both the synthetic and the metabolic product were identical. After administration of stereospecifically labelled [2S-*H]-SP, the excreted 2,3-DDHSP exhibited the same mass spectrum as the unlabelled metabolite. In contrast, the main metabolite which was excreted after administration of [ZR-'H]-SP retained more than 95% of the label. With temperatures in the g.1.c. temperature programme (see Experimental section) for the analysis of urine samples, a metabolite with a considerable longer retention time (21.9min) was observed (figure 2). T h e mass spectrum of this metabolite exhibits a molecular ion m / z = 248, which is compatible with an 0x0 derivative of SP. Comparison with the mass spectra of synthetic 15-oxosparteine ( m / z (rel. abundance, yo):248 (30.0, M'), 137 (25-3), 98 (100)) or 17-oxosparteine ( m / z(rel. abundance, %): 248 (45-8, M'), 110 (72.0), 97 (loo)), showed no identity with either compound. In contrast, full accordance with 2-oxosparteine (lupanine) was found ( m / z (rel. abundance, yo):248 (39.6, M'), 137 (100)). When synthetic
4
!
3
.I
-----I8
Time [ m i n ]
I
14
WCLL
1
17
I
20
I
23
Figure 2. G.1.c. separation of SP and metabolites excreted in rat urine. 1: Unchanged SP; 2: 2,3-DDHSP; 3: 17-ethylsparteine (internal standard); 4: lupanine; the arrow indicates retention time of 5,6-DDHSP.
Chiral quinolizidine alkaloids in the rat
851
Table 1. Cumulative urinary excretion of unchanged sparteine enantiomers and metabolites after oral administration of sparteine (SP) and pachycarpine (PA) sulphates to rats. Pachycarpine ~
~~
Unchanged PA 2,451.0
~
(4S)-Hydroxy-PA
5,6-DDHPA
2,3-DDHPA
Total
58.6 & 6.2
0.3f0.1
0.3 f0.05
61.6f 6.2
2,3-DDHSP
Lupanine
Total
16.9f6.0
6.3 f 0.8
25.2k6.5
Sparteine Unchanged SP
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2.1 f O . 5
Values are meansfSD for @-16 h excretion, expressed as percentage of dose. The doses were five rats. 5,6-DDHPA, 2,3-DDHPA=5,6-, and 94pnol/kg given to each of 2,3-didehydro-pachycarpine.
(+)-2,3-DDHSP was given to rats (n=2), 13.070 and 10.4% of the dose was recovered unchanged, and 16.4% and 16.1Yo of the dose was excreted as lupanine. Other didehydro metabolites such as 5,6-DDHSP or 11,12-didehydrosparteine were not observed during the in wiwo studies by g.1.c.-mass spectrometry. Following administration of PA, the main metabolite (4S)-hydroxy-PA was formed, and only traces of 5,6- and 2,3-didehdropachycarpine could be quantified by g.1.c. using the synthetic dextrorotary analogues as reference compounds. T h e urinary recoveries of SP and PA and its metabolites are listed in table 1. When K13CN was added to buffered (pH 7.4) aqueous solutions of iminiumstructured synthetic sparteine derivatives, the formation of different cyanosparteines was observed. T h e 13C-n.m.r. spectra of such samples exhibit the signal of the 13CN-group of the different cyanosparteines (table 2), whereas the natural abundance of the 13C resonances of the other 15 carbon atoms are not detected. If excess K12CN is added to the samples, a marked decrease of the 13C nitrile resonance, due to a rapid exchange of the cyano group, was found. After addition of K13CN to samples of rat urine, which was excreted after administration of SP, the resonance of the (%a-cyanosparteinewas observed and no respective signal of other compounds listed in table 2 was found. Resonances at 124.2, 123-0, 122.4, and 122.3ppm were also present, but these may also be obtained with blank urine samples and are therefore caused by reaction products of physiological components of the urine samples with the labelled cyanide.
Table 2.
13C-n.m.r. chemical shifts of the 13CNgroup of different cyanosparteines in aqueous buffered solution (pH 7.4). Compound
Chemical shift (p.p.m.)
6-' 3CN-sparteine 1l-'3CN-sparteine 6- "CN- 17-oxosparteine ( 2 3 - l 3CN-sparteine (17S)-'3CN-2-oxosparteine (17S)-13CN-sparteine *H,-dioxane: 6=69.26p.p.m., "CN-: 6= 116.79p.p.m.
119.79 119.86 120.13 121.31 124.01 126.92
T . Ebner et al.
852 Table 3.
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Liver sample no.
Enzyme kinetic parameters for hepatic microsomal oxidation of pachycarpine (PA). KnI
Vmex
ViUSJKSl
Metabolite
(PM)
(pmol/min per mg)
(min- ’)
1
(4S)-Hydroxy-PA 5,6-DDHPA
3.1 3.9
771 294
249 75
2
(4S)-Hydroxy-PA 5,6-DDHPA
3.9 5.9
498
128 29
1-71
Only one deuterium resonance was recorded by ’H-n.m.r. apart from the signal of C2H3CN (C2H3:6=2.078ppm) which was used as internal standard with rat urine samples after administration of [2R-’H]-SP. This resonance can be assigned to the axial methine deuterium atom in position 2 of the carbinolamine [2R-’H](2s)-hydroxysparteine (Ebner et al. 1990a). This carbinolamine derivative of sparteine is spontaneously formed when the synthetic perchlorate of [2-2H]-2,3DDHSP is added to blank urine or to buffered aqueous solutions ( p H 4-8). Biphasic Michaelis-Menten kinetics were calculated for the formation of 2,3DDHSP in the course of in vitro experiments with purified liver microsomes of male Sprague-Dawley rats. Enzyme kinetic parameters were calculated ( n = 2) for the high-affinity ( K , 2.0 f0.4 p ~ V,,,l294 , +_ 97 pmol/min per mg) and the lowaffinity component ( K , 1235 & 40 p ~ V,,, , 6346 & 884 pmol/min per mg). High substrate concentrations (concn of SP> 1 mM) resulted in the formation of very small quantities of two didehydrosparteines, identified by g.1.c.-mass spectrometry after alkaline work-up as 5,6-DDHSP and 11,12-didehydrosparteine(less than 50pmol/ml incubation mixture). These products were also found when SP was incubated with heat-denatured (30 min, 90°C) microsomes. No lupanine was formed during the in vitro experiments with rat liver microsomes and rat liver homogenates. The formation of (4S)-hydroxy-PA and 5,6-didehydropachycarpine exhibited monophasic enzyme kinetics. I n contrast to the formation of the main metabolite of S P no low-affinity/high-activity component was observed. Results of the in vitro experiments are listed in table 3 . As a measure for the enzymic activity at low, non-saturating concentrations the intrinsic clearance (CL,) was calculated from the Vmax/Km term. T h e influence of different inhibitors on the metabolism of both sparteine enantiomers was examined using the in vitro system mentioned above. (&)-Propafenone (Siddoway et al. 1987) and quinidine (Otton et al. 1988, Spiers et al. 1986) were used as inhibitors since these drugs are known to be potent competitive inhibitors of the human sparteine metabolism in vivo and in vitro. Both compounds showed marked competitive inhibition for the formation of 2,3-DDHSP from S P and (4S)-hydroxy-PA from PA. Dixon plots for (+_)-propafenoneare shown in figure 3. T h e estimated Ki values of (+-)-propafenone were 180nM for S P and 260nM for PA. T h e respective values of quinidine were 2 7 0 n ~(SP) and l 0 O n ~ (PA). When SP was added to incubations with PA (substrate concentration= 8 0 ~ ~ ) the formation of (4S)-hydroxy-PA was also inhibited. SP concentrations of 5 p ~ , ~ O P Mand , 8 O p revealed ~ a 43%, 70%, and 89%, respectively, inhibition of the formation of the main metabolite of PA.
853
Chiral quinolizidine alkaloids in the rat
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-1
0
1
2
3
2
3
Propafenone [ wMI
0.008 0.006 0.004 0.002
?
0.000 -1
0
1
r
Propafenone [ pM1 Figure 3. Competitive inhibition of 2,3-DDHSP formation from sparteine (SP) and (4S)-hydroxy-PA formation from pachycarpine (PA) by ( f)-propafenone. Each point is the mean of two determinations; substrate concentrations were 2 , s and 1 5 , 10 and 2 0 p M (PA).
0 (SP); ~ ~
Discussion T h e structure of the main metabolite of sparteine in the rat was confirmed by direct comparison with the synthetic compound using analytical and spectroscopic methods. T h e enamine base 2,3-DDHSP was identified by g.1.c. and g.1.c.-mass spectrometry, the structure of which had previously been assigned indirectly. Previous studies of other substrates have demonstrated that biodegradation products or intermediate metabolites with electrophilic properties such as iminium ions react very easily with cyanide (Murphy 1973, Ho and Castagnoli 1980). Therefore, radiolabelled 14C-cyanide has been used in in oitro studies for the trapping of iminium structured metabolites (Whittlesea et at. 1991). Synthetic cyanosparteines can be prepared by reaction of the corresponding iminium derivatives of S P with cyanide (Leonard et al. 1955, Ebner et al. 1 9 8 9 ~ )T. h e use of the 14C radiolabel, however, provides no information about the chemical structure of the products. I n contrast, the use of 13C-labelled drugs in conjunction with 13C-n.m.r. spectroscopy has been shown to be superior for monitoring drug
T . Ebner et al.
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[ 2 R -2Hl-Sparteine
I
2i
*
Oxidative metabolism
c-^t'_zi?
Excreted i n urine, pH 4-8 (Monitored by 2H-nmr)
"0
[ 2 R -'H]-(2S)-hydroxysparteine
'"2"
/
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Alkaline work-up
[2-'H1-2,3-didehydrosparteine (Measured by glc
Figure 4.
[2R -2Hl-(2S -''CN)-sparteine
- mass spectrometry)
(Monitored by ISC-nmr)
Formation, detection, and chemical transformations of the main metabolite of [2R-*HI-sparteine.
At pH 4 3 the 1,2-didehydrosparteinium ion is formed by acid-catalysed, non-enzymic dehydration.
metabolism (Meese et al. 1990, Meese and Fischer, 1990). Therefore, stable labelled I3C-cyanide and '3C-n.m.r.-spectroscopy was used for the screening of sparteine metabolites in rat urine. This method offers additional information on the structure of the metabolically formed products. T h e observation of only the one 3C-n.m.r. resonance signal corresponding to authentic (2s)-cyanosparteine provides further evidence for the structure of the main urinary excreted metabolite of SP in the rat. In contrast, urine of human extensive metabolizers of SP exhibited two resonances, according to two '3C-cyanosparteines, viz. (2s)- and 6-cyanosparteine, which were formed from the two human metabolites of SP (data not shown). In full accordance with our results obtained with the human major metabolite of SP (Ebner et al. 1990a), the main metabolite of SP in the rat exhibits the structure of (2s)-hydroxysparteine in neutral aqueous solution. T h e free enamine base 2,3DDHSP which is detected by g.1.c.-mass spectrometry has to be considered as an artefact formed during the strongly alkaline analytical work-up. No other metabolite of S P with enamine structure was found in the urine of rats by g.1.c.-mass spectrometry, or I3C- and 2H-n.m.r. spectroscopy. However, lupanine (2-oxosparteine) is a secondary metabolite of SP, which is probably formed by oxidation of the carbinolamine derivative of 2,3-DDHSP, (2s)hydroxysparteine. Lupanine was recently reported to be a second metabolite of SP in rat (Chaudhuri and Keller 1990). Similar lactame-structured metabolites were reported for other drugs (Ruenitz et al. 1979, Schwartz and Kolis 1973), the formation of which is catalysed by various cytosolic (Brandange and Lindblom 1979) or microsomal (Obach and Vunakis 1990) iminium oxidases. 5,6-DDHSP was completely absent from samples of rat urine and only traces of this compound beside other oxidation products were formed in vitro, by non-enzymic oxidation.
'
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Chiral quinolizidine alkaloids in the rat
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Thus, the assumed formation of 5,6-DDHSP by rat liver microsomes, as reported in the literature (Guengerich 1984), is not consistent with our results. PA is metabolized in the rat to a stable aliphatic alcohol, o i z . (4S)-hydroxy-PA by the same enzyme which catalyses the formation of the main metabolite of SP, excreted in the urine as (2S)-hydroxy-SP. This carbinolamine dehydrates very readily and only the enamine 2,3-DDHSP can be extracted into non-polar solvents. Since exchange of the hydroxyl group may also take place in aqueous solution, it is not possible to find out whether the hydroxyl group of (2S)-hydroxy-SP is transferred to the sparteine molecule by the enzyme directly, as it is obviously the case for the (4S)-hydroxy-PA. However, our results with the stereospecifically labelled analogues [2R-’H]- and [2S-’H]-SP (Ebner et al. 1990a, b, Ebner and Meese 1988) clearly indicate a high degree of stereoselectivity of the metabolizing cytochrome P450 isozyme for the abstraction of the axial /3-hydrogen. This is closely related to the stereoselective hydroxylation of PA to (4S)-hydroxy-PA. The axial b-hydrogens in positions 2 or 4 are abstracted exclusiveIy during the formation of the main metabolites of the respective enantiomers. In the course of the formation of the 5,6-DDHSP, the human minor metabolite, and also the 5,6didehydropachycarpine in the rat, the hydrogen atom at position 6, which is also located on the /hide of the SP molecule, is also abstracted. Thus, the enzymic attack seems to occur from the sterically less-hindered /%side. A reaction mechanism similar to the formation of the aliphatic alcohol (4S)-hydroxy-PA, i.e. direct C-hydroxylation of the carbon atom C-2 by the enzyme to yield (2S)-hydroxy-SP is therefore also probable for the formation of the main metabolite of SP. The cytochrome P450 isozyme also shows a high degree of regioselectivity, as only positions of ring A of the tetracyclic sparteine molecule are involved in metabolic reactions in mammals. The binding of the molecule is mediated by the second nitrogen (N-16) as was proposed by different authors (Meyer et al. 1986, Wolff et al. 1985). Small changes in orientation between the SP and PA molecules bound to the enzyme may cause alteration in product formation of the two sparteine enantiomers. Variations of the structure of the binding sites of the human cytochrome P-450 IID6 and rat cytochrome P-450 I I D l could explain the divergencies in the metabolic pattern for the metabolism of SP between the two species. Results about marked differences in the affinity for different competitive inhibitors of the human and rat isozyme reported for the metabolism of debrisoquine (Kobayashi et al. 1989) and metoprolol (Lancaster et al. 1990) are also consistent with differences in the geometry of the binding sites of the two enzymes. Our investigations on the metabolism of PA are the first reported case of a metabolic reaction at an aliphatic CH, group of the sparteine molecule not adjacent to nitrogen. In addition, ’H-n.m.r. spectroscopy and the use of l3C-labe1led reagents, such as cyanide, together with 3C-n.m.r. spectroscopy, is another example for the use of n.m.r. methods for the identification of metabolic products. Finally, these results indicate that both enantiomers of this quinolizidine alkaloid are metabolized by the same cytochrome P450 isozyme which has a remarkable regioselectivity and stereoselectivity for the two enantiomers.
Acknowledgements This work was supported by the Robert-Bosch Foundation, Stuttgart. Excellent technical assistance of Ms S. Hod1 and Mr J. Rebel1 is gratefully acknowledged.
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