Biotransformation of terodiline. v. stereoselectivity in hydroxylation by human liver microsomes

325

Chem.-Biol Interactions, 71 (1989) 325-337 Elsevier Scientific Publishers Ireland Ltd.

B I O T R A N S F O R M A T I O N OF TERODILINE. V. S T E R E O S E L E C T I V I T Y IN HYDROXYLATION BY H U M A N LIVER MICROSOMES

B E N G T NORI~,N ~,S I G N H I L D S T R O M B E R G

~,O R J A N E R I C S S O N b and B J O R N L I N D E K E ~

aKabiVitrum AB, R&D, S-112 87 Stockholm, bDepartment of Clinical Pharmacology, Huddinge University Hospital S-151 86 Huddinge and cACO Lgkemedel AB, Box 1827, S-171 26 Solna {Sweden) (Received October 27th, 1988) (Revision received January 23rd, 1989) (Accepted January 27th, 1989)

SUMMARY

The stereoselective hydroxylation of N-tert-butyl-4,4-diphenyl-2-butylamine (Terodiline) was studied in human liver microsomes. Formation of the two main metabolites, N.tert-butyl-4(4-hydroxyphenyl}-4-phenyl-2-butylamine (II) and N-(2-hydroxymethyl-2-propyl)-4,4-diphenyl-2-butylamine (VI), was found to be stereoselective. R-Terodiline was preferentially transformed by phenolic hydroxylation to the 2R,4S-II and 2R,4R-II forms with a pronounced selectivity for the former. The formation rate ratio 2R,4S-II/2R,4RII was about 6, obtained from two liver preparations. S-Terodiline was mainly hydroxylated to the alcohol 2S-VI although phenolic hydroxylation to the 2S,4S-II and 2S,4R-II also occured, yielding about equal amounts of the two phenols.

Key words: Terodiline - Metabolism - Mass spectrometry - Microsomes INTRODUCTION

Terodiline, N.tert.butyl-4,4-diphenyl-2-butylamine (I), an anticholinergic and calcium antagonistic drug previously used against angina pectoris, is becoming a valuable drug in the treatment of urinary incontinence [1--5]. It is eliminated from the body mainly by oxidative metabolism followed by conjugation with glucuronic acid [6]. Since terodiline contains an asymmetric centre, it exists in two enantiomeric forms. The absolute configurations of the enantiomers have been determined [7]. Aromatic hydroxylation, which is a major metabolic pathway, introduces a second asymmetric centre in the 0009-2797/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

326 molecule and consequently four stereoisomeric phenolic metabolites can be formed from racemic terodiline, which is currently used as the drug. Hydroxylations occurring in the tert-butyl moiety as well as benzylic hydroxylation do not, however, create any new asymmetric centres. In a previous study on the in vitro metabolism of terodiline in rat liver microsomes, a pronounced substrate stereoselectivity in both the aromatic and the benzylic hydroxylation was demonstrated [8]. Thus R-terodiline was preferentially hydroxylated in the aromatic rings whereas S-terodiline mainly underwent benzylic hydroxylation. The two most abundant phase I metabolites of terodiline found in l~uman urine are formed by hydroxylation in one of the aromatic rings and in the tert-butyl group [6]. The aim of the present study was to investigate the formation of these metabolites in human liver microsomes in order to establish stereoselective metabolism also in man. EXPERIMENTAL

Chemicals R- and S-terodiline hydrochloride {R-I/S-I), S-N-tert-butyl-4,4-diphenyl-1{2Hs)-2-butylamine hydrochloride (S-I-d3), N-tert-butyl-4(4-hydroxy-phenyl)-4phenyl-2-butylamine hydrochloride (II), N-tert-butyl-4(4-hydroxy-phenyl}-4phenyl(2Hs)-2-butylamine hydrochloride (II-ds), N-(2-hydroxymethyl-2-propyl)~ 4,4-diphenyl-2-butylamine hydrochloride (VI), N-(2-hydroxymethyl-2-propyl)4,4-diphenyl-l-(2Hs)-2-butylamine hydrochloride (VI-d8) and N-(2-hydroxymethyl-2-propyl)-4,4-diphenyl[G-2H~2-butylamine hydrochloride (VI-d 8) were synthesized at the Organic Chemistry Department at KabiVitrum AB as described elsewhere [8--10; T. Werner, unpublished]. II-d 5 and Vl-d 8 were used as internal standards. A pseudo-racemic mixture of terodiline hydrochloride was prepared by mixing equimolar amounts of R-I and S-I-d3. N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and trifluoroacetic anhydride (TFAA) were purchased from Pierce {Rockford, IL). NADPH, type I and Tris--HCl (pH 7.6) were obtained from Sigma Chemical Company, St Louis, MO. Other chemicals were of analytical grade and used without further purification.

Liver microsomes Three human liver specimens, HL39, woman, age 62, HL41, man, age 35 and HL45, man, age 46, were obtained from the liver bank at the Huddinge Hospital [11]. The specimens were taken shortly after circulatory arrest and frozen in liquid nitrogen. Microsomes were prepared as described by Pacifici [12]. The microsomal protein concentration was determined using Peterson's method [13]. The concentration was adjusted to 10 mg protein/ml and the microsomes were stored at - 7 0 °C until used.

Incubations Three sets of experiments were performed: (1) Incubation of R- and S-

327 terodiline (50 pM) in pooled liver microsomal preparations from three liver specimens (HL39, HL41 and HL45) at a concentration of 2 mg protein/ml. The incubation time was 30 min. Samples from these incubations were used to identify major in vitro metabolites. (2) A time-dependence study for the formation of the optical isomers of metabolites II and VI from R-, S- and pseud~racemic RS-terodiline (50 ~M) using the microsomal preparations HL41 and HL45 (2 mg protein/ml). The incubations were run for 0, 10, 20 and 30 min. (3) Determination of the enzyme kinetic parameters Vmax(obs)and K m~appJ , . for the formation of the isomers of II and VI from R- and S-terodiline (10--50 ~M) in three liver microsomal preparations (HL39, HL41 and HL45) at 2 mg prot/ml and 10 min incubation time. The microsomal incubations were performed according to a method by Spina et al. [14] and were run at 37 °C in a total volume of 1 ml of a mixture containing T r i s - - H C I (pH 7.4) (50 mM), MgCI 2 (6 mM), N A D P H (1.24 raM) and terodiline substrate (50 ~M unless otherwise stated). The mixture was preheated at 37 °C for 30 s before the incubation was started by addition of the microsomes to a final concentration of 2 mg protein/ml. The incubation was stopped by adding trichloroacetic acid (10%, 500 ~l) and rapid freezing in acetone/dry ice. The samples were kept in a freezer ( - 18 °C) until analyzed.

Sample extraction To about 0.2 ml of the incubation mixture were added the internal standards, II-d 5 and VI-d s, the mixture was alkalized with sodium carbonate buffer (3 ml, 1 M, pH 12) and the metabolites were extracted with 30% diethyl ether in pentane (3 ml). The ether-pentane was extracted with sulfuric acid (1 ml, 0.1 M) and discarded. The acidic aqueous solution was alkalized with sodium carbonate buffer (3 ml, 1 M, pH 12) and extracted with 30% diethyl ether in pentane (3 ml). The ether-pentane was dried over anhydrous sodium sulfate, transferred to a new vial and the sample was evaporated to dryness at 60°C. The metabolites in the residue were converted to their TMS and TFA derivatives as previously described [8].

Gas chromatography-mass spectrometry The samples were analyzed using an HP5970B mass selective detector (Hewlett-Packard) working at an ionization potential of 70 eV (EI). For qualitative analysis of the TMS-derivatized metabolites from the first incubation set, mass spectra were recorded in the mass range 35--650 amu. The GC separation was performed on a fused silica capillary column, 25 m * 0.20 mm i.d. containing cross-linked methyl silicone to a film thickness of 0.52 ~m. The oven temperature was programmed from 140 to 300 °C at a rate of 50 °C/min. The sample was injected in the splitless mode. Quantitative analysis by multiple ion selection (MIS) was performed on a fused silica capillary column, 25 m* 0.2 mm i.d. containing cross-linked 5% phenyl methyl silicone to a film thickness of 0.33 ~m. The oven temperature was programmed from 140 to 280 °C at a rate of 50 °C/min. For quantitation of II, the diphenylmethyl cations (if) at m/z 255 and 260

328 were monitored. When pseudoracemic terodiline was used as a substrate, ion

(ix) which appears at m/z 354 for unlabelled II, 357 for II-d 3 and 359 for II-d 5 was monitored. For quantitation of VI the same ion (ix) was monitored at m/z 266 for unlabelled VI, m/z 269 for t r i d e u t e r a t e d VI-d 8 and m/z 274 for the internal standard VI-d 8. (Scheme I). The ratio between the diastereomeric enantiomeric pairs, 2R,4R/2S,4S and 2R,4S/2S,4R, of II was determined after conversion to TFA derivatives. The separation was performed using a 15 m* 0.2 mm i.d. fused silica column containing crosslinked methyl silicone to a film thickness of 0.11 /~m. The column temperature was programmed from 120 to 300°C at a rat e of 50°C/min. Splitless injection mode was used during the first minute of the analysis. The ion (xx) at m/z 433 for I I and m/z 436 for II-d 3 were monitored (Fig. 1).

Calculations The rates of formation of metabolites II and VI were not linear with time for 30 min under the conditions used. However, the data points could be fitted to a second-order equation (r = 0.97-1.00) from which the initial rates of formation (slope at time = 0) were estimated after derivation of the equations. T h e r e is no theoretical basis for using a second-order equation to describe the rates of formation of the metabolites. However, the estimated initial rates of formation obtained from the second-order equation and the calculated rates of formation obtained from Michaelis-Menten param et ers are in good agr e e m ent (Table II).

CH3(2H3)/CH3 TMSO-<~>-~ (TMSO)~'O-~CH-CH2-~H-NH=C~_, (2H)~ "OH3 (~H~) •

e

(ii)

(ix)

ion II ll-d 3

ll-d 5 VI Vl-d 3

Vl-d a

Scheme I.

(ii)

m/z 2 5 5 255 260

ion

(ix)

m/z 3 5 4 357 359 266 269 274

329

H3C CH3q * CH3 \C/- CH~ T FA-O"~-C H-CH~ t H- N~. ~"-~H "~

~ "

CF~ (i)

T FA-O

-x'CH3 OH']~. - CH~CH-N= C\CF3 (xx)

Fig. 1. Formation of the ion (xx) following a McLafferty rearrangement. The deuterium-labeled methyl group is marked by an asterisk.

RESULTS

Analytical method Straight line standard curves were obtained for quantitation of the TMSderivatized metabolites II and VI. The precisions in the quantitative analyses of II and VI were checked by multiple analysis of one sample containing known amounts of II and VI (Table I). Good separation of the TFA-derivatized isomers of II were also obtained. Figure 2 shows an example of the separated isomers of II obtained from incubation of a pseudo-racemic mixture of terodiline. The isomeric ratios were calculated from the peak area ratios.

TABLE I DETERMINATION

O F II A N D VI - P R E C I S I O N A N D A C C U R A C Y

Metabolite

Added pmol

Found pmol (S.D.)

Rel. S.D. (%}

II VI

65 14.2

71.7 (4.5) 15.9 (1.2)

6.3 7.7

330

1.£E4

2R ,4S

IB000

2R,4R

8000 6000 O

c 20002 0 -2000 -4000

....

-6~00 4.8

2S4 4,3 Time

.... 4.4 (rnin.)

S4" ..... 4.5

4.6

4.7

Fig. 2. Separation of the isomers of II obtained from incubation of a pseudo-racemic mixture of terodiline.

Metabolism Figure 3 shows the total ion chromatograms of the TMS-derivatized extracts of the pooled liver microsomal incubates of R- and S-terodiline. In both cases t hr ee major metabolites, II, VI and X I I (Fig. 4), could be identified. The chromatograms indicate an opposed substrate stereo-selectivity in the formation of II and VI. In the case of R-terodiline, the major phase I metabolite is I I and the second most abundant one is VI. Metabolite X I I is poorly resolved from an interfering peak and thus its content can not be accurately estimated. Incubation with S-terodiline results in markedly larger amounts of VI while the amounts of II diminish. Judged from the total ion chromatograms metabolite X I I seemed to be less abundant. The formation of the stereoisomers of II and VI from R-, S- and RSterodiline was studied in the two liver microsomal preparations HL41 and HL45. The rates of formation were not linear with time, but the data points could be fitted to a second-order function (Fig. 5) from which the initial rate of formation was derived. Table II thus summarizes the initial rates of formation of metabolites II and VI from R-, S- and RS-terodiline in two preparations. The opposed substrate stereoselectivity indicated in the total

331

B.BEB

4.0EB

2R -I

2.BEB o~

XII V I

II

c

/

I 2S-I

-4B@~@

I

5

'

'

'

'

I

6 Time (mln.)

Fig. 3. Total ion chromatograms of the TMS-derivatizedextracts of 30-rain incubation at 37°C of R- and S-terodiline(50 ~M) in three pooled human liver microsomalpreparations.

ion chromatograms (Fig. 3) is now explicit. Thus, while phenol formation (II) greatly exceeds alcohol formation (VI) in the metabolism of R-terodiline, the situation is r e ve r s e d in the metabolism of S-terodiline. The ratio of the initial rates of formation of I I : I V (total) is in the former case about 5.9, but in the latter case only about 0.7. Moreover, separation of the phenols into diastereomeric pairs reveals that the formation of one isomer only, 2R,4S-II, is responsible for the major part of the phenol pool formed from R-terodiline. The apparently lower rate of formation of the metabolites from pseudoracemic mixture to the separate enantiomers (Table II and Fig. 5) is in most cases only due to the different substrate concentrations. 25 pM of each enantiomer when the pseudoracemate was incubated and 50 /~M when the pure enantiomers were used. T here is one notable exception. The

332

CH3 CH~ (~-X)-CH-C H2-~FI-N H- C- C

"=-"[~

CHart3

I

S CH3 CH20H C H-C H~-CH-NH-C- C H3 CH3

OH

CH3

CH3

~)-@CH2-CH-NH-C-CH3

Vl

Xll

CH3

[~

II

~H3 CHa

Fig. 4. Primary metabolites from human liver microsomes.

formation rate of 2R,4S-II from the pseudoracemate is cansiderably lower than that predicted from the incubations with the pure enantiomers. It should be stressed that the deuterium substituted isomer was labeled in a position considered metabolically inert. Comparisons of the metabolic activity between the microsomal preparations from the three individual livers (HL39, HL41 and HL45) show as expected great interindividual differences. Kinetic parameters were determined by applying the Michaelis-Menten kinetics for the formation of the individual isomers of II and VI. The estimated values of Kin(ape) and Vaxlobs) are given in Table III and examples of the reciprocal plots from which the constants have been calculated are shown in Fig. 6. Despite the interindividual differences seen, it is quite obvious that the formation of 2R,4S-II from R-terodiline is a metabolic route which is the most rapid and extensive. Among the different enzymes which appear to be operating simultaneously in the conversion of terodiline, one high affinity form with a relatively high turn-over seems to generate 2R,4S-II. DISCUSSION Previous studies on the metabolism of terodiline in vivo [6,10] have not taken into account differences between the enantiomers or the stereochemistry of the metabolites. In a recent study using rat liver microsomes [8] we

,

500

0

0 ~-

•

2S

2R

10

•

2O

,

30

400

min

0

100

200

100

i

20

!

10

0

2R

2S

[] •

10

min

20

Vl from RS-terodiline

0

2R,4R 2R,4S 2S,4S 2S,4R

Vl from R- and S-terodiline

30

m • [] e

min

,

IO0

200

•

a

: 20

j

200

300

400

500 -

rnin

10

-

~

.

_.~ ~ -

300

0

•~ f

2R,4R 2R,4S 2S,4S

300

400

500

0

100

200

300

B • u

II from RS-terodiline

!

30

30

Fig. 5. Formation of the isomers of metabolites II (upper left) and VI (lower left) from incubation in human liver microsomes (HL45, 2 mg prot./ml) with R- and S-terodiline (50 ~Jd), respectively, compared to their formation, II (upper right) and VI (lower right), from RS-terodiline (50/~M).

O_

Q.

E o..

o

E

(31.

400

500

II from R- and S-terodiline

¢,0 ¢,,0 ¢.0

334 TABLE II OBSERVED INITIAL AND CALCULATED RATES OF FORMATION (pmol/mg prot.min) FOR THE FORMATION OF THE ISOMERS OF II AND VI FROM R-, S- AND RS-TER0DILINE (50 ~VI) Substrate

Product

Rate of formation" HL41

R-I

2R,4R-II 2R,4S-II 2R-VI

2.3 (2.9) 17.0 (16.0) 4.3 (3.4)

2.6 (2.8) 18.2 (20.6) 10.7 (8.7)

S-I

2S,4S-II 2S,4R-II 2S-VI

4.4 (3.2) 2.7 (2.4) 6.5 (6.8)

3.1 (2.8) 3.2 (2.7) 19.9 (18.9)

RS-I

2R,4R-II 2R,48-II 2S,48-II

1.5 (--) 4.4 (-) 1.0 (--) 0.9 (-) 2.4 ( - ) 3.2 (-)

2S,4R-II

2R-VI 2S-VI ICalculated values in parenthesis.

HL45

1.6 (-) 4.6 (-) 2.0 (-) 1.7 (-) 5.8 (-) 8.7 (-)

showed t h a t the m e t a b o l i s m of terodiline, as e x p e c t e d , is indeed s u b s t r a t e s t e r e o s e l e c t i v e , and similar r e s u l t s are now shown for human liver microsomes. T h e overall metabolic p a t t e r n s of terodiline a p p e a r s to be qualitatively similar in the r a t [8,15] and man [6] but t h e r e are p r o n o u n c e d q u a n t i t a t i v e differences. The most i m p o r t a n t p r i m a r y r o u t e of metabolism in liver microsomes from both r a t [6] and man is a r o m a t i c h y d r o x y l a t i o n (II). This metabolic r o u t e is in both cases s u b s t r a t e s t e r e o s e l e c t i v e in as much as considerably m o r e phenol is f o r m e d from R- t h a n S-terodiline. M o r e o v e r , w h e n R-terodiline is the s u b s t r a t e , a m a r k e d p r o d u c t s t e r e o s e l e c t i v i t y is seen in the formation of 2R,4S-II in both liver p r e p a r a t i o n s . Thus, liver microsomes from r a t and man exhibit both t h e same s u b s t r a t e and p r o d u c t s t e r e o s e l e c t i v i t y with r e g a r d to phenol f o r m a t i o n (II). T h e high p r o d u c t s t e r e o s e l e c t i v i t y in the formation of 2R,4S-II could, as discussed in our p r e v i o u s publication [8], reflect the p r e s e n c e of d i f f e r e n t forms of c y t o c h r o m e P-450, with d i f f e r e n t catalytic p r o p e r t i e s , one of which is highly selective in forming only one of the d i a s t e r e o m e r s from R-terodiline. A l t h o u g h the kinetic p a r a m e t e r s given in Tables II and III should be r e g a r d e d as highly conditional, t h e y indicate the p r e s e n c e of such an e n z y m e with a c o m p a r a b l y low Km(app)and a high Vmax(obs~gm(app)ratio for the formation of 2R,4S-II. Similar to the situation in r a t liver microsomes [8], human liver microsomes oxidize S-terodiline p r i m a r i l y in the tert-butyl m o i e t y (Figs. 2

-'y

•

n

•

~

•

n

[] • = •

~

4,

2R,4R 2R,4S 2S,4S 2S,4R

211.3 189.0 157.8

152.9 35.4 89.5

HL41

•

u

0,00

~m~(ob~)

64.5 41.2 120.5

20.7 185.2 45.7

HL39

16.6 11.3 28.1

11.9 27.3 9.6

HL41

11.0 7.5 69.4

10.5 25.7 56.2

HL45 HL39

0.50 0.24 0.71

0.39 2.01 0.20

= • n • ~ • = • u ' 'l O,Oe+O 1,0e-2 2 , 0 e - 2 3 , 0 e - 2 4 , 0 e - 2 5 , 0 e - 2 6 , 0 e - 2

0,05

0,10

0,15

0,20

145.1 89.8 133.9

138.3 12.3 273.7

HL45

FROM LINEWEAVER-BURK PLOTS

Fig. 6. Reciprocal plots for the formation of isomers of II and VI by liver mierosomes HL45.

If"

n

8

='

129.2 174.4 169.8

51.9 92.3 231.3

HL39

Km(=pp)

9,0e+O 1 , 0 e - 2 2 , 0 e - 2 3 , 0 e - 2 4 , 0 e - 2 5 , 0 e - 2 6 , 0 e - 2

Oe+O

le-1

2e-1

3e-1

4e-1

•

2S,4S-II 2S,4R-II 2S-VI

S-I

f~

2R,4R-II 2R,4S-II 2R-VI

R-I

5e-1

Product

Substrate

CALCULATED Kmcappp(raM) AND V ~o~j(pmol/mg prot. rain) AND V J K

TABLE III

HL41

0.08 0.06 0.18

0.08 0.77 0.11

HL45

0.08 0.08 0.52

0.08 2.09 0.21

~o

336 and 3). In human liver preparations oxidation of S-terodiline is predominantly in the tert-butyl group (VI) while benzylic oxidation (XII) is of little importance (Fig. 3). In rat liver microsomes oxidation of S-terodiline primarily occurs in the benzylic position [8]. One advantage of using the pseudo-racemic terodiline is that the rate and extent of the formation of all four isomers of II from the racemate can be simultaneously determined (Figs. 2 and 5). An intriguing finding from these experiments was that S-terodiline to a considerable extent inhibits the formation of 2R,4S-II (Fig. 5). Although the concentrations used in this in-vitro study (saturation conditions) may not reflect what happens under normal dosing conditions in man, inhibition might be of importance in the pharmacokinetics of the terodiline [16,17] and one would expect R-terodiline to exhibit a shorter half-life and to generate higher plasma levels of II than RS-terodiline. Also, stereoselective interaction with other drugs is to be expected [18]. The results obtained with terodiline have their precedences in some earlier studies [19-23], the relevance of which was discussed in a previous publication [8]. Some preliminary recent experiments indicate that terodiline is metabolized by the same human enzyme system as debrisoquine [24] and thus might be subject to a metabolic polymorphism. Thus, the metabolism of terodiline is far from simple and further studies are warranted. ACKNOWLEDGEMENTS

We wish to thank Staffan Henricsson and Monica GSransson, Huddinge Hospital for supplying the human liver specimens and Dr Pinchas Moses for revising the English text. REFERENCES 1

2

3

4

5

6

T. Rud, K.-E. Andersson, N. Boye and U. Ulmsten, Terodiline inhibition of human bladder contraction. Effects in vitro and in women with unstable bladder, Acta Pharmacol. Toxicol., 46, Suppl. I (1980) 31. G. Ekman, K.-E. Andersson, T. Rud and U. Ulmsten, A double-blind, cross-over study of the effects of terodiline in women with unstable bladder, Acta Pharmacol. Toxicol., 46, Suppl. I (1980) 39. S. Husted, K.-E. Andersson, L. Sommer and J. R. (~stergaard, Anticholinergic and calcium antagonistic effects of terodiline in rabbit urinary bladder, Acta Pharmacol. Toxicol., 46, Suppl. I (1980) 20. D. Peters and the Multicentre Study Group, Terodiline in the t r e a t m e n t of urinary frequency and motor urge incontinence. A controlled multicentre trial, Scand. J. Urol. Nephrol., Suppl. 87 (1984) 21. W. Fischer-Rasmussen and the Multicentre Study Group, Evaluation of long-term safety and clinical benefit of terodiline in women with urgency/urge incontinence. A multicentre study, Scand. J. Urol. Nephrol., Suppl. 87 (1984) 35. B. North, S. Str6mberg, 0. Ericsson and B. Lindeke, Biotransformation of terodiline. IV. Identification of unconjugated and conjugated metabolites in dog and human urine, Acta Pharm. Suec., 25 (1988) 281.

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8

9

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12 13 14

15

16 17 18

19

20

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