Identification of the major urinary metabolite of alprenolol in man, dog and rat

Life Sciences Vol. 14, pp. 685-692, 1974. Printed in Great Britain

Pergamon Press

IDENTIFICATION CP T H E ~ A J O R URINARY METABOLITE OF ALPRENOLOL IN MAN, DOG AND RAT Nils-Olov Bodin Toxicology Laboratories,

Astra Pharmaceuticals

SSdert~lje,

AB,

Sweden

(Received 22 Oc~ber 1973; in final ~rm 31 December 1973) SUMMARY After oral administration of 3H-alprenolol to man, dog and rat, urinary metabolJtes of the drug have been separated by ion-exchange chromatography on Bio-Rex 70, a carboxylic acrylate resin. The major metabolite has been identified by GC-MS as 4-hydroxyalprenolol. Occurring in the urine largely in a conjugated fozm, it represents about 40 % of the excreted amount in man and dog and about 30 % in rat. Including alprenolol, which also appears largely as a conjugate, about 80 % of the amount of radioactivity excreted in human urine can be accounted for. It has been shown (1-7, a review in 8) that the biotransformation beta-adrenergic different

receptor blocking agents may proceed primarily by several

pathways;

e.g., by oxidative N-dealkylation

amination of the alkylaminoalkoxy ring system,

and by reactions

chain,

involving

extent of biotransformation

and by oxidative de-

by hydroxyla~ion

of the aromatic

the second side chain (if present)

In spite of these agents having appreciable

different

structural

similarities,

as well as the relative importance

reactions varies considerably

In the present study, metabolites

The major metabolite

fied by gas chromatography

within the group.

of the beta-blocking

drug alprenolol 1

has been isolated and subsequently

- mass spectrometry.

1Aptin@, AB H~ssle, MSlndal,

Sweden

685

the

of the

in the urine of man, dog and rat have been studied by ion-exchange tography.

of

chromaidenti-

686

Alprenolol Metabolism

Vol. 14, No. 4

MATERIALS AND METHODS

Compounds.

Tritium-labelled alprenolol

amino-2-3H-propano~,

El-(2-allylphenoxy)-3-isopropyl-

alprenolol hydrochloride, 4-hydroxyalprenolol

(2-allyl-4-hydroxyphenoxy)-3-isopropylamino-2-propanol]

[1-

hydrochlor~de,

5-hydroxyalprenolol, N-desisopropylalprenolol hydrochloride and 1-(2-carboxyphenoxy)-3-isopropylamino-2-propanol

hydrochloride were obtained from

AB H~ssle, MSlndal, Sweden. Dosing and sampling.

In a typical experiment, a male rat of the Sprague-

Dawley strain, weighing 240 g, was given a solution of ll mg/kg (0.14 mCi) of 3H-alprenolol hydrochloride by stomach tube. During 8 hours, 6.5 g of urine containing 23 % of the radioactive dose was collected° A male Beagle dog, weighing lO kg, was given lO mg/kg (0.31 mCi) of 3H-alprenolol hydrochloride.

The substance was administered p.o. in 1 ml

of water in a gelatine capsule. Urine was collected over 24 hours. The urine sample obtained weighed 225 g and contained 66 % of the radioactive dose. A male volunteer, aged 41 and weighing 72 kg, was given 1.4 mg/kg (0.32 mCi) of 3H-alprenolol hydrochloride, dissolved in 50 ml of water. Urine formed during the 4 hours following administration was collected for analysis. The amount of radioactivity recovered (in 182 g of urine) corresponds to 6 1 %

of the dose.

All urine samples were kept at -20°C until processed further. Determination of radioactivit 2.

Samples were mixed with 16.0 ml of scin-

tillation solution (toluene lO00 ml, ethylene glycol monoethyl ether 600 ml, butyl-PBD/Ciba A.G./7.OO g) and measured in a Packard Tri-Carb @ Mod. 3320. The counting efficiency was determined by an external standard channels ratio procedure. Enzymatic hydrolysis.

A methanol extract (3 x 4 ml) of a weighed (about

1 g) and then dried aliquot of urine was evaporated and the residue dis-

Vol. 14, No. 4

Alptmolol letabolism

687

solved in 1 ml of 0.5 ~ ammonium acetate buffer, pH 6.5, containing 67 mM 1-butanol. To t h l s s o l u t i o n 1 ml of ~-glucuronidase/arylsulphatase (crude solution from Helix pomatia, Type H-2, Sigma Chemical Co.) and 1 drop of chloroform were added. After incubation at 37°C for 24 hours, the sample was freeze-dried and extracted with methanol (3 x 4 ml). The extract was evaporated and the residue redissolved in a weighed amount (about 1 g) of the buffer. The resulting solution constituted the "hydrolyzed urine" sample. During this process the recovery of the radioactivity was 55-64 %. Ion-exchange chromatography.

An analytical column was prepared by dynamic

packing of a slurry of Bio-Rex 70, 200-400 mesh (Bio-Rad Labs. Inc.), into a 2.8 x 1000 mm glass column (Chromatronix Inc.). Buffer, 0.5 M ammonium acetate, pH 6.5, containing 67 mM 1-butanol, was pumped at a flow rate of 6 ml/h (Chromatronix C~P-2) through the column, which was kept at 60°C. The resulting pressure was less than lO0 psi. Samples, 0.12 ml, were applied by means of a rotary valve (Chromatronix R 6031 SV). The optical density at 254 nm could be recorded continuously (Chromatronix UV-detector Mod. 200 with 8 ~l cells). The radioactivity of the effluent, collected in fractions of 0.40 ml~ was subsequently determined. For preparative work, a 9 x 200 mm glass column was used. Samples, 0.45 ml, were eluted at a flow rate of 24 ml/h. Fractions of 2.40 ml were colle~ ted, and 0.20 ml aliquots of these were assayed for radioactivity.

The re-

maining parts of appropriate fractions were then pooled and freeze-dried. Gas chromatography - mass spectrometry.

Prior to analysis, the freeze-

dried sample, or a minute amount of the reference compound, was converted to a trifluoroacetyl derivative by addition of O.1 ml of benzene and O.1 ml of trifluoroacetic anhydride. The volume was reduced to about 25 ~l after one hour and a 3 ~l aliquot analyzed on an LKB 9000 instrument 1. A 3 x 3000 glass column packed with 1 %

OV-17 on Gas Chrom Q (80-100 mesh) operated at

1Kindly performed by Mr. H. Thorin, Astra Nutrition AB, Malndal, Sweden.

688

Alprenolol Metsbolism

170°C, was used for separation. of the separator were 220°C. meter were:

Vol. 14, No. 4

The temperatures

The operating

of the injector block and

conditions

of the mass spectro-

electron energy 70 eV, ion source temperature

current 60 ~A, and accelerating

270°C,

trap

voltage 3.5 k7.

RESULTS AND DISCUSSION The ion-exchange

chromatographic

for the separation of metabolites be adequate for this purpose. alprenolol achieved

containing an amino function,

Base-line

and N-desisopropylalprenolol,

(figure 1)o As expected,

appreciably

system, which was designed primarily

separation of 4-hydroxyalprenolol, appearing in that order, was

the benzoic acid derivative was not

retained and the 4- and 5-isomers

not be resolved.

of hydroxyalprenolol

could

Although some variation in retention volumes was observed

during the course of the experiments

(several months),

could always be recognized without difficulty. system was the length of time, about 6 hours, ration. Attempts

seemed to

to improve the performance

and by using a smaller particle

compressed when the pressure The chromatograms number of resolved,

The main disadvantage required

the Bio-Rex 70

and the column tends to become

is increased.

radioactive

a small

peaks, as well as a broad peak in fractions

9-18 (figure 1). This first peak was considered which were unretained

to contain a group of un-

due to the absence of a

positive charge and/or the presence of a negative The fact that the peak was considerably

to this group.

charge on the molecule.

diminished upon enzymatic hydro-

lysis showed that conjugates with glucuronic large contribution

the flew rate

of samples from the species studied contained

separated metabolites,

of the

to complete a sepa-

by increasing

size were unsuccessful;

resin has rather poor packing properties

the different peaks

acid or sulphuric acid made a

Vol. 14, No. 4

Alpreaolol Metabolism

I

589

I

MAN

Iv

v

I

DOG

> o o

t0-

%, . . . . .

i

'°~

j

m

RAT

~ d m ~v

,o

.

,o FR&CTION N U ~ I t

PIG. 1

Ion-exchange

chromatograms o f r e f e r e n c e compounds, o f u r i n e (_- _-), and o f (o---o) from man, dog and r a t a f t e r o r a l a d m i n i s t r a t i o n o f

d r o l y z e d 'urine ~H-alprenolol° A

2.8 x i000 mm column of Bio-Rex 70, 200-400 m e e ~ w a s operated at 600 Sample8, 0.i ml, were eluted with 0.5 M NH~Ac~ ~ 6.5~ 67 mM 1-butanol at a flow rate of 6 ~/11. Praotions of 0.4 ml were~ collected.

690

Alprenolol Metabolism

The peak corresponding all samples,

to alprenolol,

Vol. 14, No. 4

which was known to be present in

appeared at the expected position

(denoted IV). The correct-

ness of this identification was confirmed by GC-MS analysis° A well resolved peak (denoted III) at the position of the reference hydroxyalprenolol tify this peak,

was also present in all chromatograms.

larger amounts were first prepared

human urine. As was to be expected, gave inferior separation,

but the metabolite

In order to iden-

from hydrolyzed

the preparative

4-

rat and

column used for this

could be isolated in an essen-

tially pure form, as judged from rechromatography

in the analytical

system.

When analyzed by GC-MS, the trifluoroacetyl derivative of the metabolite gave a peak in the total ion current with the same retention time as that of the 4-hydroxyalprenolol practically

derivative.

identical,

The mass spectra of the two compounds were

with the base peak at ~ / ~ 308 and a molecular

(m_/A 553) consistent with the presence of a tri (trifluoroacetyl) tive (9). While an assignment

could therefore

deriva-

of the hydroxy group to the 5-position

not be ruled out from the mass spectrum obtained, shorter retention

ion

could

this compound had a

time and was easily separated on the GC column used and

be excluded.

From theoretical

considerations,

the position

para to the alkoxy chain rather than to the allyl chain was also believed to be the preferential

position for hydroxylation

In man and dog, minor peaks nolol,

(denoted II) appeared before 4-hydroxyalpre-

in rat these peaks were larger,

to identify

these metabolites

(6).

especially after hydrolysis.

Effort~

have failed so far. The very small peak

appearing last in the chromatograms

(denoted V) was concluded

to be iden-

tical with N-desisopropylalprenolol. The chromatographic ferent metabolites

and of unchanged

values for appropriate the investigation.

system used also permitted

fractions,

quantitation

of the dif-

drug by summation of the radioactivity

though this was not the main purpose of

The abundance values were calculated relative

total amount of radioactivity

recovered,

to the

but since complete recoveries

of

Vol. 14, No. 4

Alpremolol Metabolism

applied radioactivity were consistently obtained, sentative for the concentration

691

they were equally repre-

in the urine samples. However,

since the

recovery of radioactivity was incomplete in the preparation of the hydrolyzed urine samples, been partly,

products arising from unstable metabolites may have

or completely,

lost.

The values obtained are given in Table 1. TABLE 1

Relative Abundance mined by Ion-Exchange Alprenolol.

of Alprenolol

and some of its Metabolites

Chromatography

after Oral Administration

Data presented

Amounts of Radioactivity as 4-Hydroxyalprenolol (DIA), respectively. cluding Conjugates,

Species

Dose (mg/kg)

of Peaks I-V. Peaks III, IV and V were identified

(HOA), Alprenclol The Unresolved

(A) and N-Desisopropylalprenolol

Peak I contains Acid Metabolites,

Dog

Rat

lO

ll

Sampling interval

Relative abundance (% of total amount recovered) Sample I

II

III (HOA)

IV (A)

V (DIA)

94

1

5

O

O

Hydrolyzed urine

18

3

42

36

1

Urine

79

3

13

4

l

Hydrolyzed urine

33

~

41

20

1

Urine

88

5

6

0

0

Hydrolyzed urine

30

34

33

3

1

0-4

0-24

0-8

In the unhydrolyzed the first peak.

in-

while Peak II is unidentified.

Urine 1°4

of 3H-

in Pig. 1 were used to calculate Relative

(h)

Man

as deter-

samples, most of the radioactivity

was located

in

The other peaks were small and of these the 4-hydroxyalpre-

nolol peak was largest. enzymatic hydrolysis.

As mentioned above,

Simultaneously,

the first peak was re~uoed by

the 4-hydroxyalprenolol

to contain about 40 % of the total radioactivity

peak increased

in man and dog and about

692

Alprenolol Metabolism

Vol. 14, No. 4

30 % in rat. The abundance of alprenolol also increased considerably in man and dog after hydrolysis. The value found in the former was higher than expected from earlier studies and this was taken to indicate that the subject used in this study had a low hydroxylation capacity, which would make the direct conjugation pathway more important. The rat was deviant~ while peak I still constituted about 30 % after hydrolysis,

the peak II

complex (containing unidentified metabolites) increased to about the same value

as that of 4-hydroxyalprenolol.

Thus, although the total number of metabolites may well be large, the most striking feature in the biotransformation of alprenolol is the fact that one major metabolite, 4-hydroxyalprenolol, appearing largely in conjugated form, represents a large part of the excreted amount in all the species studied. Including alprenolol, which also appears largely as a conjugate, about 80 % of the total amount of radioactivity excreted in human urine can be accounted for, ACKNOWLEDGEMENT The author would like to thank Mrs. Britt Bryske for excellent technical assistance. REFERENCES 1.

P.A. BOND, Nature, 213, 721, (1967).

2.

P.A. BOND and R° HOWE, Biochem. P h a r ~ c . , 16, 1261-1280,

3.

J.W. PATERSON, M.E. CONOLLY, C.T. DOLLERY, A. HAYES and R.G. COOPER, Pharmacol. Clin., ~, 127-133, (1970).

4.

B. SCALES and M.B. COSGROVE, J. Pharmac. exp. Ther., 175, 338-347,

(1967).

(197o). 5.

P.-J. LEINWEBER, R.C. GREENOUGH, C.F. SCHWENDER, L.J° HAYNES and F.J. DI CARLO, J. Pharm. Sci., 60, 1516-1519, (1971).

6.

D.A. GARTEIZ, J. Pharmae. exp. Ther., 179, 354-358,

7.

G.L. TINDELL, T. WALLE and T.Eo GAFFNEY, Life Sciences, ll, 1029-1036, (1972).

8.

K.K. WONG and E.C. SCHREIBER, D_ru~ Metab. Revs., !,,1/, ' 10]-1}6,

9.

D.A. GARTEIZ ~ad T. WALLE~ J. Pharm. Sci., 61, 1728-1731,

(1967).

(1972) ,

(1972).