The distribution of cinnarizine and its metabolites in the rat

Life Sciences Vol . 7, Part I, pp . 989-994, 1968 . Printed in Great Britain.

Pergamon Press

THE DLSTRIBUTION OF CINNARIZINE AND ITS METABOLITES IN THE RAT F. T. N, Allewijn Jansaea Pharmaceutics, Research Laboratories Beerse, Belgium (Received 14 June 1968 ; in final form 3 July 1988) Cinnarizine, 1-benzhydryl-4-cinnamylpiperazine (trans), trade name

STUGERON, is extensively metabolized in the rat, primarily by N-dealkyla-

tion (1). The present paper concerns the distribution of the drug and its metabolites in the body tissues of the rat, Materials and Methods Specifically carbon-labelled cinnariziae (specific activity 1 mC~mmole) was synthesized (1) and dissolved in 0, O1 M tartaric acid . The site of the

radioactive label on the cinnarizine molecule is shown in Fig, 1 . Distribution and excretion were studied at 7 timQS (1~2, 1, 2, 4, 8, 16 and 32 hr) after administration by oral gavage of 2, 5 ml of the labelled compound at 4 dosages (0, 63, 2. 5, 10 and 40 mg~kg),

FIG. 1 Site of radioactive label on cinnarizine molecule The experimental animals comprised adult male Wistar rate (248 + 10 g) kept in metabolic cages in groups of 5, and with free access to food and water . For each dose-time record (Table 1), one group of 5 rate was sacrificed by decapitation, a total of 24 groups (120 animals) being used in the study,

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Sample preparation Immediately after decapitation, the blood was collected and heparinized, and the organs (liver, kidneys, heart, spleen, lungs, gonads and brain) were dissected from each rat, Corresponding tissues from the 5 animals in each

group were then pooled, Urine and faeces were collected separately from the metabolic cages, The organs and faeces were disrupted in distilled water

using Ultra-Turrax TP-18/2 homogenizera at 20, 000 rpm, Dilution factors, dependent upon the weight of the organ and its expected radioactivity level

were as follows : liver 10, kidneys 5, heart 4, spleen 5, lungs 7, 5, gonads 5, brain 4 and fee ce s 20,

1 . Estimation of radioactivity level in tissues sad excreta (Mahin and LofberA wet combustion technique (2))

Duplicate 200 pl samples of homogenate or blood were dispensed into

the bottom of small counting vials, to which were added 0, 2 ml of 70% perchloric acid (to further tissue dissolution) and 0, 4 ml of 30% H2 02 (to deco

lorize the sample and reduce quenching), The vials were then capped tightly

to minimize evaporative loss during warming, and placed in a constant temperature oven (70-80'C) for 3 hr to promote dissolution and de coloration,

Occasional agitation accelerated these processes and any slight precipitate present at the end of this 3 hr period readily dissolved in the scintillator

solution, Sample vials were then cooled to 0°C to condense volatile gases

and finally prepared for counting by addition of scintillator solution (see on), Urine samples were treated directly with scintillator solution,

2. Estimation of proportion of radioactivity in liver, brain and blood due to unmetabolized dru

Inverse Isotope Dilution Method)

Oae aliquot (10-20 ml) of heparinized blood, or homogenized brain or liver, was added to a water solution containing about 150 mg unlabelled cinnarizine and agitated for about 10 min to ensure good exchange of labelled and unlabelled compound . The mixture was then alkalinized with ammonia to about pH 10 (to ensure that the drug was present ae the base, because the salt is insufficiently soluble in CHC1 3) sad extracted by shaking with 100 ml

* Blood collection by this method is unavoidably incomplete, and the estimate accepted for the circulating blood volume was 1/19th part of the body weight i, e, 12 . 5 ml per rat,

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of chloroform for 10 min, The two layers were then separated by centrifugation at 2, 500 rpm for about 10 min, The organic layer was evaporated to dryness under nitrogen and the oily residue was dissolved in boiling methanol and allowed to crystallize as the base on cooling, The isolate was purified by repeated recrystallization from methanol until constant specific activity was attained, The relative amount of unmetabolized cinnarizine present in the original tissue sample was calculated from the formula

X =

where S

bm

x 100

= specific activity of the isolate

m = weight of unlabelled cinnarizine added b

= total radioactivity" of the original sample

X = % of this due to the unmetabolized drug

Radioactivity measuFement For the orally administered solutions, urine samples and crystalline isolates, the scintillation solution comprised 10 ml of a 4 g~l solution of 2, 5-bis-2-(5-tert,-butylbenzoxazolyl) thiophene (BBOT) in a xylene-alcohol mixture (p-xylene : m-xylene : absolute ethanol 6 : 3 : 1, by vol, ), For the faeces and tissue samples, the scintillator solution comprised 15 ml of a 7 g~l solution of BBOT in a toluene-ethyl cellosolve mixture (6 : 4,

vw),

All radioactivity levels were measured using a Packard Tri-Garb Liquid Scintillation Spectrometer (Model 3315 and~or Model 3310), Control samples (for background radiation counts) and 3 H-toluene standard samples (for assessment of counting efficiency) were assayed, and the counts per minute (cpm) for all samples were collected in a 30-1000 volt window (to reduce background noise) and converted into disir.tegrations per minute (dpm) . Results and Discussion The results are given in Table 1 . Tissue radioactivity levels did not vary in proportion to the dosage of the labelled compound administered, Accordingly, further discussion is presented in terms of the mean tissue levels at each

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time interval after treatment (see Table 1) . The administered compound was rapidly resorbed from the gastro-inteatinal tract and peak radioactivity levels occurred within 1 hr of treatment for all tissues, except for the gonads in which the peak was reached only 4-8 hr after treatment, For the liver, the peak level corresponded to 6% of the admini~tered dose and for all other tissues it was less than 0, 5% . After re sorption the drug was rapidly metabolized : within 1/2 hr of treatment 90 °fo of the radioactivity in the liver was due to cinnarizine metabolites and only 10 °fo to the drug itself, At this time metabolites were also present in the blood and brain and presumably, therefore, in the other tissues too, After 8-16 hr the tissue level of unchanged cinnarizine was negligible (less than 0, O1 % of the administered dose, even in the liver) . The rapidity of cinnarizine metabolism resembled that of the related compounds cyclizine and chlorcyclizine (2) : one hour after a 50 mg/kg dose of chlorcyclizine was administered orally to male rats, only 17% of the radio activity in the pooled tissue extract (liver, kidney, spleen and lung) was due to the nonmetabolized drug (2) . Summary The study was conducted on 120 male Wistar rats and the radioactivity levels in the blood, liver, kidneys, heart,

spleen, lungs, gonads, brain,

urine and faeces were found at 7 time intervals (1/2,

1,

2, 4, 8,

16, 32 hr)

after administration of 14 C-cinnarizine at 4 dosage rates (0,063, 2 .5, 10 and 40 mg/kg), The percentage of the tissue radioactivity due to the nonmetabolized drug was found for the blood, liver and brain, There was rapid resorption of the administered compound from the gastro-intestinal tract and peak tissue radioactivity levels were reached within 1 hr, except for the gonads in which the peak was reached only after 4-8 hr . Extensive metabolism occurred within 1/2 hr of treatment, and after 32 hr the tissue drug levels were negligible, Ackn owle d~ments I should like to thank C . J . E . Niemegeers, F, M . Lenaerts, J . Dony, P, J, Lewi and A . E . F . Chandler for their help in various stages of this work, and I, Dergent, tance,

M . Raeves, M, Pesters and J . Jacobs for technical assis-

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References 1.

W, Soudijn and I. van Wijngaarden, Life Sci, 7, 231 (1968) .

2.

D. T. Mahin and R . T, Lofberg, Anal, Biochem,

3.

R, Kuntzman, A. Klutch, I. Tsai and J. J, Burns, J . Pharmacol. Exp, Thera , 14 , 29 (1965) .

L,

500 (1966) .