Toxicology, 27 (1983) 315--320 Elsevier Scientific Publishers Ireland Ltd.

BETA-ADRENOCEPTOR STIMULANTS AND MESOVARIAN LEIOMYOMAS IN THE RAT

DAVID JACK, DESMOND POYNTER* and NEVILLE W. SPURLING

Glaxo Group Research Ltd., Ware, Herts, SG12 ODJ (Great Britain)

{Received September 28th, 1982) (Accepted January 10th, 1983)

SUMMARY

The exper iment reported here shows that mesovarian leiomyomas may be induced in rats by the administration of 2 chemically distinct adrenergic stimulants, salbutamol or terbutaline. That the induction of these benign tumours is a funct ion of adrenergic stimulation is shown by the fact that the concurrent administration of the adrenergic blocker propranolol prevented their development.

K e y words: Beta-adrenergic stimulants; Leiomyoma; Salbutamol; Terbuta- line; Propranolol

INTRODUCTION

In 1971 Nelson and Kelly repor ted that prolonged oral administration of soterenol, a po ten t long-acting beta-adrenoceptor stimulant, induced leio- myomas in the mesovaria of female Sprague--Dawley rats. As a result, clinical work on soterenol as a bronchodi la tor was discontinued. They subsequently found that mesuprine hydrochlor ide, an analogue of soterenol, also caused mesovarian le iomyomas in Charles River CD rats (Sprague--Dawley derived) and concluded that the product ion of such leiomyomas in the rat might be a general proper ty of beta-stimulants [1]. They, therefore, carried out fur ther tests on soterenol, using pigmented Long-Evans rats as well as the Sprague--Dawley strain, and salbutamol sulphate, a primarily beta-2 selective adrenergic agent, as a control substance. Both drugs, given at 20 mg/kg/day by mouth for 96 weeks, induced leiomyomas in about 25% of the Sprague--

*To whom reprint requests should be addressed.

0300-483 X/83/$03.00 ,~ 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Dawley animals and 5--10% of the Long-Evans strain. These results were clearly consis tent with their proposa l tha t beta-s t imulat ion was the cause of the mesovar ian l e i o m y o m a s bu t did no t prove it.

An alternative causat ion of the l e iomyomas was suggested by data pre- sented at the Pu lmona ry Allergy Drugs Advisory Commi t t ee o f the FDA held on 3rd May 1979 [2] . Two fur ther agonists, z interol and reproterol , were stated to p roduce l e iomyomas but others, carbuterol , fenoterol , pyr- buterol , terbuta l ine and hexoprenal ine , were repor ted to have been tested in rats with negative results. In our view, the latter evidence was weak because the tox ic i ty tests did no t take accoun t of the k n o w n variat ion in sensitivities of d i f ferent strains of rats to beta-s t imulants or, more impor- t an t ly , of the great variat ion in the bioavailabilities of beta-s t imulants given orally to rats. The exper imen t described in this paper was carried ou t to establish b e y o n d d o u b t whe the r or no t beta-s t imulat ion was the pr imary cause of mesovarian l e i o m y o m a s in the rat.

E XPERIMENTAL

Design o f e x p e r i m e n t The exper imen t carried ou t is def ined in Fig. 1. It consis ted o f 2 parts. In

the first, the incidence of l e i o m y o m a fo rma t ion in rats t reated only with sa lbutamol was c o m p a r e d with tha t in rats t reated with sa lbutamol plus

Test compounds

Test species

Route of administration Duration of test Number of animals

Salbutamol sulphate BP (Glaxo, U.K.) Terbutaline sulphate BP (Astra, Sweden). Female Charles River CD (Sprague--Dawley derived) rats. Oral -- drug distributed in the diet. At least 104 weeks. Control -- minimum 100 Treated groups -- minimum 50

Experimental design and dosage

Group Treatment level Dosage of base equivalent a

1 Control 2 Salbutamol low dose 3 Salbutamol high dose 4 Terbutaline low dose

Terbutaline high dose

Salbutamol plus Propranolol

2 mg/kg/day 20 mg/kg/day

6 mg/kg/day for 62 weeks 12 mg/kg/day thereafter 60 mg/kg/day for 62 weeks

120 mg/kg/day thereafter 20 mg/kg/day 33 mg/kg/day

aEstimated average amount ingested by each rat based on known food consumption.

Fig. 1. Outline protocol for the salbutamol/terbutaline/propranolol comparative study carried out at the Huntingdon Research Centre, England.

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proprano lo l , a specific be ta-antagonis t known to block all beta-receptors . The ra t ionale of this compar i son was tha t the incidence of l e iomyomas in the be ta -b locked group would be significantly less than in the o the r if beta- s t imula t ion was the cause o f l e i o m y o m a fo rma t ion ; any o the r resul t would require an al ternat ive exp lana t ion for t u m o u r induct ion . The second par t of the e x p e r i m e n t was in t ended to be a compar i son of the effects of equivalent be ta -s t imulant doses of sa lbu tamol and te rbuta l ine because similar incidences o f l e iomyomas would be e x p e c t e d if be ta-s t imula t ion was the causal mecha- nism. Terbu ta l ine was chosen for this compar i son because it had no t pre- viously been f o u n d to cause l e i omyomas in the rat.

Determination o f dosages The relative be ta-s t imulant po tenc ies of sa lbutamol and te rbuta i ine in

the rat were d e t e r m i n e d in 2 ways. First and mos t relevant, thei r abilities to relax mesovar ian s m o o t h muscle con t r ac t ed by potass ium chlor ide in vi tro were de t e rmined by the m e t h o d o f Apper ley et al. [3 ] ; sa lbutamol was a b o u t twice as active as t e rbu ta l ine in this test. Second, the ef fec ts of orally admin is te red sa lbu tamol and te rbu ta l ine on the hear t rates of the rat were de t e rmine d by a m e t h o d involving the use of a W + W indirec t b lood pressure record ing device on fasted rats p r ehea t ed to 40°C for 30 min, the hear t rate being der ived e lect ronical ly . Rats given 20 mg/kg sa lbutamol had an increase in hear t ra te o f 102 bea ts /min as measured 1 h af te r dosing. At 2 mg/kg the increase was 50 bea t s /min which was only marginal ly greater than tha t seen wi th saline where 38 bea t s /min increase were recorded . Terbuta l ine at 60 mg/kg gave a 94 bea t s /min increase and a 69 bea ts /min increase at 6 mg/kg. Thus sa lbu tamol was abou t 3 t imes more active than te rbuta l ine in fasted rats, a r a the r greater d i f fe rence was seen in fed animals. A dose-rat io of 3 was selected for the main e x p e r i m e n t because, al lowing for the k n o w n p o o r e r bioavailabil i ty o f te rbuta l ine in the rat, this ra t io was expec t ed to give plasma and, the re fo re , tissue levels of te rbuta l ine abou t twice those of sa lbu tamol and these levels would be biologically equivalent on mesovarian muscle.

In the event, the bioavailabi l i ty o f te rbuta l ine in the rat p roved to be less than e x p e c t e d and, as shown later, the plasma levels of the drug f o u n d dur ing the first year o f the e x p e r i m e n t were no greater than with salbutamol . The dose of t e rbu ta l ine was, there fore , doub led on week 63. Accordingly , for the first year of the expe r imen t , each dose of te rbuta l ine provided only a b o u t one half of the in t ended plasma concen t r a t i on and, therefore , a s ignif icantly lower level o f be ta-s t imula t ion than in the sa lbu tamol- t rea ted animals.

The dose o f p roprano lo l was tha t f oun d ef fec t ive ly to abolish the increase in hear t rate caused by oral sa lbutamol in the rat.

Salbu tamol and terbutaline plasma levels Salbu tamol and te rbu ta l ine plasma levels were measured by the m e t h o d

of Martin e t al. [4 ,5] using gas c h r o m a t o g r a p h y mass spec t rome t ry .

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TABLE I

INCIDENCE OF M E S O V A R I A N LEIOMYOMAS IN CHARLES R I V E R CD ( S P R A G U E - - DAWLEY DERIVED) RATS T R E A T E D O R A L L Y F O R 2 YEARS WITH SALBUTAMOL SULPHATE ALONE (GROUPS 2 and 3), T E R B U T A L I N E SULPHATE (GROUPS 4 and 5) OR SALBUTAMOL SULPHATE PLUS P R O P R A N O L O L H Y D R O C H L O R I D E (GROUP 6)

No. of rats No. o f rats with No. of rats with examined mesovarian l e iomyomas hyperplasia o f

(No. of l e iomyomas) s m o o t h muscle

Group 1: Cont ro l 105 0 0

Group 2 : 2 mg/kg /day 55 0 2 Salbutamol

Group 3 : 2 0 mg/kg /day 55 16 (23) 2 Salbutamol

Group 4 : 6 / 1 2 mg/kg /day 55 1 (1) 0 Terbuta l ine

Group 5 : 6 0 / 1 2 0 mg/kg /day 55 7 (9) 2 Terbuta l ine

Group 6 : (Sa lbutamol 20 mg/kg /day 55 0 0 (Propranolol 33 mg/kg /day

TABLE II

MEAN PLASMA LEVELS OF SALBUTAMOL OR T E R B U T A L I N E IN CHARLES R I V E R CD ( S P R A G U E - - D A W L E Y DERIVED) RATS A F T E R CHRONIC ORAL ADMINISTRATION OF SALBUTAMOL SULPHATE OR T E R B U T A L I N E SULPHATE IN THE DIET

The actual plasma levels o f individuals showed marked variation. No corre la t ion was observed be tween individually measured plasma levels and numbers or size of l e iomyomas in any rat.

Time Control (~eeks )

Mean plasma level (ng/ml) ± S.D.

Sa lbutamol Terbuta l ine

2 mg/kg 20 mg/kg 6 mg/kg 60 mg/kg 12 mg/kg 0--6 2 weeks

120 mg/kg 63--104 weeks

15 <1.0 28 <1.0 52 <1.0 68 <1.0 78 <1.0

104 <1.0

<1.6 +- 0.7 7.5 + 2.4 <1.0 12.1 ± 5.3 <1 .2 + 0.4 19.0 + 4.5 <1 .2 + 0.5 17.3 + 3.1

2.0 ± 0.1 28.8 + 7.1 2.6 +_ 0.81 20.6 + 4.0 1.9 + 0.5 21.8 + 5.6 8.9 + 7.1 50.7 ÷ 8.9 3.3 + 0.9 27.4 + 5.6 4.4 + 1.3 32.0 + 4.1 1.9 + 0.5 10.1 + 5.2 8.8 ± 9.7 51.5 ÷ 10.5

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R ESULTS

The essential results of the exper iment are shown in Table I. Mesovarian leiomyomas or smooth muscle hyperplasia occurred in Groups 2, 3, 4 and 5 which were treated with salbutamol or terbutaline and the incidence of leiomyomas was dose-related. These tumours were macroscopically and microscopically like those reported by Nelson and Kelly [6]. No leiomyomas or hyperplasia occurred in Group 6 which received propranolol in addition to salbutamol. The mean plasma levels of salbutamol and terbutaline, in blood taken from the orbital sinus, are summarised in Table II.

At week 52 the levels of propranolol and salbutamol in Group 6 were measured. Salbutamol levels were 34.36 _+ 12.75 ng/ml and propranolol 26.72 i 14.90 ng/ml. Thus propranolol did not inhibit absorption of the agonist.

DISCUSSION

The most significant new finding in this work was that concurrent ad- ministration of propranolol completely prevented the induction of meso- varian leiomyomas by salbutamol in Charles River CD rats. This leaves no doubt that the primary cause of the le iomyoma formation was the beta- stimulant action of the drug and not some other unknown specific tumour- inducing mechanism. Furthermore, one need look no fur ther than the meso- varian smooth muscle cells themselves for the site of the receptors involved since, as shown by Apperley et al. [3], these cells contain beta-2 adreno- ceptors which mediate cellular relaxation. The proliferation of mesovarian muscle caused by beta-stimulants in the rat may be an a t tempted physio- logical adaption to cont inuous relaxation of the muscle.

If mesovarian le iomyoma formation induced by salbutamol results from prolonged beta-stimulation of the target organ and since the beta-receptors of mesovarian muscle are the beta-2 sub-type and not a further beta-receptor variant, any other beta-stimulant delivering a sufficient stimulus at a tolerated dosage schedule must also induce leiomyomas. The t ruth of this deduct ion was confirmed when terbutaline, a primarily beta-2 selective agent thought not to induce leiomyomas, was found to behave similarly to salbutamol. The final incidences of affected animals, 16/55 and 7/55 for salbutamol and terbutaline, respectively, are entirely expectable after allowance is made for the fact that the plasma concentrat ions of terbutaline for the first 62 weeks of the exper iment were only about one half of those intended. The sal- butamol and terbutaline results obviously belong to the same absolute beta-stimulus/response curve.

The rat studies described in this paper const i tute the only truly controlled exper iment carried out so far on the induction of mesovarian leiomyomas by beta-stimulants and they provide an entirely rational explanation of the phenomenon. Indeed, a variety of chemical types of beta-stimulants have now been shown to induce leiomyomas and their relative activities in this

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respect are compa t ib le with their k n o w n activities at beta-2 receptors and their bioavailabilities in the rat; t hey include sulphonanil ides (soterenol , mesupr ine and zinterol) , resorcinols ( terbutal ine, reproterol ) and a saligenin (salbutamol) . Accord ing ly , it is now virtually certain tha t the negative da ta r epor ted with o ther beta-s t imulants result f rom failure to take proper a c c o u n t of their absolute potencies and bioavailabilities in the rat and /o r the use of relatively insensitive strains of rats in the exper iments .

In o ther exper iments carried ou t in our laboratories, sa lbutamol given orally in high doses in life-span studies did no t cause mesovarian l e iomyomas in the mouse or the hamster . We do no t know why the rat differs f rom the o ther species in this response to beta-s t imulants but believe it to be atypical. We certainly have no evidence to suggest tha t female as thmat ic pat ients are likely to develop mesovar ian l e iomyomas as a result of their therapy. M y o m a of the uterus occurs in a b o u t 20% of w o m e n over the age of 30 and the re t roper i tonea l region is no t an unusual site of l e iomyomas and m y o s a r c o m a s [7] . L e i o m y o m a of the ovary or mesovar ium is, however , rare with on ly abou t 30 repor ted cases up to 1972 [8] an observat ion conf i rmed by Pro- lessor Harry Fox in Manchester and by Professor Colin Berry in L o n d o n (Personal c o m m u n i c a t i o n ) . Since l e iomyomas of ovarian tissue are rare, any substantial increase in their incidence associated with the use of beta- s t imulant drugs would be expec ted to be obvious. No such increase had been observed despite m a n y years of use of beta-s t imulants for bronchia l asthma.

ACKNOWLEDGEMENTS

We wish to thank Dr. E. Hansson of Astra, Sweden for the supply o f terbuta l ine and for his helpful discussion during the c o n d u c t of the trial. We also thank Dr. L.E. Martin for the de te rmina t ions of drug in plasma and Mr. T. Hilditch for da ta on rat heart rates. This s tudy was pe r fo rmed at the H u n t i n g d o n Research Centre and we are grateful to the m a n y people involved.

REFERENCES

1 L.W. Nelson, W.A. Kelly and J.H. Weikel, Mesovarial leiomyomas in rats in a chronic toxicity study of mesuprine hydrochloride. Toxicol. Appl. Pharmacol., 23 (1972) 731.

2 F.D.C. Anon, Reports. Vol. 41 (1979) No. 20. p. TaG 10--11. May 14. 3 G.M. Apperley, R.T. Brittain, R.A. Coleman, I. Kennedy and G.P. Levy, Character-

isation of the beta-adrenoceptors in the mesovarium of the rat. Br. J. Pharmacol., 63 (1978) 345P.

4 L.E. Martin, Janet Oxford, R.J.N. Tanner and M.J. Hetheridge, The determination of terbutaline in human plasma by selected ion monitoring of the t-butyldimethylsilyl ether. Biomed. Mass Spectrom., 6 (1979) 460.

5 L.E. Martin, Janet Rees and R.J.N. Tanner, Quantitative determination of salbutamol in plasma, as either its trimethylsilyl or t-butyldimethylsilyl ether, using a stable isotope multiple ion recording technique. Biomed. Mass Spectrom., 3, (1976) 184.

6 L.W. Nelson and W.A. Kelly, Mesovarial leiomyomas in rats in a chronic toxicity study of soterenol hydrochloride. Vet. Pathol., 8 (1971) 452.

7 R.A. Willis, Pathology of Tumours, 3rd Edn. Butterworths, London, 1900. 8 M. Fallahzadeh, M.B. Dockerty and R.A. Lee, Leiomyoma of the ovary. Report of

five cases and review of the literature. Am. J. Obstet. Gynecol., 113 (1972) 394.

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