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Comparative bioassay of vasoactive drugs using isolated perfused rabbit arteries
EUROPEAN JOURNAL OF PHARMACOLOGY 6 (1969) 286-293. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM
COMPARATIVE
BIOASSAY OF VASOACTIVE
USING ISOLATED PERFUSED
DRUGS
RABBIT ARTERIES
J. M. VAN NUETEN Department o[ Pharmacology, Janssen Pharmaceutica, Beerse, Belgium
Received 17 February 1969
Accepted 25 February 1969
J.M.VAN NUETEN, Comparative bioassay of vasoactive drugs using isolated perfused rabbit arteries, European J. Pharmacol. 6 (1969) 286-293. A simple and reliable assay technique for vasoactive drugs is described. Using KCl-depolarised central artery preparations from the rabbit ear, and saphenous artery preparations from the hind leg, the following drugs were assayed for potency, rate of onset and duration of action (in inhibiting ~(Cl-depolarisation-induced vasoconstriction): papaverine hydrochloride, cyclandelate (Cyclospasmolf~, cinnarizine (Stugeron(~, xanthinol niacinate (Complamin ~) and dihydroergotoxine methanesulphonate - DEM - (Hydergin(~. The effects of the drugs on the two types of preparation were similar. For the central artery, potency decreased in the order: cinnarizine > papaverine > DEM > cyclandelate ~ xanthinol niacinate, duration of action in the order: cinnarizine > cyclandelate > xanthinol niacinate > papaverine > DEM, and speed of onset of action in the order: DEM > papaverine > xanthinol niacinate > cyclandelate > cinnarizine. In vitro Perfused arteries Rabbits
Vasodilation Papaverine Cyclandelate
1. INTRODUCTION Isolated perfused vessels have been found useful tools for the study of vasoactive drugs and various assay techniques have already been described (e.g. Waugh, 1962; Carrier and Holland, 1965; De la Lande and Harvey, 1965; Hrdina, Bonaccorsi and Garattini, 1967). The present paper describes a further method of bioassay which was found to be simple, reliable and widely applicable for comparing the potency, duration and mode of action of vasoactive drugs on peripheral arteries.
2. METHODS Vessel segments 2.5-3.5 cm long were removed from the central artery of the ear and the saphenous artery of the hind leg of freshly killed rabbits weighing 2 - 4 kg. The arterial segment was cannulated at
Cinnarizine Xanthinol niacinate Dihydroergotoxine methanesulphonate
the proximal end (internal diameter ca. 1 mm) with a BD 20 G hyperchrome stainless steel hypodermic needle (external diameter 0.9 mm) connected to PE 60 Clay-Adams polyethylene tubing (internal diameter 0.76 mm). The preparation was suspended in a 30 ml constant volume organ bath and maintained vertical by a 50 mg load. The bath contained perfusion fluid at 37°C and was gassed with a 95% 0 2 and 5% CO 2 mixture. 2. I. Apparatus The experimental scheme is shown in fig. 1. Gassed perfusion fluid (95% 0 2 - 5 % CO 2 mixture) was prewarmed to 37°C and delivered to the organ bath at a constant flow rate of 6 ml/min by Tygon tubing (internal diameter 2.38 mm). The flow rate was maintained by a Sigmamotor T 8 peristaltic pump operating at 30 pulses/min. The bath fluid was continuously replaced by the new perfusate, and constant bath volume was maintained by drainage through the overflow.
BIOASSAY OF VASOACTIVE DRUGS
287
Perfusion fluid ---I,¢
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Fig. 1. Apparatus and perfusion scheme used in the assay of vasoactive drugs on rabbit artery preparations.
In contrast to other perfusion techniques (Waugh, 1962; De la Lande and Harvey, 1965; Bonaccorsi and Hrdina, 1966; Hrdina et al., 1967) test drugs were not injected into the main perfusion stream, as direct additions of even small volumes of solution cause transient, but marked, alterations in the perfusion pressure. Instead, test drugs were introduced v/a a small supplementary perfusion tube (PE 20 Clay-Adams polyethylene tubing of internal diameter 0.38 mm) in a total volume of 0.10 ml for a period of 25 see. Afterwards, only perfusion solution was passed through the subsidiary tube. Concentrations of drug solutions were adapted in order to use a volume of 0.10 ml for all the doses. Using this technique, therefore, the rate of fluid delivery was constant throughout the experiments, and any change in perfusion pressure could be reliably attributed to the responses of the preparation.
Variations in the perfusion pressure were monitored continuously by a Statham P 23 Gb pressure transducer and, after boosting with a Brandau DD3 amplifier, the output signal was recorded on a Kipp Micrograph BD or a Heath EUW 20 ME linear recorder. 2.2. Perfusion solutions The perfusion solutions used were (in m moles): normal Tyrode solution (KCI 2.7, CaCl 2 1.8, MgCl2 1.04, NaHCO 3 l l.9, NaH2PO4 0.42, NaCl 136.9, glucose 5.55) and KC1 depolarising solution (KCI 99.83, CaC12 3.59, MgCl2 1.04, NaHCO 3 l l.9, NaH2P04 0.42, NaC1 39.33, glucose 5.55). The pH of both solutions was 7.4-7.6. 2.3. Drugs The following vasoactive drugs were assayed using
288
J.M.VAN NUETEN
the de~olarised arterial preparations: cinnarizine (Stugeron ~, Janssen) in an aqueous solution of 0.1 M tartaric acid; xanthinol niacinate (Complamin®, Wiilfing) from commercially available ampoules (150 mg/ ml); cyclandelate (Cyclospasmol®, Brocades) in polyethylene glycol (PE 200); papaverine hydrochloride in aqueous solution; and dihydroergotoxine methanesulphonate - DEM (Hydergin®, Sandoz) from commercially available ampoules (0.3 mg/ml)*. Solvent controls for each drug were also run. 2.4. Procedure After setting up a blood vessel in the organ bath, it was perfused with normal Tyrode solution for a 30 min equilibration period, during which the sensitivity of the preparation was checked by administration of 0.25 #g (central artery) or 1/ag (saphenous artery) of (-)-norepinephrine. After equilibration, the Tyrode solution was replaced by KCI depolarising solution and a further 30 min period allowed for the development of contraction. Drug solutions were then applied for 25 see in a 0.10 ml volume and the response of the depolarised artery was observed for 90 min. Recording was uninterrupted from the time of setting up the vessel in the organ bath to completion of the drug trial session.
3. RESULTS When a vessel segment was attached to the cannula, the perfusion pressure rose to 100-150 mm Hg, but within a few minutes it decreased again to a constant value of 14--41 mm Hg (mean for 130 central artery preparations = 26 mm Hg, mean for 60 saphenous artery preparations --- 25 mm Hg).
3.1. Response to KCl-depolarisation Depolarisation-indueed vasoconstriction caused a rise in the mean perfusion pressure: it reached a peak after 2 - 3 min, then fell slightly, and thereafter rose again very slowly for the duration of the experiments (fig. 2). The magnitude of the response to depolarisation varied with the site of origin of the vessels: the * A 0.63 mg dose was prepared by dissolving DEM powder in water, but it could not be assayed as the drug tended to precipitate out of solution during the experiments.
I/,0iI ,i
i
120
"~. .
Central artery ..0
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E IO0E
i~ Sal)henous artery
g ~.
_1f-
60-
~0It---@--~ 20"
-10
~
10
10
~0
Time after addition of depolarisation solution (min}
Fig. 2. Influence of KCl-depolarisation on the perfusion pressure of isolated arterial preparations. Each point represents the mean value from 130 experiments on the central artery of the rabbit ear, or 60 experiments on the saphenous artery. mean peak perfusion pressure was 141 mm for the central artery segments (range = 6 2 - 2 1 6 mm Hg), but only 89 for the saphenous artery segments (range = 44-155 mm Hg). Although the mean response to depolarisation was triphasic, conforming to the pattern recorded by Bonaccorsi and Hrdina (1966) for the isolated perfused renal artery of the rat, a sizeable percentage of the preparations showed a monophasic or biphasic response to depolarisation, and the percentage was approximately the same whether the segment was taken from the central or from the saphenous artery (fig. 3). 3.2. Response of depolarised central artery preparations to vasoactive drugs All the drugs studied had a relaxing effect upon depolarisation-induced contraction of the central artery (figs. 4-6).
BIOASSAY OF VASOACTIVE DRUGS
289
Bi~a~¢ responu
NoelpklI oc IIiII4II~I
2
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m
j
~& : 10110~ I 7 "/*
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C . a . • ~ 1 1 1 3 0 • 31.5 %
C.a. • 69/130• 53.1"1.
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=17160
•
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o 3~ Time after addition of KCI depolarisation solution (min)
1o
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Fig. 3. Stylised response curves of the isolated arterial preparations to KCl-depolarisation. Below each curve is shown the number of preparations tested that responded approximately as indicated: c.a. = centz~J artery, s.a. = saphenous artery.
100-
80
V ~
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~Cycla~delate
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-
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Fig. 4. Comparison of the potencies of the 5 drugs in inhibiting depolar~tion-induced contraction of the perfused centralartery of the rabbit ear. Each dose was added to the perfusion fluid for 25 sec, the time necessary to deliver 0.I ml of the drug solution. Each point represents the mean peak effect from 6 experiments and the vertical lines represent the standard errors of the means.
290
J.M.VAN NUETEN
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Fig. 5. Comparison of the rates of onset of peak action of the 5 drugs, and their durations of action (---time during which effect was ~ 50% of the peak value). Effect measured = inhibition of depolarisation-induced contraction of the perfused central artery of the rabbit ear. Each dose was added to the perfusion fluid for 25 see, the time necessary to deliver 0.1 ml of the drug solution. Each block represents the medium value from 6 experiments.
Papaverine had a potent inhibitory effect at the highest doses tested (figs. 4 and 6). It had a.rapid onset o f action, reaching peak effectiveness in 2 - 3 rain, but its effect was sustained for less than 20 rain (figs. 5 and 6). Cyclandelate had a slower rate o f onset o f inhibitory action than papaverine and was less potent, but it had a longer median duration of action (up to about 35 min).
Cinnarizine had a still slower rate o f onset o f peak action but, when achieved, the peak effect was sustained for a long time, and at doses o f 0.04 and 0.16 mg, it lasted for the duration o f the experiment ( > 90 min). The drug was more potent t h a n both cyclandelate and papaverine, and was active at a dose of only 0.0025 mg. DEM and xanthinol niacinate exerted only moderate and transitory inhibitory effects, even at the
BIOASSAY OF VASOACTIVE D R U G S
291
I
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:
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Fig. 6. Time-response curves for the maximum doses of the 5 drugs tested on the perfused central artery of the rabbit ear. Each dose was added to the perfusion fluid for 25 sec, the time necessary to deliver 0.1 ml of the drug solution. Each point represents the mean value from 6 experiments, and the vertical lines represent the standard errors of the means.
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Fig. 7. Time-response curves for the 5 drugs tested on the saphenous artery of the rabbit at doses identical to the maximum doses tested on the central artery of the ear (fig. 6). Each dose was added to the perfusion fluid for 25 sec, the time necessary to deliver 0.1 ml of the drug solution. Each point represents the mean value from 6 experiments, and the vertical lines represent the standard errors of the means.
J.M.VAN NUETEN
292
Table 1 Differences in drug responsivenessof depolarised saphenous and central artery preparations.
No. of experiments
Drug and dose (mg)
xanth/nol niacinate papaverine DEM cyclandelate cinnarizine
(40) (0.63) (0.16) (10) (0.16)
Median rate of onset of action (min)
Median duration of action (min)
central artery
saphenous artery
central artery
saphenous artery
2 3 3 25 43
1½ 4 10 20 30
6 10 10 34 >90
3 19 21% 68 >90
6 6 6 6 6
highest doses tested * Solvent controls never caused more than a 5% reduction in the depolarisation-induced contraction. 3.3. Responses of depolarised saphenous artery pre-
parations to vasoactive drugs The effects of the 5 drugs on the depolarised saphenous artery segments (fig. 7) were similar to those obtained with the central artery segments. Comparative results are presented in table 1. Solvent controls never caused more than a 6% reduction in the depolarisation-induced contraction.
4. DISCUSSION The assay technique described proved a reliable method for studying the response of peripheral arteries to vasoactive drugs, permitting quantitative evaluation of the potency, rate of onset and duration of action of vasodilator effects, and comparison of the drug-sensitivity of vessels from different sites of origin. Papaverine and cyclandelate exert their vasodilator effects primarily by direct musculotropic action (Bijlsma et al., 1956; Charlier, 1961; Lewis, 1965; Gifford and Moyer, 1967). Cirmarizine is believed to act on blood vessels by inhibiting selectively the calcium influx into depolarised cells and thereby reducing the availability of free Ca++ ions for the induction and maintenance of contraction (Godfraind, Kaba and Polster, 1968; Godfraind and Polster, 1968; God* See footnote on page 288.
fraind and Kaba, 1969). Xanthinol niacinate also has a musculotropic action, but it acts preferentially on the coronary vessels (Charlier, 1961) and on cardiac muscle (Kappert, 1961), so that the increased rate of blood flow through peripheral vessels in vivo is mainly an indirect effect. DEM also has a predominantly indirect effect upon the peripheral vessels, its action being exerted primarily v/a the vasomotor centre (Rothlin, 1946; Konzett and Rothlin, 1953). In the present experiments cirmarizine was by far the most potent and long-acting of the drugs tested: on the isolated central artery, it was about 4 times as potent as papaverine and about 100 times as potent as cyclandelate, and, at equivalent dosage, its duration of action was at least 9 times that of papaverine and 3 times that of cyclandelate. Further studies, to evaluate the effectiveness of all 5 drugs on the peripheral vascular system in vivo, would seem warranted.
ACKNOWLEDGEMENTS I would like to thank Mr. L.Haagen for technical assistance with the experiments, Dr. A.E.F.Chandler for his help in the preparation of the manuscript, and Sandoz Products Ltd for the gift of Hydexgin®powder.
REFERENCES Bijlsma, U.G., A.B.H.Funcke, H.M.Teersteege, R.F.Rekker,
M.J.E.Emsting and W.T.Nauta, 1956, The pharmacology of Cyclospasmol, Arch. Intern. Pharmacodyn. Ther. 105, 145.
B1OASSAY OF VASOACTIVE DRUGS Bonaccorsi, A. and P.Hrdina, 1966, Interactions between desipramine and sympathomimetic agents on the cardiovascular system, in: Antidepressant Drugs, eds. S.Garattim and M.N.G.Dukes (Excerpta Medica Foundation, Amsterdam) p. 149. Carrier Jr., O. and W.C.Holland, 1965, Supersensitivity in perfused isolated arteries after reserpine, J. Pharmacol. Exptl. Therap. 149,212. Charlier, R., 1961, Coronary Vasodilators (Pergamon Press, Oxford). Dc la Lande, I.S. and LA.Harvey, 1965, A new and sensitive bioassay for catecholamines, J. Pharm. Pharmacol. 17, 589. Gifford Jr., R.W. and J.H.Moyer, 1967, Vasodilator drugs for the treatment of peripheral vascular disturbances, in: Drugs of Choice 1968-1969, ed. W.Model (C.V.Mosby Co., St. Louis) p. 402. Godfraind, T. and A.Kaba, 1969, Blockade or reversal of the contraction induced by calcium and adrenaline in depolarized arterial smooth muscle, Brit. J. Pharmacol., in press. Godfraind, T., A.Kaba and P.Polster, 1968, Differences in sensitivity of arterial smooth muscles to inh~ition of
293
their contractile response to depolarization by potassium, Arch. Intern. Pharmacodyn. Ther. 172,235. Godfraind, T. and P.Polster, 1968, Etude comparative de m6dicaments inhibitant la rdponse contractile de valsseaux isolds d'origine humaine ou animale, Th~rapie 23, 1209. Hrdina, P., A.Bonaccorsi and S.Garattini, 1967, Pharmacological studies on isolated and perfused rat renal arteries, European J. Pharmacol. 1, 99. Kappert, A., 1961, Experimentelle und klinische Erfahrungen mit Complamin bei zerebralen und peripheren Durchblutungsst6rungen, Praxis 34, 861. Konzett, H. and E.Rothlin, 1953, Investigations on the hypotensive effect of the hydrogenated ergot alkaloids, Brit. J. Pharmacol. 8, 201. Lewis, J.J., 1965, Introduction to Pharmacology (Livingstone, Edinburgh). Rothlin, E., 1946, The pharmacology and the nature of dihydrogenated alkaloids of ergot, Bull. Acad. Suisse Sci. Med. 2, 249. Waugh, W.H., 1962, Adrenergic stimulation of depolarised arterial muscle, Circulation Res. 11,264.
COMPARATIVE
BIOASSAY OF VASOACTIVE
USING ISOLATED PERFUSED
DRUGS
RABBIT ARTERIES
J. M. VAN NUETEN Department o[ Pharmacology, Janssen Pharmaceutica, Beerse, Belgium
Received 17 February 1969
Accepted 25 February 1969
J.M.VAN NUETEN, Comparative bioassay of vasoactive drugs using isolated perfused rabbit arteries, European J. Pharmacol. 6 (1969) 286-293. A simple and reliable assay technique for vasoactive drugs is described. Using KCl-depolarised central artery preparations from the rabbit ear, and saphenous artery preparations from the hind leg, the following drugs were assayed for potency, rate of onset and duration of action (in inhibiting ~(Cl-depolarisation-induced vasoconstriction): papaverine hydrochloride, cyclandelate (Cyclospasmolf~, cinnarizine (Stugeron(~, xanthinol niacinate (Complamin ~) and dihydroergotoxine methanesulphonate - DEM - (Hydergin(~. The effects of the drugs on the two types of preparation were similar. For the central artery, potency decreased in the order: cinnarizine > papaverine > DEM > cyclandelate ~ xanthinol niacinate, duration of action in the order: cinnarizine > cyclandelate > xanthinol niacinate > papaverine > DEM, and speed of onset of action in the order: DEM > papaverine > xanthinol niacinate > cyclandelate > cinnarizine. In vitro Perfused arteries Rabbits
Vasodilation Papaverine Cyclandelate
1. INTRODUCTION Isolated perfused vessels have been found useful tools for the study of vasoactive drugs and various assay techniques have already been described (e.g. Waugh, 1962; Carrier and Holland, 1965; De la Lande and Harvey, 1965; Hrdina, Bonaccorsi and Garattini, 1967). The present paper describes a further method of bioassay which was found to be simple, reliable and widely applicable for comparing the potency, duration and mode of action of vasoactive drugs on peripheral arteries.
2. METHODS Vessel segments 2.5-3.5 cm long were removed from the central artery of the ear and the saphenous artery of the hind leg of freshly killed rabbits weighing 2 - 4 kg. The arterial segment was cannulated at
Cinnarizine Xanthinol niacinate Dihydroergotoxine methanesulphonate
the proximal end (internal diameter ca. 1 mm) with a BD 20 G hyperchrome stainless steel hypodermic needle (external diameter 0.9 mm) connected to PE 60 Clay-Adams polyethylene tubing (internal diameter 0.76 mm). The preparation was suspended in a 30 ml constant volume organ bath and maintained vertical by a 50 mg load. The bath contained perfusion fluid at 37°C and was gassed with a 95% 0 2 and 5% CO 2 mixture. 2. I. Apparatus The experimental scheme is shown in fig. 1. Gassed perfusion fluid (95% 0 2 - 5 % CO 2 mixture) was prewarmed to 37°C and delivered to the organ bath at a constant flow rate of 6 ml/min by Tygon tubing (internal diameter 2.38 mm). The flow rate was maintained by a Sigmamotor T 8 peristaltic pump operating at 30 pulses/min. The bath fluid was continuously replaced by the new perfusate, and constant bath volume was maintained by drainage through the overflow.
BIOASSAY OF VASOACTIVE DRUGS
287
Perfusion fluid ---I,¢
0 2 + CO2
Warming __ coil
Recorder--o
~
.
.
0
0
0
0
~--- Amplifier
0
Supplement.ar~ perfulion way
02
Peristaltic pump
Perfusion+ fluid te=t
drug
o
- CO2 . - ~ "91--1=
ii
30 ml O r g l n " bath
il
pill~
preparation
Fig. 1. Apparatus and perfusion scheme used in the assay of vasoactive drugs on rabbit artery preparations.
In contrast to other perfusion techniques (Waugh, 1962; De la Lande and Harvey, 1965; Bonaccorsi and Hrdina, 1966; Hrdina et al., 1967) test drugs were not injected into the main perfusion stream, as direct additions of even small volumes of solution cause transient, but marked, alterations in the perfusion pressure. Instead, test drugs were introduced v/a a small supplementary perfusion tube (PE 20 Clay-Adams polyethylene tubing of internal diameter 0.38 mm) in a total volume of 0.10 ml for a period of 25 see. Afterwards, only perfusion solution was passed through the subsidiary tube. Concentrations of drug solutions were adapted in order to use a volume of 0.10 ml for all the doses. Using this technique, therefore, the rate of fluid delivery was constant throughout the experiments, and any change in perfusion pressure could be reliably attributed to the responses of the preparation.
Variations in the perfusion pressure were monitored continuously by a Statham P 23 Gb pressure transducer and, after boosting with a Brandau DD3 amplifier, the output signal was recorded on a Kipp Micrograph BD or a Heath EUW 20 ME linear recorder. 2.2. Perfusion solutions The perfusion solutions used were (in m moles): normal Tyrode solution (KCI 2.7, CaCl 2 1.8, MgCl2 1.04, NaHCO 3 l l.9, NaH2PO4 0.42, NaCl 136.9, glucose 5.55) and KC1 depolarising solution (KCI 99.83, CaC12 3.59, MgCl2 1.04, NaHCO 3 l l.9, NaH2P04 0.42, NaC1 39.33, glucose 5.55). The pH of both solutions was 7.4-7.6. 2.3. Drugs The following vasoactive drugs were assayed using
288
J.M.VAN NUETEN
the de~olarised arterial preparations: cinnarizine (Stugeron ~, Janssen) in an aqueous solution of 0.1 M tartaric acid; xanthinol niacinate (Complamin®, Wiilfing) from commercially available ampoules (150 mg/ ml); cyclandelate (Cyclospasmol®, Brocades) in polyethylene glycol (PE 200); papaverine hydrochloride in aqueous solution; and dihydroergotoxine methanesulphonate - DEM (Hydergin®, Sandoz) from commercially available ampoules (0.3 mg/ml)*. Solvent controls for each drug were also run. 2.4. Procedure After setting up a blood vessel in the organ bath, it was perfused with normal Tyrode solution for a 30 min equilibration period, during which the sensitivity of the preparation was checked by administration of 0.25 #g (central artery) or 1/ag (saphenous artery) of (-)-norepinephrine. After equilibration, the Tyrode solution was replaced by KCI depolarising solution and a further 30 min period allowed for the development of contraction. Drug solutions were then applied for 25 see in a 0.10 ml volume and the response of the depolarised artery was observed for 90 min. Recording was uninterrupted from the time of setting up the vessel in the organ bath to completion of the drug trial session.
3. RESULTS When a vessel segment was attached to the cannula, the perfusion pressure rose to 100-150 mm Hg, but within a few minutes it decreased again to a constant value of 14--41 mm Hg (mean for 130 central artery preparations = 26 mm Hg, mean for 60 saphenous artery preparations --- 25 mm Hg).
3.1. Response to KCl-depolarisation Depolarisation-indueed vasoconstriction caused a rise in the mean perfusion pressure: it reached a peak after 2 - 3 min, then fell slightly, and thereafter rose again very slowly for the duration of the experiments (fig. 2). The magnitude of the response to depolarisation varied with the site of origin of the vessels: the * A 0.63 mg dose was prepared by dissolving DEM powder in water, but it could not be assayed as the drug tended to precipitate out of solution during the experiments.
I/,0iI ,i
i
120
"~. .
Central artery ..0
-r
E IO0E
i~ Sal)henous artery
g ~.
_1f-
60-
~0It---@--~ 20"
-10
~
10
10
~0
Time after addition of depolarisation solution (min}
Fig. 2. Influence of KCl-depolarisation on the perfusion pressure of isolated arterial preparations. Each point represents the mean value from 130 experiments on the central artery of the rabbit ear, or 60 experiments on the saphenous artery. mean peak perfusion pressure was 141 mm for the central artery segments (range = 6 2 - 2 1 6 mm Hg), but only 89 for the saphenous artery segments (range = 44-155 mm Hg). Although the mean response to depolarisation was triphasic, conforming to the pattern recorded by Bonaccorsi and Hrdina (1966) for the isolated perfused renal artery of the rat, a sizeable percentage of the preparations showed a monophasic or biphasic response to depolarisation, and the percentage was approximately the same whether the segment was taken from the central or from the saphenous artery (fig. 3). 3.2. Response of depolarised central artery preparations to vasoactive drugs All the drugs studied had a relaxing effect upon depolarisation-induced contraction of the central artery (figs. 4-6).
BIOASSAY OF VASOACTIVE DRUGS
289
Bi~a~¢ responu
NoelpklI oc IIiII4II~I
2
Q.
m
j
~& : 10110~ I 7 "/*
io
C . a . • ~ 1 1 1 3 0 • 31.5 %
C.a. • 69/130• 53.1"1.
S.I
S.S. : 3 3 1 6 0
=17160
•
28.3"/.
o 3~ Time after addition of KCI depolarisation solution (min)
1o
• SS.O*/,
;o
~o
3'o
Fig. 3. Stylised response curves of the isolated arterial preparations to KCl-depolarisation. Below each curve is shown the number of preparations tested that responded approximately as indicated: c.a. = centz~J artery, s.a. = saphenous artery.
100-
80
V ~
Papaveri~
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~Cycla~delate
/
O" i 0.0025
'
, 0.01
~ 0.0 L
/
-
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0.16
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2.5
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nigcihIt4
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Fig. 4. Comparison of the potencies of the 5 drugs in inhibiting depolar~tion-induced contraction of the perfused centralartery of the rabbit ear. Each dose was added to the perfusion fluid for 25 sec, the time necessary to deliver 0.I ml of the drug solution. Each point represents the mean peak effect from 6 experiments and the vertical lines represent the standard errors of the means.
290
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Fig. 5. Comparison of the rates of onset of peak action of the 5 drugs, and their durations of action (---time during which effect was ~ 50% of the peak value). Effect measured = inhibition of depolarisation-induced contraction of the perfused central artery of the rabbit ear. Each dose was added to the perfusion fluid for 25 see, the time necessary to deliver 0.1 ml of the drug solution. Each block represents the medium value from 6 experiments.
Papaverine had a potent inhibitory effect at the highest doses tested (figs. 4 and 6). It had a.rapid onset o f action, reaching peak effectiveness in 2 - 3 rain, but its effect was sustained for less than 20 rain (figs. 5 and 6). Cyclandelate had a slower rate o f onset o f inhibitory action than papaverine and was less potent, but it had a longer median duration of action (up to about 35 min).
Cinnarizine had a still slower rate o f onset o f peak action but, when achieved, the peak effect was sustained for a long time, and at doses o f 0.04 and 0.16 mg, it lasted for the duration o f the experiment ( > 90 min). The drug was more potent t h a n both cyclandelate and papaverine, and was active at a dose of only 0.0025 mg. DEM and xanthinol niacinate exerted only moderate and transitory inhibitory effects, even at the
BIOASSAY OF VASOACTIVE D R U G S
291
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Fig. 7. Time-response curves for the 5 drugs tested on the saphenous artery of the rabbit at doses identical to the maximum doses tested on the central artery of the ear (fig. 6). Each dose was added to the perfusion fluid for 25 sec, the time necessary to deliver 0.1 ml of the drug solution. Each point represents the mean value from 6 experiments, and the vertical lines represent the standard errors of the means.
J.M.VAN NUETEN
292
Table 1 Differences in drug responsivenessof depolarised saphenous and central artery preparations.
No. of experiments
Drug and dose (mg)
xanth/nol niacinate papaverine DEM cyclandelate cinnarizine
(40) (0.63) (0.16) (10) (0.16)
Median rate of onset of action (min)
Median duration of action (min)
central artery
saphenous artery
central artery
saphenous artery
2 3 3 25 43
1½ 4 10 20 30
6 10 10 34 >90
3 19 21% 68 >90
6 6 6 6 6
highest doses tested * Solvent controls never caused more than a 5% reduction in the depolarisation-induced contraction. 3.3. Responses of depolarised saphenous artery pre-
parations to vasoactive drugs The effects of the 5 drugs on the depolarised saphenous artery segments (fig. 7) were similar to those obtained with the central artery segments. Comparative results are presented in table 1. Solvent controls never caused more than a 6% reduction in the depolarisation-induced contraction.
4. DISCUSSION The assay technique described proved a reliable method for studying the response of peripheral arteries to vasoactive drugs, permitting quantitative evaluation of the potency, rate of onset and duration of action of vasodilator effects, and comparison of the drug-sensitivity of vessels from different sites of origin. Papaverine and cyclandelate exert their vasodilator effects primarily by direct musculotropic action (Bijlsma et al., 1956; Charlier, 1961; Lewis, 1965; Gifford and Moyer, 1967). Cirmarizine is believed to act on blood vessels by inhibiting selectively the calcium influx into depolarised cells and thereby reducing the availability of free Ca++ ions for the induction and maintenance of contraction (Godfraind, Kaba and Polster, 1968; Godfraind and Polster, 1968; God* See footnote on page 288.
fraind and Kaba, 1969). Xanthinol niacinate also has a musculotropic action, but it acts preferentially on the coronary vessels (Charlier, 1961) and on cardiac muscle (Kappert, 1961), so that the increased rate of blood flow through peripheral vessels in vivo is mainly an indirect effect. DEM also has a predominantly indirect effect upon the peripheral vessels, its action being exerted primarily v/a the vasomotor centre (Rothlin, 1946; Konzett and Rothlin, 1953). In the present experiments cirmarizine was by far the most potent and long-acting of the drugs tested: on the isolated central artery, it was about 4 times as potent as papaverine and about 100 times as potent as cyclandelate, and, at equivalent dosage, its duration of action was at least 9 times that of papaverine and 3 times that of cyclandelate. Further studies, to evaluate the effectiveness of all 5 drugs on the peripheral vascular system in vivo, would seem warranted.
ACKNOWLEDGEMENTS I would like to thank Mr. L.Haagen for technical assistance with the experiments, Dr. A.E.F.Chandler for his help in the preparation of the manuscript, and Sandoz Products Ltd for the gift of Hydexgin®powder.
REFERENCES Bijlsma, U.G., A.B.H.Funcke, H.M.Teersteege, R.F.Rekker,
M.J.E.Emsting and W.T.Nauta, 1956, The pharmacology of Cyclospasmol, Arch. Intern. Pharmacodyn. Ther. 105, 145.
B1OASSAY OF VASOACTIVE DRUGS Bonaccorsi, A. and P.Hrdina, 1966, Interactions between desipramine and sympathomimetic agents on the cardiovascular system, in: Antidepressant Drugs, eds. S.Garattim and M.N.G.Dukes (Excerpta Medica Foundation, Amsterdam) p. 149. Carrier Jr., O. and W.C.Holland, 1965, Supersensitivity in perfused isolated arteries after reserpine, J. Pharmacol. Exptl. Therap. 149,212. Charlier, R., 1961, Coronary Vasodilators (Pergamon Press, Oxford). Dc la Lande, I.S. and LA.Harvey, 1965, A new and sensitive bioassay for catecholamines, J. Pharm. Pharmacol. 17, 589. Gifford Jr., R.W. and J.H.Moyer, 1967, Vasodilator drugs for the treatment of peripheral vascular disturbances, in: Drugs of Choice 1968-1969, ed. W.Model (C.V.Mosby Co., St. Louis) p. 402. Godfraind, T. and A.Kaba, 1969, Blockade or reversal of the contraction induced by calcium and adrenaline in depolarized arterial smooth muscle, Brit. J. Pharmacol., in press. Godfraind, T., A.Kaba and P.Polster, 1968, Differences in sensitivity of arterial smooth muscles to inh~ition of
293
their contractile response to depolarization by potassium, Arch. Intern. Pharmacodyn. Ther. 172,235. Godfraind, T. and P.Polster, 1968, Etude comparative de m6dicaments inhibitant la rdponse contractile de valsseaux isolds d'origine humaine ou animale, Th~rapie 23, 1209. Hrdina, P., A.Bonaccorsi and S.Garattini, 1967, Pharmacological studies on isolated and perfused rat renal arteries, European J. Pharmacol. 1, 99. Kappert, A., 1961, Experimentelle und klinische Erfahrungen mit Complamin bei zerebralen und peripheren Durchblutungsst6rungen, Praxis 34, 861. Konzett, H. and E.Rothlin, 1953, Investigations on the hypotensive effect of the hydrogenated ergot alkaloids, Brit. J. Pharmacol. 8, 201. Lewis, J.J., 1965, Introduction to Pharmacology (Livingstone, Edinburgh). Rothlin, E., 1946, The pharmacology and the nature of dihydrogenated alkaloids of ergot, Bull. Acad. Suisse Sci. Med. 2, 249. Waugh, W.H., 1962, Adrenergic stimulation of depolarised arterial muscle, Circulation Res. 11,264.