Cardiovascular pharmacology: Report of the Main working party

Pharmac Ther Vol 5 pp 63-97 1979 Pergamon Press Ltd

Pnnted m Great Britain

CARDIOVASCULAR PHARMACOLOGY: REPORT OF THE MAIN WORKING PARTY H. BRUNNER (Rapporteur) and F GROSS Oba-Ge~gy Limited. Basle (Swttzerland), and Department of Pharmacology, Unwerslty of Heidelberg. Federal Repubhc of Germany

l

I N T R O D U C T I O N (F Gross, Heidelberg)

Pharmacological m e t h o d s in prechnical studies of drugs serve mainly the purpose of defimng the selectlve action of a new, chemical substance with r e s p e c t to its possible therapeutic use and of understanding the m e c h a m s m of action So far, these m e t h o d s have rarely been a p p h e d to toxicological studies, with the aim to predict what IS generally called drug safety H o w e v e r , as little as therapeutic usefulness can be foretold f r o m the sum of all pharmacological studies, as httle can drug safety be e s t a b h s h e d on the basis of current conventional toxicity testing Any a t t e m p t to I m p r o v e the predictability of results by pharmacological m e t h o d s f o r therapeutac as well as for unwanted effects of a new drug has to be based on the vast e x p e r i e n c e available and on the results of studies that have the aim to relate a d v e r s e reactions of drugs in man with o b s e r v a t i o n s m a d e m the p r e c h m c a l pharmacological analyszs It Is evtdent that the pharmacological m e t h o d s which are used to define and to study the p h a r m a c o d y n a m i c s of a new drug, and especially ltS s e l e c u v e action, cannot be s e p a r a t e d f r o m m e t h o d s that are applied for the safety evaluation This IS especially so in studies on the c a r d i o v a s c u l a r s y s t e m N e a r l y all drugs act on the vascular s y s t e m and m a n y affect the a c t m n of the heart either selectively or, m o r e often, in a non-specific m a n n e r F u r t h e r m o r e , a d v e r s e drug effects on the c a r d m v a s c u l a r s y s t e m h a v e not only to be considered for drugs which are intended to be used m disorders or d~sease of the heart and the peripheral circulation, but also for drugs to be used In n o n - c a r d m v a s c u l a r diseases, such as antibiotics, antldmbetlcs, antlrheumatlcs, psychotrop~c agents and others A m a j o r difficulty In the a s s e s s m e n t of p h a r m a c o i o g | c a l effects which in man m a y cause a d v e r s e reacttons IS the fact that in m o s t cases only results of acute e x p e r i m e n t s In healthy a m m a l s are analyzed, w h e r e a s quite a few undesired effects m a y only o c c u r after repeated or c h r o m c administration Methods to be used for an acute p h a r m a c o d y n a m i c analysis differ m a r k e d l y f r o m those applicable during c h r o m c e x p e r i m e n t s It d e p e n d s on the e x p e r i e n c e and the ~magmation of the individual Investigator which m e t h o d s he thinks will give the valid basis for an a s s e s s m e n t of possible risks or positively for an a s s e s s m e n t of safety In the context of the work of our group, It was n e c e s s a r y to c o n c e n t r a t e on those m e t h o d s which are m use for the prechnIcai evaluation of drug actions which m a y later be considered a d v e r s e r e a c u o n s or s~de effects Of course, drugs foreseen for the t r e a t m e n t of c a r d m v a s c u l a r diseases are studied m much more detail by means of m e t h o d s for the a s s e s s m e n t of a selective action on e~ther the heart, blood pressure, or blood distribution than drugs which are considered for the t r e a t m e n t of nonc a r d i o v a s c u l a r disorders For both groups, h o w e v e L the m e t h o d s will be the s a m e , only the dosages to obtain effects on the cardiovascular s y s t e m are generally h~gher for the non-selective than for the s e l e c t w e actions In man, a d v e r s e reactions in the c a r d i o v a s c u l a r s y s t e m concern mainly the heart (both the m y o c a r d i u m and the s p e c m h z e d tissue) and the resistance vessels In various vascular beds as well as blood pressure regulation in general As a rule, the r e s p o n s e s 63

64

H BRUNNERand F GROSS

of the cardiovascular system to noxious substances, including drugs, are bruited, and similar functional and morphological lestons may occur, irrespective of the ehcltmg mechanisms. It has also to be considered that the sites where these disturbances occur vary according to the individual disposition caused by various factors, which are unknown in most cases and can therefore not be defined The selection of methods is not complete by far, and only some of those available have been chosen and described Such a selection is not free from subjective features, and other experts might have come to different conclusions One of the main criteria for the selection of the methods presented was slmphcity and therefore we have not included procedures which need sophisticated equipment. The results of a pharmacodynamlc analysis of drug activity on the cardiovascular system has the advantage that in general a good correlation exists between results of preclimcal studies and clinical observations, especially with respect to potential adverse reactions

2. H E A RT 2 l

2.2.1

CONTRACTILITY OF H E A R T M U S C L E

In vitro M e t h o d s (A. Truog, Ciba-Geigy Limited, Basle)

There are several m v~tro systems aveulable for determining cardiac contractility. Spontaneously beating heart-muscle preparaUons such as the perfused heart (Langendorff technique), the isolated right atrmm or cardiac-cell cultures are not suitable, since contractile force can only be measured exactly If the rate of contraction is kept constant. Depending on the initial rate, a change m rate may either increase or decrease the contractile force (Fig. I). For details of the complex relation between the rate and the force of contraction see Koch-Weser and Blinks (1963), Hajdu (1969), and Rutlen and Powell (1973) Measurements of contractile force should consequently be made m preparations of cardiac tissue electrically snmulated at a constant rate. The most widely used parts of the heart are the papillary muscle, the left atrmm and atrial or ventncular strips from the cat, the gmnea pig or the rabbit The selection of the appropriate tissue and species depends mainly on the thickness of the muscle preparation, which hmits the oxygen supply Although there are differences between atrial, papillary and ventncular muscles, it is reasonable to concentrate on one of these test systems

2 1 1 1 M e t h o d s a v a d a b l e . Detaded description of the methods m use are gwen by Levy (1971) and by Parmley and Sonnenbhck (1971) The isolated muscle is placed m an organ bath containing an oxygenated buffer solution, e g. Krebs-Henseleit solution

1

j0 OI

J

I

I

I0 Interval,

I I(30

I000

sec

FIG I I n t e r v a l - - f o r c e relation tn isolated guinea pig left atria ( v a l u e s given are .f ~- sf f r o m 5 e x p e r i m e n t s ) Ordinate C o n t r a c t i o n f o r c e In g A b s c i s s a Interval b e t w e e n c o n t r a c t i o n s

Cardiovascular pharmacology

65

gassed with a mixture of 95% O2 and 5% CO2 To ensure metabohc and functional stablhty of the tissue, the bath temperature Is kept at 30-34°C (Pool et al., 1967, Parmley and Sonnenblick, 1971) Special attention should be paid to the electrical stimulation, since the type of electrode and stimulus can influence contractile force To minimize the release of endogenous transmitters (norepinephrme, acetyichohne), point electrodes should be used and the sumuh should be of short duraUon (2-5 msec) and low voltage (10-50% above the threshold value) (Levy, 1967, Jewell and Blinks, 1968) For the papillary muscle a stn'nulataon rate of 6-24 impulses per minute is recommended (Parmley and Sonnenbhck, 1971) For the left atrmm the stimulation rate should be adlusted to the rate of spontaneous contractions observed in the right atrmm of the same species under the same experimental condmons, especially at the same bath temperature Contractile force is best characterized m terms of isometric tension development and the lsotomc force-velocity relation Isometric tension development Is defined by the rate of tension development (dP/dt), the peak developed tension (P) and the time to peak tension ( T T P ) Any two of these parameters define the third, since maximum dP/dt and P / T T P are linearly related (Buccmo et al., 1967) The force velocity relation is determined by the velocity of lSOtOmc shortening at increasing afterloads (Sonnenbhck, 1962)

2 1 1 2. Assessment o f methods and szgmficance of results Prechmcal evaluation of the safety of a drug mainly lmphes the detection of negative lnotroplc effects In this context several questions arise It ~s for instance difficult to say, whether the m vitro system corresponds to a competent or a fading heart It is also not certmn, whether the 'depressed' or the 'normal' myocardlum is more sensmve to negative inotropic effects For the following reasons ~t Is preferable to use the 'normal' myocardmm The absolute degree of the negative effect will be greater m a non-depressed heart muscle Moreover, pretreatment with propranolol or a barbltm'ate usually apphed to reduce myocardial depression may Interfere with the effect of the drug under study However, there exzst more adequate methods to Induce myocardsal depressmn such as the reduction of oxygen supply, or the use of right ventncular papdlary muscles from cats m which heart failure was induced by clipping the pulmonary artery (Spann et al, 1967) Little is known about the relative risks attendant on the different mechanisms inducing negaUve motroplc effects It cannot, for instance, be taken for granted that the myocardial depression caused by the mhlbluon of the slow influx of calcmm Is equal to that elicited by parasympathomtmetscs or membrane-stabihzmg agents. Trendelenburg (1968) and Babulova et al (1973) reported that cocaine and desipramme have negative chronotroplc effects which are masked by posmve chronotropic effects The negative effects only become apparent in tissues depleted of endogenous noreplnephrme by reserpine or 6-OH-dopamme or m the presence of propranolol Similar results can be demonstrated with regard to the motroplc effect of lm~pramme (Fig 2) Two possible mechanisms were discussed the combination of a 'direct' negaUve effect with either a release of endogenous norepmephrme (;r a potentiation of a subthreshold leakage of noreplnephrme It is generally accepted that congestive heart failure m man is accompanied by a depletion of the cardiac catecholamme stores (Chldsey et al., 1965; DeQuattro et a l , 1973) Therefore the predictive value of experiments with drugs which affect either the release or the uptake of catecholammes (e g tncychc antidepressants) can be Improved, ff such drugs are mvesugated both in normal and in catecholamme-depleted ussues Babulova et al (1973) have shown that deslpramme slowly accumulates m isolated rat atria causing a concentration gradient of about I00 to 1 between the tissue and the bath fired Similar concentration gradients between the heart Ussue and the plasma were also observed m rabbits after 1 v administration of various antidepressants (Elonen et a l , 1975) It is evident that such accumulation can interfere wlth the

66

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g

F

GROSS

h~-_

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so =:

BRUNNER a n d

II11_ _11]TJJ.rlTNrlmn-rr -, - _ _

............

I

0

I

I0

I

20

I

30

I

60

mm o f f e r odchhon

P

90

I

120

of =m~promlne

FIG 2 Relat:ve changes In force of contraction after mupramme (I ~g/ml) In ~solated guinea pig left atna C ~ - - - Q No pretreatment 0 - - - 0 Animals pretreated with reserpine. 5 mg/kgi p about 20 hr before the expenment (Values given are .~ from six resp five experiments, corrected for spontaneous changes observed m six and nine control experiments respectively )

determination of negative motroplc effects and with the mterpretauon of results concerning chmcal safety evaluation 2.1.2. I n v a s w e In vlvo Methods (U. M. Bucher, Clba-Gelgy Limited, Basle and H Bahrmann, Firma Goedecke, Fre~berg ~. Br.) For in vtvo experiments the analysis of cardmc performance m terms of cardiac contractd~ty is the more d~fficult the less stable the three other determinants of cardiac performance are kept: heart rate, pretoad and afterioad It should be d~scrimmated between changes in contractde performance resulting from alterations of one or more of the four variables. Dunng m v~tro experiments the determinants of cardiac performance may be kept constant, which is the major advantage of in vitro preparatmns. The complexity of cardiovascular responses m the m vtvo preparation make simple and inexpensive m vttro techmques useful mainly for preliminary testing In vwo experiments provide reformation about the adaptations of the orgamsm to drug effects. Assessment of contractihty ms only possible if several variables are momtored simultaneously" In a closed-chest, anesthetized dog or cat, these have to include heart rate, blood pressure and cardiac output 2.1.2 1 Methods avadable. Most commonly used methods for detecting changes m contracuhty are based on measurements of intravascular and mtracardiac pressures and of cardiac output m dogs and cats Depending upon the particular method chosen, ~t may be necessary to use open-chest techmques Various methods avadable can be apphed to conscious animals If ammals have to be anesthetized, either sodmum pentobarbital or a combinatlon of chloralose, urethane and morphine are recommended 2 1.2 2. Force o f contractzon. D~rect measurement. For th~s most d~rect estimation of the inotropic state a mural strata-gauge arch ms fixed directly on the myocardium. In open-chest, amficlally respirated animals the device to measure msometric contraction ms sewn to the surface of the heart at two points with a segment of myocardium m between. For detads see Felgl et al. (1967) Indirect measurements (see also paper by Burden et al., p. 99). Isovolumetnc phase indices: Indirect estmmtes of the motrop~c state of the ventricles can be obtained from the rate of development of mtraventricular pressure. Left ventncular pressure (LVP) is measured by cardiac catheterization (e.g. by means of a catheter-Up-manometer) Maximum d P / d t can be calculated graphically from high-

Cardiovascular pharmacology

67

speed m t r a v e n t n c u l a r pressure tracings, or it can be recorded directly by a dlfferentinting device Like the rate of tension d e v e l o p m e n t m Isolated preparations, the rate changes of left ventricular pressure reflects fairly well the peak contractde force of the ventricle Besides dPIdtm~ the following radices can be m e a s u r e d m conscious or anestheUzed a m m a l s --(dP/dt)/DP at a d e v e l o p e d pressure of 40 m m Hg --Vpm or m a x i m u m (dP/dt)lP - - V m . based on total pressure --Vm~ b a s e d on d e v e l o p e d pressure

--(dP/dt)HT Ejection phase radices - - s t r o k e volume and stroke work related to L V E D V and L V E D P - - m a x i m u m acceleration of a o r u c blood flow --ejectmn fractmn - - m e a n VcF - - m e a n n o r m a h z e d systohc ejection rate Stroke volume (SV) and cardmc output (CO), are m e a s u r e d by m e a n s of elect r o m a g n e u c or ultrasomc flowmeters m open- or closed-chest a m m a l s or by m e a n s of methods based on Fick's principle (thermo-ddutlon, dye-ddutlon) B~ m e a n s of electromagnetic or ultrasomc flowmeters st ~s possible to m e a s u r e phasic flow Disadvantages include cahbratlon and the r e q m r e m e n t that the flow-probe for the m e a s u r e m e n t of stroke volume be surgically implanted on the root of the aorta As a general r e f e r e n c e for details see Braunwald et al (1976)

2 1.2 3. Assessment of methods and s~gmficance of results Of the various radices used, both m a x i m u m d P / d t and (dP/dt)/DP at a d e v e l o p e d L V P of 40 m m Hg respond to acute changes in contractihty whereas they are relatively m s e n s m v e to changes m preload and afterioad. F o r detads see Mahler et al (1975) M a x i m u m d P / d t generally ~s reached at the u m e of the opening of the s e m d u n a r valves and correlates d~rectly wRh the contractde state of the ventricle Measuring d P / d t at a d e v e l o p e d left ventricular pressure of 40 m m H g has the a d v a n t a g e of relauvely httle variation and ~s insensitive to changes m left v e n t n c u l a r and dmstohc p r e s s u r e (Davldson et al., 1974) If the aortic dmstohc pressure ~s low, ~t ~s preferable to use dP/dt/DP4o, b e c a u s e the s e m d u n a r valves m a y open before p e a k d P / d t is reached The radices are s e n s m v e to changes of heart rate, which influences the contractde state m vtvo To e h m m a t e changes m contractdlty caused by varmt~ons of heart rate, constant pacing Is n e c e s s a r y In the final analysis, cardiac output Is the m o s t important varmble (Fig 3) H o w e v e r , cardmc output Is not always a rehable index of contractihty b e c a u s e it ~s influenced by multiple, peripheral, reflexogemc m e c h a m s m s M e a s u r e m e n t s of stroke v o l u m e (SV), related to changes m left v e n t n c u l a r end-dmstohc pressure ( L V E D P ) or left v e n t n c u l a r end-dlastohc volume ( L V E D V ) m a y be more s e n s m v e p a r a m e t e r s for the a s s e s s m e n t of changes m c o n t r a c u h t y E x p e r i m e n t s wRh instrumented, c o n s c m u s a m m a l s are useful for repeated m e a s u r e m e n t or for studying long acting drugs (Fig 4) The influence of the four d e t e r m i n a n t s of cardmc p e r f o r m a n c e on the various md~ces for contracUhty ~s s u m m a r i z e d m Table 1 T o d e m o n s t r a t e the pred~ctabd~ty of e x p e r i m e n t a l results for chmcal findings on drug-reduced changes of c o n t r a c u h t y various p o s t u r e and negative motrop~c agents were stud~ed D a t a are s u m m a r i z e d m Table 2 WRh r e s p e c t to the usefulness and h m R a t m n s of the various md~ces of contracUhty ~t m a y be concluded that no single index can be considered o p u m a l to assess acute changes m the contractile state F r o m a practical point of view, m a x i m u m d P / d t or (dP/dt)DP~0 are r e c o m m e n d e d

H BRUNNER and F GROSS

68 ~250 F

Aoctlc pressure Rtin

$ec Central venous pressure J

i

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Heart rote

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Phastc aorhc flow i

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Stroke volume Cardiac output (4 sec}

)50r -~

o'-Accelerahon

~' 0 Pertpt~eral reststonce~L

__

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i

FIG 3 Records from an anesthet=zed dog (I 1 3 kg) taken at a fast paper speed during a control period and at a slow speed dunng ventdaUon w=th 2% halothane Aortic pressure, heart rate, phasic aorttc flow, stroke volume, cardtac output, max=mum acceleratmn and peripheral vascular resistance were reduced From R Hughes (1972) The hemodynamlc effects of halothane In dogs Br J Anoesth 45 416 Aortic pressure mmH(J

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(b)After 5 5hr

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FIG 4 Records from an unanestheuzed dog (12 4 kg) taken dunng a control period and at (a) I hr, (b) 5 $ hr, and (c) 24 hr after an oral dose of 4 ms/kg ¢phedrme From R Hughes (1971) Continued measurement of peripheral vascular resistance and ctrculatory function Med Mol Engng 9 603--610

2.1 3 Non-lnvaswe Methods (F Follath and M Framer, Kantonsspttal Basle) The mare advantage of the non-mvastve techmques conststs m their apphcabdlty for repeated studies of the cardiac function in man. Szmultaneous use of the systohc Ume intervals and echocardlography is preferable, smce the two methods are complementaxy by provtdmg data on the tsometnc and elect~on phases of left ventrlcular contxactmn. For the preclimcal evaluatton of new drugs m ammai expertments,

Cardiovascular

69

pharmacology

TABLE 1 Egects Caused by Changes of the Four Determinants of Cardiac Performance lnvaswe methods In v l v o Invastve Darect

lndlrect

peak Heart rate

Increase Decrease Preload Increase Decrease Afterload Increase Decrease Inotropism Positive

dP/dt DP,o

dPldt

cf ~J

?

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CO

1' ? ~

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1' Corresponds to an mcrease on thts parameter Corresponds to a decrease on this parameter N o effect on this parameter cf Contractile force measured by a stram gauge arch

E~ects of Selected Standard Drugs

TABLE

In vitro

I n Vlf)O

Non invaslve ECHO STI

Invaslve

gp atria Dtgox~n lsoproterenol Phentolamme Propranolol Verapamd Pentobarbttal Qmmdme Dtphenylhydantom Procamamzde lmlpramlne

gap muscle

strata gauge

peak dP/dt

dPIdt DP~

CO

Dd

EF +

+

+

44.

44.

+ +

+

+

+

+

+

+

+

+

4-

+

+

+

4"

~~~+/-

~g-

+

+

+

¢+

+ +

--

--

--

-4-

~ -

-

--

--

--

-,t-/--

-

_

_

_

+ / - -

+/-

+/-

+1-

+/-

Vce PEPILVET

--

+1-

+ Corresponds to a poslttve motroplc effect on th,s parameter - Corresponds to a negat,ve motroptc effect on this parameter 0 Not effecttve

however, mvas,ve hemodynamlc recommended

studies are more rehable and should be generally

2 1 3 1. M e t h o d s a v a d a b l e Echocardtography E c h o c a r d i o g r a p h y ts a n o n - i n v a s i v e m e t h o d to s t u d y t h e c a r d t a c f u n c t i o n b y reflected ultrasound The different cardiac structures, including the left venmcular walls, can be recognized Dimensional changes durmg the cardiac cycle are exactly recorded and thus allow estimaUon of end-diastolic and end-systohc volumes (Pombo et al., 1971). In a d d m o n , e j e c t i o n p h a s e r a d i c e s , s u c h a s e j e c t i o n f r a c t i o n ( E F ) a n d v e l o c i t y o f t h e c i r c u m f e r e n t i a l fibre s h o r t e n i n g ( V c e ) , c a n b e c a l c u l a t e d ( C o o p e r et al., 1972). T h e i n c r e a s e o r d e c r e a s e o f t h e l e f t a n t e r o p o s t e n o r v e n m c u l a r d i a m e t e r is a l s o a n m d t c a U o n f o r c h a n g e s m l e f t v e n m c u l a r b l o o d v o l u m e , Le t h e p r e l o a d Echocardtography has already been successfully used m animal experiments and Jr'[

5

|

~ F

H BRUNIqERand F GROSS

70

FIG 5 Example of a left ventncular echocardiogram recorded simultaneously wtth the external carottd pulse tracing m man D, = Endsystohc diameter Dd -- Enddtastohc dmmeter S -- Interventrtcular septum PW -- Left ventncular postertor wall LVET = Left ventncular eJection tune human studies, but further technical advances wdl cert~/nly enlarge the possibilities of thins method (Fig 5) Systolic time intervals (STI) The systolic time intervals provide an appropriate non-invaslve method for the assessment of the left ventncular function (Weissler, 1977) The following intervals are measured (a) The total electromechanlcai systole (QS2) (b) The left ventrlcular ejection time (LVET). (c) The left ventrlcular pre-ejectlon period (PEP), derived by subtracting the left v e n t n c u l a r election time from total electromechanicai systole ( Q S 2 - L V E T ) Systolic time intervals are not only influenced by myocardial contractility, but also by heart rate, preioad and afterload (Ahmed et a l , 1972). QS2, P E P and L V E T are usually rate-corrected according to Welssler et al (1968). The ratio pre-ejection period to left ventrlcular ejection time ( P E P / L V E T ) correlates well with changes in contractility and requires no correction for heart rate m the range of 50-I10 beats per minute. If left ventricular contractility decreases the following pattern of interval changes is seen" pre-electlon period lengthens, (indicating lower d P [ d t ) , left ventncular ejection time shortens (indicating lower stroke volume) and the ratio PEP/LVET Increases, whereas total electromechanlcal systole remains nearly unchanged. Changes obtained by non-mvaslve measurements deviate from normal simultaneously and in the same direction as hemodynamic parameters measured by mvaslve techniques H o w e v e r , they do not have perfect correlations with, e.g stroke volume and election fraction, because they may reflect different parameters, i.e. time periods rather than pressure or TABLE3 Changes m Systohc Ttme intervals Caused by Various Drugs m Man

ECHO

STI

lntervenuon

Dd

EF

Vce

PEP

PEP/LVET

DJsoxm Propranolol Pbenylephrme Phentolamme

~ T 1' ~,

1' $ J, 1'

1' $ ,L 1'

J, T t ~,

J, t ~, ~,

Decrease Increase Symbols ECHO EchocardJography, STI" Systohc tune interval, Dd End dJastohc dmmeter. EF Ejectmn fracUon, VcF Velocity of clrcumferenuai fiber shortemng, PEP Left ventncular pre-elecuon penod, LVET Left ventncular ejecuon ume

Card,ovascular pharmacology

71

TABLE 4 EHects Caused by Changes of the Four Determinants of Cardmc Performance Non-lnvaswe Methods

EC H O Dd

EF

t

Hean rate

Increase

~

Preload

Decrease Increase

I'

Decrease

~

Increase

1'

J,

Decrease Inotroplsm PosRive

~ ~

1' t

Negauve

1'

Afterload

VcF

I' --~

1'

STI PEP LVET

References

~/--. ECHO Roelandt et al (1975). Qumones et al (1976) STI Wezssler et al (1968~ no data ~ ECHO Qumones et al (1976) STI Follath (unpub) 1' ECHO Rankmg et ai (1975) STI Stafford et al 0970) 1' ECHO H,rshleffer et al (1975) STI Salzman et al (1971), Harris et al 0966) ~ ECHO & STI Hardarson et al (1974) ~ ECHO Schmz el al (1977) STI Welssler et al (1970) ~ ECHO & STI Fnshman et al (1975)

Symbols as m Table 3 The same effects measured by mvaslve methods are g.ven m Table I

v o l u m e changes. Changes in systoli~ intervals under different pharmacological mterv e n U o n s are s h o w n in Tables 3 and 4 2 1 4 Conclusions Changes m contractihty o b s e r v e d in experiments on ammals correlate well with the results o f invasive and n o n - m v a s l v e m e a s u r e m e n t s m man For zn vitro experiments, It ~s essential to use Isolated heart preparaUons sUmulated at a constant rate, e.g. left atria or papillary muscle of the g u m e a pig or cat. In vivo, besides direct m e a s u r e m e n t s of contractihty wRh a stram gauge, r e c o m mended techniques to a s s e s s contractility consist m determining dP/dtm, or dP/dt/dP,,o T h e s e m v e s U g a U o n s should preferably be carried out m the dog or the cat, o w i n g to certain peculianUes of the rat, the use of this species Is not to be recommended With agents that affect the a u t o n o m i c n e r v o u s s y s t e m or the m e t a b o h s m of transmitter substances, it m a y be n e c e s s a r y to pretreat the a m m a l s before the experiment, e.g w~th reserpme to deplete n o r e p m e p h n n e stores. In v~tro or in mvo studies of myocardial contractility after repetitive admmlstraUon of the test substance would, on the other hand, presumably only be of s o m e purpose ff the substance or its metabolites accumulated m the heart. At the end o f c h r o m c toxicity studies m dogs, ~t m a y be advisable to record heart rate, blood pressure, dp/dt and cardiac output ~mmedmtely before sacrificmg the a m m a l s In ammals n o n - m v a s l v e m e t h o d s such as echocardlography, systolic time Intervals etc are less suitable than invas~ve techniques

2 2 CARDIAC RHYTHM 2.2 1. D r u g EMects on t h e I m p u l s e G e n e r a t i o n H e a r t (G S c h o l t y s i k , S a n d o z L , m R e d , Basle)

a n d on the C o n d u c t i o n S y s t e m in t h e

A m o n g the drugs cllmcaily used in n o n - c a r d i o v a s c u l a r diseases, psychotroplc drugs have often been described as p r o d u c i n g toxic effects on the specialized m y o c a r d i a l tissue ECG abnormalmes observed m paUents included c h a n g e s m the impulse g e n e r a U o n ( t a c h y c a r d i a , b r a d y c a r d t a , a r r h y t h m l a , c a r d i a c arrest), c h a n g e s in the impulse c o n d u c U o n ( p r o l o n g a t m n o f the P R - m t e r v a l , p r o i o n g a U o n o f the Q R S c o m p l e x , A V - b l o c k , b u n d l e b r a n c h b l o c k , S T - d e p r e s s i o n o r elevataon) a n d c h a n g e s in the r e p o l a r i z a u o n ( p r o l o n g a U o n o f the Q T - m t e r v a l , striking blfid T - w a v e s , n o t c h m g o f

72

H BRUNNER and F GROSS

T-waves, elevation or weakening of T-waves). Psychotroplc drugs which reduce cardiotoxmc effects have different pharmacodynamtc profiles The cardmac effects of psychotropic drugs have been related to" --antichohnergtc effects (Vohra, 1974), ---blockade of norepmephrme reuptake (Barth et al., 1975): ---qumidine-like direct membrane effects (Carmeliet et ai., 1976); or ----changes in the mtracellular-extracellular ratio of potassium (Alvarez-Mena and Frank, 1973). Various pharmacological models have been designed in order to predict cardiac adverse reactions in patients. For psychotropic drugs, these include ECG-momtorlng following drug admintstrauon m rats (Btanchettt, 1977, Marmo et al., 1972), dogs (Dhumma Upakorn and Cobbin, 1975, Kmbbe and George, 1976) and guinea pigs (Dumovic et aL, 1976, 1977), and in vitro studies on isolated cardiac tissue (Landmark, 1971; Carmeliet et al., 1976) or isolated perfused hearts (Barth and Muscholl, 1974, Langslet, 1969). The drug-reduced ECG changes were essentially the same as those seen in patients, namely impairment of mtraventricular conducuon and repolanzauon The underlying basic mechanisms inducing such cardiotoxic effects vary and are not well defined. No standard drug inducing disturbances of heart rate, cardiac rhythm and conduction whsch may be useful as a pharmacological tool for' comparison with new potentially arrhythmogemc drugs can be recommended. Other types of drugs, e.g. anthracychne cytostattcs hke daunomycln (daunorubicin, rubidomycin) or adrlamyctn reduce severe cardiotoxtc effects (Hoff et al., 1977, Mlnow et aL, 1977). Drugs used for the treatment of cardiac diseases may also have undesired effects on impulse generation and the conduction system. Disturbances m the rhythm or conduction induced by cardiac glycosides, anttarrhythmics or drugs interfering with the autonomic nervous system can mostly be interpreted as exaggerated pharmacodynamic effects rather than toxic effects. Some groups of drugs Inducing side effects on the impulse generation and conduction system in the heart are summarized m Table 5. TABLE 5 Drugs Which Induce Cardmc Arrhythrma

(a) Drugs used In non-cardiovascular diseases Psychotropl¢ drugs --Phenothu~nes (Ray and Benson, 1975, Crane. 1970) --Thioxanthenes (Thorstrand, 1975, ABet't, 1976) --Butyrophenones (Pteschl, 1973) --Anndepressants (Burckhardt e t a / , 1976, Brown, 1974) ~Ltthmm (Wellens e t a / , 1975, Crane, 1970) AnestheUcs --.Cyc|opropane (Seuffen and Urbach, 1967) ~Halothane (Elkard and Andersen, 1977) --Ketamme (Lyons et al, 1974) ~Propanlchd (Dlckmann eta/, 1973) Cytostaucs mDaunorublcm (Hoff et al, 1977) --Adnamycm (Ulmer et al, 1977) (b) Drugs used m cardmvascular diseases ~ l ~ t a h s glycosides (Srmth. 1973) ~Anttarrhythnucs (Koch-Weser, 1973) --Sympathomtmettcs (Lelfer and Wttng, 1975)

2.2.1.1. Conclusions. With regard to the generation and conducUon of impulses, it is primarily the funct/onal state of the heart that is decisive for the occurrence of arrhythmias during drug therapy (Koch-Weser, 1973) In testing various substances, ,t may therefore be preferable to use hearts previously damaged, e g by mduced hypoxla or high doses of anesthetics The evaluation of the safety of a drug m animal experiments ts thus only possible to a limited extent In most cases the phar-

Cardiovascular pharmacology

73

m a c o d y n a m m profile ls known and therefore some conclusions on the possible toxmologm risk may be drawn by comparison For various classes of drugs certain effects on the heart may be predmted, e.g local anesthetics, anticholinergtcs, sympathomtmetics Determination of the functional refractory period of the guinea pig a m u m ts suggested as an in vivo method. In vivo E C G tracings may be recorded in various specles and the intervals measured E C G m o m t o r m g is to be recommended during long-term toxicity studles, with a view to detecting changes that only appear after prolonged admmtstrauon of a substance 2.2.2. In Vitro Methods for the Assessment of Arrhythmogenic E~ects of Drugs (E. Heeg, Pharmakologzsches Inst,tut der Umversmtat Braunschwemg) Various drugs mterfere with the formation and conduction of cardiac impulses, gwtng rise to supravenmcular and ventncular premature contractmns, d~sturbances of conduction, bradycardia or tachycardta Some of these substances have already been used for a long ume to produce 'model arrhythmms', e.g. digitalis glycosides, acomtin, thtopentone and chloroform plus haiothane or cyciopropane, together with epmephrme (review m Szekeres and Papp, 1975) There are, however, no specific techmques available for assessmg the arrhythmogemc potential of a given drug A simple, but not always rehable method ~s to momtor heart rate and rhythmicity durmg routme mvestlgaUons of cardiovascular effects (See also Koch-Weser p 125) 2.2 2.1 Methods available One simple screenmg method makes use of the 'pmredstimulation' techmque, devised by Govter (1965) for the determmatlon of the functional refractory period of the myocardmm and modified by Reuter and Heeg (1971) Since atrial and ventncular muscle can react d~fferently to drugs, both atria and papillary muscle are tested simultaneously Oven and Brasch, 1977, Korth, 1975, Scholz, 1969, Washizu, 1972). .t Myocardial preparations in Tyrode's soluUon (see below) at 30°C are sumulated with square-wave impulses (3 msec) at a basra frequency of I Hz The sumulus Impulse contmuously displaces a second impulse of the same mtenslty and duration until there is a distract mcrease m the force of contraction (Figs 6 and 7) The mterval

0 Left otnum

Shmulus lOOms

lOOms

122ms

,°t

Popd tory muscle

0

S,,mulos L.J

k.-J

lOOms

142ms

I

I

280ms

FiG 6 0 n g m a l recordmg from functional refractory period (FRP) determmauons by means of pmred electrical stzmulaUon of the left atrium (upper panel) and papillary muscle (lower panel) The upper curve m each case shows the isometrically measured force of contracuon, the stimulus impulses are shown below Upon paired sUmulatton, no chanse occurs as long as the second s u m u l u s still falls wathm the absolute refractory phase (100 msec in the atnum, 142 msec in the papillary muscle) When the absolute refractory period is exceeded, there is a distinct Increase In the force of contracuon The atrial contracuon curve is not altered, but a small secondary contracuon can be observed In the papillary muscle The FRP of the atrium is always less than half the refractory penod of the papillary muscld m the gumea pig (paper speed 50 mm/sec) Ordinate force of contracuon m g

H

74

,o u:

o

S hmulul

BRUNNER and F GROSS

\

I

L e f t atr, um

L ~ . .

-

t t

ii LJ I00 ms

~_J 122 ms

F,

~_J 122 ms

L.J 122 ms

F2

,°t o

Papdl0ry muscle

lOOms

2BOres

F,

280ms

280ms

F2

FIG 7 As Fig l, hut at slower paper speed to show the steady state F~ = force of contraction after stimulation with single impulses, F: = force of contraction after paired stimuli Ordinate force of contraction in g

Left

atr=um

u . ' i O I0~

K

looms Isopr0terefloI

I

2 6 xlO"Sg/[: IO';'mol/t

1

166ms

Isoproterenol

/

166ms

1

166m$

166ms

2 6 x IO'Sg/l : 10"Tmol./L

30I

o'20

u-" I 0

o

rOOms Isoproterenol

2 6 x l O ' ~ / t s l O "TmO(/t

FIG 8 Effect of tsoproterenol on guinea pig left atrium A c o n c e n t r a t i o n of i 0 -? mol/i h a s a p o s t u r e inotropic effect (F,) After paired stimulation no further ,ncrease in the force of contraction is o b s e r v e d (F2) T h e F R P p a r a m e t e r is the small s e c o n d a r y c o n t r a c u o n Add m o n a l p r e m a t u r e c o n t r a c t i o n s o c c u r ( J, ), which persist e v e n after the s e c o n d impulse h a s been c u t out In this e x p e n m e n t the organ is contracting s p o n t a n e o u s l y T h e external stimuli fall within the r e l a u v e refractory period and only c a u s e m,nlmal contractions (lower curve) Ordinate force of contraction , n g

Cardiovascular pharmacology

75

between the impulses at this point serves as a measure of the functional refractory period (FRP); the greater the intensity of ~he triggering impulse, the closer it Is to the absolute refractory period Stimulation with the five-fold threshold current ts therefore recommended All substances that shorten the FRP are potentmlly arrhythmogemc- among those that do so are norepmephrine, epinephrine and isoprenahne, as well as dlgltahs glycosides m toxic concentrations Popdlory muscle

~°~T ~J IOOms

Control

o°sT ~" o l

~

i

238ms

L_..J

i

238 ms

238 ms

i

Control F2

olo

I:~p,llor y muscle

u. OST " 0 "L

U K-Strophonthm 2 5xlO"ZglL=3 4 x I O ' S m o L I L

/

/

~.°5T Fz

L~-- O " L

170ms

170ms

170ms

170ms

K-Strophonthm 2 5xlO'2g/l=3 4 x I0 -5 m o [ / L

/ o

/

/

/

/

I L.J lOOms K-Strophanthm

2 5x I0"291L= 3 4x l(~mollL

(b) FICIS 9a and b Effect of a toxic concentration (3 4 × 10-5 mol/l) of K - s t r o p h a n t h m on gumea pig papdlary muscle Control Fig 9a AddstJon of K - s t r o p h a n t h m ,s followed by an mltml increase m force of c o n t r a c u o n (F,) After pa~red stlmulatmn premature contractions occur ( ~ ) the FRP ms shortened by comparison w,th the control (2nd hne) Premature contractmns continue even after the second stimulus ss cut out (lower curve)

H

BRUNNER

and

F GROSS

Popdlory muscle

/ uZ

/

/

°°I 0

I

I

I I I 1 159 ms 159 ms Irnloromm IO'3g/L • 3 2 x 10"6mol/l

I I 159ms

FIG 10 Effect o f imlpramlne (3 2 x l0 -6 tool/l) on guinea pig papillary muscle Here. too the second stimulus ,nduces premature contractJons ( J, ) [n this expenment, the Tyrode's solutmn contains double the amount o f calcium

The paired-sumulauon method provides mformatmn on the excltabthty of the myocardium, since the second stimulus impulse acts as an 'ectoplc focus'. If the excitability of the tissue is increased, the second impulse induces extra contracuons, which may continue even after parred stimulation has been stopped. This occurs, for instance, after isoprenaline (Fig. 8), K-strophanthm in a toxic concentraUon (Fig. 9a, 9b) and also imlpramme (Fig. 10). The foregoing method gwes no indication of the mechanism and site of acUon of the test substance; for this purpose, complementary electrophyslologlcal methods have to be employed. Recordings of mtracellularly measured monophaslc actmn potentmls reveal changes occurring in the course of the excitation process A reduced depolarization rate, for example, is always connected with a retardation of lntracardmc conduction velocity (Weldmann, 1974) 2.2.2 2. Concluszon. The deciswe factor for the occurrence of cardiac arrhythmlas during drug therapy ~s the functional state of the heart, in particular with regard to the formaUon and conducUon of Impulses Evaluation of the safety of drugs with regard to the arrhythmogenic potentml of drugs m animals IS consequently only possible to a limited extent. Since the general pharmacodynam~c effects of most compounds of which the safety has to be verified have already been determined in other tests, e g. assays of anticholinergic or adrenerg~c acUvlty, etc., Inferences can often be drawn from comparisons w~th substances known to act m a similar way, provided the comparisons are made m the same species and under ~denUcal conditions It may be advantageous to study the effects of certain substances in animals whose hearts have been predamaged, e.g. by hypoxia or the administration of high doses of barbiturates. It ~s suggested that the most statable in vitro method of assessing the arrhythmogenic potential of drugs is the determination of the functmnal refractory period in the guinea pig atnum or papdlary muscle. In wvo ECG recordings with segmental measurements must be made in dogs or in cats. In this connectmn, It must, however, be borne in mind that even normal dogs can show arrhythmms, especmlly if they are in a state of fear Appendix. Mtxture of the tyrode solutmn.

Na + K+ C a 2+

Mg 2+

CIHCO3HzPO4 Glucose 95% 0 , + 5% CO2

149 3 m Aq/! 2.7 m Aq/l 3 6 m J~q/I 2.1 m Aq/I 145 4 m/~q/! 11.9 m Aq/! 0 4 m Aq/l

2 0 g/!

Cardiovascular pharmacology

2.2.3. Electrocardiographic Studies (R Pharma GmbH, Hannover)

77

Budden and G. Buschmann, Kali-Chemie

In pharmacological experiments it is customary to record an ECG by one lead only, since in most cases the ECG is used to determine heart rate As the shape of the tracing depends on the lead used it is suggested to concentrate ECG evaluation on the intervals rather than on the form of the various complexes 2.2.3.1. Methods available. For a bipolar ECG lead it is preferable to record the signal m the direction of the anatomical heart axis. In anesthetized animals needle electrodes Inserted subcutaneously are used. One electrode ts placed between shoulder blades or at the right side of the neck and the other one at the caudal end of the sternum (Grauwiler, 1965). Polarity should be adjusted to give a positive P wave deflection Two different ECG tracings recorded by this method as well as the mtervals and segments evaluated are shown m Fig 11 As the S-T segment often cannot be defined and under normal recording conditions ts even lacking in the rat (Grauwiler, 1965, Zbmden and Brandle, 1975), tt is replaced by the S a T segment (Hapke et al., 1968) which can readily be determined in all ECG recordmgs of all species tested All ECG Intervals evaluated [P(Q)R and (Q)RT intervals, (Q)RS complex, S a T , and T - P segments] shorten with increasmg heart rate m dogs, cats, guinea pigs, and rats (Fig 12) Despite the different rate ranges the intervals and segments in the ECG of the gumea pig depend upon heart rate similarly as m man In contrast the anestheuzed dog shows a much less shortening of the Q - T segment than man for an equivalent increase in heart rate (Fig 13) It has to borne m mind that arrhythmlas are apt to occur m dogs even under normal circumstances but particularly if the animal Is frightened

(Q)R i ST

mV

Guinea plq

Rot

SaT FIG I 1 ECG of an anesthettzed guinea ptg (above) and a rat (below) (dtgttal-to-analog computer reconstructson) P(Q)R and (Q)RT mterva|s. (Q)R$ complex, and ST, SaT and TP segments are mdscated

78

H BRUNNER and F GROSS 020

QT 015

~

o,o

005

J

t

SoT

J

I

OlS 02 025 R-R mterval Is] I

I

400

03

3~00 I 240 I HeOrt rate [rnIRi]

I 200

FrO 12 Correlatton between R-R interval and other electrocardmgraphlc parameters m anestheuzed guinea plgs (n = 15) Ordinate duraUon of intervals m sec Absctssa R-R intervals and heart rate

(I)/./12) 040Man , / / ~ / / / / 035

/

030 p-

o

025 ~

.

.

.

.

.

.

(4)

O2O Or5 I

04 t

J 300

I

150

I

I

I

I

06 08 R-R interval Is] 1(30 75 Heart rote [mln"~]

I

i0 60

FIo 13 Correlation between R-R mterval and Q-T mterval m man (l 2) and dog (3, 4, 5) (l) Q T = 0 4 0 8 x R R ° ' ~ (own results) (2) Q T = 0 3 9 x R R °5 (Zuckermann, 1959) (3) Q T = 0 2 6 8 x R R ° ~ (own results) (4) Q T = 0 2 1 7 1 7 x R R °'3'~ (Zbmden et ai. 1977) (5) Q T = 0 116+00727x RR°25 (Reuter, 1973) Ordinate QT interval m sec Abscissa RR intervals and heart rate

As most of the intervals and segments wdi shorten considerably at an mcrease m heart rate, drug reduced electrocardiographic changes must be corrected for heart rate. Formulas, which are essentially empirical, have been given mainly for the P-Q and Q- T intervals of man and laboratory animals (Grauwder, 1965, Zuckermann, 1959). Only those ECG changes can be attributed to drug effects wh]ch are more pronounced than effects induced by changes of the heart rate. Because of the rather uncomplicated technique it is recommended to record a one lead ECG not only in acute pharmacological but also m chronic toxicology studies

Cardiovascular pharmacology

79

2.2,3.2. Significance of results.Influences of various drugs in E C G intervals m m a n and some animal specles are given in Table 6. In most cases results are sunilar. As an example the effects of adrzamycin on the ECG of the rat are shown in Figs. 14(a) and (b), which show the influence of a repeated adnamycin administration m rats Cardlotoxlc symptoms are the progresswe wzdening of the QRS complex and the development of an S-wave trough.

I ,.v I

I0 Isec

FIG 14(a) (l) ECG of an untreated rat (2) Same ammal as m (!) ECG recorded after 5 × 4 mg/kg adnamycm i p W,demng of QRS wnh appearance of S-wave trough (3) ECG of a rat treated with $ × 8 mg/kg NSC-16401 marked bradycard~a and ,ntravenmcular block From Bachmann et al (1975) Ozcm~s I00 9o

,,, 8O 0

7o 0

\

~o

i

f

w 50 a,.

,oo

ADP/O

9O 29

I

28

ORS

2T 26 25 24 23 22 20 19 18

Treotments 0

]

2

3

4

5

6

?

8

9

IO

tl

J2

Doys FIo i 4(b) Effect of 4 mg/kg adrlamycm, p on the QRS complex o f the ECG ,n rats Arithmetical mean (± SD) of szx rats From Bachmann et a! (1975)

H BRUNNERand F GRoss

80

T ^ a t ~ 6 E1ects of Drugs on the Electrocardiogram m Man and Laboratory Ammals

Man P-R Drug

interval

Ammai (Q)R-T

(Q)RS interval

Haiopendol

Chlorprornazme Imlpramme Barbiturates Morphine Antthtstammtcs Anthracyclme antlbtotlcs Qumtdme Dtphenyihydantom LIdocame Procaine amlde Propranolol Cardiac glycosides

(P

1'" t °

t"

0" 1'"

0" 0" 1'"

P-R interval

0~ jc 1' c

(Q)R-T

(Q)RS interval 0c ( 1, )c 1'c

1'" 0"

1' ~ 1'~

1`" 1'/( J, )" 0°

1`"

1'•

1' f~

O"

~. "

f"

0"

0"

0h 1,s

1'"

1'"

1' "

0/( 1' )" 1`~

(P 0/( 1`)=.b

( ~,)" ~, =.b

Consczous rat Conscmus rat Conscious rat Dog Conscious rat

1,t, 0h 1,s

Anesth cat Dog Anesth cat Anesth cat

0~

Anesth dog Anesth cat

1`"

fb ( 1' )~

Animal species

0k Or"

1' / ~/0 = prolongation/shortening/unaffected "Surawlcz and Lasseter (1970), bSchellhorn and Kaltenbach (1969). =Zbtnden et al (1977), dBrahm (1971). "Zbmden and Brandle (1975). fHeeg and Reuter (1972), "Reuter (1973). hDIonlagl~ et al (1975). hHaas and Busch (1967). mRothim and Suter 0947)

3 ARTERIAL

B L O O D P R E S S U R E (G. S p o n e r , GmbH, Mannhelm)

C

F

Boehrmger

&

Soehne

In evaluaUng the s a f e t y o f a new drug, direct m e a s u r e m e n t s o f arterial blood p r e s s u r e are n e c e s s a r y T h e species r e c o m m e n d e d f o r this p u r p o s e are the dog, the cat and the rat the first t w o b e c a u s e h e m o d y n a m , c studies c a n also be carried o u t s i m u l t a n e o u s l y or m s e p a r a t e e x p e r i m e n t s tf a n y notable c h a n g e s m b l o o d p r e s s u r e take place, the rat b e c a u s e a great m a n y p h a r m a c o l o g i c a l tests are m a d e m this species and m v e s t l g a t l o n s o f the effects o f a c o m p o u n d on blood p r e s s u r e m the s a m e species could p r o v i d e a basts f o r calculating Its t h e r a p e u u c mdex. With c e r t a m s u b s t a n c e s , e g. drugs that potentiate anesthesia, e x p e r i m e n t s on c o n s c i o u s , trained dogs or on cats or rats with c h r o m c a l l y implanted c a t h e t e r s are useful. Since a n e s t h e s i a impairs reflex blood p r e s s u r e regulation, less m a r k e d c h a n g e s in p r e s s u r e c a n generally be e x p e c t e d to o c c u r m c o n s c i o u s animals than In anesthetlzed ones. F u r t h e r m o r e , a c u t e c h a n g e s o f blood p r e s s u r e have to be distinguished f r o m c h a n g e s w h i c h are o b s e r v e d o n l y after p r o l o n g e d t r e a t m e n t It ts not n e c e s s a r y to use h y p e r t e n s w e animals tn a s s e s s m g the possible effects of s u b s t a n c e s on the blood pressure. N e i t h e r h y p e r t e n s i v e rats nor h y p e r t e n s w e dogs react tn a qualitatively different w a y f r o m n o r m o t e n s l v e a m m a l s . T h e effects o f h y p o t e n s w e drugs are at the m o s t quanUtatively m o r e p o t e n t On the o t h e r hand, the r e a c u o n s to mdtvtdual a n t l h y p e r t e n s w e agents seen m the various f o r m s of expertmental h y p e r t e n s i o n ( s p o n t a n e o u s , renal, D O C A - N a C I h y p e r t e n s i o n ) differ to such an e x t e n t that tt is h a r d l y possible to d r a w a n y c o n c l u s i o n s regarding the side effects the s u b s t a n c e might be apt to p r o d u c e m man f r o m the effects o b s e r v e d m a m o d e l o f hypertension 3 1 METHODS AVAILABLE Studies o f blood p r e s s u r e c h a n g e s aiming to predict a d v e r s e effects in m a n should c o n s i d e r the following points" - - A m m a l species. - - A n e s t h e s i a and m o d e o f a d m i m s t r a U o n . --Method of measuring blood pressure

Cardiovascular pharmacology

81

3.1 1 Animal Species No animal species is known which In all instances has a similar blood pressure response to that observed in man. This holds true for drugs which increase blood pressure as well as those which lower it For example: --Prostaglandm F2~ decreases the blood pressure in cats (Koss and Nakano, 1976), but an increase is seen in man after a single dose ---The blood pressure-increasing action after prolonged administration of carbenoxoione in man is due to its mineralocortlcoid-like activity Therefore, it might only be possible to demonstrate a blood pressure Increase m rats if they were treated over some weeks and loaded simultaneously with sodium chloride - - H o r m o n a l contraceptives, which may produce an increase In blood pressure in healthy women, are without such an effect m experimental animals, even when administered over a prolonged period - - I n acute experiments L-DOPA increases the blood pressure in rats (Henning and Rubenson, 1970) and cats (Brogden et al., 1971) and in high doses also In dogs (Goldberg and Whltsett, 1971), whereas in man postural hypotenslon is observed after prolonged administration Whether this discrepancy is related to differences In the species or the period of treatment, is unsettled so far Such discrepancies between experimental and clinical data make It advisable to investigate the effects of drugs on blood pressure at least in two species, namely m dogs or cats and in rats One of the most important reasons for using rats In blood pressure experiments is the fact that the rat is the preferred species for many pharmacological tests, e.g. in psychopharmacology, endocrinology, or m studies on the antiinflammatory activity. In order to calculate a therapeuUc ratio, it ~s useful to obtam an information on blood pressure changes in the same ammal species as that used for the assessment of the 'therapeutic' action.

3 I 2 Anesthesia and Mode of Admimstratwn It has repeatedly been discussed whether cardiovascular pharmacodynamics should be studied m anesthetized or conscious animals (Vatner and Braunwald, 1975) Anesthesm affects not only the reflex mechanisms but also various parameters monitored with the consequence that the effects on blood pressure may be influenced both quantitauvely and quahtauvely On the other hand, the suppression of reflex mechamsms may result in more distinct effects The most widely used anesthetics in dogs and cats are chloralose-urethane or long-acting barbiturates (e.g. pentobarbltone) In rats urethane or barbiturates are used. The type of anesthetic should be selected according to the type of experiment planned (e.g cardiovascular reflexes are less suppressed with chloralose than with barbiturates). If unanestheUzed dogs are used, they have to be carefully trained in order to avoid erratic responses caused by handling the animal and evoking fear reactions A good mdicator is heart rate, which should be below 90 beats per mm in a well trained dog When studies in conscious rats are planned, it is recommendable to train the animals beforehand Drugs have usually to be administered intravenously by infusion. If administered by stomach or duodenal tube, the results are uncertain m view of incomplete absorption When solubility In water is poor, solvents (e.g polyethylenglycol, 400 DMSO) may be used, but they have a hypotenslve effect by themselves

3 1.3 Measurement of Blood Pressure Direct measurement of blood pressure in anestheuzed dogs or cats is the most statable method for obtaining information about drug effects on blood pressure In contrast, direct measurement of the blood pressure m rats---especially m conscious animalsmis technically more difficult, but ts possible by means of special techniques,

H BRUNNER and F GROSS

82

e.g. measurement with catheters implanted into the aorta (Weeks and Jones, 1960), carotid arteries, or tail artery In general, redirect measurements of blood pressure in rats (Byrom and Wilson, 1938; Breuninger, 1956, Geroid and Tschirky, 1%8; Armah, 1977) are techmcally easy but have the following disadvantages: --DisconUnuous measurement; - - N e e d for heating the ammai (or at least the t=ul), or for a slight anesthesia, --Discrepancies with directly measured blood pressure. The correlation between values obtained by direct or indirect blood pressure measurements m rats has been reported by several investigators. The r-values range from 0.76 (Bunag and Riley, 1971) to 0.98 depending on the number of measurements (Pfeffer et aL, 1971; Sponer et aL, 1974; Armah, 1977). An example of correlation =s given m Fig. 15

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FIG 15 Correlatmnbetween d=rectand md=rectmeasurementof the blood pressure m rats.

By means of the tall cuff method drug effects may not be correctly measured and the results may differ quantitauvely from those obtmned with the direct method (Bunag and Riley, 1974; Proosdij-Hartzema, 1963). A possible source of error can be local vasoconstrsctlon of the tad artery by vasoconstrictors which reduce pulsaUons and flow m the taul.

3 1.3.1 Blood pressure measurement dunng chronic toxicity studies. Since for some drugs changes in blood pressure occur only after prolonged administration, it may be necessary to follow up blood pressure by repeated measurements over several weeks. These measurements of blood pressure should be included in chrome toxicity studies in rats, as well as in dogs In the latter species blood pressure could be measured by a tail cuff method (Prioli and Winbury, 1960; Baum and Rowles, 1969) because this procedure does not induce major stress. An additional possibility for the evaluation of the cardiovascular parameters after chronic treatment is their determination under anesthesm at the end of the toxicological study immediately before killing. At this time some cardiovascular parameters may be monitored, such as blood pressure, heart rate, ECG, left ventricular pressure, dpldt, pulmonary arterial pressure, flow in different vascular beds etc.

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H o w e v e r , the interpretation of such a single set of measurements could be difficult, because the values can be influenced not only by the drug, but b y the depth of anesthesia, the anesthesia chosen, duratwn of preparaUon etc. An indicator for a chronic increase m blood pressure would be the h y p e r t r o p h y of left ventricle found at autopsy 3 1 . 3 . 2 0 r t h o s t a t i c r e a c t w n s . Experiments on animals are not a sattsfactory means of detecting disturbances of orthostatlc blood pressure regulation Orthostatic reacUons are hkely to occur after the administration of substances that interfere with the sympathlco-adrenerglc regulation of total peripheral resistance (a-blockers, mhibltors of post-ganghonlc sympathetic transmission) In such cases, the possible risks attendant on the use of the substances m man can best be estimated on the basis of comparative studies in cats or dogs with k n o w n substances of the same type (See also article on antonomlc pharmacology p. 9).

3.2. SHOCK-LIKE STATES (G. M a n n e s m a n n and B. Mtlller, Schermg A. G., Berhn) In man a shock-hke state characterized by cardiovascular fadure and inadequate peripheral blood supply can be ehctted by different mechamsms - - B y a direct effect of drugs on the cardiovascular system, - - I n d l r e c t l y as a consequence of an overshooting a c u o n of the drug, e.g. e n d o t e x m shock as a c o n s e q u e n c e of a bactericidal action resulting m a destrucUon of large amounts of bacteria: - - B y ehcltmg an anaphylactic or anaphylactold response The risk of elicltmg a shock-hke state by the first two mechanisms may be derived from the p h a r m a c o d y n a m l c profile and the possible consequences which may arise after absolute or relative overdosage, special sensitivity of the paUent, neglection of warnings, etc T h e r e f o r e , addiuonal studies to produce a shock-hke state m experimental ammals which is comparable to chmcal eqmvalents are unnecessary The importance of the anaphylactlc shock as a hfe-threatenmg comphcaUon is evident from lists of adverse reacUons m man in which anaphylactlc shock ranks high Anaphylacuc and anaphylactold responses are m most cases not apparent m preclinical studies and only in exceptional cases may be supposed on the basis of the chemtcal structure Severe reacuons are rare and are related to the individual constitution of the paUent, a factor which is unpredictable so far Three main mechamsms which may be responsible for the o c c u r r e n c e of an anaphylactlc shock-like syndrome are known - - D r u g s act as antigens and when given repeatedly produce an allergic or anaphylacUc response, e.g. antisera, enzymes, peptide hormones, gelatine - - D r u g s act as haptens and are bound to proteins with which they form complete antigens, which produce antibodies After repeated contacts anaphylacUc reactions m a y be p r o v o k e d (analgesics, antiblotacs, anticoagulants, iodine-containing contrast media, local anestheUcs, etc.) - - D r u g s may have a primary anaphylactold action which is caused by degranulatton of mast cells, activation of kmm systems, etc. (antibiotics, some alcalotds, polypepudes, plasma expanders, etc ) In view of the numerous drugs which may cause anaphylacuc reactions It might be desirable to undertake prechnical studies m order to get Information on the anaphylactic or anaphylactold potential In the literature various experimental techniques are described which are considered suitable to detect such a type of drug response H o w e v e r , none of these techniques is used routinely In the evaluation of possible cardiovascular responses By standard cardiovascular methods It is not possible to obtain reformation on the potential of a drug to induce anaphylactlc shock or other allergic or anaphylactold effects

84

H BRUNNERand F GROSS 3.3. CONCLUSIONS

Direct measurements of arterial blood pressure form an essential part of the evaluation of the safety of drugs. These studies should preferably be carried out m dogs and cats or in rats; in the two first mentioned species because hemodynamic parameters can also be investigated In these animals, simultaneously or m separate experiments, If any conspicuous changes in blood pressure should be observed; and in rats because a great many of the routine pharmacological tests are performed In this species so that additional measurements of blood pressure can provide an indication of the therapeutic ratio of a drug. With certain substances, e.g. compounds that potentiate the effects of anesthetics, investigations can appropriately be carried out in conscious trained dogs or in cats or rats with indwelling catheters. Since anesthesia impan's the reflex regulation of blood pressure, changes in blood pressure are generally less marked in conscious than in anesthetized animals. The use of hypertensive animals in testing compounds for effects on blood pressure is not necessary: no qualitative differences have been observed between the responses of hypertensive rats or dogs and those of normotenstve animals. There is no satisfactory animal model available for the detection of disturbances of orthostatic blood pressure regulation, which are likely to be provoked by substances that Interfere with sympathlco-adrenerglc vasomotor regulation (alphablockers, inhlbttors of post-ganglionic sympathetic transmission). With such substances it is recommended that the potential risk to man should be estimated on the basis of comparisons in cats or dogs with known substances of the same type Repetitive measurements of arterial blood pressure during studies of the toxicity of new compounds upon chronic administration appear desirable. Repeated direct measurements cannot be made in conscious animals under these conditions as the dogs or monkeys used in toxicity studies are not specially trained. No experience has been gained with regard to repeated measurements under anesthesia. Single determlnations at the end of a toxicity study in anesthetized animals could provide some information about decrease in blood pressure, whereas chromc elevation of blood pressure should manifest itself in an increase in the relative weight of the left ventricle. Methods for the indirect measurement of arterial pressure m the rat are available. There are, however, considerable individual variations from the values obtained by simultaneous direct measurements, even though the two sets of values correlate well over a relatively wide range of pressures. The methods developed for the indirect measurement of blood pressure in the dog are by no means satisfactory, but it Rs nonetheless desirable that they should b e applied to monitor pressure In chronic experiments. 4. PULMONARY ARTERIAL PRESSURE (J. Schuster, Berlin)

Bundesgesundheitsamt,

In the pulmonary circulation, only an elevation of blood pressure Is to be regarded as a significant adverse reaction. Since such changes are not easily detectable under clinical condiuons and may be ~rreversible, an attempt should be made to obtain some indication of the likelihood of such effects. A rise in pulmonary artery pressure measured in acute experiments is of no or only little predictabdity for the mamfestation of a chronic elevation of blood pressure. Anyhow, it is advisable to include measurement of pulmonary artery pressure in a bemodynamlc analysis of a drug in dogs. However, a more indicative parameter of sustained increase in pulmonary artery pressure is the hypertrophy of the right ventricle observed at autopsy at the end of chronic toxicity studies. It is not possible to conclude from changes in systemic arterial pressure on changes In pulmonary artery pressure. Responses may be similar, • g. nitrates, sodium nitro° prusside, a-adrenoceptor-blocking agents; or opposite, e.g histamine, serotonin, bradykimn, eledotsm, hydralazme, which increase pulmonary artery pressure. Such an Increase is not indicative of a chronic elevaUon of pulmonary pressure.

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4.1. METHODS AVAILABLE For studying the effect of a drug on pulmonary circulation it is necessary to measure continuously pulmonary artery pressure, pulmonary wedge pressure (by means of a Swan-Ganz catheter) which gives information on left atrial pressure, systemic arterial pressure and cardiac output Arterial Po2 and Pc~ have to be kept at normal values, since hypoxla Induces an increase In pulmonary artery pressure In anesthetized animals artificial respiration is recommendable Both chloralose-urethane and pentobarbItal are satisfactory as anesthetics Since the anesthetic may influence the results, It IS preferable to use conscious animals whenever this is technically feasible. Measurement of the parameters mentioned makes it possible to distinguish the causes responsible for changes in pulmonary pressure (e g primary or secondary pulmonary hypertension, mixed pulmonary hypertension, etc ) 4 2. DRUGS WHICH MAY INDUCE PULMONARY HYPERTENSION In acute experiments norepInephrme transiently Increases pulmonary arterial pressure as a function of dose An escape phenomenon can already be observed during the Infusion In chronic studies (rat, s.c InJection) morphological changes in pulmonary blood vessels (edema and proliferation of lntlma and tunlca media may occur (Gunther and Schmld, 1959) Serotonin increases pulmonary arterial pressure as a function of close in acute experiments This effect is transient like that of noreplnephrine In chronic studies (rabbits, dogs) pulmonary sclerosis can be produced (Rossi and Zamboni, 1958, Carlier et a l , 1959) AmInorex increases pulmonary arterial pressure in acute experiments This Increase is not strictly dose-dependent and is long lasting After the end of Infusion the effect on pressure can persist for some time In these experiments, Aminorex causes a greater Increase In pulmonary vascular resistance than In total peripheral resistance (Fig. 16)

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In chronic experiments even after exposure for 1-2 years, only Increases In pressure can be observed which do not reach the extent seen In human beings suffering from Irreversible pulmonary hypertension. Morphological changes may occur (Backmann et al., 1974), but it IS not possible to correlate these changes with the pathogenesis of pulmonary hypertension JPT

5 1 3---~

86

H BRUNNER and F GRoss

In ammals chlorphentermm has similar effects as aminorex. Here, too, It is still open as to whether the observed morphological changes (Backmann et a l , 1974, Parwaresch et a l , 1972) may be regarded as indicators for a pulmonary hypertensive potentml of the drug In man The results quoted show that ammorex fumarate, the sclerogenous effect of which on the pulmonary arteries m man is beyond doubt, differs in its pharmacodynamlc profile both quantltatwely and qualitatively from other mdirectly acting sympathomlmetics or anorecuc agents. However, so far it cannot be stated whether a relationship exists between these acUons and the potentml of ammorex to reduce pulmonary hypertension m man At present no standard methods can be recommended for the study of drugs which may cause chronic pulmonary hypertension 4.3 C O N C L U S I O N S In the pulmonary cn.culaUon the only ~mportant side effect to be watched for IS hypertension In view of the difficulty of detecting increases in pulmonary pressure in pauents and the fact that they may be n.reversible, however, an attempt should be made in the course of every chrome toxlclty study to detect any mdlcaUons of effects on the lesser circulaUon by determining the relative weight of the right ventricle and examining the pulmonary vessels for morphological changes. Hemodynamlc studies m the dog are unlikely to furmsh any addlUonal lnformauon S ORGAN BLOOD FL O W Studies of the blood supply to the organs are designed to provide answers to a variety of questions and are consequently carried out according to different methods which, however, are with few excepUons applicable to all organs One of the most important points that has to be Investigated in evaluating the safety of new compounds is the possiblhty that they may affect the blood supply to a particular organ or vascular bed without otherwise interfering with the systemic cn.culatlon, i.e. assuming that inflow, perfusion pressure and vascular resistance in the organ m question are freely variable These measurements, which ultimately Indicate the influence of the drug on the distribution of cardiac output, have to be made under as nearly physiological conditions as possible In many cases, Individual factors that are involved m the regulation of blood flow to the organs are of special interest and have to be Investigated separately One method frequently applied conszsts in determining the vascular resistance in a given organ or vascular bed (artery to vein or vascular segment only) by recording the perfusion pressure upon perfuslon at a constant volume. Specml techmques have been developed for demonstrating changes m the mlcroclrculaUon, such as the establishment of shunts, or for measuring capillary blood flow Finally, there are methods available for the demonstrataon of changes in the distribution of blood within an organ, i.e. between the epicardmm and endocardmm or the renal cortex and medulla, and for recording locahzed changes in blood flow, e.g. in cn.cumscnbed regions of the brain or the heart. Measurements of blood flow are most often made with the aid of electromagnetic flow meters or indicators (dye or thermal dilution) The clearance substances especially statable for renal function tests fall Into the same category Other methods, e.g those based on the influx or washout curves of inert gases of radioacUve substances (N20, Xe, Th201), the dlstnbutlon of labeled mlcrospheres or local temperature measurements with thermo-elements, are so complicated and then" results so difficult to interpret that their apphcation is limited to the study of special problems. They are not described in any detail here, nor are the techmques for the measurement of capillary blood flow and the distribution of blood within an organ. With regard to changes m organ blood flow, one tends to presume that it is predominantly a decrease that matters, because in extreme cases this will result in

Cardmvascular pharmacology

87

functmnal ~mpazrment It must, however, be borne m mind that increases m blood flow can also produce side effects, e.g flushing, headaches after ddatation of mtercramal vessels or reducuons m the excretory capacity of the kidneys assocmted wRh a drastic lowering of postglomerular resistance and a decrease m the filtratmn fractmn Ddatatmn m intact vascular beds, moreover, can result m an ~mpalrment of blood flow to a post-stenotlc area (hemometakmesm). Such 'steals' always occur when resistance ~s reduced less m the post-stenoUc area than m other areas, and the blood, following the path of least resistance, drams into the healthy area; or when diffuse vasodilatatlon leads to a pronounced reductmn of perfuslon pressure at the stenosls 5.1 PERIPHERAL CIRCULATION (J Schuster, Bundesgesundheltsamt, Berhn) Peripheral circulation refers mainly to blood flow m extremmes, i e skeletal muscle and skin

5 1 1 Methods Avadable Arterial strips By means of arterial strips it is possible to study changes m smooth muscular tone reduced by drugs added to the bathing fired (Pelper et al, 1969, Pelper et al, 1971, Schmld and Pelper, 1972, Trendelenburg, 1974). Isolated perfused hind limbs Perfusmn of isolated hind hmb preparaUons at a constant rate affords a mean of determining changes in vascular reststance (Folkow et al, 1970) Spasmogemc substances increase resistance The alteratmns can be read off d~rectly from a manometer Angtography By mjectmn of a statable contrast medmm into (for example) the abdominal aorta, vasospasms are demonstrable (Lynch et al., 1973) Today, angmgraphy m a m m a l s is not only used for studies of radm-opaque diagnostic medm and vascular reactmns produced by them, but is also a suitable method of demonstrating effects of vasoactlve drugs (Redman, 1974, Diamantopoulos et al, 1975) Direct measurement of regional blood flow, is done by flowmeter (Kohn, 1936. Wetterer, 1937) It Is possible to determine e~ther the flow m the afferent arteries or the venous outflow Simultaneous measurement of the arterial and venous pressure at the sRe where the perfusion is measured allows the calculation of the regional vascular resistance Studies on the influence of vasoddators on the blood supply of post stenotlc areas need specml methods To predict the degree of insufficient perfuslon It is necessary to measure the effect of the resistance of the stenosls and the peripheral regmnal resistance For that reason pressure and perfusmn rate have to be measured proximally and distally to the stenosls 5 1 2 Results The regional perfuslon depends upon the regional vascular reslstance, perfuslon pressure and on the cardiac output The determlnaUon of the cardiac output is necessary to get lnformaUon on the particlpatmn of the vascular action a drug may have for the change m perfuslon of the organ or tissue studied, and for the calculaUon of the total peripheral resistance Examples of drugs which may cause arterial spasms - - E r g o t alcalolds Ergotamlne --Alpha-adrenoreceptor stimulants Im~dazollnes --B~ogemc amines Serotonm --PepUdes Angmtensm II (amide), vasopressm, octapressm Drugs which may worsen inadequate blood supply --Substances w~th papaverme-hke actlvRy (myotroplc spasmolytics)

H BRUNNER and F GRoss

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--N~trates and N~tntes --N~cotlmC acid and derivatives - - Hy d r o ly s a t e s of nucleic acids --Xanthmes --Calcmm antagomsts --Catecholamme denvatwes w~th vasoddatmg actw~ty - - C h o h n e denvaUves --Antlhypertens~ve drugs w~th vasoddatIng actw~ty 5 2 CORONARY CIRCULATION (F

Stroman, Chemlewerk Homburg, Frankfurt/Mare)

There are numerous reports concermng adverse reactions m the form of anginal complaints following drug apphcatlon Antlhypertenslve drugs, dmzoxlde, (Kanada et a l , 1976) or hydralazme (Balazs and Herman, 1976), beta-sympathomlmetlcs (Mancml and Mannino, 1976), contrast medm used in coronary anglography (Oprmn et a l , 1976), ergot alkaloids (Gughelmo et a l , 1976) and cardmc glycosides (Lehmann et a l , 1977) were reported to provoke anginal attacks. Even coronary vasodflators may cause hemometakmesla and their dmgnosUc use m order to verify the coronary ongm of anginal pare was suggested (Tauchert et a l , 1976) All these adverse reactions occur m patients suffering from coronary heart disease Undel" those conditions any increase m myocardml oxygen consumption either by increase m heart rate or in work load as well as any reducUon m oxygen supply (reduced perfuslon, low blood pressure, low Po.,) may provoke an attack Hence, such unwanted effects are predictable In view of these general hemodynamlc effects one may warn against the use of such drugs m coronary heart disease Prechmcal testing of newly developed drugs with respect to their posslble undesired effect on the coronary circulation has to include drugs recommended for the treatment of coronary heart disease itself as well as drugs given simultaneously to a certain percentage of paUents (e g antlhypertenslve agents, antldlabetlcs, anucoagulants, mhlb~tors of platelet aggregation or adhesiveness) In the latter group especially those drugs may be examined which during preceding examination for their cardiovascular acUon have shown effects that might increase myocardml oxygen consumption or decrease oxygen supply 5 2 1 Methods A v a d a b l e

Isolated heart preparations (e g Langendorff-heart) may be useful under special conditions, as when drugs are to be apphed directly into the coronary circulation such as contrast media. But the method gives no information on local blood supply, mchemlc regions, collateral flow, etc Furthermore, effects of neural control and peripheral circulation (after-load) are undetectable An estimate of left ventricular blood flow and oxygen consumption may be done m anesthetized dogs, measuring coronary sinus outflow (Lochner and Oswald, 1964) and the difference m arterial and coronary venous blood oxygen content Such a procedure, however, provides only overall Information and does not inform on lschemia hmlted to certain areas, which ~s assumed to be the most frequent cause of anginal attacks A detatled 'mapping' of local myocardial blood flow even under experimental pathological condmons (local lschemla, collateral blood supply, infarction) can be performed m a m m a l experiments by means of tracer mlcrospheres By using dtfferent isotopes (e g J-125, Ce-141, Cr-51, Sr-85, Nb-95) which may be dlstmgmshed by their gammaspectrum, it is possible to determine flow at certam periods during the same experiment (Schaper et a l , 1973, 1975) Radioactivity is correlated to flow by taking a reference blood sample with constant speed at the moment of m~crosphere mjecaon The advantage of the method ~s the h~gh degree of dlscnmmaUon of local flows which permits a dlfferentmtlon of eplcardmi and endocardial blood flow The dtsad-

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vantages are the restriction to only a few measurements per experiment (due to the hmlted number of available tracers that may be distinguished by their gamma spectrum), the amount of work, t~me and eqmpment necessary for the follow-up analysis A special model for coronary heart disease can be performed m thoracotomlzed dogs If the descending branch of the left coronary artery is perfused through a by-pass from the subclavian or carotid artery it is possible to increase flow resistance by a screw occluder placed at the coronary artery This device imitates the stenosls of a superficml vessel Beyond a crmcal value lschemla will develop which results in typical changes of ECGs, lead by surface electrodes situated at or near the artificmlly perfused area In blood samples taken from an accompanying veto aside of the perfused artery concentrations of Po:, Pco:, pH and lactate differ markedly from those taken from the coronary sinus If the stenosls is released slowly until the various parameters are fmrly normahzed, it may be assumed that in the perfused area capillary resistance is mimmlzed and therefore autoregulatlon suppressed Under these condmons any increase in oxygen demand (e.g by increased heart rate, Fig 17) or any reduction m oxygen supply (e.g hypotenslon) will provoke lschemta with the above mentioned signs If heart rate is increased by atrial pacing (Szekeres et al., 1976) the resulting cardmc tschemm can be reduced or prevented by mtroglycerm or beta-adrenerglc blocking drugs

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FIG 17 Eplcardml ECG records a from center of perfused area, b from borderhne, c from normally supphed myocardmm Increase m heart rate provokes ST-segment changes only in the area where the crmcal stenosls had been estabhshed

This method--like other more sophisticated methodsmhas been widely used in evaluating drugs for expected beneficial effects on coronary circulation It seems to be applicable also for the testing of unwanted effects m coronary heart disease Any results of such studies are not yet known 5.3 CEREBRAL CIRCULATION (J Schuster, Bundesgesundheltsamt, Berhn) Drugs which lower blood pressure may reduce cerebral blood flow Oxygen supply of the brain or of certain cerebral regions may be critical under those conditions

90

H BRUNNER and F Gaoss

5 3 l Methods Available 5 3.1 1 Electromagnetic flowmeter technique Changes m cerebral blood flow are measured by means of an electromagnetic flowmeter placed on either one carotid artery or one vertebral artery (Meyer et al., 1967) This method gives only quahtatlve reformation, since only the flow m one artery contributing to total cerebral blood flow is measured Total cerebral circulation may be quantltatwely determined by simultaneous measurement of the flow in the two internal jugular veins after hgatton of the vertebral, the facml and the external jugular veins (Meyer et al., 1971) The method is time consuming and expensive. Similar informauon is obtained by the venous outflow technique

5 3 1 2. Venous outflow technique (Rapela and Green, 1964). After complete stenosis of the sinus transversus the confluens smuum only contains blood from the lntracerebral vessels. Therefore, continuous monitoring of flow from the confluens smuum mforms on changes of cerebral blood flow (Traystman and Rapela, 1975) 5 3 1 3. The nitrous oxide method. This permits not only the calculation of the total cerebral blood flow, but also the determination of oxygen uptake from cerebral tissue. A review on the method and results obtained with tt for various drugs is given by Gottstem (1962) 5 3 1 4 The mtraarterial isotope clearance This method, was first recommended for Krypton-85 (Ingvar and Lassen, 1962) and allows measurements to be taken of changes in the blood flow not only of the total brain, but also of single brain regions In this way different drug actions m intact and m post-stenotlc parts of vascular beds may be evaluated. Subsequently, Krypton-85 has been replaced by Xenon-133 which has some technical advantages Xenon-133 can be rejected repeatedly into one internal caroUd artery, hence the method has also been called 'multiple bolus method' (Rtsberg and Ingvar, 1973) By means of this method it is possible to detect rapid changes of blood flow, e g. after the admimstrat~on of drugs, and by the use of a suitable detector instrument changes m regional cerebral blood flow (rCBF) can be ascertained 5 3 1 5 Heat clearance (thermal techmques) This method gives quahtative mformation on variations m cerebral blood flow. The cooling of a heated thermocouple is proportional to the blood flow and the difference of the temperature between the heated and a non-heated thermocouple Is measured (Betz, 1968) The thermocouple m form of a sonde can be introduced either into a part of the brain or may be placed m the form of a thin gold-plate electrode on the brain's surface The value of the method ts hmited, since edema formation as a consequence of the cerebral lesions may cause an impairment of the local tissue flow and m this way gwe ~rregular results Furthermore, changes of the permeability of the blood-brain barrier have been observed Quantitative evidence can only be obtained if an mdtrect cahbratlon Is done by a method which gives quantitatwe reformation on the rCBF of the region where the thermocouple is placed This method is only useful if the thermocouple is implanted and repeated measurements over a prolonged period are planned

5.4. RENAL CIRCULATION (B. Scholkens, Hoechst Aktlengesellschaft, Frankfurt/Main, see also Dl6zi and Blallaz p. 135) Measurements of renal blood flow have to be considered in a close relationship with the general hemodynamlc profile of a drug Renal blood flow ts kept relatively constant independently of perfuslon pressure changes over a range between 70 and 200 mm Hg (autoregulatlon)

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For the assessment of drug effects on renal blood flow in prechnlcal safety evaluation, factors such as dietary sodium intake have to be considered (McNay, 1977) 541

Methods Avadable

Total renal blood flow can be measured by either direct or indirect methods The direct techniques include the electromagnetic flowmeter and the Doppler ultrasonic flow transducer, which permits continuous monitoring of renal blood flow in acute studies In anesthetized animals and long term studies in conscious animals Indirect measurements are carried out with the well known clearance methods 542

Results

In consldermg the predictive value of renal blood flow measurements one has to be aware that It is difficult to differentiate between cause and consequence when observing a drug induced change m renal blood flow A change m renal blood flow may be the consequence of either a drug-reduced side effect or a direct drug action With a variety of drugs which can induce acute renal failure m patients, the change in renal blood flow seems to be of secondary nature They may Induce morphological changes at different sites in the kidney--the arteries (dlphenylhydantom, gold salts, pemciihns, thlazldes, sulphonamldes), glomeruli (hydralazlne, phenylbutazone, sulphonamldes), tubules (ammoglycosldes, ferrous sulphate, penicillin, quinine, Sallcylates, paracetarnol aescln) and the Interstztium (sulphonamides, metlucflhn, amplcilhn, rifamplcm) (Curtis, 1977) An example for the possible predictive value of pharmacological measurements of renal blood flow is radlological contrast media, which can cause renal failure in patients. Chnical and experimental studies have shown that the mtravenous injection of iodipammde may produce a marked fall in blood pressure which may contribute to renal failure, though patients with this comphcation do not necessarily exhibit hypotenslon Invection of lodipamide Into the renal artery of cats produces a fall In renal artery flow and edema of both renal cortex and medulla, the capillaries being dilated and filled with closely packed red cells Rapid intravenous Injections also cause edema of the renal cortex and medulla associated with a fall m renal blood flow and arterial blood pressure Slow intravenous injection produced no change in renal histology or renal blood flow (Lmdgren et al., I%6) Measurements of renal blood flow following experimental renal artenography in dogs show a blphaslc response There is a short period of vasodilatlon followed by a more prolonged period of vasoconstriction (Cald~cott et a l , 1970, Nroby and DIBona, 1975, Burgener et al., 1975) With the anUtumor agent mosme-dialdehyde a decrease In renal blood flow manifested as renal tubular damage could be observed in dogs Similar pathologic changes were noted in patients receiving this drug (Kaufman et a l , 1977) Antlhypertenslve drugs which reduce systemic blood pressure by vasodilatlon (e.g dlazoxlde, mmoxidfl, mtroprusslde) may induce a marked fall in systemic blood pressure and renal blood flow Kidneys with severe morphologic lesions may show an impaired capacity for autoregulatlon and renal blood flow may decrease during pressure reduction induced by systemic vasoddatlon (Kaneko et a l , 1%8, Roselio et a l , 1974) The conclusions to be drawn from these observations and considerations for routine evaluation of drugs with respect to prechmcal safety evaluation can be summarized as follows Methods of determmatlon of renal blood flow are not screening methods There is no need for routine determination of mtrarenal blood flow distribution or routme measurements of total renal blood flow for prechnlcal safety evaluation of new drugs The measurement of total renal blood flow in anesthetized animals should be reserved for special drugs according to the following criteria agents whose primary action Is in the cardiovascular field and substances with a primary pharmacodynamic action in another field of therapeutics

92

H BRUNNERand F GROSS

should be tested acutely (as far as possible m connection with a cardiovascular analysis m anesthetized animals) p r o w d e d that they interfere with kidney funcUon after acute administration or chronic routine toxicology evaluation 6 VENOUS SYSTEM The reactions of the venous s y s t e m to a p h a r m a c o d y n a m m agent can be quahtatlvely similar to or o p p o s e d to the changes reduced simultaneously in the arterial circulation If a s u b s t a n c e exerts a v a s o d i l a t m g a c t m n on the arterial clrculataon only (as does h y d r a l a z m e , for example) reflex v a s o c o n s t r i c t i o n ~s reduced in the v e n o u s circulation. S u b s t a n c e s such as nltroprusslde that act simultaneously on both s y s t e m s cause dilation of the veins as well as the arteries. Other substances, such as the nitrates, have a preferential dilating a c t m n on the v e n o u s system. Fundamentally, inadequate reaction of the venous s y s t e m to a reduction in blood pressure is frequently the cause of orthostatic side effects m patmnts, since in a d d m o n to lowering arterial vascular resistance this also dimlmshes venous return to the heart, owing to venous poohng. 6 1 METHODS AVAILABLE Direct effects on veins can best be detected in vitro m isolated v e n o u s strips W h e r e a s / n vivo effects on the arterial s y s t e m are relatively e a s y to d e m o n s t r a t e by p e r f u s m g a vascular bed with a constant volume and recording changes in perfuslon pressure, to determine the capacity of the venous s y s t e m c h a n g e s tn v o l u m e have to be recorded at a constant pressure The only available in vlvo methods are laborious ones (Mellander, 1960) that permit the detection of effects on the venous s y s t e m through p l e t h y s m o g r a p h i c m e a s u r e ments of vascular capacity It is e x t r e m e l y difficult to draw inferences regarding any side effects that might be apt to o c c u r f r o m o b s e r v a t i o n s made in animals, since the c o n d m o n s prevailing in the h u m a n clrculatmn differ f r o m those found m a m m a l s (e g a d a p t a t m n to postural changes) 7 RECOMMENDATIONS The following tests are considered to f o r m an essential part of the evaluaUon of the safety of new drugs with regard to the cardiovascular s y s t e m 7 1 FOR

THE

DETERMINATION OF INOTROPIC ACTIVITY

In vitro

Determination of effects on electrically stimulated atria or papillary muscle of the guinea pig or the cat I n ViVO

Determination of d P / d t ~ the cat or the dog

or dP/dt/dP4o or direct m e a s u r e m e n t s with strain gauge in

7 2 FOR THE DETECTION OF POTENTIAL ARRHYTHMOGENICITY In vitro

Determination of the functional r e f r a c t o r y period (FRP) in atria or papillary muscle of the guinea pig. I n ViVO

Segmental m e a s u r e m e n t s in suitable E C G tracings f r o m cats or dogs

Cardiovascular pharmacology

93

7.3 FOR THE DETERMINATION OF EFFECTS ON BLOOD PRESSURE Acute

D~rect m e a s u r e m e n t s m a n e s t h e U z e d o r c o n s c i o u s d o g s , c a t s o r r a t s Chronic

I f p r a c U e a b l e , b l o o d p r e s s u r e m e a s u r e m e n t s ( p o s s i b l y r e d i r e c t ) , b u t at l e a s t d e t e r m m a U o n of relaUve weights of right and left ventricles. M e a s u r e m e n t s o f o r g a n b l o o d flow a r e n o t n e c e s s a r y , u n l e s s o t h e r t e s t s f u r m s h md~cat~ons to t h e c o n t r a r y In g e n e r a l , an e f f o r t h a s b e e n m a d e to d r a w up a c a t a l o g u e o f p r a c u c a b l e m e t h o d s f r o m w h i c h m v e s u g a t o r s c a n c h o o s e t h o s e m o s t a p p r o p r m t e to t h e g w e n c~rcums t a n c e s W e h a v e p r e f e r r e d n o t to g~ve firm d o s a g e r e c o m m e n d a U o n s , b u t s l m p l y a d v i s e m v e s U g a t o r s to i n c r e a s e t h e d o s e up to t h e p o i n t w h e r e tt b e c o m e s p o s s i b l e to a s s e s s t h e risk b e n e f i t r a t i o In th~s l a t t e r r e s p e c t ~t m a y p r o v e n e c e s s a r y to t e s t t h e w a n t e d t h e r a p e u U c a c U o n a n d u n w a n t e d c a r d i o v a s c u l a r side r e a c t a o n s o f t h e s u b s t a n c e in q u e s t i o n m t h e s a m e s p e c i e s REFERENCES AHMED, S S, LEV1NSON,G E , SCHWARTZ,C J and E'rrir4CER, P O (1972) Systolic time intervals as measures of the contracUle state of the left ventrtcular myocardmm in man C~rculat~on 46 559 ALLERT, M L (1976) Nebenwlrkungen der Psychopharmaka Med Welt 27 572--616 ALVAREZ-MENA,S C and FRANK, M J (1973) Phenothlazme-mduced T-wave abnormalities J A M A 224 1730-1733 AMUE, J P , LESSU~t, S , COLLINS,R F and LANDMMtK,K (1977) Plasma levels and electrophystologtcal effects of acebutoiol in the dog heart m situ Acta Pharmac Toxicol 40 378-388 AMUE, J P , REFSU~ H and LANDMARK,K (1979) Acebutolol-mduced changes in refractonness and monophaslc action potential of the right venmcle of the dog heart m suu Cardiovasc Res (in press) ~ , I B (1977) Experimental hypertension Apparatus for reliable indirect determination of blood pressure m conscious rats Arznelm -Forsch/Drug Res 27(II) 10, 1882-1884 ARMOUR, J A , HAGEMAN,G R and RANDALL,W C (1972) Arrhythmtas induced by local cardiac nerve stamulataon A m J Physlol 223 BABULOVA, A, BAR~.~I, S R, BONACCORSi, A, GARATnNX, S, MORSELLI, P L and PANTAROTTO, C (1973) Correlaraon between desipramme levels and (-)-noradrenalme uptake and chronotroplc effect in isolated atriaof rats Br J Pharmac 48 464--474 BACHMANN, 17 WEBER, E and ZBINDEN, G (1975) Effects of seven anthracychne antibiotics on electrocardlogram and mttochondnal function of rat hearts Agents Actlons 5/4 383-393 BACKMANN, R, HASCIIEMIAN, A, KEMPER, F and MORGENROTH, J (1974) EmfluB yon Anoreklaka auf dle Lunge von Patten In Adlposttas-Krelslau[-Anorektlka,pp 123-129 Verlag Hans Huber, Bern BALAZS, T and HERMAN, E H (1976)Toxlc cardlomyopathles Ann Chn Lab Scz 6 467 BARTH, N , MANNS, M and MUSCHOLL, E 0975) Arrhythmms and mhlbltlon of noradrenalme uptake caused by tncychc antidepressantsand chlorpromazme in the Isolatedpcrfused rabbit heart NaunynSchmtedeberg" s Arch Pharmac 288 215-231 BARTH, N and MUSCHOLL,E (1974) The effects of the tncychc anudepressants despn'amine, doxepm and iprmdole on the isolated perfused rabbit heart Naunyn-Schraiedeberg's Arch Pharmac 2.84 215-232 BAUM,T and ROWLES,G (1969) Drug-induced changes in blood pressure as recorded by direct and redirect methods Arch Int Pharmacodyn 177 179-184 BETZ, E (1968) Measurements of local blood flow by means of heat clearance, In Blood Flow Through Organs and Tissues, p 169, BAIN, W H and HARPER, A M (eds) Livingstone, Edinburgh BETZ, E . 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94

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BUNAG, R D and RILEY, E (1974) Simultaneous measurements m awake rats of drug-induced changes m carotid and tall-cuff systohc pressure J appi Phystol 36 621-624 BURCKHARITr, D , FLEISCHHAUER, H J , MUELLER, V and NEUBAUER, W H (1976) Beltrag zur Wtrkung trlund tetrazykhscher Antldepresslva auf Herz und Krelslauf Schwezz Med Wochenschr 106 18961903 BUROENER, F A , FISCHF~, H W and WEBER, D A (1975) Nlerendurchblutung nach selekuver Injektlon yon verschiedenen Dosen l:hatrlzoat m die Nlerenarterle Fortschr Rontgenstr 123 331-334 BYROM, F and WILSON, C (1938) A plethysmographlc method for measurmg systolic blood pressure m the intact rat J Physlol Lond 93 301-304 CALDICO'I~r, W J H , HOLLENBERG, N K and ABRAMS, H L (1970) Charactensncs of response of renal vascular bed to contrast media Evidence for vasoconstriction induced by reran anglotensm system Invest Radiol $ 539--547 CARLIER, J , LF_JEUNE-LEDANT and KEIL. 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Cardiovascular pharmacology

95

H~I)U, S (1969) M e c h a m s m of the Woodworth staircasephenomenon m heart and skeletalmuscle A m J Phystol 216 206-214 HAPKE, H - J , BUDDEN, R and KONERMANN, H (1968) Herz- und Kretslaufwlrkungen von Kalzmrmnfustonen bet Wtederkauern Deutsch Tlerarzti Wschr 75 2-7 HARDARSON, T , ZIADY, G M and CURII~L, R (1974) The cardiovascular mode of acUon of phentolamme as studted by non-mvaslve methods Arch lnt Pharmacodyn 207 208--219 HARMS, W S , SCHOEN~LD, C D , BROOKS, H and WEISSLVJt, M (1966) Effect of the beta adrenergsc blockade on the hemodynamlc responses to epmephnne m man A m J Cardwl 17 484 HEEG, E and REUTE~, N (1972) Wtrkung yon Almalm, N-Propylajmahn and Chmldm auf Herz and Krelslauf narkotlsterter Katzen Arch Pharmacoi 272 297-306 HEN~ING, M and RU~ENSON, A (1970) Evsdence for a centrally mediated hypertenswe effect of L-DOPA m the rat J Pharm Phannac 22 241 HIRSHLEIFER, J , CRAWFORD, M , O'RoURKE, A and KAmJNER, S (1975) Influence of acute alterations m heart rate and systemic arterial pressure on echocardsographtc measures of left ventncular performance m normal human subjects CIrculatwn $2 835 HOFF, D D yoN, ROZENCWI.:IO, M , LAYARD, M , SLAVIK, M and MUOGIA, F M (1977) Daunomycmreduced cardtotoxtctty m chddren and adults A revtew of 1 I0 cases A m J IVied 62 200-208 INOVAR, D H and LASSEN, N A The blood flow of the cerebral cortex determined by Krypton-85 Acta Phystol Scand $4 325 IVEN, H and BRAS¢I-I, H (1977) Effects of the local anesthetics brufacmn and hdocame on transmembrane actmn potenttais, refractory period and reacUvatmn of the sodmm system m guinea p~g heart muscle Naunyn-Schmledeberg' s Arch Pharmac 297 153--161 JEWELL, B R and BLINKS, J R (1968) Drugs and the mechantcal properUes of heart muscle Ann Rev Pharmac 8 113-130 K^NADA. 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