A Pharmacological. Study of the Ephedrine Isomers

This dissertation has been 64—1295 microfilmed exactly as received

PATIL, Popat Narayan, 1934- A PHARMACOLOGICAL. STUDY OF THE EPHEDRINE ISOMERS.

The Ohio State Universityt Ph,Dt, 1963 Pharmacology

University Microfilms, Inc., Ann Arbor, Michigan A PHARMACOLOGICAL STUDY OF THE HPHEDRIHE ISOMERS

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Popat Narayan Patil, B. Sc. in Phara., M. Sc.

xxxxx

The Ohio S tate University 1963

Approved by

Adi College of Pharmacy ACKNOWLEDGMENTS

The author would Ilka to acknowledge the efforte of Drs. Arthur Tye and Jules B. LaPidus for their valuable advice and Inspiration.

The suggestions of Drs* Harold H. Wolf and John W. Nelson are also acknowledged. Special thanks to Mr* Balkrishna Modi for preparing the isomers and Mr. Dalai for the statistical analysis of the data*

ii TABLE OF CONTENTS

ACKNOWLEDGMENTS ......

LIST OF TABLES......

LIST OF ILLUSTRATIONS ......

LIST OF ABBREVIATIONS ......

CHAPTER

I. INTRODUCTION AND LITEiiATURS REVIEW

G e n e r a l ......

Literature on Optical Isom ers ......

II. MATERIALS AND METHODS

Acute Toxicity ......

Experiments in Dogs ......

Experiments in C a t s ...... ,

Isolated Perfused Rabbit Heart ...... TAIL'S or CONTENTS (Continued)

Page

IV. DISCUSSION

Comparative Potency ...... 13?

Mechanisms of Action of Optical Isomers of Efcihedrine ...... 139

Effect of D (-) Pseudoephadrine Towards Other Pressor A m in es ......

SUMMARY...... 153

3I3LI0GRAPHY...... 153

AUTOBIOGRAPHY...... 166

iv Activity Ratios of Some Optical Isomers ......

Control B.P. and H.R. in (Atropinized) Anesthetized Dogs ......

Control 3.P. and H.R. in Reserpinized Doge . . . ,

Control H.R. of the Isolated Rabbit Heart . . . .

Intravenous Acute Toxicity of the Isomers of %>hedrino in M ice ......

Relative Potencies of the Drugs Derived from Equipressor Doses ......

Epinephrine Equivalents at Equip res so r Doses of the Isomers ...... ■ ,

Duration of Pressor Action of the Isomers in Dogs

B.P. Responses tc D (-) Pseudocphedrine in Anesthetized Dogs ......

Comparative Pressor Potency of D (-) I^hcdrina and D (-) Pseudoephedrine in Spinal Cats . . .

Increase of H.R. by the Isomers (In Dogs) . . . .

Increase in the H.R. to D (-) Pseudoephedrino in Anesthetized Dogs ......

Pressor Effects to the Repeated Doses of Isomers in Dogs ......

Effect of the Isomers on the H.R. and Contractile Force (C.F.) of the Isolated Rabbit Heart , . .

Effect of D (-) Pseudoephedrine on the H.R. and Contractile Force (C.F.) of the Isolated Rabbit Heart ......

Effect of the Isomers on the Coronary Flow of the Isolated Rabbit Heart ...... LIST OF TABLES (Continued)

Page Summary - Approximate Comparative Potencies of the Isomers ...... 65 Effect of Reserpine Pre-treatment on the B.P. Effect of the Isomers and Anqphetandne ...... 73 Effect of Reserpine Pre-treatment on the H.R. E ffect o f the Isomers and Amphetamine ...... 7b Summary of the Control B.P. and H.R. and Effect of Reserpine Pre-treatment on it . . . . 75 Effect of D (-) Pseudoephedrine at the Height of Pressor Responses to D (-) Ephedrlne and Vasopressin in D o g s ...... 77 The Effect of D (-) Pseudoephedrine at the Height of Pressor Response to D (-) Efc>h- edrine in Anesthetized and Spinal C ats ...... 36 Antagonistic Action of D (-) Pseudoephedrine Towards N.M. E ffect o f D ( - ) i^phedrine in Anesthetized Cats ...... 90 Antagonistic Effect of D (-) Pseudoephedrine Against Norepinephrine on the Rabbit Aortic Strip ...... 93 Antagonistic Effect of D (-) Pseudoephedrine Against D (-) Ephedrlne on the Rabbit Aortic Strip ...... 96 Blocking the Pressor Response and I.R. Effect of D (-) Ephedrlne (0 .3 3 mg/kg) by D (-) Pseudoephedrine In Dogs ...... 100 Blocking the Pressor Action ofD ( -) Ephedrlne (0.33 mg/kg) by D (-) Pseudoephedrine in Spinal Cats ...... 103

v i LIST OF TABLES (Continued)

Table Page 23. Blocking the Pressor Response and H.R. of D (-) Ephedrlne, L (+) Ephedrlne, L (+) Pseudo ephedrine, D (+) Amphetamine, by 30 Minutes Pre-treatment of D (-) Pseudoephedrine (3.3 mg/kg) ...... 10h 29. Blood Pressure Response to D (-) Pseudoephedrine in Anesthetized Cats , 109

32. Potentiation of Epienphrine Pressor Effects by D (-) Pseudoephedrine ...... I l l

31. Potentiation of Norepinephrine Pressor Effects by D (-) Pseudoephedrine ...... l l h

32. Effect of D (-) Pseudoephedrine on C.O. Pressor Response ...... 116

33* Effect of D (-) Pseudoephedrine on the Stimulation of the Cervical Sympathe t i c Nerve and N ic tita tin g Membrane (N.M.) . . . 119 3'U Effect of Diphenhydramine (5 mg/kg) on Depressor Responses to Histamine, Iso proterenol, and D (-) Pseudoephedrine .... 123

35* Effect of D.C.I. (10 mg/kg) on the Depressor Response to D (-) Pseudoephedrine (9.9 mg/kg) . 12^

36. Effect of D (-) Pseudoephedrine in Spinal Cats Infused with Norepinephrine ...... 123

37. Effect of D (-) Pseudoephedrine after Hexamethonium C h lo rid e ...... 131

v ii LIST OF ILLUSTRATIONS Figure Page 1. A Typical Experimental Design Used for the Com parative Study of the Isomers in Dogs ...... 21

2. Graph Showing the Determination of the epinephrine Equivalent for L (+) Pseudoephedrine from Experiment Shown in Fig. 1 ...... 24 3. B arb italized Dogs Showing Depressor Response to Graded Doses o f D (-) Pseudoephedrine .... 43

4. Dose Response Curve to Pressor Effects of D (-) Pseudoephedrine in Spinal Cats and D (-) Eph- edrine Equivalent from Graphic Interpolation . . 47 5. Gradual Increase in Average Heart Rate of Seven Anesthetized Dogs to D (-) Pseudoephedrine, 3.3 mg/kg, over a Time Period of 30 Minutes . . . 52 6. Tachyphylaxis to Pressor Effects of D (-) fyhedrine (0.33 mg/kg), L (+) Efchedrine (0.99 mg/kg), D (+) Amphetamine (0.34 ®g/kg), and L (+) Pseudoephedrine (l .65 mg/kg) in Anesthetized Dogs ...... 56 7. Effect of D (-) Pseudoephedrine and D (-) Ephedrlne on the Isolated Rabbit H e a rt ...... 6l 3. Effect of Reserpine Pre-treatment on the B.P. and H.R. Effects of the Isomers in Anesthetized Dogs ...... 67

9. Effect of Reserpine Pre-treatment on the Pressor Effects in Anesthetized D ogs ...... 69 10. Effect of Reserpine Pre-treatment on the H.R. in Anesthetized D o g s ...... 71 11. Antagonistic Effect of D (-) Pseudoephedrine Against the Pressor Effect of D (-) Ephedrlne in Anesthetized D ogs ...... 78

v iil LIST OF ILLUSTRATIONS (Continued) Figure Page

12. Antagonistic Effect of D (-) Pseudoephedrine on the Pressor Effects of D (+) Amphetamine in Anesthetized Dogs ...... 30

13. The Effects of L (+) fyhedrine and L ( + ) Pseudoephedrine at the Height of Pressor Effects of D (-) Ephedrlne in Anesthetized Dogs ...... 32

14. Effect of D (-) Pseudoephedrine Against the Pressor Effects Induced by D (-) Ephedrine and Vasopressin in Reserpinized D ogs ...... 34

15- Antagonistic Effect of D (-) Pseudoephedrine Against D (-) Ephedrine in Spinal C ats ...... 8?

16. Antagonistic Effect of D (-) Pseudoephedrine Against D (-) Ephedrlne Induced Con traction of the Cat N.M ...... 91

17. Antagonistic Effect of D (-) Pseudoephedrine Against Norepinephrine Induced Con traction of the Rabbit Aortic S trip s ...... 9*+

18. Antagonistic Effect of D (-) Pseudoephedrine Against D (-) Ephedrine on Rabbit Aortic Strips 97

19. Reduction of D (-) Ephedrine Induced Pressor Effects by Pre-treatment with Different Doses of D (-) Pseudoephedrine ...... 101

20. Reduction of Pressor Effects of D (-) Sfchedrine, L (+) Ephedrlne, D (+) Amphetamine and L (+) Pseudoephedrine by 30 Minute Pre-treatment with D (-) Pseudoephedrine in Anesthetized Dogs I0j>

21. Effect of D (-) Pseudoephedrine on the Pressor Responses to Epinephrine and Norepinephrine . . 112

22. Effect of D (-) Pseudoephedrine on the C.O. Pressor Responses ...... 117

ix LIST OF ILLUSTRATIONS (Continued)

Figure Page

23. Effect of D (-) Pseudoephedrine on the N.M. Con tractions Induced by Stimulation of Cervical Sympathetic N erve...... 120

2k, Effect of Diphenhydramine on Depressor Responses to D (-) Pseudoephedrine in Anesthetized Dogs YZk

25. Comparative Effects of High Doses of D (-) Pseudoephedrine in Anesthetized Cat, Spinal Cat, and Spinal Cat Infused with Norepinephrine ...... 129

26. D (-) Pseudoephedrine a fte r Hexamethonium .... 132

27. E ffect of Acute ReseiTpine Pre-treatm ent on D (-) Pseudoephedrine (and Vice Versa); Followed by Dibenzyline ...... 135

28. Optical Isomers of Ffchedrlne and Possible Conformations (throu-^i the courtesy of Dr. L a P id u s ...... Ik2

x LIST OF ABBREVIATIONS

The terms and abbreviations given below have been used; the ab breviations therefore have not been explained every time they appear.

B. P...... Blood pressure

C. 0...... Carotid occlusion H. R...... Heart ra te N. M...... N ic tita tin g membrane

(+)» ( - )...... Indicate the actual sign of rotation of a com pound. without reference to its stereochemistry D or L ...... Used to indicate the configuration of a par ticular center in an acyclic compound Corbasll ..... 3» h-dihydroxynorephedrine D. C. I ...... Alpha-(aminomethyl) - 3, *+-dichlorobenzyl alcohol Dopamine ...... Beta-(3»^-dihydroxyphenyl)ethylamine

Epinephrine . . . 3,4-dihydroxy-alpha-( methylaminomethyl)benzyl (Adrenaline) alcohol E p i n i n e ...... b-( 2-methylaminomethyl )pyrocatechol Guanethidine . . . 2-(octahydro-l-axocinyl)ethyl guanidine sulfate Imipramine .... 5-(3-dimethylaminopropyl) -10,11-dihydro-5H- dibena(b,f)asepine hydrochloride Isoproterenol . . Alpha-( isopropylaminoethyl)-3 A-di hydroxy benzyl alcohol Methoxamine . . . Alpha-(l-aminoethyl)-2,5-dimethoxybenzyl alcohol hydrochloride

xi LIST OF ABBREVIATIONS (Continued)

Norepinephrine . . . Alpha~(aminomethyl)-3» ^-dihydroxybeniyl (Noradrenaline) alcohol R italin ...... Methyl-alpha-phenyl-2-piperdine acetate hydrochloride S ynephrine...... p-hydroxy-alpha( methylajninomethyl) benzyl alcohol CHAPTER I

INTRODUCTION AND LITERATURE REVIEW

General

Ephedrlne has bean investigated by many pharmacologists during the last thirty years. The isolation of this alkaloid from Ephedra Vul- garis, variety helvetica Hook et Thongs, was reported as early as 188? by Nagai (1). The drug was used by Chinese for centuries and was known as Ma Huang. The introduction of ephedrlne to the Western Wbrld was brought about by the systematic pharmacological investigations of Chen and Schmidt (2). They described the pressor, cardiotonic, smooth muscle relaxant action of this alkaloid and its clinical usefulness in asthmatic patients. The drug gained in popularity over epinephrine because it is absorbed orally, has a longer duration and is chemically stable. Later, Isolation, constitution and synthesis of ephedrlne and pseudoephedrine were described (3)• A pharmacological comparison of synthetic and natural ephedrlne showed that synthetic ephedrlne pos sessed qualitatively all the characteristics of natural ephedrlne, but in the spinal cat natural ephedrlne was shown to be slightly more active than synthetic ephedrlne (4). The general pharmacology of ephedrlne has been reviewed by Chen and Schmidt (5). It is well docu mented in the standard pharmacology texts (6,7), and therefore only

1 2

important references, which helped to explain the mechanism of action

of ephedrlne, w ill be presented here.

One of th e conclusions which BLaschko e t a l . (8) arriv ed a t as th e

result of in vitro studies on amine oxidase and sympathomimetic amines was that in the presence of ephedrlne, oxidation of adrenaline and

tyrandne is inhibited. Following this, Gaddum and Kwiatkowsld (9)

using an in vivo rabbit ear perfusion technique, put forward a hypo

thesis that ephedrlne is not oxidised by aalne oxidase but inhibits

the destruction of adrenaline by substrate competition. Thus the

action of ephedrlne was attributed to inhibition of aalne oxidase.

However, the theory was subjected to c ritic ism by many. R ichter and

Tingey (10) found practically no amine oxidase in the rabbit ear.

On repeating the in vitro studies, even with higher concentrations of

ephedrlne, the inhibition of amine oxidase was found to be negligible and slow. Chang Shaw Jang (U ) extended the work of Gaddum and

Kwiatkowsld. (9) by using e s se n tia lly the same techniques, and argued against the theory. He found that ephedrlne did not as a rule in

crease the duration of action of adrenaline. Corbasil is not at tacked by amine oxidase in vitro, and according to Gaddum*s theory it should not be potentiated. More firm objection to Gaddum*s theory

came from Brun (12) who used a direct microscopic observation tech nique for studying the vascular effects of adrenaline and ephedrlne in rats. He found that adrenaline Increases the strength and rate of rhythmic contractions of the mesenteric artery, while ephedrlne causes 3 a strong and prompt contraction, Bencs he concluded that ephedrlne might have a direct action.

A number of drugs and surgical procedures hare been utilised by various workers in order to elucidate the mechanism of action of ephedrine. One of such procedures was repeated doses of ephedrlne itself. Originally Chen and Schmidt (2) observed that with repeated doses of ephedrine, there was a gradual dlninuation of pressor re sponse, and tentatively explained this as being due to saturation of receptors by ephedrine. Qaddua and Kwiatkowsld. (9) explained th is phenomenon on the grounds th a t a f te r repeated doses, motor receptors are blocked by ephedrine. Many other explanations have been given fo r th is phenomenon o f tachyphylaxis. Schaumann (13) proposed a

"static action" by ephedrine on blood vessels, an effect that out lasted visible pressor action. Riechert and Quldin (1*0 suggested that failure to respond was concomitant to emptying of blood depots.

A mathematical proof of the theory of receptor saturation, by repeated doses of ephedrine and allied substances, was given by Winder et al.

(15) • The tachyphylactic responses were found to fit with quanti tative precision the hypothesis that they represent successive (more or less overlapping) stages of cumulative normal probability of receptor-point occupation by the agent according to the logarithm of i t s cumulative concentration. Recently the phenomenon o f tachy phylaxis to ephedrine (and allied substances) has been afforded a new interpretation by Cowan et al. (l6). They state the response to tachyphylactic amines is reduced in resezpinised animals and is 4

restored by norepinephrine and that norepinephrine night be the etio logical factor in the tachyphylaxis. Cats were made tachyphylactic by repeated doses of ephedrine (and allied substances), bit nor epinephrine infusion partly restored the response. A similar inter pretation was given by Valette and Masse (17) for ephedrine tachy phylaxis on isolated guinea pig uterus. The organ made insensitive

to ephedrine by pre-treatment with reserpine was reactivated by contact with adrenaline. However, the use of reserpine pre-treatment in explaining the phenomenon of tachyphylaxis was objected to by Moore and Moran (18).

Tainter (19) showed that cocainization of cats and dogs defin itely desensitizes pressor, cardiac and respiratory response to ephedrine. Burn (20) observed that ephedrine administered in doses which dilate the isolated iris of the normal cat's eye, do not dilate the isolated iris of the cat's eye if post-ganglionic sympathetic fibers have degenerated. Similarly, ephedrine did not have a vaso constrictor effect on the vessels of the cat's forelimb if post ganglionic sympathetic denervation has been performed. After these observations, cocainization and denervation became the tools for the classification of sympathomimetic amines. Fleckenstein and Bum (21) studied the effect of sympathomimetic amines after denervation of the nictitating membrane of spinal cats. It was observed that 24 hours after denervation response to all sympathomimetic amines 5 was increased, but thereafter there was a differential response. On this basis, sympathomimetic amines were divided into three groups:

Group A: Substances with not more than one OH group on the

benzene ring and without an OH group on the beta carbon atom. The sensitivity to this group was greatly reduced.

Group B: Substances having no t more than one (XI group on the benzene ring and one OH group on the beta carbon. The sensitivity to this group was moderately reduced.

Group C: Substances having two hydroxyls on the benzene ring.

The sensitivity of this group was increased.

According to this classification, ephedrine was apparently in

Group B. It was suggested that substances in this group might have direct as well as some indirect action. Group A substances were con sidered as acting indirectly through an inhibition of amine oxidase.

Group C substances were considered as d ire c t a c tin g . A somewhat similar study was done by FLeckenstein and Stockle (22) in spinal cats after denervation and cocainization. Their conclusion regarding the action of ephedrine was that it combines catechol-like direct and tyramine-like indirect action. Marley ( 23) studied a number o f sym pathomimetic amines on both the innervated and chronic synpathetic- denervated cat's iris. He classified these amines into three classes as following:

Group A: The phenylethylamines without an OH group, the my. driatlc action of which was lost after denervation. 6

Group B: Substances with an OH on either the phenyl ring or on the beta carbon atom of the side chain. For these the threshold my driatic dose for the denervated iris was usually raised.

Group C: Substances with OH groups in the 3:^ position (with or without a beta CM on the side chain), or with only one OH on the phenyl ring together with an OH on the beta carbon atom. Towards these the denervated iris was oversensitive.

Again ephedrlne was shown to have a mixed action, and i t was sug gested that it mlgjht have direct effect on effector organs as well as indirect effect. Trendelenburg et al.(2h) studied the effect of cocaine, denervation and decentralization on the response of the nictitating membrane to various syrapathomimetic amines in spinal cats. They did not classify the amines into three groups, but concluded that some amines have only direct effects (e.g., nor-epinephrine), some have only indirect effects (tyramine), while others have both direct and indirect action (e.g., ephedrine). Cocaine antagonizes all the indirect effects of tyramine and ephedrine-like amines. The use of reserpine pre-treatment, in elucidating the sym pathomimetic action of ephedrine, is a well studied chapter in its pharmacology. Originally Bum and Rand (25) hypothesized that the action of tyramine, ephedrine and related compounds might be due to release of adrenaline or noradrenaline from the store in the artery wall. This postulation was based on the fact that blood pressure and nictitating membrane responses of spinal cat to these substances were 7

reduced in the reserpinized animal and were reversed by infusion of norepinephrine* Similar reduction of pressor response to ephedrine in anesthetized dogs was observed after reserpinization by Maxwell

et al. (26). They classified the sympathomimetic amines into three groups:

Group A: [ - |0 - /V o r f V

Group B: O - f - OH

Group C: __ o r c - /

After reserpinization the pressor responses to Group A amines were irreversibly blocked. Group B amines were reversibly blocked, and amines in Group C were potentiated or not affected. Ephedrine was classified as a reversibly antagonized substance. A similar classi fication of sympathomimetic amines was made after pre-treatment with

Ritalin (27) and guanethidine (28).

The antidepressant drug imipramine has been studied by Osborne

( 29), who showed that imipramine pre-treatment will diminish the pressor responses to ephedrine and allied substances, but enhances pressor responses to catecholamines. Cairoli et al.(30) found that the positive inotropic action of the ephedrine was diminished but not abolished by reserpine pre-treatment when it was studied on 8 is o la te d p a p illa ry muscle o f c a t. Moore and Moran (18) rep o rted th a t the positive inotropic, chronotropic and pressor responses to ephedrine were not suppressed by acute or chronic pre-treatment with reserpine in anesthetized, open chest dogs. However, reserpine pre-treatment did inhibit or abolish the cardiovascular responses to norephedrine, amphetamine and allied substances. The conclusion from their study is that ephedrine is not dependent for its action upon the integrity of those tissue stores for catecholamines which are depleted by reserpine.

Fleming and Schmidt (31) studied the sensitivity of the isolated rabbit

Ileum to sympathomimetic amines following reserpine pre-treatment.

The action of ephedrine was unchanged but that of tyramine was re duced. Trendelenburg et al. (32) re-investigated the action of sym pathomimetic amines after reserpinization of the spinal cat. The dose response curve for ephedrine was shifted to the right without affect ing its maxima. They concluded that amines of this type have both direct and indirect action, and they seem to represent a gradual transition from the first to the second group rather than a distinct third group. Schuemann and Philippu (33) showed that in vitro ephedrine and allied drugs cause release of adrenaline and noradrenaline from the granules of bovine suprarenal medulla, and this has been also described in vivo (3*0 • The pressor responses of sympathomimetic amines in dogs were studied after dibenamine blockade by Nickerson and Nomaguchl (35).

They observed that pressor response to ephedrine was markedly reduced after pre-treatment with dibenamine but reversal was not seen as with 9 epinephrine. However, dibenamine blocked the oontraction of denervated n ic tita tin g membrane o f c a t. Induced by ephedrlne and a ll o th er sympathomimetic amines. Levy and Ahlquiat ( 36) studied the effect of ephedrlne on the epinephrine reversal after dibenamine. They showed that epinephrine reversal after dibenamine can be abolished by large doses of ephedrlne. All other amines except ephedrlne and methoxamine failed to do this, the displacement of dibenamine from the receptors by ephedrlne is the mechanism suggested by these authors.

Similar studies were carried out by Osswald (37)* According to him, the phenomenon of re -re v e rsa l of epinephrine a f te r dibenamine by ephedrlne might be due to the fact that ephedrine sensitizes those adrenergic receptors which are not occupied by dibenamine.

A differential action of ferrous iron on vascular responses to sympathomimetic amines were studied in dogs by Rajapurkar et al. (38).

Ferrous sulfate (6.8 mg/kg) blocked the pressor responses to all amines tested including ephedrine, except pressor responses to tyramine, vasoxyl and (-) phenylephrine.

Axelrod (39,^0) studied the metabolism of ephedrlne. He observed that In dogs, ephedrlne is rapidly demethylated to norephedrine and hence the action of ephedrlne was attributed by him to norephedrine.

A minor route of metabolism of ephedrlne Involves parahydroxylation of the aromatic nucleus to give parahydroxyephedrine. In rats parahydroxyephedrine is the major route of biotransformation of ephedrlne. Both ephedrlne and norephedrine were found to localise In various tissues. Further, he described an enzyme system in rabbit liver 10 microsomes which catalyses the demethylation of ephedrine to yield norephedrine and formaldehyde.

Literature on Optical Isomers

Cushny (4l) in his Dome lectures reviewed the earlier biological work on optical isomers. He himself first showed the differences in the pharmacological effects of (-) and (+) hyoscyamines. He also stressed the importance of configuration on the pharmacological action of tropenes. In recent years the work of Beckett and Casy (42) has become w ell known in th e f ie ld of a n a lg etics. They mapped the r e . ceptors for analgetics, and shoved that optically active analgetics have a D-configuration. Pfeiffer (43) generalised from a study on optical isomers that the greater the difference between the pharma cological activity of the an timers, the greater is the specificity of the active isomers for the response of the tissue under test. The literature on stereochemical factors in biological activity is re viewed by Beckett (44).

The optical isomers of sympathomimetic amines have been studied by various workers. The approximate (-)/(+) ratio of some amines in different pharmacological tests are summarized in Table I. In all these amines presented in Table I, the (-) isomer was observed to be most active and toxic. The effect of cocaine treatment was found to alter the pressor effect of these isomers as follows:

Drug E ffect a f te r cocaine Reference

(-) Epinephrine Potentiated Tainter (47) (+) Epinephrine Potentiated TABLE I Activity Ratios of Some Optical Isomers

Approximate (-)/(+) Ratio Drug T oxicity B.P, Bronchioles Rabbit Iris References

Epinephrine 20 20 45 5 Hoppe et a l.(45); Bennett et al. (46): Tainter (4?): Luduena et al.(48)

C orbasil 20 160 3000 X Schaumann (49); Luduena e t 31.(46) ~

Isoproterenol 3 300 to 800 (+) ineffec Luduena (50); Luduena et al 6oo tiv e (46); Lands et al.(5l): Bennett et al. (45)

Norepinephi*ine 14 27 70 2 Hoppe e t a l. (45); Luduena et al. (48,52); Bennett et a i . m

Synephrine X 60 XX Tainter and Sidenfold (53) 12

Drug Effect after cocaine Reference

(-) Corbasil Potentiated Schaumann (49) (+) Corbasil Decreased

(-) Norepinephrine Potentiated Luduena et a l.(52) (+) Norepinephrine Potentiated

(-) Synephrine Not potentiated Tainter and (+) Synephrine Not potentiated Sidenfeld (53)

It is apparent that cocainization does not potentiate all the isomers.

No explanation has been given for this phenomenon.

Hanna (5*0 studied tachyphylaxis to the optical Isomers of sym pathomimetic amines on the aortic strips of rabbits and on dog blood pressure. He could not demonstrate tachyphylaxis with any of the iso mers of epinephrine, isoproterenol, corbasil and norepinephrine, even

though qualitative differences were observed. However, optical isomers

of amphetamine and hydroxyamphetamine were observed to be tachyphy lactic and similar in potency. Besides these qualitative differences

in some of these amines, occasionally the least active isomer was found

to be antagonistic to the most active one (50, 53#55)*

Easson and Stedman (56) suggested a hypothesis to explain the

quantitative difference in the antimers of epinephrine. They assumed

that three of the groups linked to the asymmetric carbon atom in an

optically active drug are concerned in its attachment to its specific

receptors in the tissue. In epinephrine, the basic group, the aromatic

group and the alcoholic hydroxyl group are concerned with its attach ment to the receptor. In such compounds the least active isomer

behaves as i f one of the group is missing. This theory has been shown 13 to be in agreement with the Tact that the pressor effects of (+) epinephrine and epinlne are, within the lim its of experimental error, equal* A comparison of the miotic activities of a number of ure thanes has given results which are also consistent with their theory*

HLaschko (57) also pointed o u t th e same th ree p o in t attachm ent o f epinephrine through phenolic, amino and beta alcoholic groups. Fur ther, he stated that the catechol group and the basic group mainly determine the quality of response; and that the hydroxyl group, pro vided it is presented in the right position, mainly determines the intensity of attachment to the receptor. He gave the same examples of (+) epinephrine and epinlne. The work o f Schaumann (58) supports th e above theory. He ob served that (+) corbasil has approximately the activity of desoxy corbasil, and both are l/l60th as active as (-) corbasil. The re sults of Lands et a l.(5l) on antlners of isopropyl noradrenaline also favor the above hypothesis. They found that (+) isopropyl nor adrenaline and desoxy isopropyl noradrenaline differ from (-) iso propyl noradrenaline in somewhat similar degree. But both are much less active than (-) isopropyl noradrenaline. Wilson (59) studied the response of the isolated guinea pig ileum to (-) epinephrine,

(+) epinephrine and epinlne. His results also indicate that (+) epinephrine differs from (-) epinephrine in a similarmanner quanti tatively and qualitatively.

However, the theory of Easson and Stedman (56) was subject to criticism by Badger (60), who collected the data from literature and Ik showed some apparent discrepancies. Pseudoephedrines and pseudo- norephedrines were the typical examples that he quoted.

The ephedrlne molecule has two asymmetric carbons and hence four possible optical isomers are known. Besides D (-) ephedrlne, L (+) pseudo ephedrlne has been known to occur in nature (61). Freudenberg e t a l . ( 62) studied the configuration of ephedrlne and established its relationship to D (-) mandelic acid, and dose (63) investigated the conformation of the ephediines. Upon fusion with urea L (+) pseudoephedrine gave an oxazolidone and (+) ephedrlne gave an imidazolidone. He established that pseudoephedrine is threo and ephedrlne is erythro.

Hence the configuration of the four optical isomers of ephedrlne can be presented as follows:

C-NHCH3 h t h n - c

D (-) ephedrlne L (+) ephedrlne L (+) pseudo- D (-) pseudo ephedrine ephedrlne

Similarly, Pratesi (6k) established the relationship of D (.) mandelic acid for (-) epinephrine and (-) parahydroxy epinephrine.

A number of report3 have been published on the pharmacological aspects of the optical isomers of ephedrlne. Chen et al.(65) observed that the D (-) ephedrlne is the most potent pressor agent as compared to other isomers. Some relative potencies of these isomers are as follows:

Isomer B.P. in spinal cat

D (-) ephedrlne 1 15

Isomer B.P. In spinal cat

L (+) ephedrlne 1/3

L (+) pseudoephedrlne l/5

D (-) pseudoephedrlne l/35

However, L (+) ephedrlne and D (-) pseudoephedrlne did not show pressor effect in man* For mydriasis of rabbit's eye, the potency of these isomers were D (-) ephedrlne > L (+) pseudoephedrlne > L {+) ephedrlne

> D (-) pseudoephedrlne* All four Isomers stimulated virgin guinea pig uterus, but inhibited rabbit intestine In vitro.

Shlmaaoto et q l. ( 66) also compared the potencies of the optical isomers of ephedrlne on various test objects. They used minimum effec tive dose of these isomers, and their results can be summarised as follows:

Drug I.P . ID50 B.P. in Rabbit Guinea P: in Mice Rabbits in testin e auricle D (-) ephedrlne 388 1 1 1

L (+) ephedrlne 367 1 /3 1 /5 1/10

L (+) pseudoephedrlne 221 1 /3 1/3 1/10

D (-) pseudoephedrlne 253 1/15 1/10 > 1/100

Toaoji Tanagita ( 67 ) observed that the toxicity of isomers of ephedrlne was greater at high temperatures. Seiichi Machii (68) also studied the toxicity of D (-) and L (+) ephedrlne and aethyl ephedrines. He found that S, C. LD^q in mouse for D (-) ephedrlne is 1013 mg/kg and 1269 mg/kg for that o f L (+) ephedrlne, Ephedrlne and aethyl ephedrlne 16 were rapidly excreted in urine. No difference was observed among the

isomer8 in the excretion rate.

De Jongh (69) found that isomers of ephedrine showed different

effects on the isolated rat uterus. It was inhibited by D (-),

L (+) and L (+) pseudoephedrlne, but it was not influenced by D (-) pseudoephedrlne. The isolated rabbit gut was inhibited by D (-) ephedrine and was activated by L (+) ephedrlne and L (-?-) pseudoephedrine. Swanson and Webster (70) studied the action of D (-) ephedrine and L (+) pseudoephedrlne on the bronchiolar smooth muscle of the isolated lung. They suggested that epinephrine is bronchioneurotropic and D (-) ephedrine has both bronchioneurotropic action as well as musculotropic, but L (+) pseudoephedrlne is less bronchioneurotropic and more musculotropic. Avlado et al. (71) also re ported on the bronchiodilator action of D (-) ephedrine and L (+) pseudoephedrlne, and found it to be of the same intensity for both isomers. Vascular and renal effects of the ephedrlnes were investi gated by Light et al. (72). They observed that D (-) ephedrine reduced blood supply to all vascular beds studied in dogs, but D (-) pseudoephedrlne produced vaso-dilatation. Renal and vertebral ar terial flows were increased, and the carotid flow was decreased by

L (+) ephedrine and L (+) pseudoephedrlne. In rats, diuretic action was very prompt with L (+) ephedrine, while D (-) ephedrine was inactive. D (-) pseudoephedrlne had a delayed type of action of diuretic action. 17

Anderson and Chen (73) reported the hyperglycemic action of the ephedrlnes in the rabbit after I.V. of 1 cc of M/20 solution. The results are as follows;

Maximum Rise of Blood Sugar Time to reach peak Drug mg/100 cc of blood , (min.)

D (-) ephedrine 13 10

L (+) ephedrine 25 40

L (+) pseudoephedrine 20 30

D (-) pseudoephedrine 30 40

The central effects of some of these Isomers was investigated t}y Trevan

(7*0, who claimed that D (-) ephedrine is probably more active than

D (-) pseudoephedrine, but that both are much less active than (+) amphetamine.

Elaschko (75) investigated the amine oxidase inhibitory properties of the ephedrines in extracts of guinea pig liver. The results are shown below:

Drug Per cent in h ib itio n

D (-) ephedrine 49

L (+) ephedrine 30

L (+) pseudoephedrine 32

D (-) pseudoephedrine was not investigated. Swanson et al. ( 76 ) com pared pressor effects of the optical Isomers of epinephrine, desoxy ephedrine, benzedrine, isobenzedrine and norephedrine in pithed dogs.

Recently Ciuchta and Mann (77) studied the effect of D (-) ephedrine and L (+) ephedrine on the norepinephrine tachyphylaxis of 18

the venae heart. The conclusion of their study ie that D (-) ephedrine is the most potent antagonist of norepinephrine tachyphylaxis, while

L (+) ephedrine is the least, suggesting that ephedrine could block norepinephrine by acting at the same site on the receptor.

A review of the literature reveals that in recent years, parti,

cularly since the work of Bum and Rand (25), there has been a renewed interest in the mechanism of action of sympathomimetic substances.

Die effects of configuration on the physiological effects of isomers has also become a subject of considerable Interest since Beckett made his classical observations on analgetics (h 2).

Since ephedrine is a classic sympathomimetic drug and since the absolute configurations of the four ephedrine isomers are well esta blished, we felt that we had at hand a unique opportunity for investi gating the effects of configuration and conformation on the activity of a group of sympathomimetic agents.

Firstly, a systematic pharmacological study of the activity in various tissues of all four isomers was planned. Secondly, the ef fects of reserpine pre-treatment on all four isomers was to be investi gated because reserpine has been used as a tool to determine direct and indirect actions in a number of sympathomimetic agents* Thirdly, the finding of LaPidus et al.(78) that D (-) pseudoephedrine blocks the effects of D (-) ephedrine was to be further studied. Finally, an attempt was to be made to relate the pharmacological effects obtained to the configuration and possible conformations of the various isomers. CHAPTER I I

MATERIALS AND METHODS

The methods used in the study ere standard ones. They are de

scribed in detail in the references.

(1) Acute toxicity

The coiqjarative LD^ values of these isomers were obtained in white

Swiss female micet according to the method described fay Litchfield and

V&lcoxon (79)* The mice, weighing 16 to 21 grams, were starved for

six to ten hours, then isolated, and toxicity was determined intra

venously. The drug volume injected did not exceed 0.25 cc per mouse.

After the preliminary dose finding in five to ten mice, a total of 30

to **0 mice were used for each LD^ determination. The observation period for the incidence of death was 2h hours.

(2) Experiments in dogs

Study involves a total of 130 mongrel dogs of which h -6 per cent were

male. They were selected at random and weighed between 7 to 12 kilos.

This study in dogs is divided into three groups:

(a) Comparative study — The following parameters were esta

blish ed :

1. The equipressor doses of the isomers using epine

phrine as a standard.

19 20

2 . The coaparative duration of action of equipressor doses*

3 . the increase in heart rate at equipressor doses.

£*-. Hie relative tachyphylactic tendencies*

3* The ability of the isomers to potentiate epinephrine and

norepinephrine.

6 . Gross changes in respiration.

Dogs an esth etized w ith b a rb ita l sodium, 330 mg/kg ( I .P .) were used

(80). Atropine 1 mg/kg I.V. was given in order to avoid reflex brady cardia arising from pressor responses (in addition, bilateral vagotomy was performed). The right carotid artezy was cannulated and blood pressure recorded through a mercury manometer on the kymograph with the usual haemodynamic set-up. Hie femoral vein was cannulated for injection of drugs. Heart rate was recorded on a Sanborn twin viso recorder. Respiration was recorded on the kymograph through a can nulated trachea and a tambour. When the preparation became stable, the following drugs were given.

A fixed dose of norepinephrine was given, and after 10 minutes graded doses of epinephrine were given in descending scale ( 1 5 , 65, 8 0 ) a t 10 to 15 minutes in te rv a l. Then a chosen dose of isomer was given

15 minutes after the last injection of epinephrine. When the pressor response to the isomer subsided, epinephrine or norepinephrine or both were rep eated . Fig. No. 1 shows a ty p ic a l experim ent. E ffects o f epinephrine on blood pressure were subsequently plotted against the logarithm of the dose, and the epinephrine equivalent of the test FIGURE 1

A typical experimental design used for the comparative study of the isomers in dogs.

Upper tracin g - Blood pressure Lower tracing - R espiration Heart rate (beats/mln.) indicated by number above the blood pressure tracing a*—*b - - - Latent period for pressor response b«—-*c - - - Duration of pressor effect EPI =* Epinephrine (Drum was stopped for ten minutes between epinephrine injections) NOREPI = Norepinephrine Time Marker - 10 minutes

21 N O R FPL tPHEDPINF —J 0(-}ER-eCRlNE Odnyg.'^ ’bb mg/kg Q.33mgAg,

I if ,!(■ I.-i |

RESPIRATION 23 isomer was determined by interpolation (Fig. No. 2). Finally, com parative pressor potencies vere expressed in terms of epinephrlne- equivalents, as well as doses necessary for equipotent pressor action.

A minimum of four dogs were used for each conpound. Duration of pressor effect was measured in terms of time from the peak of the pressure rise to the point at which half recovery had occurred; a sort of half life of effect chosen to avoid vagueness. In the case of prolonged actions a great delay entailed during "complete" recovery

(15,80). The average o f the mean B.P. was 125 (S.D.+ 25), and the average heart rate was 155 (S.D.+ 33) beats per minute in controls.

The readings are shown in Table II. To study tachyphylaxis, the same dose of the isomer was repeated 30 minutes after the first dose; then subsequent doses were repeated whenever the pressor response to the previous dose levelled off.

(b) Reseroinlzed dogs — In order to study the effect of reserpinization on these Isomers, a procedure similar to that described by

Maxwell and co-workers ( 26) was follow ed. Dogs were given 1 mg/kg o f reserpine (S.C.) 20 to 2h hours previously, and pentobarbital (Nem butal) 10 to 20 mg/kg was used to an esth etize such anim als. Subse quently anesthetic was administered as needed for surgical anesthesia.

All other procedures were similar to those just described, except that respiration was not recorded though the trachea was cannulated. The average mean B.P. was 77 (S.D.+ 20); H.R. was 100 (S.D.+ 18) in con trol readings. The readings are sumarixed in Table III. FIGURE 2

Graph showing the determination of the epinephrine equivalent for

L (+) pseudoephedrine from experiment shown in Fig. 1.

2b 25

B p DOG: E(14) ( rmmHg) 100

80 U*)y EPHEDRINE- 1 65 mg/kg-

4 0 -

0.5 4 3 EPINEPHR | NE( mcg/kg) 26

TAELS I I Control B. P. and R. R. in (Atropinited) Anesthetized Dogs

Mean B.P. Heart Rate Obs. No. Dog No.* (d m . Hg) (beats per minute)

1 B (11) 120 220 2 S (3) 135 160 3 E ( 26) 130 135 4 E (24) UO 115 5 E (23) 130 180 6 S (50) 140 140 7 E (62 ) 120 160 8 E (63) 100 170 9 E (89) 130 155 10 E (59) 95 140 11 E ( 66) 100 180 12 E (18) 110 145 13 E (19) 160 190 14 E (24) 110 140 15 E (17) 160 180 16 E (64) 115 155 17 E (38) 140 180 18 E (65) 95 190 19 S. (21) l 6o 155 20 BL(47) 140 180 21 E (4?) 150 150 22 E (i*6) 140 140 23 E (49) 135 185 24 E (72) 125 170 25 E (9) 150 170 26 E (14) 140 160 27 E (13) 140 120 28 E (20) 120 125 29 E (67) 140 165 30 E (71) 120 130 31 E (52) 120 130 32 E (53) 110 125 33 E ( 68 ) 135 150 34 E (69) 130 200 35 E (55) 155 170 36 E (5? 120 150 37 E ( 10)UO 120 38 E ( 6) 125 165 27 TABLE II (Continued)

Mean B.P. Heart Rate Obs, No. Dog No.* (san, Hg) (beats per minute)

39 B (1) 160 170 40 E (2) 120 180 41 E (7) 130 150 42 E (61) UO 150 43 E (60) 130 140 44 E (57) U 5 175 45 E (56) 150 200 46 E (25) !£ £ m

Average 125.21 155 S.D.+ ( 25.49) ( 33)

* Number 3 from the original research notebook 28

TAO.E III

Control 6* P. and H. R. In Reserpinised Dogs

Mean B.P. Heart Rate Obs. No, Dog No.^ (mu. Hg) (beats per minute)

1 ER (23) 85 80 2 ER (21) 80 85 3 ER (25) 102 130 4 ER (22) 85 110 5 ER (20) 75 110 6 ER (19) 50 120 7 ER (1) 80 100 8 ER (10) 100 85 9 ER (11) 55 100 10 ER (13) 64 130 11 ER (12) 80 90 12 ER (9) 85 125 13 ER (8) 50 100 1* ER (7) 100 90 15 ER (3) 115 105 16 ER (5) 65 85 17 ER (2) 55 90 18 ER (4-) 120 140 19 ER (17) 80 95 20 ER (18) 50 80 21 ER (5) 70 100 22 ER (4) JO

Average 77.77 100 S.D.+ (20.07) (18)

♦Numbers from th e o rig in a l research notebook 2 9

(e) General experiments — In all other experiments with dogs, the anesthetic was either pentobarbital (Nembutal) 35 mg/kg I.P. or barbital sodium 330 mg/kg I.P. Blood pressure was recorded through the right carotid or femoral artery. Heart rate was recorded in some cases. The use of Nembutal as anesthetic was avoided when large doses of central stimulatory amines were used.

(3) Experiments in cats

Cats of both sexes, weighing 1.5 to 3*3 kilos were used. These ex periments are divided into two groups: (a) anesthetized cats and (b) spinal cats.

(a) Anesthetized cats — They were anesthetized with 80 mg/kg of chloralose (I.V.) followed by etherization. Atropine sulfate 1 mg/kg was given I.V. The left carotid artery was cannulated for blood p re ssu re and th e r ig h t n ic tita tin g membrane was hooked to a thread passing over a pulley to a lever which magnified the contractions 10 times on a smoked drum. The total load on the nictitating membrane was of 6 grams. The central end of the cervical sympathetic nerve was stimulated by means of shielded electrodes (Harvard Apparatus). When stimulated, the following parameters were used with the isolation u n it o f a Grass S tim u lato r, Model S^:

P o la rity 2 (+) Monophasic Frequency 10

Delay D uration 10 millisec Volts 1 to 7 30

Total duration of stimulus was 30 ssoonds. In every experiment, vol tage was varied until supramaximal voltage was found. It was then kept constant throughout the experiment. All drugs were injected into a cannulated femoral vein. (b) Spinal cats — After etherisation, spinal cats were prepared according to the method described by Bum (8l), Blood pressure was recorded through the le ft carotid artery. Atropine 1 mg/kg was also given. The spinal preparation was allowed to stabilise before any test drug was given. A rtificial respiration was maintained through a Harvard Pump (Model #607), set at ^K) to 50 oc/stroke and 18 to 20 strokes/minute. Infusion of norepinephrine was given by means of a Harvard infusion pump (Model #600), at the rate of 2 to 4 meg/kg/ minute. The left femoral vein was used for the infusion, and the right femoral vein was always used for injection of drugs. When de pletion of catecholamines was desired, resexpine 3 mg/kg (I.P.) was given 2k hours previously.

(4) Isolated perfused rabbit heart The isolated rabbit heart was perfused according to the method de scribed by Anderson and Craver (82). Heart contractions were recorded on the kymograph. In some Instances coronary flow was measured in a graduated cylinder. Chenoweth's solution (83) was used as the per fusion fluid, and was supersaturated with 95% oxygen and 5% COg. All drugs were injected into an indwelling polyethylene catheter, which was inserted through the injection capillary of the perfusion 51 apparatus. The temperature was maintained at 37. 5°C. The drugs were not injected until a steady amplitude of contractions had been at tained. The average control heart rate of 21 experiments was 138 beats per minute S.D.+ 19* as shown in Table IV.

(5) Isolated aortic strip of the rabbit Spirally out aortic strips of the rabbit were prepared according to the method described by Furchgott and Bhadrakom (84). After the rabbit was rendered unconscious by a sharp blow on the head, a long incision was made on the skin and thoracic and abdominal contents were reflected to expose the whole length of the aorta. An incision was then made at the descending level of the aortic arch, and a long glass rod (3 to 4 mm. in diameter) passed gradually in it. The whole length of the aorta was thus removed from the rabbit. The fat and other connective tissue was carefully separated from it. During this procedure it was kept moist throughout by means of Locke-Ringer's solution. The aorta was divided into lengths of 4 to 5 cms each. Then sp ir a ls 2 to 3 m illim eters wide were cut and mounted in an assembly of double jacketed 100 cc muscle chambers (Metro Industries).

Loeke-Ringer’s solution with the following formula was prepared fresh and used as bathing solution for the aortic strips: NaCl 9*00 grams KC1 0.42 ■ Cad2 0.24 " M ga; 0.2 " NaHC03 0.5 " Distilled water Dextrose 0 .5 " Ad. 1000 ails. The tem perature o f th e muscle chambers was m aintained a t 37«5°C. The contractions of the aortic strips were magnified ten times and were 3 2

TABLE IV Control H. R. of the Isolated Rabbit Heart

Obs. No. Rabbit No.* H. R. (beats per minute)

1 #6 132 2 #2 120 3 E (8) 130 4 #L1 110 5 E (2) 170 6 144 7 E (4) 126 8 #14 120 9 E (9) 136 10 E (13) 124 11 E (3) 168 12 E (6) 152 13 E (7) 142 14 #L5 140 15 E (11) 178 16 E (12) 138 17 E (10) 108 18 #13 124 19 #L2 144 20 E (5) 130 21 E (4)

Average 138 S.D.+ (19)

♦Numbers from the o rig in a l research notebook 33 counter-balanced to exert b gram tension. The preparations were oxy genated by 95$ 02 and 5$ C02, and allowed to stabilize at least two hours before any drugs were added to the muscle chambers.

The results were statistically analyzed. Standard error (S.E.) was calculated according to the following:

Standard Deviation - S.D.: S.D. = EX - EX2 \ n-1 S E = S.D. vnA The wt-test" was calculated according to the method described by

Yuker (85).

Drugs

For each experiment fresh solutions of the isomers were made in physiological saline by addition of dilute hydrochloric acid. The pH of the resulting solutions was kept within physiological lim its.

The stock solutions of the catecholamines were obtained from the com mercially available sources and were diluted just before use. These solutions were kept refrigerated throughout the experiments. The optical isomers of ephedrine were obtained by the method described by

LaPidus et al. (78). The following drugs were used for the experiments:

D (-) ephedrine, L (+) ephedrine, L (+) pseudoephedrine, D (-) pseudo ephedrine, (+) anqphetamine s u lfa te [th e (+) amphetamine has been shown to have D-configuration (86)J, epinephrine bitartrate (Suprarenin), norepinephrine bitartrate (Levophed), isoproterenol (Isuprel), reser pine (Serpasil), dicholoroisoproterenol (D. C. I.), diphenhydramine

(Benadryl), histamine diphosphate, hexamethonium chloride (Hexameton), phenoxyfcenzaaine (Dibenzyline), and vasopressin (Pitressin). CHAPTER I I I

RESULTS (PART A)

Toxicity

During the toxicity study, typical sympathetic stimulation was observed with all isomers. But it was more apparent and prominent with D (-) ephedrine and much less with D (-) pseudoephedrlne. The mice Injected with toxic doses first manifested increased motor activity, piloerection, mydriasis, etc. Then after the period of stimulation, they were exhausted. Mice injected with D (-) pseudo ephedrine showed a slightly different phenomenon, there was a loss of righting reflex in some, and the mice which died at toxic doses showed a very relaxed posture as compared to those that died with

D (-) ephedrine. In the case of D (-) ephedrlne, animals die late during the 2h hour observation period after injection. But in the case of the other three Isomers, if the animal dies it does so within a minute or so. The LD^q values of D (-) ephedrine and L (+) pseudo ephedrine were determined twice. The LD^q value of D (-) ephedrine varied somewhat, but that of L (+) Dseudoephedrine was almost the same. These values are a ll summarized in Table V.

Relative pressor potency

It was studied in barbitalized dogs. Due to the tachyphylaxis only one dose was used in each dog for comparing the potency of the

35 36

TA&.E V

Intravenous Acute Toxicity of the Isomers of Ephedrine in Mice

LDcq (calculated ac 95)8 cording to the method confidence Dose Bo. died of Litchfield & Wll- lim its Drug (mg/kg) No. used coxon) (mg/kg) (mg/kg)

D ( .) 80 4/10 Bfch. 93 6/10 75.4 to 110 8/10 86 98.9 130 10/10

D (-) 93 2/10 Efc>h. 110 5/10 108 91.6 to 130 9/10 118.8

L U ) 80 0/10 Ejph. 88 1/10 9 6 .3 to 98 2/10 105 114.4 110 8/10

L (+) 80 2/10 Pseudo 90 6/10 89 83.9 to Ejph. 102 8/10 94.8

L (+) n 3/12 Pseudo 90 4/12 93 80.0 to fyh. 90 7/12 97.6

D (-) 80 0/10 Pseudo 90 2/10 94.65 to E|?h. 102 4/10 105 117.6 115 7/10 37 isomers. All the isomers except D (-) pseudoephedrine showed pressor effects. The dose of D (-) ephedrine was chosen as 0.33 mg/kg (which is also 0.1 cc/kg M/50 Solution or 2 x 10*3 mM/kg). This dose gave a B.P. ris e of about 80 am o f Hg. Then the doses of other isomers were adjusted to give the same pressor effect. The dose of L (+) ephedrine was increased by three ( 0.99 mg/kg) and gave approximately the same response. The dose of L (+) pseudoephedrine was increased five times (1 .6 5 mg/kg) to give approximately the same pressor effect.

These dose increments were based upon the findings of Chen et al. ( 65).

But D (-) pseudoephedrine did not show any apparent rise in B.P.; on the contrary, it showed depressor responses on increasing doses.

Animal variation was observed to the pressor effects. The results in detail and relative potencies of the isomers are presented in Table VI.

The epinephrine equivalents were approximately equal at the equi pressor responses. This is presented in Table VII. Increased sensi tivity of the animal to epinephrine did not necessarily mean parallel increase of the pressor response to the test isomer. The pressor effect of L (+) ephedrine and L (+) pseudoephedrine were also tested at the dose of 3*3 mg/kg. The average pressor effect, in three dogs, to L (+) ephedrine was 81,7 mm of Hg and that of L (+) pseudoephedrine was 68.3 mm of Hg. The average pressor response of D (-) ephedrine

(0.33 mg/kg) in five barbitalized dogs was 7^.0, and that in three nembutalized dogs was 76.0 mm of Hg. 38

TABLE VI

R elative Potencies o f the Drugs Derived from Equipressor Doses

Rise in Obs. Dog Dose B.P. R elative No. No.* Drug (mg/kg) (mm Hg) Potency

1 E (1) D (-) Efrh. 0.33 80 2 E (2) n it 70 n if 3 E (7) 80 1 4 E (9) rt n 70 5 E (15) ti H 21 Average 7^.o S.E.+ 2.74

1 E (6) L (+) Ejph. 0.99 100 2 E (10) n It 80 3 E (22) it ft 60 1/3 4 E (9*0 if It 84

Average 81.0 S.E.+ 9.**9

1 E (13) L (+) Pseudo 1.65 90 2 E (1*0 E^phedrine ft 90 n ft 3 E (20) 75 1 / 5 4 E (93) ft ft 20

Average 83-7 S.E.+ *.33 1 E (52) (+) Anph. 0.34 100 2 E (53) ft ft 100 ft ft 3 E (55) 80 1 4 E (89) ft ft 105 5 E (90) ft ft 3 1 . Average 92.0 S.E.+ 6.75

♦Numbers from, the o►riginal research notebook TABLE VII

Epinephrine Equivalents at Equlpressor Doses of the Isomers

Rise in B.P. (mm Hg) to Epinephrine Rise in Epi. Equivalent Obs. Dog meg/kg Dose B.P. from the graph No, No.* 6 3 1.5 0,75 0.37 Drug (mg/kg) (mm Hg) (meg/kg)

1 E (1) 105 82 40 18 D ( -) Eph. 0.33 80 3.20 2 E (2) 116 96 60 4o 22 " « 70 1.65 3 E (7) 115 90 70 44 n R 80 2.30 4 E (3) 65 40 it « 80 4.40 Average 2 3 $ S. E.+ (0.35)

1 E (6) 156 110 70 30 L (+) Eph. 0.99 100 2.45 2 E (10) 120 82 *K) 10 ft ft 80 3.00 3 E (22) 120 70 • _ n n 60 2.55 4 E (93) 110 60 36 IV n 84 M l Average 2.5L S.E.+ (0.24)

1 E (13) 135 110 6o 20 L (+) Pseu 1.65 90 2.50 2 E (1*0 90 70 36 15 do Eph. R 80 4.30 3 E (20) 120 110 56 3h M n 76 2.00 4 E (9*0 1**0 110 60 n « 84 2.05 Average 2.71 S.E.+ (0.64) ♦Numbers from the original research notebook 40

Duration at equipressor response

The action of the D (-) ephedrlne was very rapid in onset; it reached a maximum in le s s than a minute. L (+) ephedrlne had a slower onset of pressor action; L (+) pseudoephedrine wa3 much slower in onset than D (-) ephedrlne and L (+) ephedrlne. The duration of pressor action of L (+) pseudoephedrlne was also longer. The re su lts are summarized in Table VIII.

Depressor action of D (-) pseudo ephedrine in anesthetized dogs

D (-) pseudoephedrine did not show any apparent effect at doses of 0.33 mg/kg. After injecting 3-3 ®g/kg» there was a transitory depressor response; then a gradual pressor response may be seen.

The pressor phase never exceeded 20 to 30 mro of Hg. At higher doses

(9.9 or 16.5 mg/kg) there was a marked depressor phase only. The duration of depressor response was dependent on dose used. The re sults are summarized in Table IX and illustrated by Fig. 3*

Comparative pressor potency o f D (-) ephedrlne

and D (-) pseudoephedrine in spinal cats

Since D (-) pseudoephedrine was depressor in anesthetized dogs.

It was not possible to compare it with D (-) ephedrlne. The work of

Chen et a l . ( 65) showed i t to be presscr in spinal ca ts. Hence i t was decided to compare the pressor effects of D (-) pseudoephedrine 41

TAILE VIII

Duration of Pressor Action of the Isomers in Dogs

■i—— . - - — — - ■

Duration (in minutes) Obs. Dog Dose (Time for the B.P. to be No. No.* Drug (■f/kg) 50# above normal) Remarks

1 E (1) D (-) %>h. 0.33 2.00 Onset of ac 2 E (2) rt n 2.40 tion is quick; 3 E (7) ft n 4.30 reaches maxi 4 E (9) n n 2.00 mum in 1 min. Average 27^7 S.E.fc (0.85)

1 E (6) L (+) 0.99 4.00 Onset of ac 2 E (10) if n 4.10 tio n i s some 3 E (22) n n 3.30 what slower n 4 E (9*0 ft 5.00 than D{-) Eph. Average 0 5 S.E.+ (0.98)

1 E (13) L (+) Pseudo 1.65 5.00 Onset of ac 2 E (1*0 3>h. n 7.00 tion is much 3 E (20) n n 6.40 slower; peak 4 E (93) n n ^ 0 0 pressor re Average 5 3 5 sponse in 2- (0.60) 3 minutes

1 E (52) D (+) Araph. 0.34 5.80 Pressor action 2 E (53) n n 4.60 reaches maxi 3 E (55) ft ti 4.10 mum in about Average 0 3 2 minutes S. E.+ (0.64)

♦Numbers from the original research notebook TABLE H

B.P. Responses to D (-) Pseudoephedrine in Anesthetized Dogs

Dose o f D (-) pseudo B.P. Obs. Dog Wt. esphedrine fall Duration No. No.* Kilos Sex A nesthetic (mg/kg) (mm Hg) Minutes

1 E ( 2 5 ) 9 * 5 B a rb ita l 0 .3 3 10 ^ 1 2 E (4 7 ) 1 1 .0 M n It 0 n n 3 E1( 4 7 ) 1 1 .0 F 5 ^ - 1

n 1 E (6 1 ) 1 1 .0 F 3 .3 25 ^ 1 2 E (6 0 ) 9 . 0 M n tt 18 1 ti it 3 E (5 7 ) 1 0 .0 M 20 1 4 E (5 8 ) 1 0 .0 F n n 22 <£1 it tt 5 E (56) 9 . 0 F 10 ^ 1 6 E (4 9 ) 9 . 0 M tt tt 30 ^ 1 n n 7 E (4 8 ) 1 0 .0 M 20 <=■1 tt if 8 E (8 2 ) 1 1 .3 F 20 ^ 1 tt n 9 E ( 6 5 ) 1 1 .0 M 10 < 1 n n 1 0 E (6 4 ) 1 0 .0 F 25 1 tt 11 E (7 9 ) 9 . 0 M Nembutal 20 1 n 1 2 E (2 4 ) 1 0 . 5 M B a rb ital 25 1 tt n 1 3 E (5 9 ) 9 .0 F 25 1 it 14 E (5 0 ) 8 .0 M n 20 1 n n 1 5 E ( 6 2 ) 8 .0 F 20 <■1 Average 2 0 .6 6 S* E*£ ( 1 .4 3 )

1 E (2 1 ) 1 0 . 0 F C hlorolose 9 .9 40 2 n 2 E ( 3 8 ) 1 0 . 0 F B a rb ital 25 1 It n 3 E (1 7 ) 9 . 5 F 30 1 . 5 4 E (1 8 ) 1 0 .0 M If n 30 2 It tt 5 E (2 6 ) 7 .5 F 60 3 It n 6 E (2 3 ) 1 2 . 5 M 50 2 It ni E (9 3 ) 7 .0 F n JO 1 Average 3 7 .8 6 S.E.+ ( 5 -0 3 )

Tt 1 E (1 9 ) 1 0 .0 K 1 6 .5 80 8

♦Numbers from the o rigin al research notebook FIGURE 3

Barbitalized dogs showing depressor response to graded doses o f D (-) pseudoephedrine.

Upper tracing - Blood pressure Lower tracing - Respiration Heart rate (beats/min.) indicated by numbers above the blood pressure tracing A , B , C , D - D ifferent dogs Time Marker - Every 10 minutes DC-)u> EPHEDRINE

B.R (m m Hg)

2001—

0 .3 3 mg/kg 3.3 mg/kg. 9 9 mg/Vg 16 5 mg/kg

RESPIRATION ^5

and D (-) ephedrlne In spinal cats. In a series of cats, pressor re

sponse to D (-) ephedrlne, 0.33 ®g/kg, was obtained (average 73*75. S.E.+ 6 .2 4 ). Then In other series of cats graded doses of D (-) pseudoephedrine were given in an attesqpt to obtain the sane pressor

effect as that of D (-) ephedrlne, 0.33 ®g/kg. But such an attempt was a failure due to the fact that It required a very high dose of

D (-) pseudoephedrine, and these doses were approaching toxic doses.

The solubility of the D (-) pseudoephedrine was also a problem. So a

dose response effect for D (-) pseudoephedrine was obtained at low

doses, and a D (-) ephedrlne pressor effect equivalent was found by

graphic interpolation of the dose response curve of D (-) pseudo

ephedrine. The approximate potency ratio of D (-) ephedrlne to D (-) pseudoephedrine is 106:1. The results are tabulated In Table X.

Fig. No. 4 shows the dose response curve for D (-) pseudoephedrine.

Comparative chronotropic effect in anesthetized dogs

All the isomers including D (-) pseudoephedrine showed increase

in heart rate. The heart rate was maximum at the maximum height of pressor response. At equlpressor doses the average increase in heart

rate was approximately equal. D (-) ephedrlne was observed to be

slightly more active than L (+) and L (-) pseudoephedrine. The average

increase in H.R. by L (+) ephedrlne (0.99 mg/kg) and L (+) pseudo

ephedrine (1.65 mg/kg) were quite similar, 93 (S.E.+ 13) and 95

(S.E.+ 9) beats per minute respectively. Fhrther, at higher doses TABLE X

Comparative Pressor Potency of D (-) Ephedrlne and D (-) Pseudoephedrine in Spinal Cats

B.P. Obs. Cat Dose r ise No. No.^ Drug (mg/kg) (mm Hg) Relative Potency

1 # 1 D (-) $>h. 0.33 70 2 * 6 n « 90 3 H2 n n 75 106 k *15 it rt 60

Average 73.75 S.E.+ ( 6.2*0

1 # 2 D ( - ) Pseudc• 0.33 5 Sfch.

1 * 5 tt 3.3 10 2 # 7 tt a 10 tt n 3 H9 11 Average 13.7

1 # 8 tt 6.6 10 1

1 # 9 tt 13.2 30 (From graphic in te r tt rt 2 *18 12 polation, the dose oJ D (-) pseudoeph., 35 Average 20 mg/kg is obtained as being equivalent to 1 #10 tt 26.tJ- 5^ 0.33 mg/kg o f D ( -) 2 #11 If n 29. ephedrlne) Average bz

♦Numbers from the origin al research notebook FIGURE h

Dose response curve to pressor effects of D (-) pseudoephedrine in spinal cats and D (-) ephedrlne equivalent from graphic Interpolation.

^ 7 B. R (mm Hg ) 100

8 0

D(-) EPHEDRINE. 0 33 mg/kg. 6 0

4 0

0 10

D (-)^ EPHEDRINE mg/kg *9

3*3 mg/kg of L (+) ephedrine and L (+) pseudoephedrine, the increase

in H.R. was also nearly equal, 113 (S.E.+ 9) and 105 (S.E.+ 9) beats

p er m inute. The re s u lts are shown in Table XI,

D (-) pseudoephedrine was investigated at different doses. At a

dose o f 0.33 rag/kg it showed a very slight increase in heart rate

(average 10 to 20 beats per minute) over a 30 minute observation period. But when the dose was increased to 3*3 rag/kg, there was a

definite increase in a heart rate, average of 55 (S.E.+ 9) beats per

minute over a 30 minute observation period. It was interesting to

note that there was a gradual increase in heart rate over a 30 minute period. When the dose was increased to 9.9 mg/kg, proportionately

there was not much increase in heart rate, average 63 (S.E.+ h) beats per minute, and at this dose level a gradual increase may or may not

be seen. At a dose of 16.5 mg/kg, the increase was only 50 beats per minute.

The control experiments were conducted in three dogs, over a period of 30 to^5 minutes. There was no change in average heart

beats per minute. The results are shown In Table XII. Fig. No. 5

shows the gradual increase of heart rate by D (-) pseudoephedrine

( 3*3 mg/kg) over a 30 minute period.

Tachyphylaxis

The tachyphylactic tendencies of these isomers were studied at

equipressor doses. D (-) ephedrine (0.33 mg/kg) was the le a s t tachy phylactic, and L (+) pseudoephedrine (1.65 mg/kg) was the most. 50

TABLE XI

Increase of H.R. by the Isomers (in Dogs)

H.R. Net Increase Obs. Dog Dose (beats/min.) in H .R . No. No.^ Drug (mg/kg) Before After (beats/min.)

1 E (1) D (-) Eph. 0.33 170 280 110 2 E (2) It ft 180 300 120 3 E (7) n ft 150 260 110 4 E (9) n ft 170 260 A v e . 10S S.E.+ 7 1 E (6) L (+) $>h. 0.99 165 290 125 2 E (10) n ft 120 210 90 3 E (22) tt ft 150 230 80 tt ft 4 E (9*0 160 235 3 1 A v e . 93 S.E.+ 13 1 E (68) n 3.3 150 270 120 2 E (69) tt it 200 320 120 3 E (70) tt it 200 300 100 A v e . 113 S.E.+ 9 1 E (13) L (+) Pseudo • I .65 120 210 90 2 E (14) Efch. n 165 270 105 3 E (20) If n 125 190 65 4 E (93) tf ft 150 270 120 A v e . 95 S.E.+ 13 1 E (67) IT 3.3 165 260 95 2 E (71) It TT 130 230 100 3 E (72) ft It 170 290 120 A v e . 105 S.E.+ 9 1 E (32) D ( + ) Amph. 0.34 125 220 95 2 E (53) ft It 125 250 125 3 E (54) ft ft 155 240 85 4 E (55) ft ft 170 260 90 5 E (90) n ft 165 240 A v e . S.E.+ 9 ♦Numbers from the original research notebook 51

TABLE XIX

Increase in the H.R. to D (-) Pseudoephedrine in Anesthetized Dogs

Dose of D (-) pseudo Obs. Dog ephedrine Net increase in the H .R . (beats/min.) No. No.* (mg/kg) 0-5 10 15 30

1 E (25) 0.33 10 10 20 2 E (47) n 10 15 20

1 E (49) 3.3 55 50 45 55 2 E (56) n 40 40 60 50 3 E (58) n 40 40 40 20 4 E (60) n 20 30 40 40 5 E (62) m 30 40 50 55 n 6 E (63) 40 60 70 85 n 7 E (65) 40 60 2 2 80 Ave. 3? 5 5 a 55 S.E.+ ( 4) ( 4 ) < 5) ( 9)

1 E (17) 9.9 80 70 60 2 E (18) If 65 65 mm 75 3 E (21) it 75 65 55 4 E (23) ft 60 60 60 5 E (38) ft 60 70 Ave. 8T o5 S.E.+ ( 4) ( 2)

1 E (19) 16.5 50 50

♦Numbers from the original research notebook FIGURE 5

Gradual increase in average heart rate of seven anesthetized dogs to

D (-) pseudoephedrine, 3.3 nig/kg, over a time period of 30 minutes.

(Each point on the graph indicates an average heart rate of seven dogs and vertical lines indicate the standard errors.)

52 HEART RATE D(-)y EPHEDRINE. (Beats/min) 3 3mg/kg . 100

8 0

6 0

4 0

0 5 10 15 20 25 3 0 TIME(min) 5*

D (-) ephedrine (0.33 »g/kg) produced a pressor response after the animals were completely tachyphylactic to the pressor effects of

L (+) ephedrine (0.99 mg/kg), and this pressor response was almost equal to that observed after six repeated injections of D (-) eph edrine itself. D (-) ephedrine also showed pressor response after animals were made tachyphylactic to L (+) pseudoephedrine. But L (+) ephedrine (0.99 mg/kg) was observed to be only a depressor after the animals were made tachyphylactic to L (+) pseudoephedrine.

In the case of L (+) ephedrine and L (+) pseudoephedrine, some observations were made in regard to the heart rate effects. In two observations, the average corresponding increase in H.R. by L (+) ephedrine (0,99 mg/kg) was 82 beat3 per minute after the first in jection and only 20 beats per minute after the fifth injection when tachyphylaxis to pressor effects was complete. L (+) pseudoephedrine in four observations increased the average H.R. by 95 beats per minute after the first injection and only 15 beats per minute after the third injection when tachyphylaxis to L (+) pseudoephedrine was complete. D (+) amphetamine also showed a marked tendency to produce a tachyphylactic response. The results are shown in Table XIII and illustrated graphically in Fig. No. 6.

Comparative chronotropic effect on

the isolated rabbit heart

The dose of D (-) ephedrine (33 racg) was selected so that it would give an increase of approximately 20 beats per minute. The TABLE X I I I

Pressor Effects to the Repeated Doses of Isomers in Dogs

Obs. Dog Dose I n j. No. 1 2 3 h 5 6 7 8 9 10 No. No.+ Drug (mg/kg) Time (min.) 0 .0 30 h5 60 70 80 90 100 110 120

1 E (1) D (-) %>h. 0.33 80 55 he 55 50 50 h5 h5 ho 36 2 E (7) tt n 80 65 60 60 50 h5 h5 ho ho w E (h?) it n 80 so 55 3 60 ho , 32 Ave. So oo# 6 5& 56*6 U .6 *3.3 J. 3 .6 35.3 S.E.+ 0 8 .9 2 h.2 3 3.1h 3.07 3.01 3.01 5.6h *.5L 3.71 1 E ( 6) L (+) Eph. 0.99 100 60 ho 18 8 (h2 D (-) $>h. 0.33 mg/kg 2 E (10) tt H 80 65 55 ho 20 (70 _ n " 3 E ( 2 2) n If 60 50 30 15 5 (*0 n n n it h E (9h) 8 h 76 £ 22 18 (36 Ave. "5T 62.7 £ 5 .7 25.2 12.7 (52 it S.E.+ 9 .6 6.58 6.6 7.0 h.35 (8.21 n l E (13) L (+) Pseu - 1.65 90 22 10 (30 D 0.33 mg/kg 2 E (lh ) do £bh. n 80 22 -25 (20 3 E (20) n n 75 10 0 n if h E (93) 2£ 22 -10 (20 Ave. 83.7 19 -5725 (23.33 0.33 mg/kg S.E.+ h.h 3.52 8 .7 6 ( h.12 1 E (52) D (+) Amph. o.3h 100 30 5 (30 D 0.33 mg/kg 2 E (53) n fl 100 ho 10 (ho n ti rt n 3 E (55) 80 £ 28 Ave. 93.3 35.3 1^.3 ( % 6 n S. E.+ 8.50 3.77 8.71 (h.07 if

♦Numbers from the original research notebook V_rx FIGURE 6

Tachyphylaxis to pressor effects of D (-) ephedrine (0.33 rag/kg),

L (+) ephedrine (0.99 mg/kg), D (+) anphetamine (0.3^ mg/kg), and

L (+) pseudoephedrine (1,65 mg/kg) in anesthetized dogs.

56 ( m m H q)

D B EPH.

DWAMPH LW EPH

U*Xi> EPH

Inj no. 1 2 3 4 5 6 7 8 * 9 10 Time 0 30 45 (rnn) 60 70 80 9 0 100 110 120 58 doses of the other isomers were then adjusted to give approximately the same response. Only the first dose in each preparation was used for oon^arative purposes, L (+) ephedrine at a dose of 33 meg did not show any chronotropic effect, but the same dose of D (-) eph edrine, 33 meg, increased the rate by 20 beats per minute. L (+)

ephedrine, 99 meg, increased the rate by only 6 beats per minute. Hence a higher dose of 165 meg was selected. This dose increased the average ra te by 16 beats per minute. The dose of L (+) pseudo ephedrine, which showed approximately the same effect as 33 meg of

D (-) ephedrine, was 132 meg. The relative potencies are expressed in Table XIV. D (-) ephedrine, L (+) ephedrine and L (+) pseudo ephedrine all increased the force of contractions of the heart. A depression followed by increased force of contraction was also seen in some experiments with all isomers, but it was more prominent with L (+) pseudoephedrine.

D (-) pseudoephedrine at a dose of 99 meg did not show any ef fect on the rate, but in the same preparation D (-) ephedrine, 33 meg, increased the rate by 22 beats per minute. Increased doses of D (-) pseudoephedrine from 165-1650 meg increased the heart rate by 6 to 10 beats per minute only. The effect was always submaxinial. Contractile force was decreased at higher doses. The effect of D (-) ephedrine,

33 meg, after D (-) pseudoephedrine may or may not be blocked. The results are shown in Table XV and illustrated In Fig. No. 7. Iscmers did not show any significant effect on coronary flow. Results are summarized in Table XVI, TABLE XIV

Effect of the Isomers on the H.R. and Contractile Force (C.F.) of the Isolated Rabbit Heart

F ir s t In je c tio n (Time 00 minutes) Second Injection (Time 30 minutes) Drug Dose E ffect E ffect Drug Dose E ffect E ffect (meg) on H.R. on C.F. Approx- (meg) on H.R. on C.F. per min. iraate per min. Obs. Rabbit + Increase Relative + Increase No. Ho.+ - Decrease Potency - Decrease

1 t 2 D ( -) Eph. 33 +20 + 2 * 6 " ft +18 + 3 # 8 ft +40 + 1 D ( - ) $>h. 33 +32 + 4 f l l ft +10 + Ave. 22 S.E.+ ( 8) > #L2 110 +46 + 1 E ( 6) " 165 +20 n ti +24 2 E ( 7) " It + 6 0 1/5 +16 + 3 E (11) " IT +12 + tt tt +14 + 4 E (12) " rt +24 L (+) Pseudo Ave. i£ Eph. 132 +20 S.E.+ ( 5) 1 £L2 L (+) Pseu.• 33 - 6 0 D (-) Hfch. 33 +30 2 fl3 do 5£h. 99 +12 + n tt +20 3 E (5) " 132 +24 + 1/4 " " +28 + 4 E (10) " tl +32 +. then - II It +32 + 5 E (4) " 330 +30 +, then - tt tl + 4 + ♦Numbers from the original research notebook TABLE XV

Effect of D (-) Pseudoephedrine on the H.R. and Contractile Force(C.F.) of the Isolated Rabbit Heart

First Injection Second Injection Third Injection (Time 00 minutes) (Time 3° minutes) (Time 60 minutes) D (-) Effect Effect Drug Dose E ffect E ffect Drug Dose E ffect E ffect Pseudo on H.R. on C.P. (meg) on H.R. on C.F. (meg) on H.R. on C.F* Rab- I^h. beats/min beats/m in beats/m in Obs. b it (meg) + Increase + Increase + Increase No. No.# - Decrease - Decrease - Decrease

1 UA 99 0 0 D (-) %>h. 33 +22 +

2 E (13)165 +6 slig h t- D (-) Pseu do S^>h. 1650 +18 - D (-) 5*>h. 33 +2^ +

3 E (1) 330 +6 slig h tf D (-) Efch. 33 +20 + D (-) Pseu do E£h. 825 +10 •

it. E (2) 660 +10 s lig h t- if « +12 +

5 E (4) " +h irregular D (-) e ffe c t %>h. 1650 +10 D (-) Efch. 33 +3^

6 E (9) 1650 0 - 33 +h +

♦Numbers from the original research notebook:

£ FIGURE 7

E ffect of D (-) pseudoephedrine and D (-) ephedrine on the isolated rabbit heart. All three tracings are a continuation of an experiment in the same heart. D (-) Eph. - - - - D (-) pseudoephedrine 165 meg and 1650 meg. D (-) Eph. — — — — — D C —) ephedrine, 33 meg. Heart Rate (beats/min.) Indicated by numbers. Time Marker - - - - Every minute.

61 D(-) EPH 33 meg TABLE XVI

Effect of the Isomers on the Coronary Flow of the Isolated Rabbit Heart

Coronary flow Difference Rabbit Dose Control coronary after the drug + Increase Obs. No. Heart No. * Drug (meg) flow (cc/min.) (cc/m in.) - Decrease

1 E (8) D (-) Efch. 33 13.0 15.5 + 0.5

2 E (3) L (+) Eph. 99 14.0 13.0 - 1.0

fl 3 E (6) 165 11.3 10.0 - 1.5

-'l E (7) I f n 11.0 10.0 - 1.0

ft tt 5 E (12) 8.0 3.3 + 0.5

6 E (5) L (+) Pseudo 132 11.0 10.5 - 0.5 Eph.

•7 n 1 E (4) 330 9.0 9.0 0

3 s (13) D ( - ) Pseudo 163 8.0 7.5 - 0 .5 Eph.

ft 9 E (1) 330 10.0 10.0 0

ft 10 E (4) 660 9 .0 s .5 - 0.5

II 11 E (9) 1630 12.5 12.0 - 0.5 ♦Numbers from the original research notebook 64

Gross affect on respiration

D (-) ephedrine, L (+) ephedrine, L (+) pseudoephedrine, and

D (+) amphetamine all increased the anqplitude and frequency of res piration. The effects were observed to be parallel with pressor effect. D (-) pseudoephedrine showed a transitory increase when a depressor response was seon. At doses of 33 rog/kg of D (-) pseudo ephedrine, there was an increase in frequency and amplitude 15 to 20 minutes after drug administration, and these respiratory effects were parallel to the gradual increase in heart rate.

All the comparative effects of the isomers, such as toxicity, pressor effect, duration, chronotropic effects in dog, tachyphylactic tendency, chronotropic effect on rabbit heart, are summarized in

Table XVII.

RESULTS (PART 3)

Modification of B.P. and H.R. effects

of the isomers by reserplnlzatlon

The B.P. and H.R. effects which were obtained during the com parative potency study served as controls for this part of the study.

Hence in reserpine pre-treated animals, the same doses of the iso mers were used; namely, D (-) ephedrine, 0.33 mg/kg; L (+) ephedrine,

0.99 ag/kg; L (+) pseudoephedrine, I .6 5 mg/kg; D (-) pseudoephedrine,

3.3 ng/kg, and D (+) anqjhetamine, 0.34 mg/kg. In all cases, there was a marked reduction of the pressor effects. The B.P. depressor TABLE XVII Summary - Approximate Comparative Potencies of the Isomers

H.R. Tachyphylactic H.R. B.P. in Duration at increase at tendency at is o la te d B.P. in LD cq in anesthe equipressor equipressor equipressor ra b b it sp in a l Isomer mice (I.V.) tiz e d dogs responses responses responses h eart cat

D ( - ) Efch. 1 1 + + + 1 1

L (+) Efch. 1 1/3 ++ + ++ 1 /5

L (-0 Pseudo 1 1/5 ++ +++ 1/* Eph.

D (-) Pseudo 1 D epressor - Submaximal - Submaximal 1/106 Eph.

D (+) Amph. 1 -H- + ++ - T

ON V-T, 66 effect of D (-) pseudoephedrine was reduced at statistically signifi cant level, (P ^O.Ol). In all experiments, after the first dose, five times the dose was repeated in the same animal in order to determine reversibility of the pressor effect. It was only seen in the case of D (-) ephedrine pressor effects, though H.R. was not proportionately increased. All other isomers were irreversibly anta gonised. AH the results are shown in Tables XVIII, XIX and XX and illustrated by Fig Nos. 8, 9, and 10.

Two spinal cats pre-treated with reserpine were given 3-3 and

13.2 mg/kg of D (-) pseudoephedrine. They showed B.P. fall of 8 and

5 mm of Hg respectively, and no pressor effects were observed.

RESULTS (PART C)

Effect of D (-) pseudoephedrine at the height

of pressor responses to D (-) ephedrine.

D (+) amphetamine and vasopressin

(a) In anesthetized dogs

In this series of experiments, D (-) pseudoephedrine, 3.3 mg/kg, was given at the height of pressor response to D (-) ephedrine, 0.33 rag/kg.

Immediately a reduction of pressor response was seen. Similar experi ments were carried out on the pressor effect induced after 0 . 3k mg/kg of D (+) amphetamine, and results were qualitatively similar. In control experiments the pressor effects of D (-) ephedrine and D (+) amphetamine returned to normal or subnormal levels in 15 to 20 minutes. FIGURE 8

Effect of reserpine pre-treatment on the B.P. and H.R. effects of the isomers in anesthetized dogs.

Heart rate (beats/min.) indicated by the numbers above the blood pressure tracing RESERPINE ..... 1 mg/kg, 2h- hours previously EPH ------Ephedrine

67 CONTROL RESERPINE B P (mm Hy; , 260 1 240 280 ! 220 175 [■200 -180 Or-160 V -140 m & n n I 120 -120 1 1 0 -100 -80 -60 -40 D( , t PM DO EPH D(-) EPH -20 0 32 mg/ky 0 3 3 mg/kg 1 65 mg/kg

r-2 to 0 390 (-240 ;-220 - - 200 i 180 . -160 130 M40 120 I -120 .300 .-80

f t . DC 2 PM LW EPH LM EPH C 99 mg kg 0 99my/kq 5 Omy/'kg

r-260 '40

180 -160 140 140 106 -’CO !tO 8 r (3 1 .\.,V 2WV EP21 LWv tPH 1 60 my, ky 166 mg, ky 8 26 my/ky

- 2 0 0 • 380 * * -160 100 85 90 90 • 1C 6 • • • vv' I t'40 DE)y EPH DEV EPH DEV EPH (■'20 3 3my/ky 3 3 mg/kg 16 5 mg/kg FIGURE 9

Effect of reserpine pre-treatment on the pressor effects in anesthe tized dogs.

AMPH ------Amphetamine s u lfa te EPH ------ephedrine RESERPINE - - - - 1 mg/kg, 2b hours previously n ------Number of dogs All doses are mg/kg Vertical bars indicate standard errors

69 B.P CONTROL RESERPINE (m m Ha.) 100r ‘

8 0 £

2 0 ±

.>15 0 n -5 n-5 n-4 n-4 n -5 n-6 n-4 n-4 n -3 ~T

- 2 0 L

CX-) EPH. LUEPH. LWy CX-ty DMAMPH D(-)EPH LWEPH. LWy D(->9 DWAMPH, 0 33 0 9 9 EPH. EPH 0 3 4 033 0-99 EPH. EPH 0-34 1 6 5 3 3 165 3-3 FIGURE 10

Effect of reserpine pre-treatment on the H.R. in anesthetized dogs AMPH ------Amphetamine su lfate EPH ------Ephedrine RESERPINE - - - - 1 mg/kg, 2k hours previously n ------Number of dogs AH doses are mg/kg Vertical bars indicate standard errors

71 HEART RATE CONTROL RESERPINE (Beats/mm) 120

100

2 0

i f i 0 n-4 n-4 n-4 n-7 n-5 n-6 n-4 n-4 n-6 n-3

D(-) EPH. LW EPH LWy D(-)y DWAMPH D(-)EPH. LMEPH LWy D(-)y DWAMPH. 0 3 3 0-99 EPH' EPH 0 34 0 33 0 99 EPH. EPH. 0 34 1.65 3-3 165 3 3 TABLE XVHI

Effect of Reserpine Pre-treatment on the B.P. Effect of the Isomers and Amphetamine

B.P. (mm Hg) Reserpiniied Dose +Rise Obs. No. Dog No.# Drug (mg/kg) - F a ll

1 ER (3) D ( - ) Bph. 0.33 +65 2 ER (7) fl ■t +30 3 ER (11) ft n +44 ER (12) If it +35 5 ER (15) n it +4o 6 ER (25) n tt +40 Average +52.33 S.E.+ 5.41 1 ER (4) L (+) Efch. 0.99 +30 2 ER (5) It tt +40 3 ER (8) rt n +10 4 ER (1?) if it +30 Average +27.50 S.E.+ 7.2? 1 ER (2) L (+) Pseudo 1 .6 5 +20 2 ER (10) 3ph. it +20 3 ER (1*0 m n +32 it n 4 ER (18) ± -1 Average +22.25 S.E.+ 7.41 1 ER (19) D (-) Pseudo 3.3 0 2 ER (20) B^ph. « -15 3 ER (21) If n -10 b ER (23) n ft -15 5 ER (25) it ft riO Average -10 S.E.+ 3.06 1 ER (6) D (+) Amph. 0.34 +20 ft 2 ER (9) n +20 3 ER (13) n n Average S.E.+ 6.17 ♦Numbers from the o rig in a l research notebook 7h

TABLE XIX

Effect of Reserpine Pre-treatment on the H.R. Effect o f the Isomers and Amphetamine

Reser- Net Increase Obs. pinized Dose H.R. (beats/m in.) in H.R. No. Dog No.* Drug Crag/kg) Before After (beats/min.)

1 ER (3) D ( - ) Eph. 0.33 105 lh o 35 2 ER (5) W t> 100 135 35 3 ER (7) tt n 90 110 20 k ER (11) It w 100 lk o ho 5 ER (12) It tt 90 120 30 ft n 6 ER (2k) 80 115 21 Ave. 32 S.E.+ 3

1 ER (h) L (+) Eph. 0.99 lh o 160 20 2 ER (5) n It 85 105 20 3 ER (8) n n 100 110 10 it k ER (17) Tt 95 120 21 Ave. 19 S.E.+ 3

1 ER (2) L (+) Pseudo 1.65 90 100 10 2 ER (10) Eph. n 85 100 15 3 ER (1*) n tt 70 90 20 k ER (18) n tt 80 80 Ave. U S.E.+ 5

1 ER (1) D ( - ) Pseudo 3.3 90 100 10 2 ER (19) tt tt 120 150 30 3 ER (20) tt IT 110 150 ho k ER (23) It H 90 90 0 3 ER (25) n tt 130 lh o 10 6 ER (26) tt tt 90 100 10 Ave. 17 S.E.+ 7

1 ER (6) D (+) Amph. 0.34 95 115 20 2 ER (9) 11 tt 125 125 0 3 ER (13) n tt 130 160 32 Ave. 17 S.E.+ 10 ♦Numbers from the original research notebook 75

TABLE XX

Summary of the Control B.P. and H.R. and Effect of Reserpine Pre-treatment on It

Control Reserpine Pre-treated No. o f Average No. of Average animals pressor animals pressor response response Dose + S.E. + S.E. Drug (mg/kg) (mm Hg) (mm Hg) P. Value

D (-) Efch. 0.33 5 74.0 + 2.74 6 42 + 5*41 > 0.001

L (+) %>h. 0.99 4 81.0 + 9.49 4 27 + 7*27 > 0.01

L (+) Pseu do Efch. 1 .6 3 4 83*7 + 4.33 4 22 + 7.41 > 0.001

D (-) Pseu (F a ll) (F a ll) do Efc>h. 3*3 15 20. 66+ 1 .4 3 5 10 + 3.06 > 0.01

D (*) Amph. 0.3*4- 5 9 2 .0 + 6.75 3 25 + 6.17 > 0.001

Control Reserpine Pre-treated No. of Average No. of Average animals increase animals increase in H.R. in H.R. Dose (beats/m in) (beats/m in) Drug (mg/kg) + S.E. + S.E. P. Value

D (-) Eph. 0.33 4 108+7 6 32 + 3 > 0.001

L (+) Efeh. 0.99 4 93 + 13 4 19 + 3 > 0.01 L (+) Pseu do Sjph. 1 .65 4 95 + 13 4 11 + 5 > 0.001 D (-) Pseu do Efcih. 3*3 7 55+9 6 17 i 7 >0 .5 D (+) Amph. O.34 5 94+9 3 17 + 10 >0.01 76

The increase in heart rate effect induced by D (+) amphetamine was not effectively reduced. The results are summarized in Table XXI and

Fig. No. 11 and 12 illustrate the antagonism of pressor effects of ■\ D (-) ephedrine and D (+) amphetamine by D (-) pseudoephedrine.

In another series of six experiments L (+) ephedrine (3.3 mg/kg) and L (+) pseudoephedrine (3-3 ®g/kg) were given at the height of pressor effect of D (-) ephedrine. An increase in pressor response was observed. It is illustrated by Fig. No. 13.

In the next series of experiments, dogs were pre-treated with reserpine. D (-) ephedrine in doses 3-3 ami 0.99 mg/kg caused a marked pressor effect, and D (-) pseudoephedrine (16.5 and 9.9 ag/kg respectively) antagonized the pressor effect immediately. But the pressor responses induced by 0.3u/kg of vasopressin were only slightly antagonized by the same doses of D (-) pseudoephedrine. All the re sults are summarized in Table XXI and illustrated by Fig. No. 1^.

(b) Anesthetized and spinal cats

Similar antagonism of D (-) ephedrine and D (-) pseudoephedrine was also seen in anesthetized cats as well as in spinal cats. The results are summarized in Table XXII and illustrated by Pig. No. 15.

Antagonistic action of 3 (-) pseudoephedrine towards

N.M. effect of D (-) ephedrine in anesthetized cats

Hie contraction o f n ic tita tin g membrane was n eg lig ib le a t the dose of 0.33 mg/kg of C (-) ephedrine. So it was increased to 0.99 mg/kg at which dose there was a marked contraction and tonus maintained TABLE XXI

Effect of D (-) Pseudoephedrine at the Height of Pressor Responses to D (-) Ephedrine and Vasopressin in Dogs

Dose o f D (-) Pseudo I^h. Rise in given at the Obs • Dog Dose B.P. height of B.P. No. No.* Drug (mg/kg) (mm Hg) (mg/kg) Effect Observed 1 3 (5) D ( -) Eph. 0.33 50 3.3 Prompt; reduction of pressor response 2 3 (9) n rr 75 II It tt 3 E (39) D (+) Amph. 0. 3^ 100 Tt " ; heart rate only slightly reduced h E (90) " tt 72 tt tt tt Res e m in i zed l ER (22) D (-) E£h. 3.3 1>0 1^.5 Prompt; reduction of pressor response. Heart rate was 110, after ephedrine it be came 2 1 0 ; then immediately it became 1 6 0 after D (-) pseudoephedrine. 2 ER (2**) Vasopressin o.3u 135 ft Slight reduction of pressor response; h e a rt ra te was 105; then after vasopressin it became lho, and after D (-) pseudo ephedrine i t was l 60. 3 ER (30) D (-) %>h. 0.99 95 9.9 Prompt reduction of pressor response; heart rate reduced 'by 20 beats/min. 4 ER (28) Vasopressin 0.3n 90 tt Very slight reduction of pressor response; h e a rt ra te was not reduced, but i t was slightly increased. n n Tt 5 ER (31) 1 2 0 Gradual reduction of pressor effect; heart rate slizhtlv increased. ♦Numbers from the original research notebook FIGURE 11

Antagonistic effect of D (-) pseudoephedrine against the pressor ef fect of E (-) ephedrine in anesthetized dogs.

A , 3 ( C----- All different dogs

Time ------— - Ten minutes

78 (mm Hy) r-240 CONTROL -2 2 0 _200 -1 8 0 -160 -1 4 0 _i20 -1 0 0

-80 D(-) EPHEDRINE . -60 0.33 mg/kg. _40 -2 0 1

-2 0 0 CONTROL - 180 B - 1 6 0 -140

100 -80 -6 0 D(-)y EPHEDRINE -4 0 3-3 m g/k g - 2 0

D(-)if) EPHEDRINE -240 ^ 3 -3 mg/kg. -22 0 200 -160 -160 -140 -120 -100

-80 t D(-) EPHEDRINE . -60 0.33rri9/kg -40 -20 FIGURE 1 2

Antagonistic effect of D (-) pseudoephedrine on the pressor effects of D (+) amphetamine in anesthetized dogs,

A , B , C ..... All different dogs Heart rate (beats/min.) - indicated by numbers above the blood pressure tracing Time ------Ten minutes

80 B.P (mm HyJ p240 CON TROL 220 -220 f'A'' V ' -200

-ISO .... 220 -160 -14C 125 —U r -ICC -PC t DW AMPHETAMINE —10 0 3 4 m g/kg 20

i - 160 CONTROL —140 155 170 190 -120 —100 - 8 0

-60 - 4 0 DB^J EPHEDRINE 2 0 3 3 m g/k g

D(-)^ EPHEDRINE I 3 3 mg/kg

-2 6 0 2 7 0 | -2 4 0 - 2 2 0 -200 -180 2 6 0 —160 155 '2 5 0 —140 ■**» [/V —120 - 1 0 0 I - 8 0 D W AMPHETAMINE —60 O 3 4 m g/kg —40 —20 FIGURE 1 3

The effects of L (+) ephedrine and L (+) pseudoephedrine at the height of pressor effects of D (-) ephedrine in anesthetized dogs. Time Marker ------Ten minutes

82 B P L M EPHEDRINE (mm Hg} 3-3my/kg

—220

— 200

— 180

—1 6 0 — 1 4 0 — 120

—100 Dr) EPHEDRINE 0 33 m g/k y — 6 0 — 4 0

—20

LWvy EPHEDRINE 3 3 m g/k g

D(-) EPHEDRINE O 33 m g/ky

1 FIGURE Ik

Effect of D (-) pseudoephedrine against the pressor effects induced by

D (-) ephedrine and vasopressin in reserpinized dogs.

Heart rate (beats/min.) - indicated by numbers above the blood pressure tracing RESERPINE ------1 mg/kg, 2k hours previously A , B ------Different dogs Time Marker ------Ten minutes

8k RESERPINE TREATED DOGS

(mm Hg ) D(-X+> EPHEDRINE —200 9*9 m g/kg.

—160 -1 4 0 -1 2 0 - 1 0 0 - 8 0 - 6 0 - 4 0 D(-) EPHEDRINE. - 2 0 0-99 mg/kg -I------L

-200 -180 D(-ty EPHEDRINE 9 9 mg/kg. -160 75 | -140 -120 -100 -80 -60 -40 VASOPRESSIN -20 0* 3 u/kg. -1 8 6

TABLE XXII

The Effect of D (-) Pseudoephedrine at the Height of Pressor Response to D (-) Ephedrine in Anesthetized and Spinal Cats

Dose of D (-) Pseudo Eph- Dose of D ( -) Rise in edrine given at Obs. Cat Ephedrine B.P. height of B.P. E ffect Ho. No.* (mg/kg) (mm Hg) (mg/kg) Observed

1 # 6 0.33 70 3.3 Conqplete reduc tion of pressor response

2 # 8 n 80 rt Partial reduc tion of pressor response

3 #10 0.99 90 6.6 Complete reduc tion of pressor response

4 UA ft 90 It Complete reduc tion of pressor response

Spinal cats

1 #13 0.33 50 3.3 Prompt reduction of pressor response

2 #15 n 60 tt Prompt reduction, but it was not to original level

3 #16 n 120 tl Prompt reduction of pressor re sponse (animal was more sensitive to D (-) Ephedrine)

♦Rumbers from the original research notebook FIGURE 1 5

Antagonistic effect of D (-) pseudoephedrine against D (-) ephedrine in spinal cats.

A , B , C AH different cats Time Marker ------Ten minutes

87 y/pLu c f . i -i* 1

~J 6y 6w c F 3N!bC3Hd3

O v — ■

09 -

0 8— 1

Obi NOD 0 0L *

fx'Ku £F 0 ’vaojH di (-)a

iM M 3 Gt7! —

l 6 u LUUJ 'i

VD ^VNIdS

w* -J 89

for a longer period. The antagonistic dose of D (-) pseudoephedrine

was also increased. The resu lts are summarized in Table XXIII and

Illu stra ted by Fig. No. 16 .

Antagonistic effect of D (-) pseudoephedrine towards

the contraction of the aortic strip induced by

norepinephrine and D ( - ) ephedrine

The contraction of the aortic strip was induced by 2 to 5 mcg/cc

of norepinephrine. V.'hen the response was maximum D (-) pseudo

ephedrine was added in the bath. The concentration of D (-) pseudo

ephedrine varied from 0.1 to 0.4 mg/cc. Antagonism was demonstrated

in most of the experiments as shown in Table XXIV and illustrated by

Fig. No. 17.

Similarly in other series of experiments, contraction of the

aortic strip was induced by 0.05 to 0.02 mg/cc of D (-) ephedrine,

and D (-) pseudoephedrine added to the bath at the height of con

traction . Antagonism could be demonstrated. In one experiment there was an increased contraction of the strip. D (-) pseudoephedrine in

a concentration which antagonised the contractions of D (-) eph

edrine and norepinephrine had a slight contractile effect in control

experiments. Results are summarized in Table XXV and illustrated by

Fig. No. IS. TABLE XXIII

Antagonistic Action of D (-) Pseudoephedrine Towards N.M. Effect of D (-) Ephedrine in Anesthetized Cats

Dose of D (-) Pseudo Eph. Dose of Maximum height given at the Obs. Cat E (-) Sph. of N.M. con height of con Effect No. No.* (mg/kg) traction (mn) traction (mg/kg) Observed 1 f 9 0.33 3 Control

2 * 6 ft k 3.3 Slight Inhibition of contraction as compared to Control.

3 #15 0.99 60 Control Very high tone was maintained and only l/^th reduction of tone in ho minutes

#13 »T h? rt ft

5 nz ti 50 13.5 Prompt reduction of contraction by 35 mm 6 fVi If 6.6 Prompt reduction of contraction by h mm * 6.6 Further reduction of contraction by 10 mm

n #10 ff 36 6.6 When given during the contraction, a com + plete contraction was prevented. Further 6.6 return of contraction to base line was much quicker. *Numbers from the original research notebook FIjURS l*>

Antagonistic effect of D (-) pseudoephedrine against D (-) ephedrine induced contraction of the cat N.M.

Control - - - - - 'Jpper tracing 3T ------Supramaximal stimulusto the cervical sympathetic nerve Time Marker - - - In minutes

TA3.E XXI';

Antagonistic Effect of D (-) Pseudoephedrine Against Norepinephrine on the Rabbit Aortic Strip

At the height Aortic Contrac of contraction Rabbit Strip Norepi. tion in E (-) pseudo E ffect No.* No.* (meg/cc) mm eph. (mg/cc) Observed

1 (a) 5 meg. 12 0.2 mg. Reduction of con traction by 4 mm.

(b) 5 " 33 0.2 " Reduction of con traction by 15 mm

2 (a) 3 " 24 0.4 " No apparent effect

(b) 5 " 20 0.4 " Reduction of con traction by 2 mm.

3 (a) 2 " 36 0.2 " Reduction of con + traction by 12 mn 0.2 « Further reduction by 2 mm.

(b) Control 0.2 " Slight contrac tion; maximum was 2 nun.

4 (a) 1 meg. 23 Control (washed) 0.2 " iNo e ffect

Cb) 1 fl 21 0.1 " Reduction of con + traction by 4 mm. 0.1 ” Further reduction by 1 mm only.

‘Numbers from the original research notebook 7IGURE 1?

Antagonistic effect o f D (-) pseudoephedrine against norepinephrine induced contraction o f the rabbit aortic strips.

Control - - - - - On separate aortic strips from the same animal.

9^ D(-)y EPHEDRINE 0 .2 mg/cc

NOREPINEPHRINE 2 mcg/cc

CONTROL D(-)V EPHEDRINE 0 2 mg/fcc L A 10 min. 96

TABLE XXV

Antagonistic Effect of D (-) Pseudoephedrine Against D (-) E^phedrine on the Rabbit Aortic Strip

At the h eight A ortic Contrac of contraction Rabbit S trip D ( - ) Ebh. tio n in D (-) pseudo E ffe c t No.* No.* (mg/cc) mm eph. (mg/cc) Observed

5 (a) Control 0.40 Gradual contrac tion observed; maximum was IB mm

6 Ca) 0 .0 5 60 0 .2 Gradual reduction of contraction; maximum was 25 mm in 20 m inutes.

(b) 0 .0 5 42 0.2 Slight temporary termination by 2- 3 mm., then in creased contrac tion (10 mm. more

7 (a) 0.02 25 0.1 Reduction of con traction by 6 ram.

(b) 0 .0 5 22 0 .0 5 Negligible re duction

(d) Control 20 Gradual contrac- tio n ; maximum was 6 mm.

♦Numbers from the original research notebook FIGURE 18

Antagonistic effect of D (-) pseudoephedrine against D (-) ephedrine on rabbit aortic strips.

Control -----D(-) pseudoephedrine did not have any effect.

97 D(-ty EPHEDRINE 0-1 mg/cc

D(-) EPHEDRINE 0 02mg^cc

i______i 10 minutes 99

Blocking of the B.P. and H.R. effects of P (-) eohadrlne

(0.33 mg/kg) bv ore-treatment with different

doses of D (-) pseudoephedrine

these preliminary experiments were done to test the blocking ability of D {-) pseudoephedrine towards D (-) ephedrine. It was observed that as the doses of D (-) pseudoephedrine were increased, there was an increased tendency for blocking D (-) ephedrine. It completely blocked the pressor, H.R. and respiratory effects to D (-) ephedrine at 16.5 mg/kg. lhe results are summarized in Table XXVI and illustrated by Fig. No. 19* Similar blocking was seen In spinal cats. Results are summarized in Table XXVII.

Blocking the B.P. and H.R. effects of D (-) ephedrine.

L (+) ephedrine. L (+) pseudoephedrine and D (+)

amphetamine by 30 minute pre-treatment with

P (-) pseudoephedrine (3.3 mg/kg) in dogs

Here again the pressor responses, which were used to compare potency of the isom ers, served as co n tro l. Hence the same doses were utilized in E (-) pseudoephedrine pre-treated animals. The pressor and heart rate effects were markedly reduced. The results are sum marized in Table XXVIII and illustrated graphically in Fig. No. 20. TABLE XXVI

Blocking the Pressor Response and H.R. Effect of D (-) Ephedrine (0.33 mg/Kg) by D (-) Pseudoephedrine in Dogs

Rise in B.P. to Dose of Time interval D (-) Efc>h. when Heart Rate D (-) Pseudo between the given after D (-) (beats/min.) Obs. Dog Ephedrine injections Pseudo Ephedrine Before After D if No. No.# (mg/kg) (lain.) (ram Hg) D (-) Efch. D ( -) Efch. ference

1 E (25) 0.33 30 40 190 230 40 2 E (46) fl n 30 200 250 50

3 E (4?) n 80 170 240 70

4 E (59) 3.3 90 30 190 210 20 5 E (23) 9.9 50 20 230 230 0

tf n 6 E (36) 38 - -

7 E (19) 16.5 30 10 220 220 0 8 E (17) 9.9 10 5 230 230 0 1 +9.9

9 E (1 8 ) 9.9 30 5 Complete block +9.9 ♦Numbers from th e o r ig in a l research notebook FIGURE 19

Reduction of D (-) ephedrine induced pressor effects by pre-treatment with different doses of D (-) pseudoephedrine. B.P. ------Upper tracing Respiration - - - Lower tracing H.R. ------Indicated by numbers above the blood pressor tracing Dog A ----- C ontrol " B ----- Pre-treated with O.3 3 mg/kg o f D (-) pseudoephedrine " C ----- Pre-treated with3 .3 mg/kg o f D (-) pseudoephedrine " D ----- Pre-treated with 16.5 mg/kg of D (-) pseudoephedrine Time Marker - - - Ten m inutes

101 b p . A B rirr Mg

LOO- CONTROL 260 'SO* -

'20 ■—

' 0 0 -

S0“ 6Cr~ ■£Ci ■ D(-) EPHEDRINE D(-) EPHEDRINE DC-) EPHEDRINE D(-) EPHEDRINE 0 33 rry.Vy 0 33 mg/kg C 33fT)j.'kg " nt— 103

TABLE XXVII

Blocking the Pressor Action of D (-) SJphedrine (0.33 mg/kg) by D (-) Pseudoephedrine In Spinal Cats

Dose o f Rise In B.P. to D (-) Ephedrine Obs. Cat D (-) Pseudo E&h. when given 30 minutes after No. No.* ( mg/kg) D ( . ) pseudo Efch. (mm Hg)

1 # 2 0.33 75 2 # 5 3.3 26 3 # 7 n 20

4 # 9 13.2 10 5 #13 tt 3h 6 #10 26. h 10

7 #U 5 The average B.P. rise to D (- ) Ephedrine In h control cats was 73.75 (S.E.+ 6.2*0 mm Hg, from Table X.

♦lumbers from the original research notebook 104

TABLE XXVIII

Blocking the Pressor Response and H.R. of D (-) Ephedrine, L (+) E£>h- edrine, L (+) Pseudoephedrine. D ( 4 -) Amphetamine, by 30 Minutes Pre-treatment of D (-) Pseudoephedrine (3.3 mg/kg)

Rise in H.R, D iffer Obs. Dog Dose B.P. (beats/ain.) ence (beats No. No.* Drug (mg/kg) (mm Hg) Before A fter per min.)

1 E (24) D (-) Efc>h. 0.33 18 165 170 5 2 E (W) 11 r» 20 240 270 30 3 E (49) w n 18 240 270 30 Ave. lS.7 2? S.E.+ 0.88 10

1 E( 60)L (+) Ejph. 0.99 10 180 215 35 2 E ( 61) w If 34 190 235 *5 3 E( 62) ti If 215 230 11 Ave. 19.66 32 S.E.+ 9.0 11

1 E (63) L <+) Pseu-. 1.65 10 260 290 30 2 E (64) do E^ph. n 10 2 0 0 260 60 3 E (65) It 12 270 270 0 Ave. 10 30 S.E.+ 0 21

1 E (56) D (+) Amph. 0.3^ 22 240 280 40 O it n A- E (57) 25 220 240 20 it n 3 E (53) 2H , 220 260 40 Ave. 27.24 33 S.E.+ 4.45 O

♦Numbers from the original research notebook FIGURE 20

Reduction of pressor effects of D (-) ephedrine, L (+) ephedrine,

D (+) amphetamine and b (+) pseudoephedrine by 30 minute pre treatment with D (-) pseudoephedrine in anesthetized dogs,

AMPH ------Amphetamine su lfa te EPH ------Ephedrine n ------Number of dogs All doses are mg/kg. Vertical bars indicate standard errors.

1 0 5 B.P CONTROL D(-)y EPHEDRINE (mm.Hq) 3 3 mg/kg . 1 0 0 f -

00 ■5-

40 rh 20

0 n-5 n-4 n-4 n-5 n-3 n-3 n-3 n-3

DHEPH LWEPH. LWy DWAMPH. D(-) EPH. LWEPH uWy D(*) AMPH 0 33 0 99 EPH 0 34 033 099 EPH 0 34 1 65 1.65 107

Blocking the B.P. and H.R. effect of D (-) ephedrine by

30 minute pre-treatment with L (+) pseudoephedrine

L (+) pseudoephedrine, 3-3 mg/kg, shoved an average pressor ef fect of 68,3 ito of Hg in three dogs. Thirty minutes afterwards D (-) ephedrine, 0.33 rag/kg* was injected; both B.P. and H.R. effects of the latter were markedly reduced. Average reduction of B.P. rise was

18.3 (S.E.+ 2.75) nun of Hg and that of H.R. increase was 7 (S.E.+ *0 beats per minute. In control experiments the average B.P. rise for

D (-) ephedrine was 7^ (S.E.+ 2.7^) mm of Hg and H.R. was 108 (S.E.+ 7) beats per minute.

Blocking the B.P. and H.R. effect of D (■) ephedrine.

0.33 me/kg. by 30 minute pre-treatment with

L (+) ephedrine. 3.3 mg/kg

In three experiments, the pressor response to L (+) ephedrine,

3*3 mg/kg, was 81.7 mm of Hg. I f D ( - ) ephedrine was given 30 minutes afterwards, the B.P. and H.R. response was much less than normally seen. The average rise for D (-) ephedrine of 7^ (S.E.+ 2.7^) mm of

Hg was reduced by 26.6 (5.E.+ ^.15) mm of Hg,

RESULTS (PART D)

B.P. effect of D (-) pseudoephedrine in anesthetized cats

Doses of 3*3 to 13.2 were administered in chloralized cats. At lower doses a biphasic response was obtained. But at higher doses It 108 was depressor only. The depressor response was immediate after the injection, and its magnitude and duration was proportional to the dose administered. Table XXIX summarizes the results.

E ffect of D (-) pseudoephedrine (9 .9 mg/kg) when

repeated after one-half hour

A total of four observations were made in three barbitalized and one nembutalized dog. The average B.P. fall to the first injection was 33.75 (S.E.+ 6.35) nun of Hg and 52.5 (S.E.+ 9.11) mm of Hg to that of the second injection. According to the "tn test, this dif ference is not significant. The heart rate did not increase when the second dose was administered, indicating tachyphylaxis. The average time required for B.P. to return to normal level after the first in jection was 1.6 minutes and 5.3 minutes after the second injection.

Potentiation of epinephrine by D (-) pseudoephedrine

In two dogs, a dose of 0.33 mg/kg of D (-) pseudoephedrine ap parently did not modify the pressor effects of 0.75 mcg/kg of epine p h rin e. The dose o f 3*3 mg/kg of D ( -) pseudoephedrine p o te n tia ted the epinephrine (1,5 mcg/kg) pressor effects. Such a potentiation was seen much better ^5 to 60 minutes after administration of D (-) pseudoephedrine; potentiation was not observable 10-20 minutes after administration. Increased doses of D (-) pseudoephedrine (9.9 mg/kg) reduced duration and magnitude pressor effects to epinephrine given 109

TABLE XXIX

Blood Pressure Response to D (-) Pseudoephedrine in Anesthetized Cats

B.P. Effect Dose o f (mm Hg) Obs. Cat D (-) Pseudo - F all Duration No. No.* E£h. (mg/kg) + Rise (minutes) Remarks

1 /2 3.3 - 10 1 Biphasic + 10

2 h Tt - 20 1 Biphasic + 20 3

3 #i If - 25 2 Depressor only

U #3 6.6 - 30 1 Biphasic + 10 3

5 & 13.2 - 60 15 Depressor only

6 t t TT - 70 10 Depressor only

♦Numbers from the original research notebook 110

10-20 minutes after. The results are summarized in Table XXX and i l lustrated graphically in Fig. No. 21.

Potentiation of norepinephrine pressor effects by D (-) pseudoephedrine

At a dose of 0.33 «g/kg of D (-) pseudoephedrine, the pressor effects of norepinephrine (0.4 mg/kg) were apparently not potentiated. But when the dose of D (-) pseudoephedrine was 3.3 mg/kg, the poten tia tio n became apparent. Again such a potentiation was seen much better 45 to 60 minutes after D (-) pseudoephedrine. In a control experiment when the epinephrine and norepinephrine was repeated over a one hour period, the pressor effects were not changed. The resu lts are summarized in Table XXXI and illustrated graphically in Fig. No. 21.

Potentiation of epinephrine and norepinephrine by D (-) ephedrine. L (+) ephedrine and L (+) pseudoephedrine

The pressor effect of epinephrine (1.5 mcg/kg) was potentiated by 0.33 mg/kg of D (-) ephedrine in one out of three experiments. In the other two experiments only the duration was increased. L (+) ephedrine, 0.99 mg/kg, potentiated epinephrine (1.5 mcg/kg) and nor epinephrine (0.4 mcg/kg) in two experiments. Such a potentiation was also seen after 3*3 mg/kg of L (+) ephedrine. L (+) pseudoephedrine potentiated both epinephrine and norepinephrine In all three experi ments . I l l

TABLE XXX

Potentiation of Epinephrine Pressor Effects by D (-) Pseudoephedrine

B.P. rise to epine Control Dose of phrine after D (-) Dose r ise in D ( -) Pseudo pseudo ephedrine Obs. 2og of Epi. B.P. Ephedrine (mm Hg) ( Tirae-min.) No. No.* (mg/kg) (rain Hg) (rag/kg) 10-20 *4-5-60

1 E (W ) 1 .5 *4-8 > • J *4-0 65 2 E (50) fl 66 tt 6U- 35 3 E (59) 1? *4-0 ■1 US 50 4 E (61) 11 58 »» 5 6 5 E (6*0 1* If 22 Avo. & S5 si: 65 S.E.+ 6.12 8.60 12.56

1 E (17) 0.75 26 9.9 26 Duration E (IS) n 2*4- T» 5 Reduced 3 E (21) n 25 11 10 11 Ur E ( 23) n UO rt 19 If Ave. 23.75 17.75 3.E.+ ^.36 7.01

♦Numbers from the original research notebook FIGURE 21

Effect <: )f D (-) pseudoephedrine on the pressor responses to epinephrine and nor* epinephrine. KPI Epinephrine NOREPI ----- Norepinephrine n------Number of dogs A ------Epinephrine and norepinephrine given 10 to 20 minutes after D (-) pseudo ephedrine 3 ------Epinephrine and norepinephrine given ^5 to 60 minutes after D (-) pseudo ephedrine All doses are in mg/kg. Vertical bars indicate standard errors.

112 BP CONTROL. D(-)t+> EPHEDRINE. ( mmHg) 3 3 mg/kg 1 0 0 r © B

* 40 £

n-5 n-4 n-5 n-4 n-3 n-4

EPI. NORER. EPI. NOREPI EPI NOREPI. 1.5 0 4 15 0 4 1.5 0 4 114

TABLE XXXI

Potentiation of Norepinephrine Pres3or Effects by D (-) Pseudoephedrine

3.P. rise to norepi. Control Dose of after D (-) pseudo Dose o f r is e in D (-) Pseudo ephedrine

Obs. Dog Norepi. ^—' * rP ♦ Ebhedrine (mm Hg) ( Time-min.) No. No.* (mg/kg) (nutt Kg) (mg/kg) 10-20 45-60

1 E (24) 0.4 50 3.3 4 8 5 6 2 E (48) " 40 If 4 0 6 6 II 3 E (50) " 40 3 5 7 5 4 E (59) n 40____ tt 4 8 P— Ave. 42.50 ^ 7 .7 5 6 5 7 5 0 S.E.+ 2.94 3 .5 4 4 . 4 9

1 E (17) 0.4 45 9 . 9 45 Duration 2 E (IS ) " 30 « 20 Increased 3 E (21) " 40 n 4 0 ft 4 E (33) " 60 « t* Ave. £ 3.75 § h o S.E.+ 7.34 1 3 . 4 7

1 E (19) 0.4 38 16.5 4 8

♦Numbers from the original research notebook 113

Potentiation of isoproterenol (2 mcg/kg) by

D (-) pseudoephedrine (9.9 mg/kg)

In two experiments Isoproterenol produced an average control 3.P. fall of ^7 mm of Hg. Then after D (-) pseudoephedrine it produced an average 3.P. fall of mm of Hg. The duration was also increased after D (-) pseudoephedrine.

Effect of D (-) pseudoephedrine on

bilateral c.o. pressor effect

D (-) pseudoephedrine, in doses of 3.3 9.9 mg/kg, did not block the c.o. pressor effect. Cn the contrary, there was some in crease of this reflex pressor effect. The results are summarized in

Table XXXTT and illustrated by Fig. No. 22.

Effect of D (-) pseudoephedrine on the stimulation of

the cervical sympathetic nerve and N.M. of the cat

In two control experiments, stimulation o* the cervical sym pathetic nerve over ^3 to 60 minutes did not show any significant change In the contractions of the N.M. The cats which were treated with 3*3 showed a slight reduction of the contraction ofN.M. after ^3 minutes. Increased doses of 6.6 and 13.2 mg/kg did not re duce the contraction of the N.M., but the duration of relaxation was increased. D (-) pseudoephedrine itself did not show ar^y apparent e f f e c t on N.M. contraction. The results are tabulated In Table XXXIII and illustrated by Fig. No. 23. 116

TABLE XXXII

Effect of D (-) Pseudoephedrine on C.O. Pressor Response

Dose of Rise In B.P. to c.o. D (-) Control rise after D (-) pseudo Pseudo in B.P. ephedrine Obs. Dog Efch. to e.o . (mm Hg) No. No.* Anesthetic (mg/kg) (mm Hg) (Time - min.) 10 15 30 45

1 E (79) Nembutal 3.3 50 50 - - -

2 E (82) B arbital ft 32 30 30 23 38

3 E (85) Nembutal tl 60 85 80 30 85

4 E (86) Nembutal 9.9 36 36 50 68 75

5 E (93) B arbital n 45 36 - -

♦Numbers from the o r ig in a l research notebook FIGURE 22

Effect of D (-) pseudoephedrine on the c.o. pressor responses.

C.O. - - - - - bilateral carotid occlusion for 30 seconds Time - - - - - Ten n in u tes

117 BP ' mm Hy) r--4G -J03 —OT)

— oC ) - ‘•-0 I V J20 - ’O' -6 —TO i t CO W-K*) EPHEDRINE CO CO CO CO - 4 0 3 3 mg/kg -20 _J______I______1 .

o 119

TABLE m i l l

Effect of D (-) Pseudoephedrine on the Stimulation of the Cervical Sympathetic Nerve and N ic tita tin g Membrane (N.M.)

Hei^vt of N.M. Contractions (mm) Dose of D (-) Control After D (-) Pseudo ^>h. Obs. Cat Pseudo Eph. (tim e-m in.) No. No.* (mg/kg) 10 30 ^5 60

1 # 6 Saline 68 70 66 66 67

2 t 7 H i+0 *+o 39 -

3 # 2 3.3 52 55 50 **0

b # 5 n 60 58 b6 ^5 33

5 # 3 6.6 53 51 50 52 &

6 * U 13.2 y \ 3 6 35 Ul kb

7 *L3 ft 60 - 52 - -

♦Numbers from the original research notebook FIGU RE 2 3

Effect of D (-) pseudoephedrine on the N.M. contractions induced by stimulation of cervical sympathetic nerve.

Upper and lower tracings were made from two dif ferent cats. •Stimulation of the cervical sympathetic nerve. Time Marker ----- In minutes.

1 2 0

122

Effect of diphenhydramine (5 mg/kg) on the depressor

response to histamine (2 meg/kg) isoproterenol (2

mcg/kg) and D (-) pseudoephedrine (9>9 mg/kg)

At these doses, histamine, isoproterenol, D (-) pseudoephedrine produced an approximately equal depressor response. After diphen hydramine, depressor response to histamine was markedly reduced,but

responsesto isoproterenol and D (-) pseudoephedrine were somewhat potentiated. The results are presented in Table XXXIV and illus

trated by Fig. No. 2h.

Effect of D.C.I. (10 mg/kg) on the B.P. depressor

response to Isoproterenol (2 mcg/kg) and

D (-) pseudoephedrine (9.9 mg/kg)

The depressor effect to isoproterenol was markedly reduced or blocked by D.C.I., but the depressor response to D (-) pseudoephedrine was not affected. The effect was essentially the same as in control

animals. The results are summarised in Table XXXV.

In Dog E (27), D.C.I., 5 mg/kg, caused a marked increase in H.R.

Control r a te was 115 beats per m inute; a fte r D .C .I. i t became 190 beats per minute. In Dog E (26) the dk>se of D (-) pseudoephedrine

(9.9 mg/kg) caused an increase of H.R. from 125 beats per minute to

170 beats per minute; but when D.C.I., 10 mg/kg, was given, the H.R. was reduced to 150 beats per minute only. In Dog E (92), D.C.I.,

10 rag/kg, had very little effect on heart rate; it conpletely blocked TAELE XXI7

Effect of Diphenhydramine (5 mg/kg) on Depressor Responses to Histamine, Isoproterenol, and D (-) Pseudoephedrine

Control Depressor Responses Depressor Responses After ( m Hg) Diphenhydramine (5 mg/kg) Obs. Dog Histamine Isoproterenol D (-) Pseudo E^ph, Histamine Isoproterenol D (-) Pseudo Efch. No. No.* (2 mcg/kg) (2 mcg/kg) (9.9 mg/kg) (2 mcg/kg) (2 mcg/kg) (9-9 mg/kg)

1 E (7?) 44 — Values 10 - - taken 2 E (?8) 35 from • 45 35 Table 3 E (87) 44 36 IX 8 40 65

4 E (38) 36A — 10 44 70 Ave. 41733 3^.33 3 ? M 9.33 43.00 56. 6^ S.E.+ 2.75 1.28 5.03 0.70 1.57 11.14

♦Numbers from the o r ig in a l research notebook

fO VjJ FIOITCE 2'4

Effect of diphenhydramine on depressor responses to D (-) pseudoephedrine in anesthetized dogs.

HISTAMINE ...... 2 mcg/kg ISU Isoproterenol, 2 mcg/kg D (-)¥ 0?H - - - - D (-) pseudoephedrine, 9.9 mg/kg n ------number of dogs Vertical bars indicate standard errors. HISTAMINE ISU. Dt-X*? EPH HISTAMINE. ISU. CX-Xf) EPH

n-3 n-3 n-7 n-3 n-3 n-3

2D-

40 i

80 CONTROL DIPHENHYDRAMINE B P. FALL ( m m H g ) (5 mg/kg) 126

TABLE XXXV

Effect of D.C.I. (10 rag/kg) on the Depressor Response to D (-) Pseudo ephedrine (9.9 mg/kg)

Control Fall in 3.P. FaU in 3.P. after D.C.I. mm Hg mm Hg; LO mg/kg Isoproterenol I ) {-) Pseudo Isoproterenol D (-) Pseudo Obs. Dog Ephedrine Ephedrine No. No.* 2 mcg/kg 9.9 mg/kg 2 mcg/kg 9.9 mg/kg

1 E (92) 42 These 5 40 values 2 E (29) 50 taken 0 30 from "5 J E (28) 50 Table 5 40 IX 4 E (2?) ^0____ Ave. 47.33 y f t t 3.30 35 3.E.+ 3.30 5.03 2.10 3.40

♦Numbers from the original research notebook 127 the H.R. effect of isoproterenol and reduced the H.R. effect of D (-) pseudoephedrine; e.g., after D (-) pseudoephedrine, H.R. only in creased from 150 beats per minute to 160 beats per minute.

Effect of D (-) pseudoephedrine in spinal cats

Infused with norepinephrine

Three spinal cats were infused with norepinephrine at the rate of

2 to k mcg/kg per minute. During the infusion, different doses of

D (-) pseudoephedrine were administered. In almost all the cases,

3.P. fall was observed. Its duration and magnitude was dependent on the dose of D (-) pseudoephedrine. Table XXX’/I summarizes the re sults. Fig. Ho. 25 shows the comparative effects of D (-) pseudo ephedrine in anesthetized, spinal, and spinal cats infused with nor epinephrine.

Effect of D (-) pseudoephedrine after

hexamethonium C 5 mg/kg)

This was studied in dogs anesthetized with either nembutal or barbital. Fifteen or 20 minutes after the hexamethonium, D (-) pseudo ephedrine was given in doses of 3*3 or 6.6 mg/kg. In all experiments, a rise of 3.P. was obtained. Its magnitude varied from animal to animal. Under similar conditions, the pressor effects of I (-) ephedrine, 0.33 «g/kg, were increased. The results are summarized in

Table XXXVII and illustrated in Fig. Mo. 26. 128

TABLE XXXVI

Effect of D (-) Pseudoephedrine in Spinal Cats Infused With Norepinephrine

Rate of First Injection of Second Injection of Norepi. D (-) Pseudo Eph. D (-) Pseudo E£h. Obs. Cat infusion Dose Fall in B.P. Dose Fall in B.P. No. No.* (mcg/kg/rain) (mg/kg) (mm Hg) (rag/kg) (mm Hg)

n 1 #21 c. 3-3 20 13.2 40

2 #23 3 n 26 26.4 100

3 #22 4 6.6 24 13.2 4 5

♦Numbers from the original research notebook FIGURE 25

Comparative effects of high doses of D (-) pseudoephedrine in anes thetized cat, spinal cat, and spinal cat infused with norepinephrine.

Time Marker - - - - Ten minutes

129 (mm Hg) ANESTHETISED CAt SRNAL CAT j-200 INFUSED WITH NOREPINEPHRINE - 1 0 0 -160 -1 4 0 -120 -100 -8 0 - 6 0 - 4 0 V 1-20 EPHEDRINE ______1______26 4 my «9 , ,V EPHEDRINE 26 ■imgi'tiy TABLE XXXVII

Effect of D (-) Pseudoephedrine after Hexamethonium Chloride

Dose of Dose of Sxpt. Dog hexamethonium D ( - ) Pseudo Efch. E ffects t o . No.* A nesthetic (mg/kg) (mg/kg) Observed

1 3 (1) Nembutal 5 - Fall of blood pressure, which per sisted for 2 hour observation fl If 2 B (3) - p erio d .

3 B ( 2 ) rr Tf 3.3 Gradual rise in blood pressure, which persisted for 1 hour ob servation period.

Ur B (73) B arbital ft If Gradual rise in B.P. which persisted fo r 1 and § hour. H.R., which was 120 beats/min., gradually increased to 210 at end of observation period.

fl It 5 3 ( 76 ) 6 .6 Gradual rise in B.P. which persisted fo r 3 hour observation period. H.R., which was 90 beats/m inute, changed to 170 beats/minute at end of observation period.

♦Numbers from the original research notebook FIGURE 2 6

D (-) pseudoephedrine after hexamethonium.

Upper Tracing ------Blood pressure Lower Tracing ----- Respiration Heart Rate (beats/m in.) Indicated by numbers above the blood pressure tracing Time Marker ----- Ten minutes

1 3 2 ,

Hexamethonium Cl C*-** EpheQnne t bmg /kg) (3 3mg /kg 1

Respiration

i IJb

Effect of acute pre-treataent of reserpine Cl mg/kg) on

the B.P. effects of D (-) ephedrlne and vice versa

The observations were made on a total of seven dogs. The animals were injected with D (-) pseudoephedrine (3 .3 mg/kg); then 10 minutes

afterwards, reserpine was injected. An average pressor response of

70 mm of Hg was observed. It was gradual in nature and prolonged in duration (about 30 minutes). If a second dose of reserpine was then injected, it was depressor only. Dibenzyline (5 mg/kg) reduced the pressor responses promptly. Similarly, if reserpine was injected one hour after D (-) pseudoephedrine, the same pressor effect was ob

served, but it was of greater intensity.

In another series of animals, reserpine was injected first; then

10 minutes afterwards, D (-) pseudoephedrine (3 .3 mg/kg) was Injected.

A prompt pressor effect was observed (average of 100 mm of Hg in two

experiments). This pressor effect was also promptly reduced by di benzyline, 5 mg/kg. If D (-) pseudoephedrine was repeated after the

first pressor reaction, it also became a depressor (see Fig. No. 27). FIGURE 27

Effect of acute reserpine pre-treatment on G (-) pseudoephedrine (ar.< vice versa); followed by dibenzyline.

A , B ------Different dogs Time Marker ------Ten minutes

135 5 0mg/kg. DIBENZYLINE 5 0mg/kg. 3 3 mg/kg 3 DIBENZYLINE DIBENZYLINE EPHEDRINE 1.0 mg/kg RESERPINE D(-Xj) 1.0 mg/kg. 3 ■ 3 mg/kg3 ■ 3 e p h e d r i n e RESERPINE. > > D (-)4 140 140 -200 - 6 0 —120 — —220 —200 —180 —160 CD € CHAPTER IV

DISCUSSION

Molecular architecture has a profound influence on biological activity. In asymmetric molecules, it is the absolute configuration which is most important in understanding the action of drug3 at the receptor level. The optical rotation associated with asymmetric molecules has nothing to do with its pharmacological effect. The four optical isomers of ephedrine, essentially four different mole cular species, whose absolute configurations are known (62,63), pro vided some very interesting pharmacological data.

Comparative potency

The LD^q values did not differ significantly, although the pressor a c t iv it ie s d iffe re d over a wide range. This was somewhat unexpected as the m oct active pressor Isomer in other a e ries o f compounds has been shown to be more toxic than corresponding antimers (h5.h9. 5O).

The intravenous values reported here compare well with previous in t raperl tonal determinations by Shinamoto et a l . (66). The com parative pressor potencies of D (-) ephedrine, I ( + ) ephedrine and 1 (+) pseudoephedrine were quite similar to those reported by Chen et a l.

(65), except that D (-) pseudoephedrine was observed to be a depressor in anesthetized dog3. The depressor action of D (-) pseudoephedrine found here is al30 contrary to the report of Chiaajnoto et al. (66),

1 3 7 138 who claimed it to have l/l 6th of the pressor effect of D (-) ephedrlne.

These apparently conflicting reports may be due to the fact that dif ferent criteria were used for studying the potency of these two diasterio-isomers, Shiaamoto et al. ( 66) used the minimum e ffe c tiv e doses for such comparative purposes. The validity of such methods is questioned. According to the results obtained in the present study, i t was shown th a t a t low doses D ( -) pseudoephedrine may show a s lig h t pressor effect in anesthetized dogs, but when the doses were increased,

D (-) pseudoephedrine was depressor only. The results obtained over wide dose ranges are more valid. Hence it is a depressor agent rather than a pressor one in anesthetized dogs and cats. This conclusion is also supported by previous findings that D (-) pseudoephedrine in creases the blood flow to all parts of the body of the anesthetized dog (7 2 ).

Due to the depressor effects of D (-) pseudoephedrine in the anesthetized dog, it was not possible to con^are it with other iso mers. Hence it was coi^ared in spinal cats where it was found to be pressor. The pressor potency of D (-) ephedrlne to D (-) pseudo ephedrine was shown to be 106:1. This value does not agree with the one th a t was observed by Chen e t a l . ( 65), who reported it as 35:1.

Here again these differences in values are due to the fact that Chen e t a l . ( 65) conpared the values of these at one dose level, while here it was conpared over a wide range of doses. We believe that the comparative pressor value of 106:1 is more valid. 139

The duration of pressor effect was just the reverse of the pressor potency. The least active isomer was observed to have the longest duration of action at equipressor doses. The latent period for pressor action was maximal with L (+) pseudoephedrine. A similar latent period of 30 minutes was also observed for H.R. effects of D (-) pseudo ephedrine (3*3 mg/kg). The importance of these observations cannot be assessed u n til and unless a drug k in etic study is made. However, similar facts which were observed previously are worth mentioning here. Among the four isomers, the least active pressor isomer has the most powerful hyperglycemic effect, and the most active pressor isomer has the least hyperglycemic action. An interval of about 30 minutes is required to observe the full hyperglycemic effect of the least active pressor isomer, D (-) pseudoephedrine C73)• Light et al. (72) also observed a long latent period for the diuretic effect of D (-) pseudo ephedrine. All these findings suggest a slow metabolic change. The rates of demethylation of these isomers will be of great interest in regard to these observations.

Mechanism of action of optical isomers of ephedrlne

It is apparent from the results that D (-) ephedrlne has less tendency for tachyphylaxis to its pressor effects as compared to L (+) ephedrlne, L (+) pseudoephedrine and D (+) amphetamine. This observation is quite consistent with the previous study of Winder et al. (15), that D (-) ephedrlne has less tendency for tachyphylaxis as conpared to other phenylpropylamines. In the present study, doses of D (-) ephedrlne and D (+) amphetamine ware equimolar, but that of L (+) ephedrlne and

L (+) pseudoephedrine were 3 and 5 times higher respectively. So it may be argued that early development of tachyphylaxis to L (+) ephedrine and I< (+) pseudoephedrine might be due to early receptor saturation due to relatively larger doses. But the fact remains that even after an animal is conqpletely tachyphylactic to L (+) ephedrine,

L (+) pseudoephedrine or D (+) amphetamine, D (-) ephedrine can still produce a pressor effect. These results can be explained on the basis that D (-) ephedrine has both direct and indirect action (* « g ., d ire c t action on effector structure, as well as liberation of catecholamines), while L (+) ephedrine, L (+) pseudoephedrine and D (+) amphetamine are only indirect actors (as they are irreversibly antagonised by reserpine pre-treatment). The receptors which are responsible for in direct action become saturated by L (+) ephedrine, L (+) pseudoeph edrine and D (+) amphetamine, and this is manifested by development of tachyphylaxis. But the direct sites are not saturated because

I) (-) ephedrine can still show some pressor effects, even though the animal is completely tachyphylactic to L (+) ephedrine, L (+) pseudo ephedrine or D (+) amphetamine. In contrast to D (-) ephedrine, when

L (+) ephedrine was repeated after complete tachyphylaxis to L (+) pseudoephedrine, it was depressor only.

The question arises as to what makes D (-) ephedrlne so potent a pressor agent, as compared to the other isomers. The answer is not simple, but it can be explained best by the recent, thought-provoking theory of Paton ( 87 ). It states that drug action on a receptor lh l depends on the rate at which it associates (k^) ani dissociates (k 2) with the receptor. Belleau (86) has proposed that sympathomimetic amines act at receptors by ion pair formation. First, the positive charge on the nitrogen reacts with the anionic site on the receptor; then such an ion pair is stabilized by possible hydrogen bonding through the hydroxyl group on the beta carbon of the phenylethylamine.

When the molecular model of D (-) ephedrine is viewed according to the above theory, then the steric repulsion between the cis oriented methyl group with the phenyl ring is quite apparent (Fig. No. 28), and subsequently ion pair formation will not be stabilized. This will lead to rapid dissociation of such molecules from the receptor (e.g.,

D (-) ephedrine will have a high k2 value in terms of Paton's theory

(87). Hence, it will have higher activity. Such will not be the case with D (-) pseudoephedrine; in this isomer the ion pair formation

can be relatively stable. Hence, there is a possibility of a low k2 value, and subsequently less action. These speculations are highly theoretical; hence their use in explaining the drug action is done with some reservations.

It seems from the work of Axelrod (89) that not much importance

should be attached to Gaddum's theory (9) fo r the mechanism o f action of ephedrine, because amine oxidase does not play an important role in

the metabolism of catecholamines. Subsequently, the problm has been

approached differently. The use of reserpine pre-treatment has become well recognized as a method for elucidating the mechanism of the

direct and the indirect acting amines. It was well established FIGURE 28

Optical isomers of ephedrine and possible conformations (through the courtesy of Dr. LaPidus). CH:

H — C—NHCiHt hC-- 'OH I D (-) Ephedrine H— C—Oh ch- 1 (nrythro;

H 'NHC rl-

C Ho i

CH3 HN —C—H D(-) Pseudoephednm 'OH H — C -OH H three*'

C H' 'NHCH;

L '• t- ; he'lripp

NHL H

CH, I H —C~NHCHt

I 3 i. ■■') Pseudoepnedrme CH HO — C—H ; t pf P' }

H NHCH previously and confirmed in the present study that D (-) ephedrine has both direct and indirect modes of action (21,23,31,32). Further, the sympathomimetic amines were classified into three groups after reserpine (26), Ritalin (27), guanethidine (28), cocaine (21) pre-treatment but asymmetry of molecules was never considered in these studies. Without minimizing the importance of this work, it can be stated that the configuration of asymnetric synpathomimetic amines is of the ut most importance. For example, the L-configuration in L (+) ephedrine and L (+) pseudoephedrine seams to result in an indirect mode of action since both compounds are irreversibly antagonized by reserpine pre-treatment. In D (-) pseudoephedrine, the heart rate effect was blocked by reserpinization; hence there is a possibility of some indirect action. But it also seems to conpete for direct receptors

(as will be discussed later). In this connection, it Is very inter esting to point out the observations which were made by Burn (25) that reserpinization in spinal cats completely blocked the pressor effects of D (+) phenolethanolamine, but not those of D (-) phenolethanolamlne

From these observations it seems logical to conclude that it is the D-configuration of the hydroxyl group which is (at least partly) re sponsible for direct action.

Further, the results could be considered according to the theory of Easson and Stedman (56) and Blaschko (57) that there is a possi bility for three point contact with a receptor by D (-) ephedrine,

D (-) pseudoephedrine and D (-) phenolethanolamine through the benzene ring, the beta hydroxyl group and the amino group. But in the case of 1*5

L (+) ephedrlne, L (+) pseudoephedrine and L (+) phenolethanolamine, due to different orientation of the beta OH group, only a two point contact can occur with the receptor. This is Illustrated by Fig. No. 28. When the data is considered according to this theory, then the question arises whether a three point contact is associated with direct-acting amines and a two point contact with Indirect-acting amines. Such a possibility is quite obvious from the results ob tained. Of course such an interpretation should be restricted to the phenylethylamines only. The depressor action of D (-) pseudoephedrine in anesthetized dogs and cats was of interest, and hence the possible mechanism of this depressor action was studied. The first approach was to try to block the depressor action with D. C. I., which has been shown to block depressor response to isoproterenol (90). Ihe attempt was unsuccessful. It is unlikely therefore that the depressor response is due to stimulation of beta receptors. The antihistaminic agent diphenhydramine did not block the depressor response to D (-) pseudo ephedrine. It is therefore unlikely that the depressor response is histaminic in nature. Further, such a depressor response could not be cholinergic because it was obtained in atroplnized animals. In the spinal cat, no depressor effect was observed; but at higher doses a pressor effect was seen. On the isolated aortic strip, a slight contraction was seen. In both instances, in the spinal cat as well as the ao rtic s tr ip , when the tone of these smooth muscles was main tained at a high level, a depressor effect was observed. It seems 1 ^ 6

from these results that depressor action of D (-) pseudoephedrine de pends on a relatively high sympathetic tone in the blood vessels*

Previously, Burn (25) has proposed that there is a continuous leakage of neurohormone, from its stores in sympathetic nerve endings,

to the receptors so that smooth muscles of blood vessels maintain a high sympathetic tone. The action of D (-) pseudoephedrine then can be Interpreted as that of a competitor for the neurohormone, nor

epinephrine, at the receptor site. In anesthetized animals, where blood vessels retain a good sympathetic tone, administered D (-) pseudoephedrine competes with norepinephrine, and B.P. fall is ob

served due to occupation of these norepinephrine receptors by D (-) pseudoephedrine, which lacks intrinsic activity. The theory that

affinity and intrinsic activity are not necessarily parallel has been persuasively argued by Stephenson (9l) and Ariens et al. (92).

In a reserpinised animal where sympathetic tone is low, the de pressor action of D (-) pseudoephedrine was reduced. In this case

there are a relatively small number of norepinephrine molecules to displace from receptors, and the resultant depressor effect is there fore less. In spinal cats, due to the spinal section, the animal has a very low sympathetic tone. In this case, the administered D (-) pseudoephedrine may still be conpeting for norepinephrine receptors, but another factor, the liberation of catecholamines, may be over

shadowing such competition at receptors. Similar competition for norepinephrine receptors by dopamine has been described by Bum and I®*?

R&nd (93)* Along these lines, it is worthidiile to mention the findings of Chiuchta et al. (77) that D (-) ephedrine compotes more effectively for norepinephrine receptors in the venous heart than L (+) ephedrine, and this might be due to the D-configuration. D ( -) pseudoephedrine fulfils such a requirement for competition of norepinephrine in the present study. But these correlations are hi^ily speculative. On the isolated perfused rabbit heart, D (-) pseudoephedrine showed some depressant effect. Hence the possibility, that part of the depressor action of D (-) pseudoephedrine in anesthetized animals is due to its depressant effect on the heart, cannot be eliminated.

During the study of these isomers, it was observed that D (-) pseudoephedrine, in spite of its causing a fall in B.P., increased the heart rate. Then the obvious question was, does D (-) pseudoephedrine liberate catechol amines? The following observations suggest that there is a great possibility of such a phenomenon.

(1) The heart rate effect was markedly reduced by reser

pinization.

(2) The increase in B.P. in spinal cats can be blocked by

reserpinization.

(3) Depressor effect in anesthetized animals was converted

to pressor effect after pre-treatment with hexamethonium.

The work of Domino and Rech (9*0 demonstrated th a t pre

treatment with hexamethonium is able to make perceptible

the blood pressure rise due to release of catecholamines

by reserpine. \ b S

(h) Acute pre-treatment with either D (-) pseudoephedrine or

reserpine and subsequent treatment with the other one of

the pair produced a marked pressor effect, which could be promptly reduced by adrenergic blocking agents* This raises the possibility of enhanced release of catechol

amine stores by these agents under the influence of each other. A similar phenomenon was observed with reserpine

and 7-(N-beta-methylphenethylaminoethyl) theophylline Hd

by Tdnosky et al. (95)* It might be argued that the pressor effect of reserpine after D (-) pseudoephedrine might be due to amine oxidase inhibitory pro perties. Previously such a phenomenon has been observed with amine oxidase inhibitors ( 9 6,9 7 )» but such a possibility is unlikely be cause compounds with alpha methyl groups have been shown to be immune to amine oxidase (8).

Effect of D (-) pseudoephedrine towards other pressor amines

Previous studies of LaPidus et al. (78) showed that D (-) pseudo ephedrine could block the pressor effects of D (-) ephedrine in the anesthetized dog, and proposed that D (-) pseudoephedrine might act at the same receptors as D (-) ephedrine. Further, it is reported here that the same phenomenon can be observed in anesthetized cats, spinal cats and rabbit aortic strips. It seems from these results that the central mechanism is not an important factor in the prompt 149 reduction of the preeeor effects of D (-) ephedrlne by D (-) pseudo ephedrine* Such an antagonistic action of D (-) pseudoephedrine towards ephedrine is also extended to other organs, such as nicti tating membrane. Since D (-) pseudoephedrine does not affect the contraction of nictitating membrane induced by stimulation of cervi cal sympathetic nerve, it seems more likely that such antagonism results at the effector site. The antagonistic action of 0 (-) pseudo ephedrine could not be well demonstrated to the chronotropic effect induced by D (-) ephedrine. The D (-) pseudoephedrine also probably contributed some action of its own on this effect, since it is well known th a t antagonists g enerally have some weak a g o n istic a c tio n .

The antagonist action of D (-) pseudoephedrine towards D (-) ephedrine depends on the dose used, and it also depends on the organ used. A possible explanation for the prompt reduction of the pressor effects of D (+) amphetamine and D (-) ephedrine by D (-) pseudoephedrine is th a t the f i r s t two compounds ex ert some in d ire c t e f f e c t which r e leases catecholamines that cause a pressor effect by acting at direct effector sites,and that D (-) pseudoephedrine given at the peak of pressor response competes effectively at these effector sites to reduce the response. The competition of D (-) pseudoephedrine for norepinephrine receptors was discussed previously.

The next question to be considered is whether D (-) pseudo ephedrine w ill antagonise the direct component of D (-) ephedrine*s response. To show such antagonism, dogs were reserpinized, resulting in loss of indirect effect, and relatively large doses of D (-) 150 ephedrine were given in order to produce the ease pressor effect that would be obtained in normal anesthetized dogs. It is assumed that in these reserpinized dogs, most of the pressor effect of ephedrlne is now due to a direct component of its action. In these experiments also, the D (-) pseudoephedrine promptly reduced the pressor effects of D (-) ephedrine, indicating that D (-)pseudoephedrine does indeed antagonize the direct pressor effects of D (-) ephedrine. In similar reserpinized dogs, D (-) pseudoephedrine did not (or very slightly) antagonized the pressor effect of vasopressin, suggesting that D (-) ephedrine*s action is specific for the receptors of sympathomimetic amines.

D (-) pseudoephedrine not only reduced the ongoing pressor ef fects of D (+) amphetamine and D (-) ephedrine, but pre-treatment with it also tended to prevent the pressor effects of these agents, as well as L (+) ephedrine and L (+) pseudoephedrine. According to the results obtained at the dose levels studied, D (-) pseudoephedrine did not show any preferential blocking of these above mentioned amines. The mechanism of such blocking action of D (-) pseudo ephedrine may not be the same as in the prompt reduction of pressor response.

This study shows antagonistic action of D (-) pseudoephedrine towards other tachyphylactic amines, such as D (-) ephedrine and

D (+) amphetamine. Recently it has been postulated that the develop ment of tachyphylaxis is due to reduction of norepinephrine release 15L following repeated doses of the agent (98)* D (-) pseudoephedrine seems to be a very weak liberator of norepinephrine, and even such action is very gradual over the period of 30 minutes or so. But even then It is able to block tachyphylactic amines very effectively. So in the development of tachyphylaxis, blocking may be the phenomenon of primary importance, and the release of catecholamine may be sec ondary. Arien's concept about homologous series is well known (99). It is worth testing the least inactive member of the homologous series, for an an tag o n istic action towards the most active compound. Loss of activity is the loss of intrinsic activity, but affinity is main tained; hence antagonism results. Similarly, in optical isomers, the least active isomer has been reported to be antagonistic to its an ti mer ( 50, 53. 55). but this is the first example of two diasterio-isomers, namely D (-) ephedrine and D (-) pseudoephedrine, being antagonistic to each other. The other two diasterlo-isomers, namely L (+) eph edrine and L (+) pseudoephedrine, had considerable intrinsic pressor effect, and no antagonism was perceptible. Ritalin has been reported to block the pressor effects of eph edrine and amphetamine (and allied substances), but it potentiates the pressor response of catecholamines (100,101). Further, it is found to exhibit a pressor effect after hexamethonium (101). Some of these pharmacological properties of Ritalin are very similar to those observed for D (-) pseudoephedrine. On such a basis, the 152 proposed similar configurations of Ritalin and D (-) pseudoephedrine are quite thought-provoking (78)*

In this study some light was thrown on the mechanisms of sym pathomimetic action of the four ephedrine isomers and the antagonistic action of D (-) pseudoephedrine in particular. However, further studies on the metabolism, distribution, excretion and physico chemical properties of these compounds, as well as a closer investi gation of dose-response effects, are indicated to give us a fuller understanding of the whole problem. SUMMARY

(1) In mice (I.V .), LD^q values showed no essential difference in all four optical isomers of ephedrine.

(2) In anesthetized dogs, the relative pressor potencies of E (-)

ephedrine, L (+) ephedrine, L (+) pseudoephedrine are 1, l/3i*d,

and l/5th respectively. D (-) pseudoephedrine at doses of 0.33

and 3*3 mg/kg showed a slight rise of blood pressure following

a transitory f a ll. At higher doses, 9.9 and 16.5 aig/kg, i t was

depressor only. Similar results were obtained in chloralized cats to D (-) pseudoephedrine.

(3) In spinal cats, D (-) pseudoephedrine showed slight pressor ef

fects, approximately l/l06th of that of D (-) ephedrine.

(h) In anesthetized dogs, at equlpressor responses the least active

isomer produced the longest duration of pressor effect. L (+)

pseudoephedrine showed more latent period for its pressor effect

and also lasted longer, but D (-) ephedrlne had a very quick

onset of action and a 3hort duration.

(5) For all isomers, in anesthetized dogs, the heart rate effects

were approximately equal at equipressor rosponses. E (-) pseudo

ephedrine showed increase in heart rate at all doses. At 3.3

1 5 3 15s*

mg/kg, a latent period of about 30 minutes was necessary for its

full effect. The heart rate effects were always subraaximal.

(6) In anesthetized dogs, at equipressor doses, the tachyphylactic

tendencies were as follows:

D (-) ephedrine C. L (+) ephedrine^ D (+) amphetamine

L (+) pseudoephedrine

Animals made completely tachyphylactic to L (+-) ephedrine, D (+-)

amphetamine and L (+) pseudoephedrine showed pressor effects

when D (-) ephedrine wa3 injected. D (-) ephedrine has the

least tendency for tachyphylaxis. No tachyphylaxis was observed

to the depressor effect of D (-) pseudoephedrine when doses of

9.9 mg/kg was repeated. However, tachyphylaxis to the heart rate

e ffe c t was observed.

(?) On the isolated perfused rabbit heart, approximate chronotropic

potencies of D (-) ephedrine, L (+) ephedrine and L ( + ) pseudo

ephedrine are 1, l/5th and l/3rd respectively. D (-) pseudo

ephedrine over a wide range of doses showed slight increases in

heart rate, but effects were always submaximal. D (-) ephedrine,

L (+) ephedrine and L (+) pseudoephedrine all can cause increased

force of contraction and subsequent depression may be seen.

C (-) pseudoephedrine did not show any increase in force of con

traction, and temporary depression was seen at higher doses.

(8) In reserpine pre-treated dogs, B.P. and H.R. effects of the iso

mers were markedly reduced. These effects were reversible for 155

D (-) ephedrine but Irreversible for L (+) and L (+) pseudo

ephedrine, D-configuration of the beta hydroxyl group may be

(at least partly) responsible for effects at the direct ef

fector site through three point contact. This will not be the

case in the other two isomers, where the hydroxyl group has L-

configuration, allowing orily the possibility of two point con

tact with the receptor and hence indirect action only. D (-)

pseudoephedrine may compete for the direct effector site through

the three point contact.

(9) In anesthetized dogs, the pressor effects induced by D (-)

ephedrine and D (+) amphetamine were promptly reduced by D (-)

pseudoephedrine given at the height of pressor effects. Such

antagonism is dose dependent and could be demonstrated in the

chloralized cat as well as the spinal cat.

(10) Pre-treatment of anesthetized dogs with D (-) pseudoephedrine

could also reduce or block the pressor effects of D (-) eph

edrine, L (+) ephedrine, L (+) pseudoephedrine and D (+)

amphetamine. Such a blocking action of D (-) pseudoephedrine

towards the pressor effects of D (-) ephedrine was also ob

served in chloralized cats and in spinal cats.

(11) On the isolated rabbit aortic strips, the contraction induced

by norepinephrine or f (-) ephedrine could be reduced by D (-)

pseudoephedrine when given at the height of contraction. 156

(12) In anesthetized cats, contraction of the N.M. induced by D (-) eph

edrine could be promptly reduced by D (-) pseudoephedrine when

given at the height of contraction. This antagonism is also dose

dependent.

(13) In reserpinized dogs, D (-) ephedrine induced pressor effect could

be easily reduced by D (-) pseudoephedrine, but that of vaso

pressin was very slightly affected, Indicating that the anta

gonistic effect of D (-) pseudoephedrine is not a generalized

effect. It is suggested that D (-) pseudoephedrine antagonizes

pressor effects of D (-) ephedrine and other agents at direct as

well as at indirect sites.

(14) In anesthetized dogs, D (-) pseudoephedrine (3*3 mg/kg) poten

tiated epinephrine and norepinephrine. This potentiation was best

seen JO to 45 minutes after administration of D (-) pseudoephedrine,

(15) In spinal cats, the higher doses of D (-) pseudoephedrine which

showed pressor effects could be made depressor by infusion with

no repine ph rin e.

(16) In anesthetized cats, N.M. contraction induced by stimulation of

cervical sympathetic nerve was only slightly affected by D (-)

pseudoephedrine.

(17) In anesthetized dogs, pressor effects induced by bilateral c.o.

were not blocked by E (-) pseudoephedrine. On the contrary, a

slight Increase was seen. 15 7

(18) The B,P. depressor responses of D (-) pseudoephedrine at 9.9

mg/kg were not blocked by diphenhydramine or D.C.I.

(19) D (-) pseudoephedrine was found to exhibit pressor effect in

animals which were pre-treated with hexamethonium. Heart rate

increase was also seen along with the pressor effect. The mag

nitude and duration of the pressor effect varied over a wide

range.

(20) D (-) pseudoephedrine and reserpine (acute pre-treatment) under

the influence of each other produced pressor effects which were

promptly reduced by dibenstyllne. BIBLIOGRAPHY

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101. Maxwell, R.A., Plummer, A.J., Povalski, H., Schneider, F. and Coombs, H. "A comparison of some of the cardiovascular ac tions of methylphenidate and cocaine," J. Pharmacol. Exptl. Therap., 126. 250, 195?. AUTOBIOGRAPHY

I* Popat Narayan P a til, was born at Chinchkhede (Bombay-state), on October 26, 193^* I received my high school education at Shiwaji

High School in Ehulia, Thereafter, I went to Poona U niversity, where

I completed two years of stutfy at Ferguson College. Gujarat Univer sity granted me the degree Bachelor of Pharmacy in the year 1956.

I was admitted to Graduate School a t The Ohio State U niversity in

1958. In I960 the university granted me the degree of M. Sc. I was a research assistant and research fellow during ay stay at The Ohio

State University.