The pharmacology of MS 222 (ethyl-m-aminobenzoate) in squalus acanthias

Comp. Gen. Pharmac., 1974, Vol. fie PP. 23 to 35. Pergamon Press. Printed in Great Britain

23

T H E P H A R M A C O L O G Y OF MS 222 (ETHYL-m-AMINOBENZOATE) IN SQ,UALUS ACAaVTHIAS VINCENT

G. S T E N G E R *

AND THOMAS

H. M A R E N

Department of Pharmacology and Therapeutics, Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida, U.S.A., and Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, U.S.A.

(Received22 January 1973) ABSTRACT 1. Ninety-five per cent of the drug was cleared via the gills in 2 hours with a clearance of lO. 7 ml. per kg. per minute. Plasma t4 was 56 minutes, renal excretion being ~ 5 per cent of injected drug and metabolites. The volume of distribution was that of body water. 2. About 5 per cent was metabolized to m-aminobenzoic acid and m-acetylaminobenzoic acid, which were excreted via the kidney at a rate similar to glomerular filtration. A small fraction was also converted to the N-acetyl derivative which was excreted across the gill. 3. Given intravenously, the drug did not produce anaesthesia, but when administered via the gills anaesthesia occurred rapidly. 4. The immobilization of the fish appears to be a consequence of high arterial blood levels. The evidence suggests that anaesthesia is due to events at the neuromuscular junction. A DIRECT c o r r e l a t i o n b e t w e e n t h e gill m e m brane permeability of various drugs and their p h y s i c o c h e m i c a l p r o p e r t i e s was s h o w n b y M a r c h , E m b r y , a n d B r o d i n in 1968. L i p i d soluble d r u g s (e.g., M S 222) w e r e c l e a r e d rapidly, whereas lipid-insoluble compounds (e.g., b e n z o l a m i d e ) crossed t h e gill in m i n u t e o r u n d e t e c t a b l e a m o u n t s . A possible m e a n s o f m e a s u r i n g c a r d i a c o u t p u t was suggested b y t h e r a p i d i t y o f gill c l e a r a n c e o f M S 222. T h i s r e p o r t deals w i t h t h e e x c r e t i o n a n d m e t a b o l i s m o f this d r u g (Table I) in t h e dogfish. METHODS BIOLOGICAL All fish were caught by trawl in Frenchman's Bay, off the coast of Salisbury Cove, Maine, and maintained in sea water live-cars. Studies on drug plasma levels, their tissue distribution, and renal excretion were done in this situation. The gill clearance and cardiac output studies were performed in the laboratory, using the divided *Present address: Department of Obstetrics and Gynecology, Milton S. Hershey Medical Center o f Pennsylvania State University, Hershey, Pennsylvania i7o33 , U.S.A.

box as previously described in detail (Maren and others, 1968 ) . Fish of both sexes were used, weighing x-3. 5 kg. Small polyvinyl catheters were placed in the caudal artery and into the duct of Cuvier (or sinus venosus). The urinary papilla was catheterized and a balloon attached for continuous collection of urine. Drug was injected into the caudal artery by direct puncture distal to the sampling catheter. Two types of experiments were performed. I n the first the fish were studied in the free-swim state, with repeated paired blood sampling from the artery and vein and urine taken at intervals. I n the second group the fish were placed in the divided box in the laboratory about 3 ° minutes after drug injection. Two litres of cooled, aerated sea water perfused the gills in the anterior chamber of the box at a rate of x 1. per minute, this being the normal gill water perfusion rate (Millen, Murdaugh, Hearn, and Robin, I966 ). A t 5minute intervals the sea water was sampled and paired blood samples obtained. Drug output by the gills-and the arteriovenous differences were obtained. As previously noted by March and others (x968), deterioration o f the physiologic parameters and impaired COs excretion occurred after Io-12 minutes in the box in most experiments. Doses of MS 222 of 25-35 rag. per kg. were well tolerated by the fish. Higher doses produced toxicity with the LDs0 being ioo rag. per kg (based on our observation o f 14 fish).

24

STENGER AND ~AREN

CHEMICAL

The heparinized blood samples were treated as follows : Each sample was run in duplicate on o. 5 ml. of whole blood or plasma* diluted to 4 ml. with water, and precipitated with z ml. trichloroacetic acid (TCA, 15 per cent). After xo minutes the sample was centrifuged for IO minutes at 34oo r.p.m. A 2-ml. aliquot of supematant was analysed by the standard Bratton and Marshall (1939) method for primary arylamino compounds. The analysis of drug in the sea water and urine was done using a larger aliquot of sample and replacing the TC.,A step with 6 M HC1 for acidification. The tissue samples were homogenized with water in a Waring blender, precipitated with TCA, centrifuged, and a 2-ml. sample of the supematant analysed in the above manner. In studies using possible metabolites of MS 222 (ra-aminobenzolc acid, ethyl-m-acetylaminobenzoate, and m-acetylaminobenzoic acid) the Bratton and Marshall method was modified by increasing the time for diazotization to Io minutes and the coupling time to 5 minutes (Hunn, Schoettger, and Wilford, 1968 ). The W-acetyl compounds were analysed following 45 minutes of acid hydrolysis at xoo° C. The esters were detected separately using the Hestrln reaction (i949). The metabolites were identified on thin-layer chrornatography~f by their fluorescent characteristics under ultra-violet light. The solvent systems used to separate and identify the compounds were (a) ethanol/chloroform ( I : x ) and (b) ethanol/ chloroform/acetic acid (49 : 49 : 2). The RF values obtained using the two systems, designated as (a) and (b) are as follows: MS 222, (a) 0.74 , (b) 0-66; ethyl-ra-acetylaminobenzoate, (a) 0.78 , (b) 0"69; m-aminobenzoic acid, (a) 0"56, (b) 0.04; and ra-acetylaminobenzoic acid (a) o.6I, (b) 0"07. RESULTS I. Pm'SmAL AND Cn'~mCAL PROPERTmS Ethyl m-aminobenzoate was used as the m e t h a n e sulphonlc acid salt. T h e structure is shown in Table L T h e molecular weight is 26I'3, water solubility I I per cent, pK a 3.80 (by acid titration), a n d water solution gives a p H of 2.8o. T h e base, ethyl m-aminobenzoate, is a colourless oil with a molecular weight of 165.2 , water solubility 0.38 per cent, pKa 3.52 *These gave essentially identical results. Hematocrit of fish is about 20 per cent and the drug is not concentrated in or excluded from red cells. tEastman Chromogram 6o6o--Silica Gel with fluorescent indicator.

~o?'llp. Gen. Pharma¢.

(by acid titration), a n d water solution gives a p H of 4"8o. T h e chloroform/buffer partition coefficient was determined u n d e r various conditions of shaking and different samples o f the salt a n d base. T h e range was I2O- 3I 2. T h e buffer used was an artificial e l a s m o b r a n c h p l a s m a solution. T h e d a t a reported are those o f the salt (M.W. 261.3) since this is the form used for injection a n d in m a k i n g up all chemical standards. I n order to correct d a t a to the free base, multiply concentration a m o u n t s b y 0.63 . 2. GILL EXCRETION

T h e p r i m a r y route of excretion of M S 222 is t h r o u g h the gill. D a t a are given in Table IL Gill clearance studies in the box a b o u t 3 ° minutes after injection (the fish being allowed to swim a b o u t freely in the live-car until the study period in box) give us a m o r e representative clearance, in t h a t d e c a y represents only gill clearance a n d not redistribution at that time. Using only the first 5-minute study period in calculating the m e a n of the fish in Table II, a gill clearance of Io. 7 ml. per kg. per m i n u t e is obtained. T h e d a t a on fish 17 a n d 38 (column 7) indicate a relatively steady state. I n these two fish the range for gill clearance is 7"913"9 ml. per kg. per minute. 3. R E N A L CLEARANCE

Renal excretion accounts for about 5 per cent of the total drug injected (Table gl). Diazotizable amine in the urine was not entirely M S 222, there being some cleavage of the ester bond to yield m-arninobenzoic acid. Some acetylation o f M S 222 was also observed. F u r t h e r details are given in section I I. 4. CARDIAC OUTPUT I n these experiments it is possible to estimate the cardiac o u t p u t b y the application o f the direct Fick principle for regional blood-flow. T h e gill blood-flow (equal to cardiac output, since the gills are ' in series'

MS o22 IN

1974, 5

Squgllls acanthias

25

TableL--STRUCTUREOF MS 222 AND M.eTABOr.ZTES COMPOUND

STRUCTURE

NH2

NAME

Ethyl-m-aminobenzoate

~ _ COOC2Hs

MS 222 is the methane sulphonic acid salt, this structure x t-ISOsCH3

NH2

II

/~'x, C O O H

m-Aminobenzoic acid

0

II

III

NHC--CH~

Ethyl-m-acetylaminobenzoate

--C00C2Hs

IV

0 U NHC--CH3

~

-COOH

with the heart and peripheral circulation) can be measured using the following equation : -

Ljr0where Q. = quantity of drug in sea water over the 5-minute study period (in lag.); F = gill flow or cardiac output (ml. per minute) ;

f vs= integrated level of drug in the V0

central venous system between o and 5 minutes (gill afferent) in gg. per ml.;

I

A s = integrated level of drug in the arterial A 0 tree between o and 5 minutes (gill efferent) in lag. per ml. At the time of cardiac output measurement the decay of drug in either the artery or vein was so small from the beginning to the end-of the 5-minute period that a mean value could be used with reasonable accuracy. T h e data collected on 4 fish are presented in Only the first study periods were used in calculating the mean. T h e mean cardiac output for this group is 23. 5 ml. per kg. per minute ( column 8).

TableII.

TableII,

m-Acetylaminobenzoic acid

Progressive deterioration in both gill clearance and cardiac output occurs in some fish when they are kept in the box for prolonged periods. Fish No. 17 shows no deterioration, whereas fish No. z9 shows a marked reduction in gill clearance and blood-flow. 5. COEFFICIENT OF EXTRACTION

T h e percentage of drug loss from the blood in one passage through the gill capillary network is referred to as the coefficient of extraction. This can be expressed in the following equation : - C.E. (coefficient of extraction) = (Venous level drug--Arterial level drug) Venous level drug × I oo It will be noted that this expression is independent of the a m o u n t of drug excreted. This coefficient, from the data in Table II, is 45 per cent, which is the same as the relation of gill clearance/cardiac output × xoo. 6. HALF-LIFE IN FREE-SWIMMING FISH

T h e decay of drug was measured in the free-swimming fish in the sea water live-cars.

~6

Comp. Gen. Pharmac.

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u +_ ~ - - ~ .

~.~

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MS 222 IN Squalus acanthias

I974, 5

Fig. I is a representative experiment. It was apparent that to get a meaningful half-life measurement (t~) the samples must be taken after a 3o-4 o minute equilibrium period. Prior to this, the changing t~, (Fig. I) is due to two factors: ( x) drug diffusing rapidly from the plasma to tissues and (2) simultaneous loss of large amounts via the gills. T h e early periods of rapid decay of drug from the circulation and loss from the gill also give a false extrapolation of the volume of distribution. 500 400 :300

I00

with the t~ just described, but fits with the metabolic findings of section I I. 7. VOLUME OF DISTRIBUTION T h e volume of distribution cannot be obtained in the conventional way (by extrapolating back to zero time along the decay curve after distribution has been achieved), since a very large proportion of injected drug is lost from the high plasma concentration before complete distribution occurs. T h e situation is unusual in that very rapid expansion of the drug from volume of distribution of plasma (about 4 per cent) to that of total body water (60-70 per cent) occurs at the same time that a large fraction of drug is lost from the body by gill diffusion.* T h e volume of distribution can, however, be obtained from the relation between clearance (C) and half-life (t~) according to the following expression (Butler, I958 ) : -

E o,l OJ C~l o')

27

volume distribution = C t,/lnm 30

Substituting the mean clearance (Table 11, column 7) and the mean h~ilf-llfe (Table III, column 4) we have the following : volume distribution = (Io. 7 ml. per kg. per minute) (56 minutes)/o. 7 volume distribution = 856 ml. per kg.

IO

This volume of distribution approximates that of total body water and points to equal distribution throughout it. TissuE DISTRIBUTION T w o fish were given 3 ° mg. per kg. of MS 222 and, after 60 minutes in the freeswimming state, were sacrificed. Various tissues were sampled and analysed for drug. T h e results from each fish were similar and the data were averaged and are presented in Table IV. Most tissues had almost the same drug concentration as blood and plasma, exceptions being brain, C.S.F., extradural fluid, rectal gland, and kidney. This confirms our value for the volume 8.

o l

i 20

i 40

i 60

i 80

I .. i00

T I M E IN MIN.

I.--Decay of intravenous MS 222 i n plasma. Single representative experiment in freeswimming fish. FIG.

T h e complete half-life data are shown in

Table IlL T h e drug dose of 25-35 ~g. per kg. was used and the period of study varied from 3o to i2o minutes (column 3). T h e mean central venous half-life decay of 56 minutes (column 4) compares to 73 minutes (column 5) of the artery. Close analyses of data after 12o minutes showed persistent plasma levels of about 2 gg. per ml. This was incompatible

*This situation is not commonly found in mammals, except for the gases, since drugs which diffuse rapidly into tissues are not usually excreted rapidly.

28

Comp. Gen. Pharmac.

STENGER AND W R E N

Table IlL--PLAsMA DECAY OF M S ~22 m THE (Squalus acanthias) FISH NO.

FREE-SWIMMING DOGFISH

WEIGHT TIMEINTERVAL (kg.) STUDIED(minutes) Vein

3 4 5 6 8

2.6 2"7 2"3 2.2 2"6

40-+ 80 40-+ 80 40-+ 80 6o-+z2o 3o~*2o

4° 80 36 60 42

(l,l-) Artery

90

2" I

4 ° ~ 120

80

2I 4°

*'9 1.7

40-+,00 4 o-+Ioo

58 55

-80 48 xoo 52 90 80 58

Mean

2. 3

56

73

--

Table IV.--TmstrE DISTRIBUTIONOF MS 222 m THE DOGFISH (Squalusacanthias)* Tisstrz Whole blood (artery) Plasma (artery) Whole blood (vein) Plasma (vein) Muscle Liver Gill Spleen Stomach Bile

DRUG (~g. per ml.)

TISSUE

x.9 2-0 3"o 2.6 3.o 3"9 2"8 2"3 i "9 2"5

Brain Cerebrospinal fluid Extradural fluid Rectal gland Ovary Uterus Foetus Kidney Pancreas Peritoneal fluid

DRUG (l~g. per ml.) < 0. 5 0"5 o'5 o'5 1.6 2"o x.3 39"5 I "5 2.8

*Average of 2 fish given 30 mg. per kg. and sampled at 6o minutes. of distribution. T h e concentration in muscle suggests that the d r u g is distributed t h r o u g h b o d y water. T h e r e is a special feature noted in liver, since this organ contains only 20 per cent water: the concentration found there is greater than that represented in the water c o m p a r t m e n t , almost certainly suggesting concentration in the lipids. 9. GILL ABSORPTION OF M S 222 Table V, p a r t II, shows that w h e n drug is given by immersing the fish in sea water containing 5o-xoo gg. per ml., m u c h higher concentrations are achieved in the arterial blood and G.S.F. than following relatively large parenteral doses of d r u g (Table V, part I). This appears to be due to the fact

that in the parenteral case, the d r u g is very rapidly diffusing across the gill, m a k i n g access to the brain impossible a n d m a r k e d l y reducing arterial concentrations. Table V, p a r t III, further emphasizes the fact that arterial levels of d r u g decline very rapidly w h e n the fish is p u t in sea water free of drug. Fig. 2 shows the time relations in these experiments. T h e relationship between these facts of d r u g distribution a n d the question of ' anaesthesia ' following M S 222 are discussed in the next section. Io. L O C A L VERSUS G E N E R A L ANAESTHESIA FOLLOWING M S 222 T h e data on drug distribution (Table V and Fig. 2) led to the unexpected observation that anaesthesia could not be correlated

[974,

5

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Squalus acanthias

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30

Comp. Gen. Pharmac.

STENOER AND MA~N 150 EXP. 48

EXP. 30

1.8 kg male

1.5kg male

I00

E

50

¢Xl

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0 o~ 150 EXP. 26

EXP. 49

1.9 kg male

:3.0 kg female

I00

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20

30

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0

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20

30

40

TIME IN MIN.

Fio. 2.--Absorption of MS 222 from sea water containing xoo ~tg. per ml. for 5 minutes. O - - @ ,

Artery; 0 - • -[7, vein; O, cerebrospinal fluid; m, brain; ]]/], sea water exposure.

with the concentration of MS 222 in C,S.F. or brain. This is more clearly shown in Fig. 3. When drug is absorbed through the gills from a constant outside concentration (Ioo lag. per ml.), a C.S.F. concentration of about 2o lag. per ml. is achieved rapidly, and is maintained for at least 3° minutes, even though exposure to drug is terminated at 5 minutes. This is consonant with the fact that C.S.F. turnover in this species is of the order of 5-I o hours (Oppelt, Pattak, Zubrod, and Rail, i964). Admittedly, outward diffusion of such a lipid-soluble drug might be faster than this, but we have no other data on the subject. Despite this relatively constant level in C.S.F. and brain, anaesthesia notably lightens at io minutes and terminates at 20 minutes. Fig. 3 shows, however, a precipitous drop in plasma concentration of drug during recovery from anaesthesia. The data suggest that anaesthesia may be equated with an arterial plasma concentration about 2o lag. per ml. (fish 51, Table V).

What then is the pharmacological basis for the immobilization produced by MS 222? Further work with a homologue of the drug again showed clearly that the brain was not involved, and gave a tentative answer to the question. Our colleague, Dr. Thomas F. Muther, synthesized the isobutyl homologue of MS 222 (called IBA) by the method of Adams, Rideal, Burnett, Jenkins and Dreger (I926) and found it to be eight times as potent as MS 222 in 'anaesthetizing' goldfish from external solutions (Maren and Muther, x968 ). We then found that, unlike MS 222, IBA could ' a n a e s t h e t i z e ' dogfish by intravenous (35 mg. per kg.) or intramuscular (60 mg. per kg.) injection. In this situation, however, there was essentially no drug ( < 2 lag. per ml.) in C.S.F. T h e reason that neither MS 222 or IBA reaches the C.S.F. or brain after parenteral administeration clearly seems to be that diffusion across the gill occurs so rapidly that appreciable access to the cranial circulation

MS 222 IN Squalus acanthias

I974, 5

120

31

m

I00

80 E ~ 60 Od

~

40 o

20

A

m

I

o O

i:!

o

o

o

0

5 I

1

IO

15

20

25

30

35

TIME IN MIN.

FIO. 3.--Relation of distribution of MS 022 t o anaesthesia. Line connects arterial plasma levels of drug. Arrows show immersion and withdrawal of fish from solution to drug. I , Induction of anaesthesia ( ,v i oo lag. per ml.) ;////, period of surgical anaesthesia; [3, period of beginning recovery; : : : :, complete recovery from anaesthesia. @, Arterial concentrations; 0, venous concentration; O, cerebrospinal fluid concentration; I , brain concentration. is not attained (see Discussion). IBA causes ' anaesthesia ' in this situation (in contrast to MS 222) because it is intrinsically m o r e active (.Maren and Muther, i968 ). T h e basis for the anaesthesia seems to be peripheral, and p r o b a b l y akin to neuromuscular blockade (Maren, I969). M E T A B O L I C F A T E OF MS 222 Table I shows the structures of derivatives of MS 222 that we considered as metabolic products of its sojourn in S. acanthias. Since we have shown that a m a j o r portion is rapidly excreted as such by the gill within an I I.

hour of administration, it is evident that metabolism and renal excretion will be minor events. Table VI shows that the total urinary excretion of all four components is a b o u t 5 per cent of the administered dose. Consistent with the data of Tables VI and VII, compounds I I and I V - - t h e f r e e - G O O H c o m p o u n d s - - m a k e up most of this 5 per cent. This is borne out b y the d a t a of Table VII. A few experiments were done with compounds I I and I I I themselves. I I was injected at doses o f 3-3 ° mg. per kg. I t was found i m p e r m e a n t at the gill and cleared by

32

Comp. Gen. Pharmac.

STENOER AND ~ a U q

Table VI.--URINARY R E C O V E R Y

OF AMINES A N D ESTERS A F T E R INJECTION OF 200 rag. OF M S

222 T O

S. acant.hias rag. PER PERIOD HOURS I. Free Amine Compounds I, II o- 5 5-I2 ~2-24 24-48 Total

2. Esters Compounds I, I I I

3. Total Amines Compounds I - I V

I 0.8 0"7 0.8

x.3 0. 7 <0"5 <0. 5

3" I 3"5 ,-8 i "3

3"3

1.9

9"7

Columns i and 3 from Bratton-Marshall analysis; column 2 from Hestrin reaction. the kidney with U / P ratios of i - 6 (like inulin), and a plasma half-life of 37 hours. The drug in tissues and plasma was free amine only, except in the kidney, which showed some uptake of drug and also some acetyl compound (IV). Urine showed about 2o per cent of drug in the acetyl form. Compound I I I was injected at 3o mg. per kg. and studied in the divided box, like MS 222. Gill clearance was 2-8 ml. per minute per kg., a range slightly less than that of the parent compound (Table H). No drug was found in the urine. We conclude from these experiments that MS 222 is hydrolyzed to a very small degree in vivo to form compound I I , m-aminobenzoic acid which is excreted slowly by the kidney. Acetylation of MS 222 also proceeds to a small degree to yield compound I I I , which is excreted rapidly across the gills, and Table VII.--FRAcTIONATION O1~ URINE OF DOGFISH RECErV'rNO IOO m g . i.v. M S 222

~g. PER PERIOD

HOURS o-2 4-I7 x7-42

I

II

37° I65 62

55 4Io 308

IIl

<20 25 <20

IV

75 6oo 1o5

Roman numerals refer to compounds of Table L Esters I and I I I were extracted into chloroform from buffered urine at pH 7"4. Both phases were analysed by the Bratton-Marshall method before and after hot acid hydrolysis.

compound IV, which is slowly excreted by the kidney. Thin-layer chromatography showed authentic MS 222 in the urine for 4 hours but none thereafter. Chromatographs also confirmed Table VII by showing the increasing prominence of the carboxy-metabolites I I and I V with increasing time. DISCUSSION AND CONCLUSION MS 222, developed and used initially as a local anaesthetic (Sandoz, i92o ) in humans has found extensive use in marine biology and the fish industry as a general anaesthetic agent and tranquillizer in cold-blooded animals (Bovd, undated). Although this drug has been written a b o u t extensively, little data are available on the p h a r m a c o logical behaviour in either warm- or coldblooded a n i m a l s - - m o s t reports being limited to toxicological studies. Metabolism of this drug in the dogfish is quantitatively minimal although interesting. Ninety-five per cent is excreted rapidly across the gill within the first 2 hours after injection, essentially unchanged but with possibly a small ( < xo per cent) fraction as the aV-acetyl derivative. T h e remaining 5 per cent is excreted by the kidney, following cleavage of the ester bond to yield m-aminobenzoic acid and its aV-acetyl derivative. These acids are excreted at about the rate of glomerular filtration, and so persist in the body for several days. This highly lipid-soluble base moves across the gill m e m b r a n e in either direction at

I974, 5

MS '~22 IN

Squalus acanthias

such a rapid rate that the movement would appear to be flow limited rather than membrane limited. Evidence to support this would be the apparent correlation of the coefficient of extraction of drug with the rate of perfusion (cardiac output) of the gills, i.e., the slower the gill perfusion with blood (low cardiac output), the greater the extraction coefficient, and vice versa. In support of rapid inward movement is the fact that the arterial blood drug level was equal to the level in the water perfusing the gills within 2 minutes (our earliest measurement). It could therefore seem unlikely that any significant membrane limitation is present with this compound. A new and unexpected finding is that MS 222 fails to reach the brain after systemic injection. In mammalian pharmacology it is axiomatic that lipid-soluble unbound drugs reach the C.N.S. very rapidly. The difference here is clearly the gill, which interposes a diffusion channel between the arterial blood and the brain. A puzzling quantitative problem is that no drug (or less than perhaps 2 per cent of arterial concentration, see Table IT, fish 34) appears in C.S.F., but A-V extraction of MS 222 at the gill is only about 45 per cent. This has several possible explanations : (i) that major shunting occurs across the gill such that the blood going to the caudal portion of the fish contains drug and the blood going cephalad and perfusing the brain is completely cleared of drug-unlikely since no anatomical support exists; (2) metabolism of the drug occurs between the gill and brain--again unlikely since the drug going to the brain in either instance goes via the same vessels; (3) the pseudobranch clears blood of remaining drug going to the brain; and (4) a physiological mechanism whereby the blood going to the brain perfuses the gill slower than or more extensively than blood going to the distal portion of the body. After carefully dissecting an injected dogfish and reviewing several classic anatomical works on the elasmobranch fishes (O'Donoghue and Abbott, i928; Daniel, I934) it seems probable that both (3) and (4) play a role in clearing the drug from the brain

33

circulation. All postgill blood perfusing the brain goes through the anterior dorsal aorta, hyoidean efferent artery, and the pseudobranchial artery. Furthermore, the latter two are supplied exclusively by the most cephalad pair of gills and the former is a very small vessel by comparison. Most of the blood going to the brain comes from the most cephalad paired gills and perfuse the pseudobranch prior to entering the skull. Even though there is anastomosis between the efferent collector loops, the dynamics of movement from one gill to the next is unknown. Sampling blood from these vessels has been technically impossible up to now so that confirmation of this theory is lacking. The time course of' anaesthesia ' following immersion of fish into MS 222 solutions and removal is best correlated with the level of drug in arterial blood. 'Anaesthesia' disappears when arterial levels drop below 5 ~tg. per ml., even when the C.N.S. concentration remains high. Another and more active drug of this series, the isobutyl homologue of MS 222, does produce anaesthesia by the parenteral route, but no drug reaches the brain. We conclude that anaesthesia is not ' general ' but ' local ', and is probably due to neuromuscular blockade. The structure of MS 222, of course, is basically that of a local not a general anaesthetic. The mean cardiac output calculated from these experiments is 03-5 ml. per kg. per minute, which agrees well with the data of Burger and Bradley (i 95I), Robin, Murdaugh and Miller (i966), and Peirce, Peirce, and Peirce (I967) , all reporting mean values between 2o--25 ml..per kg. per minute by three different technical methods. The clearance of MS 222 can then be used to measure cardiac output, and a simplified techniqu.e for cardiac output could be developed based only upon the plasma decay of MS 222, since this is directly a function of gill clearance and blood-flow. The conventional method of estimating the volume of distribution, by extrapolating back to zero time along the decay curve after distribution has been achieved, cannot be used, since a very large proportion of injected

34

STENGER AND MAREN

drug is lost from plasma before distribution occurs. This m a y be a unique situation with highly lipid-soluble drugs in the fish. A comparable situation would be loss of gaseous anaesthetics through the lungs of mammals. A drug that is completely cleared by the kidneys in one passage could only be removed at one-quarter the rate that would be possible if cleared by the lungs (i.e., in m a n where 2o-2 5 per cent of cardiac output goes through the kidneys). It becomes evident, therefore, that the most efficient method for elimination of a foreign material from the body of either m a m m a l s or fishes is via lung or gill systems where a diffusion pathway exists; for gases into air from m a m mals or for very lipid-soluble compounds into water from fish. SUMMARY The pharmacology of MS 222, a highly lipid-soluble drug, has been described in the spiny dogfish, Squalus acanthias. Ninety-five per cent of the drug is cleared across the gills within 2 hours with a clearance rate of io. 7 ml. per kg. per minute. T h e plasma half-life decay is 56 minutes with the renal excretion being ~ 5 per cent of injected drug and metabolites. T h e volume of distribution was found to be approximately that of body water. Cardiac output was estimated, using the direct Fick principle for regional bloodflow, to be 23"5 ml. per kg. per minute. T h e mean extraction coefficient of MS 222 across the gills was 45 per cent. Possibly a small fraction of the drug is converted to the W-acetyl derivative which is excreted across the gill. About 5 per cent of drug is metabolized to m-aminobenzoie acid and m-acetylaminobenzoic acid, which are excreted by the kidney at about the rate of glomerular filtration. T h e drug given intravascularly does not produce anaesthesia, but when the gills are perfused with sea water containing drug, anaesthesia occurs rapidly. T h e difference in the two situations is that in the latter case it is possible to build a high arterial blood level without encountering toxicity. T h e immobilization of the fish appears to be a consequence of these arterial bIood concen-

Comp. Gen. Pharmac.

trations, and the evidence from several different types of experiment suggests that the anaesthesia is due to events at the neuromuscular junction, and is not central in origin. ACKNOWLEDGEMENTS

We thank Dr. Lawrence E. Broder for his devoted assistance. This work was supported by National Institutes of Health Grant GM AI 16943. REFERENCES ADAMS, R., Rm~AL, E. K., BURRNETT, W. B., JENKINS, R. L., and DR~GER, E. E. (x926), 'Chemical constitution, physiological action, and physical properties in a series of alkyl paraaminobenzoates ', 07. Am. Chem. Sot., ,t8, 1758177o. Bov~, F. J., ' M S 222 Sandoz. The anaesthetic and tranquillizer of choice for fish and other cold-blooded organisms '. Basle, Switzerland: Sandoz Ltd. BRATTON,A. C., and MARSHALL,E. K.,jun. (i939) ' A new coupling component for sulfanilamide determinations ', 07. Biol. Chem., 128, 537-55 o. BURGER, J. W., and BRADLEY, S. E. (195I), ' The general form of circulation in the dogfish, Squalus acanthias ', 07. Cell Comp. Physiol., 37, 389 402. BUTLER, T. C. (I958), 'Termination of drug action by elimination of unchanged drug ', Fedn Proc. Fedn Am. Socs Exp. Biol., xT, x158-i 162. DANmL, J. F. (1934), The Elasmobranch Fishes, pp. i6o-I97. University of California Press. HESTRIN, S. (x949), ' T h e reaction of acetylcholine and other carboxyllc acid derivatives with hydroxylamine, and its analytical application ', 07. Biol. Chem., x ~ , 249--26I. HUNN, J. B., SCHOETTOER,R. A., and WILFOR~, W. A. (I968), ' Turnover and urinary excretion of free and acetglated MS 222 by rainbow trout, Salmo gairdneri ', .7. Fish. Res. Bd Canada, 25, 25-3 I. MAREN, T. H. (I969), 'Further observation on the pharmacology of esters of m-aminobenzoate in dogfish: local vs. general anaesthesia', Bull. Mt Desert Island Biol. Lab., 9, 32-33 • MA~N, T. H., EM~R'G R., and BRODER, L. E. (I968), ' T h e excretion of drugs across the gill of the dogfish, Squalus acanthias ', Comp. Biochem. Physiol,, 26, 853-864. M~R~N, T. H., and MUTHER, T. F. (1968), 'Preliminary observation on a new shark anesthetic, isobutyl m-aminobenzoate', Bull. Mt Desert Island Biol. Lab., 8, 42-43. MILLEN, J. E., MtrRDAUGH, H. V., jun., I"~ARN, D. C., and RosIN, E. D. (I966), ' Measurement of gill water flow in Squalus acanthias using the dye-dilution technique ', Am. 07. Physiol., 2xx, II--I 4.

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O'DoNoOHUE, C. H., and ABBOTT, ~'.. (I928), ' The blood vascular system of the spiny dogfish, Squalus acanthias Linnd, and Squalus sucklii Gill ', Tram. R. Soc. Edinb., 55, 823-89 o.

OPPELT, W. W., PATLAK, C. S., ZUBROD, C. G., and R.ALL, D. P. (1964) , 'Vcntricular fluid production rates and turnover in clasmobranchii ', Comp. Bioch~'m.Physiol.,xz9 171-177.

PEIRCE, E. C., II, PEIRCE, E. M., and PEIRCE, E. C., I I I (I967), ' Effects of tricaine mcthanesulfonate (MS222) on the circulation of Squalus acanthias ', Bull. Mt Desert Island Biol. Lab., 7, 45-47.

35

ROBIN, E. D., MURDAUGH, H. V., jun., and MILLEN, J. E. (1966), 'Acid-base fluid and electrolyte metabolism in the elasmobranch ', 3. Cell Physiol., 67, 93-ioo. SANDOZ, M. (192o), 'Preparations et proprifitds physiologiques de la Tric~ine ', Bull. Soc. Vaud. Sd. Nat., 53~ 199-

Key Word Index: Squalus acanthias, anaesthesia, MS 022 metabolism, gill absorption and excretion, renal clearance, cardiac output, volume of distribution, lipid solubility, drug half-life, coefficient of extraction.