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Unreliability of the rat stomach fundus as a predictor of hallucinogenic activity in substituted phenethylamines
Life Sciences, Vol. 35, pp. 1343-1348 Printed in the U.S.A.
Pergamon Press
UNRELIABILITY OF THE RAT STOMACH FUNDUS AS A PREDICTOR OF HALLUCINOGENIC ACTIVITY IN SUBSTITUTED PHENETHYLAMINES Davld E. N i c h o l s , Dama Schooler, M1ng C. Yeung, Robert A. Oberlender and Joseph E. Zabik Departments of Medlclnal Chemlstry and Pharmacognosy and Pharmacology and T o x i c o l o g y School of Pharmacy and Pharmacal Sciences Purdue U n i v e r s l t y West L a f a y e t t e , Indlana 47907 (Received in final form July 12, 1984)
Summary A s e r i e s of t h r e e I s o m e r i c 2 , 4 , 5 - s u b s t i t u t e d monoethoxy dlmethoxy phenyllsopropylamines were compared f o r t h e l r c o n t r a c t i l e e f f e c t In the rat fundus and as p o t e n t i a l antagonists to the e f f e c t of s e r o t o n l n in the fundus. The three isomers were also evaluated f o r t h e l r d l s c r i m i n a t i v e s t i m u l u s p r o p e r t i e s in rats t h a t had been t r a l n e d to d i s c r i m i n a t e I n j e c t l o n s of s a l i n e from LSD t a r t r a t e (0.08 mg/kg). The drug d l s c r i m i n a t l o n s t u d i e s r e v e a l e d t h a t the 2,5d i m e t h o x y - 4 - e t h o x y s u b s t l t u t i o n was most potent in r a t s , c o n s i s t e n t w i t h the r e p o r t e d c l l n l c a l actlvlty of t h i s i s o m e r i n man. By c o n t r a s t , of the t h r e e Isomers examined, t h i s was the weakest i n eliclting a contractlon i n the f u n d u s . None o f the compounds antagonized serotonin Induced c o n t r a c t l o n s , and i t was not p o s s i b l e to determine pAp v a l u e s . Q u e s t l o n s are r a i s e d a b o u t t h e d e t e r m i n a t l o n of pA~2 values f o r p a r t l a l agonists and i~t is concluded that the fundus Is not a reliable model f o r p r e d l c t i o n of h a l l u c i n o g e n i c a c t i v i t y of phenethylamines. One of the major obstacles faced by i n v e s t i g a t o r s who study h a l l u c i n o g e n i c drugs Is the lack of an in v i t r o t e s t system where a c t i v i t y in the model is a r e l i a b l e i n d l c a t o r of a c t l v l t y in man. T h i s i s p r o b a b l y p a r t l y due to the c o m p l e x i t y of e f f e c t t h a t h a l l u c l n o g e n i c drugs produce and p a r t l y due to the fact that a locus of actlon, at l e a s t f o r t h e p h e n e t h y l a m i n e type h a l l u c l n o g e n s , has not yet been i d e n t i f l e d . For some y e a r s the r a t stomach fundus has been used to s t u d y compounds t h a t have a s e r o t o n l n a g o n l s t e f f e c t ( I - 3 ) . T h l s p r e p a r a t l o n was o r i g l n a l l y d e v e l o p e d by Vane (4) and c o n t a l n s s e r o t o n i n r e c e p t o r s w h i c h e l i c l t a c o n t r a c t i o n of the fundus when s t i m u l a t e d . A b i l i t y to c o n t r a c t the rat fundus has been used as an In v i t r o model w h i c h shows some c o r r e l a t i o n with hallucinogenic p o t e n c y i n man, p a r t i c u l a r l y for indolealkylamlnes (5). However, s l m p l e c o n t r a c t i o n of the fundus o f t e n does not c o r r e l a t e to hallucinogenlc activlty. T h i s may be due to the e x i s t e n c e of two t y p e s of s e r o t o n i n r e c e p t o r in t h e f u n d u s ; one t h a t i s s e n s i t i v e to b l o c k a d e by p h e n o x y b e n z a m i n e (POB) and one t h a t is r e s i s t a n t t o POB (6). To c i r c u m v e n t t h l S p r o b l e m , Glennon et a l . (7,8) have employed the s t r a t e g y of d e t e r m l n i n g pA2 values f o r halluclnogens a c t i n g as antagonists to the c o n t r a c t i l e e f f e c t of s e r o t o n l n in the rat fundus. 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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As an in v i v o model f o r h a l l u c i n o g e n i c d r u g s , the t w o - l e v e r drug d i s c r i m i n a t i o n paradigm has come i n t o widespread use (9-11). I t is s e n s i t i v e and a c t i v i t y in t h i s model g e n e r a l l y c o r r e l a t e s w e l l w i t h human potency (12). I t seemed w o r t h w h i l e t h e r e f o r e , to compare a s e r i e s of compounds of close s t r u c t u r a l s i m i l a r i t y f o r t h e i r a b i l i t y to e l i c i t a c o n t r a c t i o n in the fundus, a n t a g o n i z e s e r o t o n i n - i n d u c e d c o n t r a c t i o n s in the f u n d u s , as measured by pA 2 values, and generate a d i s c r i m i n a t i v e s t i m u l u s s i m i l a r to LSD in the t w o - l e v e r drug d i s c r i m i n a t i o n model. The f o l l o w l n g t h r e e compounds were t h e r e f o r e prepared and studied. These have been p r e v i o u s l y reported by S h u l g i n , and only compound 2 shows s i g n i f i c a n t h a l l u c i n o g e n i c a c t i v i t y in humans (13).
.3S R30~ OR1 OR2
I R2 = R3 = CH3; RI = CH2CH3 2 R1 = R3 = CH3; R2 = CH2CH3 3 RI = R2 = CH3; R3 = CH2CH3 M a t e r i a l s and Methods
Rat Fundus. Male, Sprague-Dawley r a t s , 250 to 350 grams in w e l g h t , were used. Rats were f a s t e d o v e r n i g h t b u t were a l l o w e d f r e e access to w a t e r . Anlmals were s a c r i f i c e d by d e c a p i t a t i o n . F o l l o w i n g the method of Vane (4), 3 x I0 mm s t r i p s of fundus were suspended in 25 mL organ baths c o n t a i n i n g Tyrode's s o l u t i o n , w i t h 0.i micromolar scopolamine, maintained at 37°C and oxygenated w i t h 95% 02-5% CO2. S t r i p s were p l a c e d under I g i n i t i a l t e n s i o n and c o n t r a c t i o n s were measured using a Grass FT03 f o r c e - d i s p l a c e m e n t transducer and r e c o r d e d u s l n g a Gould p r e a m p l i f i e r and r e c o r d e r . A complete dose-response curve to s e r o t o n i n was obtained f o r each t i s s u e . C o n t r a c t i o n was measured as a percentage of the maximal c o n t r a c t i o n obtained w i t h sehotonln. Only one t e s t drug was used in each p r e p a r a t i o n . For a n t a g o n i s t s t u d i e s , a c o n c e n t r a t i o n of t e s t a n t a g o n i s t from .01 to I m i c r o m o l a r was added to the b a t h , a c o m p l e t e dose-response curve to s e r o t o n l n was obtalned, and an EC50 determined. When the a n t a g o n i s t e l i c i t e d a c o n t r a c t i o n in the fundus, the c r e s t of the response served as the new b a s e l i n e (14). Dru~ D i s c r i m l n a t i o n . M a l e , Sprague-Dawley r a t s , weighing a p p r o x i m a t e l y 200 grams at the beginning of the study, were t r a i n e d to d i s c r i m i n a t e s a l i n e injections from LSD t a r t r a t e (0.08 mg/kg, 1.p.). We have d e s c r i b e d t h i s t r a i n i n g procedure p r e v i o u s l y (15). Standard Coulbourn operant chambers w i t h two l e v e r s , s e p a r a t e d by a food p e l l e t d e l i v e r y system were employed. Food pellets (45 mg B i o s e r v , d u s t l e s s ) were used as r e i n f o r c e m e n t . Chambers c o n t a i n e d a w h i t e house l i g h t and masking w h i t e n o l s e and were c o n t a i n e d in ventilated sound-attenuated cubicles. C o n t r o l and d a t a a c q u i s i t i o n were c o n t r o l l e d by a microcomputer in an a d j o i n i n g room. Drugs w~re d i s s o l v e d in s t e r i l e p h y s l o l o g i c a l s a l i n e so t h a t animals received 0.I mL of s o l u t i o n per I00 g of body weight. Rats were deprived to a p p r o x i m a t e l y 85% of f r e e - f e e d i n g w e i g h t and were housed i n d i v i d u a l l y in a t e m p e r a t u r e c o n t r o l l e d room (25 C) w i t h an 0600-2000 l i g h t s on, 2000-0600 l i g h t s o f f schedule. Rats were t r a i n e d on an FR32 schedule w i t h 15 minute maintenance sessions. Drug t r e a t m e n t s were administered 30 minutes p r i o r to t e s t i n g . Test sesslons l a s t e d u n t i l t h e r a t e m i t t e d 32 r e s p o n s e s on e i t h e r l e v e r or u n t i l 5 m i n u t e s had passed. I f the r a t d l d not e m i t 32 responses on e i t h e r l e v e r w i t h i n 5 minutes he was scored as d i s r u p t e d and was not included in c a l c u l a t i o n s .
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Animals were scored as drug positive i f they selected the LSD-appropriate l e v e r (i.e. i f they emitted 32 responses on the drug lever). These quantal data were analyzed using the method of L i t c h f i e l d and Wilcoxon to determine an ED50 i f g e n e r a l i z a t i o n occurred, t h a t i s , i f greater than 80% of the sample population responded on the LSD-appropriate lever. Results The results of the assay of compounds I - 3 in the rat fundus are given in Table I. Figure I shows the dose-response curves for the contractile effect in the fundus. Early on, i t was apparent t h a t compounds I and 2 had a s i m i l a r potency, so a large number of preparations were used in order to obtain a clear indication of r e l a t i v e potency. Even so, a 3 pt x 3 pt parallel line bioassay procedure (16) f a i l e d to detect a s i g n i f i c a n t d i f f e r e n c e in the potency of these two. That is, the 95% confidence interval of the potency ratio included 1.0. The p a r a l l e l l i n e bioassay indicated no s i g n i f i c a n t d i f f e r e n c e in parallelism among the three test compounds. TABLE I
Assay for Contractile Effect in the Fundus Compound Serotonin I 2 3
ED50 (95% c . i . ) 5.3 3.2 4.2 9.4
x x x x
IZ 20 w 0 n-" w 0 "~ 9
6 22 21 22
/,/o /dl" /
40
(3.9-7.7) (2.2-4.6) (3.3-5.5) (6.9-12.7)
,f"
Z _o I 0 0 I-o n.- 8O I.z 0 0 6O _1 X
I0-8M IO-6M 10-6M IO-TM
n
/
//
o//, ' %
/ / ..o , . y o"
- L O G DRUG CONO (MOLAR)
FIG. 1
Contractile effect of test compounds in the rat funds; 17 = compound I , n=22; ~ = compound 2, n=21; 0 = compound 3, n:22; l: serotonin, n=6.
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The drug d i s c r i m l n a t i o n data are shown in Table I I . All compounds produced a d i s c r i m i n a t i v e stimulus s l m i l a r to the t r a l n i n g drug, LSD. However, compound 2 was most potent, consistent with the report that i t possesses human a c t i v i t y (13). The c l i n i c a l data on these compounds were quite b r i e f and i t is not clear that compounds I and 3 would lack hallucinogenic effects in man, i f tested at hlgher doses. I t is important to note that the r e l a t i v e potency in the drug d l s c r i m i n a t i o n assay for the three test compounds is exactly reversed from that observed in the fundus preparation. TABLE I I Results of Drug Discrimlnation Testing Compound i
Dose (mg/kg)
Drug P o s i t i v e / NumberTested
ED50 (95% c . i . )
2 4 8 12 20
0/6 2/6 416 4/6 5/5
6.43 (3.93-10.52)
2 4 8
3/12 3/6 7/7
3.37 (2.22-5.12)
4 8 12
3/12 4/11 7/8
7.05 (5.12-9.69)
Attempts to determine pA2 values were disappointing. Each compound was tested in f i v e separate preparatlons, in concentrations from 0.01 micromolar to i micromolar. In no case was any antagonism to the c o n t r a c t i l e e f f e c t of s e r o t o n i n observed. Indeed, dose-response curves to serotonin, generated in the presence of any of the t h r e e t e s t drugs, were superimposable upon those obtained in the absence of potential antagonist. Discussion Our results c l e a r l y show that a b i l i t y to e l i c i t a contractlon in the rat fundus is not a r e l i a b l e p r e d i c t o r of h a l l u c i n o g e n i c a c t i v i t y , at l e a s t for phenethylamines. In f a c t , the fundus indicated that the least potent compound was 2, whereas t h i s is the one which possesses hallucinogenic a c t i v i t y in man (13). On the o t h e r hand, the t w o - l e v e r drug d i s c r i m i n a t i o n assay in r a t s c o r r e c t l y i d e n t i f i e d 2 as most potent. The f a c t t h a t I and 3 also produced g e n e r a l i z a t i o n may i n d i c a t e t h a t these compounds would be halluclnogenic i f administered in high doses. Nevertheless, several r e p o r t s I n d i c a t e t h a t the Interoceptive cue produced by LSD or mescaline w111 generalize to s t r u c t u r a l l y r e l a t e d but n o n h a l l u c i n o g e n i c drugs (17-19). Thus, the drug d i s c r i m i n a t i o n procedure can indlcate false positives when used for screening purposes. Some previous studies of the stimulus propertles of these compounds have been r e p o r t e d by Glennon et al. (14,20,21). When l - ( 2 , 5 - d i m e t h o x y - 4 m e t h y l p h e n y l ) - 2 - a m i n o p r o p a n e (DOM, STP) was used as the t r a i n l n g drug (I mg/kg), generalization to compunds 2 and 3 was reported, with 2 being the more potent. However, when 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e was used as the t r a i n i n g stimulus, these workers did not obtain generalization to 1, 2, or 3. This would seem to i n d i c a t e t h a t 5-methoxy DMT may be more l i m i t e d as a
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t r a i n i n g s t i m u l u s when screening f o r p o t e n t i a l phenethylamlne type compounds.
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in
The lack of a suitable In v l t r o assay for potentlal hallucinogenic drugs Is a c o n t i n u l n g problem. The use of pA2 values by Glennon and his coworkers f o r several years encouraged us to e v a l u a t e t h a t procedure. Our f a i l u r e to observe antagonism to the c o n t r a c t i l e e f f e c t of serotonin in the fundus by any of the three test compounds was very surprising, p a r t i c u l a r l y in view of the reported pA2 f o r compound 2 of 6.78 (14). A pA2 of t h l s magnitude should indicate a reasonably potent antagonist. We b e l l e v e t h a t the f a i l u r e to observe an a n t a g o n l s t e f f e c t f o r these compounds can be a t t r i b u t e d to certaln methodological problems inherent in the application of the pA2 approach to substltuted phenethylamines. Foremost among these is the fact that nearly a l l hallucinogenic phenethylamine type compounds are agonists in the fundus, and e l i c i t contraction. Reported pA2 values, for compounds that we have tested in the fundus, are usually in the range where we have observed s i g n i f l c a n t c o n t r a c t i o n of the fundus. We have not had dlfficulty a n t a g o n l z i n g the e f f e c t of s e r o t o n i n in the fundus w l t h other antagonlsts such as 2-bromo-LSD. An inspectlon of Figure 1 shows that a pA determination would involve the use of concentrations of 2 lower than about 0.3 micromolar, but s t i l l in excess of the reported pA2 concentration of 0.17 mlcromolar. I t seems impossible to us t h a t an accurate and s i g n l f i c a n t pA2 could be d e r i v e d w i t h i n t h i s narrow range of " a n t a g o n l s t " c o n c e n t r a t i o n . Concentrations of the antagonist lower than those correspondlng to the pA2 should probably not be used. Using the 3 pt x 3 pt, completely randomlzed deslgn p a r a l l e l l i n e bioassay (16), there was no s i g n i f i c a n t d i f f e r e n c e In the potency of I and 2, y e t the ED50 r a t l o was about 1.3. I t seems l i k e l y t h a t s i g n l f l c a n t s h i f t s in the EDbo w i l l not be detectable with precision u n t i l the s h l f t IS close to two-fold. From a t h e o r e t i c a l s t a n d p o i n t , the pA2 corresponds to the d l s s o c i a t i o n c o n s t a n t , KD f o r the a n t a g o n i s t . For a ~ull a g o n i s t , the EDen is always s m a l l e r than the KD (22) w i t h the ED50 only approachlng KD w i ~ very weak p a r t i a l a g o n i s t s . Thus, we assume t h a t the KDS, and hence PA2 v a l u e s , would correspond to bath c o n c e n t r a t l o n s much higher than the ED50. That i s , f o r compound 2 the pA2 would be greater than -Iog(ED50), i.e. greater than 5.37, i f i t were appropriate to determine a pA2 for a compound with an agonlst p r o f i l e . In summary, n e i t h e r the potency in e l i c i t l n g c o n t r a c t i o n in the r a t fundus, nor the use of pA2 values can be considered a r e l l a b l e model f o r predicting halluclnogenic a c t i v i t y for substituted phenethylamines. Acknowledgment Thls work was supported in part by PHS grant DA02189 from the Natlonal I n s t i t u t e on Drug Abuse and by Chemical Pharmacology Tralnlng Grant GM 709504. Instrumentatlon for the drug d i s c r i m i n a t l o n studleS was p a r t i a l l y funded by Biomedical Research Support Grant 2-S07-RR05586-15. References 1. 2. 3. 4.
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b. 6. 7. 8. 9. I0. ii. 12. 13. 14. lb. 16. 17. 18. 19. 20. 21. 22.
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Pergamon Press
UNRELIABILITY OF THE RAT STOMACH FUNDUS AS A PREDICTOR OF HALLUCINOGENIC ACTIVITY IN SUBSTITUTED PHENETHYLAMINES Davld E. N i c h o l s , Dama Schooler, M1ng C. Yeung, Robert A. Oberlender and Joseph E. Zabik Departments of Medlclnal Chemlstry and Pharmacognosy and Pharmacology and T o x i c o l o g y School of Pharmacy and Pharmacal Sciences Purdue U n i v e r s l t y West L a f a y e t t e , Indlana 47907 (Received in final form July 12, 1984)
Summary A s e r i e s of t h r e e I s o m e r i c 2 , 4 , 5 - s u b s t i t u t e d monoethoxy dlmethoxy phenyllsopropylamines were compared f o r t h e l r c o n t r a c t i l e e f f e c t In the rat fundus and as p o t e n t i a l antagonists to the e f f e c t of s e r o t o n l n in the fundus. The three isomers were also evaluated f o r t h e l r d l s c r i m i n a t i v e s t i m u l u s p r o p e r t i e s in rats t h a t had been t r a l n e d to d i s c r i m i n a t e I n j e c t l o n s of s a l i n e from LSD t a r t r a t e (0.08 mg/kg). The drug d l s c r i m i n a t l o n s t u d i e s r e v e a l e d t h a t the 2,5d i m e t h o x y - 4 - e t h o x y s u b s t l t u t i o n was most potent in r a t s , c o n s i s t e n t w i t h the r e p o r t e d c l l n l c a l actlvlty of t h i s i s o m e r i n man. By c o n t r a s t , of the t h r e e Isomers examined, t h i s was the weakest i n eliclting a contractlon i n the f u n d u s . None o f the compounds antagonized serotonin Induced c o n t r a c t l o n s , and i t was not p o s s i b l e to determine pAp v a l u e s . Q u e s t l o n s are r a i s e d a b o u t t h e d e t e r m i n a t l o n of pA~2 values f o r p a r t l a l agonists and i~t is concluded that the fundus Is not a reliable model f o r p r e d l c t i o n of h a l l u c i n o g e n i c a c t i v i t y of phenethylamines. One of the major obstacles faced by i n v e s t i g a t o r s who study h a l l u c i n o g e n i c drugs Is the lack of an in v i t r o t e s t system where a c t i v i t y in the model is a r e l i a b l e i n d l c a t o r of a c t l v l t y in man. T h i s i s p r o b a b l y p a r t l y due to the c o m p l e x i t y of e f f e c t t h a t h a l l u c l n o g e n i c drugs produce and p a r t l y due to the fact that a locus of actlon, at l e a s t f o r t h e p h e n e t h y l a m i n e type h a l l u c l n o g e n s , has not yet been i d e n t i f l e d . For some y e a r s the r a t stomach fundus has been used to s t u d y compounds t h a t have a s e r o t o n l n a g o n l s t e f f e c t ( I - 3 ) . T h l s p r e p a r a t l o n was o r i g l n a l l y d e v e l o p e d by Vane (4) and c o n t a l n s s e r o t o n i n r e c e p t o r s w h i c h e l i c l t a c o n t r a c t i o n of the fundus when s t i m u l a t e d . A b i l i t y to c o n t r a c t the rat fundus has been used as an In v i t r o model w h i c h shows some c o r r e l a t i o n with hallucinogenic p o t e n c y i n man, p a r t i c u l a r l y for indolealkylamlnes (5). However, s l m p l e c o n t r a c t i o n of the fundus o f t e n does not c o r r e l a t e to hallucinogenlc activlty. T h i s may be due to the e x i s t e n c e of two t y p e s of s e r o t o n i n r e c e p t o r in t h e f u n d u s ; one t h a t i s s e n s i t i v e to b l o c k a d e by p h e n o x y b e n z a m i n e (POB) and one t h a t is r e s i s t a n t t o POB (6). To c i r c u m v e n t t h l S p r o b l e m , Glennon et a l . (7,8) have employed the s t r a t e g y of d e t e r m l n i n g pA2 values f o r halluclnogens a c t i n g as antagonists to the c o n t r a c t i l e e f f e c t of s e r o t o n l n in the rat fundus. 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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As an in v i v o model f o r h a l l u c i n o g e n i c d r u g s , the t w o - l e v e r drug d i s c r i m i n a t i o n paradigm has come i n t o widespread use (9-11). I t is s e n s i t i v e and a c t i v i t y in t h i s model g e n e r a l l y c o r r e l a t e s w e l l w i t h human potency (12). I t seemed w o r t h w h i l e t h e r e f o r e , to compare a s e r i e s of compounds of close s t r u c t u r a l s i m i l a r i t y f o r t h e i r a b i l i t y to e l i c i t a c o n t r a c t i o n in the fundus, a n t a g o n i z e s e r o t o n i n - i n d u c e d c o n t r a c t i o n s in the f u n d u s , as measured by pA 2 values, and generate a d i s c r i m i n a t i v e s t i m u l u s s i m i l a r to LSD in the t w o - l e v e r drug d i s c r i m i n a t i o n model. The f o l l o w l n g t h r e e compounds were t h e r e f o r e prepared and studied. These have been p r e v i o u s l y reported by S h u l g i n , and only compound 2 shows s i g n i f i c a n t h a l l u c i n o g e n i c a c t i v i t y in humans (13).
.3S R30~ OR1 OR2
I R2 = R3 = CH3; RI = CH2CH3 2 R1 = R3 = CH3; R2 = CH2CH3 3 RI = R2 = CH3; R3 = CH2CH3 M a t e r i a l s and Methods
Rat Fundus. Male, Sprague-Dawley r a t s , 250 to 350 grams in w e l g h t , were used. Rats were f a s t e d o v e r n i g h t b u t were a l l o w e d f r e e access to w a t e r . Anlmals were s a c r i f i c e d by d e c a p i t a t i o n . F o l l o w i n g the method of Vane (4), 3 x I0 mm s t r i p s of fundus were suspended in 25 mL organ baths c o n t a i n i n g Tyrode's s o l u t i o n , w i t h 0.i micromolar scopolamine, maintained at 37°C and oxygenated w i t h 95% 02-5% CO2. S t r i p s were p l a c e d under I g i n i t i a l t e n s i o n and c o n t r a c t i o n s were measured using a Grass FT03 f o r c e - d i s p l a c e m e n t transducer and r e c o r d e d u s l n g a Gould p r e a m p l i f i e r and r e c o r d e r . A complete dose-response curve to s e r o t o n i n was obtained f o r each t i s s u e . C o n t r a c t i o n was measured as a percentage of the maximal c o n t r a c t i o n obtained w i t h sehotonln. Only one t e s t drug was used in each p r e p a r a t i o n . For a n t a g o n i s t s t u d i e s , a c o n c e n t r a t i o n of t e s t a n t a g o n i s t from .01 to I m i c r o m o l a r was added to the b a t h , a c o m p l e t e dose-response curve to s e r o t o n l n was obtalned, and an EC50 determined. When the a n t a g o n i s t e l i c i t e d a c o n t r a c t i o n in the fundus, the c r e s t of the response served as the new b a s e l i n e (14). Dru~ D i s c r i m l n a t i o n . M a l e , Sprague-Dawley r a t s , weighing a p p r o x i m a t e l y 200 grams at the beginning of the study, were t r a i n e d to d i s c r i m i n a t e s a l i n e injections from LSD t a r t r a t e (0.08 mg/kg, 1.p.). We have d e s c r i b e d t h i s t r a i n i n g procedure p r e v i o u s l y (15). Standard Coulbourn operant chambers w i t h two l e v e r s , s e p a r a t e d by a food p e l l e t d e l i v e r y system were employed. Food pellets (45 mg B i o s e r v , d u s t l e s s ) were used as r e i n f o r c e m e n t . Chambers c o n t a i n e d a w h i t e house l i g h t and masking w h i t e n o l s e and were c o n t a i n e d in ventilated sound-attenuated cubicles. C o n t r o l and d a t a a c q u i s i t i o n were c o n t r o l l e d by a microcomputer in an a d j o i n i n g room. Drugs w~re d i s s o l v e d in s t e r i l e p h y s l o l o g i c a l s a l i n e so t h a t animals received 0.I mL of s o l u t i o n per I00 g of body weight. Rats were deprived to a p p r o x i m a t e l y 85% of f r e e - f e e d i n g w e i g h t and were housed i n d i v i d u a l l y in a t e m p e r a t u r e c o n t r o l l e d room (25 C) w i t h an 0600-2000 l i g h t s on, 2000-0600 l i g h t s o f f schedule. Rats were t r a i n e d on an FR32 schedule w i t h 15 minute maintenance sessions. Drug t r e a t m e n t s were administered 30 minutes p r i o r to t e s t i n g . Test sesslons l a s t e d u n t i l t h e r a t e m i t t e d 32 r e s p o n s e s on e i t h e r l e v e r or u n t i l 5 m i n u t e s had passed. I f the r a t d l d not e m i t 32 responses on e i t h e r l e v e r w i t h i n 5 minutes he was scored as d i s r u p t e d and was not included in c a l c u l a t i o n s .
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Animals were scored as drug positive i f they selected the LSD-appropriate l e v e r (i.e. i f they emitted 32 responses on the drug lever). These quantal data were analyzed using the method of L i t c h f i e l d and Wilcoxon to determine an ED50 i f g e n e r a l i z a t i o n occurred, t h a t i s , i f greater than 80% of the sample population responded on the LSD-appropriate lever. Results The results of the assay of compounds I - 3 in the rat fundus are given in Table I. Figure I shows the dose-response curves for the contractile effect in the fundus. Early on, i t was apparent t h a t compounds I and 2 had a s i m i l a r potency, so a large number of preparations were used in order to obtain a clear indication of r e l a t i v e potency. Even so, a 3 pt x 3 pt parallel line bioassay procedure (16) f a i l e d to detect a s i g n i f i c a n t d i f f e r e n c e in the potency of these two. That is, the 95% confidence interval of the potency ratio included 1.0. The p a r a l l e l l i n e bioassay indicated no s i g n i f i c a n t d i f f e r e n c e in parallelism among the three test compounds. TABLE I
Assay for Contractile Effect in the Fundus Compound Serotonin I 2 3
ED50 (95% c . i . ) 5.3 3.2 4.2 9.4
x x x x
IZ 20 w 0 n-" w 0 "~ 9
6 22 21 22
/,/o /dl" /
40
(3.9-7.7) (2.2-4.6) (3.3-5.5) (6.9-12.7)
,f"
Z _o I 0 0 I-o n.- 8O I.z 0 0 6O _1 X
I0-8M IO-6M 10-6M IO-TM
n
/
//
o//, ' %
/ / ..o , . y o"
- L O G DRUG CONO (MOLAR)
FIG. 1
Contractile effect of test compounds in the rat funds; 17 = compound I , n=22; ~ = compound 2, n=21; 0 = compound 3, n:22; l: serotonin, n=6.
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The drug d i s c r i m l n a t i o n data are shown in Table I I . All compounds produced a d i s c r i m i n a t i v e stimulus s l m i l a r to the t r a l n i n g drug, LSD. However, compound 2 was most potent, consistent with the report that i t possesses human a c t i v i t y (13). The c l i n i c a l data on these compounds were quite b r i e f and i t is not clear that compounds I and 3 would lack hallucinogenic effects in man, i f tested at hlgher doses. I t is important to note that the r e l a t i v e potency in the drug d l s c r i m i n a t i o n assay for the three test compounds is exactly reversed from that observed in the fundus preparation. TABLE I I Results of Drug Discrimlnation Testing Compound i
Dose (mg/kg)
Drug P o s i t i v e / NumberTested
ED50 (95% c . i . )
2 4 8 12 20
0/6 2/6 416 4/6 5/5
6.43 (3.93-10.52)
2 4 8
3/12 3/6 7/7
3.37 (2.22-5.12)
4 8 12
3/12 4/11 7/8
7.05 (5.12-9.69)
Attempts to determine pA2 values were disappointing. Each compound was tested in f i v e separate preparatlons, in concentrations from 0.01 micromolar to i micromolar. In no case was any antagonism to the c o n t r a c t i l e e f f e c t of s e r o t o n i n observed. Indeed, dose-response curves to serotonin, generated in the presence of any of the t h r e e t e s t drugs, were superimposable upon those obtained in the absence of potential antagonist. Discussion Our results c l e a r l y show that a b i l i t y to e l i c i t a contractlon in the rat fundus is not a r e l i a b l e p r e d i c t o r of h a l l u c i n o g e n i c a c t i v i t y , at l e a s t for phenethylamines. In f a c t , the fundus indicated that the least potent compound was 2, whereas t h i s is the one which possesses hallucinogenic a c t i v i t y in man (13). On the o t h e r hand, the t w o - l e v e r drug d i s c r i m i n a t i o n assay in r a t s c o r r e c t l y i d e n t i f i e d 2 as most potent. The f a c t t h a t I and 3 also produced g e n e r a l i z a t i o n may i n d i c a t e t h a t these compounds would be halluclnogenic i f administered in high doses. Nevertheless, several r e p o r t s I n d i c a t e t h a t the Interoceptive cue produced by LSD or mescaline w111 generalize to s t r u c t u r a l l y r e l a t e d but n o n h a l l u c i n o g e n i c drugs (17-19). Thus, the drug d i s c r i m i n a t i o n procedure can indlcate false positives when used for screening purposes. Some previous studies of the stimulus propertles of these compounds have been r e p o r t e d by Glennon et al. (14,20,21). When l - ( 2 , 5 - d i m e t h o x y - 4 m e t h y l p h e n y l ) - 2 - a m i n o p r o p a n e (DOM, STP) was used as the t r a i n l n g drug (I mg/kg), generalization to compunds 2 and 3 was reported, with 2 being the more potent. However, when 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e was used as the t r a i n i n g stimulus, these workers did not obtain generalization to 1, 2, or 3. This would seem to i n d i c a t e t h a t 5-methoxy DMT may be more l i m i t e d as a
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t r a i n i n g s t i m u l u s when screening f o r p o t e n t i a l phenethylamlne type compounds.
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in
The lack of a suitable In v l t r o assay for potentlal hallucinogenic drugs Is a c o n t i n u l n g problem. The use of pA2 values by Glennon and his coworkers f o r several years encouraged us to e v a l u a t e t h a t procedure. Our f a i l u r e to observe antagonism to the c o n t r a c t i l e e f f e c t of serotonin in the fundus by any of the three test compounds was very surprising, p a r t i c u l a r l y in view of the reported pA2 f o r compound 2 of 6.78 (14). A pA2 of t h l s magnitude should indicate a reasonably potent antagonist. We b e l l e v e t h a t the f a i l u r e to observe an a n t a g o n l s t e f f e c t f o r these compounds can be a t t r i b u t e d to certaln methodological problems inherent in the application of the pA2 approach to substltuted phenethylamines. Foremost among these is the fact that nearly a l l hallucinogenic phenethylamine type compounds are agonists in the fundus, and e l i c i t contraction. Reported pA2 values, for compounds that we have tested in the fundus, are usually in the range where we have observed s i g n i f l c a n t c o n t r a c t i o n of the fundus. We have not had dlfficulty a n t a g o n l z i n g the e f f e c t of s e r o t o n i n in the fundus w l t h other antagonlsts such as 2-bromo-LSD. An inspectlon of Figure 1 shows that a pA determination would involve the use of concentrations of 2 lower than about 0.3 micromolar, but s t i l l in excess of the reported pA2 concentration of 0.17 mlcromolar. I t seems impossible to us t h a t an accurate and s i g n l f i c a n t pA2 could be d e r i v e d w i t h i n t h i s narrow range of " a n t a g o n l s t " c o n c e n t r a t i o n . Concentrations of the antagonist lower than those correspondlng to the pA2 should probably not be used. Using the 3 pt x 3 pt, completely randomlzed deslgn p a r a l l e l l i n e bioassay (16), there was no s i g n i f i c a n t d i f f e r e n c e In the potency of I and 2, y e t the ED50 r a t l o was about 1.3. I t seems l i k e l y t h a t s i g n l f l c a n t s h i f t s in the EDbo w i l l not be detectable with precision u n t i l the s h l f t IS close to two-fold. From a t h e o r e t i c a l s t a n d p o i n t , the pA2 corresponds to the d l s s o c i a t i o n c o n s t a n t , KD f o r the a n t a g o n i s t . For a ~ull a g o n i s t , the EDen is always s m a l l e r than the KD (22) w i t h the ED50 only approachlng KD w i ~ very weak p a r t i a l a g o n i s t s . Thus, we assume t h a t the KDS, and hence PA2 v a l u e s , would correspond to bath c o n c e n t r a t l o n s much higher than the ED50. That i s , f o r compound 2 the pA2 would be greater than -Iog(ED50), i.e. greater than 5.37, i f i t were appropriate to determine a pA2 for a compound with an agonlst p r o f i l e . In summary, n e i t h e r the potency in e l i c i t l n g c o n t r a c t i o n in the r a t fundus, nor the use of pA2 values can be considered a r e l l a b l e model f o r predicting halluclnogenic a c t i v i t y for substituted phenethylamines. Acknowledgment Thls work was supported in part by PHS grant DA02189 from the Natlonal I n s t i t u t e on Drug Abuse and by Chemical Pharmacology Tralnlng Grant GM 709504. Instrumentatlon for the drug d i s c r i m i n a t l o n studleS was p a r t i a l l y funded by Biomedical Research Support Grant 2-S07-RR05586-15. References 1. 2. 3. 4.
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b. 6. 7. 8. 9. I0. ii. 12. 13. 14. lb. 16. 17. 18. 19. 20. 21. 22.
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