C00021849

[PAGE 2]
Desr Doctor

Enclosed in quadruplicate is an uusolicited proposal - to provide for the developoent of techniques and a facility for screeaing chemicals for their potential property of inducing temporary behavioral changes.

We have proposed a three-yoar progras witb the first year being devoted primarily to the developzent of techaiques. ueing known cowpounds as modele. The following two yaars would be devoted to screening copounds with soe provisioa for coastantiy improving techniques. We realize that such a project can be funded only o year at a time.

While ve cannot be certain, we balleve that by use of our alectronic data processiog equipoeat we asy be able to analyze the activity profile of screenad compounds in order to provide a more reliable guide to further compound davalopment,

If ve may clarify or smplify any polrt in the propossl, please do not besicate to let us know.

Sincerely yours,

Enclosures

C00021849'

49

Noveaber 26, 1965

[PAGE 4]
INTRODUCTION

i. This proposal concerns the screening of chemical compounds for their ability to alter the functional integrity of the complex processes of the central nervous system. The ideal compound, in addition, would be effective orally at lou doses, water soluble, and would bave neither taste nor odor.

The drugs which are classified by various authors as hallucinogens, psychomimetic drugs, psychopharmacological agents, or tranquilizers may serve as models on which the screening procedures are to be based, although, the idesl compound may not fit into these categories.

The pharmacology and various techniques of study of psychopharmacological agents (hallucinogens, psychomimetic agents, etc.) have been reported and reviewed by numerous authors (l-l2). From the array of techniques and methods available, the screening procedure which follous, we believe, constitutes sufficient depth and versatility to be most effective.

METHODS

A. TOXICITY SCREENING: In order to establish the potency of a material, mice have been chosen as the first experimental animal. In addition, by careful observation, especially in regard to characteristic actions in mice of various accepted psychopharmacological agents, one may be alerted to new possibilities.

C00021849

[PAGE 5]
will be held in temperature-controlled quarters for one week prior to compound administration in "shoe box" type cages with free access to water and Purina Laboratory Chow. The compound dissolved in water or other suitable vehicles vill be administered orally at graded doses to four mice per dose level (after range finding) to determine the oral LDso and minimum effective dose. The signs which will be noted sange from the most subtle, such as social interactioa, to death. Ihe degree of effect will be noted as slight, moderate, or sevete. The signs to be recorded sre listed below:

Social Interaction Depression Alert Analgesia

Respiration Ataxia Rapid Narcosis Slow Labored Prostration Arrhythmic :. -uunatha-s-

Ocular Lacrimation Hyperthermia Swollea Lids Salivation Ptosis Hiosis Mouth Dry Mydriasis Defecation

Reflexes · Urination Corneal Piloerection Pinnal Righting Vasoconstriction Tail Vasodilation Myotactic Hypersensitive Cyanosis

Tremors Localized General Intermittent

Behavior Unusual Straub Iail Vocalization Circling Hyperactive Hyperactive Stretching Stretching

Convulsions Clonic Tonic Death

C00021849

[PAGE 6]
The mice will be observed frequently following administration of che test materisl so that the onset and duration of activity will be known.

B. SPONTANEOUS MOTOR ACTIVITY.(Mice): The use of photocell devices is a widely accepted procedure and many reports concerning the effects of psychopharmacological agents are available in the literature (l2).

Mice purchased, acclimated, and housed as previously described vill be used as experimental subjects.. A maximum of three doses (maximum tolerated to no effect) will be selected on the basis of previous results and the compound will be administered to four treated mice per dose level., The mice will be placed in an. for 12 hours and the spontaneous activity -1

"| at 15-minute intervals will be obtained. The spontaneous activity of six control (vehicle) mice will be recorded at .ths same time.

C. SPONTANECJS ELECTRICAL CEREBRAL ACTIVITY: The effects of psychopharmacological agents on the electrical activity of the brain have been reviewed by Evarts (13,14) (n respect to psychomimetic agents, and by Brazier (l5) in respect to various drugs acting on the central nervous system. The preparation we propose to, use has been reported by Renaldi and Himwich (le,17) to be sensitive to 1 to 5 mIcrograms/kg of lysergic acid..

For each compound tested two rabbits will be curarized, artifically respirates, and fitted with surface electrodes. Increasing intravenous doses will be given at I5 minute intervais. The spontaneous cerebral electricai activity (EEG) will be recorded. The minimum (cumulative) effective dose, the type of response, and the duration of effect vill be reported.

C00021849

3

[PAGE 7]
A minimum of two dogs per compound will be used. The animels vill be fasted overnigh!, anesthetized with phenobarbital sodium and suitably prepared for right vagal stimulation and to record blood pressure, EKG, and intestinal activity. After stabilization, control recordings of the above parameters will be obtained. 1

After parameters return to normal., the test compound will be administered intravenously at doses selected on the basis of previous data. Changes in baseline values will be noted. The animal will

C00021849

[PAGE 8]
fas before and changes

in responses ln ttrms of the parameters listed above will be noted.

This procedure will be repeated lo every animal vith increasing doses of the test compound.

E. BEHAVIOR PROFILE (Cats)

Irwin (l) along with Norton and DeBeer (23) have suggested. the of standardized multidimensional bbservatlons thet áre es useful in the evaluation of drugs acting on the central nervous system. Using the procedures outlined below in cets, which were seleçted because of unusual signs elicited after treatment vith certain type of CS agents, we propose to establish s behavior profile for each compound.

A group of four cats acclimated co the procedure vill be tested once weekly in a quiet temperature-controlled room. The animals.vill 'be restrained in overlapping areas by leashes. Observatlons willbe made one-hour before àdministration of the drug and hourly thereafter, using as criteris: wakefulness, upright positlon, total locomotion, spatial activity, contact seeking, Brooming, pley, fearfulness, restlessness, vocalization,auditory response, aggressiveness, body tone, limb weakness, scaxia, nictitans relaxation, pupil size, heart rate, and respiration rate. The drug will be administered orally to.a minimum of two sets of four cats in graded doses (based on previous results) with one cat receiving no active ingredient.

F. BEHAVIORAL EFFECTS (RhesUs NOnkeVS):

(l) Effect on Social Behavior: First the order of dominance in grcup-housed monkeys will be definitively ascertained. Following this, compound will be administered orally to one monkey at a

C00021849

be treated with

[PAGE 9]
time froa various levéls of the social structure. The effect on the behavior nf the treated monkey toward other members of its society will be observed. Other behavioral effects that will be observed fnclude the order in which the monkeys eat and drink and the effect on the small cliques which are normally present.

(2) Effect on Aggressiveness: The "taming" effect of these compounds will be measured in rhesus monkeys by the method of Heise and Boff (24). This method consists of measuring aggression (forward lunging at the experimenter, baring teeth, and attacking expęrimenter's glove) and activity (general activity level or response to visual or auditory stimuli or ataxia). After drug administration a ratio, aggression score (per cent of own control)/activity score (per cent of own control), is determined. Taming is defined as, a ratio of 0.5 or less; a ratio of about 1.0 indicates that drug action or aggresslon is not specific. 3

G. OPERANT BEHAVIOR (Squirrel Monkeys)

The use of changes in opzrant behavior induced by drugs has in recent years increased markedly (25-30). Two broad types of operant behavior are differentiated on the basis of the type of reinforcement, rewerd versus punishment (30,31). Sidman (1l) has suggested that behavior controlled by different modes of reinforcement (food reward versus electrical shock negative reinforcement) may be controlled by different levels of drive. We propose to train animals to a multiple Fixed Ratio (FR) - Fixed Interval (FI) schedule (3l). Half of the animals will be reinforced with food and the other half reinforced by avoiding electrical shock (8). During the Fixed Ratio portion of the schedule, the aninal is teinforced after responding (lever pressing) a given number of times. In the Fixed Interval portion (usually taken to

C000218491

6

[PAGE 10]
Indicate temporal discrimination, 31, 32) the animal is reinforced by the first response after the lapse of a given time interval. The two components are cued by different visual stimulus and a third visual stimulus is presented in the reinforcement period or when responses elicit no effect on the reinforcement of the animal. pesined to

Four squirrel monkeys (2 for each mode of reinforcement) trained to relative responding stability vill be edministered the cest material orally, 30 minutes before testing begins (additional times will be allowed if a delayed onset of action has been noted previously). Ibe animals will be·tested the day of administration and daily thereafter until the behavior returns to normal. .Three dose levels per compound will be tested. The analyses of data will consist of FR responding rate, FI respondingrate, a mathematical index o[ FI performance (33), and responses in reward or time out portious of the schedule.

DISCUSSION

The first year would be devoted to obtaining.equipment, trainins snimals and running a serles of positive controls. It is estimated that 40 to 60 compounds could be scresned per year. This rate of screening ir the second and third year of the contract would allou additional time for research directed toward improving the techoiques of the screen, additional basic work on compounds of inserest, or further work (LDso and subacute toxicological studies) so as to permit limited use in man. permit llml. -L-asrnlogical

The procedures as described herein will provide basic pharmacological background in respect to acute toxicity, site ef action, duration of action, species difference, and side effects. Behavioral or psychopharmacological parameters related to mood, drive, visual and temporal discrimination, memory, social interaction, aggressiveness, . and motor coordination will have been evaluated.

C000218491

1

[PAGE 11]
LITERATURE CITED

2. Masserman, J. H.: Behavioral pharmacology. 1n: Neuro-Psychopharmacology l:97-107 (Bradley, P. B., P. Demker, and C. Radoucohomas, eds.) Amsterdam,Elsevier (l959)

4. Hunt, A. F.: Methods for studying the behavioral effects of drugs. Ann. Rev. Pharme=o1.1:125-144 (1961)

12. Cook, L. and R. T. Kelleher: Effects of drugs on behavior. Ann. Rev. Pharmaccl. 3:205-222 (1963)

C000218491

[PAGE 12]
,n -

…

CLe r. Tho nf n ér i-se:

C00021849