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Dear Sirs:

Enclosed in quadruplicate is a proposal for the extension of work being cenducted wncer Contract 1.Task Order No. for the period February 28, 1969, through February28,1970.

This proposal provides for a substantial increases in the level of effort in orcer to accomplish corc acvarced pharnacological characterizations of compounds. showing interesting properties by the prinary screening procecures. Since the advanced tcchnicues will cmphasize the use of nonhunen prinates, it is anticipated that the results cbtained will have good predictive value.

Sincerely yours,

Enclosures

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February 26,.1969

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TLCHNICAL PROPOSAL

submits this unsolicited proposal for continuation of the, program on piaroacolcgical scrcening of potentially useful new agcnts.

AISTPACT

Each new couvound will be examincd by primary screening in mice using acute toxicity scrcen and loccmctor activity tcsts, as well as by effect on te pysical anc neurolcgical exaninaticn in the cat. Prcnising cancicato materialo will he evaluated by secondary screening, incluáing a motivation test and a sequential rcsponse test using iccded rats. Further tests will be cone with vory promising materials to cvaluate cffect on social behavior of sauirrel monkeys, mcchaniem of action, cffects of antaconists. and dctailed anaiysis of offects on behavicr using suitable animal species with emphasis on primates.

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TECHIICAL PROPOSAL

submits this unsolicitoá proposal

for continuation of the program on pharnacological screening of potentially useful new agents.

HISTORY:

Since renewal of contract No. Task Order No. 01 has through the'contracting agency arranged vith a pharmaceutical conpany for the acquisition of compounds for scrcening. At the tine of preparaticn of this proposal, 48 compcunás received from this source have been screeneā by primary screening methods including mouse toxicity screen, spontaneous locomotor activity screen, and evaluation of behavicral and neurological profile in cats.

.Secondary screening is now in progress. At least eight of the initial conpounds show pronising activity as listed in our seconá interin report. The initial secondary screening program consists of a motivational test aná a sequential response test, both using noodcd rats. For the latter test, tuo apparatuses were constructed, and are now in operation. Additionally, selected pronising compounds are administered to selected monkeys for evaluation of the effect on physical, neurological, and behavioral profile as with the cats. Bahavior is evaluated by the effect on such characteristics as curiosity, aggressiveness, and fear. Seven squirrel monkeys (Sairire sciuresus) have been obtained, and are trained in a tost situation to evaluate changes in social behavior and performanca in situations which invclve both positive and ncgative reinforcement according to the method of Plotnik (Plotnik, R., Jour. conp. physiol. Psycol. 66 (2),

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36s-77, 1968). In each of two groups of threo monicys a hiorarchal pattern of aggression or subnissiveness has emerged. The monkoys are ranked accordirg to this charactcristic. Changes in aggressiveness, dominance, or subnissiveness inauced by the most promising candidate drugs are evaluated in subnissive or aggressive monkeys, respectively.

PROPOSAL FOR THIPD YEAR:

Each compouad will be examined by primary scrcening methods in mice and cats as described below.

I. PRIIARY SCRECUING PHASE

l. Acute Toxicity Screen - Mice s.

This procedure respresents the first stop in eventual accoptance or rejection of a compound. The scrcen is dcsigned to eliminate from consiceration compounds which co not posses sufficient biological activity, and to indicate types of prcmising activity. Criteria used in the selection of a compound include:

(a) Ratio of lethal cose to effcctive dose (safety factor)

(b) Speed of onset of pharnacolcgic signs

(c) Duration of action

(a) Type of action

(e) Completeness of recovery from the effects

(f) Degree of severity of signs observable

The effects sought are piarmacologic signs which are reauily reversible in a progressive series of tests. Ccmpounás are acninistered intravenously, and toxic signs are noted by gross observation or by manipulation and are recordod fron among those listed in a List of Reaction Signs and Standard Ters

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for Toxicity and Symptomology Feporting. If tho $I D _ { 5 0 } / : I D _ { 5 0 }$ ratio is l0 or grcater, the compound is tested ky intravenous injecticn in cats and administcred in behavior tests for secondary screening.

Froa previous experience, it has been found that with trained observers, the touse toxicity screen detccts aigrificant reaction signs (a prezerred term to toxic sign) for ccmpounds lnon to be mentaily or paysically incapacitating tc man. The test is perfored in a "partially blind" manner; i.e., the cbserver is not informed of the structures of the compound to be scrcened, but is responsible for preparing solutions and dilutions, and injecting the mice. Technicians usually work in pairs, one person recording the data and, whenever necessary, helping to cbserve the mice, while the cther techaician sorves as the regular observer. From tima to time, kncwn or standard compounds are introduced into tho routine screening as unknowns to chcck the reliability and rcprocucibility of the screening technique.

Prenaration of Compound fer Inicctions:
In the abscnce of soluiility informaticn, very small,
unrcighed amounts of comound are tricd in the following solvents and in tie order listed until a suitable one is found. lieat nay bc used to aid solution.

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2. Locomotor Activity in :ice

The spontaneous loconotor activity test measures the Crive of an animal to move. This test is recognized as measuring a basic paraneter for tho scrcening of potential trarquilizers and sedatives (Jacobsen 1964) and excitants (Chen 1964). Tho apparatus used for this tost, the

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For interpretation of the data, special attention is given the following parameters:

(a) Dosaga levels at which maximal, minimal, or no cffect is seen.

(b) Time of onset of drug effect at each dosage level.

(c) Duration of a change in locomotor activity of treatment groups as conpared to that of control groups.

REFEPENCES:

Jacobsen, E., Tranquillizers and Sedatives, Chapter lo, Evaluaticn of Drug ictivities, Vol. I, ed. by Laursnce, D. R. anù Bacharach, A. L., 2P 215-237, Acañemic Fress, 1.Y., 1964

Chen, G., Antidepressives, inalcptics, and Appetite Suppressants, Chapter ll, Lvaluaticn of Drug activities, Vol. I, PD 239-260, Acacemic Press, N.X., 1964

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3. Physical and :curological Exanination with Echavioral Observations of Cats Created vith Candicate Compcunis

In order to assess the overall physical, neurological, and behavioral effccts of candidate compounds, a systonic examination of physical signs, sensory, motor and rcflex reactions, and behavioral rcactions to the observer is ccnúucted in intact cats (kcGrath, 1960, and Norton and deEeer, l95E). Each animal is exanined before recciving drug, and periolically thereafter.

The examination consists of orservation of heart rate, respiration rate, boay temperature, pupillary dianeter (constricted or dilated), pupillary response to light, sensory response using a sharp needle to scratch or poke the skin (superficial) or pinching tho toe pads (acep), rotor activity (walking), spinal rcflexes (flexor reflex, kncc jerk, cxtensor thrust, scratch reflex, crossed cxtonsor reflex, spinal visceral reflex), and attitude and nostural rcacticns (attituda reflexes: tonic neck placing reacticns, and hopping rcacticns). The behavior of each animal and its reaction to the ocserver are noted during the exanination pericd. Drugs are acninistered intravenously (on a weight Lasis) in a Cosage of 0.l of the LD50 level in micc. Cats are exanined in most instances at aporoximate intorvals of 0.5, 1.0, 2.0, 4.0, 6.0, and 21 hours arter comnound acministration.

Urusual observaticns are presented in tl:e tables which accompany the detailed rcports. Although only positive significant effccts are usually noted, the presencs of a normal sign may be emphasizec in order to indicate that an unusual effect was cspecially scught.

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Thus when cilated yupils are recordcd, the pupillary light reflex is especially noted. Whcre both cilatation of pupil and paralysis of the light reflex occur, a parasympatholytic offect may be assuned to be prescnt. The hopping rcaction seems to be an especially sensitive one for tcsting prcpriocepticn. Therefore, this test is inúividually recoruec. In sone cases, 'sedation or over-activity and hyperexcitability arc noted. When the animal becomes hyperexcitablo, it scmotimes is impossiile to do a complete exanination. Eowever, such observations which can be made are noted in these inutances.

FEFERENCES:

NcGrath, J. T., Neurologic Examination of the Dog, 2nd Lá. Lea and Febiger, Phila., l960

Norton, S. and deBeer, E. I., Effects of Druçs cn the Behavioral Patterns of Cats. Annals of the Hew York Acaceny of Sciences, C4, Pp 249-257, 1956

II. SECONDARY SCREENING FILASE

I. otivation Test

his test is used for moro dotailed study of prcmising ċrugs, and is conducteá according to the method ofDarry and Niller (1965). üooded rats are trained to run £zom a stert box covn a straight alleyway to a goal box. Thc response is measuzed under tires motivational concitions: focd approach, shcck:. avoidance, aná shock escape. Separato groups of !:coded rats, approxinately equally distributed by weight and age are intially rancomly assigned to groups, and trained to apprcximately coual performance. Thus the motivational concitions ars highly

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comparable (sane rosponse,approxinately ccual pcrfortance)yet incependent of each othor (separate groups).

Six rats aro trained to gach motivation. Each of the rats is run through six trials daily fox five ¿ays per week. Start and run tines are recorded electronically to the nearest 0.0l second. The dosage schedule is as follows: On Nouday, no injocticns are adninistered, but the rats run through the trials to "war up" for the weck. On Tucsday, saline injccticns are aáninistcred aná "control rcadings" obtained. Cn Wednesday aná "hursday, ¿rug injections erc administered intravenously to two rats of each group, and saline to the renaining fcur, and trial runs are then concucted with all rats after an elapsed.time pericá previously determincd to be that of probable peak drug effect.

Conzounds wnich show exceptional activity will be further evaluated by the Secuential Response kethod in hoodcá rats (Policora, 19o3). Two units of the device for concucting these tests have been recently completed in our nanufacturing facility.

Secuential Pesponse Behavior Tost

The sequential rcspcnse behavioral test (Policora, V. J., Jour. Exper. Anal. Behav. 6, 271-277, l963) is a mathod uscful for the stucy of detrimental crug effects cn comolcx bchavior using hooded rats as excerimental subjects. The test compartment is cylindrical in shape, and contains at the pcriphery, four response podals separated by 9o angles. Above the response pedals are a siqnal licht and a fountain to yield liquid rewards, after a corrcct sequence of pecals is pressed by the experimental subject. Subjects aro maintaincd at a stable respcnse lovel by 2 or 3 lour water deprivation, one-half hour

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ad lib water, cnd frequert practice sessions. Baseline data control are obtained by. aúninistration of saline solution, in place of árug, and obtaining characteristic resncnse and rewarc rates at that tire...

REFEPENCES :

Barry, li., and Willcr, N. E., Comparison of Drug Effccts on Approach, Avoidance, and Escape Kotivation, Jour. Cemar. Physiol. Psyclol. 52, 18-24, 19€5

Polidora, V. G., A Sequential Response Kethod of Stucying Complex Bchavior in Animals and Its Applicaticn to the Neasurement of Drug Effccts. Jcur. Exper. Analysis Behav. 6, 271-277, 1953

III. TERTIARY SCREENING PHASE

1. Social Behavior in the Squirrel :onkey

The effect of very promising drugs will be evaluatod on sccial behavior of squirrel monkeys using the methcd of Plotnick (Jour. Comp. physiol. Psychól. 66 (2), 369-377, i968). The apparatus censists of twa boxes with plexiglass sides connected by a tunnel, which is separated fron thie boxes by guillctine doors. The floors of cach box and the tumncl contain parallel rods, vhich nay Le electrificd. Focd pollots can be aiministered at the end of cach Lox.

In this test groups of squirrel nonkeys are trained to rospond to a cue (light, Eor negative reinforcoment, scune for positiva rcinforconent) by running tiroug: the tunnsl to the cucosite comnartrent. Cbservations are nadc of running orcer and aggressiva interecticns, to dcterined the hierarchy cf Coninance-subnissicn. Fcod ccprivaticn is maintained to furnish the drive for positive food reinzcrccnent. The effects of cancidate crugs on agyressive and suinissive beiiavior. vill

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2. Drug Classification Stucios Suitable studios will be concucted to detormine clans of Crug action, rechanism of action, andi anticotes of promisirg test materials.

Such tosts in rats and other species will include offect of crugs on tlireshold to produce convulsions witi netrazol-or . eloctric shock, ability to proiuce catelepsy, potentiatien cr Llcckage of irug effect by neurohucral agents and potentiation or blcckage of known active naterials ry test ccmpcuncs. Discussion of thc applicability of thesa tcsts is given in soveral chapters of inimal Behavior and Lrug Action, ed. by Steinbcrý, i., Little Brown & Co., l964, especially the-discussicn by Janssen (2. 302).

1 3. Advanccd Behavioral Analysis on lonhunan Primatcs kethod development using prinates as tcst .subjocts ill continue for the purpose of application of suitable tosts for a complete cescription of belavioral and central nervous system effccts : or promising Crugs.

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subnitted: February 2G, 1969

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COST BREAKDOIN

THIRD YEAR

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COST ESTIXATE TECHICNL PROPOSAL- THIRD YEAR

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