Fel.:,rua 1970' OPIES COPX OF Dear five copies of the third uarter Enclosed are Drogress report for the ontractt 2 -present c overs t.,,ie -07Ct-015-p-r I... lg(g to 1969., The report includes a state.-aient of expenditure of funds and CO-mPletiOn Of the assigned task. Please let us know if there are any questions 6 or co-,%-nents as to its ccntentso Sincerely'yours, THIRD QUARTERLY REPORT ABSTRACT Areas of activity during the third quarter are listed in this paragraph and elaborated below. Progress continued on an accelerated level on the@basic toxicity and be- havioral screening programs and additionally included cardiovascular pharmacodynamic testing of a group of compounds and iso2lation and testing of extracts of several oriental plant products. A total of 53 compounds were received for 'screening during this time,interval. In addition, a natural products chemist began work on Preparation of extracts of oriental plants reportedly of medicinal value according to Chinese folklore. Several of, these plant extracts have been tested for biological activity and toxicity 4in mice; work has proceeded to the point of separation of several active fractions. The visual discrimination apparatus has been received ft and assem led. ..To date 61 per cent of the funds have been expended and 61 per cent of the task completed. 2 PRIMARY $CREENING 36 compounds were During the current reporting period, tested in mice by the acute toxicity screen and locomotor activity tests. Practically all of pot dd showed these com n a suitable minimum safety ratio between t2he LB@ 50 and MED so to warrant further testing. Five compounds showed out- standing safety ratios.(1000 or greater).. These were and In addition 10 compounds showed ratios between 100 and 1000. However, all compounds were tested in pats At a dose (mg/k4) 1/10 the mouse LD 250 level, for the effect on physical, neuror logical, and behavioral status. Ten of these compounds showed significant activity. SECONDARY AND ADVANCED BEHAVIORAL SCREENING Thirty-four compounds were evaluated in hooded rats by the motivation and sequential response behavioral tests. The dosage (mg/kg) administered was 1/20 the mouse LD 501 or lower -if overt toxic signs were e2vident in preliminary test rats. In both the motivation and the SRBA tests a number of compounds showed some psvdhopharmacologic activity, as evidenced by an increase or decrease in start or run speeds or a decrease in the number of rewards and @an increase in the percentage of errors. ror further eval- uation, test compounds were administered to monkeys, and 3 the physical, neurological, and behavioral effects, including changes in shuttle order behavior in the shuttle box test, were determined. This is further described in detail in a separate section below. Sixteen compounds were studied in monkeys. PHARKACODYNAM.IC SCREENING Pharmacodynamic screening was c3zried out on 18 compounds in anesthetized cats. The pro-tocol. --zi thf-- pzo.---ed-ire and the results obtained a.:e des.-.ribed belzw. Pharmacodynami-- S=reenin2 in Anesthetized Cats Compou2nds'were screened,for pharmacodynam4&c activity in cats anesthetized wi4--h CC-chlo-ralose (80 mg/kg, i.p.). Arterial blood pressure, heart zgte, respiratory movements,, and EKG's were recorded on..an E and 1.1 Physiograp4.__Effects on responses to the 3dm4-ni.stra*-"Lo to 2va§al- stimulation and to carotid artery oc--lusion were measured. Challenging drugs were usually administered at five-m.nute intervals or after arterial pressure had ret-arn,:@d to the control level. All injections were made via polyei@-.-hylene cannula inserted into the femoral vein, Each compound was usua'L.!y tested in one ani.Tal. The initial dose was either 0.3 or 1.@' mg/kg. Subsequent doses were in- creased or decreased by one-.bal-f of a loaarithmic interval,, depending on whether a response was eli--ited. The results of these experi-rents are surr@marli@ed in the Appendix. Intrinsic activity w;=-@s obser7ed with 15 of.the 4 18 compounds tested, One compound caused an increase in blood pressure and 12 compounds caused de-zreased blood 4 pressure. The duration of each of the blood pt-essure changes was less than five minut6s. EKG vol.caae increases were observed after alminisl-.ration of None of the compounds a,lfected the responses to any of the challeng- ing drugs or pro:edaresl, BEHAV2IORAL TITSTTNr-t OF SQUIRREL MONKEY Certain personnel. a-ldiz.-Lons,, proced-i.ral-implp-mentaticns7 and equipment modificat-ions were inztltuted to facilitate r-he operation of the s -4uir@r-el monkey testing pr-ograir. Personnel Addi.ti-@n-s A beba-vicral scientis'll- was added to the staff and assilned to the squirrel monkey testing procedure.. L T)MI I E rf,tM o--i efficien--y of the t-estirg pro--ed-.,Zres and to insuze the validity of r-=su.it-s obts-Ined a,-e listed below: a, The tes-l' 2 containing the shuttle box was isolated from the data--recording area and the investigator by a boo*:h n,.ade of sheets of opaque black plaslica Both the investigator@s and testing areas were then sim2la@ly separated fe-o-m th-- gsneral room-environ- Thus, @@he room Izhl--s could remain lit during testing procedures so that the untreated moakeys wo,;ld be exposed to a normal light-dark cycle. b. A,onel-way m,@.-rox was placed between the area of the i..nvesttga-..,@r and that o4 the tes-- subject:. so that the monkc-ys ppfforrri8ed theiz tasks while being visuall.y isoiated from the influence of the in- vestiga,oc or g?n?ral r--Iom movemenl but fully. ob@erved by th- in-;---estigaro- 5 c. General fl-.o-,escent lighting was placed in the area of the sbuttle apparatus to provide an overall il- luminance so the monkeys could readily see the visual cues and the investigator coul&d easily determine the monkeys' iden--2,-fication,and behavior patterns. d. A mechanical door closing syst-em for the shuttle boxes was added so that the entire testing operation, after the monke was placed in the shuttle box, re- y mained v-isu-3@lly isolated from the monkeys. This 2 further redu@--ed intra-test disturbances and vari- ability, The abov-= modifications were designed to isolate as mu,--h 'tuatio-,is, both visual and auditory This is si essential for the propacation of m2otivational data and response cons,.stency. A styr.-foiz! weighing cage.and a plexiglass injection tiibe were constructed to reduce mechani--al injuries. due --o handl.ing contact,,and infection from abrasion, Procediiral Chanass - tertain dhanoes were mad2e to maximize ttie data preeently ob,--ained and to provide for an increase in data in the .4uture. a.-,- Scheduled training sessions w@Br@m'ihitiAted.- Thi@i56",@7il'l contitue -in",-il the pr'oje!=ted full complement of trained monkey grolip.s is attain6ed, At present., one shut.tle group of three monkeys trained to both a positive and negative 6 reinforcement schedule and one group of three trained to a negative regime are available for group shuttle testing. In addition, seven males, which can be trained on a positive reinforcement schedule to replace any established monkeys or which can be used for the visual discrimination testing program, are in the colony. b. Seven female squirrel mon2keys were purchased and condi- tioned to the shuttle box apparatus. These are used daily to determine a dosage-level of test compound (mg/kg) which produces minimal behavioral effects and can thus be applied in a shuttle operation. These monkeys are important in the detection of behavioral effects.observed in the shuttle apparatus or in the home cage, caused b2y varying drug dosage levels. -The procedure on individuals includes the observation of many behavior 7; patterri'd and riiotivAt'!E6'nif-iEi-ei@ 4 mation about a specific drug effect which may be present. (1) Observational Procedure and Criteria. The individual. is observed and subjected to the stimuli presentat2ions every five minutes in the shuttle box for a 30 minute control period-prior to dozing. The first dose is injected, and the animal is placed back in the shutt e box and observed for one hour. Subsequent increasing dose levels proceed hourly until the maximum determined dose is reached '(cumulatively). The individual is then observed in its home cage after four 2hours and after 24 hours. The behavioral activities being cataloged during this procedure'are: (1) reactions to observation and.handling by the investigator (flight, attack; or passiveness); (2) grasping ability (to bars and manipulanda); (3) feeding behavior (eating or not); (4) reaction to blinking @ight (fixation, orientation rt resp0onse); (5) reaction to mild shock (i rk, tremor., hop, etc.); 7 (6) reaction to sound of buzzer (alert, orientation, etc;,); (7) shuttle response (ability to shuttle after light cue, door open,, and shock.if'used); (i) level of self-mani ulative behavior (groominqi self-inspection, p etc (9) locomotor coordination, speed, ability 2 (natural, under stimulation and during shuttlei; (10) level of general activity; (11) amount of sleep;' (12) behavior during crouching (alertness, head orientation responses, eye@fixation). (2) IpjeCtion Procedure. All injections of test compounds are g3.ven intravenously via a caudal vein. The total dose level is determined by extrapolation using as a base the lower limit of the 95 per cent confidence limits of the LD 50 for mice. The dose to monkeys was calculated using the conversion formula of Paget and area; Barne-s which is based-on tt±f &ce- reference to Dr. Elton Homan's equivalent surface area 2 2 dosage conversion factor card as reported b Freireich y The maximum dose level (hig/kg) administered to a squirrel monkey as extrapolated irom the dose to a mouse to yield an equivalent-dose based on surface area is determined by multiplying the mouse dose (mg/kg) by a factor2 (0.45). Aliquots of this total dose are then given in one hour intervals until effects are observed or the extrapolated dose level is reached. REFERENCES 1. PaggE.- G.E.,, and Barnes, i.M., 1564. Chap, 6. Toxicity Tests in Evaluation of Drug Activities, Pharmacometricso P.R. Laurence and A.L. Bacharach (eds.)., Vol.-l9i, Acac3-emic---,@ Press, pp. 135@-167. 2. Freireich, E.G., et al., 1966 Quantitative Comparison of Toxicity of Anti-cancer Agents in Mouse, Rato Dog, Monkey, And Man. Cancer Chemotherapy Reports, 50(4),, 219-244. 8 NATURAL PRODUCTS 1. Chinese Toxic Compoundso The names of 47 intact plants roots,, tubers, and flowers were selected from a medieval Chinese medical and pharmaceutical reference (Li Shih-Chen, 1551). Botanical names of these plaiits are as follows: 1. Rheum officinale 2. Phytolacca esculenta 2 3. Peucedanum Japohicum Thunb. 4. Potettille cryptotaemiae maxim 5. Euphorbia adenochlora Mort et Dene 6. @uphorbia sielaldiana morren et Decaisne 7. Euphorbia heliscopia L. 8. Galarhoeus siebaldianus Hara. 9. Semen Euphordiae Lathyris 10. Hyoscyamus agrestis Kitai2l 11. Un'itsu (unknown in Western terminology) 12. Richinus communis L. 13a Orixa japonica Thunb. (Dichroa febrifuga) 1,4. And ropog oD. @,S=-gt= 15. Veratrum nigrum L. 16. Leucothoe Grayana Maxim 17. Aconitum Fischri Reich 182. Tenyuh. (unknown in Western terminology but similar to Aconitum Fischori) 19. Aconitum Chinese Sieb. (daughter root)- 20. Aconitum Chinese Sieb (small root) 21. Aconitum Sinesis 22. iatropha Janipha 23. Rhizoma Arisaematis 24. Arisaema ringeus Schott var, Sieboldi Engl. 252. Hydrosme Rivieri Engl. 26. Pinellia Tuberifera 27. Polygonum bistorta L. 28. Podophyllum Versipelia Hee. 29. Pardahthus sinentis 30. Iris tectorum Maxim 314 Hosta Sieboldiana Engl4 32. Kap-=-pferris GalAnga 33. Zachoso (hot in WeStern term-4ino@logy)----- 34. SI-ramonium Datura Alba 35. Rhododendron Sinense Sa. 36. Daphne Genkwa S. et. Z4 9 37. Wikstroemia Japonica Miq- 38. Baddlea Japonica Hemsl. 39 Illicium anisatum L.- 40: Shimia Japdnica Thunb. 41. Rahunculus sceleratus L. 42. Ranuhculus acdr. L. vAr. Japohicus Maxim, 43. Aconitum Lycoctomum L. 2 44. Urtica Thunbdrgiana S. et Z. 45. Gleditschia glauca IiK 46. Alodasia Macrorhiza Schott 47.1 Rhus Toxicodendron L. var. Radicans Miq, Plants numbered 41 13-.17,, 22s, 28 29,, 30, 31,, 34# 35, 37,, 38 p I r 42., 43, and 44 have been checked by 2the Natural Product Section of the National Cancer institute and were negative in anti-tumor tests. Howevet, several fruit-bearing tuphorbia species showed activity against Sarcoma 180, Walker 256, and Lewis lung carcinoma, Several of the active principles are in the process of fractionation, but in only one case was the Active agent isolated and this was reported to.-be tannin. Didhroa,-f-@24;7-@ -root was@-active gg_z@i XB cell culture. This plant is being fractionated, but the active agent has not yet been isolated. The rhizome of one species of Arisaema was active aginst Lewis lung carcinoma, but the active compound has not been identified4 Twenty-four species selected from the above list of materials were ordered from a Chinese pharmaceutical dit2tr3'.butorl, Becaute the is encounter- ing some delay in acquisition, these-24 species also have been ordered from other sources in In addition to the plant materials described above@, a Chinese pharmaceutical-and extracts of black tea, q reen tea, and aronia fruit were tested for toxicity in mice. Sri, 10 some a Chinese pharmaceutical, was obtained from The chemical composition of the material.is unknown. Ten milliliters of distilled water was thoroughly mixed with 1,125 grams of this compound, and the material was injected in mice. No effect was observed. In the appended compnter printout tables, this material is re2ported as No. 300000. The tea extracts showed some activity in mice; however,@ it is likely that the effects of caffeine masked any pharmaco- logid action by other ingredients. The tea extracts.are designated in the appended computer printout as follows.- Extradts of green tea: Nos. 110000, 120000, and 130000 Extradts of black tea: Nos. 140006 and 150000. 2 Fruits of the aronia plant (malus Halliana Kochne) were harvested during the winter from trees located after all the leaves had fallen and only brownis red fruits remained4 The fruits were immed- iately frozen and stored at.-150 with dry ice in a stytofoam cabinet, Frozen fruits (32.51 grams) were blended first and- then homo2genated with an electric blender for 10 minutes and extracted twice with 100 milliliters of distilled water by stirring under nitrogen for two hours at room temperature. The filtrates were combined and concentrated to dryness using a freeze-drying procedure. The residue of the filtrate was re- ith 100 per cent.acetone (100 milliliters). extracted twice w 7 rated to dr yness The acetone extracts were combined and evapo under a vacuum. soft The residues from the water extract fraction and the acetone extract fraction were each made up to a volume of 32 milli- liters with distilled water, Each milliliter contained the extractable material equivalent to one gram of freshly weighed .fruit. Ali4udts (40 ml/kg, 16 ml/kg, and 5 ml/kg) of these solutions were injected intravenously in Dublin (DUB/ICR strain) albin2o mice via a tail vein, The effects of the injections are presented in the computer assembled tables (fraction numbers,, 220000 acetone, 210000 - water) and show that an active compound was soluble in acetone, The mice died during, or immediately after intravenous injection of a dose of 16 ml/kg of the acetone fraction* The previous experiments showed that at least one toxic compound 2 was distributed in the,acetone fraction, so the compound(s) was (were) further separated and purified with a series of organic solvents since the appearance of the acetone soluble material(s) in a particular solvent fraction would provide information concerning the polarity and chemical properties of the compound(s Another quantity (61.23 .grams) of fruits.lwas blended with an 2 electric blend er and extracted twice with 200-milliliter aliquots of n-hexane ('dielectric constant 1.9), chloroform (4.8), diethyl ether (4,3), acetone (20.7), ethyl alcohol (24.3), and distilled water (78.5). The extractions were carried out At room temperature. Insoluble material was filtered from the extraction fractions usi2ng Whatman Oo. 1 paper, Weight Per Cent of Biological Fraction Fresh Weight Activity 9 n-hexane 0.322 0.52 chloroli@-orm 0.521 0 85 ++ ethyl ether 1.157 1:-'88 5 acetone-1 0.456 0674 aceton6-2 5.668 9.80 +++ ethyl alcohol 2.0917 3.41 distilled water 1.331 2.17 + 12 ive comp As shown above, at least two act ounds were extracted from the aronia fruits. The designations ltacetone-l" and ol'acetbne-2" indicate different steps of fractionation; the residue following the diethyl'ether extraction was extracted twi th 200 milliliters of 100 per cent actone. The acetone ice wi extracts were filtered and the filtrate was evaporated to dry- hess. The sap of the fruit is miscible in acetone, and there- fore it was possible to extract a polar compound in the acetone. The residue was taken otce completely to dryness in a desiccator @and then re-extkadt6d with acetone (acetone-1). The residue o2f the acetone extract was freely soluble in water (acetone 2). One-tenth aliquotz of each of the residues from each extract were dissolved in 6.2 milliliters of distilled watet from which portions (40 ml,ikg, 16 lul/kg, or 5 ml/kg) were injected in 0 o-lvent extract fractions were suspended---in m-i- -c npolar s 0.5 per cent methylcellulose atd injected 2in the same volume as the polar fractions. The nonpolar fractions were n-hexane, chloroform, diethyl ether, and acetbne-1. The water fraction contained an active compound which resembles the active compound in the acetone-2 fraction. The results of biological testing are shown it the appended computer prinout tables. The acetone-2 extract is.No. 214200 and the water extract is No. 216006. At least two biologically active compounds were se-parated from the aronia fruits by the described procedur6s. one is nonpolar and the other is a polar compound. No difference in biological act,-@,,.ity was observed after the pH of the extracts was adjusted 3 with sodium-hydroxide to 6.8 from 2..S 3.0 in the acetone, ethyl alcohol, or water fractions. The active compound in the acetone-2 fraction was purified further. Four hundred sixty-nine milligrams of the acetone-2 fraction residue was dissolved in 0.5 milliliter of distilled water. The water solution was streaked onto six TLC p:@ate2s,, which were Brinkman precoated and had a 0.25 mn thickness of Silica Gel-G containing CASO 4 as a binder. The TLC plates were developed at room temperature with an ethanol (96 per cent):water:ammonia solution (23 per cent), 100:12:16 v/v, solvent system ('Braum and Genneu, 1962',.. j. Chromatg. 7, 56) . Chromatographic separation con2tinued...for-eight.hours. The plate was divided into 10 parts on the basis of Rf values. Each part was scraped off with a spatula and suspended in -.-..di ti-l.lecL,watqr ter extr lyophilized and s -.The wa acts were the residue was- dissolved in 3.1 milliliters (two times con- centrated solution of 6.2 milliliters*) fr2om which portions were used in the bioassay. The Separation of Active Comt)ound of Acetone-2 Fraction Using Thin-Laver ChromatogrEaaphny Fraction Weight of material Weight Biological No.1 With -Binder of Binder Activi@y 1 167.1 29.8 + 2 132.22 29.8 3 126 0 29.8 4 79:7 29.8 5 47.2 29.8 + @6- 45.8 29.8 45.4 29.8--@ 7 8 48.8 29.8 9 2 47.6 29.8 10 45.0 29.8 TOTAL 784.8 298.0 Correspond to Rf values. Recovery of experiment procedure was (784.8 - 298 c 486.8 486.8/569 x 100 = 85.5% Ori e-Tlyi;;l waiah+- of thp, matet-ial was--:io9 ma from 6 2 a of fresh wei-aht 14 The active compounds located in the acetone-2 fraction have not been completely purified yet, so they have not been identified. This will be done in the future, as will the purification of the active compound which is soluble in chloroform. T@e arohia extracts which were.active, showed an interesting activity, suggestive of that produced by several typesof compounds, such as s2timulants, morphine-like drugs, or hallucinatory agents. GOALS FOR NEXT PERIOD Work will continue on all aspects of the behavioral screening as described in this report. Additional compounds w-.11 be obta-ined-for testing. Training of monke s in the visual y task discrimination will be initiated. Natural product.isolation will be continued and extracts teste1d. Pharmacodynamic screen ng in anes'thetized cats will also be continued. Submitted: February 13, 1970