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Dear Sir:

lis pleased to submit in duplicate an unsolicited proposal entitled "Studies on the Mechanism of Action of Compounds Affecting the Central Nervous Systcm Using Tissue Culture Techniques."

This study would be under the direction of 1 Ph.D. Curricula vitac for Dr. and others are appended as part of the technical proposal.

. The cost to conduct this study is The breakdown of costs appears in the cost proposal which is appended.

· If there are any guestions concerning this proposal, please do not hesitate to contact us. We look forward to working with you on this project.

Sincerely yours;

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March 27, 1972

Enclosures

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USING TISSUE CULTURE TECHNICUES

Today there are many compounas known to have a pharmacological effect upon the central nervous system. However, the exact. biochemical lesion(s) or effect(s) which triggers this xesponse remains, for the most part, unknown. Until such time as the mechanism of action of these compounds is known, (i) accurate screening tests to measure the activity of these end other test conpounds cannot be evised, (ii) a person who has been exposed to these-materials cannot be identified, and (iii) the effects of these materiais cannot be countered.·. The use o new analytical techniques in a tissue culture system provides a unigue assay systen for studying the mechanis of action of a compound with an effect upon the central nervous systen. Tissue culture systens are much more readily controllable, more reproaucible, more easily manipulated, cheaper, and can yield more data in a shorter period of time.

Because of the immense background data in man and animals on the pharmacologic effect of narcotics, these analgesic and addictive druqs will be stuaied as type-specific instances of compounds which affect the central nervous system. The sane methodolocy and techniques are directly apolicable to any other type of conpound desired. There have been many theories developeá as to what changes occur within the body following auministration of a narcotic and/or development of the tolerant-dependent state (l,2). The best current explanation is that there is intcrference with several biochemical

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STUDIES ON THE MECHANISX OF ACTION

OF COXPOUIDS AFFLCTING THE CENTRAL NERVOUS SYSTEM

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reactions including a change in protein and nucleic acid synthesis as well as changes in the pathway of glucose metabolism.

There have been several reports in the literature demonstrating the feasibility of using tissue culture systcns to study the action of central nervous system affecting conpounds. These reports cover studies on the initial effect caused by administration of a narcotic (3) as well as the changes inducea-during development of the tolerant-cepencent state. The following secticns describe our experimental approach using narcotics as type-specific drugs.

EXPERINENTAL APPROACH

A. Cell Lines and Growth Conditions

In a study such as this it is most inportant that several different cell lines be used so that common reactions can be aistinguished fro reactions specific for only one cell type. Also, there is the probability that certain cell lines may not show a reaction whereas others may. We propose to use human cell lines at first. If these are unsuccessful, we may have to switch to animal cell lines.

We will use human aminion (as a primary isolate), WI-38 (as a low passage Ciploia line), and Heia (as a. heterploid, established line) as the cell lines. The alternate animal choices would be chick embryo, LLC Mk, monkcy kidney, and L-929 mouse fibroblast, respectively. The primary isolates will, of course, be ud immediately. The other cell lines

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will be grown on Eaglcs Basal media plus 20% hunari or fetal bovine serum. For most of the experiments, whcre microscopic observation is involved and where the techniques are best known, the cultures will be grown and maintained as monolayers.

It has been reported that hunan serun albuin reacts with-and binds with narcotics (5). Apparently this is not a serious problem as previous tissue culture studies have indicated an eřfect by morphine in serum-containing media.

B. Exposure to Narcotics

In addition to using several different·cell lines it will be most important to use several aifferent narcotics which differ in"their analgesic and/or addictive properties. We propose to use las a strong narcotic with addictive properties. We propose to use 1

as weak narcotic capabilities and onists.

with limited additive as narcotic antag-

have been shown to be' toxic when added to HeLa cells in cultuze (3). Cell growth was inhibited and cytoplasmic vacuolization was induced. However, at certain levels the cells were made dependont upon 1 After 20 passages withdrawal of from the culture

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meiia lead to a decrease in the growth rate, cellular changes and death. If was again added to duplicate cultures the cells regained their prior appearance and growth reswmed.

C. Parameters to be ∴easured

(1) Growth Rate - The growth rate will be determined by the time required to form a confluent monolayer.

(2) Celi Size - Cell size distribution stuäics as a function of trcatment will be periormcd using an cular micrometer. Soue idca of cell nuabcrs will be obtained by the use

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of an ocular grid and physical counting. It is póssible that certain narcotics will influence ccll size and cell numbers without causing a shift in the overall growth rate.

(3) . Cytology - Careful attention will be paid to changes in cellular morphology as a result of treatnent. Cytoplasmic vacuolization and the presence of irregular shapes have previously been noted as a result of exposure to narcotics (3,4). Any changes in cell morphology will be carefully documented. and photomisrographs obtained as a permanent record.

(4) Karyotype - Although the literature on the mechanism of action of narcotics indicates that the effect is mediated by metabolic shifts rather chan genetic injury, there is always a possibility that narcotics have some sort of a.direct effect upon chromosomal structures. Chromosonal analysis, both as to nuaber and morphology, will be mace by the colchicine tranping method of woorhead, et al (9). The results will be expressed as bar graphs of the percentage distribution of cells in the population as a function of chromosome nunber as well as photographs of the grouped chromosones.

(5) Synthesis of Macromolecules - Narcotics are known to affect the synthesis of protein and RNA in the brain (l,i0,ll). If the synthesis of protein is blocked by the addition or actinomycin, puronycin or 8-azaguanine, there is a marked inhibition in the development of tolerance in vivo. Clouet and tein as a mcasure of protein synthesis after exposure to the narcotic.

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The above specižically mentioned enzymes as well as certain other enzymes of general importance to'the cell (e.g. lactic.dehydrogenase,. esterase, acid a.d alkaline phosphatase, etc.) will be assayed in the cell-free supernatants prepared from the various'cell cultures after exposure to the various narcotics.

(7) Changes in Isoenzyme Distribution Pattern - Certain key metabolic reactions are catalyzed by more than one species of protein :clecule. These fanilies of aifferent proteins all catalyzing the sane reaction are called isoenzymes. Whar studied in sufficient detail they each appear to have slight . different physical properties and therefore allow the cell to exercise fine control over its metabolism by stimulating cne pathway and inhibiting another even though both pathways pass through.a comnon intermediate. The literature is full of studies on isoe.zyme forms of lactic, malic, isocitric, and glucose-6-phosphate dehydrogenase (6 and its references). There is some evidence for the presence of isoenzymes of phenylethanolanine-N-methyltransferase (20) and otherN- and Oethyltransferases(19).

Host of the enzymes whose specific activity is determined will be subjected to isocnzyne distribution studies.

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In this way an increase or decrease in specific activity can be attributed to a change either in one or more of its isoenzyme forns or across the boará to all isoenzyme forus. This data will be quite important when it comes to deciding whether the effect of the narcotic was general (e.g., an effect on pzo tein. synthesis) or specific (e.g., an effect cn one particular isoenzyne forn).

The data fron all of these different assays will then be sunmarized by cell line, narcotic used,"and whether the cell was treated with the narcotic or was in the tolerantdepencert state. The simplicity of the tissue culture syste coupled with its reproducibility will allow decisions to be made es to what functions and/or metabolic pathways within the cell are affected following narcotic administration versus development of the tolerant-cepenâent state. Knowing which pathways are affected by acaictive "Iversus nonaddictive will allow definitive test systems to be developed and standardized for testing new conpounds. The mechanisn of action of a narcotic and indeed the nechanism of pain itself may be studied in much greater detail by thesa procedures. The establishnent oi a reliable tissue culture screening technique for the monitoring and assaying of compounds active upon the central nervous systen will greatly facilitate future study. This methodology may then be readily applied to the study of any other phaimacologically active : material.

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t of Nare FuncticnTime Hours Rate Totel

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