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3 ANTICHOLINE=ICS A1?ROP Id, SCOPOL - INE, ~ BLAND T~EUTIC . _ ,. . _ ANTICHOLI~RGIC COMICS . SITES OF ACTION Anticholinergic drugs inhibit the actions of acety~chollne (ACh) on autonomic effecters that are innervated by poatganglio~c cholinergic nerves and on smooth muscles that lack cholinergic innervation; that is, they antagonize the muscarinic actions of ACh. They are therefore know as antimuscarinic agents or muscarinic cholinergic blocking agents. Because the main actions of all members of this class of drugs are qualitatively similar to those of the best-known member, atropine, the terms "atropln~c" and '.atropine-like" are also appropriately used. In general, antimuscarinic agent o have little effect on the actions of ACh at nicotinic receptor sites. Thus, at autonomic ganglia9 where tranamission noneally invot~res an action of ACh on nicotinic receptors, atropine produces partial block only at relatively high doses. At neuromuscular Junctions, where the receptors are nicotinic, extremely high doses of atropine or related truga are required to cause any degree of blockade. However, quaternary ammonium analoguea of atropine and related drugs generally have some degree of nicotinic blocking activity and consequently are more likely to interfere with ganglionic or neuromuscular transmission in doses only slightly greater than those that produce muscarinic block. In the central nervous system (CHSy, cholinergic transmission appears to be predominantly nicotinic in the spinal cord and both muscarinic and nicotinic at subcortical and cortical levels in the brain (23. Accordingly, many or most of the CNS effects of atropine-like drugs at ordinary doses are probably attributable to their central anticholinergic actions. At high or toxic doses, the central effects of atropine and related drugs consist, in general, of stimulation followed by depression; these are probably due to a combination of antimuscarinic and other actions. There is an increased release and turnover of ACh in the CNS associated with the ato~iniatration of antio~uacarinic drugs; this may result in the activation of nicotinlc receptors in the brain and contribute to the central effects of this class of drugs (3~. Because quaternary compounds penetrate the blood-brain barrier only poorly, antimuscarinic drugs of this type show little in the way of central effects. *.Yost of the information in this section is from Chapter 7 of Goodman and Gilman All. 51

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PERIPHERAL EFFECTS Parasympathetic neuroeffector Junctions in different organs are not equally sensitive to antimuscarlaic agents. However, the relative acnaitivity of various parsaympathetically innervated organs to blockade by atroplaic agents varica little among the bugs. Small doses inhibit salivary and bronchial accretion ant sweating. Larger dosea canae the pupils to dilate, inhibit accommodation of the eyes, and block vagal effects on the heart, so that the heart rate is increased. Still larger close a inhibit the parasympathetic control of the urinary bladder and gastrointestinal tract, thus Inhiblting micturition and decresaing intestinal tone and motility. Even larger doses are required to inhibit gastric accretion ant motility. Because only the primary phase of gastric accretion is controlled by the vague, the remaining hormonally controlled accretion is unaffected. Thus, doses of any antimuscarinlc drug that reduce the tone and motility of the stomach ant the duodenum and inhibit gastric accretion also lovariably affect salivary accretion, ocular accommodation, and micturition. The drugs produce reveralbly the functional equivalent of resection or paralysis of poatganglionic cholinergic nerves. The actions and effects of particular antimuscarinic agents usually differ only quantitatively from those of atropine' a belladonna alkaloid, which is considered in detail as the prototype of the group. CENTRAL EFFECTS Atropine stimulates the medulla and higher cerebral centers. In doses uset clinically (0~5-loO me), this effect is usually confined to milt vagal excitation. The rate ant occasionally the depth of breathing are increased, but this effect is probably the result of bronchiolar dilatation and the consequent increase in physiologic teat space.' With toxic toses of atropice, central excitation becomes more prominent, leading to reatleasnesa, irritability, disorientation, hallucinations, or telirlum. With still larger doses, atimulation is followed by depression, coma, ant metullary paralysis. The latter may be primarily responsible for a fatal outcome. Even moderate Loses of atropine may tepreas some central motor mechaniama that control muscle tone and movement. This effect has been used to advantage in the management of the tremor ant rigidity of Parkinaonism. Scopolamine in therapeutic doses normally causes drowsiness, euphoria, amnesia, fatigue, and dreamless Sleep with a reduction in rapit-eye-~ovement (REM) sleep. However, the Same toses of acopolamine occasionally cause excitement, reatlesaness, hallucinations, or delirium, especially in the presence of severe pain. Dose a of atropine required to inhibit peripheral responses to choline as t era or anti cholineat erase ( anti-ChE ) agent a produce almost no tetectable central effects. This may reflect difficulty of penetration of the drug into the CNS. In animals, atropine antagonizes the action of ACh applies locally to the cerebral cortex ant Spinal cord. However, atropine also depresses the effects of noncholinergic stimuli, indicating that the t rug has central actions other than blocking cholinergic synapses. 52

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The rise in body temperature due to the belladonna alkaloids is usually significant only after large doses. Nevertheless, in infants and small children, moderate doses induce ~ atropine fearer." In atropine poisoning in tyrants, the temperature may reach 43C or higher. Suppression of sweating is doubtless an important factor in the production of the fever, especially when the environmental temperature is high, but other mechanisms also may be important when large toses are taken. It has been suggested that atropine may exert a central effect on temperature regulation; however, animals that do not Sweat, such as togs, to not exhibit fever after atropine. POISONING BY BELLADONNA ALKALOIDS - The deliberate or accidental ingestion of bellatonna alkaloida or other drugs with atroplaic properties is a major cause of poisonings. Infants and young children are especially susceptible to the toxic effects of atropinic drug e (4~. Delirium ant toxic psychoses, without undue peripheral manifestations, have been reported in adults after instillation of atropine eyetrope. Fatalities from intoxication with atropine ant scopolamine are rare, but they sometimes occur in children, in whom lO mg or less may be lethal. Idiosyncratic reactions are more colon with scopolamine than with atropine, ant ordinary therapeutic toses sometimes cause alarming effects. Table 1 lists undesirable responses or symptoms of overdose associated with various doses of a tropine. razes Prom lntOxt.~a~^n let -h I__ __ SYMPTOMS AND SIGNS OF AIROP INK POISONING _ Symptoms ant Signs of atropine poisoning develop promptly after ingestion. The mouth becomes try and the person experiences a burning sensation; swallowing ant talking are difficult or impossible; and there is market thirst. The vision is blurred, and photophobia is prominent. The akin is hot, dry, and flushed. A rash may appear (more likely in children), especially over the face, neck, and upper part of the trunk; tesquamation may follow. Body temperature rises, especially in infants. The pulse is weak and very rapid although tachycardia may be less pronounced in infants and old people. Palpitation is prominent, and blood pressure may be increased. Urinary urgency and difficulty in ~icturition are sometimes noted. Abdominal distention may develop, especially in infants. The patient is restless, excited, and confused and exhibits weakness, giddiness, and muscular incoordination. Gait and speech are disturbed. Nausea and vomiting sometimes occur. The behavior and mental signs may suggest an acute organic psychosis. Memory is disturbed, orientation is faulty, halluct~ations (especially visual) are common, the sensorium is clouded, and mania and delirium are not unusual (5~. The diagnosis of an acute schizophrenic episode or alcoholic delirium has been mistakenly made, and some patients have been committed to psychiatric institutions for observation and diagnosis. The syndrome often lasts 48 h or longer and may be punctuated by convulsions. Depression and circulatory collapse occur only in cases of severe intoxication; blood pressure decreases, respiration becomes inadequate, and death due to respiratory failure follows after a period of paralysis and coma (6~. 53

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TREATMENT When intoxicating doses of atropine have been taken orally, gastric ravage and other measures to limit intestinal absorption ahoult be initiated without delay. Physostigmine, long overlooked as a posathle antidote to atropine polaoning, is the rational therapy. For example, the slow intravenous injection of 1~4 fig of physostigmine (005-l.O fig in children) rapidly abollahes the delirium and coma causes by large doses of atropine. Because physostigmine is metabolized rapidly, the patient may again lapse into coma within I-2 h, and repeated doses may be needed (4,5,7~. HECHANISH OF Al ION The mayor action of the antimnacarluic agents is a competitive antagonism of the actions of ACh ant other mnacarinic agonists. The antagonism can be overcome by increasing sufficiently the concentration of ACh at receptor sites of the effector organ. The receptors affected are those of peripheral structures that are either stimulated or inhibited by muscariae ~ that i89 exocriae glands and smooth and cardiac-muscle. Much evidence supports the notion that atropine and related compounds compete with muscarluic agonists for identical binding sites on mnacarinlc receptors (2,8,9). ABSORPTION FA EXCRETION . The belladonna alkaloids are absorbed rapidly from the gastrointestinal tract. They also enter the circulation when applied locally to mucosal aurfacea. Only limited absorption occurs from the intact akin. Atroplne disappears rapidly from the blood and is distributed throughout the entire body. Moat is exere ted in the urine within the first 12 h, in part unchanged. Only about 1Z of an oral dose of scopolamine is eliminated as such in the urines Traces of atropine are found in various aecretiona, lucluting milk. The total absorption of quaternary ammonium derivatives of the alkaloids after an oral dose is only about 10-25 percent (10,11); neverthelesa, some of these compounta can cause mydriasis and cycloplegia if applies to the eye. PHARMACOLOGICAL PROPERTIES OF SYNTHETIC ATROPINE DERIVATIVE' After reports in the late 1950e of the psychotomimetic effects of a number of basic esters of subetitutet glycolic acids related to acopolamine and atropine, aevera1 hundred compounta were synthesizer at Edgewood and by its contractors and by academic and Industrial researchers. These compounds were tatentet to be more active centrally than peripherally. Because quaternary salts of these anticholinergic compounta usually penetrate the CNS only with great difficulty, such derivatives were of little latereat in the testing program. The compounds prepared ant tester in the Etgevood program are listed in Table 2, ant their structures are shown in the master file (Appendix B). The basic atructures of these compounds, which are substituted glycolates, are as follows:

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x o R1 - - C R2 - O - R3 (basic structure of glycolates), where X ~ - H. -CH2OlI, -OCH3, -C1, -Br, -l, ~O2CCH3, R1 ant R2 ~ hydrogcs, phenyl, subetituted phenyl, alkyl, cycloallyl, e thylenic, olef inlc ~ thienyl, ant R3 - acyclic aminoallyl, cyclic aminoall~l, bicyclic amino aligl (e.g., tropine or scopine for atropine ant scopolamine, respectively) . Table 3 listo the relative potencica (comparing central vith peripheral effects) of - ny of these terivatives. Note that BZ and EA 3443 e~ibit extensive ceneral effecto for a long periot; the studies at Etges~oot emphasizet these chemicale, as toes this report. In a comparison of a aeriea of structurally related anticholinergic compounde, there was Ie88 tiscrepancy 1~n potency with respect to peripheral than central actio. Because tata on their effecto on man are limitet, most of the i~oreation was deri~red from ani~1 beha~rioral stuties, which in general pretict psychoto~metic actions in man. BZ and other quinuclitinyl glycolates containing at least one pheny} ant a cycloall~yl group in the actd motety were the most potent ant the longest-acting both in animale ant in man. The corresponding piperidyl glycolates were one-fifth to one-half as active as the quinuclidiny! esters. Atropine is equipotent (although of shorter duration) to BZ in producing peripheral anticholinergic effects, but its central actions are much less pronounced . ROLE OF DRUG DISPOSITION IW EX~LAINING DIFFERE:NCES . AMONG ANTICHOLINERGIC COMPOU~ AIROPItIE Numerous reports deali~ with the disposition of labeled atropine in antmals and are reviewed by Wille tn Appendix I. Investigatore have been o~y partially successful in their effort o to compare the phaneacologic actions and relative potencies of the ~rarious anticholinergic coapounde in the CNS with their distribution in brain or their binding to brain components. For Instance, BZ has been reported to adeorb to isolated brain mitochondria about 3 times as avidly as atropine (12~. The affinity of BZ for peripheral muscarinic receptors was also greater than thee of atropine (~), and atropine competed with BZ for specific binding sites ir~ the brain. Because BZ is known to be more potent than atropine and its duration of action ia appreciably loDger (13), theae trug effects court be due to differences ln ehe affinities of the two drugs for specific receptor 8ite8. BZ Snyder's group (14,15) used autoradiography to deter~ine precisely ehe location of binding of [3H]BZ in rat brain. In the cerebral cortex, the greatest binding of the benzilate was in ehe 55

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occipital cortex and the cortex of the cingulate gyrua, with decreasing binding in the cortex in the piriform area and the frontal pole. In the hippocampua, three strata of the archipallium were found to have approximately equal abilities to bind BE, whereas the white matter of the alvaua bound less. The high binding of BZ to the striate body was particularly striking in autoratiographs because of the comparatively low binding activity of the adjoining globes pallituaO Thalamic and hypothalamic nuclei exhibited less bleeding of BZ9 as dlt the inferior ant superior colllculi. Still lower in the binding of this ligand were cerebellar cortex, medulla oblongata-pona, and the cerebral pedunclea. Nerve tracts Berg., the optic chlasma, the cervical apical cord, and the corona radiata--exhibitet the lowest degree of binding. The areas of the brain that retained the greatest concentrations of the label after intravenous injection of [3~1BZ into eats (16) were motor cortex, sensory cortex, caudate nucleus, lateral geniculate, and medial geniculate. Smaller concentrations were retained tn thala~us' hippocampus, hypothalamus, medulla oblo~gata, colLiculi, cerebellar cortex, the pyramids of the medulla, cerebral white matters and cerebeilar white matters Both cholinomimetic and anticholinergic compounds reduced the retention of the label after 13~IBZ treatment in come areas of the brain. The cholinomimetic compounds used were 1,2,3 9 4-tetrahydro-5-aminoacridine (~HA) and S-diethylaminoethyI- diethylphosphorothionate, both inhibitors of cholinesterases. For the anticholinergic compounda, displacement of the label probably was related to the substitution of one drug by another. Z~irblis and Kondritzer (12) found that a mitochondrial fraction prepared from rat brain adsorbed 3-4 timea as much BZ as a eropine in the physiologic range of pa. The optimal pH f or adsorption of BZ was about 8~0, whereas that for atropine was about 9.7; however, at their own optimal pH values, the adsorptions of the two anticholinergic compounds were about the settee THA, Ca2+ ant poll- decreased the adsorption of the benzilate by mitochondrta. Yamamura et al. (17,, Studied the adsorption of [3H]BZ on the particulate fraction of homogenates of various regions of the brain of monkey after incubation with [3H]BZ in phosphate buffer at a pH of 7.4. The regions that were the most avid adoorbers of the benzilate, in order of decreasing uptake, were putamen, caudate nucleus9 occipital cortex, circulate gyros, postcentral gyrus, pyrifonm cortex, frontal cortex, superior colliculi, thalamus, and inferior colliculi. This distribution of adsorptive sites among various parts of the monkey brain locubatet in vitro tiffera from that for intravenously inJec ted [38JB2 in the cat (16~. The two studies do agree that the caudate nucleus is one of the highest areas of localization of [3H]BZ. Yaesmura et al. (17) found that uptake of the label by various areas of brain was correlated with choline uptake and with choline acety~transferase activity. Interruption of the septal hippocampal tract and elimination thereby of cholinergic afferents to the hippocampua reduced by about 70% the activity of choline acetyltransferase in homogenates of the hippocampua, but did not alter the bindlug of [3H]BZ by particles of such homogenates (8). The iDVe8tigaeot8 proposed that - presynaptic muscarinic sites that are innervated by the septal hippocampal nerve fibers do not bind the labeled benzllate, whereas postaynaptic muscarinic sites do bind it. 56

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Ket chum (18) provided information that auggeata that the brain is the only mouse organ that contains 3E 48 h after intraperitoneal injection of [38]BZ. Perfusion of [3H]BZ through rat liver hat indicated that this organ destroyed the compound quickly; 90Z of the compound preaene originally in the perfusing fluid disappeared from the perfusate within 30 min. and less than 0.2X of the unaltered compound wee found in the bile. The identity of the products wee not determined. The effects of BZ and Ditran, compared with atropine and scopolamine, have been examined to elucidate the ~echaniama by which these compounds produce their effects. All these compounds have an extremely high affinity for muacarloic receptor aites, so the higher potency and longer duration of action of BZ Gust be related to other properties. The long-lasting effect a (up to 7 d) of a single injection of BZ in cats (19) raiasd the issue of possible irreveralble interaction of BZ with brain sites. With another anticholinergic drug, [3H]pyrrolidylmethylphenylcyclopentylglycolate (20), a small amount of radioactivity wee still measurable 3 d after a alogle injection. Considering the high affinity of 8Z for mnacarinic receptors (Kt - 10-1l M), it would not be surprising to find 8Z still attached to brain receptors 5-7 d later, especially because pharmacologic effects of BZ are evident in man after 7-8 d. The prolonged action of BZ, compared with its somewhat shorter turatlon of binding to CNS structures, is not yet readily explainable. ANIMAL TOXICOLOGY OF ~ICHOLINERGIC =~U=S . IMMEDIA S BZ . BZ has been chosen as the paradigm of the various anticholinergic teat compounds used at Edgewood. It wee uset in a large number of volunteers and is probably one of-the best documented. Because it is also one of the longeat-actlug of all the drugs, it is also more likely to cause persistent adverse effects. It wee one of the first glycolatea used in relatively high close a to man, so more years have elapsed in which long-range consequences might have occurred. The pharmacology and toxicology of BZ have been studied extensively in animals. Mach of the information vea derived through a contract with Hacleton Laboratories, Falls Church. A summary of the preclinical pharmacology and toxicology in rata, dogs and monkeys is presented here; a fiat of other toxicity studies is given in Appendix F. Additional background information is contained in Appendixes G. H. I. J. K and L. Rats. Repeated intravenous administration of BZ to male albino rata was carried out for 20 daily injections during a 4-wk period. The doses included 0.01, 0.1, and 1.0 ag/kg. The moat significant pharmacologic sign noted wee mydricais. The daily pattern of mydriasia noted after each injection suggested metabolic inactivation of 8Z by rata within 24 h. No tolerance to the mydriatic response was, however, apparent during the course of the study. At the high dose there appeared to be a progressive 57

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diminution In the intensity of pharmacotoxic signs (other than mydrlasia); that suggested development of tolerance or decreased reaponaivenesa to LIZ. Other measured indicators of pharmacologic effect included body weight, food consumption, hematology, "rosa ant microscopic pathologic aigns ant organ weights. No significant effect attributable to the repeated admintatratlon of BZ was found. There were no indicationa of liver disease or malfuncelon directly attributable to BZ in chronic-toxicity studies. In some rats, there were a few small foci of periportal lymphocytlc infiltration; but these occurred as often in aaline-treated animal" Deaf. Repeated lutravenoua administration of BZ to male and female mongrel toga wee carried out for 20 d over a 4-wk period. The dosages were 0.01, 0.1, ant 1.0 mg/kg-d. The indicatora of effect were "rosa phanmacotoxic sigma, tally body weight, hematology, biochemistry, urinalysis, organ weights, organ-to-body weight ratios, ant "rosa and microscopic pathology. A minimal number of effects was produced by low dosages between the eleventh ant thirteenth injection says. Both the intermediate and high dosages produced mydrisaia, ptosia, decreased activity' ataxia. and weakness of limbs. Other signs were leas frequently observed. AD Increase in acdimentation rate occurred at 4 ok in one low- and one high-dosage animal. Liver weight was high in one male from each test group. Testicular atrophy was observed in one male each from the intensediate~ and high-dosage groups. A dose of 10 mg/kg induces bradycardia in all of five dogs ant resulted in death due to cardiac arrest in two of the animals (21). Dogs given daily intravenous doses of 100,ug/kg for 14 consecutive days had the same LD50 as togs that had not been so exposed, but the interval between injection of the tally dose and the appearance of ataxia increased from 4 min to 14 sin during the period of pretreatment. In dogs trained in a conditioned-escape routine and then given graded intravenous doses of BZ, doses up to and including 12.5 ug~kg had no effect on performance. However, a dose of 25 ~g/kg resulted in failure to escape by four of four dogs tested (22). None of the experimenta summarized in this report demonatrated any persistent effect of exposure to BZ in surviving togs. Monkeys. Repeated intravenous atminiatration of BZ at 0.01 0.1, and 1.0 mg/kg-d to male and female monkeys was carried out _ 20 d over a 4-wk period. The indicators of effect were gross pharmacotoxic signs, daily body weight, hematology, biochemistry, organ weights, organ-to-body weight ratios, and gross and micro acopic pathology. The moat frequently observer signs were mydriasis at all dosages and decreased activity and ataxia at the two highest tosages. Blood auger of one monkey given an Lntermedlate dosage and serum transaminase of one monkey given a high dosage were increased at 4 ant 2 wk. respectively. The testicular weight and organ-to-boty weight ratio were high for one intermediate-dosage animal. All other findings were within control limits. Cycloplegia persisted in monkeys for more than 7 t after exposure to BZ, whereas most other effects hat disappeared after 2 d (18). S8

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EA 3443 l Ha zleton Laboratoriea conducted preclinical toxicity studies with EA 3580, EA 3392, and EA 3443. The follo~ri~ infomation was abstracted for EA 3443. A total of 20 daily intravenoua inJectiona of EA 3443 in male and female monkeys at O.O1, 0.1, and 1.0 eg/kg produced no hematologic or biochemical abnormalities. Organs examined microscopically preacnted no significant alterations (23). A total of 20 daily intravenous injections of EA 3443 in mongrel dogs (two dogs per dose) at O.O1, O.l, and I.O ~g/kg produced no hematologic, urinary, or biochemical changes. Histopathologic findings were essentially normal (24~. EA 3834 Prompted by a report of hematuria in a subject receiving EA 3834, research was begun on the effects of these drugs on ureteral and bladder motility (25). Microacopic hematuria wee shown to occur within several minutes after intravenous administration of EA 3634 and usually cleared within 1 h. The dogs had ureteral or bladder catheters tn place and often had a few red blood cells in the urine before administration of the agent. However, the red cells became much more numerous after adminletration.. No additional information is available. ~ The hematuria was most likely the result of a reduction in renal blood flow. It is not clear that the hematuria was produced by EA 3834. LONG-TERM Am DELAYED EFFECTS ~ . . ~ Atropine Atropine sulfate was administered intramuscularly to rabbits daily for 100 d. At a dose estimated to be 5: of the Logo, sigma of toxicity were amen. Pathologic signs included weight loss, edema of most organs, hepatitis, pulmonary thrombosis, inhibition of spenmatogenes1s, Ohmic atrophy, and toxic changes in the gall bladder, spleen, and pancreas. The rabbits' survival of daily administration of atropine beyond the LD50 was related to their ability to maintain food intake (26~. Clidinium ~ Quart zany Clidinium was administered ineragastrically to dogs at 1,5 ant 25 mg/kg, 5 d/wk for 52 wk (27~. The animals were observed for aurvlval, behavior, and general physical condition. Blood, liver and kidney function were evaluated. Organs were examined ~leroscoplcally at the conclusion of tests. Toxic signs of antlcholinergic exposure were not seen. He~atologlc characteristica were not significantly altered by these prolonged exposures. Clinical-chemistry measures and results of "rosa and eicroacopic evaluations of internal organs were within nomal ranges. Administration of Clidinium for ~ yr at 5.0, 25, and 50 mg/kg in the diet of rats did not result in drug-related toxicity. Blood counts, clinlcal-chemlstry measures, and results of gross and microscopic studies remained within normal limits (28~. 59

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Other Data No study of the long-tee administration of BZ to animals has been uncovered. The only long-term toxicity study with an eater of benzylic acid was with the eater with diethylamlooethanol; no significant pathemsa were found in rats during this lifetime feeding study, but the lifetimes of the rata fed the benzilate may have been shortened somewhat. Feeding of the qu ternized ester of 3-quinuclidinol to rata for a year (27) and garaging of dogs with this compound 5 d/wk for a year (29) produced no significant pathemea. With respect to genotoxicity, BZ has bees found to have weak mutagenic activity in yeast cells In culture (30) and to produce gape and breaks in chromosomes of bone marrow cells; it has not been proved to produce a heritable change in mammalian species. Diethylaminoethylbenzilace was more toxic cytogenetically than BZ, respite its lower anticholine Tic activity. Unfortunately, there is too little information on these compounds to assume or exclude genotoxic effects. Although verioua teat ayatema have been used (polot mutations, chromocomal observations, and dominant-lethal effects), moat observed effects probably resulted from general drug toxicity, rather than genotoxlcity. The drugs have been tested at only a few concentrations, and lnterpretatlon of existing data toes not permit estimation of the genotoxlcity of BZ or even of atropine or scopolamine. HUlAN TOXICOLOGY OF "TICHOLI~GIC COPOUTS . . .. DO SING CONS MENTIONS Atropine Textbooks generally suggest that death from atropine poisoning may be expected after lugeation of doses above 100 ma. However, a review of the literature for the last 100 yr yielded a probit satiate of about 450 mg/peraon, based on several hundred cases of accidental poisoning, including some 40 fatalities. Becanae the oral route is somewhat lesa effective than the intravenous or intramuscular (and probably the inhalation) route, a conservative figure of 75 mg/peraon (approximately 900,ug/kg) is a working estimate for the human LDso. Other Antlchollnergic Compounds 1 Acute toxicity data on the 21 glycolates studied in man in a cool climate ahow a fairly consistent ratio between the lethal dose in animals and the heart-rate-increasing dose in man. Extrapolation of the animal data therefore allows an estimate of the risk associated with human exposure. Deaths have occurred in seemingly healthy people when atropine has been given at doses only 50 times greater than the heart-rate-incresaing dose. Thus, the estimated LD,o in man for BZ is about 200 ug/kg, wheresa that for EA 3834 is about 60 ~g/kg. This repreacats safety margins of about 35 for BZ ant 100 for EA 3834, where safety margin is the ratio between LDso and rDso. 60

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In a wam climate, these numbers need correction, because the heat i088 from sweating is diminished. It is speculated that fatal hyperthermia or heatstroke could occur at 408e8 very Ci086 to the IDso for BZ and not more than 2 or 3 times the IDso for EA 3834. In the Edgewood tests, no volunteer received BZ at more than 10: of the Logo. Probably no more than 30 or 40 subjects received more than 3Z of the I.D,o. Few subjects received more than one dose of a glycolate. In the case of BZ, seven subjects were given the ID50 on two OCC88~0DS, 2-3 wk apart. Wo subjects received 8.0 fig/" twice each, in a crossover double-blind study of the efficacy of physostigmlne as an antidote. Eight subjects were given ~ ~g/`g daily for 7 ~ (with no appreciable effecte)' and four received 2 g/kg on three consecutive days (filth cumulative effects approaching incapacitation on the third day.) Thus, fewer than 10% of all subjects received more than a single dose. The figures for ocher glycolates are lower. The total exposure of any person to glycolate never exceeded 3 times the LD50 dose in the entire series of tests. USE OF ANTIl~O`rES Anticholinergic agents are typically used as treatment for anticholinesterase poisoning and rice versa. PhY808ti8~1De, tetrahydroaminoacridine, and other cholinesterase inhibitors were used successfully as antidotes in several dozen subjects. The demonstration of physostigmine's effectiveness led to the first controlled study of its ability to reverse delirium due to scopolamine (31~. Physostig~ine has been used to overcome anti cholinergic t oxic ity . LKlME DIATE EFFECTS Acute clinical findings were recorded by involved physicians and nurses when Edgewood volunteers underwent tests with 21 anticholinergic chemicals shown in Table 2-2. About 1,750 subjects were tested with agents in this pharmacologic class, 102 of which were summarized for review. Selection for inclusion in this summery was based on high dose, high frequency of administration, or additional physiologic 8tre88; it was the intent to focus on the extreme cases. The summaries identify the compound administered, tose, route of exposure, and significant acute reactions or clloical findings. Peripheral and Behavioral Effects In the brain, the arousal system that maintains alertness during waking hours is depressed by the action of BZ; this leade to drowaines., which may progress to stupor or even coma at higher doses. Simultaneously, the coord1natiD-g motor systems of the cerebellum seem to be affected, and that causes an unsteady Bait and pronounced clumsiness. There is a subjective sensation of dizziness which may arise in part from disturbances of the vestibules systems in the brainstem. line modulation of muscle tone is affected, possibly owing to an action on the Renshaw interneurons in the spinal cord, and the results are an abnormal increase in tendon reflexes and a tendency toward stiffness and Jerkiness in muscle 61

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Overall Evaluation of Immediate Adverse Effects In general, in all the teatlog programs there were few violent reaponaea or lnjuriea, largely because of surveillance ant safety precautions taken at Etgewoot. The only other laboratory abnor~lltles encountered in the Etgewoot records involved an locrease in liver enzyme values of two volunteers within about 2 wk of exposure to CAR 302,368. These abnormalities refereed after a few weeka, ant it wee unclear whether the changes resulted frog' the drug at~lalatratlon. Several cases of pyuria after exposure to EA 3580, or related drugs, and runnlog of an obstacle course, were reported, but the drug etiology is unclear. LONGING A~ DEMUD EFFECTS . Delayed effects of the acute chollnerglc drugs were extremely rare. One person reported by Clapper et al. (32) experlencet "flashbacks" while still at Edgewood Arsenal. Edgewood records showed that he received EA 3834 latravenoualy. Medical recorta on this Subject obtained from the VA were volumluous9 but dlt not disclose any significant flr~tlngs. Six-month follows atudiea performed by Hart and Balter (33) on related glycolatea fallen to te~onatrate Significant cognitive changesO Volunteers in a prospective psychometric study who had receiver EA 3167 Showed no evidence of residual drug effects. Two nonvolunteers--one military (a chemist) and one civilian pharmacologlst)--were accidentally exposes to unknown doses of EA 3167, the cost peralstent of the glycolates studied In each case, sub Active ant objective observations of perfoneance over a period of 6-12 ma inculcated that mlld-, but nontrivial, lolpair~ent of cognitive function could be tlacerned for approximately 6 Do, after which seemingly full recovery enamel. These patients were of Superior intellect ant had occupations that required optimal cognitive function for them to be successful. It la possible that in lesa demanding aasignments they might not have been aware of residual deficits. Slmllar persistent decrements were not reported by BZ volunteers. An attltlonal anecdotal report (L. G. Aboot, personal coomunicatlon, 1982) concerns an evaluation of the paychologic state ant well-being of three aublecta who had been exposed to BZ during the precasting 10 ye. Subject A hat an oral dose of approximately 5 mg turlug the summer of 1969; Subject B. an oral dose of approximately 10 mg in June 1972; ant Subject C, an oral dose of 5-10 fig in October 1976. All three were graduate atutents who hat aurreptltlously undertaken aelf-experlmentatlon ant who hat been engages in laboratory experiments with the drug. All experlencet hallucluationa, delirium, confualon, amnesia, and the full spectrum of paychologlc, neurologlc, add autonomic reaponaes assoclatet with the bug. Within 2-4 h after lngeatlon, all three were hoapltalized In the psychiatric ward at the University of Rochester, Strong Memorial Hospital and retained for various periota (3-6 A). Regarding the possible long-range effects of the drug experience, all three Subjects were having tlfflcultlea both adjusting to graduate education and in their personal lives immediately before taking BZ. Subjects A ant C hat entertained the possibility of either troppln8 out or transferring to another (a

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university, largely becanae of inadequate academic performance. Within a few months after the drug experience, all three Remonstrated definite improvement in both academic performance and paychologic well-being. They all appeared to be more out-golog, coamunlcative, and enthusiastic about graduate school. This assesamene was shares by a number of faculty members and fellow students. They all eventually received Ph.D. degrees with distinction. They are now pursuing an active, productive research and postgraduate education. They all appear well-adjuated and well-motivated and seem to be leading productive, aelf-fulfilling careers. None of the subjects has experienced any adverse sequclae that might be attributable to the drug experience. In the absence of additional reports of long-range consequences in subjects who had received the antlcholinergic teat compounds, data have been sought on delayed adverse reactions to other anticholinergic drugs used therapeutically, such as Ditran and atropine. since the introduction in 1958 of Dltran for the treatment of depressed patients (34), the number of persons receiving the drug for Repression and other psychiatric dlaortera has exceeded 2,000 (34-37). During 1957-1963, Ditran wee uset to at lesat 200 people, many of whom were private patients. The treatment lavolvet the intramuscular atmlaiatratlon of 10-30 mg of Ditran to a deprecaed patient--a dose that generally produces the full spectrum of behavioral and necrologic effects attributable to the centrally active anticholinergics. The patient 'e psychiatric condition wea- closely observed and evaluated for the next few months. The progress of some of the private patients was followed for years after treatment, in some cases for 5 yr or more. In no instance wee there any complaint of aequelae or delayed psychopathologic symptoms. The drug ameliorated depression for a period of a few months in a number of patients. Hundreds of patients have received huge doses of atropine and scopolamine (up to 250 me), sometimes given three times a week for up to 4 no, and this form of therapy continues in Eastern Europe today. A chronic behavioral syndrome of toxicity appears unlikely and single or even multiple exposures to the anticholinergic drugs used in the volunteers, frequently at low doses, are deemed losufficient to stimulate a persistent toxic syndrome. Of course, individual susceptibility to acute effects, which may trigger a long-term effect, cannot be excluded. The question of occasional long-lasting or late-appearing residual effects is not easily anaweret. There is very little evidence that these might occur in man, in that so many persons have been exposed to these agents. The new case of glomerulitia several years after exposure in all likelihood wee coincidental. Animal studies have been equivocal, but they have suggested the possibility of acute effects on the kidney ant liver in some inatancea. "Flashbacks" are difficult to explain, but they say result from a heightened sensory awareness after exposure to a drug. They have been of little clinical consequence, even though reported to be associated with a number of mint-altering drugs, including marijuana. It should be emphasized that careful screening of volunteers for testing with the anticholinergic agents wee probably essential, to avoid psychiatric complications after the hallucinatory episode. 67

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If a subject has latent paychopachology, the drug experience may precipitate the latent tendencies and create problems later. In one such instance, a subject receiving Ditran at a university medical school required psychotherapy for 2 mo after the drug episode. Psychiatric interviews and psychologic tests before the drug section indicated that this subject had borderline paranoid and homosexual tendencies; otherwlae, he appeared normal. The subject was amnesic during the manifestation of these symptoms; but the complications enaued after other tivinlty tutenta in 0a dormitory reminded him of his aberrant behavior under the influence of Ditran. The severe embarrassment resulting from this confrontation may have - precipitated the anxiety and lose of self-confidence that required psychotherapy. In summary, one cannot be certain that lo~g-tenm adverse effects never follow exposure to anticholinergic agents, but it seems most unlikely. MORTALITY The teethe of 222 among some 6~620 soldiers have been analyzed. Table 2-4 shows the standardized mortality ratios (SMRa) comparing the observer numbers of teethe and the numbers expected for the U.S. population categorized by age and calendar year. Moat of the SMUT for the various test groups are somewhat leca than unity, although many of the groups are relatively appall. The values for BE, scopolamine, atropine, and "antlcholinergic only are 0.87, 2.50, 1.76, ant 1.06, respectively. It is interesting that atropine ant scopolamine, truga with a long history of therapeutic use, had SMRa exceeding unity, whereas the four candidate chemical warfare agents (BZ, EA 3834, EA 3443, and EA 3580) had SMRs less than unity. This suggests that exposure to the test anticholinerglc chemicals okay not be related to a aubaequent increase in mortality. For anticholinergic with or without other drugs, an SHR of 0.73 is statistically aignificant a" probably represents a selection artifact, inasmuch as volunteers for these studies were especially acre ened for good health and thus would be expected to have lower than average mortality. The mortality data do not reveal a cause for concern that significant numbers of the men exposed to the anticholinergics suffered an untimely death. Because the participants were all especially selected for these atudica on the bsaia of their health records, there were relatively few destine in all groups. It is imposalble to determine how ouch leca than 1.0 the She values should have bees. Comparison of the values of the various groups would not be expected to reveal treatment effect a unleca these were sizable, which was not the case. However, a small drug effect might be meaked by this type of data analysis. For the aame resaona, further subdivision of the deaths by cause toes not provide useful data. Mortality is discussed in greater detail in Chapter 4. SUGARY The Panel has examined the assembled data on the possibility of lon8-term adverse effects aasoclatet with the testing of various anticholinergic compounds in human volunteers at Edgewood Arterial.

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Although there are extensive deacrlptions of the acute CNS effects of the compounds, there is a paucity of information on delayed adverse effects. Animal experiments have not demonstrated significant long-term effects; and chronic administration of other anticholinergic drugs, such as atropine and scopolamine, which have been used therapeutically in man in high doses for years, has not been associated with chronic adverse effects. Indeed, there are anecdotal reports of improvement in behavioral patterns after such treatment. Because the CNS effects of the anticholinergic test compounds appear to be qualitively similar to those of atroplne or scopolamine (although they may differ quantitatively in potency and duration of CNS effects), no additional long-ten toxicity is anticipated. The apparent lack of an effect on mortality of the teat aubJecta also suggests that such an effect is unlikely. Morbidity data are required to determine whether the subjects have had more health problems than those anticipated in a corresponding control group. This information is not yet available. In a few persons, some relatively short-term adverse effects were reported after conclusion of the chemical testing (hematuria, hepatitis, and hallucinogenic flashbacks). It is unclear whether these effects resulted from the drug adminiatration or occurred randomly and coincidentally. The extremely low incidence of such reports makes it doubtful that the anticholinergic test substances played a role in inducing these effects. However, the data are not sufficient to answer this question with certainty. CONCLUSIONS No firm evidence has been seen that any of the anticholinerglc test compounds surveyed produced long-range adverse human health effects in the doses used at Edgewood Arsenal. More intensive study is required to confirm this conclusion. The high frequency of uncontrolled variables makes evaluation of behavioral effects difficult. On the basis of available data, in the Judgment of the panel, it is unlikely that a~iniatration of these anticholinergic compounds will have long-ter~ toxicity effects or delayed sequellae. An ongoing morbidity study should provide more definitive information once it is completed. 69

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Tams 1 BISECTS 0, A=OPtNE IN EtELA~ION TO DOSE Dose ~ a;g Effects 0.5 Slight cardiac aloulag; cone dryness of couth; tshibltion of s - sting 1.0 Deflcite dryDese of mouth; thirst; lacrease In heart rates couetimes preceded by alowlog; mild dilatation of pupil 2~0 Rapit heart rate; palpitation; marlteddr~rness of - couth; dilated pupils; awe blurring of near vision 5.0 All the absve sy~pt~ Caulked; speech tlaturbet; difficulty in swallowing,, restIeseness and fatigue; headache; 4~9 ho: And, dlfflc~ty in ~icturitioD; reduced intestl - ] peristalsis 10.0 Above ysIptoma more earlier; rapid and weals pulse; iris practically obliterated; vision very blurred; skin flushed, hots 6~. an6 scarlet; ataxia, restlesenese, and excite~elat; ~alinclDati0~8 and delirium; come 70

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T-able 2 Summary of Exposures to knticholinergice and Records Selected for Review of Acute Effects of Anticholinergic Chemicale. ~x No. Agent code No. volunteere Total expocuresb No. records selected B1 BZ (EA 2277)C 292 358 36 B2 EA 3443C - 101 101 2 5 B3 EA 358QC 130 133 27 B4 Scopolamine 534 636 42 B5 Atropine 444 738 34 B6 EA 3167C 2 4 4 B7 Ditran 9 13 12 B8 EA 4929 (benzetimide) 18 18 10 B9 27349 50 50 10 B10 226,086 21 21 8 B11 302,196 52 56 15 B12 301,060 29 - 29 9 B13 302,282 8 8 8 B14 302,368 5 5 5 B15 302,537 18 18 8 B16 302,668 39 39 11 B18 Benactizine 16 26 9 B21 ~Scopolamine 72 114 21 B22 M~Atro (atropine methyl nitrate) 18 - 50 10 B23 EA 3834 144 173 33 B25 TAB, BAT 24 24 6 1 anticholinergics 1,7526 2,614 343 ~emicale are identified in the master file (24pcndix B). ~me volunteers recei~red aore than one ch~icel. 3167, long~lasting; EA 3580, potent; EA 3443, skin penetrant; 8Z, chemical-agent candidate . 71

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Table 3 Human Pharmacologic Characteristics of 14 Glycolic Acid Esters (Based on Edgewood Arsenal Studies, 196001971) Sourcesa No. Compound of information subjects _ MEDb ID,oC Duration,t Relative Potenc (mg/kg ~ h eras era ~ ~ . . _ ~ 226~086 EA report 21 (~5) (2rO) (18-36) (2-4) (lOoQ) EA 3443 EATR 4066 101 1~2 364 48-72 203 602 EA 3580 :!31, 265, 269 136 104 3~9 16-24 206 6GG EA 3167 248 24 2~8 401 (96-240) (2-4) (5~6) BZ 228, App K 354 203 502 48-72 5~4 4~0 EA 3834 25, 254' 255 171 200 5~7 10-15 100 307 27349 EATM 114-2 50 3~0 6~0 10015 (2~0) 304 301 ~060 250 29 300 (6~0) (12 ~20) (4~0) (3c4) 302~668 259 39 (400) 8~9 10-15 0~9 2~4 302~282 273 20 4~4 12~5 6~10 (2~0) 1~] SCOp 166 63 7 9 ~4 2 0 e 2 5- 10 0 .8 1 0 302~196 261 56 (12~0) 28~9 4-6 0~7 0 7 Ditran 166 22 5400 100~0 4-8 0~2 0~2 MeScopolam. 166 66 500 (400) 5-10 3~6 005 Atropine 166 602 63~6 152~4 8-12 1~0 0~1 aRefers to reference numbers in Appendix I except EA numbers. bDose required to produce 25X decrement in Number Facility (NF) in 50X of volunteer population (when administered intravenously or intramuscularly). CDose required to produce 90X decrement in NF scores in 50: of volunteers. dAverage time in hours from administration intravenous or intramuscular to near recovery (consistently above 75X of baseline on NF) in subjects receiving Il),o. Peripheral potency is in comparison to atropine (based on EDso to produce maximal heart rate of at lesat 100 beats/mint). Central potency is in comparison with scopolamine (based on ID,o). Numbers in parentheses are approximations based on limited information. 72

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o o . - o ~ - ~ 1 1 o ~ c ~ ol To `. en 73 1 1 1 1 1 o ~ o o 1 1

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REFERENCES 1. Goodman, L. and Gilman, A. 1980: Pharmacologic Basis of Therapeutics, editors of Sixth Edition; Macmillan Pub. Co. U.S .A. 2. Brimblecombe, R O W. 19 74: Drug Ac tions on Cholinerg to Systems ~ University Park Press, Baltimore O Weiner, N. Neurotransmitter systems in the central nervous system. In Drugs and the Developing Brain. (Vernadakis, A., and Weiner, N., eds.) Plenum Press, New York, 1974, pp. 105-131. 4. Rumack, B. H. 1973: Anticholinergic poisoning: treatment with physos~igmine. Pediatrics, 52, 449-451. 5. Ketchum, J. S.; Sitell, F. R.; Crowell, E. G., Jr.; Agh~ania'I9 G. K.; and Hayes, A. H., Jr. 1973: Atropine, Scopolamine, and Ditrart: Comparative pharmacology and antagonists in mane Psychophamacologla ~ 2 8, 121-145 ~ 6. Shader, R. I., ant Greenblatt, 1). J. 19720 Belladonna alkaloids and synthetic anti cholinergics . Uses and toxicity . In, Psychiatric Complications of Medical Drugs. (Shader, R. I., ea.) Raven Prese, New York, 103-1470 Forrer, G. R. ant Miller, J. J. 1958: Atropine coma: a somatic therapy in psychic t ry, Am. J. Psychia try, 1IS, 45 5-458 0 8. Yamamura, H. I. and Snyder, SO H. 1974: Muscarinic Cholinergic Receptor Binding in the Longitudinal Muscle of the Guinea Pig Ilium with [3H] Quinuclidinyl Benzilate. Hol. Pharmacol. 10: 861~670 Hulme , E . C .: Bird sail, N. J. HI. ; Burg en, A. S . V.; and Meht a, P. 1978: The binding of antagonists to brain muscarinic receptors. Hol. Pharmacol., 14, 737-750. 10. Le~rine, R. Il. 1959: The intestinal absorption of the quaternary derivatives of atropine and scopolamine. Archo Int. Phar~acodyn. Ther., 121, 146-169. Il. Jonicman, J. El. G.; Van Bork, L. E.; Wi~sbeek, J.; De Zeeuw, R. A.; and Orie, N. G. M. 1977: Variations in the bioavailability of thiazinamium methyIoulfate. Clin. Pha~acol. Ther., 2l, 457-463. Z~irblis, P. and Kondritzer, A. 1966: Adsorption of H3BZ and Ci4-atropine to the mitochondrial fraction of the rat brat n. Edgewood Arsenal Te ch . Rept . 4042 . 13 . Sidell, F. R. undated: ~ sugary of the investigations in man with LIZ conducted by the U.S. Army, 1960~l969. CSL 000~137. ~ /

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14. Snyder, S. H., Chang, K. J., Bihar, M. J., Aura, 11. I. 1975: Biochemical Identification of Muralist Muscarlaic Cholincrgics Recep~cor. Fed. Proc. 34 :193~5~1921. 15. YamP-ura, H. I., Chang ~ K. J., Kuhar, M. J., S~yd-er, S. tl. 1975: Cholinergic ^acarinia Receptor: Biochemical and I"ight Autoradiographlc Localization in the Brain. Croat. Chea. Acta. 47 :475~486. 16. Becker, M. J. 1964: Biochemical Studies of BZ and EA 3443. Final Report, Contract No. DA-~-104-C~6631. 17 . Ya~ura , I}. ~ ., Kuhar. ~ . J., Greenberg, D ., Snyder , S . H. 1974: ~scarinic Cholinergic Receptor Binding: Regio"1 Distribution in Hockey Brain. Brain Res. 66:541-546. . IS. Ketchup, J. 1963: TIae human aseeseeent of BZ. Chemical Research and Development Laboratories Tech. Hemo 20~29. 19. Lowy, K., Abood, L. G. and Raincs, H.: 19?6. Behavioral effects and binding affinities of two seereoisometric paychoto ieetic glycolate ~ . J. Ikurosci, Res. 2, 157-165 . 20. Abood, L.G. and RinaldI, F.: 1959. Studies with a trivium labeled psychotomimetic agent . Paychophaneacol . ~ :~19~123. 21. Shallek, W. and Smith, T. H. F. 1952: ElectroeDcephalographic analysis of side effects of spa~olyelc drugs. J. Phan~col. Exp. therap. 104: 291-298. 22. McNa~ra, B. P. 1960: Research in Toxicology Division on Effects of CS4030 in animals. Chemical Warfare Laboratories Special Pub. 2-28. 23. Mennear, J., Samuel, G., Rehinge, H. McCarthy D., OlI, 3., Marshall, E. and a-` th, B. Preclinical Pharmacology Study - Monkeys (EA3443~. Contract DA I8-10~8-"C-78~] (C73, Index 105). 24. Mennear, J., Jennings, tl., Kultell, K., Hall, D., Poland, R. and Lamb, R. Preclinical Pharmacology Study - Dogs. Contract DA-~-108-405-W~-826. 25. Shiner, P. T., taJ., M. C., Agent EA3834-renal evaluation as of 3~. 1970, Clinic investigation Bra such, Clinical Medical Sciences Dept., Hed. Res. Label 26. Boyd, C. E. and lloyd, E. M. 1962. The chronic toxicity of atropine administered intramuscularly to rabbits. Tox. Appl. Pharaoh. 4:457~467. 27. Bagdon, R.E., 1965: One year chronic toxicity studies of quartsan tr1 dogs, Dec. 13, 1965. Boffmann-LaRoche, Inc. (Prepared by Food and Drug Res. Ida be. Inc. and submitted to Hoffmann LaRoche Dec. 8, 1965 for use in connection with new drug application to FDA for human use). 75

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28. Bagdon, R.E. and Garner, Z., 1966: One freer chronic toxicity studies of clidiniue bromide in rate. April 20. Boff~-n~LaRochc, Inc. 29. Carson, S. 1965: Chronic (local) ore1 dosage studies with Quartzen in dogs. Report from Food and Drug Research Lobe. Inc. to BoffmanaI - oche, Inc. 30. Sram , R. J., tic erova , M. and Berdote , Z . 1975 : Mutagenic ac tlVi ty of 3=hInuclityl - =ilete. 31. Crowell, B. B., Jr., and Ketchup J. S. 1967: The treatment of scopolao~ine-induced delirium with physosti~ir~e. Etgevood Arsenal Itch. Rept. 4115. 32. Clapper, A. J., McCarroll, J. B. and McColloch, M. 1972: Long-tere' follownp of aedice1 volucteera. Edgewood AreeDe1 Tech. Bept 0 461 I. 33. Hart, Jut. ant Balter, L.: 1966. ~ study of possible resit~1 EFFE CTS of EA 3443 and EA 3S80 on coo NitIve &bllley tuj. Aberd een Proving Ground ~ Hary lent . 34. Aboot, L.G. and Meduna, L.J. 1958. Some effects of a new psychotogen in depressive states. J. Ikrv. [tent. Dis 127, 546-554 ~ Meduna, I. J. and Abood, L. G. J. Neuropsych. I:20~22, 1959. Studies of a New Drug (DItran) in Depressive States.' 36. Finkelatein, B. A.: 1961. Dieren, a psychotherapeutic advance ~ review of 103 cases. J. Ncuropsychiat, 2, 144-148 3 7. English, D. C.: 1962. Reintegration of affect and psychic emergence with Ditrsn. J. ~uropaychiat0 3, 304-310. 76