Pharmacology of Benzodiazepines: Laboratory and Clinical Correlations

Pharmacology of Benzodiazepines: Laboratory and Clinical Correlations GERHARD ZBINDEN AND LOWELL0. RANDALL Research Division. Hoffmann-La Roche Inc., Nutley. N e w Jersey

I . Introduction . . . . . . . . . . . . . . . . . . . . I1. Pharmacologic Profile of the Benaodiazepines A . General Remarks . . . . . . . . . . . . . B. Neuropharmacologic and Psychopharmacologic Effects . . . . C . Pharmacologic Comparison between Bensodiaaepines and Other Psychotropic Drugs . . . . . . . . . . . . I11. Clinical Profile of the Bensodiascpincs . . . . . . . . A . General Remarks . . . . . . . . . . . . . B . Effects in Normal Subjects . . . . . . . . . . C . Clinical Uses . . . . . . . . . . . . . . IV . Relationship Between Clinical Observations and Animal Pharmacology . A . General Remarks . . . . . . . . . . . . . B. Chemical Structures and Pharmacologic Properties of Clinically Tested Bensodiazepines . . . . . . . . . . . . . C . Psychosedative Effects . . . . . . . . . . . D . Sleep-Inducing Effects . . . . . . . . . . . E . Stimulation . . . . . . . . . . . . . . F. Muscle-Relaxant Effects . . . . . . . . . . . G . Anticonvulsant Effects . . . . . . . . . . . H . Autonomic Effects . . . . . . . . . . . . I . Endocrine Effects . . . . . . . . . . . . . J . Drug Dcpendence . . . . . . . . . . . . . K . Drug Interactions . . . . . . . . . . . . . V . Metabolism . . . . . . . . . . . . . . . VI . Toxicology . . . . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

213 215 215 216 227 247 247 247 248 250 250 251 251 261 265 266 267 270 271 273 276 279 281 283 284

.

I Introduction

The rapid development of the newer psychotropic agents represents one of the dramatic chapters of modern medicine . Many pharmacologists who originally studied these chemicals with the classical methods were surprised by the fast and widespread acceptance of these drugs by psychiatrists. a section of the medical profession not known to rely heavily on drug therapy . The pharmacologists’ skepticism essentially vanished. however. when the clinicians described the use of psychotropic drugs strictly as a symptomatic therapy of mental diseases and thereby made a clear distinction against the exaggerated claims of enthusiasts 213

214

GERHARD ZBINDEN AND LOWELL 0. RANDALL

who were expecting a cure for schizophrenia, mental depression, and psychoneuroses. Even more convincing were the statistics soon released by large mental institutions which demonstrated impressively that symptomatic treatment of mental diseases with psychotropic drugs can be of significant benefit for a substantial number of patients (Brill and Patton, 1962; Gerz, 1965). There is no doubt that the essential early discoveries of the usefulness of modern psychotropic drugs were made by clinicians who recognized the “tranquilizing” properties of the hypotensive drug reserpine, the “psychoenergizing” effect of iproniazid, an antitubercular drug, the “antipsychotic” effect of chlorpromazine, an antihistamine, and the “antidepressant” properties of a weak chlorpromazine analog, imipramine. As the first clinical observations were confirmed, pharmacologists took another look a t these chemicals and in every case were able to demonstrate many pharmacologic properties different from those of other known central nervous system (CNS) -active drugs, such as barbiturates, amphetamines, and narcotics. As a matter of fact, the intensive work proved to be so fruitful that the experimental psychologists and neuropharmacologists can now describe the action of psychotropic drugs in a much more distinctive fashion than the clinicians. Under clinical conditions, a puzzling overlapping of therapeutic actions, even with compounds with distinctly different pharmacologic properties, is not uncommon. For example, mental depression may be alleviated not only by stimulant agents such as monoamine oxidase inhibitors and antidepressants of the imipramine type, but also by psychosedatives or even neuroleptic agents. Despite the undeniable fact that the action of psychotropic drugs can now be describcd in more detail by pharmacologic tests than by clinical qualities, it should be remembered that ‘(tranquilizer,” “psychic energizer,” “antianxiety drug,” ‘(antidepressant,” “neuroleptic,” and “antihallucinatory agent” arc essentially clinical terms coined to describe the symptomatic actions of these agents in patients with emotional disorders and mental diseases. It is thus important to restrict these terms to such compounds which have a proven therapeutic effcct, excluding those substances which, in one animal test or another, imitate the actions of a true psychotropic drug. Since psychotropic drugs act primarily on the CNS, it is understandable that their pharmacologic analysis reveals a broad spectrum of action affecting almost every function of the body. These effects are sometimes very specific and may be directly related to a drug’s action in man, e.g., the hypnotic effect of the barbiturates or the anticonvulsant properties of diphenylhydantoin. More frequently, howcvcr, the relationship between a pharmacologic observation and a clinical activity of a psycho-

PHARMACOLOGY OF BENZODIAZEPINES

215

tropic drug can only be suspected. This is particularly true for observations obtained with operant conditioning techniques, analysis of the electroencephalogram (EEG) using implanted electrodes, and drug interaction experiments, as well as with enzyme inhibition and alterations of brain amines. The discovery of the benzodiazepines, a new chemical class of psychotropic agents with an interesting pharmacologic spectrum (Sternbach and Reeder, 1961 ; Randall et al., 1960, 1961), provided good opportunity for comparison of pharmacologic observations and therapeutic effects in man. It occurred a t a time when the major tranquilizers of the phenothiazine and reserpine type and the psychosedatives and muscle relaxants of the meprobamate type were already extensively studied. Thus, much information was available and could be used as a basis for a comparative evaluation. In this review an attempt will be made to describe the principal pharmacologic and therapeutic effects of the benzodiazepines and to correlate experi,mental findings in animals with clinical observations in normal human subjects and patients with a variety of psychoneurotic, mental, and neurological disorders. Based on experience with chlordiazepoxide and diazepam, the first benzodiazepines introduced in clinical practice, and the still limited observations with a number of newer analogs (for structural formulas see Section IV,B), i t will be shown that therapeutic efficacy and side effect liability can, in part, be directly related to the pharmacologic effects in normal animals or especially prepared animal models. For other clinical observations, however, the pharmacologic basis remains speculative. The comparison with other psychotropic agents will provide further data which may shed some light on the significance of animal testing for the predication of the therapeutic value of psychotropic drugs. II. Pharmacologic Profile of the Benzodiazepines

A. GENERALREMARKS The pharmacologic subtleties of psychotropic drugs are often overshadowed by their gross effects on animal behavior. Thus, sedation and hypnosis appear to be common effects of the so-called CNS depressants. Superficial evaluation of these findings would indicate a rather limited degree of specificity of action. This may explain why there are still doubts in the minds of pharmacologists and clinicians whether the newer tranquilizers and antianxiety agents have indeed distinctive features which would justify their preferential use over older soporifics and the barbiturates in particular (Sharpless, 1965). I n order to obtain a more detailed evaluation, the effects of benzo-

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GERHARD ZBINDEN AND LOWELL 0. RANDALL

diazepines on the CNS will be ranked and presented according to the absolute dose in milligrams pcr kilogram of body weight required t o induce a significant change of behavior or of nervous functions. It is assumed hereby that those drug actions that are dernonstrablc a t the lowest dose are likely to represent characteristic fcatures of a compound and that the effects occurring only after multiplying the mini,mum effcctive dose are of lesser importance or to be considered as side effects due to overdosage. The clinical usefulness of a psychotropic drug is dependent on the ratio between the dose which has a beneficial, calming, relaxing, or antipsychotic effect and the dose inducing oversedation, drowsiness, and slccp. Thus, any behavioral or neurologic action discovered in animal experiments must be related to the dose a t which neurotoxic effects or hypnosis occur. Psychopharmacologists also consider i t essential that a psychopharmacologic effect can be demonstrated over a t least two, better three or more, doublings of thc dose. This climinates insignificant observations of drug cffects which may be obtained by a fortuitous selection of the test dose but cannot be demonstrated any more when this dose is either decreased or increased. Species differences also have to be watched for since CNS-active drugs sometimcs produce paradoxical effects in one particular species. The marked excitation of cats by morphine is a wellknown example. I n the benzodiazepine series, species differences are not prominent although the cat has provcd to be morc sensitive to the muscle relaxant properties than any other species. Some differences also have been observed with respect to the excitatory effects in micc, rats, and monkeys, but thcse seem to be characteristic only for ccrtain benzodiazepine derivatives and not typical for the class (see Section I1,C).

B. NEUROPHARMACOLOGIC A N D PSYCHOPHARMACOLOGIC EFFECTS A summary of the actions of chlordiazcpoxide and diazepam on the CNS and selected functions regulatcd by the highcr nervous centers is presented in Tables I, 11, 111, and IV. It is limited to chlordiazcpoxide and diazepam bccause they are the most widely studied representatives and show all characteristic properties of thc benzodiazepine series. 1. Mice

The most characteristic neuropharmacologic cffects in mice (Table I) are found a t the lower and upper end of the dosage scale. Both drugs are highly active in preventing pcntylenetetrazole-inducedseizures, an effect which is easily de,monstrated at levels considerably bclow the sedative and muscle-relaxant dose. The high activity against chemically induced convulsions is characteristic for benzodiazepine derivatives, which are more potent in this test than any other anticonvulsant agent (Sternbach

217

PHARMACOLOGY OF BENZODIAZEPINES

TABLE I PHARMACOLOGIC PROFILE OF CHLORDIAZEPOXIDE AND DIAZEPAM IN MICE E D d per os, mg/kg Description of test Inhibition of pentylenetetrazole convulsions Ataxia (rotabar device) Ataxia (rotating wire mesh cylinder) Antagonism of morphine-induced stimulation Inhibition of maximal electroshock Inhibition of fighting behavior due to electrical stimulation Analgesia test (phenyl-quinone writhing) Inhibition of strychnine convulsions Inhibition of minimal electroshock Muscle relaxation (inclined screen test) Analgesia test (hot plate) Loss of righting reflex for 3 minutes or more “Hypnotic effect” Acute toxicity (LDso)

Chlordiazepoxide Diazepam

References

9.3 12.5

1.9 3.9

Ranziger (1965), Gluckman (1965) Gluckman (1965) Klupp and Kahling (1965)

13.5

4.1

Gluckman (1965)

3.7-8.0

0.4-1.37

40

10

Banziger (1965), Gluckman (1965) Randall et al. (1961, 1965a)

41

12.5

Sternbach et al. (1964)

87

16

Randall et al. (1965a)

91.7

64

Banziger (1965)

100

25

Randall et al. (1961, 1965a)

100

100 225

17 .O-29.9 3 . 4 - 6 . 4

370 620 720 k 51

Sternbach et al. (1964) Baneiger (1966)

Sternbach et al. (1964) 530 620 +_ 30 Banziger (1965)

Effective dose 50%.

et al., 1964; Swinyard and Castellion, 1966). This was also demonstrated

in rats, rabbits (Banziger, 1965), cats (Roldan and Escobar, 1961), monkeys (Chusid and Kopeloff, 1961, 1962), and man (Kaim and Roscnstein, 1960). Other forms of chemically and electrically induced seizures are also inhibited, but somewhat higher doses are needed. The outstanding observation a t the upper end of the dosage scale is the high dosage required to induce sleep. Only a t doses close to the LD,, are the animals paralyzed; but they still respond to painful stimuli and react with muscle twitching and squeaking if surgical procedures are attempted. Intermediate doses cause muscle incoordination and relaxation which probably also account for the weak effects observed in the analgesia tests. The characteristic fighting of pairs of mice receiving a n electric current through the grid to the feet (Tedeschi et al., 1959) is abolished by benzo-

218

GERHARD ZBINDEN AND LOWELL 0. RANDALL

diazepines. The dosage required is close to that found to block electroconvulsive seizures, thus considerably above the minimum effective dose (MED) in the most sensitive test. It is unlikely therefore that this experiment measures “taming” which is one of the most characteristic properties of the beneodiaeepines observed at low doses in many higher animal species (Heuschele, 1961; Flyger, 1961; Hubbell, 1965).

2. Rats Treatment of rats with benzodiazepines causes sedation and muscle relaxation without hypnosis over a broad dose range. This observation is illustrated in Fig. 1. It depicts the behavior of a rat in a continuous

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FIG.1. Cumulative records of the effects of increasing doses of diazepam on continuous avoidance of a representative rat. Method of recording: Each time the rat pressed the avoidance lever, the cumulative pen moved upward a short, fixed distance, and every 15 minutes the pen was reset downward automatically. Consequently, the response rate during each 15-minute interval is directly proportional to the slope of the pen tracing. Offsets of the cumulative pen mark the occurrence of a foot shock. Each offset of the horizontal line below the cumulative records indicates that the rat failed to turn off a shock (escape failure), Shock rate is significantly increased a t 8 mg/kg. Escape failure occurs for 1% hours a t 64 mg/kg. (Courtesy of C. L. Scheckel.)

219

PHARMACOLOGY OF BENZODIAZEPINES

avoidance situation which demands regular level pressing to postpone a foot shock. After treatment with diazepam, the animal shows signs of tranquilization as demonstrated by a small but significant increase in the number of shocks taken. At the same ti,me, the animal retains its ability to turn off the shock over two to three doublings of the dose. At higher doses the response rate decreases due to the drug's muscle-relaxant and sedative effects. This effect is in marked contrast to the effects of pentobarbital (Fig. 2) and ethyl alcohol for which the incapacitating doses are PENTOBARBITAL W c

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FIG.2. Effects of pentobarbital on continuous avoidance behavior in a rat. Shock rate increase occurs at 20 mg/kg S.C. At 40 mg/kg complete inability to respond for a period of over 2 hours. Method of recording same as Fig. 1. (Courtesy of C. L. Scheckel.) very close to the tranquilizing dose. This observation indicates that the effect of chlordiazepoxide and diazepam observed in various operant conditioning procedures (Table 11) may be to a large extent an expression of the general "slowdown" of the animals which have to react physically to external stimuli (Heise and McConnell, 1961) or are trained to press a lever continuously without a warning stimulus (Heise and Boff, 1962). For the understanding of the drug's action, these tests are therefore of lesser importance than those which demonstrate significant efficacy a t levels considerably below the sedative dose. The outstanding example is the attenuation of the marked stimulation which occurs if animals pre-

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220

GERHARD ZBINDEN AND LOWELL 0. RANDALL

PHARMACOLOGIC PROFILE O F

Description of test Attenuation of tetrabenasine stimulation in iproniazid-pretreated rats Attenuation of conflict behavior Continuous avoidance, shock rate increase Inhibition of maximal electroshock Attenuation of irritability in “septa1 rats” Appetite stimulation (acute) Inhibition of pentylenetetrasole convulsions Continuous avoidance, avoidance rate decrease Discrete trial “trace” avoidance, 25y0 noise response failure Continuous avoidance, escape failure Discrete trial “trace” avoidance, 10% response failurc Antagonism of caffeineinduced increased motility Reduction of locomotor activity Inhibition of minimal electroshock Acute toxicity ( *SE)

Definition of dose.

TABLE I1 CHLORUIAZEPOXIDE

AND

DIAZEPAM IN RATS

Activity (mg/kg) and route of administration Chlordiazepoxide 0.45 i.p.

< 2 . 5 i.p. 3 . 7 i.p. 4 . 2 i.p. 5 . 8 p.0. 11.0 i.p. 12.5 p.0.

Diazepam

References

0 . 0 5 i.p. Randall et al. (1965a) < 2 . 5 i.p. 0 . 9 i.p. 10.0 i.p.

Geller (1964); H e i e (1964) Randall et al. (1961, 1965a) 12.0 p.0. Banziger (1965) 1 6 . 0 i.p. b

Randall et ul. (1961) Randall (1960)

13 . O p.o.

12.0 p.0.

Banziger (1965)

13.7 i.p.

13 . O i.p.

1 5 . 0 i.p.

8 . 0 i.p.

Randall el ~ l . (l965a) Heise and McConnell (1961)

1 8 . 0 i.p.

67.0 i.p.

5 0 . 0 i.p.

3 0 . 0 i.p.

-100 p.0.

-12 p.0.

60 p.0.

152 p.0.

>goo p.0.

42 p,o.

1315 f 122 p.0.

Randall et al. (1961, 1965a) Heise and McConnell (1961) Klupp and Kahling (1965) Randall et al. (1961) Banziger (1965)

2425 f 330 Bansiger (1966) p.0.

a hlED = Minimum effective dose; ED, = Effective dose 50%; LDia = Lethal dose 50%,. b Slight and inconsistent effect.

22 i

PHARMACOLOGY OF BENZODIAZEPINES

treated with the monoamine oxidase inhibitor iproniazid are injected with the amine releaser tetrabenaxine (Heise arid Boff, 1962) (Fig. 3 ) . It is probable that this stimulation is due to a central release of norepinephrine, the metabolism of which is delayed by previous administration of a monoamine oxidase inhibitor. This assumption is supported by the fact

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FIG.3. Effects of iproniazid, tetrabenazine, and the combination on rate of lever pressing of rats in the continuous avoidance procedure. Top record: Avoidance response rate during a 5-hour control session. Second record: Iproniazid alone had no significant effect on behavior. Third record : Tetrabenazine alone produced nearly a complete loss of responding. Fourth record: In rats pretreated with iproniazid, tetrabenazine produced marked stimulation, shown by the increased rate of lever pressing. (Courtesy of C. L. Scheckel.)

that no stimulation occurs if the brain norepinephrine has been reduced by administration of a-methylmetatyrosine or if norepinephrine synthesis has been disrupted by either a-methylparatyrosine which prevents conversion of tyrosine to dihydroxyphenylalanine (dopa), or disulfiram, which inhibits the dopamine-norepinephrine conversion (Scheckel and Boff, 1966). Figure 4 shows that a very small amount of chlordiazepoxide,

222

GERHARD ZBINDEN AND LOWELL 0. RANDALL

injected 2 hours before tetrabenazine in a rat pretreated with a monoamine oxidase inhibitor, completely blocks the excitation and increased response rate induced by tetrabenazine. The same effect is observed with 0.05 mg/kg of diazepam i.p., a dose 200-fold smaller than the MED for shock rate increase in the continuous avoidance procedure. Other benzodiazepine tranquilizers are also highly active in this test (Randall e t al., 1965s). Continuous avoidonce (rats)

20 0

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FIG.4 . Effect of chlordiazepoxidc on the stimulation induced by iproniazid and tctrabenazinc,. Top rccord: Control behavior. Second record: A small dose of chlordiazepoxide had no effect on normal avoidance behavior. Third record: It was neccwary t o incrcasc the dose of chlordiazepoxide to 60 mg/kg to markedly suppress normal avoidance behavior. Fourth rccord : Stimulation produced by iproniazid and tetrsbcnazinc (cf. Fig. 3). Bottom record: Administration of a small dose of chlordiazepoxide 2 hours prior to tetrabenaeine, in rats pretreated with iproniazid, conipletrly blockcd the stimulation. (Courtesy of C. 1,. Scheckel.)

Attenuation of vicious and aggressive behavior in rats with electrolytic lesions in the septum is demonstrated a t rather low dose levels (Schallek e t al., 1962). Similar doses also inhibit pentylenctctrazoleinduced seizures and maximal clectroshock. It takes ahout five to ten times larger doses to have a noticeable effect on locomotor activity (Randall e t al., 1961). McDonald e t al. (1963) and Jacobsen (1964) also observcd that large doses of chlordiazepoxide were necessary to decrease normal motor activity i n rats, t o lower open field activity, and to reduce the response to activity wheel running. If doses of 50-150 mg/kg S.C.were

PHARMACOLOGY OF BENZODIAZEPINES

223

given repeatedly, the effect on motor activity disappeared within a few days, so that there was no loss of motor response to bell stimulation or changes in activity wheel running (McDonald e t al., 1963). A rather unique effect is the appetite stimulation first observed with chlordiazepoxide in rats and dogs. If the rats are starved, 12.5 mg/kg p.0. of chlordiazepoxide will increase food intake over a 4-hour period by 50%. I n starved dogs, a single dose of only 1 mg/kg has the same effect (Randall, 1960).

3. Cats The most prominent effect of the benzodiazepines in cats is muscle relaxation (Table 111). It occurs at very low doses and can best be observed in animals suspended by the scruff of the neck. The cats are relaxed and limp but this muscle-relaxant effect does not interfere with normal motions and playfulness. At higher doses marked ataxia occurs. There is no relaxation of the nictitating 'membrane. Relaxant effects may be caused by depression of either the large-fibred alpha motor system or the small-fibred gamma motor system. Granit e t al. (1955) found that these systems could be separated by two types of decerebration: ( 1 ) Following anemic decerebration cats become rigid because of hyperactivity of the alpha system. (2) Following intercollicular decerebration rigidity occurs because of hyperactivity in the gamma system. Many benzodiazepines have a pronounced effect on both types of rigidity (Fig. 5 ) but, as shown in Table 111, diazepam depresses rigidity due to intercollicular decerebration a t a dose below that depressing rigidity following anemic decerebration. The same is true for several other benzodiazepines tested in a similar manner (Schallek, 1966). This indicates that the principal relaxant effect is on the gamma rather than the alpha motor system. This conclusion is supported by Tardieu e t al. (1964) who de,monstrated that diazepam selectively blocked the tonic stretch reflex in cats decerebrated by the intercollicular technique and suggested that depression of gamma tone was involved in this action. .Jimenez-Pabon and Nelson (1965) also observed reduction of muscle tone by diazepam in cats decerebrated by intercollicular section. Ghelarducci et al. (1965) observed that nitrazepam, another benzodiazepine analog, depressed gamma rigidity at a dose which did not affect the alpha motor system. Additional aspects of the relaxant effects of diazepam and other drugs were studied in the cat by Ngai et al. (1966). Comparison of drug effects on the knee jerk and on the crossed extensor reflex indicated whether the principal action was in monosynaptic or in polysynaptic pathways, while comparison of drug effects on spinal and on decerebrated preparations

224

GERHARD ZBINDEN AND LOWELL 0. RANDALL

I’HARMACOLOGIC PROFILE OF

TABLE I11 CHLORDIAZEPOXIDE

AND

DIAZEPAM I N CATS

Act,ivity (mg/kg) and route of administration Description of test Muscle-relaxant effect Block of spinal reHex

Definition of dosea MED

MED

Chlordiazepoxide

Diazepam

2 . 0 p.0. 2.0-3.0 i.v.

0 . 2 p.0. 0 4 i.v.

Inhibition of E : D B 0 f SE Not tested 0.85 f 1 04 i.v. decercbrate rigidity (intercollicular preparation) EDso f SE Inhibition of 8.0 f 3.7 i.v. 0.37 i 0.7 i.v. pressor rcsponse to hypothalamic st imiilation Sedation and b 10.0 p.0. 5 . 0 p.0. ataxia Inhibition of EI):,o f SE >20.0i.v. 0.55 k 5 . 0 i . v . intcstinal response to hypothalamic stimnlation Inhibition of EDw rf- SE 26.0 f 10.0 i.v. 2 . 5 f 3 . 3 i.v. decerebrate I igidity (anemic preparation)

hlEl)

=

References Randall et al. (l965a) Randall (1960), Randall el al. (1961) Schallek (1966)

Schallek el at. (1964)

Schallek et al. (1964) Schalleketal. (1964)

Schallek et al. (1964)

Alinimum effective dose; EL)5o= Effective dose 50%. of the cats.

* Effective dose in two-thirds

indicated whcthcr relaxant effects were exerted on spinal or on supraspinal levels. The investigators found that crossed extensor reflexes were reduced or abolished by the following drug doses in milligrams per kilogram: Ihiy

Uiazepam Chlordiazrpoxide Mephenesin Meprobamate

Decerebrated Cat

Spinal Cat

0.05-0.2 10-30 10-50 2040

3-10 40-120 30-100 80-120

PHARMACOLOGY O F BEN ZODIAZEPINES

225

It was also noted that doses of benzodiazepines which depressed the crossed extensor reflex had no effect on the knee jerk. These data indicate that diazepam and chlordiazepoxide are more active on the crossed extensor (polysynaptic) reflex than on the knee jerk (monosynaptic) reflex. The two benzodiazepines are more active on the decerebrated than

FIG.5. Action of Ro 5-2092 on decerebrate rigidity in cat prepared by anemic technique. Top picture shows maximum rigidity before drug ; bottom picture shows complete relaxation 15 minutes after injection of 20 mg/kg i.v. Cat 40, Jan. 8, 1W1. (Courtesy of W. Schallek.) (Chemical formula of Ro 5-2092 is shown in Table XIS.)

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GERHARD ZBINDEN AND LOWELL 0. RANDALL

on the spinal preparation, indicating that under thc conditions of this experiment the primary action of the drugs is on supraspinal levels, most likely on the brain stem reticular formation. 4. Monkeys

Early experiments in vicious and aggressive monkcys suggested that chlordiazepoxide had a marked taming effect (Randall e t al., 1960). This was found to be characteristic for most benzodiazepine tranquilizers (Sternbach et a!., 1964). A calming effect on aggression and abolishment of fear is generally observed at dose levels considerably below those causing decrease of activity, ataxia, and drowsiness. Based on these findings, benzodiazepines have been widely used in taming aggressive and excited zoo animals (Heuschele, 1961 ; Flyger, 1961 ; Hubbell, 1965). Taming of wild animals has been claimed for most psychosedatives and tranquilizers (Hanson and Stone, 1964). A more detailed discussion of this effect will therefore be presented in Section II,C,3, where the effects of different types of psychotropic drugs are compared. I n the continuous avoidance situations, squirrel monkeys behave very similarly to rats (Table I V ) . Increase of the number of shocks taken occurs at a low dose. It takes much larger amounts to have an effect on avoidance rate or to lead to escape failure. Bringing the animal to an “anxicty situation” permits demonstration of behavioral effects of chlordiazepoxide and diazepam a t very small doses, which suggests again that a more specific drug effect is being unmasked. The experimental procedure is called “delayed matching” and demands that the animal (Rhesus monkey) , which is restrained in a ‘Lmonkeychair,” remember the color of a light and match that color by pressing an appropriate lever in order to obtain a food reward. Every two correct matching responses increase tlic delay between presentation of the correct color and the subsequent opportunity to iiiatch this color and to obtain the reward. Incorrect responses decrease the delay. The delay intervals which can be presented to the monkey extend from 1 to 105 seconds. Under control conditions each animal will adjust to its own limit of delay. With drug therapy the animals will either improve their performance and achieve significant longer delay intervals or their ability to “remember” the correct color will deterioratc. As they make more incorrect responses, the delay interval will shorten (Scheckcl, 1962, 1965). After treatment with chlordiazepoxide the limit of delay is significantly increased a t doses as low as 0.156 mg/kg. In the animal whose behavior is shown in Fig. 6, the avcrage ascending limit of delay for the control period is 45.7 seconds. It incrcases to 67.6 seconds after 0.156 mg/kg of chlordiaxepoxide, to 72 seconds after 0.312 mg/kg, and to 59.7 seconds after 1.25 mg/kg of the drug. A larger dose of 10 mg/kg depresses the monkey’s response indicating sedation. Other

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PHARMACOLOGY OF BENZODIAZEPINES

benzodiazepines have a similar effect. Chlorpromazine, on the other hand, does not improve the delayed matching performance (Scheckel, 1963). Observation of the monkeys which are subjected to the delayed matching procedures leaves little doubt that these animals are under considerable emotional stress, which is reflected in a very tense appearance and seemingly anxious behavior. Improvement of their performance under Delayed matching -

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PO

I

0

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2

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2

3

FIG.6. Effect of small doses of chlordiaeepoxide on a Rhesus monkey in a delayed matching procedure. Each panel shows performance during a 3-hour session. (Each point shows the result of a single trial. When the animal made correct responses on two consecutive trials at one delay, the delay presented on the next trial automatically increased one step. Incorrect responses or the lack of an observing response decreased the delay one step. Horizontal lines in each panel are drawn through the average limit of delay. Note that the ordinate (duration of delay interval) is logarithmically scaled. (Scheckcl, 1963.) (Courtesy of C. L. Scheckel.) the influence of benzodiazepines, therefore, is most probably due to an antianxiety effect rather than an improvement of short-term memory, which would also increase the limits of delay.

C. PHARMACOLOGIC COMPARISON BETWEEN BENZODIAZEPINES AND OTHERPSYCHOTROPIC DRUGS I n order to explore the clinical significance of the pharmacologic observations obtained with a new class of psychotropic agents, it is useful to look for striking similarities and differences in comparison with other drugs whose therapeutic efficacy in man is generally acknowledged. The

228

GERHARD ZBINDEN AND LOWELL 0. RANDALL

TABLE IV PHARMACOLOGIC PROFILE OF CHLORDIAZEPOXIDE A N D DIAZEPAM IN MONKEYS

MED (mg/kg) and route of administration Description of test,

Chlordiazepoxide

Improvement of performance in 0.156 p.0. delayed matching procedure 1. 0 p.0. Inhibition of aggression (cynomolgus monkeys) Continuous avoidance (squirrel 1 . 0 p.0. monkeys) shock rate increase Antironvulsant effect in normal 1.14.4i.v. and epileptic monkeys challenged with pentamethylenetetrazole Stimulation No stimulation 20 p.0. Ataxia Continuous avoidance (squirrel monkeys) avoidance rate decrease Continuous avoidance (squirrel monkeys) escape failuie

Diazepam < 0 . 5 p.0.

References

1.O p.0.

Scheckel (1962, 1965) Rnndalletal. (1965a)

1.O p.0.

RandalletaL (1905a)

-

Chusid and Kopeloff (1961, 1962)

21 p.0.

2.5-5 p.0. Sternbach et al. (1964) 20 p.0. Sternbach et al. (1964) 25 p.0. Randall et aL(1965a)

29 p.0.

33 p.0.

Randall elal. (1965a)

fact that the pharmacologic action which is common to two chemically different compounds results in a similar therapeutic effect would support the assumption that the pharmacologic test employed was indeed representative for the common clinical properties of the two agents. Also, if striking diffcrences in clinical efficacy between two drugs can be duplicated by pharmacologic observations, it appears justified to draw a t least some cautious conclusions about the predictive value of the animal testing proccdures. It is beyond the scope of this revicw to describe and compare the pharmacologic spectrum of all clinically important sedatives and tranquilizing agents. This discussion will be limited therefore t o a comparison of some of those pharmacologic actions which were recognized in the preceding chapter as being most typical for the benzodiazepine class. 1. Anticonvulsant Efiects

Benzodiazepincs are distinguished by an exceptionally high activity against pcntylenetetrazole-induced convulsions. This is demonstrated in

229

PHARMACOLOGY OF BENZODIAZEPINES

TABLE V ANTICONVULSANT EFFECTSOF SELECTED PSYCHOTROPIC DRUGSIN MICEO Musclerelaxant effect Anticonvulsant effects EDmmg/kg p.0. as measured in inclined Pentylene- Maximal Minimal Strychnine screen test EDSO mg/kg tetrazole electro- electroconvulshock shock shock sions p.0. Chlordiazepoxide Diazepam Nitrasepam Diphenylhydantoin

18.0 2.2 0.69 >800.0

92.0 12.0 30.0 15.0

150.0 127.0 357.0 600.0

87.0 16.0 24.0 800.0

Phenobarbital Meprobamate Trime thadione

75.0 150.0 400.0

18.0 200.0 >80.0

90.0 167 . O 800.0

50.0 500.0 >800.0

Chlorpromazine Chlorprothixene

42.0 600.0

150.0 264.0

113.0 200.0

80.0 93.0

100.0 25.0 15.0 inactive at, 400.0 120.0 256.0 inactive a t 500.0 17.0 17.0

Results obtained in the laboratories of R. F. Banziger and L. 0. Randall, in part published by Randall (1960, 1961), Sternbach et al. (1964), and Baneiger (1966).

Table V which shows the oral ED,, in mice of chlordiazepoxide, diazepam, and nitrazepam, a newer benzodiazepine analog which has considerable promise in certain convulsive disorders in man. These compounds are also very active in raising the threshold for pentylenetetrazole-induced seizures in cats (Requin e t al., 1963; Lanoir et al., 1965). Maximal electroshock (Swinyard et al., 1952) and strychnine convulsions are inhibited a t doses close to the levels causing marked muscle relaxation and incoordination as determined in the inclined screen test (Randall et al., 1960). Even higher doses are needed to block minimal electroshock (Swinyard et al., 1952). Of the compounds listed in Table V, trimethadione also exhibits its highest activity against the pentylenetetrazole-induced seizures (Goodman e t al., 1946). Phenobarbital and diphenylhydantoin are particularly effective against maximal electroshock, meprobamate shows weak activity in all but the antistrychnine tests, and chlorpromazine and chlorprothixene are effective only a t dose levels considerably above the muscle-relaxant dose. These data, which agree essentially with those reported by Hanson and Stone (1964) and Swinyard and Castellion (1966) , demonstrate that there are considerable differences with regard to the effects of various psychotropic compounds on different types of experimental seizures. Significant differences between

230

GERHARD ZBINDEN AND LOWELL 0. RANDALL

benzodiazepines and diphenylhydantoin or phenobarbital were also observed by Eidelberg et al. (1965) who reported that diazepam and other benzodiazepines effectively blocked cocaine-induced convulsions in rats a t doses which caused only minimal reduction of spontaneous activity. Diphenylhydantoin and phenobarbital were inactive. It is interesting that in this test chlorpromazine, reserpine, and Dibenamine demonstrated activity similar to that observed with the benzodiazepines. The effects of various psychotropic drugs on the electroencephalographic responses to brain stimulation are discussed in Section II,C,7,a. 2. Conditioned Behavior

Modern operant conditioning techniques permit the observation of animals under controlled conditions for prolonged periods of time with the option to introduce various environmental stresses which make the subjects more susceptible to drug actions. I n the continuous avoidance procedures of Sidman (1953) as modified by Heise and Boff (1962), animals are trained to postpone a n electric foot shock by pressing a lever a t regular rate and, in case of avoidance failure, to terminate a shock by pressing a second lever. In this procedure depressant drugs often lower the rate of lever pressing, increase the number of shocks received, and may cause failure of pressing the lever to turn off shock (escape failure). Compounds may be classified on the basis of the ratio of the minimum dose which causes escape failure to the minimum dose which just increases the number of shocks taken (avoidance failure) (Heise and Boff, 1962). This “dose range ratio’’ reflects the range of doses over which a drug has a measurable depressant effect without causing complete inability to respond. As shown in Table VI, high dose range ratios are obtained with benzodiazepine tranquilizers. Lower dose range ratios due to failure to separate behavioral effect from paralytic or hypnotic effect are shown for barbiturates, meprobamate, and other hypnotics and sedatives (Heise and Boff, 1962). The continuous avoidance procedure satisfactorily distinguishes tranquilizing agents of the benzodiazepine type from sedative-hypnotics. However, it fails to do justice to the obvious differences on animal behavior of benzodiazepines and neuroleptics of the phenothiazine type. Another procedure cklled “discrete trial trace avoidance” (Heise and McConnell, 1961) was therefore developed by which phenothiazines and benzodiazepines can clearly be distinguished. I n this procedure, rats are trained to respond to a 5-second noise stimulus by pressing a lever. Failure to press the lever is followed by a 5-second noise and shock but interspaced between warning stimulus and shock is a 5-second silent

231

PHARMACOLOGY OF BENZODIAZEPINES

TABLE VI MINIMUM EFFECTIVE DOSESA N D DOSE RANGERATIOIN CONTINUOUS AVOIDANCEPROCEDURE^ Rats

Compound

Shock rate Route increase of MED admin. mg/kg

Chlordiazepoxide Diazepam Nitrazepam Phenobarbital Methyprylon Hexobarbital Emylcamate Pentobarbital Chlormezanone Meprobamate Chlorpromazine

p.0. i.p. p.0. i.p. i.p. S.C.

s .c .

S.C.

i.p. S.C.

i.p. i.p. p.0. p.0. S.C.

i.p. Trifluoperazine

S.C.

Escape failure MED mg/kg

THE

Squirrel monkeysb Shock rate Dose Route increase of MED range ratio admin. mg/kg

5.2 >60.0 18.0 3.8 4.2 20.0 >5.2 >120.0 67.0 10.0 5.5 14.0 0.81 19.0 61.0 2.1 30.0 1.8 41.0 25.0 42.0 75.0 1.7 1.2 78.0 56.0 ls.o 1.1 12.0 1.1 70.0 62 . O 105.0 103.0 1 .o 1.2 300.0 250.0 8.2 1.5 5.4 0.62 3.4 0.21 2.1 1.8 1.1 0.05 1.9 0.03

p.0. c

p.0.

p.0. p.0. p.0.

Dose range ratio

1 .o

29.0 29.0

1 .o

3 3 . 0 33.0

-

p.0.

Escape failure MED mg/kg c

c

c

c

c

c

80.0

80.0

C

e

C

c

C

c

c

c

1.2 c

10.0

20.0

c

c

c

>200.0

>200.0

-

E

c

2.0 c

c

c

2.5 c

c

1.5

1.3 c e

c

c

a Data from Heise and Boff (1962) and Scheckel and Boff (unpublished). Median of 3 4 rats per drug. MED = Minimum effective dose. Data from Scheckel and Boff (unpublished). Median of 5-7 monkeys per drug. No data available.

“gap.” In rats trained t o make more than 90% of their responses during the initial noise period when not drugged, the number of responses during the “gap” period is significantly increased by administering chlordiazepoxide (Heise and McConnell, 1961) and other benzodiazepines (Randall e t al., 1961, 1965a). Chlorpromazine has a distinctly different effect. If these animals fail to respond to the noise stimulus, they almost never press the lever during the following silent gap period while still maintaining the ability to terminate the shock when it occurs. It is probable that the high incidence of ((gap” responses after treatment with benzodiazepines is due to a lengthened latency of response to the noise. It is also seen after treatment with meprobamate and phenobarbital, whereas neuroleptics of the chlorpromazine type seem to have a selective blocking effect on the responses t o a discrete warning stimulus.

232

GERHARD ZBINDEN AND LOWELL 0. RANDALL

3. Taming Taming of vicious and aggressive animals is a characteristic property of tranquilizing drugs which is obvious t o the experimenter and the personnel handling the animals, but is most difficult to assess. The effect includes abolishment of active aggression in those animals who attack spontaneously, a noticeable change of response to provocation if animals are attacked, teased, or brought into unfamiliar surroundings, and finally an abolishment of fear in those animals who demonstrate their aversive feelings by avoiding any contact with man. Any assessment of taming effects therefore has to take into account the different ways an experimental animal manifests its aggressive behavior. Most of the experimental work has been conducted on monkeys, but there are marked differences with respect to expression of aggressiveness evcn within this species. Squirrel monkeys, for example, are quite vicious when attacked, but do not show much spontancous aggressiveness. Cynomolgus monkeys, on the other hand, are often unpredictably vicious but aggressiveness varies much with age and adjustment of individual animals to laboratory surroundings. Golden marmosets show extreme fear and attack only when they have no chance to retreat or hide. These differences are highlighted by the findings of Gluckman (1965) who reports only slight effects of various benzodiazepines on aggressiveness in monkeys described as “attackers” whereas more striking taming properties were demonstrated in animals exhibiting mostly a fearful behavior. With these difficulties in mind, Heise and Boff (1961) havc devised a checklist which permits fairly objective ratings of the animals’ aggressiveness as well as their general activity. These studies indicate that both chlordiazepoxide and diazepam as well as most of the other benzodiazepine tranquilizers tested reduce aggressive behavior of monkeys. The animals can be handled without protective gloves, do not bite even when pulled or prodded with a stick and, in case of the golden marmoset, lose their fear and can be caught and petted (Randall e t al., 1960, 1961; Bagdon and de Silva, 1965). Using the checklist of Heise and Boff (1961), it could be established that chlordiazepoxide and diazepam have a safety margin of approximately 20 between the antiagressive dose and the ataxic dose. Chlorpromaaine, beginning at 0.63 mg/kg, reduced both activity and aggression scores to less than 50%. The animals often withdrew from the observer and continued to bite when handled or provoked. Meprobamate had a slight taming effect but only a t doses which caused severe ataxia and sluggishness. Barbiturates had no effect a t lower doses. Severely sedative and ataxic doses did not prevent the

PHARMACOLOGY OF BENZODIAZEPINES

233

monkeys from occasionally attacking the observer (Randall et al., 1960, 1961 ; Scheckel, 1966). I n lower animals, aggressiveness has to be provoked by various experi,mental procedures. I n mice, fighting can be induced by applying electric current to the feet, but for all tranquilizing drugs tested, the dose necessary to block this type of fighting behavior is rather high (Sternbach et al., 1964). Another method uses mice which are isolated for a t least 6 weeks. After this period a small victim mouse is introduced in the home cage of the isolated mouse which usually starts to fight with the intruder. I n this test both chlordiazepoxide and chlorpromazine abolish aggressive behavior but for chlordiazepoxide one-eighth of the minimal dose causing neuromuscular deficit is needed whereas with chlorpromazine, aggressiveness is suppressed a t one-third of the neurotoxic dose. Meprobamate and pentobarbital do not show differential effects between doses which abolish the aggression and doses which produce neuromuscular impairment (Cole and Wolf, 1966). This experiment therefore is in general agreement with the observations in monkeys. 4. Conflict Behavior

An excellent experimental method to induce fear and apprehension is based on the principle of bringing the animals into a situation in which a food reward to a conditioning signal is regularly or occasionally coupled with punishment. I n a procedure developed by Geller and Seifter (1960), hungry rats were trained to press a lever the reward for which was a supply of milk a t approximately 2-minute intervals. At a 15minute interval a 3-minute tone was introduced during which each leverpressing response was rewarded with food but a t the same time also punished with shock. Before drug administration the animals rarely pressed the lever during the tone; after administration of benzodiazepine tranquilizers such as chlordiazepoxide, diazepam and oxazepam, however, the rats continued to work during the tone period and accepted the punishment (Geller et al., 1962; Geller, 1964). No such attenuation of conflict behavior was observed with chlorpromazine and other phenothiazines. On the contrary, if a low shock was administered, chlorpromazine actually reduced the number of shocks accepted during the tone period. I n this test, meprobamate, pentobarbital, phenobarbital, and emylcamate had similar effects as the benzodiazepines (GeHer, 1962, 1964; Geller et al., 1962). Another method which permits a more detailed evaluation of conflict behavior was developed by Scheckel and McConnell (1963). Rats were trained to press a lever for food reward when a high tone was presented

234

GERHARD ZBINDEN AND LOWELL 0. RANDALL

(approach trials) and to press a second lever also for food reward when a low tone was presented, but 10% of the responses to the low tone produced foot shock in addition to the food reward (approach-avoidance trials). The response latency was measured as the time from presentation of the tone until the correct lever was pressed. It was separately computed for low tone and high tone. Under control conditions the response latency to the high tone averaged 1.7 seconds while the response latency to the low, conflict approach tone averaged 6.9 seconds. Figure 7 demonApproachI - avoidance T,l,ll< trials trials -Approach

@-+

V

100

O X - 27

‘II 2

OI

C

\

5

ox- 33

\\\

\

\

J,

8

‘*

.- 60 ?

--

V

0

L

---Q

m 19

6

w

L

-

1

37

75

80

b a

15

Chlordiozepoxide (mg/kg,tp)

30

40

20

C

1,9 37 75 15 Chlordiozepoxide(mg/kg,ip)

30

FIG.7. Effect of chlordiazepoxide on approach and approach-avoidance (conflict) behavior in rats. A t doses of 3.7-15 mg/kg there was a selective reduction in response latency (left graph) to the conflict tone (open symbols), and no change in response latency to the approach tone (solid symbols). Right-hand graph shows similar effects when response failures were measured. (Courtesy of C. L. Scheckel.)

strates that chlordiazepoxide a t 3.7 to 15 mg/kg i.p. ,markedly reduced the latency to the conflict tone without altering the latency of response to the high tone. Simultaneously, the percentage of response failure to the low conflict tone was reduced while the percentage of response failure to the high tone (simple approach) remained low until the dose was raised to 30 mg/kg. This high dose level caused neuromuscular impairment and a high percentage of failure to respond to either tone. Diazepam a t 0.93-15 mg/kg i.p., meprobamate a t 25-100 mg/kg i.p., and pentobarbital a t 10-80 mg/kg i.p. had similar effects. Chlorpromazine, however, given a t 0.25 mg to 2 mg/kg i.p. did not show any attenuation of conflict behavior. As a matter of fact, response latency to the low conflict tone increased somewhat with increasing doses. Failure to respond to the high tone (simple approach) occurred only when the dose was raised to 2 mg/kg (Fig. 8). I n this procedure amphetamine a t 0 . 3 7 5 3 mg/kg had a similar effect as chlorpromazine.

235

PHARMACOLOGY OF BENZODIAZEPINES

From these studies it is concluded that antianxiety drugs of the chlordiazepoxide type reduce “passive avoidance,” i.e., they reduce the tendency of the animal to withhold a response which has a potentially aversive consequence. The phenothiazine-type tranquilizers, on the other hand, are ineffective in reducing passive avoidance but are highly effective in reducing what may be called ‘(active avoidance behavior.” An active avoidance would refer to the situation in which the animal must make a specific response in order to avoid punishment. Analysis of *--aApproach-avoidance trials

w Approach trials Rats -

OX-41 ax-33 OX-40

n

loor 80-

-$ 6 0 5 !a

--

0

0

L

%

40-

0

m

[L 0)

0

20

C

025

05

10

Chlorpromazine (mg/kg,sc )

20

-

0--

C

025

05

10

20

Chlorpromazine (rng/kg,sc)

FIG.8. Effects of chlorpromazine on approach and conflict behavior in rats. This drug did not attenuate conflict, and all behavior was suppressed by the highest dose (2.0 mg/kg). (Courtesy of C. L. Scheckel.)

whether a drug pri,marily affects active or passive avoidance behavior may therefore provide a means to qualitatively separate the “classical” tranquilizers from the antianxiety agents. Various experimental modifications for the study of drugs in animals subjected to conflict situations have also been reported by Liberson et al. (1963) , Lewis and Feldman (1964), Feldman (1964a,b) , and Feldman and Green (1966). Their findings in general confirm the impression that benzodiazepine tranquilizers attenuate fear and apprehension when the animal is confronted with a conflict situation. Further investigation of these methods led to thc development of experimental neurosis as a tool for evaluating psychotropic drugs. Jacobsen (1965) trained cats to perform a feeding cycle of pressing a lever, opening a feed box, and

236

GERHARD ZBINDEN AND LOWELL 0. RANDALL

eating fish cakes. The conflict situation consisted of applying an air-blast instead of the food until the animal refused to perform for food. During the conflict the cats sharpened their claws, licked themselves, rubbed against the wall, rolled on the floor, and crouched in a corner. This conflict-induced behavioral disturbance and the concurrent failure to press the lever were effectively blocked by chlordiazepoxide a t 2.5-10 mg/kg S.C. and the animals responded normally as they did during the preconflict state. 5 . Experimental Anxiety and Stress The external stimuli which cause apprehension and fear in an experimental animal can be increased from mild anxiety induced by operant conditioning procedures and the more intensive anxiety connected with conflict situations (Section II,C,4) or delayed matching situations (Section II,B,4) to an obvious state of severe fear or panic with or without pain or actual tissue injury, Severe external stress may also lead to secondary functional or structural changes such as release of free fatty acids, hypertension, gastric ulcers, or even death. A useful method described by Lang and Gershon (1963) is based on the observation that injection of yohimbine into human subjects causes a state simulating considerable anxiety with tenseness, restlessness, and irritability (Holmberg and Gershon, 1961). Intravenous injection of the alkaloid in conscious dogs is also followed by behavioral changes suggestive of severe anxiety accompanied by rise in arterial blood pressure and heart rate. Chlordiazepoxide, 2 mg/kg, injected intravenously 15 minutes before the administration of 0.5 mg/kg of yohimbine significantly reduced behavioral changes as well as the rise in blood pressure and heart rate. Similar effects were achieved with 0.5 mg/kg of chlorpromazine. Amylobarbitone and meprobamate had no significant attenuating effects whereas emylcamate reduced blood pressure rise but did not significantly alter behavioral changes. Single and repeated dosing with imipramine led to marked potentiation of the blood pressure rise and significant worsening of the yohimbine-induced behavioral changes. This observation was duplicated in imipramine-treated human subjects in whom yohimbine injection can cause a state of acute panic (Holmberg and Gershon, 1961). It is possible therefore that the model anxiety induced by yohimbine in dogs and man could be useful as a tool for assessment of the therapeutic potential of antianxiety agents. The value of even more drastic measures to induce severe anxiety is less obvious, although “antistress” effects can be demonstrated with various sedatives and tranquilizing agents if the experiments are appropriately designed. For example, the incidence of gastric ulcers induced by

PHARMACOLOGY OF BENZODIAZEPINES

237

forced restraint of rats of 5-6 hours’ duration is significantly reduced by 50 >mg/kgof chlordiazepoxide (Haot et al., 1964). If the severity of the stress is increased by restraining the rats for 20 hours in a water bath a t 25’ C, most CNS depressants lose part or all of their activity or have to be given a t high doses to maintain the protective effects (Takagi et al., 1964). Another severe stress which leads to marked increase of plasma-free fatty acid levels is repeated electroshock. If rats are treated with 50-volt electroshocks of 2 seconds duration every minute for a total of 1.5 hours, the plasma-free fatty acid levels increase from an average of 489 & 18 peq/liter to 729 & 28 peq/liter (Khan et al., 1964). In this situation tranquilizing agents of various types, including chlordiazepoxide, benzquinamide, reserpine, chlorpromazine, meprobamate, and hydroxyzine inhibit significantly the stress-induced increase of free fatty acid levels. Pentobarbital and ethanol are partially active (Khan et al., 1964). This experiment again illustrates the “antistress” action of CNS depressants but is not discriminative enough to demonstrate the finer differences in the action of psychotropic drugs. The same is true for another experimental procedure which employs whole body mechanical vibration in mice. If various psychotropic drugs are given prior to the procedure, mortality is reduced with CNS depressants and increased with CNS stimulants. Large doses of CNS depressants are necessary for a protective effect. Although there are some differences in degree of protection between a more active group consisting of chlordiazepoxide, reserpine, pentobarbital, and phenobarbital, and a less active group including hydroxyzine, chlorpromazine, and meprobamate, the experimental procedure does not allow any detailed differentiation in the action of the various classes of depressants (Aston and Roberts, 1965). Tranquilizing agents such as chlordiazepoxide, diazepam, Insidon, and chlorprothixene also decrease the lethal effect of whole body irradiation (Locker and Ellegast, 1964). It is probable that this protection is due to a decrease of total hody metabolism and hypothermia. From these selected examples, it is concluded that inhibition of anxiety reactions and secondary effects of various external injuries by CNS-depressant drugs is more difficult to demonstrate and requires much larger doses as the severity of the stimulus is increased. Simultaneously, much of the specificity of the drug’s effect is lost and secondary effects such as hypothermia and general depression of the whole body metabolism become the determining factors. Thus, severe stresses are generally more efficiently counteracted by neuroleptics of the phenothiazine type which have a pronounced effect on body temperature and intermediary metabolism (Kollias et al., 1962). For the evaluation and understanding

238

GERHARD ZBINDEN AND LOWELL 0. RANDALL

of the psychotropic effects of these agents, such procedures are therefore of limited usefulness. 6. Stimulation During evaluation of a series of benzodiazepines it was observed that one analog, nitrazepam, caused marked hyperactivity in ,mice a t doses below those causing sedation (Sternbach e t al., 1964). It was further noted that this effect could be demonstrated with various benzodiazepine derivatives in the continuous avoidance procedure. After administration of these agents, the animals increased their rate of lever pressing much in the same way as may be seen with amphetamine and methylphenidate. For example, the dose of diazepam which increased the rate of lever pressing varied between 2 and 10 mg/kg in rats and 3 and 15 mg/kg in squirrel monkeys. Higher doses caused sedation as indicated by decreased rate of lever pressing. This stimulant effect was also observed with several other benzodiazepine derivatives, but not with chlordiazcpoxide (Randall et al., 1965a). Other sedative agents such as barbiturates and ,meprobamate had no stimulant effect in the continuous avoidance procedure. The antidepressant imipramine did not produce acceleration of lever pressing, but amitriptyline had a weak stimulant effect a t 8 mg/kg i.p. Some, but not all, animals treated with a low dose of chlorpromazine also showed increased response rate (Scheckel and Boff, 1964). This spontaneous stimulation observed with many benzodiazepines and chlorpromazine in the continuous avoidance procedure must be distinguished from the increase of response rate often present in conditioning procedures involving food reinforcement, where many sedatives, including benzodiazepines, barbiturates, and meprobamate show pronounced “stimulant” activity. In this situation, however, phenothiazines have an opposite effect (Kelleher et al., 1961; Hanson and Stone, 1964). Following the observation of a stimulant effect of certain benzodiazepines, these drugs were evaluated in animal tests designed to measure more specifically “antidepressant” action of the imipramine type. The procedure employed is described by Scheckel and Boff (1964). It uses rats trained to avoid an aversive foot shock in the continuous avoidance situation as modified by Heise and Boff (1962). Imipramine and amitriptyline and their demethylated derivatives induce marked stimulation when combined with a behaviorally inactive dose of tetrabenazine, cocaine, or d-amphetamine. In this test chlordiazepoxide and diazepam did not show any imipramine-like effect. Some of the newer benzodiazepine analogs, however, markedly potentiated cocaine and d-amphetamine and produced stimulation when combined with behaviorally inactive doses of tetrabenazine. Although these compounds have

PHARMACOLOGY O F BENZODIAZEPINES

239

not yet been explored clinically, the experimental findings indicate that many benzodiazepine derivatives have stimulant properties a t doses below those causing sedation. Thus potentiation of centrally induced excitation may occur a t appropriate dose levels. Chlorpromazine and promazine do not show imipramine-like effects in these drug interaction studies. Meprobamate and barbiturates are also inactive. 7. Site of Action

a. General Remarks. Neuropharmacologists try to determine what parts of the CNS are involved in the various effects of centrally active drugs. Their goal is t o establish a distinct pattern of activity for each class of psychotherapeutic agents and to pinpoint the brain centers which are stimulated or inhibited preferentially a t the lowest dose. The most important experimental tool used in these studies is the EEG obtained from implanted electrodes combined with electric or chemical stimulation of selected brain centers. Simultaneous observation of behavioral changes or physiological responses of various peripheral organs provide added information. By recording the EEG in different centers of the brain before and after administration of psychotropic drugs, i t is possible to find differences of a quantitative and qualitative nature which indicate the probable site of action. An alteration of evoked or spontaneous electric events in an area of the brain by a psychotropic drug does not necessarily mean that the compound’s therapeutic action in man is mediated through this particular brain center. However, the electrophysiological findings help to explain certain differences in the therapeutic usefulness and side effect liability between groups of different psychoactive drugs. In the subsequent paragraphs several experimental procedures will be described in which the neuropharmacological properties of selected benzodiazepine tranquilizers are compared with those of severaI other psychotropic drugs. I n reviewing these data, it must be kept in mind that the rcsults of electrophysiological studies of the brain depend to a large extent on technical and methodological details. These are location of the electrodes, type and extent of anesthesia, route of drug administration, animal species and method of stimulation, to name only a few. Thus, differences between findings of various laboratories are inevitable. b. Central Control of Autonomic Functions. The effects of benzodiazepines on autonomic responses to ccntral stimulation were first studied by Carroll et al. (1961). They observed that chlordiazepoxide greatly attenuated pressor responses obtained by stimulation of cortex, amygdala, hypothalamus, or brain stem in the cat. Cardiac arrhythmias following stimulation of cortex and amygdala were completely blocked.

240

GERHARD ZBINDEN AND LOWELL 0. RANDALL

TABLE VII HYPOTHALAMIC STIMULATION IN IMMOBILIZED CATS INHIBITION OF PRESSOR AND INTESTINAL RESPONSES BY CNSDEPRESSANT DRUGS EDGO, mg/kg i.v.0 No. of cats 16 9 9 13 9

7

Drug Chlordiazepoxide Diazepam Nit~raeepam Chlorpromaaine Mebutamate Phenobarbital

Pressor response

Intestinal response

11 .o

>20.0

0.56 0.71 0.95 9.8 16.0

0.64 0.69 >10.0 >20.0 28.0

Calculated by graphing dose-response curves. Data obtained from the laboratory of W. Schallek. Some of these data, calculated by another method, appeared in Schallek et al. (1964).

As shown in Table VII, diazepam and nitrazepam have an even more marked effect on the rise in blood pressure. In addition, they effectively counteract the inhibition of gastrointestional motility following hypothalamic stimulation in the cat (Schallek e t al., 1964). Chlorpromazine also blocks the blood pressure rise but has only a weak effect on the intestinal response to hypothalamic stimulation. Phenobarbital and meprobamate reduce the hypertensive response only a t high doses and have a weak effect on the intestinal response. An important question is whether the effects of the benzodiazepines are exerted centrally or peripherally. Chai and Wang (1965) observed that in anesthetized cats diazepam a t 0.1 mg/kg i.v. reduced pressor responses to hypothalamic and medullary stimulation but did not alter responses to stimulation of the stellate ganglia. Signs of ganglionic block occurred with doses of 1 4 mg/kg. These data suggest that diazepam has a depressant effect on central cardiovascular control mechanisms. I n additional studies Chai and Wang (1966) injected diazepam, 0.01 to 0.02 mg/kg, into the vertebral or carotid artery of the cat. Pressor responses from the hypothalamus were reduced to a greater extent than those from the medulla. This was true even when the drug was administered by the intravertebral route and presumably reached the medulla first and in highest concentration. These findings were confirmed and amplified by Schallek and Zabransky (1966). They demonstrated that while the benzodiaeepines reduced pressor responses to hypothalamic stimulation, they had no effect on pressor responses to medullary stimulation in immobilized cats. I n contrast, chlorpromazine a t 10 mg/kg i.v. significantly reduced the blood pressure rise following stimulation of

PHARMACOLOGY O F BENZODIAZEPINES

241

both the medullary and the hypothalamic pressor centers. These data indicate that the effects of benzodiazepines on cardiovascular responses to central stimulation are primarily exerted through the hypothalamus, while chlorpromazine seems to have also an effect on medullary pressor centers which are not significantly inhibited by the beneodiazepines. Diazepam and nitrazepam and several of the newer derivatives markedly blocked intestinal responses to hypothalamic stimulation, an effect which, surprisingly, was not shown by chlorpromazine, suggesting a more specific effect of the phenothiazines on certain sympathetic responses. c. Psychodepressant Effects. Evidence that psychodepressant effects of benzodiazepines may be correlated to changes in the limbic system was first suggested by the marked calming action in experiments with “septa1 rats” (Section 11,B12). Rats with lesions in the septal area of the brain become extremely vicious. A second lesion in the amygdala restores this behavior to normal. This suggests that in the intact animal the septum dampens hypothalamic activity associated with emotional states while the amygdala facilitates this activity (for refs. see Schallek et al., 1962). It is possible therefore that the benzodiazepines which attenuate the irritability of septal rats a t doses below those affecting control rats act by a depression of the amygdala. Confirming evidence was subsequently accumulated using a variety of experimental approaches. i. Changes in spontaneous electric activity. Experiments performed in unanesthetized cats with implanted electrodes indicate that the earliest changes of the spontaneous EEG following administration of benzodiazepines occur in the limbic system. Chlordiazepoxide, 5 mg/kg i.p., for example, produces a statistically significant slowing in the electric activity of the amygdala, hippocampus, and septum, but not of the cortex which shows slow electric activity only when the chlordiaeepoxide dose is doubled. At 1 mg/kg i.p. EEG changes are limited to the hippocampus and the amygdala (Schallek et al., 1962). With diazepam, the first electroencephalographic evidence of drug action is seen a t 0.5 mg/kg i.p. exclusively in the hippocampus (Randall et al., 1961). Other central nervous system depressants affect spontaneous electric activity of the brain in a distinctly different manner. Phenobarbital acts primarily on the cortical EEG, whereas meprobamate produces prominent slow waves in both hippocampus and cortex (Schallek et al., 1962). Chlorpromazine acts primarily on reticular formation and thala,mus (Killam et al., 1957) and a t higher doses also on the hippocampus. The affinity of chlordiazepoxide and diazepam to the limbic system was also demonstrated on spontaneous electric activity in rabbits (Monnier and Graber, 1962). Immediately after intravenous injection of 30

242

GERHARD ZBINDEN AND LOWELL 0. RANDALL

mg/kg of chlordiazepoxide, slow waves and sleep spindles appeared in all leads. After 1 hour, spiking and spontaneous discharges were observed only in the hippocampal leads. Arrigo et al. (1965) observed similar “monomorphic activity” in the hippocampus of the rabbit following 10 mg/kg of diazepam i.v. ii. Evoked potentials. Morillo et al. (1962), using the cerveau isol.6 preparation of the cat, studied responses evoked in the hippocampus for single shocks delivered every second to the amygdala. These responses were markedly depressed by 5-10 mg/kg of chlordiazepoxide i.v. I n an extension of these experiments, Morillo (1962) observed that diazepam and nitrazepam a t 2 mg/kg i.v. strongly depressed the responses evoked in the ventral hippocampus by stimulation of the ipsilateral amygdala. However, the responses evoked a t the same point by stimulation of the contralateral hippocampus were either unchanged or moderately facilitated. The author concluded that these drugs may simultaneously inhibit the amygdala and facilitate the hippocampus. He suggested that the amygdala normally exerts facilitatory influences on the hypothalamus. Reduction of these influences by the drugs may result in “modulating the emotional output.” iii. Behavioral responses to brain stimulation. The arousal response to hypothalamic stimulation in the unanesthetized rabbit was studied by Monnier and Graber (1962). The threshold was increased by 30 mg/kg of chlordiazepoxide i.v. Arousal responses from both hypothalamus and amygdala of the rabbit were tested by Arrigo et al. (1965). Thresholds in both areas were increased by chlordiazepoxide and by diazepam. The authors concluded that the hypothalamus may play the central role in the mechanism of action of these drugs. Supporting evidence for the role of both amygdala and hypothalamus in the psychodepressant effects of diazepam comes from unpublished experiments of Schallek and Kuehn (Table VIII). The thresholds for arousal responses from reticular formaTABLE VIII EFFECTSOF DIAZEPAM 5 mg/kg p.0. ON AROUSALTHRESHOLDS I N CATS No. of experiments 8 3 4 4

Stimulated area

Change in arousal threshold, volts. Mean SE

Value of ‘p’ drug vs. dextrose

Reticular formationa Pyriform cortex Anterior amygdala Anterior hypothalamus

+ 0 . 2 8 f 0.23 +1.13 f 0.37 0.20 + 2.25 t 3 . 1 5 0.23

< O 05 <0.04

<0.01 <0.01

Data on reticular formation from Schallek and Kuehn (1965). Other data from unpublished experiments by Schallek and Kuehn. (1

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243

tion and cortex were moderately increased by diazepam while larger increases occurred in thresholds from amygdala and hypothalamus. A number of different types of behavioral responses to brain stimulation in the cat were studied by Kid0 and Yamamoto (1965). Chlordiazepoxide, 20 mg/kg P.o., raised the thresholds of the following responses. The areas stimulated are added in parenthesis. Searching Rage Hissing

(anterior hypothalamus) (posterior hypothalamus) (central gray of mesencephalon)

There were no changes in the threshold of two other responses Somatic movement Feeding

(reticular formation) (amygdala or septum)

Rage responses induced in the cat by stimulation of posterior hypothalamus or septum were abolished by nitrazepam (Hernandez-Peon and Rojas-Ramirez, 1966) . The hissing response induced by stimulation of the anterior hypothalamus in the cat was studied by Baxter (1964). There was little change in threshold during the 105-minute test period following the first injection of chlordiazepoxide but the threshold rose dramatically on subsequent injections. This suggests that in addition to an immediate action on the CNS some long-term effects may be involved in the action of these drugs. d. EEG Response to Electric or Chemical Stimulation. The neuropharmacologic approach to anticonvulsant testing is based on electric stimulation of specific brain areas believed to be involved in different forms of epilepsy. Repetitive stimulation of these areas induces afterdischarges in the EEG which resemble the seizure discharges seen in clinical epilepsy. In unanesthetized animals the behavioral effects accompanying these after-discharges may resemble behavior during clinical seizures. Drug effects can be observed by comparing the electroencephalographic responses to stimulation before and after therapy and the preferential site of action may be deduced by stimulating various brain centers. i. Stimulation of Limbic System. Kaada et al. (1953, 1954) observed that stimulation of the amygdala or hippocampus in unanesthetized cats induces staring, dilatation of the pupils, facial twitching, and drooling. This behavior resembles that seen during psychomotor (temporal lobe) seizures in man. Schallek and Kuehn (1960), Morillo et al. (1962), and Schallek et al. (1962, 1964) have used stimulation of these brain areas to study drug effects in freely moving as well as in immobilized cats. Ex-

244

GERHARD ZBINDEN AND LOWELL 0. RANDALL

peri,ments in unanesthetized cats with chronically implanted electrodes permit the study of both behavioral and electrographic effects of brain stimulation (Schallek et al., 1964). I n order to test different compounds on comparable levels, all drugs wcre tested a t the minimum dose producing ataxia in a t least two of three cats. Effects of drugs on afterdischarges are shown in Table IX. Changes in behavioral effects of stimulation paralleled changes in the EEG. The following compounds produced depressant effects on seizure discharges which were statistically significant a t the 1-5% level: Amygdala: phenobarbital, chlordiazepoxide, diazepam, nitrazepam. Hippocampus: phenobarbital, diazepam, nitrazepam. I n contrast, chlorpromazine showed a stimulant action (lowered afterdischarge threshold) in the amygdala. TABLE IX STIMULATION OF LIMBICSYSTEMI N FREELYMOVING CATS" ~~

~

~~

~

EEG After-discharge* No. of experiments

Amygdala Dose my/kg Threshold Duration p.0. (volts) (seronds)

Drug

10

4

10 C

3

Phenobarbital

10

5

Phenobarbital

20

5

Chlordiazepoxide

10

3

Iliazepam

5

3

Nitrazepsm

1.0

~

~~

Threshold (volts)

Duration (seconds)

4.35 f0.57 -0.36 50.16 $0.17 k0.44 $0.56' f0.44 +2.50* 50.67 -0.90 k1.03 -0.08 k0.74 -0.08 k0.46

61.8 f11.8 +15.4 i9.82 +65.0 f32.1 -17.0 i 7 .0 -45.0* i19.7 -4.20 56.72 -43.3+ 514.2 -43 .O0 535.3

-

Saline controls Mean preinjection values Mean change on injection Chlorpromazine

~

Hippocampus

2.98 k0.36 -0.10 50.09 -1.00' f0.48 $0.50" f0.29 +1,95* f0.78 +2.70* 5 0 .56 $0.08 50.69 -0.17 f0.44 ~

51 .0 k12.5 +20.4 k8.66 +3.75 k8.56 -15.3 k3.74 -43.'4* f21.8 -63.0* k24.0 -23.3" k1.35 -62.0' f46.6 ~~

~~~

Part of this material appeared in Schallek el al. (1964). (Courtesy of W. Schallek.) Data show mean k standard error of mean. The first line shows mean preinjection values, the other lines show mean change following injection. Statistical significance, drug vs. saline: *p < 0.01, f p 0.01-0.02, Op 0.02-0.05, no symbol p > 0.05. This dose of chlorpromazine caused emesis in two other cats. a

PHARMACOLOGY OF BENZODIAZEPINES

245

Higher dose levels were tested in experiments on immobilized cats. This type of experiment permits drugs to be tested on the electrographic but not the behavioral effects of brain stimulation. Reports from various laboratories agree that chlordiazepoxide, diazepam, and nitrazepam markedly decrease the duration and often also the amplitude of the after-discharge following electric stimulation of the septum, hippocampus, and amygdala (Schallek and Kuehn, 1960; Schallek et al., 1962; Horovitz et al., 1963; Requin et al., 1963; Lanoir et al., 1965). At higher doses seizures may be almost completely abolished (Requin et al., 1963). This effect of the benzodiazepines can be distinguished from that of other tranquilizing agents such as chlorpromazine and reserpine which lower the after-discharge threshold of the amygdala in immobilized rabbits (Kobayashi and Ishikawa, 1965). The effect of benzodiazepines on the amygdala can also be demonstrated with chemicalIy induced seizures. This was demonstrated by Hernandez-Peon et nl. (1964) and by Hernandez-Peon and Rojas-Ramirez (1966),who observed that diazepam and nitrazepam abolished seizure discharges induced in the amygdala of the cat by implantation of acetylcholine. Moreover, Eidelberg et al. (1965) reported that cocaine-induced seizures in cats originated in the amygdala and were accompanied by behavior resembling that seen in temporal lobe epilepsy in man. Increased 40 per second “spindling” in the electric activity of the amygdala induced by subconvulsant doses of cocaine was abolished by diazepam and other benzodiazepines. ii. Stimulation of thalamus. Hunter and Jasper (1949) found that stimulation of the intralaminar nuclei of the thalamus of the cat produced behavioral arrest accompanied by a 3 per second spike slow wave pattern in the EEG. Both behavioral and electrographic effects resembled those seen in petit ma1 epilepsy in man. Trimethadione is clinically effective in petit ma1 epilepsy. Schallek and Kuehn (1963) found that this agent blocked after-discharge in the central lateral nucleus which is part of the intralaminar thalamus of the cat, while it had no effect on the medial dorsal nucleus which is outside the intralaminar area. It also had no effect on after-discharges in hippocampus or cortex. Diphenylhydantoin and phenobarbital, which are clinically effective in grand ma1 epilepsy, showed a depressant effect on all areas tested. These data suggest that specific blocking of after-discharges in intralaminar nuclei may predict clinical utility in petit ma1 epilepsy. Table X shows effects of drugs on after-discharges in the central lateral nucleus of the thalamus and on the ectosylvian or suprasylvian gyri of the cortex. These experiments were performed on cats immobilized with deeamethonium. Chlordiazepoxide and diazepam had a significant effect on after-discharge in the thalamus at 10 mg/kg i.v. or more,

246

GERHARD ZBINDEN AND LOWELL 0. RANDALL

STIMULATION OF

No. of cats 6

5 5 4

3 4 4 5 5 3 4 5 5

TABLE X THALAMUS AND CORTEX

Drug

Saline contrrols preinjection r.hange on injection Chlordiazepoxide Diazepam Nitrazepam Chlorpromazine Diphcnylhydantoin Phenobarbital Trimethadione

IN IMMOBILIZED

Dose mg/k i.v.

Thalamus

-

14.0 f 1 . 0 m a -3.0 f 0 . 5

10 40 1 10 1 10 10 20 10 40 100 400

CATS"

Threshold for Afterdischargeb

+8.0 +l0.6 +l0.0 +11.7 f13.3 -5.9 +0.62 +3.7 -0.83 $11.9 43.1 $12.5

f 0.5* f 2.1* f 4.3 f 2.2*

k 3.0*

f 0.05+ f 2.6 f 2.3* f 0.83 f 1.2* k 0.5* f 2.1*

Cortex 2.85 f 0 . 5 8 m a -0.33 f 0.14 +0.32 -0.24 -0.15 -0.10 -0.17 +0.12 +2.2 +5.8

+0.12 -I-0.80 +0.10 -0.36

f 0.70 f 0.40 f 0.19 f 0.35 It 0.31 f 0.08 f 1.3 f 1.0* f 0.10 f 0.35+ f 0.30 k 0.0s

* CourteAy of Dr. W. Schallek.

* The first

line shows mean preinjection values; the remaining lines show mean change on injection. All data in milliamperes, mean f SE. Data in part from Schallek and Kuehn (1963) and Schallek et al. (1964). Statistical analysis, change in response for drug VB. change in response for saline: *p < 0.01, +p = 0.01-0.02, "p < 0.05.

whereas nitrazepam proved to be effective a t much lower doses. The data suggest that nitrazepam may be useful in petit ma1 epilepsy. Chlorpromazine again lowered the threshold for after-discharge. iii. Stimulation of the cortex. Various authors found that diphenylhydantoin which is clinically effective in grand ma1 epilepsy depressed the cortex in the cat and monkey (for refs., see Schallek and Kuehn, 1963). This suggests that cortical stimulation may be a screen for agents useful in grand ma1 seizures. The data in Table X indicate that the benzodiazepines do not affect seizure discharges in the cortex, whereas diphenylhydantoin and phenobarbital have a marked effect. By using other methods, however, several authors have demonstrated that benzodiazepines have anticonvulsant effects also in cortical regions. For example, Chusid and Kopeloff (1962) induced chronic epilepsy in monkeys by application of alumina cream to the cerebral cortex. The high amplitude slow waves and spikes in the EEG of these monkeys were diminished by chlordiazepoxide a t 1.1 mg/kg i.v. and completely abolished by 35 mg/kg i.v. In the cat, chlordiazepoxide, 3 mg/kg i.v., doubled the

PHARMACOLOGY OF BENZODIAZEPINES

247

threshold for the induction of seizure discharges by electric stimulation of the cortex (Roldan and Escobar, 1961). After-discharges in the cortex were diminished or abolished by chlordiazepoxide or diazepam (Requin et al., 1963; Hernandez-Peon et al., 1964). 111. Clinical Profile of the Benzodiazepines

A. GENERALREMARKS By the end of 1966 there were over 2300 papers published in the scientific world literature describing the therapeutic use of benzodiazepines in a wide variety of mental and neurological diseases and emotional disorders. The authors of this review feel neither obliged nor competent to pass judgment on the necessity and value of these drugs since their clinical assessment will take many more years and will undoubtedly continue to oscillate between enthusiastic acceptance and indifference. For the purpose of this study it is assumed that the application of these drugs by a large segment of the psychiatric profession and other medical specialists is a valid indication of potential therapeutic usefulness. For several therapeutic applications such clinical practices are already backed by controlled studies, whereas in other areas, such a s epilepsy, the evaluation of the benzodiazepines will require many more years. In other indications, however, where subjective responses make a controlled appreciation of drug therapy most difficult, no absolute proof of a drug’s therapeutic effect can ever be expected. This is particularly true when psychotropic drugs are prescribed merely as an adjunct to a comprehensive patient care program. The pharmacologic evaIuation, therefore, cannot be used as an argument to support therapeutic claims, but should be viewed as a basis which can reinforce clinical observations and, hopefully, also point to new areas worthy of clinical exploration.

B. EFFECTS IN NORMAL SUBJECTS At therapeutic doses benzodiazepines have little effect on normal human subjects. Various testing methods were developed to demonstrate an alteration of motor and mental performance and feeling of well being. I n one experiment on healthy students, 15 mg of chlordiazepoxide or 6 mg of diazepam per day given for 2 days, and one-third of these doses on the day of testing did not alter mental performance in nine tests using delayed auditory feedback and attentive motor performance as measured with a pursuit meter. The testing procedure, however, was able to demonstrate significant impairment of mental and attentive performance when the subjects received 15 ml per 150 pounds of body weight

248

GERHARD ZBINDEN AND LOWELL 0. RANDALL

of ethanol, corresponding to about 3 ounces of whiskey (Hughes et al., 1965). Two careful observers could not find any abnormal behavior in volunteers who had received either three times 10 mg of chlordiazepoxide or placebo capsules (Hoffer, 1962), whereas a slight influence on psychomotor performance could be demonstrated in male volunteers after 39’3 days of treatment with 15 mg per day of diazepam (Lawton and Cahn, 1963). At a somewhat higher dose, chlordiazepoxide (60 mg) given to young, healthy women caused a significant depressant effect on the flicker fusion frequency but in several other tests did not reduce psychological performance to any great extent. The drug significantly improved performance in the “cancellation test” as measured by the number of errors. Furthermore, standing steadiness was also significantly better than after placebo treatment (Holmberg and William-Olsson, 1963). An improvement of well being and friendliness was seen in male students treated with 37.5 mg of chlordiazepoxide. This was tested in a card sorting procedure and was highly significant when compared with placebo treatment (Holmberg and William-Olsson, 1964). I n another study, a group of students who took diazepam found themselves tired, apathetic, lethargic, or bored, but the drug did not reduce precision of free-hand copying of a complicated geometrical figure. Central stimulants such as amphetamine and trany Icypromine, on the other hand, caused noticeable sensory motor disturbance (Reed et al., 1965). Distinct pharmacologic effects of various degrees are seen if large doses of benzodiazepines are ingested. This was shown in an experiment in which 0.5 mg/kg of body weight of chlordiazepoxide had no effect in healthy volunteers, whereas signs of ataxia, motor incoordination, and sleepiness were observed when the dose was tripled (Burger, 1963). After taking 50 mg of chlordiazepoxide on the first day, 150 mg on the second, and 500 mg on the third day, a volunteer experienced mild euphoria, loss of appropriate and inappropriate anxiety, mild fatigue, and loss of equilibrium. After increasing the dose t o 1000 mg/day for 9 more days, irritability became worse and ataxia and dysarthria were noted. Similar symptoms were also observed in several patients who had made a suicide attempt by ingesting large doses of chlordiazepoxide (Zbinden et al., 1961). With other benzodiazepines, overdosage in healthy subjects also causes ataxia and muscle weakness, whereas with some derivatives sedation, drowsiness, and sleepiness are more prominent.

C. CLINICAL USES Up t o this writing, three benzodiazepines, chlordiazepoxide, diazepam, and oxazepam, have been introduced in clinical practice in the United

PHARMACOLOGY OF BENZODIAZEPINES

249

States. The list of indications for which these agents are mostly prescribed includes four major areas, namely: ( 1 ) anxiety, (2) muscle spasms, (3) convulsive disorders, (4) acute and chronic alcoholism. The effect on psychomotor agitation and anxiety is responsible for the broadest use of benzodiazepines since tension, nervousness, apprehension, and fear not only constitute some of the most frequently observed symptoms in psychoneurotic disorders, but are present to a larger or lesser degree in almost every patient suffering from a somatic or a mental disease. Thus, these agents are prescribed as preanesthetic medication, for anxiety, apprehension, and fear in patients with cancer, stroke, and cardiovascular diseases and in the large group of psychosomatic disorders as well as many organic diseases with heavy psychological overlay, e.g., asthma, angina pectoris, irritable colon, gastric ulcers, and various skin diseases. They also find a wide application in many forms of depression, obsessive-compulsive behavior, hysteria, personality problems, and in patients with psychosis where excitation or anxiety is prevalent. I n psychotic patients, benzodiazepines are often combined with other psychotropic drugs, in particular phenothiazines and antidepressants of the imipramine type. Neuromuscular disorders of various origins are frequently treated with benzodiazepines which seem to be useful, particularly in severe cases of cerebral palsy with athetosis, multiple sclerosis, hemiplegia, and paraplegia, and overriding fractures. Tetanus (Weinberg, 1964; Moriarty and Bertolotti, 1965) and stiff-man syndrome (Howard, 1963; Rogers, 1963) have recently been added to the list of promising indications. The use of the benzodiazepines in convulsive disorders is still in the exploratory phase. However, patients with certain forms of seizures, particularly myoclonic seizures, petit mal, and psychomotor seizures seem to benefit from these agents. Intravenous administration of benzodiazepines is widely accepted as the therapy of choice for status epilepticus (Gastaut et al., 1965). Acute alcohol withdrawal symptoms are another indication for benzodiazepines which is accepted by most physicians. The use of these agents for maintenance therapy of chronic alcoholics is favored by many specialists, but pharmacotherapy with benzodiazepines can only be regarded as an adjunct to a comprehensive treatment program of alcohol addiction. The benzodiazepines so far available in the clinic have no “antipsychotic” effects as reported with the phenothiazines. While they are useful in certain forms of depression, particularly when anxiety is prevalent, they do not possess the antidepressant, stimulant, or psychoenergizing properties of the imipramine-type agents, the monoamine oxidase inhibitors and amphetamine. They are not hypnotics in the

250

GERHARD ZBINDEN AND LOWELL 0. RANDALL

classical sense of the term, although they are widely used in neurotic sleep disorders. As pointed out in Section III,A, the authors of this review do not intend to evaluate the clinical usefulness of the benzodiazepines. The brief listing of the diseases and symptoms for which benzodiazepines are presently prescribed by a large segment of the medical profession serves only as a background against which the pharmacological properties of benzodiazepines will be evaluated. A more extensive appreciation of the therapcutic usefulness of benzodiazepines can be found in several reviews, such as those by Hines (1960), Detre and Jarecki (1961), Gross and Kaltenback (1961), Hayman (1961) , Sussex (1962), and Svenson and Gordon (1965). IV. Relationship between Clinical Observations and Animal Pharmacology

A. GENERALREMARKS The attempt to relate clinically observed therapeutic effects of drugs to pharmacological findings in animals is not motivated solely by a theoretical interest in mechanisms of action. A more practical reason is the desire to determine those pharmacological effects which appear to be directly responsible for thc therapeutic properties or the undesired side actions, so that new, more potent or more specific compounds of the same or a different chemical class may be assayed and selected for clinical trials. A comparison between clinical effects and animal pharmacology is more difficult with psychotropic drugs than with many other agents because a substantial part of the therapeutic effects deals with entirely subjective experiences of individual patients. Thus, the qualitative differentiation of these drug effects is much more dependent on clinical judgment than on objective measurements. Quantitative differentiations depend to a great extent on the type and severity of a patient’s mental and emotional disorder, his age, and other modifying factors. This explains why the therapeutic dose range is generally very wide, so that psychotropic drugs have to be titrated to every patient’s individual needs. I n the subsequent sections an attempt will be made t o compare the pharmacologic properties of a number of benzodiazepines with the clinical information available to date. The compounds to be discussed are chlordiazepoxide, diazepam, and nitrazepam on which there is a considerable amount of clinical information available. In addition, a number of newer analogs will be included which have not been extensively used in man but which were studied by clinical pharmacologists and medical investigators to the extent that their general clinical properties can be

PHARMACOLOGY OF BENZODIAZEPINES

25 1

described with a certain degree of accuracy. Since the clinical spectrum of these benzodiazepine derivatives tested so far is broad and often overlapping, attention will be focused primarily on the pharmacologic differences which were originally responsible for the selection of these derivatives for clinical exploration.

STRUCTURES AND PHARMACOLOGIC PROPERTIES OF B. CHEMICAL CLINICALLY TESTED BENZODIAZEPINES Tables XIa and X I b contain the structural formulas of a number of benzodiazepines which appeared interesting in the pharmacologic screening and were subsequently administered to human subjects. The extent of the clinical trials of the newer derivatives varied from clinical pharmacology studies for tolerance and preliminary efficacy evaluation (Phase I and early Phase I1 clinical trials) to more extensive investigations in selected neurological and psychiatric indications. A few derivatives have been tested in Phase I11 clinical studies involving most of the therapeutic uses described in the preceding section. It is noteworthy that the list contains a major metabolite (“lactam” Ro 5-2092) of chlordiazepoxide as well as a major metabolite (oxazepam) of diazepam (Randall et al., 1965a). Results of several pharmacologic tests conducted with these benzodiazepines are summarized in Tables XI1 and X I I I . I n order to permit an easier comparison, the activity of chlordiazepoxide calculated as effective dose 50% or minimum effective dose is used as standard and given the value 1. The tables contain the relative potency of all other benzodiazepine derivatives in comparison with chlordiazepoxide, with the exception of the avoidance rate increase for which the active dose ranges in milligrams per kilogram are listed. Several of the newer testing procedures such as attenuation of conflict behavior and tetrabenazineinduced stimulation in rats and delayed matching procedures in monkeys as well as neurophysiological experiments in cats, are quite involved and are therefore not yet completed with all benzodiazepine analogs listed.

C. PSYCHOSEDATIVE EFFECTS 1. Clinical Pharmacology Most of the benzodiazepines selected for clinical exploration exhibited, a t least to a certain degree, a chlordiazepoxide-like depressant effect on psychomotor excitation in man. This was demonstrated in Phase I clinical pharmacologic studies which were conducted on 15 to 70 subjects per compound. Included were groups of adult volunteers in a prison setting and groups of patients suffering from mild to moderate degrees of anxiety and tension. Ro 5-3636 was given to four patients only. Drug

252

GERHARD ZBINDEN AND LOWELL 0. RANDALL

TABLE XIa STRUCTURAL FORMULAS OF BENZODIAZEPINES TESTED IN HUMAN SUBJECTS NHCH,

d y0

c1

c1

Chlordiazepoxtde HCI

RO 5-2092

-N

Diazepam

Oxazepam

6” Ro 5-2904

6’

RO 5-4200

Nitrazepam

/

RO 5-3440

RO 5-3021

RO 5-4023

Ro 5-4520

administration was started a t low doses and increased gradually. An attempt was made to determine the minimum effective dose causing a drug-related effect in a few patients (up to 25%). I n most cases these were calming and relaxing effects noted by the patient, the physicians, or the nursing staff, but with some compounds the first indication of drug action was often a side effect such as sleepiness, drowsiness, and unsteadiness. Further increase of the dose permitted the determination of a

253

PHARMACOLOGY OF BENZODIAZEPINES

TABLE XIb STRUCTURAL FORMULAS OF BENZODIAZEPINES TESTEDIN HUMANSUBJECTS

Br

RO 5-3350

i-’

R O 5-3590

RO 5-6901

Ro 5-4933

RO 5-5807

Q Cl R O 5-4864

c1

q-

0

RO 5-4556

4 3

c1

f:

c1

-N

Ro 5-6227

RO 5-2181

RD 5-3636

Ro 5-3785

Ro 5-4964

. 2 HC1

level which caused drug-related effects, either therapeutic or in form of side effects, in approximately 50% of the subjects. Finally, a leveI was reached which caused clearly drug-related effects in the majority of the patients. The maximum tolerated dose was determined in those subjects who did not show marked response to the dose levels which affected the majority of the patients of the group. This bioassay procedure is

N

TABLE XI1

PHARMACOLOGIC POTENCIES OF 23 BENZODIAZEPINES I N VARIOUSPHARMACOLOGIC TESTSIN MICE A N D RATS (RELATIVE P O T E N C I E S I N COMP.4RISON WITH CHLORDIAZEPOXIDE)" Compound (generic name or laboratory designation) Chlordiazepoxide RO5-2092 Diazepam Oxazepam Nitrazepam RO 5-2181 RO 5-2904 RO5-3027 RO 5-3350 RO 5-3448

Anticonvulsant effect, mice Penty- Maximal leneelectr. tetrazole shock 1.0 1.3 5.8 11.6 11.6 1.1 6.2 20.0 11.4 26.6

1.o 0.58 4.7 1.1 1.0 0.75 6.0 2.3 0.88 2.0

Minimal electr. shock

Muscle relaxation, mice

1.0 0.23 1.4 0.39 0.26 0.46 4.6 0.80 0.75 0.54

1.0 1.0 3.3 0.44 6.7 0.33 10.0 1.0 3.3 2.5

Continuous avoidance, rat Fighting mouse test 1.o 1.o 4.0 1.0 8.0 1.0 4.0 20.0 4.0

4.0

Shock rate increase 1.0 0 0.42 0.51 5.2 0 0.47 1.1 3.8 7.0

Fscape failure 1.0 0 0.27 0.26 1.2 0 0.29 1.8 3.2 4.5

Avoidance rate increaseb 0 0 2-10 0 2.06 0 0 0 0 0

"Hypnotic" effect,c mouse 1.0 0.54 1.6 <0.37 0.84
Chlorprothixene potentiation,d mouse 1.0 >7700.0 1.8 77.0 >7700.0 2.8 260.0 >7700.0 3.6 >7700.0

E a w

H

z

3 d

F

0

r

?

RO 5-3590 RO 5-3636

RO5-3785 Ro 5-4023 Ro 5-4200 Ro 5-4528 Ro 5-4556 Ro 5-4864 Ro 5-4933 Ro 5-4964 RO 5-5807 Ro 5-6227 Ro 5-6901

f

11.4 0.8 0.03 50.0 80.0 8.0 5.0 0 2.2 0 16.0 1.2 5.0

0.23 0.30 0.46 0.07 2.5 2.1 0.81 0.1 1.o 0 2.0 0.09 0.37

0.31 0.11 0.11 0.11 0.27 0.46 0 0 0.35 0 1.1 0 1.1

1.o 0.25 0 0 100.0 5.0 1.0 If 2.0 0 0.22 0.40 0.50

4.0 1.0 0 2.0 50.0 8.0 1.o 0 1.0 0 0.50 1.0 2.0

0.47 0 0.06 0.07 0.14 0.15 1.1 0.30 14.0 0.56 0.62 0.53 3.5 10 0 0 0.22 0.9 0.26 0.67 No data No data -0.07 0 0.42 0.64

0 120-240' 0 0 0.15-0.3 1.2 0 0 0 0 No data 0 0

<0.37 <0.37 <0.37 <0.37 <0.37 0.41 0.44 I

3.3 < O . 74 <0.37 0.75 0.57

Data from laboratories of L. 0. Randall, C . L. Scheckel, and R. F. Banziger. Dose range in milligrams per kilogram i.p. at which stimulation is observed. Mice remain in lateral position for a minimum of 3 minutes. Based on ED%to induce sleep for a minimum of 3 minutes in combination with 25 mg/kg of chlorprothixene. Observed in 50y0 of the animals. Higher doses cause convulsions, salivation, Straub tail reflex.

>7700.0 >7700.0 0.28 >7700.0 2600.0 2.3 0.34 I

77.0 0.07 1.05 0.65 110.0

+d

F

0

z

K 23*

$ 0

r

z

td H

t?

Y

B 'd

3

m

TABLE XI11

PHARMACOLOGIC POTENCIES OF 23 BENZODIAZEPINES IN VARIOUS PHARMACOLOGIC TESTSIN RATS,CATS,AND MONKEYS IN COMPARXSON WITE CHLORDIAZEPOXIDE) (RELATIVEPOTI~NCIES ~

Compound (generic name or laboratory designation) Chlordiazepoxide RO5-2092 Diazepam Oxazepam Nitrazepam Ro 5-2181 RO5-2904 Ro 5-3027 Ro 5-3350 Ro 5-3448 Ro 5-3590 Ro 5-3636 Ro 53785 Ro 54023 Ro 5-4200 Ro 5-4528 Ro 54556 Ro 5-4864 Ro 5-4933 Ro 5-4964 Ro 55807 Ro 5-6227 Ro 5-6901 a

Discrete trial “trace” avoidance, rat

Inhib. of preesor resp. to Noise Muscle hypothal. response Response relaxation, stim., cat cat failure failure 1 <0.06 1.9 1.0 0.65 ND 2.0 2.7 4.0 10.0 0.21 0.19 ND 6.8 5.5 0.27 2.1 ND ND ND ND <0.62 0.48

1

<0.21 1.7 <0.62 <0.83 ND 1.25 1.7 <1.7 2.5 <0.42 0.42 ND <3.3 6.2 1.4 2.3 ND ND ND ND <0.62 <0.83

1

0.40 10.0 2.0 20.0 0.2 20.0 20.0 10.0 20.0 20.0

1 3.3 19.6 NDb 15.5 1.6 3.4 20.8 3.7 ND ND

1

0

0.1 40.0 100.0 4.0 0.5 ND 0 0 0.1 0.40 1

ND 79.0 35.4 ND ND ND ND ND ND >11.0 1.4

Inhib. of decereContinuous avoidance, monkey brate rigidity, Avoidance Shock Avoidance cat Taming, rate rate Escape rate (anemic) monkey decrease increase failure increase. 1

2.7 10.4 ND 3.6 ND 4.2 62.0 1.9 4.0 ND ND ND ND ND ND ND ND ND ND ND ND ND

1 1.05 0.84 0.52 ND <1.0 ND 1.8 2.6 1.9 ND 0.42 ND ND ND ND

1 0.40 1 0 12.5 0.10 1.o

12.5 1.0 0.40 0.20 0 0 0.40

2.0 1 0.20 ND 0 0 0 0 0.20

Dose range in milligrams per kilogram p.0. a t which stimulation is observed.

<0.2

1.4 ND ND 0 0.52 0.52 b

1

0.07 1 <0.02 ND <0.05 ND 0.4 0.25 0.33 ND 0.04 ND ND ND ND 0.02 0.1 ND ND 0 0.05 0.03

1 1.45 0.88 <0.72

ND <1.45 ND 3.9 3.6 4.3 ND 0.29 ND ND ND ND <0.3 1.4 ND ND 0 0.72 0.72

ND = No data available.

0 5-10 3-15 10-20 ND 20.0 ND 0.25-2.5 0.05-2.0 0.25-5.0 ND 0

ND ND ND ND 0 5-10 ND ND 0 0 2-20

PHARMACOLOGY OF BENZODIAZEPINES

257

similar t o the one described by Nodine (1962). Since the same subjects were often involved in the evaluation of several benzodiazepine analogs, the procedure provided a sufficient body of information on which to base a reasonable judgment of potency and psychosedative effectiveness of each benzodiazepine derivative. A summary of these studies in which the compounds are ranked according to potency is given in Table XIV. The ranking of the compounds is by necessity somewhat arbitrary, but a clear distinction can be made between highly potent compounds, analogs of moderate activity, and compounds of low potency. Oxazepam was not tested in these studies. Its potency is judged to be similar to that of Ro 5-6901. I n Table XV the ranking of these benzodiazepine analogs which is based on potency in the bioassay in man is compared with the rankiug in various animal tests. It demonstrates that the ten animal screening tests quite reliably separated the highly active group from the compounds of intermediate and those of low potency. The correlation between pharmacologic potency and efficacy in humans is satisfactory with all tests but seems best with the antipentylenetetrazole test in mice and the muscle relaxant effect in normal cats. All tests are most reliable in predicting the compounds with weak psychosedative properties. With all experimental procedures occasional inconsistencies are noted. Among the twenty-two compounds tested there are several whose general pharmacologic potency corresponds well with their ranking for clinical efficacy in humans. Ro 5-3448, RO 5-3027, Ro 5-4528, Ro 5-6901, and the four derivatives with the lowest potency are good examples. Other derivatives show unusually marked or weak activity in one or several tests. For example, in the group of potent derivatives the most potent compound, Ro 5-2904, only ranks tenth and thirteenth, respectively, in the antipentylenetetrazole and escape failure tests. Another potent sedative, Ro 5-4023, is very weak in the antimaximal and antiminimal electroshock and in the inclined screen tests. There are no obvious clinical correlates for these pharmacologic differences. An interesting compound is Ro 5-2092 (“lactam”), a major metabolite of chlordiazepoxide in man. Clinically, this drug behaves just about like chlordiazepoxide but it is inactive in the continuous avoidance situation in rats and nine times weaker than chlordiazepoxide in attenuating tetrabenazine-induced stimulation in rats pretreated with a monoamine oxidase inhibitor. It is considerably weaker than the parent compound against maximal and minimal electroshock, but much more potent in the chlorprothixene potentiation test. I n monkeys, Ro 5-2092 has a stimulant effect in low doses which is not observed with chlordiazepoxide, whereas both compounds have similar depressant effects in the continuous avoidance behavior situation a t higher doses. From these data it is clear

CLlNIC.4L

Compounds

High potency RO5-2904

RO 5-3448 RO 5-3027

Ro 5-4200 Ro 5-4023 Intermediate potency Ro 5-4528 RO5-3350 RO 5-3590 Ro 5-2092 Nitrazepam Diazepam Chlordiaaepoxide RO5-6901 Ro 5-4933 Ro 5-4864 Ro 55807 Ro 5-2181 Ro 5-4556 Low potency RO 5-3636 RO 5-6227 RO 5-3785 Ro 5-4964 a

b

No. of patients treated

TABLE XIV BENZODI.%ZEPINE DERIVATIVES"

MED mg/day

Dose causing drug effectb mg/das

1 1.5 1.5

3 6 3 6 7.5

1.5 3

3 6

23 70 35 15 30 25 64 46 36 24 30 25 18

2.5 3 6 6 10 10 10 10 15 15 10 15 15

20 30 15 25 20 50 50 25 30 30 50 30 30

4

0.25 0.5

Approx. effective dose 50% mg/day

35 40 29 22 33

25 35 32

N u1

PHlRMACOLOGY O F VARIOUS

1

20 40 40 200

20 75 120 -

Max. tolerated No. of patients dose tolerating mg/day max. dose 21 9 15 6 20

40 60 40 30 30 75 100 100-200 40 40 120 50 120

100 800 60 100 120 100 200 200 80 200 160 240 160

20 100 240

30 200 480 600

-

Data obtained from W. B. Abrams and collaborators. The ranking is similar to the one presented by Whitman (1966). In majority of subjects. Psychotic subject. d Mild sedation in 2 of 32 patients at 600 mg.

1 1 3 15 2 1 1 C

7 2 3 6 1 11 1 1

3 1 1

4 1 2 d

m

1 *3:

5 U

N

E

3

Z

5

tl

r

3

F

P Z

g

r r

COiWARISON Compound (generic name or laboratory designation) RO 5-2904 RO5-3448 Ro 5-3027 Ro 54200 Ro 54023 Ro 54528 Ro 5-3350 Ro 5-3590 Ro 5-2092 Nitrazepam Diazepam Chlordkzepoxide Ro 5-6901 Ro 5-4933 Ro 5-4864 RO5-5807 RO5-2181 Ro 5-4556 Ro 53636 Ro 5-6227 Ro 5-3785 Ro 54964

OF POTENCY OF

TABLE XV VARIOE3 BENZODIAZEPIKES I N THE HUMAN

Ranking in human bioassay 1 2 3

4

5 6 7 8 9 10 11 12 13 14

15

16 17 18 19 20 21 22

BIO.\SSAY A N D I N PHARMlCOLOGIC

TESTS

Ranking in various animal tests“ Test n u m b e d I

I1

111

IV

V

VI

VII

VIII

IX

X

10 3 4 1 2 9 7 7

1 6 4 3 20 5 11 18 14 8 2 8 16 8 21 6 13 12 17 19 15 22

1 8 6 13 16 9 7 12

2 7 9 1 22 4 5 9 9 3 5

5 5 2

10 2 5 1 5 9 4 10 22 3 12 7 12 15 22 ND 22 7 18 17 16 14

13 1 4 10 12 11 3 22 22 5 14 6 9 7 22 ND 22 2 16 22 15 8

7

3 3 3 1 2 10 8 3 15 3 8 11 11 22

4 9 1 3 9 4 4 12 9 1 4 4 12 22 ND 22 15 12 22 22 22 22

15

6 11 18 12 14 22 5 17 12 19 16 20 22

15 14 2 5

3 11 22 3 9 22 16 22 16 22

9

14 8

NDc 18 16 9 17 15

22 22

1 10 3 5 5 12 3 5 12 10 12 22 19 12 12 12 12 22 22

1 5 3 2 12 4 13 16 10 8 9 11

ND

ND

ND ND 6 14 15 ND ND

ND 18 17 14 11 15 18 22

~

Compounds with t.he same activity are given the same ranking. Inactive compounds are ranked twenty+econd. I = htipentylenetetrazole test in mice; I1 = Antimaximal electroshock in mice; I11 = Antiminimal electroshock in mice; IV = Muscle relaxation in mice (inclined screen test); V = Fighting mouse test; VI = Continuous avoidance, shock rate increase, rat; VII = Continuous avoidance, escape failure, rat; VITI = Discrete trials “trace” avoidance, noise response failure, rat; IX = Muscle relaxaND = No data available. tion, cat; X = Taming of monkeys. a

b

260

QERHARD ZBINDEN AND LOWELL 0. RANDALL

that the pharmacologic action of chlordiazepoxide can be distinguished from that of one of its major metabolites. Although the two compounds behave similarly in patients, the pharmacologic findings indicate that the therapeutic properties of chlordiazepoxide are not solely due to this metabolite. The comparative activities of diazepam and one of its metabolites, oxazepam, have been discussed by Randall et al. (1965a). Another noteworthy compound is Ro 5-4864. This derivative was originally selected for clinical trials because it appeared to have a selective action against maximal electroshock in mice. On retesting, this effect could not be confirmed. There was no activity in the fighting mouse test, the continuous avoidance test in rats, and the hypnosis test in mice with and without chlorprothixene. At higher doses the drug had some muscle relaxant and marked convulsant properties. The latter effect is rare among the benzodiazepines tested so far. In humans, the clinical pharmacologists noted tranquilization and drowsiness in the majority and ataxia in few of the patients treated with 75-100 mg per day. Thus, there was no correlation between clinical findings and neuropharmacologic screening in rodents, but studies in monkeys indicated that Ro 5-4864 was about as effective as chlordiazepoxide in the continuous avoidance procedure. It is possiblc, therefore, that Ro 5-4864 is converted to an active metabolite in monkeys and man but not in rodents. 2.

Clinica 1 Investigation

Further study of the more promising compounds in patients with moderate to severe degrees of anxiety, tension, irritability, and hypochondriasis indicated a considerable difference in the therapeutic usefulness. As pointed out above, most benzodiazepines tested so far possess some antianxiety effects. The clinical usefulness of a given compound, however, is dependent on the separation of psychorelaxant and sedativehypnotic properties. Such a separation was demonstrated in various mimal tests, particularly the continuous avoidancc procedure in rats and monkeys and the discrete trial trace avoidance situation in rats (Section II,C,2). It was found that benzodiazepines are characterized by a wide margin between the dose causing avoidance failure and the incapacitating dose leading to escape failure when a shock is received (high dose range ratios) (Heise and Boff, 1962). The question thus arises whether or not the magnitude of the spread between dose leading to avoidance failure and dose causing escape failure would provide an indication as to the clinically demonstrable separation of anxiolytic and hypnotic-sedative effects. As far as the rat experiment is concerned, this is definitely not the case. The potent benzodiaeepine derivatives, for example, which very

PHARMACOLOGY OF BENZODIAZEPINES

261

often caused oversedation a t therapeutic doses in man, were generally found to have dose range ratios a t least as high but often much higher than chlordiazepoxide and diazepam. These two drugs, however, exert their anxiolytic, muscle-relaxant, and anticonvulsant effects a t doses which do not, in the majority of the patients, induce sleep, oversedation, and ataxia (Svenson and Hamilton, 1966; Tobin e t al., 1960). From this comparison it is concluded that a high dose range ratio in rats is a characteristic pharmacologic property of benzodiazepines but does not permit any conclusions as to the therapeutic index of a benzodiazepine derivative in man. Limited data in squirrel monkeys show that the dose-range ratio of the potent sedatives Ro 5-3027 and Ro 5-3448 are markedly lower than those of chlordiazepoxide and diazepam, indicating a better correlation with the clinically observed situation.

D. SLEEPINDUCING EFFECTS During the clinical evaluation of several benzodiazepine analogs, a number of compounds were found which caused marked sedation and often sleep in doses almost identical with the ones which have beneficial psychorelaxant or muscle-relaxant effects. The most pronounced hypnotic action was found with the compounds listed in Table XVI. The table also shows that the classical methods of testing hypnotic effects is inadequate for this class of drugs since these highly active agents were mostly less potent than chlordiazepoxide and diazepam in causing loss of righting reflex in mice. A better correlation with the clinical experience is seen if the mice are treated simultaneously with a small (nonhypnotic) dose of chlorprothixene (Banziger, 1966). The muscle-relaxant effect in normal cats also correlates quite well with the hypnotic properties observed in man. I n the continuous avoidance procedures, however, the potent sedatives behaved like chlordiazepoxide and diazepam (Section IV,C,2). I n man, oversedation and sleep were induced particularly with the group of compounds which were so powerful that adjustment of the dose was almost impossible. This action, however, must be distinguished from sleep-producing properties of less potent analogs such as nitrazepam, Ro 5-6901, and diazepam which are prescribed for those forms of insomnia in which anxiety and other psychoneurotic symptoms are largely responsible for the sleep problem (Aivazian, 1964; De Lemos e t d., 1965; Peck and Sherrington, 1966; Le Riche et al., 1966). These agents are generally not effective in severe forms of insomnia which may still respond t o barbiturates. This difference is also borne out in neurophysiological experiments. The sedative and anesthetic properties of the barbiturates are

TABLE XVI BENZODIAZEPINE DERIVATIVES WHiCH HAVETHE MOSTPRONOUNCED SEDATIVE EFFECTIN MAN COMPARISON WITH RESULTS OF SELECTED ANIMALTESTS'

Compound (generic name or laboratory designation)

Dose causing oversedation in most subjects mg/day

H D d in Mice mg/kg P.O.

HD6$ in Mice in comb. with 25 mg/kg chlorprothixene mg/kg p.0.

0.5-2.0 1.0-2.0 1.0-3.0 1.54.0 6.0-10.0

330.0 >1000.0 31.5 478.0 > 1000.0

<0.001 0.003 0.03 <0.001 <0.001

GI

Continuous avoidance, rat

Muscle relaxation in cats MEDc mg/kg p.0.

Shock rate increase MED mg/kg i.p.

Escape failure MED mg/kg i.p.

2.0 0.02 0.1 0.01 0.05

3.8 0.3 9.0 0.6 3.7

10.0 32.0 62.0 4.0 60.0

m a X + N

W

2

4

9

RO 5-3027

Ro 5-4200 RO 5-2904 Ro 53448 Ro 5-4023 Reference substances Chlordiaaepoxide Diazepam

-

370.0 225.0

7.7 4.2

2.0 0.2

Data from the laboratories of L. 0. Randall and R. F. Banziger. HDB = Dose required to cause loss of righting reflex in 50% of the mice for a t least 3 minutes. MED = Minimum effective dose.

4.2 10.0

18.0 67.0

3 F 2 RF ? a

*s 2-

r

F

263

PHARMACOLOGY OF BENZODIAZEPINES

attributed to selective depression of the brain stern reticular formation (Killa'm, 1962). Various experiments dealing with the effects of benzodiazepines, however, indicated that these agents produced less depression of the reticular activating system (Soulairac et al., 1965; Schallek e t al., 1965; Schallek and Kuehn, 1965). EEG activation was recorded in immobilized cats after stimulation of the sciatic nerve. Chlorpromazine, diazepam, nitrazepam, and pentobarbital were tested, each a t 10 mg/kg i.v. Pentobarbital was the only compound to produce a statistically sig10:35 A.M.

-

PRE-DRUG

RP-RO RO-

LO

I

0.2 MA

P

11~20A.M.

-

2 5 MIN. AFTER DIAZEPAM

10 MG/KG I.V.

RP- R O

1305 PM.

CAT

32

-

2 HR. 10 MIN. POST-DRUG

26 FEE. 1964

FIG.9. Diazepam, 10 mg/kg i.v., causes slight rise in threshold for EEG activation produced by sciatic stimulation in immobilized cat. Stimulation parameters, pulse duration 1 msec, frequency 100 cyclsecond, duration of stimulation 5 seconds. EEG leads are right parietal-right occipital, right occipital-left occipital. Top panel before drug: E E G activation a t 0.2 ma outlasts stimulation period. Middle panel 25 minutes after drug: EEG activation limited to stimulation period; amplitude is higher than in predrug pattern. Note appearance of fast waves in E E G before stimulation. Lower panel, 130 minutes after drug: Little or no activation a t 0.2 m a ; delayed activation a t 0.4 ma. (Courtesy of W. Schallek.)

264

GERHARD ZBINDBN AND LOWELL 0. RANDALL

nificant increase in activation threshold (Figs. 9 and 10) (Schallek e t al., 1965). Similar differences were noted in unanesthetized cats with chronically implanted electrodes in which behavior arousal induced by reticular stimulation was observed (Schallek and Kuehn, 1965). Several other studies on the effects of barbiturates and benzodiazepines on thalamic recruiting response and the electrical activity of the cortex support the view that the neuropharmacologic actions of these two classes of drugs

-

l o t 4 8 AM. PRE-DRUG

4 H -

RF-RP

I I 55

AM

-

1°Y

0.2 MA

35

MIN

AFTER PENTOBARBITAL

LF-RF

I V 2 0 0 uv ;

I

200 u v

RF-RP

I

3.0 MA

CAT 23

10 MG/KG

I

7 FEB. 1964

FIQ. 10. Pentobarbital, 10 mg/kg i.v., completely blocks EEG activation in immobilized cat. Arrangement as in Fig. 9. Top panel before drug: EEG activation at 0.2 ma. Middle panel 35 minutes after drug: No activation at 3.0 ma. Note decreased frequency and increased amplitude of EEG. Lower panel 3 hours after drug: EEG activation reappears at 0.2 ma, although amplitude is greater than in predrug pattern. (Courtesy of W. Schallek.)

do not coincide (Monnier and Graber, 1962; Requin e t al., 1963; Arrigo e t al., 1965). Thus, clinical experience and neurophysiological data indicate that facilitation of sleep by benzodiazepines is essentially due to a reduction of emotional activity caused primarily by dcpression of amygdala and hippocampus (Schallek et al., 1965). A similar conclusion was reached by Hernandez-Peon and Rojas-Ramirez (1966) who at-

PHARMACOLOGY OF BENZODIAZEPINES

265

tributed the action of these drugs to “pharmacologic depression of the limbic emotional circuits.’’

E. STIMULATION A direct stimulant effect which manifests itself in an increased rate of lever pressing in the continued avoidance situation in rats and monkeys is seen with several benzodiazepines (Section II,C,6). It occurs only a t low dose levels. If the dose is increased, a slowing of the response rate occurs. As shown in Tables XI1 and XIII, five benzodiazepine derivatives demonstrated a stimulant effect in rats and nine derivatives had a similar effect in monkeys. Diazepam stimulated both rats and monkeys and nitrazepam also had a stimulant effect in mice (Sternbach e t al., 1964). From clinical studies it is clear that these agents do not possess a comparable stimulant action in man when given continuously a t doses which control psychoneurotic symptoms. Under special circumstances, however, stimulant effects have been observed. For example, with diazepam treatment, hyperactivity, agitation, and overstimulation occurred as side effects in an occasional patient (Krakowski, 1963; Hare, 1963), but the same was observed with chlordiazepoxide (Tobin e t al., 1960; Hollister e t al., 1961) which has no stimulant action in animals. It is also seen with phenothiazines (Maculans, 1964; Smith and Chassan, 1964) and, rather frequently, with placebo medication (Hare, 1963; D e Lemos e t al., 1965). I n reviewing the clinical literature, one gains the impression that diazepam is more useful than chlordiazepoxide in certain forms of depression, such as anxiety depression and reactive depressions (Towler, 1962 ; Stanfield, 1963; Gold and Dribben, 1964; Svenson and Gordon, 1965). This, however, is probably not related to a direct stimulant effect since chlordiazepoxide as well as diazepam seem to produce improvement of mood and lessening of depression essentially as a result of decreased anxiety and tension (Yochelson, 1961). In a direct and very carefully conducted comparison of chlordiazepoxide and diazepam, Feldman (1962) found that in hypoactive patients diazepam had an energizer-type effect which was also manifested in the type of side effects, such as an exacerbation of delusions observed in some anergic schizophrenics. This never occurred with chlordiazepoxide and might be a reflection of the stimulant qualities of diazepam. In evaluating the conflicting clinical experience, it should be noted that in experimental animals stimulant effects of certain benzodiazepines were only present a t low doses. At high doses all derivatives caused CNS depression. Since these agents are slowly excreted in man, the chances that a stimulant effect may manifest itself during continuous administration of rather large doses are remote. It might be worthwhile, therefore, to investigate if a more consistent

266

GERHARD ZBINDEN AND LOWELL 0. RANDALL

stimulant effect could be demonstrated in man by administering small and infrequent doses of those derivatives which had the most pronounced stimulant effect in rats and monkeys.

F. MUSCLE-RELAXANT EFFECTS Recent pharmacologic (Friend, 1964) and clinical (Svenson and Gordon, 1965) rcviews of the literature have demonstrated that benzodiazepines have muscle-relaxant properties and are clinically effective in various forms of muscle spasms (Section II1,C). Several questions, however, arc still under discussion. While there are good indications as to the site of action of the benzodiazepines in animal models (Section II,B,3), clinical investigators have not yet identified with certainty the principal mechanism of action responsible for the antispasmodic effects in man. There is also no agreement whether a sufficient separation of sedative and muscle-relaxant properties has been achieved, and which animal test most reliably predicts muscle-relaxant properties in man. The difficulty in determining the mode and site of action of musclerelaxant drugs stems from the enormous complexity of the central facilitating and inhibiting mechanisms involved in the control of ,muscle tone (Magladery, 1964; Kane, 1964). Experimental and clinical observations indicate that in cases of upper motoneuron lesions the gamma motoneurons are released from descending inhibition, Their overactivity then results in muscle spasm (Rushworth, 1964a). Granit (1964), who developed the concept of gamma and alpha rigidity (Granit et al., 1955), however, cautioned against an unqualified acceptance of this theory with regard to clinical interpretations, since under pathological conditions several systems may be involved in any one pathophysiologieal event. This is underlined by the fact that in addition to the anatomical lesions in the CNS, many other factors contribute to the clinical syndrome of musculoskeletal spasticity, among them pain, anxiety, and other emotional disorders, diseases of the muscles and joints, the patient’s nutritional state, the extent of his immobilization, etc. (Leavitt et al., 1963; Kane, 1964). I n evaluating the effectiveness of drug therapy, an influence on any one of these factors may result in muscle relaxation. I n the case of the benzodiazepines, the marked antianxiety effect certainly contributes to the muscle-relaxant action observed, for example, in dystonic-athetoid cerebral palsy children. However, clinical experience indicates that a t least part of the beneficial effect is due to a neuropharmacologic action, either on polysynaptic transmission within the spinal cord (Friend, 1964) or on supraspinal structures (Phelps, 1963; Payne and Ishmeal, 1963). This conclusion is supported by the observation that nonhypnotic doses of diazepam suppressed startle reflex in patients with

PHARMACOLOGY OF BENZODIAZEPINES

267

cerebral palsy (Marsh, 1965) and relieved severe muscle spasms in patients with stiff-man syndrome (Howard, 1963; Rogers, 1963) and tetanus (Weinberg, 1964; Moriarty and Bertolotti, 1965). Control of such conditions cannot be explained merely by a psychosedative effect. The principal site of action of diazepam and other benzodiazepines in man, however, has not been identified. I n discussing the available evidence, Rushworth (1964b) suggests three possibilities, namely: (1) the gamma system and its central connections whereby descending facilitation of these groups of motoncurons would be blocked; (2) the Ia afferent nerve fibers which would lead to a block of the afferent facilitation of alpha-motoneurons; and (3) the muscle spindle sensory endings themselves. More recent pharmacologic data indicate that research with benzodiazepines should be concentrated on the gamma-system and the supraspinal center of the brain stem reticular formation (Section II,B,3) . Information gained from clinical evaluation of several benzodiazepine analogs in humans indicates that there is poor correlation between the clinical effects in spasticity and the muscle-relaxant properties as assayed in the inclined screen test in mice. I n the inclined screen test in mice, diazepam has an ED,, of 30 mg/kg whereas Ro 5-4023, which is also quite a potent muscle relaxant, has an ED,, of >500 mg/kg. Ro 5-3350 has only weak spasmolytic effects in man, but the ED,, in mice equals that of diazepam. A somewhat better correlation is found when the results obtained in decerebrated cats are compared. In this test the two potent muscle relaxants, diazepam and Ro-5-4200, show a very high activity, particularly in the intercollicular preparation, the ED,, being about one-fourth of the one observed with Ro 5-3590, a weak muscle relaxant in man. In the anemic prcparation, diazepam is considerably more active than chlordiazepoxide, which parallels the clinical impression. While these findings indicate a satisfactory correlation between animal test and clinical experience, the number of compounds tested in spastic disorders is not yet large enough to permit a definite evaluation as to the predictive value of the decerebrated cat preparation.

G. ANTICONVULSANT EFFECTS The difficulties in evaluating the therapeutic effectiveness of new anticonvulsant agents are many. One of the difficult problems is the fact that many epileptic patients are reasonably well controlled with the existing agents and any change in medication can precipitate an exacerbation of the syndrome. Physicians are therefore often reluctant to switch medication. A new drug is thus either added in small doses to a basic anticonvulsant drug regimen or given to patients who are not controlled with existing agents (Schwab e t al., 1960). Long observation periods are

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GERHARD ZBINDEN AND LOWELL 0. RANDALL

needed to assure that a drug effect is real and that no resistance develops. The normal variations in seizure intensity and frequency which are partly also dependent on the patients’ emotional ups and downs further complicate the assessment of drug therapy. Drug effects on E E G often do not correspond with clinical improvement (Markham, 1964; Gibbs and Anderson, 1965), so that the only really objective measurement of a drug’s CNS effect is of questionable value in interpreting therapeutic efficacy. Boyer (1966) recently summarized clinical studies in which the anticonvulsant effects of benzodiazepines are reported. He concludes that these drugs have a definite therapeutic effect in many forms of seizures. I n this review the question will be discussed of whether or not the various qualitative and quantitative pharmacologic differences between benzodiazepines and other anticonvulsant drugs and those found between various benzodiazepine analogs (Sections II,C,l ; II,C,7d) correspond with clinically demonstrable differences in effectiveness against various forms of epilepsy. The anticonvulsant screening in mice (Sections II,B,l ; II,C,l) has indicated that benzodiazepines are particularly effective against pentamethylenetetrazole-induced seizures which is also, with a different order of magnitude, characteristic for trimethadione (Swinyard and Castellion, 1966). From this similarity one might conclude that benzodiazepines could be particularly useful in petit ma1 epilepsy. This view is supported by neuropharmacologic findings in cats (Section II,C,7d) which showed that benzodiazepines and trimcthadione increased the threshold for afterdischarge after stimulation of the central lateral nucleus of the thalamus (Schallek and Kuehn, 1963). I n these tests nitrazepam proved to be considerably more potent than chlordiazepoxide and diazepam (Table X) . Other studies with implanted electrodes in cats (Section II,C,7d) showed that benzodiazepines also markedly inhibited EEG after-discharges after stimulation of the limbic system. Sincc stimulation of these centers induces behavioral changes resembling psychomotor seizures in man (Kaada et al., 1953, 1954), the benzodiazepine effect might indicate potential usefulness in temporal lobe epilepsy. Benzodiazepines were also found to supprcss seizures induced by stimulation of the cortex, indicating potential usefulness in grand ma1 seizures (Section II,C,7d). I n some of the studies, however (Schallek and Kuehn, 1963; Schallek et al., 1964), the benzodiazepines appeared less effective in this area than phenobarbital and phenylhydantoin (Table X ) . From these data it might be predicted that benzodiazepine derivatives have broad spectrum anticonvulsant properties in man, with particular effectiveness in psychomotor and petit ma1 seizures. Of the compounds tested clinically, nitrazepam appeared to be the most promising because of its highest activity in de-

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pressing after-discharges following stimulation of the thalamus and the limbic system. Three benzodiazepine analogs, chlordiazepoxide, diazepam, and nitrazepam, have been studied extensively in epileptic patients but only few comparisons of effectiveness against different forms of seizures have been made. Thus, only very preliminary conclusions can be drawn. From the review of Boyer (1966), it can be concluded that all three benzodiazepines are broad spectrum anticonvulsants and may be useful as adjunctive treatment, occasionally also as the sole medication, in all forms of seizures. Published data and results of unpublished clinical studies indicate that chlordiazepoxide is the least potent anticonvulsant agent of the three. With diazepam, best results have been observed in petit ma1 and minor motor seizures. Its effect in grand mal, and Jacksonian focal motor and temporal lobe seizures is moderate. I n this indication diazepam appears to be somewhat more useful than nitrazepam, particularly because it does not seem to induce grand ma1 and petit ma1 seizures as often as nitrazepam. Nitrazepam increased frequency and severity of grand ma1 and petit ma1 seizures or other seizure forms rather frequently (Liske and Forster, 1963; Gibbs and Anderson, 1965). On the other hand, nitrazepam has shown promising effects in myoclonic seizures with hypsarythmia as well as focal spiking, polyphasic spiking and slow waves, and other electroencephalographic abnormalities (Liske and Forster, 1963; Markham, 1964; Gibbs and Anderson, 1965; Millichap and Ortiz, 1966). In this condition, which is most difficult to treat, nitrazepam seems to be supcrior to all previously used anticonvulsant drugs and corticotropin (Millichap and Ortiz, 1966). Although dia2epa.m has been found effective (Weinberg and Harwell, 1!365), it is probable that the action of nitrazepam is more consistent. For example, of sixteen children with hypsarythmia who were controlled with nitrazepa;m, seven relapsed when switched to the maximally tolerated dose of diazepam (Gibbs and Anderson, 1965). In comparing the clinical effectiveness with the results of the pharmacologic studies, it becomes evident that as predicted by animal cxperiments, benzodiazepines have a broad-based anticonvulsant effect which is not limited to any one or a few brain areas. The relatively weak activity against grand ma1 and focal motor seizures can be related to observations in cats where there was no effect on after-discharge upon cortical stimulation. It Is difficult to explain the occasional induction of grand ma1 seizures, particularly by nitrazepam, since lowering of seizure threshold was not observed with these compounds in any of the brain centers studied. This is in contrast t o the effect of chlorpromazine which is known to lower seizure threshold to electric stimulation in various

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areas of the brain. As indicated earlier, nitrazepam did have an unexplained stimulant effect in mice (Sternbach e t al., 1964) which was not observed with any other berizodiazepine analog. It is questionable, however, whether this effect has anything to do with the relative frequency of grand ma1 seizure production in epileptic patients. The marked effect of nitrazepam against myoclonic seizures was not predictable from animal studies. Myoclonic seizures belong to the group of generalized seizures, probably arising a t thc mesenccphalic or diencephalic level (Dreifuss, 1966). The etiology has not yet been identified in many of these patients whereas in others a number of illnesses and developmental defects of the brain may be the underlying cause (Markham, 1964). There is no animal model as yet which imitates this type of epilepsy. It is hoped that further clinical studies with other benzodiazepine derivatives will provide additional data for comparison between animal experiments and clinical effects in myoclonic seizures. From experiments with cats it was predicted that benzodiazepines, in particular nitrazepam, might have good activity against temporal lobe epilepsy (Section II,C,7d). The clinical experience so far indicates that psychomotor seizures sometimes respond to treatment with benzodiazepines (Bercel, 1961; Liske and Forster, 1963; Milliehap and Ortiz, 1966) but the effect is not as consistent and pronounced as that observed in myoclonic seizures. The same is true for pctit ma1 seizures which are sometimes reduced or abolished by benzodiazepines (Liske and Forster, 1963; Markham, 1964) but again no specific action against this type of epilepsy can be postulated. The overall effect of benxodiazepines on psychomotor and petit ma1 seizures appears to be more favorable than the one on grand ma1 seizures (Bercel, 1961; Liske and Forster, 1963) which is in good agreement with findings in various animal tests.

H. AUTONOMIC EFFECTS Benzodiazepines have very wcak, if any, ,effect on the peripheral autonomic ncrvous system. In the standard animal testing procedures, only very weak or no anticholinergic action can be demonstrated (Madan et al., 1963; Gluckman, 1965). Consequently, the typical anticholinergic side effects in man, such as tachycardia, dry mouth, blurred vision, pallor, difficulties in urination, and constipation are rarely observed (Hollistcr, 1961, 1964)’. A fleeting and weak depressant effect on blood pressure and heart rate in anesthetized cats and dogs was observed after parenteral administration of rather large doses (Sternbach et al., 1964 ; Madan et al., 1964; Gluckman et al., 1965) but no alteration of the blood pressure responses to serotonin, acetylcholine, carotid occlusion, or central vagus stimulation was demonstrated (Stcrnbaeh e t al., 1964).

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Benzodiazepines had no ganglionic blocking effect in cats. I n cats and dogs the vasoconstrictor response to norepinephrine was slightly potentiated a t low doses of chlordiazepoxide and somewhat reduced when the dose was increased. These effects were transient and did not appear to be of any significance for clinical use (Moe e t al., 1961). Oral administration of chlordiazepoxide or oxazepam to unanesthetized dogs had a negligible effect on blood pressure and heart rate (Gluckman, 1965). There was no change of the heart norepinephrine and serotonin lcvels in rabbits treated for 4% days with 50 mg/day of chlordiazepoxide (Moe e t al., 1961. In clinical pharmacologic studies Steen and Martinez (1964) found no effect of intravenous injections of 0.5-1.5 mg/kg of chlordiazepoxide on blood pressure and pulse rate in healthy volunteers. I n another experiment on eight healthy male subjects, the circulatory response in the supine position and in the 60" head-up tilt was not altered after intravenous injection of 20 mg of chlordiazepoxide (Dobkin and Criswick, 1961). Clinical experience also indicates that orthostatic hypotension occurs only in exceptional cases. This was seen in one chronic alcoholic patient who received the enormous dose of 200-500 mg of chlordiazepoxide per day to prevent delirium tremens. Systolic blood pressure dropped 45 mm Hg upon standing and heart rate increased by 50 beats per minute (McCurdy and Kane, 1963). In contrast to the benzodiazepines, phenothiazine tranquilizers have a pronounced effect on peripheral autonomic control mechanisms, a difference which is readily recognized clinically when the two types of drugs are compared in double blind studies (Hare, 1963; Smith and Chassan, 1964) or on a statistical basis (Cares and Buckman, 1963; Hollister, 1964). I. ENDOCRINE EFFECTS The adrenal cortex, testis, ovaries, and thyroid and their respective hormonal secretions are undcr the control of tropic hormones secreted by the pituitary gland. The pituitary gland and its secretions are in turn under the control of neurohumoral substances secreted by neurohumoral cells in the hypothalamus (Harris, 1955). Consequently, it should not be surprising that drugs which affect the hypothalamus either directly or indirectly can manifest endocrine side effects. Such changes can be rather easily produced in animal experiments but the correlation with clinically observed side effects in man is not satisfactory. For example, single doses of 25 mg/kg of chlorpromazine depressed markedly the uptake of l3II by the thyroid gland in rats (Wiseman, 1962). Similar effects were seen with reserpine and phenothiazine tranquilizers (Mayer et al., 1956; Raddle and KaIow, 1960; Stumpf et al., 1963). This effect is

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probably due to inhibition of thyrotropin secretion (Stumpf et al., 1963) but has not been a problem clinically. A few studies on the effects of benzodiazepines on the hormone output of the pituitary and on secondary changes of endocrine target organs have been reported in the literature. Boris et al. (1961) using standard endocrine techniques did not find any effect of pharmacologically active doses of chlordiazepoxide on testis and prostate weight in immature rats, ovarian hypertrophy in parabiotic rats, the estrous cycle in mature rats, ovulation in rabbits following mating, thiouracil-induced enlargement of the thyroids in rats, seminal vesicle and prostate weight in castrated rats, uterine weight in ovariectomized rats and progestational activity in estrogen-primed rabbits. These results were essentially confirmed by Superstine and Sulman (1966). These authors, however, demonstrated pituitary depression with guanethidine, reserpine, and phenothiazines (Sulman, 1959). With larger doses of chlordiazepoxide (10-20 mg/rat b.i.d., S.C. for 15 days) Braitenberg and Golob (1964) observed a reduction of the number of vaginal smears showing estrus. This was considercd the result of an inhibition of pituitary hormone secrction due to a depressant action on the hypothalamus. Similarly, a partial reduction of gonadotropin-stimulated superovulation in immature mice was observed with large doses of chlordiazepoxide ( 4 mg per mouse). I n this experiment, reserpine alkaloids and phenothiazines proved to be considerably morc active (Purshottam, 1962). RiZany other drugs, including barbiturates, antiadrenergics, anticholinergics, morphine, and ether, are also known to interfere with the estrous cycle and to prevent ovulation in animals if administered a t sufficiently high doses (Everett, 1961). From these experimental findings it is evident that CNS-depressant drugs can inhibit secretion of pituitary hormones if given a t appropriate doses. Reserpine alkaloids and phenothiazines seem to be considerably more potent than the benzodiazepine analogs tested so far. Correlation of these experimental findings with clinical effects is difficult because minor or major disturbances of the endocrine balance are frequently associated with the mental and emotional diseases for which these drugs are prescribed. Thus, it is often impossible to determine whether changes such as irregularities of menstruation and ovulation are indeed caused by drug therapy. Clinical experience with many psychotropic drugs shows that clearly drug-rclated hormonal disturbances are rare. Whitelaw (1956, 1961) described irregularities of menstruation, amenorrhea, and delayed ovulation aftcr treatment with chlorpromazine as well as chlordiazepoxide. Other investigators (Schwartz and Smith, 1963) did not find any effect of prolonged administration of chlordiazepoxide on menstrual regularity and ovulation. Moreover, normalization of ovulation

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and menstrual abnormalities induced by emotional disturbances occurred under chlordiazepoxide therapy (Moore, 1962). From these observations it can be concluded that hormonal imbalance may be produced as well as normalized by psychotropic agents. Experience with many CNS-depressant drugs shows, however, that under clinical conditions and with therapeutic doses, inhibition of pituitary hormone secretion rarely reaches a degree which could have serious consequences. Psychotropic drugs also have an effect on secretion or activity of the antidiuretic hormone. However, the reports in the literature on this subject are conflicting (Boris and Stevenson, 1966). Recent studies by Boris and Stevenson (1964, 1966) have shown that tranquilizing agents of various chemical structures inhibited urine secretion in water-loaded rats, which indicates antidiuretic activity. The doses needed were considerably higher than those necessary to produce significant behavioral depression. Of the two benzodiazepines tested, chlordiazepoxide was active only a t 64 mg/kg and diazepam was inactive. When experiments were conducted in dehydrated rats, phenothiazines, chlorprothixene, tetrabenazine, dibenzazepine antidepressants, and monoamine oxidase inhibitors blocked partially or completely the antidiuretic effect of dehydration. This could be due to inhibition of secretion of antidiuretic hormone. Chlordiazepoxide, diazepam, meprobamate, and reserpine were inactive. There is no clear clinical correlation which could be rclated to these experimental findings, although it was reported by Parrish and Levine (1956) and Cohen (1957) that chlorpromazine causes diuresis in man and concomitantly a decrease in the urinary excretion of an antidiuretic substance. From these findings it may be concluded that the block of antidiuretic hormone secretion suggested by animal experiments can indeed have clinical consequences if chlorpromazine is administered by the parenteral route. No such effect is to be expected from the benzodiazepines tested so far.

J. DRUG DEPENDENCE Physical and psychic dependence is a possibility to be watched for with all CNS-depressant and -stimulant drugs. Much work has been done to predict addiction liability in man from animal experiments. Thc widest choice of ani,mal tests is available for the determination of physical dependence on morphine-like compounds (Halbach and Eddy, 1963) and an excellent correlation between the effects in animals and direct addiction tests in man has been found. Particularly useful is the experiment in morphine-dependent monkeys in which the capability of a drug to suppress abstinence symptoms is measured (Seevers and Deneau,

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1963 ; WHO Scientific Group on the Evaluation of Dependence-Producing Drugs, 1963). A similar approach of detecting physical dependence on barbiturates and the group of newer hypnotics and antianxiety agents was proposed by the WHO Scientific Group on the Evaluation of Dependence-Producing Drugs (1963). Dogs made tolerant and dependent on 100 mg/kg/day of sodium barbital are used. The test drugs are administered to these dogs after withdrawal of the barbiturate and it is determined whether or not these agents are able to prevent the typical abstinence symptoms. In this experiment, a large number of hypnotics, sedatives, and antianxiety drugs were found to be effective, including several barbiturates, chloral hydrate (500 mg/kg q 12 h) , paraldehyde (1-1.5 ml/kg q 12 h) , chlordiazepoxide (100 mg/kg q 12 h) , carisoprodol (200 mg/kg q 6 h ) , and meprobamate (150 mg/kg q 6 h) (Deneau and Weiss, 1964). Physical dependence on barbiturates develops regularly if patients take larger than the therapeutic amounts for prolonged periods of time. It was suggested therefore that the above-described animal experiment would give an indication as to the ability of a compound to produce barbiturate-like physical dependence. I n the case of carisoprodol, the experiment gave a false positive result, since it was impossible to produce abstinence symptoms in man after 20 days of treatment with several times the therapeutic dose. Chlordiazepoxide also suppressed barbiturate abstinence syndrome in dogs but a dose which is about one hundred times higher than the minimal effcctive dose in dogs was necessary. The induction of physical dependence with benzodiazepines in man has been studied by various investigators. I n an experiment with eleven schizophrenic patients chlordiazepoxide was abruptly withdrawn after therapy with 300-600 mg/kg (eighty to twenty times the usual therapeutic range) for 2-6 months. Depression, aggravation of psychoses, agitation, insomnia, loss of appetite, and nausea appeared 2-8 days after discontinuation of the drug. Major motor seizures were seen in two patients, one of whom had a prior history of a convulsive episode. The authors concluded that these symptoms were probably due to a withdrawal reaction (Hollister et al., 1961). Hollister e t al. (1963) also reported probable withdrawal reactions in patients after abrupt discontinuation of therapy with up to 120 mg/day of diazepam. An additional case of grand ma1 seizure was observed in a mentally retarded child after discontinuation of chlordiazepoxide therapy in the dose range of 40-60 mg (Pilkington, 1961). I n a clinical experiment conducted on twenty-five alcoholic patients, chlordiazepoxide was administered for 14 days a t 50 mg t.i.d. and then withdrawn suddenly. NO withdrawal symptoms occurred (Burke and Anderson, 1962). With-

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drawal reactions were also not seen in five patients who discontinued chlordiazepoxide therapy after the uncontrolled administration of doses from 80-160 mg/day had caused excessive side effects (Lemere, 1960). A patient who took a t least 900 mg of diazepam in a suicidal attempt, however, experienced one grand ma1 attack with generalized convulsions and coma on the sixth day after ingesting the drug (Lingjaerde, 1965). Death occurring after withdrawal of diazepam in a patient with dystonia musculorum progressiva and bizarre lipidosis of the caudate nucleus and putamen was related to excessive hypothalamic discharge due to withdrawal of the drug. This reaction and the inability of the patient to respond normally to fever may have caused hyperthermia, exhaustion, and death (Relkin, 1966). Most investigators studying physical dependence with benzodiazepines conclude that abstinence symptoms, if they occur, are rather mild. The rare incidence and the delayed occurrence of such symptoms may be partially due to the rather slow excretion of these drugs (Hollister et al., 1961, 1963). More important than the development of physical dependence after prolonged administration of excessive doses is the problem of psychic dependence. It is the distinct quality of a drug to cause psychic dependence which will in actual practice determine whether or not a significant number of patients is likely to overuse or abuse a compound and then develop tolerance and physical dependence. Factors which favor development of psychic dependence are rapid onset of action, euphoriant effect, absence of unpleasant neurological or other side effects, and ability of a drug to produce rapidly a state of total oblivion. Animal tests which measure the tendency of a drug to produce psychic dependence are only now being developed. The most promising approach is a device which permits monkeys to self-administer drugs by intravenous injection. The animals can activate an injector device by pressing a lever. Preliminary findings indicate that monkeys will become completely dependent on morphine, barbiturates, and alcohol, and will continue to self-administer these agents until they are completely incapacitated, drunk, or asleep. With chlordiazepoxide, no psychic dependence developed. The animals pushed the injector lever off and on and abstained from taking the drug sometimes for days (Deneau, 1966). Clinical experience so far is in good agreement with these experimental observations. Review of the literature indicates that benzodiazepines have a weak tendency to produce psychic dependence and only a few cases have been reported (Czerwenka-Wenkstetten e t al., 1965a,b; Schremly and Solomon, 1964). Several investigators commented specifically on the lack of psychic dependence with benzodiazepines (Bowes, 1960; Cohen and Harris, 1961; Moore, 1962; Burke and Anderson, 1962;

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Kelley, 1962; Katz et al., 1962; Hoff, 1963; Dorfman, 1964; Burdine, 1964; Friend, 1966). I n the treatment of chronic alcoholics with benzodiazepines, voluntary reduction of dose is frequently observed (Lawrence et al., 1960; Kissen, 1961; Armour, 1963). The rare occurrence of psychic dependence is probably due to the fact that the presently available benzodiazepines have a relatively slow onset of action, do not produce euphoria, and cause ataxia a t higher doses, a side effect which is unpleasant and embarrassing (Kinross-Wright et al., 1960) . Despite these properties, certain individuals may well choose to experiment with benzodiazepines beyond medically advisable intake. Good medical practice, therefore, demands that psychoneurotic patients, chronic alcoholics, and other addiction-prone individuals be properly supervised and informed if treatment with benzodiazepines is deemed necessary (Lingjaerde, 1965). K. DRUGINTERACTIONS I n animals, antagonism to the CNS-depressant effects of large doses of chlordiazepoxide was demonstrated with many analeptics and stimulants, e.g., megimide, amphetamine, caffeine, and pentylenetetrazole (Zbinden et al., 1961 ; Frommel et al., 1963). Sedative and tranquilizing agents generally increased the depressant effect of the benzodiazepines. The degree of potentiation was only slight to moderate with methyprylon, chloral hydrate, glutethimide, and chlorpromazine but generally quite marked with various barbiturates (Frommel et al., 1960, 1961, 1963; Zbinden et al., 1961 ; Dobkin, 1961 ; Taccardi, 1962; Fujimori, 1965). The combination of morphine and chlordiazepoxide did not have any additive effect (Frommel et al., 1964). In humans the interaction of CNS-depressant drugs may become important if tranquilizing agents are used as preanesthetic medication (Elliott, 1962). In a double blind trial designed to evaluate the potentiating effect of diazepam, however, no prolongation of the effects of various anesthetic agents was determined (Tornetta, 1963). Moreover, no effect of chlordiazepoxide and diazepam on the respiratory response to meperidine was noted (Sadove et al., 1965). It is probable that summative effects may occur if benzodiazepines and barbiturates are administered simultaneously a t higher doses (Kane and McCurdy, 1964). Of particular practical importance is the interaction of sedatives and tranquilizing drugs with ethyl alcohol. In experimental animals, high doses of chlordiazepoxide had a moderate effect on the CNS depression induced by alcohol. For example, the 50% hypnotic dose (HD,,) in mice for alcohol was found to be 3640 f45 mg/kg i.p. After pretreatment with 25 mg/kg of chlordiazepoxide i.p., the HD,, of alcohol was 2320 t

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57 mg/kg (Zbinden e t al., 1961). Chlordiazepoxide, 5 mg/kg, administered prior to a nonhypnotic dose of 2 mg/kg of ethanol i.p. induced loss of righting reflex in rats of 46 f 4.3 minutes (Madan e t al., 1962). Ethyl alcohol is a poor hypnotic agent in mice and rats and has to be givcn in enormous doses to produce an observable effect on righting reflex. For the study of drug-alcohol interaction, an experimental procedure which measures a less drastic effect is to be preferred. An interesting attempt to evaluate tranquilizing properties of chlordiazepoxide and alcohol combinations was reported by Hughes e t al. (1963). These authors found that the tranquilizing effect of chlordiazepoxide measured in a conditioned avoidance behavior situation in rats was reversed by a small dose of alcohol. It is noteworthy also that the percentage of trials in which the rats were totally unresponsive and did not react to either warning stimulus or shock was consistently higher in animals which received chlordiazepoxide alone as compared t o those rats which had received drug plus alcohol. A similar trend which may be interpreted as an antagonistic effect was observed in rats subjected to discrete trial trace avoidance procedures (Section II,C,2). I n three of five rats the combined effect of chlordiazepoxide and a small dose of alcohol on noise response failure was less than that of chlordiazepoxide alone (Heise and McConnell, unpublished data). As expected, chlordiazepoxide and diazepam have no effect on the rate of the .metabolic breakdown of ethyl alcohol in rats and dogs (Khan et al., 1964; Seidel and Soehring, 1965). Experimental studies in human volunteers are in good agreement with the observations in animals. As shown in Table XVII, there is no significant potentiation of alcohol effects on mental and motor performance and social behavior if the experimental studies are conducted with therapeutic doses of chlordiazepoxide or diazepam and if moderate amounts of alcohol are used. The situation may be different if large doses of benzodiazepines are ingested, particularly if they are taken together with excessive amounts of alcohol (Smith, 1961 ; Gilbert, 1961). It is interesting to note that in the double blind study of Hughes et al. (1965) chlordiazepoxide and diazepam appeared to antagonize the subjective effects of ethanol. A similar phenomenon was also reported by Goldberg (1965) who claimed that in experimental studies involving driving skills chlordiazepoxide antagonized the subjective and objective impairment of performance due to alcohol. The experimental details of this study are not published as yet. These two observations may be related to the findings in animals in which a trend indicating the possibility of antagonistic effects has been noted. Further studies on drug interaction are necessary to clarify this problem.

TO EXPERIMENTAL STUDIES DESIGNED

TABLE XVII I N V E S T I G A T E ALCOHOL POTEXTIATION BY

Investigator type of study

No. of subjects

Hoffer (1962) Double blind cross-over

6

Miller et al. (1963) Double blind cross-over

8

Lawton and Cahn (1963) Double blind cross-over

20

3 oz. Vodka 100 proof

Hughes et al. (1965) Double blind cross-over

18

45 ml of ethanol per 150 Ibs. body weight

Alcohol, dose 6 oz. Canadian rye whisky within 2 hours 4 oz. Scotch whisky 94 proof

BENZODIAZEPINES I N MAN

Drug, dose

Observation

Chlordiazepoxide 30 rng/day for 1 day Chlordiazepoxide 10 mg q.i.d. for 4 days Diazepam 15 mg/day for 335 days

KOpotentiating effect as reflected in social

Chlordiazepoxide 15 mg/day or Diazepam 6 mg/day for 21.5 h Y S

behavior or subjective experience

No difference between effect of placebo and chlordiazepoxide on social behavior or performance Slight influence on psychomotor performance with diazepam (four psychological tests) with or without alcohol. No evidence for potentiation No additive effects of drugs with alcohol observed in nine mental performance tests. No significant drug-alcohol interaction in attentive motor performance. I n one test pattern a synergistic effect of diazepam with alcohol

N

W

2 Z

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From these experimental findings it can be seen that drug-alcohol interaction experiments may lead t o different conclusions, depending on the dose used. With large doses of drug and alcohol, marked sedation and hypnosis result. With small amounts, there is no additive or potentiating effect. Under special circumstances, the depressant effect of alcohol may be antagonized by the benzodiazepines. V. Metabolism

Detailed metabolic studies have been conducted only with a limited number of benzodiazepine derivatives. But even these few examples demonstrate that the biotransformation of this class of drugs does not follow a uniform pattern. Chlordiazepoxide is demethylated to a metabolite identified as Ro 5-0883, deaminated to the “lactam” Ro 5-2092, and finally converted to the “open lactam” (Fig. 11) (Koechlin e t al., 1965). The latter compound, which is pharmacologically inert (Randall e t al., 1965a), is excreted in the urine as such or in the form of alkali-labile conjugates. Repeated administration of 20 mg of chlordiazepoxide b.i.d. for 14 days to adult subjects produced serum levels of about 2 pg/ml of chlordiazepoxide, and 1 pg/ml of the demethylated metabolite Ro 5-0883, and 1 pg/ml of the “lactam” Ro 5-2092. I n man the half-life of chlordiazepoxide in plasma is 22-24 hours, in dogs 10-14 hours. The metabolism in dogs is similar to that in man. I n rats chlordiazepoxide disappears rapidly from the blood (half-life 4-6 hours); the major metabolite detected in the urine is a basic derivative of unknown structure, absent from the urine of man and dog. Gastric secretion of the metabolites and hepatic secretion of the intact drug were demonstrated in rats (Koechlin et al., 1965). While ring opening is the characteristic pathway of disposition for chlordiazepoxide in man, no such process appears to occur with diazepam. The metabolites observed after administration of 3H-labeled diazepam in man are shown in Fig. 11. Peak levels were reached a t 2-4 hours. The fall-off curves showed a fast component with a half-life of 7-10 hours and a slow component with a half-life of 2-6 days. During the rapid phase of metabolism, diazepam was converted to the N-demethylated product Ro 5-2180. The latter product is slowly metabolized over several days and appears in the urine in the form of conjugated metabolites (Schwartz et al., 1965). Repeated daily doses of 30 mg of diazepam caused a progressive increase of diazepam levels in plasma. The metabolite Ro 5-2180 appeared 24-36 hours after the first dose and thereafter the levels increased rapidly, approximating those of diazepam. There was no accumulation of oxazepam in the blood. Upon discontinuation of the drug, Ro 5-2180 persisted in the blood longer than diazepam (deSilva

280

GERHARD ZBINDEN AND LOWELL 0. RANDALL H N-CH, a N = c > C H z c1

/lor demethylation

d

i

gtion

NH, COOH

I

C=N

Deniethylated metabolite Ro 5-0883

demeth ylation

"Lactam" Ro 2-2092

Diazepam

"Opened lactam"

h ydroxylation

H N-CO

CI

Oxazepam

Glucuronide

FIG.11. Metabolic pathways of chlordiaeepoxide and diazepam in man.

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et al., 1966). I n dogs, diazepam is excreted rapidly (half-life 2 hours).

The same metabolite (Ro 5-2180) as in man is found in the blood. The major urinary metabolite was identified as conjugated oxazepam. Metabolism in the rat was found to be different from that in man and dogs. The urine also contained conjugated polar metabolites but their structure is not known (Schwartz et al., 1965). Metabolic aIterations of nitrazepam also occur without ring opening. The compound is converted to the 7-amino and 7-acetamino metabolite (Rieder, 1965; Randall et al., 196513). The half-life in man is 7-10 hours (Rieder, 1965). Nitrazepam is excreted in the urine mostly in form of the two above-mentioned metabolites. After single oral doses of oxazepam to human subjects, the drug persists in the plasma for a t least 48 hours. Oxazepam is excreted principally in the urine in form of the glucuronide. The feces contain a small amount of unchanged drug. Similarly in the dog and pig, two-thirds of the dose of radioactive oxazepam appears in the urine as glucuronide and one-third in the feces. After intramuscular injection in rats, twothirds of the dose appears in the feces and one-fifth in the urine in the form of a t least seven unidentified metabolites (Walkenstein et al., 1964). From this summary it is clear that the benzodiazepines investigated so far are metabolized similarly in man and dogs. Excretion occurs mainly through the kidney, but gastric and hepatic secretion with excretion in the feces is also found. The metabolism in rats seems to differ considerably from the biotransformation in man and dogs. Tissue distribution was studied in rats with chlordiazepoxide and diazepam. The lowest tissue levels were found with both agents in the brain and by far the highest in the liver. A high concentration of diazepam was found in the adipose tissue, whereas low levels were detected in the perirenal fat with chlordiazepoxide (Koechlin et al., 1965; Schwartz et al., 1965). VI. Toxicology

The most frequent side effects observed with benzodiazepines in man are directly related to the CNS-depressant and muscle-relaxant properties and consist of oversedation, drowsiness, and mild ataxia (Hollister, 1961 ; see Section IV,C). If large doses are ingested or if high therapeutic doses are given to old patients, these symptoms may become quite severe. Thirty-eight+ cases of suicidal attempts with chlordiazepoxide and 19+ cases with diazepam are described in the medical literature (Lemere, 1960; Hines, 1960; Kinross-Wright et al., 1960; Nobili and Cerquetelli, 1960; Clarke et al., 1961; Jenner and Parkin, 1961; Yennington and Synge, 1961; Thompson and Glen, 1961; Smith, 1961; Zbinden et al., 1961 ; Stanfield, 1961 ; Galeano-Munoz and Bedo, 1962; Krauthammer,

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1963; Singh and Gupta, 1963; Hare, 1963; Collard and Kerf, 1963; Ehlers, 1963; Bravo and Jensen, 1964; Juul, 1964; Voellmy, 1964; Maculans, 1964; Angst, 1965; Loew, 1965; Czerwenka-Wenkstetten et ul., 196513; Cleckley, 1965; Harder, 1965; Spark and Goldman, 1965; Herzka and Haber, 1965; Retterstol, 1965; Lingjaerde, 1965; Hillyer, 1965). Doses up to 2250 mg of chlordiazepoxide (Zbinden et al., 1961) and 900950 mg of diazepam (Lingjaerde, 1965) were taken either alone or in combination with other CNS-depressant drugs or alcohol. Symptoms were mild to moderate in 'most cases and consisted of ataxia, drowsiness, dysarthria, and sleep. Coma with hypotension was occasionally observed, particularly when other CNS depressants or alcohol were ingested simultaneously. Neurological symptoms such as hyporeflexia, pathological reflexes, and spasticity were rarely seen. The pupils were normal in most patients and pupillary reaction to light was sluggish only in a few. Recovery was uneventful and without sequelae in all cases. One fatal case of chlordiazepoxide overdosage is mentioned in a survey paper by McBay (1966). The drug was found in the intestine and a plasma level of 19 pg/ml was determined. No other details about this patient are given. Information obtained from suicidal cases indioates that acute toxicity of chlordiazepoxide and diazepam in man is low. The compounds have weak depressant effects on the autonomic nervous system. Therefore, cardiovascular functions and respiration are generally not markedly impaired even after large doses have been taken. There are no acute hepatotoxic or nephrotoxic effects. The acute poisoning with these substances does not depress the bone marrow and rarely causes gastrointestinal upset. I n chronic toxicity studies in rats, dogs, monkeys, and chickens (Randall et al., 1960; Zbinden et al., 1961; Randall et al., 1961) chlordiazepoxide and diazepam did not exhibit specific organotoxic properties. I n clinical practice a few instances of drug allergy or idiosyncrasy have been noted, e.g., sporadic cases of agranulocytosis (Kaelbling and Conrad, 1960; Wilcox, 1962), thrombocytopenia (Heyssel, 1961), an acute asthmatic attack (O'Grady and Pokorny, 1964), facial edema (LoefflerSchnebli, 1961), fixed drug eruption (Gaul, 1961), and hepatic dysfunction (Caccioppo and Merlis, 1961; Bloom et ul., 1965). The occurrence of such toxic reactions, however, has been rare. It should be noted that there is no typical pattern for the metabolic detoxification of various benzodiazepine derivatives studied so far (see Section V). One can therefore not expect that the toxic properties of all derivatives of the benzodiazepine class would be similar to the ones described for chlordiazepoxide and diazepam. Any new derivative to be introduced in human therapy, therefore, must be investigated in animals and its characteristics

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with regard to neurotoxicity, tendency for sensitivity reactions, and specific organotoxic effects should be established in detailed human studies. VII. Conclusions

I n the past 8 years following the introduction of the benzodiazepines in human therapy, a considerable body of scientific information has been accumulated in the field of therapeutic application and basic pharmacologic properties of this new class of psychotropic drugs. This review represents a first attempt to correlate on a broad basis the knowledge obtained in animal experiments with the clinical experience in man. It is clear that in the atmosphere of exploration and discovery which is always created by a new class of drugs, clinicians and basic scientists often tend to pursue their respective research goals without too much concern about the results of other disciplines. There comes the time, however, when stock has to be taken, other views have to be acknowledged, and discrepancies have to be reconciled. Drug effects on conditioned animal behavior, for example, are one of the areas where basic scientists have gained an enormous amount of data, but have not been able to correlate many of these results with clinically observed drug actions in man. The new and very interesting findings on the maintenance of muscle tone through an interplay of the alpha and gamma motoneurons have greatly advanced our knowledge about motor reflexes and experimental induction of spasticity of isolated muscles in cats. This knowledge has not yet been applied fully to the spastic patient who suffers not only from upper motoneuron lesion but also has muscular atrophy, osteoarthrosis of the joints, decubital ulcers, and a severe emotional problem which reinforces all other causes of spasticity. The short history of benzodiazepines has proved that progress can be achieved if the clinician uses techniques and concepts elaborated in basic studies and if the experimental biologist is prepared to derive guidance and perspective from the observation of drug effects in patients. I n dealing with drugs which act on emotions and mind, it will always be difficult, and often impossible, to demonstrate a clinically recognized therapeutic action in an animal experiment, but experimental procedures can be made more specific and more meaningful if one tries to measure selectively essential steps in a pharmacologic drug action rather than an overall effect on gross animal behavior. This review has shown that meaningful results can be achieved if the animals are brought into a state of measured anxiety, if the recordings are made with sensitive equipment placed right a t the centers of preferential drug action, if seizures are induced in brain centers known to be important for human

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epilepsy, and if muscle-relaxant effects are not only measured on normal reflexes but in animals with a type of muscle spasticity which resembles the musculoskeletal spasticity following upper motoneuron lesion in man. By learning as much as possible about the site and mode of action of psychotropic drugs, their distribution in tissues, and their effects on the electric and chemical activity of nerve cells, we are perhaps coming closer to a n understanding of certain pathological processes which are the cause of those mental, emotional, and neurological disturbances which are temporarily alleviated or eliminated by these chemicals. The studies with benzodiazepines have shown that their most specific action becomes evident if anxiety and excitation is produced by central release of norepinephrine in animals which arc unable to rapidly metabolize the free neurohormone. Is the antianxiety effect of the benzodiazepines therefore a central antiadrenergic phenomenon? And if so, what is the role of brain norepinephrine in the chain of events which leads to psychoneurotic fear and hysteria? These and many other questions must be asked and answered if the experimental and clinical investigations with benzodiazepines and other psychotropic drugs are to lead to a better understanding of mental and emotional diseases and help to find more specific and better agents t o combat them. ACKNOWLEDGMENTS The authors gratefully acknowledge the contributions of W. B. Schallek, C. L. Schcckcl, R. Banzigw, W. B. Abrams and their rollaborators. They also thank Mrs. E. Hollis, Miss J. Klukowicz, Miss E. Rolleri, Mrs. M. Jones, and Miss C. Imperato for secretarial help.

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