Biological Aspects of Depression: A Review of The Etiology and Mechanisms of Action and Clinical Assessment of Antidepressants

BIOLOGICAL ASPECTS OF DEPRESSION: A REVIEW OF THE ETIOLOGY AND MECHANISMS OF ACTION AND CLINICAL ASSESSMENT OF ANTIDEPRESSANTS By S. 1. Ankier Charterhouse Clinical Research Unit Limited London EClM 6HR, England

and B. E. Leonard Phormacology Department University College Galway, Republic of Ireland

1. Introduction

Depression is an illness which, in the United States alone, affects some 400,000 patients and has been rated as the tenth major cause of death (Hollister, 1978). For over two decades, it has been widely assumed that depressive illness is associated with an abnormalityin brain noradrenaline and/or serotonin metabolism (Schildkraut, 1965;Bunney and Davis, 1965). Since the initial hypothesis was proposed, numerous studies have been conducted on body fluids of depressed patients and on postmortem brain material from suicides, attempting to validate the hypothesis and determine the precise mechanism whereby the abnormality in central biogenic amine metabolism occurs. Studies of the changes in the major metabolites of brain noradrenaline (3-methoxy-4-hydroxyphenylg1yco1, MHPG), serotonin (5-hydroxyindole acetic acid, 5-HIAA), and dopamine (homovanillic acid, HVA) have helped only partially to validate the hypothesis. Thus it has been reported that a reduction in the cerebrospinal fluid (CSF) concentration of MHPG before treatment is associated with a favorable response to tricyclic antidepressants, whereas a slightly elevated CSF concentration of this noradrenaline metabolite is associated with a poor response to such drugs (Maas et al., 1972; Beckmann and Goodwin, 1975; Hollister et al., 1980). However, not all investigators could replicate these findings (Coppen et al., 1979; Maas et al., 1982). Despite the recent study by Maas et al. (1984) in which it was shown that a low urinary excretion of MHPG was associated with a greater response of patients INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 28

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with bipolar affective disorder to tricyclic antidepressants, the consensus of opinion casts doubt on the usefulness of neurotransmitter metabolite studies in predicting the response of depressed patients to different types of antidepressants. This has been reviewed elsewhere (Leonard, 1982). A major change in research emphasis has occurred during the last decade following the detailed studies of Sulser and colleagues, who established that the delay in the onset of response to antidepressants, or electroconvulsive therapy (ECT), was correlated with a decrease in the functional responsiveness of the postsynaptic P-adrenoreceptors (Vetulani et al., 1976; Sulser, 1978). The discovery of P-adrenoreceptors on the human lymphocyte membrane led F’andey et al. (1979) to study changes in the activity of this receptor system in depressed patients; these investigators found that the responsiveness of the P-adrenoreceptors was decreased in the depressed patient. Other investigators have, however, shown that the numbers of f3-adrenoreceptors are increased in the untreated patient and return to control values following effective treatment (Healy et al., 1983). Clearly there is a significant divergence between these findings, and while the results of the study by Healy et al. (1983) would support the concept of P-adrenoreceptor desensitization (“down-regulation”) following effective treatment which has been shown to occur in the frontal cortex of the rat brain after chronic antidepressant administration (Sulser, 1978), the significanceof these findings to our understanding of the etiology of depression is still uncertain. A somewhat similar situation has arisen when attempts have been made to evaluate changes in ap-adrenoreceptors on the platelet membrane as a possible marker of noradrenaline autoreceptors. Thus various groups of investigators have shown that the density of these receptors is either reduced (Wood and Coppen, 1981), unchanged (Daiguji et al., 1981), or increased (GarciaSevilla et al., 1981; Healy et al., 1983) in the untreated-depressed patient. In contrast to these disparate results obtained from studies on depressed patients, the effects of chronic antidepressant treatments on the density of as-adrenoreceptors in rodent tissues have been more consistent and have shown that chronic drug treatment is associated with a decreased density of a*-adrenoreceptors in brain and other tissues innervated by the sympathetic system (Crews et al., 1978a,b, 1981). As it is uncertain whether the ap-adrenoreceptor on the platelet membrane is similar to the pre- or postsynaptic as-adrenoreceptor in the brain or even that the presynaptic a2-adrenoreceptorsfunction as autoreceptors on central noradrenergic terminals (Laduron, 1984), the relevance of the clinical findings to the etiology of depression remains an open question. Despite the well-established evidence that many clinically effective antidepressants reduce the reuptake of [’Hlserotonin into synaptosomes from rat cortex following acute administration and have a similar effect on the uptake of serotonin into platelets of nondepressed subjects, their

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effects on platelet serotonin uptake of depressed patients is qualitatively different. Thus several groups of investigators have shown that the platelet serotonin uptake in depressed patients is reduced and increases following effective treatment (Coppen et al., 1978; Tuomisto et al., 1979; Born et al., 1980; Healy et al., 1983). It is interesting that this increase in serotonin transport into the platelet occurs irrespective of the acute effect of the antidepressant on serotonin transport into rat cortical synaptosomes, which suggests that there may be a correlation between altered serotonin transport and the symptoms of depression. There is also evidence that the activity of the serotonin 2 (5-HT2) receptor on the platelet membrane is reduced in the untreated depressed patient, but returns to control values following effective treatment (Healy et al., 1983).This finding is supported by a number of experimental studies in which it has been shown that the postsynaptic response to ionophoreticallyapplied serotonin in most limbic areas of the rat brain was enhanced by chronically administered antidepressants (De Montigny and Aghajanian, 1978; De Montigny et al., 1981; Wang and Aghajanian, 1980). The acute and chronic effects of antidepressants on central serotoninergicfunction has been extensively reviewed by Willner (1985). The purpose of this review is to assess critically the evidence supporting the amine hypothesis of depression in the light of the recent studies on amine metabolites and receptor function and thereby to define more precisely the etiological basis of the illness. In addition, the possible role of other neurotransmitters and neuromodulators will be examined. However, such an approach would be incomplete unless an attempt were also made to define the mode of action of the various classes of antidepressants in current use and to examine the methods used to assess their efficacy in clinical trials. No attempt will be made to assess the limitations of the various animal models of depression, which are being used for the initial identification of putative antidepressants, as this has been the subject of several critical reviews elsewhere (Leonard and Tuite, 1981;Jancsar and Leonard, 1983; Cairncross et al., 1979; Vergnes and Karli, 1963; Porsolt et al., 1978).

II. Biochemical Changes in Depression

A. POSTMORTEM MATERIAL

While the precise relationship between suicide and depression is unclear (Goldberg and Huxley, 1980), it is widely accepted that primary or secondary depression is a major contributory cause for suicide. It is not unreasonable to predict, therefore, that direct evidence implicating the

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involvement of biogenic amines in the etiology of depression should come from postmortem studies. Results from studies conducted prior to 1976 showed that the concentration of serotonin andlor 5-HIAA in the brain stem or other regions innervated by the serotonergic system was reduced (Shaw et al., 1967; Bourne et al., 1968; Pare et al., 1969; Lloyd et al., 1974; Birkmayer and Riederer, 1975), whereas studies by Beskow et al. (1976) and Cochran et al. (1976), which had been carried out on more discrete regions, showed no change in either serotonin or its metabolite. There is no consistent evidence from postmortem studies that either noradrenaline or dopamine concentrations are affected. Of the more recent studies of postmortem brain, the study of Meyerson et al. (1982) is of particular interest. These investigators compared the brains of suicides with those obtained from victims of homicide in the same region of the United States. Using standard receptor ligand techniques to determine receptor numbers in different regions of the brain, they showed that the densities of muscarinic receptors and [3H]imipramine-bindingsites were increased in the cortex of the suicide victims. It was of interest that the density of the P-adrenoreceptors, which are thought to play a crucial role in depression, were unchanged. Interpretation of these results is complicated by the fact that a substantial minority of suicide victims are not endogenous depressives (Goldberg and Huxley, 1980) in addition to uncertainty of the relationship between receptor number determined by ligand-binding studies and the functional status of the various receptors. This is discussed elsewhere. The major difficulties arising from studies of the brains of suicides are due to ( 1 ) the difficulty in assessing the effect of postmortem change on the metabolism of the neurotransmitters, (2) the presence of drugs that may affect the metabolism of the neurotransmitters being investigated, and (3) the precise diagnostic classification of the patients at the time of death. It is difficult to see how such factors may be controlled satisfactorily. Until this is achieved, the relevance of results obtained from postmortem findings to the etiology of depression must be treated with caution. In a recent review, Rossor (1984) has critically examined the problems relating to postmortem brain studies.

B. AMINEMETABOLITESIN BODYFLUIDS Despite the inconclusive evidence from studies on brains from suicides,

it might be anticipated that an analysis of neurotransmitters and their

metabolites in the CSF and urine of depressed patients would provide a direct assessment of the relationship between the symptoms and neurotransmitter status.

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Differences in the concentration of 5-HIAA in the CSF of depressed patients compared with nondepressed controls have been replicated by several groups of investigators. Thus Sjostrom and Roos (1972) found that both 5-HIAA and the major dopamine metabolite, HVA, were reduced in the CSF of depressed patients. Later Asberg et al. (1976) reported that not all depressed patients had a reduced CSF 5-HIAA concentration, and they postulated that a subgroup of patients exists whose symptoms are primarily attributable to a reduction in the concentration of brain serotonin. The introduction of probenecid to block the efflux of acid metabolites from the CSF enabled investigators to assess the turnover of at least some of the biogenic amines in the patients’brain. Using such a technique, several groups of investigators have shown that the rate of accumulation of 5-HIAA was significantly less in depressed patients than in the control group (Bowers, 19’72; Goodwin and Post, 1973; Van Praag et al., 1973). Most studies also reported that the accumulation of HVA was reduced, which suggested that the turnover of dopamine was diminished in some depressives. The probenecid technique cannot be used to assess central noradrenaline turnover, as the efflux of the principal CNS metabolite, MHPG, is not impeded by this drug. Studies of central noradrenergic function in depression have, therefore, been largely restricted to an analysis of MHPG in the urine. Maas et al. (1973) examined the urinary concentration of MHPG in a heterogeneous group of depressive patients and found that there was a subgroup of patients with subnormal excretion of the metabolite. These investigators later concluded that the reduced MHPG excretion occurs in patients with primary affective disorders (Maas et al., 1984). Several questions arise when one attempts to evaluate the MHPG studies. First, the lifestyle of the patient (e.g., exercise, changed diet, and circadian rhythm) may play a determining role in changing the CSF or urinary MHPG concentration. Second, it is assumed that MHPG is derived primarily from noradrenergic nerve terminals in the brain so that the concentration of this metabolite in the CSF reflects central noradrenergic activity In an attempt to answer the first question, Sweeney et al. (1978) showed that in female depressives there were no significant effects of physical activity on urinary MHPG levels. These investigators did show, however, that a relationship existed between changes in the concentration of MHPG in the urine and the degree of anxiety, which might suggest that those patients with lower baseline MHPG levels were those who were more prone to anxiety under stressful conditions. Unfortunately, no nondepressed controls were included in this study, and as the urinary excretion of this metabolite can vary fourfold in normals and is subject to a diurnal rhythm (Hollister et al., 1978), great caution must be exercised in extrapolating from MHPG excretion data.

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Furthermore, these investigators showed that in normal subjects the individual excretion pattern varied considerably and was uncorrelated with diet, physical activity,or the prevailing affective state (Hollister et al., 1978). As regards the source of CSF MHPG, there is evidence from animal studies that this metabolite arises, at least in part, from noradrenaline contained in nerve terminals adjacent to the cerebral ventricles and that a considerable portion of this metabolite is dependent on the activity of the neurons comprising the locus coeruleus (Ader et al., 1979). In man, however, while MHPG in the lumbar CSF is mainly of central origin, there is also a considerable contribution from the spinal cord (Chase et al., 1973; Ziegler et al., 1977),which further emphasizes the need to exercise caution in drawing conclusions regarding the pathogenesis of affective disorders from such studies. Several investigators, already referred to above, have shown that the concentration of CSF 5-HIAA in depressed patients shows considerable variation. There is also ample evidence to show that a wide variation occurs in the urinary excretion of MHPG. These variations in the concentrations of amine metabolites occur despite the apparent homogeneity of the patient population as assessed by standard-rating scales. Thus Sacchetti et a f .(1979)reported a wide variability in the concentration of this metabolite in the urine of 25 primary depressed patients, and they suggested that the age of onset of the disease may be one factor accounting for the variability; a positive correlation was found between motor retardation and low MHPG excretion. These investigators also reported that depressed patients with normal or elevated MHPG concentrations tended to respond to clomipramine and amitriptyline, which suggests that the ability of such drugs to reduce serotonin reuptake may be related to a primary defect occumng in serotonin rather than noradrenaline metabolism. However, it must be stressed that no control group was used in this study. In contrast, nondepressed controls were used in a study of depression in female patients by Maas and colleagues (De Leon-Jones et al., 1975; Maas et al., 1973), who reported that the MHPG excretion was reduced during depression. However, neither Goodwin and Post (1973) nor Schildkraut el al. (1978) found any change in the excretion of this metabolite in unipolar depressives. While such studies may be indicative of a tendency toward defective brain noradrenaline function in depression in a subgroup of patients, the results are by no means unequivocal. The range of values quoted [from 910 & 99 pg/24 hr by Sacchetti et al. (1979) to 1950 +- 177 fig/24 hr by Schildkraut et al. (1978)l are well within the limits found by Hollister et al. (1978) for normal subjects (900-3500 pg/24 hr), and it is noticeable that several of the clinical studies reporting changes in MHPG excretion do not contain adequate control data. Furthermore, there is evidence that prolonged stress can significantlyelevate the MHPG excre-

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tion and decrease P-adrenoreceptor sensitivity even in normal controls (Mackenzie et al., 1980). Maas et al. f1984), in a detailed study of 104 patients with affective disorder, determined the CSF concentrationsof MHPG, HIAA, and HVA before and following treatment with amitriptyline or imipramine in an attempt to determine whether changes in the pretreatment concentrations of any of these metabolites could predict subsequent therapeutic response. The results of this study showed that a reduction in the MHPG concentration was associated with a better response to drug therapy in the bipolar group of patients only, whereas a slightly raised concentration of this metabolite predicted a poor response. Studies on unipolar patients revealed that a reduction in the HIAA concentration was related to the subsequent response to drug treatment, which supports the hypothesis that there are at least two biochemical subtypes of affective disorders, one type being associated with a subnormal noradrenergic system, while the other reflects an abnormality in serotonergic transmission (Maas, 1975; Asberg et al., 1976). While such findings support the view that irregularities in amine function may underlie different types of affective disorders, other investigators have produced evidence which conflictswith this view. Thus Montgomery ( 1982) studied the antidepressant response of the “specific”noradrenaline and serotonin uptake inhibitors maprotiline and zimelidine, respectively, on subgroups of endogenously depressed patients who had reduced or normal basal CSF HIAA concentrations. No correlation could be found between the clinical response of the patients to either antidepressant and the pretreatment HIAA values. Thus patients with initially a low CSF HIAA (“serotonin deficient”) responded equally well to either drug as did those with initially normal CSF HIAA concentrations. Veith et al. (1983) have also failed to demonstrate differences in response to antidepressant treatments based on differences in pretreatment MHPG concentrations. These investigators showed that a reduction in the pretreatment urinary MHPG concentrations did not predict those patients who subsequentlyresponded to desipramine (which shows some selectivity in reducing noradrenaline reuptake) from those respoqding to amitriptyline, a drug which impedes the reuptake of both noradrenaline and serotonin. Mendlewicz and co-workers (1982) in their study of the comparison between mianserin and amitriptyline on monoamine metabolites in the CSF of depressed patients also failed to find a relationship between therapeutic response to treatment with either drug and the pretreatment CSF metabolite concentrations. These studies lead one to conclude that the concentrations of monoamine metabolites in body fluids is unlikely to be of value as markers of the depressed state or of response to drug treatment. Nevertheless, it is

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not without interest that changes in the plasma noradrenaline concentrations have been shown to reflect the mood of the patient (Wyatt et al., 1971; Shimizu and Fujita, 1981; Luck et al., 1983). If it is assumed that the concentration of plasma noradrenaline reflects the activity of both central and peripheral sympathetic systems, then the determination of changes in plasma noradrenaline concentrations, coupled with an assessment of peripheral receptor function, might provide a useful link between neurotransmitter status and the mood of the patient.

C. PERIPHERAL AMINE RECEPTORS The results of experimental studies on the chronic effects of antidepressant drugs on postsynaptic adrenoreceptors have led to a greater insight into the adaptive changes that occur following drug administration and have also enriched the methods that have been developed for the selection of new putative antidepressants (Olpe, 1982). However, has such an approach led to a greater understanding of the state-dependent biochemical changes that might occur in the depressed patient? If antidepressants decrease the functional activity of the adrenoreceptor-linked cyclase system, then it may be postulated that this receptor-enzyme complex is hyperactive in the untreated patient. Furthermore, as the activity of the postsynaptic receptor system reflects changes in the concentration of neurotransmitter in the synaptic cleft, it must also be assumed that the mechanisms governing the release of the neurotransmitter are abnormal in the depressed patient. As the release of noradrenaline from the nerve terminal is regulated, at least partially, by the presynaptic a-adrenoreceptors (Stiirke, 1977), it seems likely that the activity of this receptor system is also abnormal. While there is broad agreement that chronic antidepressant treatment attenuates postsynaptic adrenoreceptor activity in the limbic cortex of the rat brain, presumably as a consequence of a decrease in the activity of the inhibitory a*-synapticadrenoreceptors which are located presynaptically, studies on changes in monoamine receptor activities on the lymphocyte and platelet membrane are equivocal. Thus different groups of investigators have found that the density of a*-adrenoreceptors (which are presumed to be similar to the presynaptic a-adrenoreceptors on the neuronal membrane) is unchanged, decreased, or increased on the platelet membrane of untreated depressed patients (Healy et al., 1983, for review of literature). Garcia-Sevilla and colleagues (1981), who showed that the apadrenoreceptor density was increased in untreated depressives, also showed that the density of these receptors returned to control value following effective antidepressant treatment therapy, suggesting that the

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change in a2-adrenoreceptoractivity was a state-dependent process. These findings have been largely replicated in a more extensive study by Healy et al. (1983). Studies on the changes in P-adrenoreceptor activity on lymphocytes of depressives have also yielded equivocal results with F‘andey and colleagues (1979), showing a diminished response of the receptorlinked isoprenaline-stimulated cyclase in untreated patients, which presumably reflects a diminished P-adrenoreceptor activity, whereas Healy et al. (1983) have shown an increased (3-adrenoreceptor density, which may indicate an increased receptor activity. Undoubtedly, one of the main difficulties in drawing any firm conclusions from such conflicting findings lies in the heterogeneous population of patients used (some studying manic-depressives during the depressive phase of the illness, whereas other studied postpartum or endogenously depressed patients), the differences in the techniques used to assess the density of the adrenoreceptors, and the possible effects of circadian fluctuation on changes in the receptor sensitivity. Another possible explanation for the variation in the results obtained by different groups of investigators for the changes in the density of presynaptic a-adrenoreceptors in the depressed patient before and following therapy could be associated with a variation in the nutritional status during the different phases of the study. It is well known that anorexia and a loss in body weight are frequent symptoms of the disease; increased appetite is generally taken to be a sign of clinical improvement and many of the conventional (“tricyclic”)antidepressants increase body weight as a consequence of the changes in the intermediary metabolism of carbohydrates which they induce. There is clinical evidence that an inverse relationship exists between platelet a2-adrenoreceptor density and the plasma catecholamine concentrations (Davies et al., 1982); fasting has also been shown to induce a fall in the concentration of plasma noradrenaline (Jung et al., 1979). More recently, Luck et al. (1983) have shown that the plasma noradrenaline concentrations in a group of malnourished patients with anorexia nervosa were significantly lower than in age- and sex-matched controls; the decrease in plasma noradrenaline was associated with a rise in the platelet cy2-adrenoreceptordensity. From this study, it may be concluded that the nutritional status of the depressed patient must be critically assessed and taken into account when attempts are made to correlate changes in platelet a2-adrenoreceptor density with the psychiatric status of the depressed patient. In the studies of changes in platelet adrenoreceptor activity in the depressed patient already referred to, such factors as the loss in body weight and nutritional status of the patient before and following therapy have largely been ignored. One of the major problems which arises when attempts are made to interpret the changes in tritiated ligand binding to the platelet or lym-

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phocyte membrane concerns the relevance of such ligand-binding data to the functional role of the receptor. Apart from the study by Kafka et al. (1981), none of the platelet a2-adrenoreceptor-binding studies concurrently measured the response mediated by the receptor. 1x2-Adrenoreceptors, by modulating the release of noradrenaline, indirectly inhibit cyclic adenosine monophosphate (CAMP)synthesis at the postsynaptic receptor site, while prostaglandin El (PGEI) has the opposite effect. Thus the inhibition by noradrenaline of PGEl-stimulated cAMP synthesis can be used as an index of responsiveness of the ap-adrenoreceptor (Kafka et al., 1977). While two earlier studies reported no difference in the inhibition by noradrenaline of PGEl-stimulated cAMP synthesis, Murphy et al. ( 1974), Wang et al. (1974), and Sever et al. (1984) have recently shown that, in a group of 23 selected depressives, although the ol2-adrenoreceptor density (assessed by [SH]dihydroergocriptinebinding) was increased before treatment, the PGEl-stimulated cAMP response and its inhibition by noradrenaline were significantly reduced compared to the controls. Thus there was an apparent dissociation between the 1x2-adrenoreceptor-binding data (suggesting increased numbers of a*-adrenoreceptors) and the functional change which suggested hyposensitivity of the receptors. These changes were not correlated with the plasma noradrenaline concentrations which did not differ significantly from the controls. The results of such studies emphasize the need for caution when extrapolating from ligand-binding sites (which reflect binding sites which may not be associated with physiologically active receptor sites) to physiologically responsive receptor sites. Whether changes in platelet membrane receptors are a true reflection of those occurring in the brain remains an open question, but detailed studies by Checkley (1980) on the neuroendocrine responsiveness to different adrenoreceptor agonists in depressed patients suggest that central adrenoreceptors are functionally subnormal in the untreated patient. This implies that the changes in a2-responsivenessin the platelet may be a reflection of similar changes in the brain. Whether these changes reflect the density and activity of pre- or postsynaptic a2-adrenoreceptors is unknown.

D. ARE RECEPTORSON BLOOD CELLSUSEFULMARKERSOF CNS ADRENORECEPTORS?

In addition to the difficulties of interpreting changes in adrenoreceptor density on platelets and lymphocytes, which have already been mentioned, recent experimental evidence has thrown doubt on the existence of autoreceptors in the mammalian central nervous system. This chal-

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lenges the theoretical basis of the receptor adaptation hypothesis of depression and the mode of action of antidepressants. Experimental evidence suggests that most presynaptic receptors are not autoreceptors (i.e., receptors which are sensitive to the transmitter originating from the nerve terminal on which it is located), but are receptors which respond to a different neurotransmitter released from an adjacent neuron. Evidence for the existence of a2-adrenergic autoreceptors is largely indirect and derived from experiments on stimulation-evoked release of exogenous neurotransmitter; for example, determining the tritium release following the electrical stimulation of brain tissue which had been preloaded with [’H]noradrenaline (Langer, 1981). However, as Laduron (1984) has concluded in his critical review of the evidence for the existence of adrenergic and dopaminergic autoreceptors, there is no evidence from such studies that the released tritium originates from the same intraneuronal compartment as the endogenous transmitter. Furthermore, it has been shown that tritiated ligands, most of which are basic compounds, are trapped via different intracellular compartments of intact cells which have a slightly acidic pH (Maloteaux et al., 1983). An additional complication arises with the possibility that a number of peptide cotransmitters coexist with the “classical”neurotransmitters, such as noradrenaline, and it is not unreasonable to assume that some of these cotransmitters may modulate noradrenaline release independently of any presumed action of noradrenaline on its autoreceptor. Furthermore, in vitro binding studies have failed to locate clonidine-binding sites at presynaptic sites (Tanaka and Starke, 1979), which suggests that the drug, which has been widely used as a marker for the cr2-adrenoreceptors, has failed to demonstrate the presence of such receptors on the presynaptic noradrenergic neuron. From such experimental studies, it may be concluded that changes in the density of a*-adrenoreceptors on the platelet membrane of the depressed patient before and during treatment do not necessarily represent presynaptic changes. It therefore seems more likely that change in a2adrenoreceptor activity is an epiphenomenon unrelated to the etiology of the illness. Despite well-established experimental evidence showing that all clinically effective antidepressants, following chronic administration, reduce the functional activity of postsynaptic P-adrenoreceptors in the frontal cortex of rat brain (Sulser, 1983),the search for suitable models of the padrenoreceptor complex in the peripheral tissues of depressed patients, which may be used as an indication of central receptor function, has met with limited success. It is well established that platelets (Steer and Atlas, 1982; Kerry and Scrutton, 1983) and lymphocytes (Williams et al., 1979) have type 2 P-adrenoreceptors on their membranes. So far, studies on changes in P-adrenoreceptor density in depression

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have been restricted to those receptors found on the lymphocyte membrane. However, such studies have not taken into account the nonuniformity of the lymphocyte population. Thus the numbers of B- and T-type lymphocytes vary independently of one another during the day (Richie et al., 1983). In addition, there is currently no agreement regarding the density of P-adrenoreceptors on the lymphocyte subtypes (Pochet and Delespesse, 1983), while evidence is now emerging that the receptors on lymphocytes undergo circadian changes which are not necessarily related to those governing the number of circulating cells (Titinchi et al., 1984). It may be concluded from such studies that extrapolation from changes in P-adrenoreceptors on lymphocytes to those in the brain of the depressed patient should be made with considerable reservation. It is possible that studies of the p-adrenoreceptor on the platelet membrane may provide more reliable information particularly as this type of membrane contains other types of amine receptors in addition to P- and ap-adrenoreceptors; this has led some investigators to conclude that the platelet is a useful model of the central neurons (Campbell, 1981).

E. PERIPHERAL MARKERSOF CENTRAL SEROTONERCIC FUNCTION It is well established that many effective antidepressants impede the reuptake of ['H]serotonin into cortical synaptosomes from rat brain and into platelets of nondepressed subjects who have received a single dose of antidepressants such as clomipramineor zimelidine. Such findings suggest that the transport system governing the uptake of serotonin into the synaptosome and platelet is similar, suggesting that the platelet may provide a useful model of the nerve terminal in the brain of the depressed patient. Several investigators have found that the transport of ['H]serotonin into the platelets of depressed patients is reduced before treatment, but returns to normal following effective therapy irrespective of the presumed mechanism of action of the antidepressant (i.e., whether a specific noradrenaline or serotonin uptake inhibitor); ECT has also been shown to be equally as effective as antidepressants in normalizing the serotonin uptake (Healy et al., 1983, 1984; Born et al., 1980; Tuomisto et al., 1979). The sensitivity of the serotonin receptor on the platelet membrane also appears to be subnormal in the untreated patient and returns to control values following effective drug treatment. There is experimental evidence to suggest that the serotonin receptor on the platelet membrane is of the 5-HTp-type(Lampugnani et al., 1982),which is widely distributed in limbic regions of the brain and whose activity is increased following

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chronic antidepressant treatment. These findings therefore suggest that the sensitivity of 5-HT2 receptors is decreased in the untreated patients and returns to normal values following effective treatment. Other investigatorshave failed to find changes in peripheral serotonin receptor sensitivity in depressed patients. Thus Wood et al. (1984b) reported no difference in the serotonin-induced aggregation response in the platelets obtained from drug-free depressed patients and their controls. The reason for the difference in the findings of Wood et al. (1984b) and Healy et al. (1983)is uncertain apart from the different methods used to prepare the platelet-rich plasma. The question also arises concerning the relevance of platelet serotonin receptors and those found in the brain. Thus although central serotonin 2-type receptors have a high affinity for [’Hlspiperone, Schacter and Grahame-Smith (1982) failed to show that this ligand could bind to the human platelet membrane. Leysen et al. ( 1984) have, however, convincingly argued that both the ligand-binding sites and the platelet aggregation responses are mediated by 5-HT2-type receptors. E ENDOCRINE MARKERSOF DEPRESSION Carroll et al. (1976a) and others (Meltzer and Fang, 1983; Asnis et al., 1981) have demonstrated clearly that hypersecretion of cortisol occurs in the depressed patient and that it is not readily suppressed by the administration of 1 mg of dexamethasone. Furthermore, the circadian rhythm which underlies the secretion of cortisol is blunted in the depressed patient. These observations have led to the development of the dexamethasone suppression test (DST) for the diagnosis of depression. The widespread application of the DST in recent years is confirmation of its usefulness and of the need to supplement standard clinical criteria of diagnosis with objective biochemical markers of the depressive state. The use of the DST has invariably shown that “false-positives”can occasionally arise in patients suffering from senile states, alcoholism, and anorexia nervosa (Ballin et al., 1983). Whether such abnormal endocrine profiles are attributable to secondary symptoms of depression or due to such factors as the nutritional status of the patients awaits elucidation. Carroll and colleagues, who have made the major contribution to the development and use of the DST, have recently received its application and reliability (Carroll et al., 1981). An interesting application of the DST has been its use in the identification of the subtypes of depressive disorder. Schlesser et al. (1980)have shown that nonsuppression of the cortisol level by dexamethasone can

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assist in separating those patients with primary unipolar depression (45% nonsuppression) from bipolar (85%) and controls (6%).These investigators further used the DST to differentiate familiar subtypes of unipolar depression; it was also found that nonsuppression, following dexamethasone administration, is correlated with a good response to antidepressant therapy, providing further evidence for a biological difference between these groups. Other investigators have studied the impaired growth hormone response to different types of physiological and pharmacological challenge and the hyporesponsiveness of the thyroid gland to thyroidstimulating hormone (TSH) stimulation. Such studies have also shown that bipolar (manic-depressive) patients differ from unipolar (endogenous) patients. This work has been reviewed by Brown et al. (1984) and substantiates the view that changes in neuroendocrine responsivenessmay be used to differentiate between the subtypes of depression. Despite the widespread interest in the use of endocrine markers in the study of affective disorder, little attention has been paid to the possible involvement of the nutritional status of the patient in affecting the endocrine response. This problem has been addressed by Fichter et al. (1984) in their study on the neuroendocrine disturbances that occur in depression, anorexia nervosa and starvation. These investigators found that weight loss, catabolic state and reduced caloric intake induced major changes in the response to dexamethasone suppression thyroid-releasing hormone (TRH) and growth hormone (GH) responsiveness to physiobgical challenge. This finding raises a serious question regarding the specificity of such tests as biological markers for depression. Is it possible to link the changes in glucocorticord secretion with changes in the adrenoreceptor status of depressed patients? So far studies have been limited to the interactions between circulating glucocorticoids and the adrenoreceptor-linked cyclase system in the rat brain. Thus Sulser and colleagues (1983) have shown that a decrease in circulating corticosteroids following adrenalectomy is associated with an increase in the responsiveness of the receptor-linked cyclase system to noradrenaline; this change was not associated with the density of P-adrenoreceptors as assessed by their affinity for [SH]dihydroalprenalol.By contrast, other investigators [e.g., Wagner et al. ( 1979)jhave shown that estradiol decreases the cortical adrenoreceptor density and reduces the sensitivity of the cyclase unit, suggesting that different steroid hormones have different modulating effects on different populations of adrenoceptors in different brain regions! These effects could possibly be affected by changes in the regulatory processes in the cell nucleus which are altered by the binding of the appropriate steroid to its nuclear receptor.

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In addition to changes in the cortisol response to various types of drug treatment, which have been well documented and have helped to validate the usefulness of the dexamethasone suppression test as a diagnostic marker of depression, other changes in pituitary-adrenal function have also been found in the depressed patient. 1. Growth Hormone (GH) Physiological stimuli such as insulin induced hypoglycemia and amino acids (e.g., dopa and 5-hydroxytrypotophan)have been shown to stimulate GH secretion in control subjects. Depressed patients exhibit a blunted GH response to both insulin and 5-hydroxytryptophan(Takahaski et al., 1974; Gruen et al., 1975). Other investigators have shown that an apparent abnormality in the GH response to dopa could not be substantiated when factors such as the age and sex of the patients were adequately controlled (Sacher et al., 1975). An abnormal GH response to TRH or luteinizing hormone (LH) challenge in depressed patients has been reported by Brambilla et al. (1978), which supports the hypothesis that the pituitaryadrenal axis is functioning subnormally in the depressed patient. Matussek et al. (1980) have shown that the elevation of the plasma GH concentration caused by infusion of clonidine is reduced in the depressed patient, an effect which may reflect decreased responsiveness in central ap-adrenoreceptors. Patients with obsessive-compulsive disorders also exhibit a similar response to clonidine (Siever et al., 1984), so that it still remains to be proved whether a blunted GH response is specifically related to depression.

2. Luteinazing Hormone (LH) Preliminary studies have shown that the concentration of plasma LH is reduced in depression (Altman et al., 1975; Rubin et al., 1981) and increased in mania (Benkert, 1975). Such changes may be a reflection of an alteration in the state of libido in patients with depression of mania (Winokur et al., 1969). While there is a paucity of detailed studies on changes in LH secretion in depression, Whalley et al. (1985)have recently studied changes in plasma LH, cortisol, and prolactin levels in young males with mania or schizophrenia. Their results showed that plasma LH concentrations were raised as were plasma prolactin and cortisol concentrations; no changes occurred in the plasma testosterone and sex hormone-binding globulin concentrations in either patient group as compared with the controls. Perhaps more detailed studies on changes in LH concentrations before and after treatment of depressed patients may identify a useful marker for the illness and of response to treatment.

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3. Prolactin Baseline plasma prolactin concentrations have been shown to be elevated in both bipolar and unipolar depressed patients (Cuenca et al., 1978), with an abnormal circadian rhythm in the secretion of prolactin reported in such patients (Halbreich et aE., 1979). Nielsen et al. (1980) have shown a correlation between changes in the plasma prolactin concentration and the response of the depressed patient to antidepressant treatment, suggesting that this hormone may provide a useful state-dependent marker.

OTHER THAN BIOGENIC AMINES G. NEUROTRANSMITTERS 1. Peptides and Depression Increasing evidence has accumulated in recent years to suggest that neuropeptides produced in the hypothalamus and in the extra hypothalamic regions of the mammalian brain may have a significant effect on brain function. Such "peptidergic" pathways probably play an important role in behavior (Guillemin, 1977). In recent years, interest in the possible role of vasopressin in central neurotransmitter processes has arisen largely as a consequence of the effects of this peptide on the restoration of memory in rats, following the extinction of a conditioned avoidance response (de Wied et al., 1977). Preliminary studies with a derivative of vasopressin (Gold et al., 1979) showed that three out of four depressed patients improved after such treatment. Until these studies are repeated using the double-blind procedure, the validity of such a finding is doubtful. The concentration of endorphins in the cerebrospinal fluids of depressed patients has also received considerable attention in recent years, and a hypothesis has been advanced that depression is associated with a hypoactive endorphinergic system, whereas mania is associated with a hyperactivity of this system (Emrich, 1982). The body of experimental evidence, so far, provides little support for this hypothesis despite the claim by Emrich (1984) that the activation of specific opiate receptors in the brain by, for example, P-endorphin, may have some beneficial effects in depression; the partial opiate receptor antagonist buprenorphine has been shown in an open trial to produce a 40% reduction in the depression score within 4 days of the start of treatment.

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2. GABA and Depression The y-aminobutyric acid (GABA) agonist progabide has been shown to be effective in the treatment of depression in two double-blind studies (see Bartholini et al., 1984), its therapeutic efficacy being similar to imipramine. The finding that the concentration of GABA in the CSF of depressed patients is lower than in controls (Gold et al., 1980) and that tricyclic antidepressants inhibit the reuptake of GABA thereby facilitating its effect (Harris et al., 1973)helps support the view that one of the actions of antidepressant drugs is to correct a deficit in the GABAergic system. If the therapeutic action of GABA-mimetic drugs can be fully substantiated, the conventional amine hypothesis of depression will have to be amended to account for the modulatory role which GABA and possibly other amino acids and peptides has on central noradrenergic and serotonergic processes. The possible mechanism whereby progabide produces its antidepressant effect has been investigated by Zivkovic et al. (1982),who found that the chronic administration of progabide reduces noradrenaline turnover in rat brain. This change is not associated with a decrease in the density of P-adrenoreceptors or in the activity of the postsynaptic adrenoreceptor cyclase. Thus progabide has a qualitatively different effect to conventional antidepressants on noradrenergic transmission. Bartholini and Morselli (1983) have speculated that GABA receptor agonists act by changing the firing rates of noradrenergic and serotonergic cells so normalizing central neurotransmission in the depressed patient; presumably this could be brought about by the drug-activatingGABA heteroreceptors located on such cell bodies. It is not without interest that conventional antidepressants such as amitriptyline and atypical antidepressants such as citalopram produce changes in GABA receptor density following their chronic, but not acute administration. Thus Pilc and Lloyd (1984) have shown that different types of antidepressant increase the GABA-B receptor density after chronic administration; the monoamine oxidase (MAO) inhibitor pargyline had a qualitatively similar effect to the other types of antidepressants tested. These studies suggest that antidepressants may owe at least part of their activity to a modulation of GABA-B receptors, thus providing a link between the GABAergic and monoaminergic system. a. Histamine and Depression. Many clinically effective antidepressants have potent antihistaminic properties which may contribute to their mode of action. Although it is generally considered that the antihistaminic effects of antidepressants contribute primarily to the sedative rather than the antidepressant effect of these drugs, Wood et al. (1983, 1984a) have

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shown that the accumulation of ['4C]histamine by platelets from female depressives was significantly decreased compared to controls. In a more extensive study, these investigators have shown that the platelet histamine accumulation rate was lowest in both depressed male and female patients and slightly higher in those being treated with lithium, compared to ageand sex-matched controls (Wood et al., 1984a). The meaning of these results is currently a matter of speculation. However, as the uptake of histamine in the human platelet is by passive diffusion, it is possible that these changes reflect a nonspecific abnormality in the transport of small molecules across active membranes. In support of this view, Pettergrew et al. (1982) have reported that a membrane abnormality occurs in the intact erythrocytes and lymphocytes of manicdepressives. 6. S-Adenosylmethionzne (SAM) and Depression. Studies undertaken since 1975have suggested that the methyl donor, SAM, has antidepressant properties. Initial studies were undertaken on schizophrenic patients as part of a program to evaluate the transmethylation hypothesis of schizophrenia. Two double-blind placebo controlled studies in depressed patients showed that SAM was as effective as clomipramine or amitritpyhe (Muscettola et al., 1982);Del Vecchio et al., 1978; Kufferle and Grunberger, 1982). Apart from a slight increase in the anxiety case, SAM appeared to be reasonably free from side effects. Preliminary studies on the antidepressant properties of SAM in the United Kingdom tended to confirm the Italian studies (Charney et al., 1981). The possible mechanism by which SAM brings about its effect is unclear, but Ordonez and Wurtman (1974) have examined the interrelationship between folate and the SAM metabolism in rat brain and suggested that the increase in the availability of folate as a cofactor for biogenic amine synthesis might contribute to the effect of SAM on central neurotransmission. In addition, experimental studies have shown that SAM increases the turnover of serotonin and noradrenaline in rat brain (Curcio et al., 1978) and also increases the concentration of HIAA in CSF of depressed patients (Agnoli et al., 1976). Whether these effects of SAM are secondary to the changes in membrane phospholipid methylation, which is thought to be the initial pathway for the transduction of receptormediated signals through the neuronal membrane (Hirata and Axelrod, 1980),is unproved. However, it seems possible that changes in neurotransmitter turnover, receptor sensitivity to biogenic amine neurotransmitters, or in endocrine function may be mediated by changes in membrane activity; such changes may be modulated by chronic SAM administration.

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Clearly, more double-blind studies are needed to confirm the efficacy of SAM as an antidepressant and also to elucidate its precise mechanism of action in modifying membrane processes.

111. Mechanisms of Action of Antidepressants

A. STRUCTURE-ACTIVITY STUDIES ON NORADRENALINE-UPTAKE INHIBITORS Because of their long clinical availability, it is not surprising to find that the most extensive studies on structure-activity relationships have been made on the tricyclic antidepressants. Thus Salama et al. (1971) studied the effects of several different tricyclic antidepressants on ['Hlnoradrenaline uptake into rat cortical slices and showed that some of the most potent noradrenaline-uptake inhibitors contained a dihydrodibenzazepine ring (e.g., desipramine with an IC50 of 7.1 X lo-' M). The inhibition of noradrenaline uptake was found to be decreased if the number of carbon units in the side chain was increased or decreased from the optimal value of three: branching of the side chain also reduced the potency With regard to substitutions on the side chain N , Salama et al. (1971) found that compounds in the dibenzocycloheptatriene (protriptyline) series which contained-NH2, NHCH3, and N(CH& were equipotent; N-ethyl,N-isopropyl, and N-butyl derivativeswere only weakly active. Horn et al. (1971) studied the amine uptake inhibitory properties of the amitriptyline series and showed that the secondary amine (nortriptyline) had only one-tenth of the potency of the tertiary amine (amitriptyline)in inhibiting noradrenaline uptake into hypothalamic synaptosomes; others have shown that the difference between these drugs w a s only fourfold (Salama et al., 1971; Maxwell et al., 1969). In addition to the effects of changes in side-chain substitution on noradrenaline uptake in vitro, Maxwell et al. (1969)noted that if the rings of a tricyclic antidepressant were coplanar, then the compound was only a weak inhibitor of amine reuptake, whereas uptake inhibition was dramatically increased if the phenyl rings were held at dihedral angles >90" and <180".These investigators suggested that the low potency of coplanar tricyclic compounds could be attributed to the projection of the ring system into the binding site for the nitrogen atom. Later studies showed that several bicyclic antihistamines also had weak noradrenaline-uptake

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S . I. ANKIER AND B. E. LEONARD

inhibitory properties in vitro, showing that the tricyclic ring was not essential for uptake inhibition (Maxwell et al., 1974).This has been verified more recently with the establishment of the bicyclic compound viloxazine as an effective antidepressant. Studies on the optical isomers of desipramine and other noradrenaline uptake inhibitors (Maxwell et al., 1970a) suggest that the phenylethylamines and the amine-uptake inhibitors bind by a single phenyl ring to a hydrophobic surface, while the N binds to a negatively charged area within the same plane as the phenyl ring. A hydrophobic area is believed to be adjacent to the negatively charged area in the receptor site; this hydrophobic area receives any methyl substitution on the N-side chain. A detailed discussion on the structure activity relationship for amine reuptake in the tricyclic antidepressant series has been published by Maxwell and White (1978). For optimal interaction with the noradrenaline uptake system, derivatives of phenylethylamine must occupy the fully extended (antiperiplanar) conformation (Maxwell et al., 1970b; Bartholow et al., 1977). As most derivatives of phenylethylamine are conformationally mobile, a variety of conformations can be assumed by the drug in the region of the transport site for noradrenaline. Determination of the nature of the amine-uptake site may help in evaluating the relationship between the structure and activity of specific noradrenaline-uptake inhibitors. The most widely accepted model of the transport site is based upon that described by Bogdanski and Brodie ( 1969),which suggests that a carrier noradrenaline-Na+ complex is transported to the inner surface of the neuronal membrane, where the Na+ and noradrenaline dissociated form the carrier, an effect facilitated by the low amine and Na' concentrations. The low amine concentration is maintained by monoamine oxidase, while the sodium ions are removed by the Na' ,K+-ATPasepump. The mechanism may be represented diagrammatically as follows: +

IInner membrane1

NA

-MA0 Astorage +met abol i t e s

+ Nd K+

NA, noradrenaline; Na. sodium; K + , potassium ions; 0 ,carrier.

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203

Despite the attraction of the hypothesis linking catecholamine uptake to Na+,K+-ATPaseactivity, studies attempting to validate this hypothesis (following the administration of antidepressant and other types of psychotropic drugs to rats) have proved inconclusive. Thus Leonard and McNulty (1977) found that, although amitriptyline inhibited the activity of this enzyme in vitru, when the drug was administered acutely or chronically to rats, the enzyme activity was increased in several brain regions. If inhibition of amine reuptake by amitriptyline is linked to membranebound ATPase, one would anticipate that the enzyme activity would be decreased, not increased, following administration. It would appear that changes in Na+,K+-ATPasefollowing antidepressant treatment are coincidentally, rather than causally, linked to the amine transport site. It is not the purpose of this article to review extensively the relationships between chemical structure and pharmacological activity of the various classes of antidepressants, but to give an overview of the less controversial aspects of the subject. Detailed aspects of the structureactivity requirements for noradrenaline-uptake inhibitors have been published by Patil et al. (1975) and Ross (1984), while the conformational preferences of drugs, specifically inhibiting noradrenaline uptake, have been assessed by Rutledge et al. (1984). In addition to the conformational structure, chirality may also play an important role in determining the M A 0 or catecholamine-uptakeinhibitory properties of an antidepressant. For example, the cis-isomer of tranylcypromine has only one-third the potency of the trans-isomer in inhibiting M A 0 in vivo, even though it appears to be equipotent with the trans-isomer as a monoamine oxidase inhibitor MA01 in vitro (Zirkle et at., 1962;Zeller and Sarkar, 1962).Among the tricyclic antidepressants, the bridged derivative maprotiline and its side-chain-hydroxylated derivative oxaprotiline are potent and selective inhibitors of noradrenaline uptake into the rat heart and brain in vitro (Pinder et al., 1977; Waldmeier et al., 1977). Detailed studies of the (R)( -) and (S)-( +) enantiomers of oxaprotiline (Waldmeier et al., 1982)show that the pharmacologically activity form of the drug resides in the (S)-( ) configuration. This corresponds to the (S)-configurationof noradrenaline and presumably inhibits the transport site for the amine. Further studies by Delini-Stula et al. (1983) have shown that the stereospecific action of oxaprotiline is maintained after subchronic administration as only the (S)(+) form antagonizes the central depressant effects of clonidine. These effects are paralleled by a reduction in P-adrenoreceptor sensitivity, as indicated by a decrease in the P-adrenoreceptor density in rat cortical membrane coupled with a decrease in functional activity of the adenylate cyclase system. Despite the well-established pharmacological differences

+

204

S. I. ANKIER AND B. E. LEONARD

between the oxaprotiline enantiomers, preliminary clinical studies suggest that both enantiomers are equally effective as antidepressants (DeliniStula et al., 1983). If confirmed by more extensive studies, this finding emphasizes that caution must be taken in extrapolating from experimental studies to the therapeutic activity in depressed patients. A detailed review of the differences between the pharmacological properties of chiral stereoisomers of various classes of antidepressants has been made by Nickolson and Pinder (1984) and by Ross (1984). Caution must be exercised in extrapolating from in vitro data to the chronic administration of amine uptake inhibitors. For example, compared with some of the newer nontricyclic antidepressants, drugs such as imipramine and amitriptyline show only slight selectivity in inhibiting serotonin compared to noradrenaline uptake after acute administration. Following chronic administration, both drugs produce secondary amine metabolites (desipramine and nortriptyline, respectively), which show some selectivity for inhibiting noradrenaline reuptake. Similarly,the serotonin uptake inhibitor clomipramine is metabolized to its desmethylated derivative during chronic administration. Therefore, both noradrenaline and serotonin reuptake will be impeded by the parent compound and its metabolite in this situation. Even with the newer nontricyclic antidepressants such as nomifensin, which show considerable specificity in inhibiting noradrenaline uptake in vitro, there is evidence from in vivo studies that one of its principal metabolites, 4-hydroxynomifensin, selectively impedes the reuptake of serotonin (Leonard et al., 1984). Thus it seems unlikely that the specific uptake inhibitory properties of many antidepressants have any relevance to their activity on amine transport following their chronic administration.

B. STRUCTURE-ACTIVITY STUDIES ON SEROTONIN-UPTAKE INHIBITORS It is well established that the tertiary amine tricyclic antidepressants, such as imipramine and amitriptyline, are 5-10 times more potent in inhibiting serotonin than noradrenaline reuptake. Horn and Trace (1974), in their study of the effects of a series of tricyclic compounds on the uptake of 5-r3H]HT into rat hypothalamic homogenates, found that altering the length of the side chain by one or more carbon units (from its optimal length of three units) decreased the uptake inhibitory effect 7- to 8-foId, while OL or @-methylsubstitution in the side chain reduced the potency 17- to 33-fold, respectively These investigators also showed that

BIOLOGICAL ASPECTS OF DEPRESSION

205

chlorine substitution into position three on the tricyclic ring increased the potency !&fold,while dimethylamino-substitution in this position did not affect the potency. Substitution of a sulfur atom for the 2C bridge in imipramine and in clomipramine, which give promazine and chlorpromazine, respectively,reduced serotonin-uptake inhibition by 30- to 50-fold, respectively.

C. STRUCTURE-ACTIVITY STUDIES ON MONOAMINE OXIDASE INHIBITORS The general structure, which describes most of the known monoamine inhibitors (MAOIs), can be described by the formula Aryl-X-N-R2

I

RI

where aryl is a phenyl, an indole, or a substituted analog; X is an aliphatic group containing one or more C or 0 groups; N is an amino, amide, or hydrazine N; R1 is -OH or -CHs; and R2 is hydrazine, propargyl, or cyclopropyl. In the most potent irreversible MAOIs, the R2 group binds irreversibly to the enzyme. In some MAOIs, a cyclopropyl group may be introduced into the X position (e.g., tranylcypromine), thereby markedly increasing the inhibitory potency. P-Carbolines may be considered as cyclized indolealkylamines and conform to the structural features of this general formula. The requirement for the aromatic ring in all MAOIs, together with an N-group, suggests that the inhibitor must bear some resemblance to the substrate if it is to bind to the active center of the enzyme. The most potent MAOIs contain an aromatic group separated from the amine-N group by at least one carbon unit. It is thought that the initial enzymeinhibitor interaction is initially weak and reversible; the attachment to the enzyme surface becomes irreversible if other features of the MAOIs molecule provide favorable binding properties. Fujita ( 1973) has suggested that the aromatic moiety is bound to an electron-rich noncatalytic site of the enzyme and that changes in M A 0 inhibitory potency, as a result of substitution on the aromatic ring, is usually dependent on the steric effects of these substitutents. A detailed review of the structure-activity relationships of different types of MAOIs has been written by Maxwell and White

(1978).

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S. I. ANKIER A N D B. E. LEONARD

D. CHRONIC EFFECTS OF ANTIDEPRESSANTS ON NEUROTRANSMI-M-ER FUNCTION Antidepressants are often classified in terms of their effects on the presynaptic sites on the neurons of biogenic amines. Thus until the discovery of such atypical antidepressants as iprindole, mianserin, and trazodone, antidepressants were classified as monoamine oxidase inhibitors or amine reuptake inhibitors. Such a classification was undoubtedly helpful in the development of a plethora of drugs based on chemical modifications of imipramine or phenelzine. This was perpetuated by the use of animal models which could only select molecules with MA01 or reuptake inhibitory properties, but which could not identify antidepressants such as mianserin or trazodone that do not act in this way in vim. It may be argued that the very success of MAOIs and amine reuptake inhibitors helped to provide the basis for the amine hypothesis of depression. Recently, a number of serious deficits have been found in the amine hypothesis, some of which have already been referred to. Thus a delay occurs between the initial administration of the antidepressant and the time of onset of the therapeutic effect. This delay in onset occurs irrespective of the chemical nature or acute pharmacological profile of the drug. As a delay also occurs with nonpharmacological treatments such as electroconvulsive shock therapy or REM sleep deprivation, it may be presumed that all effective antidepressant treatments bring about adaptational changes in one or more central neurotransmitter systems which take several weeks to become established. The lack of correlation between the acute effects of antidepressants on the amine reuptake processes with the antidepressant response is further compounded by the lack of correlation between the potencies of the drugs in inhibiting amine reuptake and their therapeutic potencies. Furthermore, several drugs were developed on the basis of their amine uptake inhibitory properties in animal models of depression only to be found ineffective when administered to depressed patients. This can be exemplified by the phenyl propylamine derivative gamfexine which was shown to be 30 times more potent than imipramine in reversing reserpine-induced ptosis in mice (Gershon et al., 1967). Similarly, the benzylammonium compound BL-KR 140 was shown to reverse reserpine-induced hypothermia in mice, a routine method used in screening compounds for their potential antidepressant activity, but failed to show any antidepressant activity in initial clinical studies (Hekimiran et al., 1968). At least ten compounds of varying structure and potency in animal models of depression have been reported to be therapeutically ineffective (Kelwala et al. 1983), thereby serving as a warning regarding the extrapolation of the acute effects of psychotropic drugs on

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207

neurotransmitter systems in animals to their therapeutic effects following chronic administration. A further limitation of the amine hypothesis of depression arises when one considers the effect of amine precursors on depressive symptoms. Thus if the depression arises as a consequence of a deficit in brain biogenic amines, one would anticipate that the administration of the amine precursors of the catecholamines and indole alkylamines (L-dopa and L-tryptophan, respectively) would lead to a rapid reversal of the symptoms. Clinically, the response of depressed patients to either treatment has been found to be equivocal. It must, therefore, be concluded that antidepressant treatments bring about their therapeutic effects indirectly and not directly by modifying the reuptake or metabolism of biogenic amine neurotransmitters. This subject has been reviewed critically by Sulser (1982). In the last decade, there has been a major switch in research emphasis from the pre- to the postsynaptic neurons regarding the mode of action of antidepressant drugs. The initial studies of Vetulani et al. (1976) and Banerjee et al. (1977) indicated that various chronic antidepressant treatments changed the number and responsiveness of postsynaptic P-adrenoreceptors, while acute treatments were without effect. These findings were of considerable conceptual value because they established for the first time a single biochemical event which could be specifically induced by both pharmacological and nonpharmacologicaltreatments, irrespective of the acute effects of the treatments. Subsequent studies have shown that, in addition to tricyclic antidepressants, MAOIs and ECT, “atypical”antidepressants such as iprindole and mianserin also reduce the functional activity of the P-adrenoreceptor. On the other hand, barbiturates, anticonvulsants, benzodiazepines, antihistamines, and butyrophenones have no effect. This has been reviewed by Sulser (1983). It is interesting to note that chlorpromazine has an “antidepressant”profile in this biochemical model. While this may be an example of a false-positive, it should be remembered that this drug has been shown to have antidepressant properties (Overall et al., 1966). Although the effects of antidepressant treatments on the functional activity of cortical P-adrenoreceptors has proved to be of value in studying known antidepressants, it remains to be established whether this will provide a completely reliable model for the selection of novel antidepressants. In this respect, it has been shown that the specific serotonin-uptake inhibitor, fluoxetine, which has been shown to be an antidepressant in several clinical studies, does not change the density or functional activity of cortical P-adrenoreceptors (Mishra et al., 1979). Relatively high doses of trazodone (Clements-Jewery, 1978) and buproprion (Ferris et al., 1983) have however been shown to decrease 13-adrenoreceptor density.

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S. I. ANKIER A N D B. E. LEONARD

Moreover, studies on the effect of the triazolobenzodiazepine alprazolam on the functional activity of cortical adrenoreceptors suggests that this atypical antidepressant may have a similar neurochemical profile to those regarded as tricyclic antidepressants. The relevance of the desensitization of cortical P-adrenoreceptors for the selection of antidepressants is supported by electrophysiologicalstudies in which the subsensitivity of postsynaptic P-adrenoreceptors has been established. Thus subsensitivity of postcerebellar Purkinje cells (Siggins and Schultz, 1979) and cortical pyramidal cells (Olpe and Schellenberg, 1980) has been reported following the microiontophoretic administration of noradrenaline to rats which had been treated with chronic, but not acute, antidepressants; MAOIs, "typical," and "atypical" antidepressants were found to be effective. Other studies in which the rate of firing of noradrenergic neurons in the rat hippocampus were studied showed that only chronic treatment with desipramine resulted in a suppression of the firing rate (Huang, 1979). Thus electrophysiologicaland biochemical studies largely support the hypothesis that chronic antidepressant treatment reduces the sensitivity of postsynaptic P-adrenoreceptors. Furthermore, the time necessary for the reduction in p-adrenoreceptor sensitivity to occur (about 14 days) approximates to that needed for the antidepressant effect to become apparent in patients. In addition to their effects on P-adrenoreceptors, most antidepressants have also been studied for their effects on other biogenic amine receptor densities. Consistent changes in the receptor densities only become apparent following the chronic ( 14-21 days) administration of antidepressant drugs. The results of studies on a selected group of typical and atypical antidepressants on serotonergic, adrenergic, muscarinic, and dopaminergic receptor sites are summarized in Table I. The effects of the antidepressants are compared with those of methysergide and chlorpromazine, psychotropic drugs with well-established actions on central neurotransmission but which do not exhibit antidepressant properties. In addition to their ability to reduce the density of postsynaptic padrenoreceptors, the typical and atypical antidepressants listed in Table I have been shown to cause a subsensitivity in the responsiveness of the Padrenoreceptors to noradrenaline; mianserin and zimelidine, which do not affect the P-adrenoreceptor density, reduce the functional sensitivity of these receptors, whereas fluoxetine is without effect (Sulser and Mobley, 1981). This lack of effect of the therapeutically active antidepressants on P-adrenoreceptor density suggest that a change in the P-adrenoreceptor density is not a prerequisite for the functional desensitization of these receptors. It may be speculated that these antidepressants act on the coupling mechanism between the receptor site and the adenylate cyclase subunit. Because of the well-established finding that all clinically effective anti-

TABLE I EFFECTS OF CHRONIC ADMINISTRATION OF ANTIDEPRESSANTS ON THE DENSITY OF BIOCENIC AMINERECEPTORS IN RAT BRAIN"^^ Receptor type ~

Tricyclic antidepressants Amitriptyline Imipramine Desipramine Atypical antidepressants Iprindole Fluoxetine' MA01 Pargyline Serotonin antagonist Methysergide Neuroleptic Chlorpromazine

~~

~

Serotonin 1 5-[$H]HT

Serotonin 2 [ 'HISpiperone

a-Adrenergic ['H]WB4 101

P-Adrenergic ['HIDHA

Muscarinic [$H]QNB

Dopamine ['HISpiperone

NC'

p < O.Old

NC

p < 0.01

NC NC NC

p < 0.85d p < 0.05 p < 0.05

NC NC NC

NC NC NC

NC

p < 0.01

NC NC

p < 0.05

NC

NC

NC NC

NC NC

p < 0.05

p < 0.01

NC

p < 0.05

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

NC

p < 0.01f

p < 0.01

"From Green and Nutt (1983). [3H]WB4101, a,-adrenoreceptor antagonist, dimethoxyphenoxyethylamino methyl benzodioxan; ['HIDHA, the p-adrenoreceptor antagonist, dihydroalprenolol; ['HIQNB, the muscarinic receptor antagonist, quinuclidinyl benzilate. "C, No change relative to controls. dDecrease in receptor density relative to controls. 'After Wong and Bymaster (1981). /Relative to controls.

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S. I. ANKIER A N D B. E. LEONARD

depressants reduce the functional responsivenessof the P-adrenoreceptors in rat brain, it has been suggested that changes in the postsynaptic receptors must reflect an increase in neurotransmitter release, presumably as a consequence of a decrease in the sensitivity of the noradrenergic autoreceptors. This hypothesis is supported by the finding that the atypical antidepressant mianserin (Fludder and Leonard, 1979) and tricyclic antidepressants (Crews and Smith, 1978b) decrease the sensitivity of the noradrenergic autoreceptors following chronic treatment. Since the autoreceptors inhibit the release of noradrenaline, the blockade or desensitization of these receptors by chronic drug treatment would facilitate transmission and so decrease the sensitivity of the postsynaptic receptor sites. This simple hypothesis has been challenged by the observation that not all antidepressants which decrease the functional activity of postsynaptic P-adrenoreceptors decrease the density of the presynaptic autoreceptors (Sugrue, 1981). Furthermore the results of experiments in which the density of a*-adrenoreceptors, presumed to be similar to the noradrenergic autoreceptors, were determined using ['Hlclonidine are conflicting with both a decrease (Smith et al., 1981) and an increase (Tsukamoto et al., 1982) in a2-adrenoreceptor numbers being reported following the chronic administration of antidepressants. With the plethora of changes in the densities of various neurotransmitter receptors following antidepressant administration, involving histamine 1- and 2-type receptors (Green and Maayani, 1977; Kanof and Greengard, 1978) and dopamine autoreceptors (Chiodo and Antelman, 1980) in addition to serotonergic and noradrenergic receptors (Wang and Aghajanian, 1980; Garcia-SeviIIa et al., 1981) it is difficult to draw any conclusions regarding the primary site of action of antidepressants on neurotransmission in the rat brain. In addition to the uncertainty regarding the existence of autoreceptors, few investigators appear to distinguish between ligand-binding sites and receptor sites, which has undoubtedly added to the confusion when attempting to interpret the mechanism of action of antidepressants on rat brain. As Sulser (1982) has emphasized in his review, to qualify as a receptor, the dual function of "recognition and response" must be demonstrated. Thus irrespective of its affinity and number of specific sites, a binding site for a ligand cannot be designated a receptor unless a close relationship between occupancy and biological response can be demonstrated; this has been considered in detail by Hollenburg and Cuatrecasas (1978). Furthermore, it must be emphasized that changes in sensitivity reflect the functional responsiveness of the cell to a neurotransmitter, and the number of receptors as determined by ligand-binding studies need not nec-

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essarily parallel the changes in cellular responsiveness. This is not surprising since not all receptors need be occupied for an optimal response to be elicited. Presumably all available receptors would be occupied by a ligand and, therefore, incorrect conclusions could be drawn regarding the relationship between receptor number and the subsequent physiological response of the cell. So far only the studies in which the changes in receptor-linked adenylate cyclase activity have been monitored, following chronic antidepressant treatment, appear to have addressed this question rigorously. Whether all antidepressant treatments produce their effect through a final common pathway which involves the functional desensitization of postsynaptic P-adrenoreceptors is still a matter of conjecture. Several studies have shown that chronic antidepressant treatment also results in the increased responsiveness of serotonin receptors in rat brain; such an effect occurs following the chronic administration of tricyclic antidepressants, mianserin, adinazoiam or ECT, MAOIs (particularly the MAO-A inhibitor, clorgyline, but not the MAO-B inhibitor, deprenyl), and of such specific serotonin-uptake inhibitors as zimelidine and indalpine (De Montigny et al., 1984). These changes in rat brain appear to simulate the functional change in serotonin receptor responsiveness in platelets from depressed patients in which the aggregation response to serotonin is reduced to approximately 50% of the control value before the start of treatment, but returned to control values following effective drug treatment (Healy et al., 1983, 1985). How the changes in noradrenergic and serotonergic receptor function, following chronic antidepressant treatment, can be linked is a matter of conjecture but studies by Brunello et al. (1982) have shown that desipramine cannot reduce the density of f3adrenoreceptors in the rat cortex and hippocampus following specific lesions of the serotonergic system with 5,7-dihydroxytryptamine.This was confirmed by Sulser ( 1983), which suggests that both the serotonergic and noradrenergic systems are involved in the regulation of the functional activity of noradrenaline-coupled adenylate cyclase in the cortex of the rat brain. Presumably a serotonin heteroreceptor located on the noradrenergic terminals could play a key role in linking these systems. Whether the other classical neurotransmitters (such as acetylcholine, dopamine, and GABA), the putative transmitters (phenylethylamine,histamine, and adrenaline), and the peptide cotransmitters (as exemplified by the enkephalins, endorphins, and vasopressin) play any major role in modifying the responsiveness of the noradrenergic pathway to the chronic effects of antidepressant treatments is unknown.

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IV. Clinical Assessment of New Antidepressants

There is increasing need for more well-designed and carefully controlled clinical studies to assess the ever-growing list of putative antidepressants (Ankier, 1986). Each time a new clinical trial is being planned, it is crucial to consider the specific aim of the study, the study design, and also to anticipate and resolve logistic problems. These considerations lead to the preparation of a protocol which should provide a complete specification for the study management and be accompanied by examples of record forms, rating scales, grading cards, and any other relevant information or documentation. As such, the protocol is a formal document agreed between the study sponsor, the clinical investigator, the patient, and the scientific community and assists in accurate communication between all those involved. Protocols are constructed under specific headings, and those items to be considered for antidepressant studies are basically the same as those used for studies on any other therapeutic class of drug. The preparation of a protocol is helped by considering headings in published checklists (Lionel and Herxheimer, 1970; Chaput de Saintonge, 1977;Warren, 1978; Peterson and Fisher, 1980; Friedman et al., 1981). Reference to the guidelines for the adequate reporting of some methodological and patient variables (Kupfer and Rush, 1983a,b) will further help to ensure that essential considerations for the conduct of antidepressant trials are not overlooked. It is also useful to develop a standard “in-house”version as a “master” reference which should be updated in the light of one’s and others’ experiences. Having drafted an early outline of the protocol, discussions with all members of the clinical trial “team,” to include a medical biostatistician, are necessary with the trial coordinator being responsible for distilling advice and information given both on theoretical and practical aspects. The protocol will require frequent redrafting until agreement is achieved. individual and collective commitment to the success of the study must also be established at this stage. This important process is normally very demanding, and it is essential to allocate sufficient time to this activity when scheduling the timing of a trial. Important factors requiring careful consideration when preparing a protocol for an antidepressant study, as well as some more general points of particular importance, are now discussed.

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A. THEINVESTIGATOR The design and performance of any scientifically valid clinical trial is the joint responsibility of the sponsor and the clinical investigator. The sponsor must choose the best available study coordinator (also known as the monitor) as their representative. The coordinator should be suitably qualified, have administrative ability, and possesses personal qualities of tact, enthusiasm, and leadership. The study coordinator’s choice of an appropriate investigator is most important, and this may be approached in different ways. For example, the investigator may already be known through publications, a previously successful cooperation, or by a good reputation. The trial coordinator should make every reasonable effort to ensure that the investigator chosen has appropriate facilities, is both interested in and motivated by the project, has the time to be involved personally, will cooperate, keep information confidential and that the investigator has access to an adequate number of suitable patients to meet the study objective. The investigator should be trained adequately, experienced, competent, and reliable. The “professional investigator,”who is observed to be overcommitted to several concurrent trials and who, by his/ her attitude, reveals commercial rather than scientific motivation, should normally be avoided. Such judgments are very difficult to make but become easier with experience. The study coordinator should remember that their own good reputation also depends on the successful conclusion of valid clinical trials. Lastly, the personal relationship between the investigator and the study coordinator cannot be overestimated. The successful completion of a clinical trial is not simply a mechanical process, but depends on the harmonious interaction of those people involved.

B. PATIENT POPULATION TO BE STUDIED Ordinary sadness is a normal disturbance of mood caused by a stress or loss. Sometimes this mood disturbance is outside the limits accepted as “normal” and leads to a syndrome involving depressive symptomatology, which for research purposes may be classified as “reactive”or “endogenous’’ depression using the Newcastle rating scale (Kiloh et al., 1972). Reactive (anxious, neurotic, or situational) depression appears suddenly and is precipitated by acute and stressful events, for example, a bereavement. Endogenous depression (sometimestermed “psychotic”depression

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when more severe symptoms are present) is thought to be associated with biochemical abnormalities which may be genetically determined. Most endogenously depressed patients suffer from depression alone (unipolar), but some may experience depression with intermittent episodes of mania (bipolar manic-depression). Moreover, patients with endogenous depression may experience a life-disturbing event compficating their illness with neurotic symptoms (called “mixed” depression) (Klerman, 1975; Hamilton, 1979; Kiloh, 1980; Klein et al., 1981). These features should be recorded at trial entry.

Diagnosis of Depression No one classification of depressive illness is ideal, although several useful sets of criteria have been evolved. A study organized by the Medical Research Council (1965) used a carefully defined set of criteria which has subsequently proved a useful research tool. The primary manifestation and major symptom of the depressive illness was specified as being a persistent alteration of mood (with or without diurnal variation)exceeding customary sadness, being evident to the examiner, and which is accompanied by one or more of the following symptoms: self-depreciation and a morbid sense (or delusional ideas) of guilt, sleep disturbance, hypochondriasis, retardation of thought and action, and agitated behavior. Other classifications of primary depression based on the collective opinions of leading researchers have been developed, for example, the St. Louis or the Feighner criteria (Feighner et al., 1972). Subsequently, the Research Diagnostic Criteria (RDC) were developed (Spitzer et al., 1978). It is derived from the St. Louis criteria, but considers both inclusion and exclusion criteria as well as the symptoms, signs, duration or course of illness, and the severity of impairment. In some cases, certain symptoms or symptom clusters are of diagnostic significance only if they persist beyond a certain stated duration. Diagnostic terms are frequently defined in the criteria themselves to avoid possible ambiguity. The schedule for Affective Disorders and Schizophrenia (sADS) is a structured interviewing procedure with rating scales designed to elicit information so an RDC diagnosis may be made (Endicott and Spitzer, 1978). Thus a probable case of “major depressive disorder” is defined when a patient has been persistently depressed or sad for at least a week and also has four of the following symptoms: (1) loss of appetite or body weight; (2) difficulties in sleeping; (3) complaints of tiredness or lack of energy; (4) agitation or retardation; ( 5 ) loss of interest or pleasure in activities that had been pleasurable; (6) feelings and thoughts of self-reproach or guilt; (7) complaints of indecision, slowed thinking, or difficulty with concentration; or

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(8) suicide preparations or attempts or recurrent thoughts of death or suicide. For a “definite”case of depression the depressed mood must be associated with five of the symptoms lasting at least 2 weeks. Recently the Diagnostic and Statistical Manual (DSM 111)of the American Psychiatric Association has become established as an FDA requirement. Affective Disorders are divided into “Major Affective Disorders” (bipolar and major depression) to include a full affective syndrome and “Other Specific Affective Disorders,”which include a partial affective syndrome of at least 2 years duration. This latter class includes cyclothymic and dysthymic disorders. A third class, “AtypicalDisorder,”has been added to define depressions that do not fulfill the criteria for the duration or severity of the two specific subclasses.The DSM 111 has the merit of taking into account the personality,precipitatingstress,and the physical condition of the patient. It provides clear-cut categories defining all types of affective disorders without splitting them into psychoses, neuroses, and personality disorders. There are other diagnostic systems currently accepted as useful for defining depression. For example, the Depression Component of the Minnesota Multiphasic Personality Inventory (Graham, 1977), the Present State Examination (Wing et al., 1974)or the W.H.O. International Classification of Diseases, 9th edition, or the so-called ICD-9 (197’7).It is suggested that the DSM 111 and one other currently recognized diagnostic criterion be used in an antidepressant study. Since anxiety states and depressive syndromes are represented by overlapping clusters of symptoms (Foa and Foa, 1982), the degree of anxiety should be assessed. Meanwhile, it is hoped that the search to identify specific physiological, biochemical, or pharmacological markers will succeed in providing an objective screening procedure for the diagnosis of depressive subtypes.

C. INITIAL SEVERITY The minimum acceptable severity of the patients’ depression should be assessed at trial entry using at least one clinician-ratedmethod. Criteria chosen tend to be pragmatic, which ensures an adequate number of patients are entered into the study, but they should remain relevant. Kupfer and Rush (1983a,b) recently reported on the views of a group of clinical investigators attending an international conference on the origins of depression. They could not arrive at a consensus about the use of any particular rating scale to assess the severity of depression. It was noted that many available scales are alleged to measure the severity of depressive

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symptomatology but that a cross-scale comparison study was needed to confirm if each was measuring the same phenomena. Although relatively time-consuming to administer correctly and consistently, the (HAM-D) (Hamilton, 1967) is widely used for this particular purpose with a total score of 17 often being used as a suitable but arbitrary minimum measure of severity at entry. The severity of the patients’ illness may also be described using a single global scale with say a three, five, or even a seven point scale and/or by the use of a visual analog scale either by the investigator or completed by the patient. Whatever techniques are used to assess the initial severity of illness, they should also be used to measure drug response during the study for comparative purposes. While the debate about the relative merits of various scales continues, the use of several approaches is advocated.

D. WHERETO TEST AN ANTIDEPRESSANT Antidepressants may be prescribed for hospital in- or outpatients or for general practice patients, hence the need to conduct valid clinical studies in both settings. However, most patients suffering from depression are managed in general practice (Wheatley, 1973) with only 2% being referred to a psychiatrist (Watson and Barber, 1981).Recruitment in general practice studies, therefore, has the possibility of achieving large numbers of patients, and since they are unlikely to be receiving concomitant medication, the possibility of complications arising from drug-drug interactions is minimized. Unlike the hospital psychiatrist, the type of depression most often seen by the general practitioner tends to be reactive. Although the general practitioner may not normally separate depression from anxiety states perhaps due to the enormous pressure on their time, clear operational definitions must be adhered to. It is often the policy in general practice for ambulant working patients to be maintained on an antidepressant drug at a dose below those at which side effects occur; however, it is important that antidepressants are tested in this setting using adequate doses as defined in the protocol. From an ethical point of view, efficacy must be assessed properly, utilizing the smallest number of carefully selected patients (Wheatley and Little, 1982). The hospital psychiatrist is trained extensively in the accurate diagnosis and in carefully rating depression and also has more time than the general practitioner to investigate and monitor patients intensively. If side effects occur during a hospital inpatient study, compliance is less likely to be compromised since reassurance can be given by staff who are more readily accessible. There are, however, problems with recruiting suitable

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cases for antidepressant studies in the hospital setting. Thus most patients referred to hospital by the general practitioner will already be on medication (Bouras et al., 1983) and probably be drug treatment failures. They may even be suffering from social, physical, or personality problems. It may be more sensible and easier for depressed patients to be recruited for a clinical study as soon as their illness is diagnosed and hence much future psychopharmacological research might well be conducted by psychiatrists attached to general practice (Little et al., 1978). The alternative approach is for collaborative multicenter hospital studies, and this possibility is considered below. E. INFORMED CONSENT

A clinical study on an antidepressant must be a compromise between an ideal scientific experiment and an investigational procedure in which both moral and legal principles are observed. Thus although the random selection of patients is a scientific ideal, the individual should be given the right not to provide informed consent. Although to obtain “informed consent” is considered necessary by ethics committees and regulatory authorities, Hamilton (1983) has described it as a “lawyers’ myth.” To inform a patient fully about a study, there will be a considerable amount of detail, some of it of a highly technical nature which needs to be communicated. A severely depressed patient will not be able or competent to make a decision about risks versus benefits before entering a clinical trial, since their ability to comprehend information may be poor and/or their judgment impaired. If the information given to the patient is condensed so as to enhance comprehension, then paradoxically they cannot be said to be in a position to give fully informed consent. There may be doubt about the validity and also the duration of consent given by a depressed patient. Then again perhaps a patient appears to consent but, subsequently, they may develop feelings of guilt and doubts. It may be prudent, therefore, for a responsible relative or friend of the patient to be present when informed consent is being sought and to obtain their signature as a witness. Moreover, the investigator has the obligation to evaluate the original consent of the patient throughout the trial and to permit a patient to withdraw from a study, if the patient wishes to at any time. Regardless, it remains the professional responsibility of the trialist to be reasonably convinced that the possible benefits of a new treatment are likely to outweigh its disadvantages, as compared with other treatments or even none at all (Hamilton, 1983). In any event, if valid clinical data is to be obtained, the cooperation of the patient is essential. Such cooperation also enhances

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compliance by what has been described as an “attention-placebo”effect (Myers and Calvert, 1984).

E MULTICENTERED STUDIES The difficulty of recruiting sufficient patient numbers for antidepressant studies in a reasonable time is not new (Little et al., 1978). One unsuccessful approach was to set up a special hospital clinic to which patients were specificially referred (Bouras et al., 1983). Thus the collaboration of several centers in a national or international multicenter study, using an identical core protocol, has remained necessary for statistical purposes. Such studies have the merit that a wide cross section of depressed patients can be studied relatively quickly. To mitigate against a local bias and to ensure an homogeneous recruitment of patients, some stratification may be necessary. Perhaps when improved methods of quantifying antidepressant efficacy emerge, the number of patients required for assessment will be reduced. At the outset, decisions about timing, details of the protocol, the data analysis required, and publications must be established between study sponsor and the collaborating investigators. In the case of a long-term study, the selection, commitment, and training of suitable personnel may need to be repeated due to a “turnover” of staff. Copeland and Gourlay (1973) have reported on the differences in the use of terms and definitions occurring even within one culture. Thus the understanding by each investigator on any special procedures and in the proper use of the chosen rating scales must be checked, unified, and cross-validated before patient recruitment commences. These measures will help reduce the variability between centers which could mask subtle differences between treatments. There is a need for strong central coordination particularly to ensure adequate communication between all members of “the team.” The central coordinator must visit each center regularly to monitor performance (Ferris and Ederer, 1979; Mowery and Williams, 1979). It is also sensible to have a coordinator at each center with responsibility for monitoring adherence to the protocol, avoiding a “drift”in performance, supervising the quality of the data being collected, and maintaining and promoting continued enthusiasm.

G. COMPLIANCE It may be incorrect to assume that patients have taken their medication as planned. Moreover, patients may not attend when required, record forms may not be completed adequately, or laboratory procedures may

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not be followed correctly. The reasons for such noncompliance may be related to many factors, such as insufficient attention to details in the planning and preparation of a study, transportation difficulties making patient attendance a problem, complex dosing regimens, inadequate labeling of the medication, or by inaccurate, complex, or incomplete instructions given to the patient by the investigator. The noncompliance might also be due to a clinically significant reason such as an unpleasant or unacceptable characteristicof the medication or even to the patient feeling better and incorrectly believing that medication may be discontinued. Even though potentially useful pharmacological effects may be obscured, the data on all patients randomized to the study should normally enter the statistical analysis for efficacy. This “pragmatic approach” or “analysis by intention to treat” is preferable, since noncompliance itself may be an integral feature of the treatment being studied and this would be relevant to its clinical usage (Schwartz and Lellouch, 1967; Schwartz et al., 1980). Provision should be made to monitor and enhance compliance. It may often be helpful to involve a member of the patient’s family A patient record card could be kept or the investigator could make a “pill”count on returned and unused medication. Unfortunately, these approaches are open to criticism, since the patient could claim an accurate drug compliance while actually having discarded unused medication before visiting the investigator. Another approach is to perform a plasma, urine, or saliva assay, although detection of low concentrations of drugs may present technical problems, and venepuncture may be ethically unacceptable if performed solely to assess compliance. Moreover, assays are open to misleading conclusions, since patients might only take medication prior to an interview in the knowledge that a check on compliance was to be performed. Assay results may also lead to false conclusions about drug compliance due to large differences reported between different subjects receiving the same dose of an antidepressant (Coppen and Perris, 1976). One strategy reported to improve compliance is to provide comprehen1976),although Myers sible information to the depressed patient (Leyet d., and Calvert (1984) showed that the cognitive or educational content is less important than an attention-placebo effect. Reasonable procedures to monitor compliance should be established, and the interpretation of study results should take into account the degree to which the study was adequately performed.

H. USEOF PLACEBO Patients referred to a psychiatrist by their general practitioner frequently enter the hospital while on medication. This may mask a valid diagnosis of the patients’ condition and hence lead to inception of an

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incorrect treatment after inclusion in a clinical study. A drug-free period is thus justified during which time a placebo might be given. This out” procedure, usually for about 7 days long, allows the initial severity of the patients’ depression to be assessed and also helps avoid possible drug interactions with the study medication. It identifies “placebo-responders: that is those patients who dramatically improve on placebo, who should not enter the active medication phase of a clinical trial. The possibility exists that a depressed patients’ clinical condition could deteriorate during the placebo “screeninglwash-out”period with the risk of suicide being a major consideration. Therefore, during the placebo period the patients’ well-being must be monitored carefully, and an “escape” to active medication must be permitted in the protocol on ethical grounds. It is possible that an apparent deterioration of a patient’s symptoms during the placebo period may not be due to the depression getting worse, but rather to symptoms, such as increased anxiety and sleep disturbance, caused by an acute benzodiazepine withdrawal phenomenon (Hallstrom and Lader, 1981; Petursson and Lader, 1981). A careful note of medication taken by the patient before consideration for the trial is therefore necessary. The use of placebo (negative control) as a parallel treatment to an established medication (positivecontrol) and a test medication is a further problem (Joyce, 1982). It can be argued that it is unethical to perform an antidepressant study without a placebo control group, since such a design cannot adequately assess the pharmacological efficacy of a test medication, particularly as a placebo response has been reported in about 30% of depressed patients (Medical Research Council, 1965). However, it may be improper to deny an established medication to patients with severe depression or who are potentially suicidal. Where doubt exists about the efficacy of the established medication, then the use of placebo would appear justified, particularly if an advantage in terms of side effects were also anticipated for the test medication. One possible compromise is in the use of an “active”placebo. This approach has been used (Russell et al., 1978; Richards et al., 1982; Ather et al., 1985)on the basis that a benzodiazepine, such as diazepam, is known to be effective in reducing the symptoms of anxiety associated with depression, but with only limited activity on the core symptoms of depression. Dose-response studies (Wittenborn, 1977), which compare fixed “high” and “low” dosages of the test medication, avoid using a placebo group altogether. However, a valid inference may only be possible if the chosen high dose is significantly more effective than the chosen low dose. I. ASSESSING SEVERITY OF DEPRESSION

To compare the efficacy of two or more treatments across different patients, clinicians, studies, and settings requires reliable means to assess

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the change in severity of the patients’ depression. Since suitable objective laboratory-based data are impossible to collect in the depressed patient, rating scales have been developed in an attempt to quantify such subjective information. The main symptoms are affective (e.g., sadness and anxiety) and biological (e.g., insomnia, lack of energy, and loss of appetite), and interview techniques are highly effective when combined with self-rating assessments. Rating scales may be completed by an observer, who will make an evaluation based on clinical judgment, or can be completed by the patient. These rating scales exist in an attempt to categorize the depressive syndrome into items to which numbers may be attributed so that a total score can be derived. Reliable assessment of the behavior of patients is easiest for the observer, who will have standards against which to evaluate the intensity of a symptom, but most difficult for the patient, who may lose insight into their own condition. Thus some symptoms, such as agitation, hypochondriasis, or depersonalization, are likely to be rated inaccurately by the patient. Therefore, self-rating scales are likely to be most useful in assessing patients with mild to moderate depression, in which full insight is retained, although they make the assumption that definitions of terms, such as “pessimism,”“mood,”or “guilt,”are stable across different cultures or across patients from different socioeconomic backgrounds. The choice of a rating scale, although often done in an arbitrary and ad hoc fashion, is critically important. It should reflect the degree of severity of the depression and be reliable both between raters and between test and retest. If a rating scale is too short, it will tend to have a low reliability, while if it is too long, it may become very difficult to use. However, for a study where the number of patients available is limited, then the scale chosen should have the highest validity and reliability, regardless of the difficulties involved, although it should not be used in isolation from other means of assessing the patient’s clinical condition. The patient should be carefully supervised initially by the investigator in the use of self-rating scales,and it is important for investigators themselves to be trained in the proper use of any scaIe chosen for a clinical study. The apparent simplicity of some rating scales makes them vulnerable to error, as they appear easy to administer and usually yield quantifiable data. Visual analog scales have been developed to circumvent the limitations in the use of language for measurement of a feeling (Clarke and Spear, 1964; Aitken, 1969). They have been reported to be particularly suitable for the measurement of change (Zealley and Aitken, 1969) and have been further validated (Luria, 1975). Basically, the visual analog scale is a continuous line 100 mm long with both ends labeled on which the rater (usually the patient) makes a mark bisecting the line to define their feelings within a chosen time frame, such as ‘‘normall$ “over the last week,”or “at

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that particular moment." A score is derived from the distance of the mark (to the nearest millimeter) from one end of the line or the other. The exact wording of the questions asked and the labels used are important, since patients and psychiatrists often mean different things by the use of certain words. The patient has only their own experience by which to judge the severity of the symptom or syndrome being rated and might overreact once they appreciate what they are expected to do. Patients may not be able to project onto the scale what they are asked to rate, and for the elderly patient, conceptualization of the scale dimensions may be impossible. It is also possible that the medication under investigation could impair the patients' ability to assess an effect. The observer scales may suffer from observer bias, particularly in the general assumption that patients are more ill before they commence a clinical trial than at the end of the study (Seldrup, 1977).Other factors, such as whether the line should be horizontal or vertical or whether the subject should have knowledge of their previous recording and use that to represent the midpoint for the current assessment (Seldrup and Beaumont, 1975), may influence the results obtained, Often such scales are developed ad hoc, so proper validation is essential. Scales assessing the overall current global severity, based on discrete categories to grade continuous phenomena, are used to represent the investigator's general impression of the severity of the patient's illness. Assessment of therapeutic or global improvement compared to the start of active treatment, also using categories, is used to monitor the progress of a patient in a clinical study. Most rating scales are prepared in a written form and should be used only if they are of a length that will engage the concentration and secure the cooperation of the depressed patient being assessed. They may be inappropriate for the elderly, those of low intelligence, or the illiterate. They cannot be used in very severe degrees of depressive illness, since patients may not be accessible to questioning or may be incapable of giving meaningful answers. ECT may be most suitable for such patients who should really be excluded from a clinical study comparing the efficacyof chemical antidepressants. The HAM-D, originally described in 1960, was revised subsequently (Hamilton, 1967) and is now widely used in clinical research. There are 21 items used to assess responses either on a three-point (e.g., middle or delayed insomnia, general or gastrointestinal somatic symptoms, genital symptoms, insight, and loss of weight) or five-point (e.g., depressed, guilt, suicide, initial insomnia, work and interests, retardation, agitation, psychic or somatic anxiety, and hypochondriasis) basis, depending on the feasibility of grading the responses coarsely or finely. The depressive symptoms are presumed to be part of a depressive syndrome and not secondary to

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some other condition such as schizophrenia. A total score is calculated based on the patient’s condition, which should not be assessed more than once a week to avoid minor fluctuations; items such as loss of weight and loss of libido are unlikely to alter in shorter periods. The total score does not always reflect the quantity or even the quality of the mood disturbance. Thus, although a total score may have decreased sufficiently to indicate an apparent improvement, highly significant symptoms of depression, such as the possibility of suicide, may actually have become more of a risk. Many items are concerned with psychomotor symptoms and on the somatization of mood, making the HAM-D more appropriate for rating endogenous depression. Cognitive disturbances are represented, but less so than are affective changes. Rarer events (or diurnal mood variation, which reflects the type of depression), such as depersonalization and derealization, obsessive-compulsive symptoms, and paranoid symptoms, are excluded, although other workers have reported these items to be useful (Guy, 1976; Lader, 1981).Several other modifications of the HAMD have appeared in the literature (Hedlund and Vieweg, 1979),so that it is important to specify which version has been used in any study. Clinical improvement has been defined by some workers as a reduction of initial score by 50% or a reduction of the total score to less than 10. In some studies, individual items have been analyzed or symptoms have been grouped into rational clusters for the purposes of defining changes in the patients’ condition (Hedlund and Vieweg, 1979).The approach to be used should have some consistency with real practice and be decided upon before the study commences. The HAM-D was designed to be administered independently by two trained raters with adequate clinical experience in assessing the severity of depression in patients already having been diagnosed as suffering from depression. When used in this way, the interrater reliability of the total score has been found to be high. It is not a diagnostic tool and was never designed to measure change, and yet the latter purpose is exactly that for which it has become a standard instrument. Bech et al. (1981) examined the consistency of the HAM-D and concluded that the original 17-item scale was inadequate. They developed a melancholia subscale (itemsof depressed mood, guilt, work and interests, retardation, psychic anxiety, and general somatic symptoms) claimed to be capable of comparing patients on a one-dimensional depression continuum, which would save the time of investigators. Other scales have been used to rate the severity of depression, with the HAM-D being the principal validating instrument, and some of the more widely used ones are considered below. A subset of items from the regular sADS make up a version designed

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to measure change. It is known as the sADS-C and is used at follow-up evaluations when sADS had been used initially for diagnostic purposes (Endicott and Spitzer, 1978). The Beck Depression Inventory (Beck et aZ., 196l), which contains items referring to symptoms and attitudes, is now administered as a self-rating scale with the subject reading the questions to themselves (Bech et al., 1975). This scale places more emphasis on cognitive or psychodynamically orientated aspects than the HAM-D, which itself emphasizes physiological correlates, with less than a third of the Beck items referring to somatic or behavioral features of depression. Since cognitive disturbances are prominent in “mild”or “neurotic”depressions and psychomotor and somatic disturbances are prominent in “severe” or “psychotic” disturbances, a self-administered scale such as the Beck may be a more accurate measure in the neurotically depressed, while the HAM-D may be more appropriate in the psychotically depressed (Prusoff et al., 1972). Bech et al. (1975) found that the total scores of the HAM-D and Beck scales were comparable both for baseline ratings and for change ratings, although both scales failed to differentiate adequately between moderate and severe depression measured by a global clinical assessment. Bailey and Coppen (1976) reported that HAM-D and Beck scales compared well in about two-thirds of the depressed patients studied, but that the Beck showed less percentage change over time than did the HAM-D. The Wakefield self-assessmentdepression inventory (Snaith et al., 1971) was derived from the Zung self-rating depression scale (Zung, 1965),which lacks items on guilt, insight, and retardation, and has been reported to correlate well with the HAM-D. The Wakefield has the merit of brevity and simplicity with 12 questions each being scored 0, 1, 2, or 3, according to the response obtained. However, Kearns et al. (1982) have suggested that the Wakefield inventory should be discarded as a research instrument, since its ability to discriminate between different grades of severity was found to be low when compared with several available depression rating scales. In fact the Wakefield self-assessmentdepression inventory has been superseded. An item analysis and the addition of 10 further items has resulted in the establishment of the Leeds scales for measurement of depression and of anxiety (Snaith et al., 1976).Zigmond and Snaith (1983) recently developed a self-assessment mood scale specifically for detecting and assessing the severity of depression and of anxiety in the setting of a hospital medical outpatient clinic. The concepts in emotional and somatic illness were carefully separated, and the scale is thus unaffected by the presence of bodily illness. The modified Zung (1974)self-rating depression scale contains 20 items, has good concurrent validity with the HAM-D for mildly to moderately depressed patients, but not so for the more severely depressed patient (Carroll et al., 1973). This may be attributable to the

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lack of such items as retardation, guilt feelings, and hypochondriasis in the Zung scale, although it is doubtful whether patients with marked features of this kind are capable of rating themselves accurately (Lader, 1981). A rating instrument developed recently to assess the patients’response to treatment and concerned exclusively with the psychic symptoms of depressive illness (i.e., the patient’s report of mood) is the MontgomeryAsberg scale (Montgomery and Asberg, 1979). It is derived from the comprehensive psychopathological rating scale (Asberg et al., 1978) and comprises the 10 items found to show the largest change with treatment and the highest correlations to overall change. The items chosen for the rating scale are (1) apparent sadness, (2) reported sadness, (3) inner tension, (4)reduced sleep, (5) reduced appetite, (6) concentration difficulties, (7) lassitude, (8) inability to feel, (9) pessimistic thoughts, and (10) suicidal thoughts. The rating itself is based on a clinical interview, moving from broadly phrased questions about symptoms to more detailed ones which aIlow a recise rating of severity. For each of the 10 items on the Montgomery- sberg scale, the rater must decide whether the rating lies on the defined scale steps (0,2,4, or 6) or between them (1,3, or 5). There are clear definitionsguiding the rater as to the definition of each symptom and examples of the value to be ascribed to a particular rating. It is claimed that the Montgomery-Asberg scale is a more precise measure of change than the HAM-D so that significant differences between treatments might be revealed with smaller numbers of patients. This is an important ethical point, since it would mean that fewer patients will need to be exposed to possibly inferior treatment. In a comparative study, this rating scale performed about equally as well as the established HAM-D (Kearns et al., 1982). The two scales, however, have different applications in clinical trial work with, for example, the use of the HAMD being more appropriate when a broad assessment of the psychic, behavioral, and somatic features of depression is important. On the other hand, the Montgomery-Asberg scale, which is exclusively concerned with the psychic symptoms of depressive illness, is particularly suited for investigations on patients suffering from concurrent physical illness or who are likely to experience marked somatic side effects from the antidepressant being tested or from a concurrent medication. Unlike the HAM-D, the Montgomery-Asberg scale is suitable for rating the severity of depression at time intervals of less than 1 week. Hence it is a useful instrument for assessing the onset of action of an antidepressant in which relatively frequent measurement of the patients’ condition is indicated early on during active treatment. Computers are now in regular use for assessment of psychiatric pa-

x

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tients. An automated system modified for the self-rating of depression which utilizes a microcomputer has been developed (Carr et al., 1981). It is based on the HAM-D, since existing scales tended to concentrate on mood disturbance. The computer displays the questions asked by the investigator with three to five possible answers. Such computerized techniques enable the collection of information from patients to include “delicate” questions, such as about suicide or sex, and encourages patients to admit to symptoms and feelings that would perhaps otherwise be denied. Also it enhances the speed of data collection and processing, enables an investigator to monitor relatively large numbers of patients reliably and accurately, and establishes a standardized format between centers. The system is simple enough for relatively untrained staff to operate and supervise. Since data are stored on small disks with distant computers not being involved, the information collected remains confidential. Cost is still a limiting factor to routine use in large multicenter studies, and the production of “numbersby a computer” may appear to be more convincing than a simple rating scale. The value of an experienced clinicaljudgment is still a powerful instrument for measuring changes in the severity of depression. Although there may be professional antipathy from experiences with mainframe computers (Carr and Ancill, 1983), this new technical development is likely to be useful for taking the patients’ history, assessing the severity of depression, and evaluating new rating scales. Work on psychological, physiological, biochemical, and/or pharmacological changes during depression may help to identify a test, or group of tests, which will provide specific and objective measurements of the patients’ condition. Meanwhile, there being no ideal scale for antidepressive research which would be suitable for all kinds of patients and situations, well-validated and familiar scales known to be reliable should be used with new ones (for example the Hospital Anxiety and Depression Scale; Zigmond and Snaith, 1983, for patients being assessed in a general medical clinic) also being tried out. There is a real problem in choosing any particular rating instrument for a trial to quantify depression in view of the large number available with each having their own individual sources of error. In general, it is sensible for both investigator and patient rated scales to be used for the assessment of the severity of depression in a clinical trial. At least two appropriate investigator rated scales should be used and complemented by a patient rated scale, a global scale, andlor a visual analog scale. The choice of investigator scales depends on factors such as the severity of the depression as well as whether assessment of psychomotor symptoms and somatic aspects of mood and/or cognitive aspects is important to achieve the study aim. Thus, whereas the HAM-D is particularly useful for the former, other scales such as the Beck may be

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more useful for the latter purpose. Finally, since the accurate completion of rating scales is essential for the success of antidepressant studies, particular attention should be paid to the careful design of clear and simple record forms and with the provision of grading cards to assist in their correct completion. Factors, such as the print chosen, the layout adopted, and the phrasing of questions, particularly for visual analog scales designed on an ad hoc basis, are all relevant factors (Wright and Haybittle, 1979). J. ASSESSING ADVERSEEVENTS Although antidepressants are well tolerated by most patients, they can produce undesirable effects. This may lead to poor patient compliance resulting in an apparent treatment failure. The severity of side effects usually diminish after the first few days of treatment, and the outcome of a study could be affected if this possibility is explained to the patient. Assessing side effects is notoriously difficult, since many preexist a study as symptoms of the depression itself (e.g., fatigue, dry mouth, dizziness, nausea, and drowsiness)or emerge during treatment without being drug related (Klein at al., 1981). The use of a placebo-treated control group is, therefore, important, although symptomatic complaints may be reported even in healthy volunteers taking placebo (Green, 1964; Reidenberg and Lowenthal, 1968). Nevertheless, the use of a placebo allows assessment of the extent to which possible side effects are drug induced. However, although comparisons with a placebo remain the most useful approach to assess the incidence, severity, and frequency of side effects, this technique provokes both ethical and legal problems (Lasagna, 1979). It should be remembered that, although drug-drug interactions may be minimized by the use of exclusion criteria, when a patient enters a clinical study, the recent (prestudy) withdrawal of a medication might itself precipitate an adverse reaction. Moreover, for hospital inpatients, there is always the possibility that communication between patients may produce a “halo” effect with an “epidemic-like spread of reports of an adverse effect (Lasagna, 1981). These considerations emphasize the importance of carefully noting recent medication and preexisting complaints and of using controls to evaluate adverse reactions which occur during a clinical study. There are two fundamental approaches to eliciting adverse subjective feelings in a clinical study (Lasagna, 1981).First, an open-ended question such as, “Did you experience any problem with the medication you took?”, which will evoke a volunteered response. This approach tends to elicit the

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more important side effects. Second, a checklist with specifically anticipated events (and also a few not to be anticipated, as a control), plus an open-ended unspecified category, may be administered. This latter approach has the disadvantage of collecting some relatively trivial data, probably of no clinical significance, although one cannot be certain about this in the absence of a placebo control. It has been suggested that both types of approaches should be used with side effects being rated on severity (1, mild; 2, moderate; 3, severe; and 4,very severe), frequency (1, once; 2, infrequently; 3, often; and 4, continually), and also on the investigator’s opinion regarding the relationship of the event to medication (0, not related; 1, possibly related; and 2, definitely related). Since all these factors have mutual effects, a total side-effects score may be derived by multiplying together the individual scores obtained (Priest, 1976; Pugh et al., 1982) to represent the overall intensity, persistence, and relevance of any given side effect.

K. STATISTICAL CONSIDERATIONS There are two statistically valid options in designing a clinical study, namely, to perform a sequential analysis or to use a fixed sample size determined in advance. A sequential analysis is not generally suitable for antidepressant studies, since there is not usually one major criterion on which to choose a treatment success or a failure. Consequently, a fixed sample size is used which, for ethical and economic reasons, should be the minimum number of cases required to achieve a valid conclusion. Determination of sample size involves the statistical concepts of Type I and Type I1 errors, the probability of the statistical errors (aand f3, respectively), and the size of the therapeutic effect regarded as clinically important. The Type I error arises when it is incorrectly claimed that the treatments differ (i.e., the null hypothesis is true and the investigator rejects it incorrectly). The Type I1 error occurs when the treatments actually do differ, but the clinical study failed to demonstrate this difference, (i.e., the null hypothesis is false, and the investigator fails to reject it). Unlike the a-probability, which defines the probability of incorrectly rejecting the null hypothesis and which can be specified exactly for each experiment, the @-probabilitycan only be specified by making assumptions about the size of the experimental effect. It is the “power” (defined as 1 - p) or sensitivity of an experiment which expresses the probability that a study will find a treatment difference, if one exists. The precision of a clinical trial can be enhanced by increasing the aprobability level, thus changing the magnitude of the effect under study,

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or by increasing the group size. The investigator is normally unwilling to increase the a-probability level beyond the conventional maximum and accepted value of 5% although it may sometimes be possible to enhance the apparent magnitude of an effect under study by reducing uncontrolled sources of variability, such as the biological heterogenicity in diagnostic groups (Buchsbaum and Rieder, 1979). It is the manipulation of sample size which is the usual way of increasing the power of a clinical study, having first made a value judgment on the required difference to be regarded as clinically relevant. In general, the smaller the difference judged to be clinically significant, the greater the number of patients required. However, in practice, the size of a clinical trial is a compromise between statisticalconsiderations, particularly the required power (usually 80% is suitable) to find the difference decided upon as being clinically significant, and practical considerations, such as the budget, manpower, time constraints, and the anticipated recruitment rate (Altman, 1980, Gore, 1981). It seems to be common experience that the latter is likely to be an overestimation of the truth, and yet, inappropriate enthusiasm is not unknown to result in a study being initiated at a center where the required number of patients cannot actually be recruited in a reasonable time period. Hence a more realistic recruitment rate should be established by monitoring the situation during the period that the study is being setup. If it is discovered that sufficient patient numbers are not available, then clearly the study should not be performed or a multicenter approach be employed. Although the actual power chosen for a particular study is arbitrary rather than optimal (Rothpearl et al., 1981),the various considerations involved in deciding its value help avoid serious problems of interpretation between statistical significance and clinical importance when the study results become available. Since antidepressant studies involve evaluations at regular intervals over several weeks, much data are generated. Separate significance tests should not be performed at each time point, since such multiple testing will lead to confusion and probably spurious conclusions. Pocock (1983) illustrated this point by citing a published example involving 45 hospitalized patients with primary depression in which over 100 significance tests had been used to describe the results. It is more appropriate to use an analysis of variance technique with appropriate significance tests being applied only to identify sources of statistical difference if an overall difference is found. Alternatively,a two sample t-test for treatment difference between scores at baseline value and the final assessment or baselins value and the last two assessments divided by two could be used. The latter option avoids placing too much emphasis on the score obtained on the final day. A further possibility, which gives equal "weight" to each time

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point, is to use the difference between baseline score and the mean of all scores on treatment. Whichever approach is to be used, the decision should be taken before the study commences, and this should be specified clearly in the protocol.

V.

Conclusions

Ten years ago, Leonard (1975) reviewed the possible neurochemical basis of depression and the neuropharmacologicalaspects of its treatment. In the past decade, several conceptual leaps have been made with respect to our understanding the possible etiology of the disease. In experimental studies, this is reflected in the change of emphasis from behavioral and neurochemical studies following the acute administration of antidepressant drugs to chronic studies in which subtle changes in neurotransmitter function have been assessed in discrete brain regions. The major advantage of this approach lies in the correlation between the changes in neurotransmitter receptor function and the onset of the response to treatment. Whether these associations are causally or coincidentally related still remains to be proved. The second major conceptual advance has involved studies of receptor function in depressed patients both preceding and following antidepressant treatment. Despite the previous enthusiasm for studies in which amine metabolites were determined in the cerebrospinal fluid, details of which were reviewed by Leonard (1975), the conclusions based on such studies have been at best equivocal. Recent studies on postmortem brains from suicide victims have also failed to provide convincing evidence in favor of specific and discrete changes in neurotransmitter functions. Such studies are likely to be of only limited value until a mechanism is developed for the rapid analysis of receptor activity and neurotransmitter concentrations shortly after the death of adequately diagnosed patients. The neuropharmacologist is, therefore, left with studying peripheral markers of neurotransmitter function (as exemplified by changes in the endocrine responsiveness,pharmacological responses to the administration of indirectly acting sympathomimetic arnines, changes in receptor activity on platelets and lymphocytes, and in neurotransmitter uptake into platelets) in the hope that the results reflect neurotransmitter function in the brain of the patient. The results from the changes in ['H]serotonin uptake into platelets and in the amine receptor responsiveness on the platelet membrane (as assessed by determining the aggregatory rate to serotonin and noradrenaline) appear to provide the experimenter with useful state-dependent markers of depression.

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The fact that the reduction in the serotonin-uptake rate into platelets of depressed patients has been reported by investigators in different countries may lead to a useful biochemical test for depression. While more sophisticated experimental and clinical studies have been undertaken in the last decade, uncertainty surrounding the precise mode of action of antidepressant drugs has increased largely due to the realization that any change in one pathway will initiate a change in all other pathways with which it is in contact. These interrelationships in the neurotransmitter systems subserving the limbic regions, cortex, nucleus accumbens, and the basal ganglia of the rat brain have been reviewed by Leonard (1984). The recent establishment of internationally approved criteria for the diagnosis of depression has undoubtedly been of major technical importance in a field in which objective data are virtually impossible to collect. The widespread use of effective antidepressant drugs has helped to restrict the number of patients being hospitalized to those who are more severely ill and/or who are nonresponsive to usual drug treatment. Therefore, most clinical trials of antidepressants are, of necessity conducted initially on hospitalized depressed patients. This trend could lead to an atypical subpopulation of depressed patients being selected for study. Perhaps it is more relevant to study depressed nonhospitalized patients, since this is where most patients with depression are found initially. Since there is no ideal rating scale suitable to quantify depression in all types of patient and different situations, it is important to include well-validated and familiar scales. In the future, the use of computers to assess depression is likely to become more commonplace. Despite all the difficulties that have arisen in proposing a comprehensive hypothesis of depression, a disorder in neurotransmitter function in which the biogenic amines are the prime candidates would still seem to be the most reasonable hypothesis available. Perhaps the next decade will provide new insights into the neurobiology of depression that will enable a more valid conclusion to be reached. References

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