Prq Nan-Psychophammco 1. & BioL Psychtat. 1991. Vol. 15. pp. 357-367 0276-5646/91 $0.00 + .50

Prtnted In Great Britain. All rights reserved 0 1991 Pergamon Press plc

EARLY DECREASE IN DENSITY OF MONONUCLEAR LEUKOCYTE f3-ADRENOCEPTORS IN DEPRESSED

PATIENTS FOLLOWING AMINEPTINE TREATMENT: POSSIBLE RELATION TO CLINICAL EFFICIENCY

PASCALE MAZZOLAl, REGINE JEANNINGROSl, JEAN M. AZORIN2,

LAURENCE ALIGNE2 and RENE TISSOTll 2

'Unite de Psychiatric Biologique, C.N.R.S., Marseille, France.

'Clinique de Psychiatric et de Psychologie Medicale, C.H.U. Timone, Marseille, France.

(Final form, September 1990)

Mazzola, Pascale, Regine Jeanningros , Jean M. Azorin , Laurence Align& and Rene Tissot : Early decrease in density of mononuclear leukocyte i3- adrenoceptors in depressed patients following amineptine treatment: Possible relation to clinical efficiency. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1991, =:357-367.

1. The effect of chronic amineptine treatment (2OOmg/day) on iS-adrenoceptor density of intact mononuclear leukocytes ( MNL 1 was examined in unmedicated major unipolar depressed patients.

2. Pretreatment parameters of (-I- [12511 -iodopindolol specific binding did not differ significantly from age- and sex-matched healthy controls as the patients were only moderately depressed.

3. All patients showed a highly significant clinical improvement as assessed by the AMDP-depression scale after one week of amineptine (D7), while 2 patients relapsed after one month of treatment (D28) and were considered to be non-responders.

4. The maximal density of %-adrenoceptors ( BIMX 1 was significantly decreased at D7 (by 33%) compared to pretreatment level ( DO ) in the treatment responders and remained lower at D28, although the difference was no longer significant. No alteration in B-receptor affinity (Kd) was detected during the treatment.

5. These results indicate that treatment with amineptine, an anti- depressant drug known to selectively inhibit the dopamine uptake system, can rapidly affect MNL iS-adrenoceptors.

6. Moreover, the present findings show that the reduction in MNL is- adrenoceptor density, which is associated with a stable clinical improvement, may provide a predictive index for successful anti- depressant treatment.

uvwora: Amineptine treatment, B-adrenoceptor, Depression, Mononuclear leukocyte

. . Abbrevlatlons: AMDP Depression scale (AMDP-D), day (D), maximal number of binding sites (Bmax), mononuclear leukocytes (MNL), platelet poor plasma (PPP)

357

358 P. Mazzola etal.

Amineptine is a tricyclic antidepressant drug with peculiar biochemical

properties. Unlike most other tricyclic agents, amineptine selectively

inhibits dopamine uptake from synaptosomal preparations of rat cortex (Ceci

et al., 1986; Bonnet et al., 1987). Chronic treatment with amineptine

induced down-regulation of B-adrenoceptors in the rat cortex, a feature

common to the majority of antidepressant agents which has been postulated

related to their clinical efficiency (Sulser et al., 1978; Maj et al.,

1984). In the absence of direct methods for studying l3-receptors in the

human central nervous system, it has been demonstrated that leukocytes

provide a useful peripheral model for investigating S-receptors in

depressive illness (Bunney and Murphy, 1975; Landman et al., 1983; Pandey

et al., 1987). A decrease in lymphocyte &-adrenoceptor density in

untreated depressed patients has been found when compared to healthy

controls (Extein et a1.,1979; Pandey et al.,1985 and 1987; Carsten et al.,

1987; Magliozzi et al,. 1989). Recently, we have found a significantly

lower number of iodopindolol binding sites in intact mononuclear leukocytes

in patients suffering from major unipolar depression. This decrease in

maximal binding was correlated with symptom severity in different DSM-III

depression subtypes (Jeanningros et al., 1990). So far, only one report

has been made on the effect of antidepressant treatment on peripheral 15-

adrenoceptors. Trazodone or amitriptyline have been shown to induce a

decrease in lymphocyte &-adrenoceptor binding in depressed Patients who

responded to treatment (Healy et al., 1983 and 1985).

In this study, we investigated the influence of chronic treatment with

amineptine, a drug known to rapidly improve different types of depressive

syndromes, on intact MNL B-adrenoceptors in unipolar major depressed

patients. In addition, the affinity of amineptine for MNL i3-adrenoceptors

was studied both in patients before treatment and in sex- and age-matched

controls.

Ten major unipolar depressed patients (6 men and 4 women) diagnosed

according to the DSM-III 'criteria (American Psychiatric Association 1980)

Leukocyte p-receptors and amineptlne treatment 359

with a mean age (f S.E.M.) of 44.1 f 3.4 (range: 30 to 66 years) gave

informed consent to the ethically approved research procedures. All

depressed patients had a AMDP-D score of f0 or more with a mean score f

S.E.M. = 26.7 f 3.4. Criteria for exclusion were medical illnesses such as

asthma , congestive heart failure, hypertension, atopic eczema or the use

of drugs (e.g. steroids, %-agonists and E-antagonists) that may alter beta-

receptor function. The control population consisted of ten physically and

mentally healthy volunteers, matched for age and sex.

All patients were treated with amineptine (200 mg/day) after an

antidepressant wash-out period of 10 days, and received no other

psychotropic medication, except benzodiazepines (equivalent to 5-20 mg

Diazepam).

The clinical state of the depressed patients was evaluated using the

depression items from the AMDP scale (AMlIP-D) before treatment and after

the first and the fourth week of treatment. Patients whose scores at the

end of the trial period had been lowered by at least 50% from their

pretreatment level, were considered as responders. Two patients stopped the

treatment before day 28 on their request although one of them had clearly

improved . Upon entering the study, both patients and volunteers underwent

mononuclear leukocyte S-receptor determination. Following the onset of

amineptine treatment, binding assessments were carried out at day 7 (D7)

and day 28 (D28).

Between 7:30-8:00 a.m., after the subjects had rested in bed for 30 min,

blood samples (50 ml) were withdrawn into glass tubes, anticoagulated with

heparin (10 UI/ml), and immediately processed at room twerature. After a

first centrifugation at 215 x g for 10 min, the pellet was preserved and

the platelet rich plasma layer was carefully aspirated and centrifuged at

2,000 x g for 15 min to produce platelet poor plasma (PPP). PPP and the

pellet were pooled, diluted 1:2 with 20mM Hepes, 154 mM NaCl, pH 7.5 and

then carefully layered on Ficoll-Hypaque gradient (density 1.077 g/ml:

Sigma). The mononuclear' leukocytes were isolated by centrifugation

360 P. Mazzola et al.

according to Boyum's method (1968). Finally, the cells were washed with the

same buffer and counted in a Neubauer cell. The lymphocyte yield from this

isolation procedure was greater than 85% with less than 3.5 platelets per

MNL. Cell viability as demonstrated by tryptan blue exclusion was greater

than 95%.

MNL cells ( 2 x 105 - 6 x lo5 ) were incubated in duplicate in

siliconized polypropylene tubes. The incubation buffer consisted of 8mM

Hepes, 62 mM NaCl, pH 7.35 containing 9 concentrations varying from 12 to

380 pM (-) [lz51] -iodopindolol with or without 1 p (-)propranolol to

define non-specific binding. Samples were incubated for 30 min at 37 'C,

then the incubates were diluted by adding 3ml of 154 mM NaCl containing

1OmM Tris HCl, pH 7.4 at 4OC and rapidly filtered through Wathman GF/C

filters. The incubation tubes and the filters were washed with 3ml or 6 ml

of the ice cold buffer respectively. The binding fulfilled conventional

criteria for saturability. The saturation data were regressed as specific

bound/free versus bound by Scatchard analysis (Scatchard, 1949). The

regression coefficients were above 0.90, indicating monophasic Scatchard

plots. Specific binding was at least 75% of total binding at the Kd value.

Inhibition by amineptine (1 pM - 0.3 mM) of 280 pM (-) [12511iodopindolol

binding was assessed on intact mononuclear leukocytes in depressed patients

and controls. IC,, values (concentration that inhibits 50% of specific

binding) were calculated by computer analysis.

The Wilcoxon T test was used for comparisons between paired samples.

Correlation was evaluated by the Spear-man Rank correlation.

Before treatment, the mean Bmax value of [12sIl -iodopindolol binding in

the depressed patients was the same as that for the age- and sex-matched

controls both in the responders and non-responders.

Leukocyte p-receptorsand amineptinetreatment 361

Table 1

Depression Evaluation and Iodopindolol Binding on MNh Cells

in Patients before and during Amineptine Treatment.

AMDP-D score Bmax (sites/cell)

Controls

(n=lO) 1835 f 153 -

All depressed

DO (n=lO)

D7 (n=lO)

D28 (n=8)

26.7 f 3.4 1653 f 150

10.6 f 1.5' 1596 f 247

12.7 f 4.Sa 1344 f 260

Responders

DO (n=7)

D7 (n=7)

D28 (n=6)

28.4 f 4.2

10.3 f 2.lb

6.3 f 1.6a

1818 f 163

1205 f 120agd

1383 f 344

Non-responders

DO (n=3)

D7 (n=3)

D28 (n=2)

22.7 f 6.6 1270 f 214

11.3 f 2.0 2509 f 470

32.0 f7.0 1228 f 319

All data are mean f S.E.M.

Scores were evaluated using the depression items from the AMDP scale.

a p<0.03; b p<0.02; c ~~0.01, significantly different from DO value;d p<0.03

significantly different from control value, Wilcoxon T test.

After the first week of treatment, all the patients had a clear clinical

improvement and mean AMDP-D score decreased by 60% compared to the

pretreatment value. However, out of the 8 patients who underwent the

treatment for 4 weeks, 6 patients fully improved, while 2 patients relapsed

and were considered to be non-responders (Fig. 1).

When all the depressed patients were considered, mean Bmax value at D7

and D28 of amineptine administration did not differ significantly from the

362 P. Mazola etal.

baseline level _ However, at D7, the responders exhibited a large and

significant decrease in Bmax f34%), while the non-responders showed a non-

significant Bmax increase (49%) compared to their baseline level (Table 11.

It is worth noting that non-responders had the lowest pretreatment Bmax

values (Fig. 21.11-1 all patients, individual changes in ANDP-D score after

one month of treatment were highly correlated with respective Bmax changes

during the first week of amineptine treatment (Fig. 3).

Bmax Values at D28 were lowered by 24% in the responders compared to DO

values but the difference did not reach statistical significance. Two

patients exhibited a large increase in Bmax between D'7 and D28 (Fig. 2).

Kd values in depressed patients before treatment did not differ from the

age-and sex-matched controls ( mean f S.E.M. = 43.8 f 10.1 vs. 47.6 f 7.6

pM, n=lO, respectively, NS) and remained stable throughout the treatment.

Amineptine failed to inhibit I-) 112511 -iodopindolol binding, even at a

concentration of 0.3 mM, and the IC50 value could not be determined.

The unaltered maximal density of [1251]-iodopindolol binding sites on

mononuclear leukocytes observed in moderately unipolar depressed patients

before treatment compared to controls is in line with our previous

studies. We have shown that the decrease in the number of S2-adrenoceptors

depends on depression severity rather than on its nosographical adherence

(Jeanningros et al., in press). The advantages of using both iodopindolol

as a ligand and intact cells to assess S-adrenoceptor binding in leukocytes

have been discussed in this recent report.

This is the first report of an early decrease in f52-adrenoceptor density

in intact mononuclear leukocytes from depressed patients successfully

treated with amineptine, a selective dopamine uptake inhibitor. The

rapidity of this peripheral effect contrasts with the usual long latency

observed at the central level in Rat following chronic antidepressant

Leukocyte p-receptors and amineptine treatment 363

50

40

E

8 m 30

cl

d, 20

0 0 7 14 21 28

Days of treatment

Fig-l. Individual variations of AMDP rating scores for depression in

depressed patients before treatment (n=lO), and after the first (n=lO) and

the fourth (n-8) week of amineptine treatment. At D7 and at D28, the AMDP-D

scores were significantly lowered as compared to the DO (p<O.Ol and pCO.03,

respectively, Wilcoxon T test). Black triangles represent the treatment

responders and white squares represent non-responders.

z

1300-

In

300 : I I I 1 0 7 14 21 28

Days of treatment

Fig.2. Individual variations of Bmax values in depressed patients before

treatment (n=lO) and after the first( n=lO), and the fourth (n=e) week of

amineptine treatment (ZOOxng/day). At D7, Bmax value of treatment responders

(black triangles) were significantly lowered (pCO.03, Wilcoxon T test). The

non-responders are represented by white squares.

P. Mazzola et al.

r= 0.82; ~~0.01; n=8

-80 -60 40 -20 0 20 40 60 80 100 120

Bmax variation (%I from DO to D7

Fig.3. Relationship between early Bmax changes (after one week of

amineptine treatment) and late AMDP-D score changes ( after one month of

treatment) in depressed patients. Data are relative variations in %.

administration. with amineptine, cortical B-adrenoceptor number decreased

after 2 weeks (Ceci et al., 1986). However, it is unlikely that changes in

circulating &-receptors reflect changes associated with g-receptors at the

central level. In a recent study, Chalezka-Franazeck and Vetulani (1986)

reported that chronic administration of imipramine in rat for 2

months did not affect [3H] -dihydroalprenolol binding to lymphocyte

membranes, though this treatment reduced such binding to the cerebral

cortex.

The few reports on the influence of antidepressant therapies on leukocyte

IS-receptor density have led to inconsistent results. Cooper et al., (1985)

failed to find any significant difference in the number of lymphocyte [3H]-

dihydroalprenolol binding sites after ECT therapy even when the depressed

patients recovered. In contrast, Healy et al., (1983 and 1985) showed that

a three week administration of trazodone or amitryptiline reduced the

density of lymphocyte &-adrenoceptors in moderately depressed patients.

In agreement with the present results, this decrease was only observed in

Leukocyte p-receptors and amineptine treatment 365

treatment responders. However, Bmax changes in the Healy's study only

occured after 3 weeks of treatment and lasted more than 6 weeks. This

difference in the time course of the antidepressant effect could depend on

the action pattern of the antidepressant drug used. Amineptine is

characterized by the rapidity of its clinical efficiency. The Emax decrease

occurred after the first week of amineptine administration as seen by high

patient improvement. After one month of amineptine, Bmax changes were not

similar in all the responders, despite their full clinical improvement.

Five responders exhibited a Bmax decrease and 3 exhibited a Bmax increase

between D7 and D28.

The drastic decrease seen after seven days of amineptine treatment was

only observed in patients who exhibited a stable clinical improvement after

one month of treatment. The non-responders failed to show a similar change

despite a clear but transient improvement at D7. The fact that in all

depressed patients the variation of their clinical state following one

month of treatment was linearly proportional to changes in their i3-receptor

density after one week of treatment is a crucial point. Indeed, the early

reduction in MNL %-adrenoceptor number could be an easily determinable

predictive index for successful antidepressant treatment. Further data are

needed to confirm these results and to examine what parameters may

influence the time course of this effect, e.g. the type of the

antidepressant agent, nosographical category or depression severity.

The present data show that chronic treatment of depressed patients with

amineptine, a drug known to selectively inhibit the dopamine uptake system,

can rapidly decrease the maximal number of mononuclear leukocyte i3-

adrenoceptors. This peripheral effect, which was associated with a stable

clinical improvement, could represent a predictive index of therapeutic

success.

This research was supported by Servier Laboratories of France. The

authors wish to thank H&&ne Rescigno for her technical collaboration.

366 P. Mazzola eta1

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Inquiries and reprint request should be addressed to :

R&ine Jeanningros, Ph. D. Unite de psychiatric biologique, C.N.R.S. Faculte de medecine 27 Bd Jean moulin 13385 Marseille cedex 5, France