Changes in platelet 3H-imipramine binding with chronic imipramine treatment are not state-dependent

478

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Changes in Platelet 3H-Imipramine Binding with Chronic Imipramine Treatment Are Not State-Dependent Edward M. DeMet, Robert H. Gemer, Kate M. Bell, Curtis D. Kauffmann, Aleksandra Chicz-DeMet, and Stacey Warren

One month of imipramine treatment increased both the Kd and B,, of platelet -‘Himipramine binding in I1 endogenous unipolar depressed patients. Continued treatment (13 weeks) of 5 patients subsequentl~l lowered the B,, values of 2 patients who had initially shown the largest increases, so that binding was no longer signi~cant~y elevated after 13 weeks. The observed hinges in I(d but not in B,,, could be explained by the carryover of tightly bound drug to the binding assay, although neither of the measures were correlated with plasma imipramine levels, Posttreatment B,, {4 weeks) values were inversely related to plasma cortisol levels, although a weak but positive correlation was found before treatment. No signijicant change was found in plasma cortisol with treatment. Clinical responses were not related to cortisol or B,, changes, although optimal improvement was associated with extreme values (high and low) of pretreatment B,,,. The present results, obtained with imipramine, and similar results obtained after nortriptyline and electroconvulsive shock by others, suggest that at least some antidepressants ma) induce transient changes in the B,, of platelet binding that are independent qf qflective state.

Introduction Since the original proposal that platelet 3H-imipramine (“H-IMI) binding may be a specific biological marker of affective disorders (Langer et al. 1980), many studies have confirmed that the number of binding sites (B,,,) is reduced in major depression (Asarch et al. 1980; Briley et al. 1980; Paul et al. I98 I), although others have not (Mellerup et al. 1982; Whittaker et al. 1984). The question of whether B,,, differences in depressed patients reflect a genetic trait or are due to changes in affective state is less clear. Trait specificity is generally supported by studies that have found decreased binding in specific

From the Department of Psychiatry and Human Behavior, University of California. Irvine (E.M.D.. K.M.B., A.C.-D.,S.W.); the Psychiatry Service, VA Medical Center. West Los Angeles. Los Angeles, (R.H.G.): and the Psychiatry Sew&, VA Medical Center, Long Beach, CA (C.D.K.1. Supported in pan by NIMH Grant ROI-MH40468. Address cowespondence to Dr. Edward DeMet, Department of Psychiatry and Human Behavior. Medical Sciences Bldg. I, Rm. D435. University of California, Irvine, CA 92717. Received June I, 1988: revised December 14. 1988

‘0 198Y Society of Biological

Psychiatry

0006-7223189/SO3,50

Non-State-Dependent

Changes

in 3H-1MI Binding

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depressive subtypes (Lewis and Mc~hesney 1985; Baron et al. 1986) or in diseases associated with a risk of major depression, e.g., anorexia nervosa (Weizman et al. i986a) or nocturnal enuresis (Weizman et al. 1986b), as opposed to nonfamiliai depression (Lewis and McChesney 1985) or other psychiatric disorders, e.g., alcoholic disease (Ahtee et al. 1981), Alzheimer’s dementia (Suranyi-Cadotte et al. 1985a), schizophrenia (Wood et al. 1983), and hypertension (Kamal et al. 1984). A twin study failed to show an increased concordance of B,,, values in monozygotic versus dizygotic twins, but only studied normal controls and cannot be generalized to depressed patients (Fried1 and Propping 1984). Studies that have examined the state dependency of B,,, values have been inconclusive. Electroconvulsive therapy (ECT) has been reported to improve symptoms, but to have no effect on platelet binding, although the same patients had normal B,,, values when sampled as euth~ics a year later (Langer et al. 1986). Conversely, others found increased B,,, values within 1 week of ECT, which had returned to baseline when resampled after a year (Wagner et al. 1987). Several studies have followed state changes after treatment with drugs. Two to three weeks of treatment with imipramine, amitriptyline, or maprotyline reduce depressive symptoms with no change in platelet binding (Langer et al. 1982; Langer and Raisman 1983; Suranyi-Cadotte et al. 1982, 1985b), although the latter found that B,,, values were increased to normal levels with complete symptomatic recovery and the values remained elevated for 4 weeks after imipramine discontinuation. in contrast, others have noted B,,, increases after as little as 1 week treatment with alaproclate, zimelidine, and nortriptyline, with a return to baseline after 3 weeks of nortriptyline (Wagner et al. 1987). Desipramine has been claimed to produce a state-dependent increase in &,,, after 17-28 days (Arora and Meltzer 1988). However, similar B,,, increases have also been reported in normal controls after 16 days of desipranline (Cowen et al. 1986), which were presumably independent of affective state. Another study also found B,,, increases in normal controls treated with amitriptyline (Braddock et al. 1984). Although B,,, increases in control subjects appear to be state independent, recent studies suggest that platelet imipramine binding may be regulated by plasma cortisol levels (Arora and Meltzer 1986; Roy et al. 1987), which in turn may be altered by antidepressant treatment in both patients and controls. AS hypercortisolemia is common in depression (Carroll et al. 1981) and tends to normalize in remission (Roy et al. 1984), commonly observed increases in patient B,,, values after treatment may be directly related to changes in affective state. The present study compares platelet binding, plasma cortisol levels, and clinical measures of affective state in unipolar depressed patients prior to and following chronic imipramine treatment.

Methods Subjects were 8 male and 3 female psychiatric inpatients in the University of California Irvine Medical Center and in the VA hospitals at Long Beach and West Los Angeles, CA, who had remained free of psychiatric medication for at least 4 weeks prior to testing. The average age was 37.5 +- 11.0 SD years (range 18-55 years). All of the patients satisfied DSM-IIIR and Research Diagnostic Criteria (RDC) for endogenous unipolar depression (with melancholia) and had initial H~ilton depression scores greater than 20 on the 17-question scale (HamiIton 1967). A comparison group of 11 normal controls was matched to the patient sample with regard to gender and time of year (within 1

BIOL PSYCHIATRY 1989;25:478-488

E.M. &Met et al.

month) and was approximately matched with regard to age (mean age 32.5 k 6-7 year% range 21-40 ye&. Normal controls had no personal DSM-IIIR axis 1 or axis 2 disorders and had no first-degree relatives with an axis 1 disorder. Both patients and controls were excluded who had physical impairments or histories of alcohol or drug abuse. Informed consent was obtained from all participants. Whole blood samples were collected between 9:00 and IO:00 AM by antecubital venipuncture using acid-citrate-dextrose (ACD) as the anticoagulant. Platelets were isolated by the method of Corash (1980), except that the ~ent~fugation time was increased from 3 min to 18 min and the force decreased from 600 X g to f 00 X g in order to produce more reproducible fractions Sedimentation under those conditions is mathemat~~aily equivalent to the original procedure (Stokes Iaw) (Bull 197 1) and gave similar recoveries and platelet distributions. Platelets were collected separately from platelet-rich plasma (PI@) and supernatants in order to permit cortisol and imipramine analyses from the undiluted supernatants. The platelet pellets were resuspended in 10 ml of 50 mM Tris buffer (150 mM NaCl, 20 mM EDTA, pH 7.5), and platelet profiles were characterized using a Coulter ZM-C2S6 channelyzer. The platelets were then pelleted at 10,000 x g for I5 min and resuspended in hypotonic buffer (5 mM Tris, 5 mM EDTA, pH 7.5) before freezing and storage at -70°C for subsequent analysis. Frozen platelets were thawed at 4°C and ultrasonically disrupted on ice (6OW X 10 set) until Coulter counts verified greater than 99% dis~ption. Platelet membrane fragments were collected and washed in an equal wlume of70 mM Tris buffer (pH 7.5) by ~ent~fugation for 10 min at 39,000 x g. Platelet ‘H-IMI (specific activity 87 Ciimmol) (New England Nuclear, Boston, MA) binding was evaluated using the assay conditions of Langer et al. (1980). Specific binding was determined in paired samples in the presence and absence of 100 p.M desipramine (final concentration) at 16 ‘H-EM1 concentrations ranging from 0.2 to 5.5 nM. Each assay tube contained 58 +g of membrane protein. The assay conditions were consistent with a World Health Organization (WHO) standard, except that the incubation time was extended from 1 hr to 3 hr to ensure equilibrium, the platelets were ultrasonically rather than mechanically disrupted, and 16 different concentrations, rather than 8 paired concentrations, were evaluated, Extension of the incubation time and the use of 16 versus 8 paired concentrations showed no significant difference in comparison samples, although ultrasonic treatment produced smaller memb~ne fragments with higher specific binding than mechanical disruption. CompIete platelet breakage was verified following ultrasonic disruption, but not mechanical treatment (ca. 80% breakage), and the generally higher B,,, values obtained by the present method were presumably due to a tack of cellular protein in the membrane p~p~ation (Freidl et al. 1983). Protein values were determined by the method of Bradford (1976), but were adjusted to give equivalent values to the method of Peterson (1977) in order to conform to a WHO standard. The Irvine Iaboratory is the U.S. representative in a WHO protocol that studies .$I-IMI binding, and assay consistency was verified in exchanged samples with the other pa&ipating centers. The between-assay variance of repeated measures of platelet pools from our laboratory was CV = 10.9% for iu, and CV = 3.7% for B,,, determinations. Plasma cortisol and imip~mine Levels were dete~ined using commercially available ~mmunofluores~ence assays on an Abbott TDx clinicaf analyzer. The ~mipramine assay employed is equally sensitive to both imipramine and its desipramine metabolite, Statistical analyses were performed using BMDP programs PID, P3D, P6D, and p6R (University of California, Los Angeles) using an AMT-286 computer. Platelet binding

Non-State-Dependent

BIOL PSYCHIATRY 1989;26:478-488

Changes in “H-lM1 Binding

481

5000

4000 r\ z \ : t

3000

X t m 2000

1000

BA&lNE4

13

W;EKS TIME

OF

WEEKS

TREATMENT

Figure 1, Effects of chronic imipramine treatment on the B,,, of platelet ‘H-imipramine binding in endogenous unipolar depression.

parameters were estimated (Cambridge, UK).

using the EBDA and LIGAND

programs of Elsevier Biosoft

Results The maximum number of imipramine binding sites (B,,,) and the affinity of binding (&) were determined in 11 endogenous depressed patients prior to and following 1 month of treatment with imipramine-HCl (150-300 mg). All of the patients had pretreatment Hamilton depression scores in excess of 20 (mean 26.6 +- 4.5 SD), and all had posttreatment scores of 10 or less (mean 5.3 ? 3.7). The average pretreatment B,,,, was 1716 ‘_ 879 fmol/mg, and the Kd was 1.46 ‘- 0.63 nM. Baseline B,,, values were 71% (paired t = 2.29; df = 10; p = 0.04; n = 11) of those obtained from matched normal controls (mean 2409 2 877 fmol/mg). No significant differences were found between patient and control Kd values (control mean 1.59 t 0.54 nM), and neither B,,,, nor Kd were significantly correlated with age in either the patients or controls. B,,, values of the patient groups were increased by 33% (paired t = 2.66; df = 10; p = 0.024; n = 11) after 4 weeks of imipramine treatment (Figure 1). This trend was evident in 7 of the I 1 patients, although 3 showed moderate decreases and 1 had a large decrease. Treatment also resulted in an 89% increase in Kd (2.66 ? 0.89 nM; paired t = 3.99; df = IO; p = 0.003; n = ll), which was evident in 9 of the 11 patients. A reanalysis of 5 of the

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25

Figure 2. Scatchard plot of ‘Himipramine binding by p~ateIe&s from a normal control (0) and by a split sample exposed in vitro to 100 ngiml imipramine (0).

IO’00

0 3H-IMI

BOUND

20’00 Cfmollmg~

patients following 13 weeks of treatment showed that 2 who had previously exhibited the largest B,,, increases after 4 weeks had lower than baseline levels after the longer treatment (Figure 1). Although the other subjects showed moderate increases (including one that had initially decreased), the net change was not significantly different from baseline (paired t = 0.87; df = 4; p = 0.450; n = 5). A change in Kd value is generally attributed to the presence of drug carryover. The potential effects of drug carryover on platelet binding were investigated using split whole blood samples from a normal control that were incubated in the presence and absence of 100 ng/ml imipramine at 22°C for 30 min prior to the usual preparation and binding procedures. The results (Figure 2) show that the Kd of binding was increased from 0.95 to 2.3 1 nM by the imipramine treatment. The figure clearly reff ects the purely competitive nature of this effect and shows that B,,, changes in the patient sample cannot be accounted for by drug carryover. Platelet %I-IMI binding was also evaluated as a function of plasma cortisol levels. No signi~c~t correlations were found between baseline cortisol and baseline Kd (r = - 0.123), or plasma imipramine levels (I. = - 0.435), although nonsigni~cant trends were noted between cortisol and B,,, at baseline (r = 0.537) and between delta cortisol and plasma imipramine (r = 0.538) after treatment (4 weeks). In contrast, posttreatment cortisol levels were linearly related to posttreatment B,,, (r = -0.745; p = 0.007) and were even more highly correlated with the difference in B,,, between baseline and treatment (A&,,,,; r = -0.852; p < O.~I), as shown in Figure 3. No significant difference was found between paired pre- and posttreatment (4 weeks) cortisol levels (t = 0.52; df = IO), although pretreatment values (mean 13.17 _t 5.24 SD) were slightly higher than those after treatment (mean 12.42 L 3.14 SD). Treatment efficacy was estimated as the difference between Hamilton-D scores at

bob-agate-Dependent Changes in ‘H-IMI Binding

6

I

I

f

1

i

8

IO

I2

14

16

[CURTISOL]


Figure 3. ~e~atioas~ipbetween the B,,, of ~~~t~~~t ~~-jrn~~rarni~e binding and plasma cortisol levels after 4 weeks of imipraminetreatment.

baseline and following 4 weeks of treatment (AHAM-D). Alternatively, a percent improvement was calculated as the AHAM-D divided by the baseline value. In every case, the percent improvement was better correlated with the bio~~ernjc~~ measures than was the AI-IA?&D. No signi&znt linear correlations were found between cfinical improvement and either B,, or cortisol, although treatment responses (9%irn~rove~~e~t~tended to be correlated with p~t~atmeut B,,, values (f = 0.517), Instead, the results fF@~e 42 appeared to indicate the presence of a B,,, o~~~rn~rnwith diminished responses at higher and lower plate& binding. Discussion The current study confirms previous reports that the B,,, of -‘H-IMI binding in depressed patients is decreased by ca. 30% relative to normal controls (Paul et al. 1981). One month’s treatment with imipram~n~-~Cl increased B,,, to control levels and improved depressive symproms, al~o~g~ no correfation was found between these changes in individual patients. Similar B,, increases after imipr~ine treatment (4 weeks) were reported in an earlier study that also failed to correlate bi~hemical and clinical n~easures ~Su~nyi-~adotte et ai. f985bf. The latter found even larger B,,, increases after 4 weeks of i~~ipramine d~s~o~~~uation and suggested that increases lag behind clinical improve-

484

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90

tz

W P 9

80

0 a n P

60

1000

2000 Bmax

Figure 4. Relationship of pretreatment weeks of imipramine treatment.

3000


values with subsequent

clinical improvement

after 4

but are nevertheless related to affective state. The present study also found B,,, increases in 3 of 5 patients after 13 weeks of treatment. However, these increases were modest in comparison with the larger sample at 4 weeks, and 2 patients who had initially showed the largest increases subsequently had B,,, values that were below baseline levels. Therefore, platelet binding in the overall sample was not signi~cantly elevated after 13 weeks of treatment, in spite of the fact that all 5 patients remained in full remission. Similar decreases to baseline foIlowing initial increases have been reported after successful treatment with nortriptyline and electroconvulsive shock (Wagner et al. 1987). A general lack of correlation between clinical improvement and B,,,increases following treatment with a variety of antidepressants (Suranyi-Cadotte et al. 1985b; Wagner et al. 1987; Arora and Meltzer 1988) may be due to a lag in binding responses resulting from platelet maturation (half-life of 1 week) (Friedman 197 1) or to indirect, but statedependent, modulators of platelet binding, such as imipramine-like endocoids (SuranyiCadotte et al. 1985b) or plasma cortisol (Roy et al. 1987). Antidepressants are known to diminish serotonin (5HT) induced increases in plasma cortisol (Meltzer et al. 1984), and hyperco~isolemia is a common feature among depressed patients, which tends to normalize with successful treatment (Roy et al. 1984). As animal studies with adrenalectomized rats have demonstrated a dependence of platelet “H-IMI binding on adrenal ments

No~~tate-candent

Changes in 3H-IMIBinding

BIOL PSYCHIATRY 1989:26:478-488

485

function (Arora and Meltzer 1986), B,,, increases after treatment could reflect stateinduced changes in circulating corticosteroids. Roy et al. (1987) found a significant negative correlation between B,,, values and plasma cortisol at 4:OOPM and 11:OOPM following dexamethasone administration. The same study also found that female, but not male, dexamethasone (DST) nonsuppressors had lower B,,, values than female controls, although another study failed to relate B,,, and nonsuppression (Tang and Morris 1985). The present study did not observe a correlation between B,,, changes and baseline cortisol levels, but did find a significant negative correlation between cortisol and both B,,, levels and B,,, changes after 4 weeks of ~eatment. A lack of co~elation between basefine B,,, and cortisol is consistent with the results of a larger study in which we tested 34 untreated depressed patients (to be published elsewhere). The present study also failed to observe significant decreases in plasma cortisol after treatment, although B,,, values were increased. As a higher correlation was found between posttreatment changes in B,,,, than in posttreatment B,,, per se, the results suggest that imipramine treatment was responsible for B,,, increases, but that platelet binding was not directly dependent on cortisol levels. The results also show that pretreatment decreases in B,,, relative to controls were not due to hypercortisolemia. Therefore, imipramine treatment appears to have increased B,,, values in a manner that was independent of affective state. A possible mechanism for this effect lies in the known action of a variety of antidepressants and other tricyclic compounds as inhibitors of platelet aggregation (Va~derhoek and Feinstein 1979: Kindness et al. 1980; Ishigooka et al. 1980; Rao et al. 1980). An inhibition of aggregation is apparently due to a suppression of calcium mobilization within cells and probably involves a specific interaction with anionic phospholipids (Lasslo and Quintana 1984). Decreased calcium mobilization could in turn inhibit calcium-dependent phospholipases (Wang and Cheung 1979), which have been proposed as possible regulators of the number of ?HIMI binding sites (Wennogle et al. 1981). A direct action of antidepressants on platelets could also be due to drug carryover to the binding assay. Significantly elevated Kd values were found in spite of extensive washing. Similar increases have been reported by others following treatment with chlorimipramine (Poirier et al. 1987) and amitriptyline (Braddock et al. ISSit), which, like imipramine, have a high affinity for the 3H-IMI binding site (Melle~p et al. I982), whereas ~onsigni~~ant increases have been found after desipramine (Cowen et al. 1986; Arora and Meltzer 1988), which has a lower affinity (Mellemp et al. 1982). However, a comparison of split samples in the presence and absence of therapeutic imipramine levels clearly showed that imipramine carryover cannot account forBmaxchanges following treatment 1 The present study also examined the potential use of baseline binding measurements as predictors of treatment response. B,,, values were not linearly related to therapeutic outcome. However, a tendency was noted such that optimal responses were obtained at ca. 1300 fmolimg (approximately half the value observed in our normal controls). Similar results were reported by Baron et al. (1986), although two other studies found that low B,,, values were associated with a poor prognosis (Hrdina et al. 1985; Wagner et al. 1987). In summary, the present results obtained before and after chronic imipramine administration suggest that B,,, increases following treatment may be due to drug effects that are independent of affective state. As similar increases have been reported by others following treatment of normal subjects with some other antidepressants, it appears possible that these agents may also provoke non-state-dependent changes.

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Drs.

The authors express their appreciation to Evan Schaffer, M.S., James ~eishmann, L.V.N.. and 72th and Laura Seed for their assistance in the present work.

Mark

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males and

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Wood PL, Suranyi-Cadotte BE, Nair NPV, LaFaille F, Schwartz G (1983): Lack of association between ‘H-imipramine binding sites and uptake of serotonin in control, depressed and schizophrenic patients. Neuropharmacology 22: 121 l-1214.