Zinc supplementation augments efficacy of imipramine in treatment resistant patients: A double blind, placebo-controlled study

Journal of Affective Disorders 118 (2009) 187–195

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Journal of Affective Disorders j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j a d

Research report

Zinc supplementation augments efficacy of imipramine in treatment resistant patients: A double blind, placebo-controlled study Marcin Siwek a, Dominika Dudek a, Ian A. Paul e, Magdalena Sowa-Kućma d, Andrzej Zięba a, Piotr Popik c,d, Andrzej Pilc c,d, Gabriel Nowak b,d,⁎ a b c d e

Department of Psychiatry, Collegium Medicum, Jagiellonian University, Kraków, Poland Chair of Pharmacobiology, Collegium Medicum, Jagiellonian University, Kraków, Poland Faculty of Public Health, Collegium Medicum, Jagiellonian University, Kraków, Poland Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, MS, USA

a r t i c l e

i n f o

Article history: Received 22 June 2008 Received in revised form 18 February 2009 Accepted 18 February 2009 Available online 10 March 2009 Keywords: Unipolar depression Imipramine treatment Zinc Placebo Supplementation

a b s t r a c t Background: One of the main problems in the therapy of depression is the limited efficacy of antidepressants and the limited utility of augmentation strategies. Zinc, a non competitive NMDA receptor antagonist exhibits preclinical antidepressant efficacy. Moreover, a preliminary clinical report suggests augmentation of antidepressant therapy by zinc in depression. Methods: A placebo-controlled, double blind study of zinc supplementation in imipramine therapy was conducted in sixty, 18–55-year old, unipolar depressed patients fulfilling the DSMIV criteria for major depression without psychotic symptoms. After a one week washout period, patients were randomized into two groups treated with imipramine (~ 140 mg/day) and receiving once daily either placebo (n = 30) or zinc supplementation (n = 30, 25 mgZn/day) for 12 weeks. Results: No significant differences in CGI, BDI, HADRS and MADRS scores were demonstrated between zinc-supplemented and placebo-supplemented antidepressant treatment nonresistant patients. However, zinc supplementation significantly reduced depression scores and facilitated the treatment outcome in antidepressant treatment resistant patients. Conclusion: Zinc supplementation augments the efficacy and speed of onset of therapeutic response to imipramine treatment, particularly in patients previously nonresponsive to antidepressant pharmacotherapies. These data suggest the participation of disturbed zinc/glutamatergic transmission in the pathophysiology of drug resistance. © 2009 Elsevier B.V. All rights reserved.

1. Introduction One of the main problems in the therapy of depression is the limited efficacy of antidepressants and the problematic usefulness of augmentation strategies (Nemeroff, 2007; Nierenberg et al., 2008; Carvalho et al., 2007). Only about 50% of treated patients reach remission criteria (Warden et

⁎ Corresponding author. Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Poland. Tel.: +48 12 6623 215; fax: +48 12 6374 500. E-mail address: [email protected] (G. Nowak). 0165-0327/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jad.2009.02.014

al., 2007; Kirsh et al., 2008). In addition, the dosage required to achieve remission often results in multiple unwanted effects. These data clearly argue the need for novel antidepressant therapies and/or augmentation strategies. Recent studies suggest, that disturbances of glutamatergic transmission (especially NMDA receptor hyperactivation) are involved in the pathogenesis of mood disorders (Sanacora et al., 2004a,b). Considerable evidence has accumulated over the past 20 years indicating that direct and indirect NMDA receptor antagonists exhibit antidepressant-like effects in preclinical animal paradigms sensitive to antidepressants and in models of depression in rodents. Of particular note are the

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recent reports that NMDA receptor antagonists also display clinical efficacy (Berman et al., 2000; Prescorn et al., 2008; Zarate et al., 2006). Similarly, there are clinical reports suggesting augmentation of antidepressant therapy by non specific NMDA receptor antagonists (e.g. amantadine, riluzole) in refractory depression (Dziedzicka-Wasylewska et al., 2002; Sanacora et al., 2004a,b). Zinc is an important modulator of glutamatergic transmission. The main activity of zinc in the glutamatergic system is an attenuation of NMDA receptor function—directly or via modulation of AMPA receptors and metabotropic glutamate receptors (Szewczyk et al., 2008; Nowak et al., 2005). Preclinical studies have demonstrated the antidepressant— like effects of zinc administration in animal tests/models of depression. Thus, zinc is active in preclinical antidepressant screening paradigms such as the forced swim test and tail suspension test in rodents (Kroczka et al., 2000; Kroczka et al., 2001; Nowak et al., 2003c; Rosa et al., 2003; Franco et al., 2007). Moreover, zinc has antidepressant-like properties, in animal models of depression such as olfactory bulbectomy, chronic unpredictable stress and chronic mild stress in rats (Nowak et al., 2003c; Cieslik et al., 2007; Sowa-Kućma et al., 2008). Recent data has also revealed that zinc enhances the efficacy/potency of citalopram or imipramine in preclinical paradigms sensitive to antidepressants. Thus, subeffective doses of zinc administered in combination with subeffective doses of antidepressants resulted in antidepressant-like effects in the forced swim test, tail suspension test and chronic unpredictable stress model (Cieslik et al., 2007; Cucha et al., 2008; Rosa et al., 2003; Szewczyk et al., 2002, 2009). Conversely, in rodents, chronic antidepressant (imipramine, citalopram) treatment slightly increases the zinc level in the hippocampus and decreases it in the cortex, cerebellum and basal forebrain, although the calculation of the hippocampus/ other brain regions zinc concentration ratio demonstrated a significantly higher level of zinc in hippocampus after treatment of both drugs (Nowak and Schlegel-Zawadzka, 1999). Chronic electroconvulsive shock (ECS) also induced a robust (32%) increase in zinc concentration in hippocampus and small (11–15%) increases in other brain regions (Nowak and SchlegelZawadzka, 1999). Chronic ECS was also reported to increase hippocampal mossy fiber sprouting, which was demonstrated histochemically by the zinc–selenium method (Vaidya et al., 1999; Lamont et al., 2001). This method actually determines concentrations of synaptic zinc, so this (Vaidya et al., 1999) and the other reports (Lamont et al., 2001) indicate an increase in hippocampal zinc synaptic concentration induced by ECS. Recently, we have also demonstrated an increase in hippocampal synaptic zinc concentration after chronic zinc treatment (Szewczyk et al., 2006). Complementing these data are clinical investigations that have indicated an alteration of blood zinc level as a potential marker of depression (Maes et al., 1994, 1997, 1999; McLoughlin and Hodge, 1990; Nowak et al., 1999). As noted above, there is much preclinical data to provide “proof of concept” that antagonists of the NMDA receptor can have significant antidepressant efficacy. However, the dissociative effects of full antagonists such as ketamine limit their introduction into general clinical practice. As a result, strategies which augment inhibition of the NMDA receptor without full antagonism may represent more viable clinical approaches. We recently reported in a preliminary clinical

study that zinc enhanced the efficacy of several antidepressants in patients with major depression (Nowak et al., 2003a). We now report that zinc augments the efficacy and speed of onset of therapeutic response to imipramine treatment, particularly in patients previously nonresponsive to antidepressant pharmacotherapies. 2. Methods 2.1. Patients and treatments Patients admitted to the Department of Psychiatry Collegium Medicum Jagiellonian University or to Affective Disorder Outpatients (single center study, January 2005–April 2006) and fulfilling the DSM-IV criteria for major depression, with a moderate or severe depressive episode without psychotic symptoms (or drug or alcohol abuse/dependence) were accepted for the study. The patients were randomized 1:1 by independent staff member into two groups: one receiving placebo (group I, n = 30) and second receiving zinc supplementation (group II, 25 mg Zn2+ once daily, Zincas Forte, Farmapol, Poznan, Poland, n = 30). Placebo was prepared by Farmapol (Poznan, Poland) as identical to the Zincas tablets and consisted of all ingredients of Zincas besides zinc hydroaspartate. All study researchers and subjects were blind to group allocation for the duration of the study. Both groups were treated with imipramine (Polfarma, 100–200 mg once daily). All patients had a one week washout period without any pharmacotherapy. 2.2. Assessments The Hamilton Depression Rating Scale (HDRS, 17 items) (Hamilton, 1960), Beck Depression Inventory (BDI) (Beck et al., 1961), Clinical Global Impression (CGI) and Montgomery–Asberg Depression Rating Scale (MADRS) were used to assess efficacy of antidepressant therapy, and patients' status was evaluated before the treatment and 2, 6 and 12 weeks after its commencement. A therapeutic response was defined as: “much” or “very much improved” in CGI scale plus at least 50% reduction of MADRS (MADRS/CGI criteria) scores or HADRS scores (HADRS/CGI criteria). Remission was defined as: “very much improved” on the CGI scale plus scores on MADRS (MADRS/CGI criteria) ≤ 10 or HADRS (HADRS/CGI criteria)≤ 7 or BDI (BDI/CGI criteria)≤ 9. Staging of depression based on prior treatment response was made according to 6grade treatment resistant criteria by Thase and Rush (1997). Scores were rated by well trained and experienced psychiatrists. Supplementation and assessment of the patients' status were performed according to a “double blind” procedure. The study was approved by the Ethical Committee of Collegium Medicum, Jagiellonian University, Kraków and the informed consent was obtained from all participants. Trial Registry: Zinc supplementation of imipramine therapy; number: NTC00693680; URL: http://www.clinicaltrials.gov/. 2.3. Statistics Assessments of depression were evaluated with General Linear Model mixed design ANOVA with the TEST NUMBER as repeated factor, and TREATMENT and ANTIDEPRESSANT

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TREATMENT RESISTANCE as between factors, followed by Duncan's post-hoc test (Statistica 8 and SPSS 16). Depression assessment data were distributed normally and the variances were homogenous; missing data were excluded from analyses. Some of the data reported in tables did not meet criteria for normal distribution, and thus they were assessed with the X2 test, Fisher's Exact Test, Mann Whitney U or Wilcoxon tests, where appropriate. Data were deemed significant when p b 0.05.

Table 2 Number of patients in groups Pl and Zn in each measuring points (group Pl— imipramine and placebo-treated patients; group Zn—imipramine and zinctreated patients).

3. Results

(3 in placebo and 2 in zinc group). At the end of the study, groups consisted of 25 placebo-treated and 27 zinc-supplemented patients (Table 2). The frequency of unwanted side effects was similar in both treatment groups (58.3%—Table 3). Only trial completers were included in the analysis of zinc and imipramine treatment effects.

3.1. Patient characteristics Group characteristics at the beginning of study are presented in Table 1. The mean age (±SD) in the placeboand zinc-treated groups was 45.7 ± 6.1 and 46.2 ± 5.8 years, respectively, and did not significantly differ (p = 0.73, t-test). There were no significant differences in the male/female ratio between placebo (9/21) and zinc group (11/19) (X2 Test). Similarly, demographic parameters (education, employment, marital status, cigarette smoking, treatment site) were not significantly different between placebo and zinc groups (Table 1). 3.2. Exclusions Some patients were excluded during the trial because of unwanted side effects and need to alter therapeutic strategy (2 in placebo and 1 in zinc group) or due to compliance failure

Table 1 Demographic features of patients included in trial and comparison between individual groups (χ2 and t-tests); (group Pl—imipramine and placebo-treated patients; group Zn—imipramine and zinc-treated patients; groups Pl+ Zn—all patients included in trial).

Age

Group Pl + Zn Group Pl (n = 60) (n = 30)

Group Zn (n = 30)

Group Pl vs Zn

45.9 ± 5.9

46.2 ± 5.8

t-test p = 0.73 χ2 test

45.7 ± 6.1

Sex Female 40 (66.7%) 21 (70.0%) Male 20 (33.3%) 9 (30.0%) Education Elementary or 20 (33.3%) 9 (30.0%) professional Secondary 29 (48.3%) 15 (50.0%) Higher 11 (18.3%) 6 (20.0%) Employment status Employed 22 (36.7%) 14 (46.7%) Unemployed 38 (63.3%) 16 (53.3%) Annuitant 46 (76.7%) 22 (73.3%) Marital status Single 5 (8.3%) 3 (10.0%) Married 50 (83.3%) 26 (86.7%) Separated/Divorced 5 (8.3%) 1 (3.3%) Cigarettes smoking No 29 (48.3%) 16 (53.3%) Yes 31 (51.7%) 14 (46.7%) ≤20 cigarettes/day 27 (46.7%) 13 (43.3%) N 20 cigarettes/day 4 (6.7%) 1 (3.3%) Place of therapy performed in recruitment day Outpatient department 17 (28.3%) 10 (33.3%) Clinical department 43 (71.6%) 20 (66.7%)

19 (63.3%) p = 0.58 11 (36.7%)

Weeks of treatment

Group Pl Group Zn

13 (43.3%) p = 0.79 17 (56.7%) 14 (46.7%) 3 (10%) 7 (23.3%) p = 0.57 23 (76.7%)

6

12

28 29

27 27

25 27

The mean of the final imipramine doses were similar in both groups: 140.2 ± 17.1 and 137.1 ± 15.8 mg/day for placebo and zinc, respectively (p = 0.47, Mann Whitney Test U).The mean ± SD of the CGI-S and BDI in the beginning of therapy were 5.0 ± 0.8 and 35.9 ± 4.9, respectively, and did not significantly differ between placebo + imipramine and the zinc + imipramine groups. There were also no differences between these groups assessed by HDRS and MAS score at baseline (22.9 ± 3.3 and 37.0 ± 5.6, respectively). The analysis of BDI scores (Fig. 1) revealed that treatment with imipramine and zinc resulted in lower scores than the treatment with imipramine and placebo (main factor of treatment). Moreover, antidepressant treatment non-resistant patients showed lower scores than antidepressant treatment resistant patients (main factor of treatment resistance). All groups demonstrated a gradual decrease in the scores over the time of treatment (main factor of the test number). A significant interaction between test number and antidepressant treatment resistance suggest treatment resistance differently affected BDI

Table 3 Frequency of individual side effects observed during the trial in groups: Pl, ZnI, Pl + Zn and comparison between groups (χ2-test) (group Pl— imipramine and placebo-treated patients; group Zn—imipramine and zinctreated patients; groups Pl + Zn—all patients included in trial). Group Pl + Zn

14 (46.7%) 5 (16.7%)

2 (6.7%) p = 0.35 24 (80.0%) 4 (13.3%)

2

30 30

3.3. Efficacy of treatment and supplementation

11 (36.7%) p = 0.85

8 (26.7%) p = 0.11 22 (73.3%) p = 0.54 24 (80.0%)

0

Patient excluded from trial by the reason of side effects All side effects Dry mouth Dizziness/Orthostatic hypotonia Drowsiness Constipations Tachycardia Urination disturbances Nausea Headache Visual disturbances

Group Pl

Group Zn

Group Pl vs Zn χ2 test

5 (8.3%)

3 (10.0%)

2 (6.7%)

p = 0.64

35 (58.3%) 30 (50%) 19 (31.7%)

16 (53.33%) 14 (46.7%) 11 (36.7%)

19 (63.3%) 16 (53.3%) 8 (26.7%)

p = 0.43 p = 0.61 p = 0.41

8 (13.3%) 17 (28.3%) 5 (8.3%) 3 (5.0%)

5 (16.7%) 7 (23.3%) 3 (10.0%) 1 (3.3%)

3 (10.0%) 10 (33.3%) 2 (6.7%) 2 (6.7%)

p = 0.45 p = 0.57 p = 0.64 p = 0.55

3 (5.0%) 3 (5.0%) 2 (3.3%)

2 (6.7%) 2 (6.7%) 1 (3.3%)

1 (3.3%) 1 (3.3%) 1 (3.3%)

p = 0.55 p = 0.55 p = 1.0

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Fig. 1. Effect of zinc supplementation on response to imipramine in antidepressants resistant and non-resistant patients measured by Beck Depression Inventory (BDI). Data represent the mean+ SEM BDI scores of 9 treatment resistant patients treated with imipramine + placebo, 16 treatment nonresistant patients treated with imipramine + placebo, 12 treatment resistant patients treated with imipramine + zinc and 15 treatment non-resistant patients treated with imipramine+ zinc. ANOVA demonstrated the following values: treatment: F(1,48) = 6.05, P b 0.025, treatment resistance: F(1,48) = 5.86, P b 0.025 and test number: F(3,144)= 178, P b 0.001. The interaction between test number and treatment resistance was also significant: F(3,144)= 3.57, P b 0.025. Post-hoc Duncan's test revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline, however the “antidepressant treatment resistant group treated with imipramine and placebo” did not demonstrate an improvement at week 6 as compared with week 2 test, while other treatment groups did (# symbol). At week 6, all groups showed significant reduction of scores as compared with “treatment resistant group treated with imipramine and placebo.” The difference between placebo and zinc supplementation in antidepressant treatment resistant groups was seen also at week 12 (⁎ symbols). Analysis of covariance using Greenhouse– Geisser estimate revealed no interaction between test number and age, sex, education, smoking, length of disease, number of hospitalizations, final drug dose, and treatment resistance; only the test number× treatment interaction was significant: F(2.467,96.195)= 5.713, P = 0.02.

measure along with testing. The post-hoc analysis revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline; however, the “antidepressant treatment resistant group treated with imipramine and placebo” did not demonstrate an improvement at week 6 as compared with week 2 test, while other treatment groups did. In addition, at week 6, all groups showed significant reduction of scores as compared with “treatment resistant group treated with imipramine and placebo.” The difference between placebo and zinc supplementation in antidepressant treatment resistant groups was seen also at week 12. These comparisons suggest lower BDI scores of imipramine and zinc treatment (as compared with imipramine and placebo) in antidepressant resistant patients at weeks 6 and 12. The analysis of CGI-S (Fig. 2) scores revealed that treatment with imipramine and zinc resulted in lower scores than the treatment with imipramine and placebo (main factor of treatment). All groups demonstrated a gradual decrease in the scores over the time of treatment (main factor of the test number). In addition, the significant interactions between test

number and treatment as well as between test number and antidepressant treatment resistance suggested that these factors affected the CGI-S responses over the time of treatment. The post-hoc analysis revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline as well at week 12 as compared with week 6, however the “treatment resistant group treated with imipramine and placebo” did not demonstrate an improvement at week 6 as compared with week 2 test, while other treatment groups did. In addition, at weeks 6 and 12, all groups showed significant reduction of scores as compared with “treatment resistant group treated with imipramine and placebo.” These comparisons suggest lower CGI-S scores of imipramine and zinc treatment as compared with imipramine and placebo in antidepressant resistant patients at weeks 6 and 12. The analysis of HDRS scores (Fig. 3) revealed that treatment with imipramine and zinc resulted in lower scores than the treatment with imipramine and placebo (main factor of treatment). All groups demonstrated a gradual decrease in the scores over the time of treatment (main factor of the test number). In addition, the significant interactions between

Fig. 2. Effect of zinc supplementation in response to imipramine in antidepressants resistant and non-resistant patients measured by Clinical Global Impression Scale (CGI). Data represent the mean + SEM CGI scores of 9 treatment resistant patients treated with imipramine + placebo, 16 treatment non-resistant patients treated with imipramine + placebo, 12 treatment resistant patients treated with imipramine+zinc and 15 treatment non-resistant patients treated with imipramine+ zinc. ANOVA demonstrated the following values: treatment: F(1,48)=7.56, P b 0.01 and test number: F(3,144)= 143.55, Pb 0.001. The following interactions were significant: test number ×treatment: F(3,144) =3.13, Pb 0.05 and test number × treatment resistance: F(3,144) = 4.64, P b 0.01. Post-hoc Duncan's test revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline as well at week 12 as compared with week 6, however the “treatment resistant group treated with imipramine and placebo” did not demonstrate an improvement at week 6 as compared with week 2 test, while other treatment groups did (# symbol). At weeks 6 and 12, all groups showed significant reduction of scores as compared with “treatment resistant group treated with imipramine and placebo” (⁎ symbols). Analysis of covariance using sphericity estimate revealed no interaction between test number and age, sex, education, smoking, length of disease, number of hospitalizations, final drug dose, and treatment resistance; however, the test number × treatment interaction: F(3,117) = 6.07, P = 0.01 and test number × treatment resistance interaction: F(3,117) = 4.205, P = 0.007 were significant.

M. Siwek et al. / Journal of Affective Disorders 118 (2009) 187–195

Fig. 3. Effect of zinc supplementation in response to imipramine in antidepressants resistant and non-resistant patients measured by Hamilton Depression Rating Scale (HDRS). Data represent the mean + SEM HDRS cores of 9 treatment resistant patients treated with imipramine + placebo, 16 treatment non-resistant patients treated with imipramine + placebo, 12 treatment resistant patients treated with imipramine + zinc and 15 treatment nonresistant patients treated with imipramine+ zinc. ANOVA demonstrated the following values: treatment: F(1,48) = 6.40, P b 0.025; test number: F(3,144)= 238.4, P b 0.001. Three interactions were also significant: treatment × treatment resistance: F(1,48)= 4.74, P b 0.05; test number× treatment: F(3,144)= 3.30, P b 0.05 and test number× treatment resistance: F(3,144)= 4.46, P b 0.01. Posthoc Duncan's test revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline, however the “treatment resistant group treated with imipramine and placebo” did not demonstrate an improvement at week 6 as compared with week 2 test, while other treatment groups did (# symbol). At weeks 6 and 12, all groups showed significant reduction of scores as compared with “treatment resistant group treated with imipramine and placebo” (⁎ symbols). Analysis of covariance using Greenhouse–Geisser estimate revealed no interaction between test number and age, sex, education, smoking, length of disease, number of hospitalizations, final drug dose, and treatment resistance, however, the test number × treatment interaction was significant: F(2.344,91.425) = 6.522, P = 0.001.

test number and treatment as well as between test number and antidepressant treatment resistance suggested that these factors affected the HDRS responses over the time of treatment. A significant interaction between treatment and antidepressant treatment resistance suggest zinc supplementation differently affected HDRS measure in the treatment resistant and non-resistant patients. Post-hoc Duncan's test revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline, however the “treatment resistant group treated with imipramine and placebo” did not demonstrate an improvement at week 6 as compared with week 2 test, while other treatment groups did. Conversely, this group showed lower HDRS scores at week 12 as compared with the week 6, while other groups did not. In addition, at weeks 6 and 12, all groups showed significant reduction of scores as compared with “treatment resistant group treated with imipramine and placebo”. The difference between placebo and zinc supplementation in antidepressant treatment resistant groups was already seen at week 2. These comparisons suggest lower HDRS scores of imipramine and zinc treatment as compared with imipramine and placebo in antidepressant resistant patients at weeks 2, 6 and 12.

191

The analysis of MADRS scores (Fig. 4) revealed that treatment with imipramine and zinc resulted in lower scores than the treatment with imipramine and placebo (main factor of treatment). All groups demonstrated a gradual decrease in the scores over the time of treatment (main factor of the test number). A significant interaction between test number and antidepressant treatment resistance suggest that treatment resistance differently affected MADRS measure along with testing. A significant interaction between treatment and antidepressant treatment resistance suggest that zinc supplementation affected MADRS measure in treatment resistant and non-resistant patients. Post-hoc analysis revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline as well as an improvement at week 6 as compared with week 2 test. Except the group of resistant patients supplemented with placebo, none of other groups showed reduced MARDS scores between week 12 and week 6. Moreover, at week 2 test, the group “resistant to antidepressants and treated with imipramine and placebo” demonstrated significantly higher score than the “resistant to antidepressants treated with

Fig. 4. Effect of zinc supplementation on response to imipramine in antidepressants resistant and non-resistant patients measured by Montgomery–Asberg Depression Rating Scale (MADRS). Data represent the mean+SEM MADRS scores of 9 treatment resistant patients treated with imipramine +placebo,16 treatment non-resistant patients treated with imipramine+placebo, 12 treatment resistant patients treated with imipramine+zinc and 15 treatment non-resistant patients treated with imipramine+ zinc. ANOVA demonstrated the following values: treatment: F(1,48)=8.41, Pb 0.01; test number: F(3,144)=267.7, Pb 0.001. Two interactions were also significant: treatment× treatment resistance: F(1,48)= 5.88, P b 0.025 as well as test number×treatment resistance: F(3,144)=3.73, Pb 0.025. Post-hoc Duncan's test revealed that all groups demonstrated significant improvement at week 2 as compared with the baseline as well as an improvement at week 6 as compared with week 2 test (# symbols). At week 2 test, the group “resistant to antidepressants and treated with imipramine and placebo” demonstrated significantly higher score than the “resistant to antidepressants treated with imipramine and zinc.” At weeks 6 and 12, the group “resistant to antidepressants and treated with imipramine and placebo” demonstrated significantly higher scores than all other groups (⁎ symbols). Analysis of covariance using Greenhouse–Geisser estimate revealed no interaction between test number and age, sex, education, smoking, length of disease, number of hospitalizations, final drug dose, and treatment resistance; the interaction of the test number× treatment was significant: F(2.449,95.510)=3.361, P=0.03.

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Table 4 The percentage of patients who fulfilled criteria for response and remission after 6 and 12 weeks of treatment with imipramine and supplemented with placebo (Pl) or zinc (Zn). Non-resistant %

Resistant p

Pl

Zn

N = 17

N = 16

76.5 82.4

68.8 62.5

41.2 47 52.9

37.5 43.8 43.8

N = 16

N = 15

75 87.3

86.7 86.7

62.5 56.3 62.5

73.3 60 73.3

% 2

(test χ )

p (test χ2)

Pl

Zn

N = 10

N = 12

0.89 0.38

30 30

58.3 75

0.18 b 0.05

0.83 0.85 0.6

20 20 10

50 41.6 50

0.14 0.28 b 0.05

N=9

N = 12

0.41 0.95

44.4 30

75 75

0.15 0.056

0.52 0.83 0.52

22.2 30 22.2

41.2 50 50

0.35 0.45 0.2

6 weeks Response HADRS/CGI MADRS/CGI Remission BDI/CGI HADRS/CGI MADRS/CGI 12 weeks Response HADRS/CGI MADRS/CGI Remission BDI/CGI HADRS/CGI MADRS/CGI

imipramine and zinc.” At weeks 6 and 12, the group “resistant to antidepressants and treated with imipramine and placebo” demonstrated significantly higher scores than all other groups. These comparisons suggest lower MADRS scores of imipramine and zinc treatment as compared with imipramine and placebo in antidepressant resistant patients at weeks 2, 6 and 12. Zinc supplementation significantly improved response and remission at 6-week of treatment in treatment resistant patients evaluated by MADRS/CGI, while the effect on response at 12-week was statistically marginal (Table 4). Altogether, in antidepressant treatment resistant patients, the lack of zinc supplementation did not reduce measures of depression as much and as fast as in all other tested groups. Since in all four measures of depression the treatment resistant, zinc-supplemented group showed as rapid and profound reduction of scores as in non-resistant groups (regardless of the treatment), while placebo-supplemented group did not, one may conclude that zinc supplementation facilitates and improves the clinical outcome of treatment resistant depressive patients. 4. Discussion The data presented here demonstrate that the addition of zinc hydroaspartate supplements to conventional antidepressant pharmacotherapy such as imipramine improves both the efficacy of antidepressant response as well as the speed of onset of therapeutic effects. Moreover, this effect is almost entirely due to the effect of zinc hydroaspartate supplementation in previous antidepressant nonresponders. Although beneficial effects of zinc supplementation on antidepressant response were suggested by the earlier pilot study of Nowak et al. (2003a), the present study represents the first large scale clinical trial of zinc augmentation therapy. Further, the

present study has provided the necessary clinical population to separately assess the effects of zinc supplementation in antidepressant responsive and nonresponsive populations. Finally, these data indicate that the combination of imipramine with zinc is safe and well-tolerated. Treatment resistance to antidepressant pharmacotherapy remains one of the principle issues in the treatment of major depressive disorders. As recently noted, despite pressing need, there are relatively few adequate double blind trials of any augmentation therapies (Nemeroff, 2007). The behavior of zinc augmentation therapy is unusual inasmuch as the data indicate that addition of zinc hydroaspartate to imipramine could differently influence antidepressant treatment, depending on the patient's history of resistance to previous pharmacotherapies. Zinc hydroaspartate did not facilitate or augment imipramine action in patients who had a history of response to conventional antidepressant pharmacotherapies. However, in striking contrast, zinc hydroaspartate addition to imipramine in patients who had failed to respond to at least one previous adequate pharmacotherapy of the current depression episode, facilitated and augmented imipramine action. This was observed as a larger reduction of depression intensity and higher response (and remission) rate at the end of the treatment. In the above-mentioned study of Nowak et al. (2003a), patients receiving an antidepressant in combination with zinc hydroaspartate (AD + zinc) showed statistically significantly greater (vs. AD + placebo-treated subjects): 1) reduction in HDRS score at weeks 6 and 12 of the treatment, and 2) reduction in depression intensity, measured by Beck Depression Inventory at week 12 of the active treatment. There were some limitations of that study including: 1) a small final number of patients (6 subjects in AD + zinc group and 8 in AD + placebo group); 2) diverse antidepressant treatment regimens (clomipramine, amitriptyline, citalopram, fluoxetine) and; 3) lack of data on patients' response to previous treatment of the current depression episode. The conclusion that zinc is able to augment antidepressant treatment in patients not responding to a previous adequate pharmacotherapy of the current depression episode should be viewed with some caution because of the relatively small group size. Specifically, the final number of patients in the subgroup that had not responded to a previous adequate pharmacotherapy was relatively small (group fulfilling this criterion and receiving imipramine and zinc comprised 12 patients while imipramine + placebo-treated group included 9 subjects). Nonetheless, it is encouraging that the ability of zinc to facilitate the therapeutic effects of imipramine was evident in all four rating systems. Moreover, our data would suggest that zinc augmentation not only accelerates therapeutic response, but also appears to enable pharmacotherapy to achieve an efficacy comparable to that observed in previous antidepressant responders. It is possible that the neurobiological difference between patients with a history of antidepressant non-response and those who previously responded to antidepressant may be a consequence of previous antidepressant discontinuation or switching. For example, Harvey et al. (2002) propose alteration in NMDA receptors as a one of the possible targets involved in the treatment resistant patients. Based on clinical data of antidepressant treatment interruption-

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induced stress exacerbation (Michaelson et al., 2000), they demonstrated an imipramine-withdrawal-induced increase in the density of NMDA receptors in the rat hippocampus (Harvey et al., 2002). Moreover, since increased stress may account in the treatment resistance phenomena, additional evidence may be derived from our previous work demonstrating enhancement of glycine affinity to glycine/NMDA site in the cortex induced by forced swim challenge in rats (Nowak et al., 1995). Brain extracellular (synaptic) zinc concentration rapidly increases following peripheral (i.p.) acute or chronic zinc administration to rats (Opoka et al., 2008, our unpublished data). Thus, zinc supplementation may augment inhibition of pathologically hyper-active NMDA receptors in non-responder patients. The demonstration that the volume of the hippocampus of multiple depressive episodes patients is reduced compared with first depressive episode patients or controls (MacQueen et al., 2003), and which may by related to ongoing stress [e.g. (Bonne et al., 2008; Lee et al., 2002)] also points to neuronal damage, perhaps driven by NMDA receptor hyperactivity. Zinc functions as a reversible antagonist of the NMDA receptor complex (Smart et al., 1994). Moreover, preclinical studies have indicated that zinc, like other antagonists of the ionophore, glutamate, glycine and polyamine recognition sites of the NMDA receptor has antidepressant-like properties (Kroczka et al., 2000, 2001; Nowak et al., 2003c; Rosa et al., 2003; Franco et al., 2007; Cieslik et al., 2007; Sowa-Kućma et al., 2008). In addition, there is evidence for the ability of zinc to augment the effects of tricyclic antidepressants in preclinical studies in mice (Cieslik et al., 2007; Cucha et al., 2008; Rosa et al., 2003; Szewczyk et al., 2002, 2009) and rats (Cieslik et al., 2007). However, since zinc can interact with serotonergic, nicotinic acetylcholinergic and AMPA receptors and can inhibit the glycogen synthase kinase-3 (GSK-3) (Cucha et al., 2008; Szewczyk et al., 2008, 2009) it is premature to assert that the adjunctive benefits of zinc are entirely due to actions at NMDA receptors. There is also growing evidence that major depressive disorders are associated with reduced serum levels of zinc (Maes et al., 1994, 1997, 1999; McLoughlin and Hodge, 1990; Nowak et al., 1999). Moreover, it has been reported that serum zinc levels are particularly low in treatment resistant major depressives (Maes et al., 1997) and that serum zinc levels normalize with successful antidepressant treatment (Maes et al., 1997; Schlegel-Zawadzka et al., 2000). The relationship between serum zinc level and its concentration in discrete brain structures has not been established. Central interactions between zinc and antidepressant drugs have been analyzed only in animal models (Nowak and Schlegel-Zawadzka, 1999). Studies in rats have shown that augmentation of antidepressant efficacy of imipramine administered in combination with zinc hydroaspartate was accompanied by a drop in total content of zinc and desipramine, an imipramine metabolite, in the brain (Wyska et al., 2004). Conversely, long-term imipramine or citalopram treatment in rats caused a significant 10% increase in zinc level in the hippocampus as well as a significant decrease in the forebrain, cortex and cerebellum (Nowak and Schlegel-Zawadzka, 1999). Moreover, imipramine administered repeatedly significantly diminished IC50 value of zinc to inhibit [3H]dizocilpine binding to the NMDA receptor ion channel in rat cortex, indicating the

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sensitization of NMDA receptor to the inhibitory effects of zinc (Szewczyk et al., 2001). Finally, while zinc concentrations is not altered in brain samples from suicide victims, the IC50 value of zinc to inhibit [3H]dizocilpine binding is increased in suicidal hippocampus (Nowak et al., 2003b). Taken together, these data suggest that augmentation of antidepressant effect of imipramine in combination with zinc is attributable to redistribution of zinc (and possibly imipramine) in the central nervous system and to pharmacodynamic interaction between imipramine and zinc occurring directly and/or indirectly at NMDA receptor, thereby influencing glutamatergic neurotransmission. Open label trials with the NMDA receptor modulators riluzole and lamotrigine indicate the antidepressant efficacy of reducing NMDA receptor activity in treatment resistant patients (Zarate et al., 2005; McElroy et al., 2004). Moreover, two placebo-controlled clinical studies have demonstrated the efficacy of the NMDA receptor antagonist, ketamine, to relieve symptoms of major depressive disorder in a previously treatment resistant population (Berman et al., 2000; Zarate et al., 2006). However, the dissociative properties of available NMDA receptor antagonists such as ketamine limit the utility of this treatment, particularly in non-inpatient settings. Although, very recently, Preskorn et al., have published exciting data with an NMDA NR2B receptor antagonist which demonstrates antidepressant activity without dissociative effects (Prescorn et al., 2008). Nonetheless, these data combined with results of the present study suggest a contribution of NMDA receptor complex to pathophysiology underlying treatment resistance in major depressive disorders. Further investigations on the use of NMDA receptor modulators in patients responding unsatisfactorily to previous therapies are clearly warranted. However, in view of zinc's ability to interact with both NMDA and AMPA receptors, it is of interest to note that antidepressant effects of some NMDA receptor antagonists may also involve the activation of AMPA receptors (Dybała et al., 2008; Maeng et al., 2008). In summary, this placebo-controlled, randomized double blind study confirms our previous report of the benefit of zinc supplementation of antidepressant therapy. Notably, zinc supplementation has no influence on the efficacy of imipramine therapy in patients with a prior history of positive response to conventional antidepressant therapy. In contrast, zinc supplementation can robustly enhance the effect of imipramine in previous treatment nonresponders by increasing the efficacy of this conventional antidepressant. Role of funding source This study was supported by the Funds for Statutory Activity of Collegium Medicum, Jagiellonian University Krakow and the Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland, but had no further role in study design, in the collection, analysis and interpretation of data, in writing of the report and in the decision to submit the paper for publication. Conflict of interest No conflict declared.

Acknowledgement The authors thank “Farmapol” Sp. z o.o., Poznań, Poland for the generous gifts of Zincas and placebo.

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