L-Carnosine combination therapy for major depressive disorder: A randomized, double-blind, placebo-controlled trial

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L-Carnosine Combination Therapy for Major Depressive Disorder: A Randomized, Double-Blind, Placebo-Controlled Trial Behin Araminia , Mohammadreza Shalbafan , Amirhosein Mortezaei , Elham Shirazi , Salomeh Ghaffari , Erfan Sahebolzamani , Seyyed Hosein Mortazavi , Behnam Shariati , Mehrdad Eftekhar Ardebili , Ali Aqamolaei , Sina Naderi , Shahin Akhondzadeh PII: DOI: Reference:

S0165-0327(19)30993-0 https://doi.org/10.1016/j.jad.2020.02.020 JAD 11651

To appear in:

Journal of Affective Disorders

Received date: Revised date: Accepted date:

16 April 2019 15 January 2020 6 February 2020

Please cite this article as: Behin Araminia , Mohammadreza Shalbafan , Amirhosein Mortezaei , Elham Shirazi , Salomeh Ghaffari , Erfan Sahebolzamani , Seyyed Hosein Mortazavi , Behnam Shariati , Mehrdad Eftekhar Ardebili , Ali Aqamolaei , Sina Naderi , Shahin Akhondzadeh , L-Carnosine Combination Therapy for Major Depressive Disorder: A Randomized, Double-Blind, Placebo-Controlled Trial, Journal of Affective Disorders (2020), doi: https://doi.org/10.1016/j.jad.2020.02.020

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Highlights 

We assessed the safety and efficacy of L-carnosine on depressive symptoms of patients with major depressive disorder (MDD).



Over 6 weeks, patients who received L-carnosine showed significant and rapid-onset improvement in Hamilton Depression rating Scale (Ham-D) scores.



Treatment with L-carnosine seems to be well tolerated with no serious adverse event.

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L-Carnosine Combination Therapy for Major Depressive Disorder: A Randomized, DoubleBlind, Placebo-Controlled Trial Running head: L-Carnosine as an adjunct to citalopram in MDD

Behin Araminia1, Mohammadreza Shalbafan1, Amirhosein Mortezaei2, Elham Shirazi3, Salomeh Ghaffari4, Erfan Sahebolzamani2, Seyyed Hosein Mortazavi2, Behnam Shariati3,

Mehrdad

Eftekhar Ardebili3, Ali Aqamolaei2, Sina Naderi2, Shahin Akhondzadeh2 The first three authors contributed equally in to this study. 1

Mental Health Research Center, Iran University of Medical Sciences, Tehran, Iran.

2

Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences,

Tehran, Iran. 3

Mental Health Research Center, School of Behavioral Sciences and Mental Health, Tehran

Institute of Psychiatry, Iran University of Medical Sciences, Tehran, Iran. 4

School of Persian Medicine, Iran University of Medical Sciences, Research Institute for Islamic

and Complementary Medicine, Tehran, Iran.

Corresponding author: Shahin Akhondzadeh, PhD, Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences, South Kargar Street, Tehran 13337, Iran. Fax: (+9821)-55412222, Tel: (+9821)-55419113, E-mail: [email protected]

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ABSTRACT Background: Evidence for antidepressant effects of L-Carnosine was shown in some experimental studies. In this study we tried to evaluate the efficacy and tolerability of LCarnosine combination therapy in treatment of patients with major depressive disorder (MDD). Methods: Fifty-eight patients with MDD (DSM-V) and Hamilton Depression Rating Scale (HAM-D) score ≥ 19 were randomized to receive either 400 mg twice daily L-Carnosine or placebo in addition to

citalopram (maximum dosage of 40 mg/day)

for six weeks in a randomized

double-blind, and placebo-controlled study. Patients were assessed using the HAM-D scale at baseline and weeks 2, 4, and 6. Results: Fifty-two patients completed the trial. General linear model repeated measure showed significant difference for time × treatment on HAM-D score [F=3.17, df=2.39, p-value=0.03]. Significantly greater improvement was detected in HAM-D score of the L-Carnosine group compared with the placebo group from baseline to weeks 2, 4 and 6 [Ps= 0.013, 0.028 and 0.023; respectively]. Patients in the L-Carnosine group experienced significantly greater response and remission rate than the placebo group [Ps= 0.023 and 0.012; respectively]. There was no significant difference between the two groups in baseline parameters and frequency of side effects. Limitations: Short follow-up period and small population size were two important limitations of this study. Conclusions: L-Carnosine combination therapy with citalopram can effectively improve symptoms of patients with major depressive disorder. Rapid-onset antidepressant effects of LCarnosine were also shown which need further investigation. Key words: Glutamate; Major Depressive Disorder (MDD); L-Carnosine; Randomized controlled trial. 3

1. Introduction Major depressive disorder (MDD) is a chronic disabling psychiatric disorder which is within the top five causes of global burden of disease according to The World Health Organization (WHO) reports (Collins et al., 2011). Several problems still remain in spite of the fact that significant improvements have occurred in treatment of MDD. . Rate of complete or partial response to a standard treatment of an episode of major depression is about 50 percent. Lack of response might increase the risk of suicide and morbidity (Carvalho et al., 2007). Switching treatment to another

medication or augmentation therapy are usually used to manage resistant patients. It is necessary to develop new efficacious augmentative medications due to clinically significant lag time to onset of therapeutic efficacy and incomplete response to first-line antidepressant monotherapy in patients with MDD (Fava, 2009). Although some studies have shown positive effects of combination therapy of some medications in achieving quicker and greater response, more definite studies have to be performed in order to support this notion (Salardini et al., 2016). The monoaminergic systems (serotonin, norepinephrine, and dopamine) have received the greatest attention in the neurobiology of MDD for the past recent decades. Effects of some glutamate modulatory drugs on some psychiatric disorders like OCD and AttentionDeficit/Hyperactivity Disorder have been studied recently (Baziar et al., 2019; Naderi et al., 2018). Recent studies also show that glutamate, an excitatory neurotransmitter in the brain, plays an important role in the pathophysiology and treatment of this disease (Baziar et al., 2019; Hashimoto, 2009; Naderi et al., 2018). Thus, it is reasonable to hypothesize that medications with anti-glutamatergic effects may be able to be used in treatment of MDD. One of these possible agents is L-Carnosine proposed to have a putative antidepressant effect (Hipkiss, 2015).

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L-Carnosine is a natural antioxidant dipeptide composed of β‐alanine and L‐histidine (Boldyrev et al., 2013). It has a neuromdulatory effect on glutamatergic neurons via up regulation of the glutamate transporter 1 and reduction of glutamate concentrations in the central nervous system (Ouyang et al., 2016). L-Carnosine was shown to improve cognitive functions in the elderly (Rokicki et al., 2015) and has potential therapeutic effects in treatment of autism (Rossignol, 2009). Furthermore, it has been used in other neurologic and mental disorders like obsessive compulsive disorder, Parkinson’s disease, schizophrenia, Alzheimer's disease, AttentionDeficit/Hyperactivity Disorder and cognitive impairment in the elderly (Arabzadeh et al., 2017; Boldyrev et al., 2008; Chengappa et al., 2012; Corona et al., 2011; Ghajar et al., 2018; Rokicki et al., 2015). Based on the available data, we hypothesized that onset of treatment effects should be earlier and extent of symptom reduction in depression should be more significant in patients who received L-Carnosine due to its modulatory properties on the glutamatergic neurotransmission and its acceptable safety. To the best of our knowledge, this is the first double-blind placebo-controlled study investigating the pure anti-depressant effects of L-Carnosine in patients with major depressive disorder.

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2. Methods 2.1 Trial design and setting This study was conducted as a two-center, randomized, double-blind, parallel-group study at the outpatient clinic of Iran Psychiatric hospital and Tehran Psychiatric Institute (both affiliated to Iran University of Medical Sciences) from April 2018 to January 2019. The institutional review board (IRB) of Iran university of medical science (IUMS) approved the study (approval no: IR.IUMS.FMD.REC 1396.9411286001). The trial was conducted according to the Declaration of Helsinki and its subsequent revision. After explanation of the procedures and purpose of study for each patient, the written informed consent was obtained from all eligible patients. The patients were free to withdraw from the trial and return to their standard treatment without any concern regarding their relationship with their health care provider. The trial was registered at the Iranian registry of clinical trials (www.irct.ir; registration no: IRCT20170123032145N2). 2.2 Trial participants Eligible patients were men and women aged 18-60 years at the outpatient clinic who met the criteria for diagnosis of major depression disorder (MDD) based on Diagnostic and Statistical Manual of Mental Disorder, fifth edition (DSM-V) (Structured Clinical Interview). The patients had a score of at least 19 on the 17-item Hamilton Rating Scale for Depression (HAM-D) and a score of 2 or more on item 1 of HAM-D to be included in the trial. Exclusion criteria were as follows:

receiving

any

antidepressant

drug

during

the

previous

month,

receiving

electroconvulsive therapy (ECT) during the past 2 months, presence of psychosis or diagnosis of other mental disorders, suicidal ideation (score > 2 on the suicide item of the HAM-D), depression due to other disease, alcohol or substance (with the exception of nicotine) abuse, any

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uncontrolled medical condition such as history of thyroid disease, cardiovascular problems and liver disorders, pregnancy or lactation. The patients were also excluded if they had a history of treatment with citalopram during the past 6 months without any proper response. 2.3 Interventions Eligible patients were randomly divided into groups to receive either 400 mg L-Carnosine (ACER, Tehran, Iran) twice daily or Placebo capsules in the same manner for six weeks. All participants received 10 mg/day and 20 mg/day citalopram (citalopram, Sobhan Darou, 20 mg) in the first and second week of the trial respectively and for the rest period of the trial, the dosage of citalopram raised to maximum of 40mg/day due to the response of the participants to the treatment. Patients were not treated with any other psychiatric medication during the course of this trial. Weekly capsule

counts were justified against participant reports of medication intake to calculate the proportion of dispensed medication doses that were actually ingested. 2.4 Outcomes All patients were evaluated at baseline and at weeks 2, 4 and 6 using HAM-D which is a validated 17 item rating scale and is applied extensively for assessing the severity of depressive symptoms (Hamilton, 1960). It had been used in several clinical trials in Iran (Abbasi et al., 2015; Modabbernia et al., 2012). The primary outcome of this study was to evaluate L-Carnosine in improvement of depressive symptoms, compared to placebo, using a general, linear, repeated measured model. Two groups also were compared with HAM-D score changes from baseline to each time-point, early improvement (≥20% reduction in HAM-D score in the first 2 weeks), response to treatment (≥50% reduction in the HAM-D score), remission rate (HAM-D score≤7)

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and time needed to response to treatment (Kashani et al., 2017). Two experienced psychiatrists conducted all assessments with an inter-reliability > 90% on the HAM-D score. 2.5 Safety All patients received complete explanation that they could leave the study whenever they wanted and return to the standard treatment. During the course of the trial, the patients were asked to inform their physicians or research team about any unexpected changes in their behavior immediately. Physical examination was performed for each patient and their vital signs were recorded at the screen session. All patients, systematically were asked about adverse effects via a checklist at each visit session (Amiri etal., 2008; Khajavi et al., 2012; Shahmansouri al., 2014; Zeinoddini et al., 2015). 2.6 Sample size Assuming a mean difference (MD) of 3 in the HAM-D score between the two groups of this study with a standard deviation (SD) of 3 (based on a pilot study performed previously), a power of 80% and a 2-taild significance level of 5%, and a sample size of 30 (15 patients in each group) was calculated. Considering an attrition rate of 20%, 40 patients were needed. Ultimately, a total number of 50 patients (25 patients in each group) were selected. 2.7 Randomization and drug allocation A computerized random number generator was used for generation of randomization codes (blocks of 4, allocation ratio 1:1). Sequentially numbered and sealed opaque and stapled packages were used to conceal allocation. Separate people were in charge of random allocation and clinical assessment of patients. The participants, the physician who referred the patients, the psychiatrists who rated the participants and prescribed the medication and the statistician were 8

all blinded to the allocated treatment. L-Carnosine and placebo capsules were completely similar by their size, shape, color and odor. 2.8 Statistical analysis IBM SPSS statistic 25 (IBM corporation, Armonk, NY, USA) was used to analyze the data. Categorical variables were reported as number of patients and as a percentage. Continuous variables were presented as mean ± standard deviation (SD). At the baseline, continuous variables in two groups were compared using independent sample t-test. A two-factor repeated measures analysis of variance (ANOVA) was used to compare the HAM-D score of the two groups during the trial. In this test, two treatment groups were considered as between-subjects factor and three measurements were considered as within-subjects factor. The results of Greenhouse-Geisser were reported whenever Mauchly`s test of sphericity was significant. To compare score changes from baseline between two groups of study at each time-point, independent sample t-test was used. The χ^2 test or Fisher`s exact test was used to compare categorical variables. Comparison of time needed for response to treatment between two groups was conducted using the Kaplan-Meier estimation with log-rank test. In all analyses, p-values < 0.05 were considered significant statistically.

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3. Result 3.1 Participants After screening 90 participants for the eligibility criteria, 32 participants were excluded and 58 patients were included in the trial and randomized to receive either citalopram plus L-Carnosine (n=29) or citalopram plus placebo (n=29). Fifty-two patients completed the study and participated in all follow-up visits and their data were used in analysis. Three patients in the LCarnosine and the placebo group gave up the study after the first post-baseline visit due to withdrawn consent. (Fig. 1) There were no statistically significant differences in baseline HAMD scores between the L-Carnosine and the placebo arms [26.38 ± 3.63 vs 26.65 ± 3.76; respectively, df=50, t= -0.26, p-value= 0.79)]. Other baseline characteristics of patients were summarized in Table 1 and there was no significant difference between the two groups. (Table 1) 3.2 Outcomes Two factor repeated measured ANOVA demonstrated significant effect for time × treatment interaction on HAM-D scores [Greenhouse-Geisser corrected: F=3.17, df=2.39, p-value=0.037]. (Fig. 2) A significant difference was observed in time [Greenhouse-Geisser corrected: F=377.86, df=2.39, p-value<0.001] showing that both groups improved significantly during the trial. Significantly greater improvements were observed in the L-Carnosine group compared with the placebo group at the end of the trial [week 6: 20.38 ± 3.57 vs 17.23 ± 5.83, t(50)=2.35 , pvalue=0.023]. (Table 2) Early improvement was greater in the L-Carnosine group (80.8%) compared with the placebo group (42.3%) and this difference was statistically significant [pvalue=0.009]. As demonstrated in Table 3, the response rate (≥50% reduction in the HAM-D score) and remission rate (HAM-D score ≤ 7) were significantly greater in the L-Carnosine

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group at the end of the study. The response rate in the L-Carnosine group was 100% compared with 76.92% in the placebo group at week 6 [p-value= 0.023]. The remission rate was significantly greater in L-Carnosine group compared with the placebo group at the end of the trial [73.0% vs 34.6%; respectively, p-value= 0.012] (Table 3). Kaplan-Meier estimation demonstrated that the patients in the L-Carnosine group responded significantly faster to treatment [4.69 ± 0.22 vs 5.30 ± 0.19 weeks; respectively, long rank p-value= 0.006]. Moreover, the time to remission was significantly shorter in the L-Carnosine group [5.69 ± 0.14 vs 6.00 ± 0.00 weeks; respectively, long rank p-value= 0.003]. 3.3 Adverse effects A number of the most probable side effects were recorded. (Table 4) The frequency of adverse effects was not significantly different between the two groups and no serious adverse event was recorded during the trial.

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4. Discussion The current study demonstrated that L-Carnosine was an effective adjuvant to citalopram in treatment of MDD. HAM-D scores improved significantly in the L-Carnosine group compared with the placebo group at weeks 2, 4, and 6 of the trial. Moreover, remission rate was significantly greater in the L-Carnosine group at the end of the study. The use of L-Carnosine combination therapy also appeared to be safe and well tolerated and no serious adverse event was reported in this study. Synaptic dysfunction of glutamatergic circuits and disturbance in brain concentration of glutamate, the principle excitatory neurotransmitter of central nervous system, has been shown to be involved in pathophysiology of variety of neurodegenerative diseases and mood and anxiety disorders (Miladinovic et al., 2015). Although intra synaptic function of glutamate is fundamental in information processing and synaptic plasticity, extra synaptic glutamate cause neuronal malfunction and eventually death. Consequently, the amount of glutamate in the different synaptic areas must be controlled. So there are several transporters and antiporters on glial cells, mostly astrocyte, and neuronal endings that remove the glutamate from synaptic cleft and prevent its harmful effects (Danbolt, 2001; Hardingham and Bading, 2010; Kalivas, 2009; Pittenger et al., 2008). The many lines of evidence have suggested that alteration in expression of these glutamate transport genes may play a critical role in pathogenesis of neurobehavioral and affective disorders, including MMD (Chandley et al., 2013; McCullumsmith and MeadorWoodruff, 2002; Medina et al., 2013; Zink et al., 2010). Furthermore, brain imaging techniques have also supported the possible role of glutamatergic neurotransmission in the neuropathology of mood disorders (Auer et al., 2000; Luykx et al., 2012).

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Recent research has increasingly revealed that the application of an effective treatment with glutamatergic modulators, like ketamine, amantadine, riluzole and lanicemine, which have neural plasticity enhancing property along with neurochemical and neuroprotectional support, would be of benefit in MDD patients and could show promising results (aan het Rot et al., 2010; AtesAlagoz and Adejare, 2013; Duman et al., 2016; Salardini et al., 2016; Wilkinson et al., 2017). In addition, in 2015, Alan R. Hipkiss

proposed that L-carnosine supplementation, a natural

dipeptide with antioxidant activities, which has been found to decrease the extracellular glutamate concentration, could have a putative antidepressant effect (Hipkiss, 2015; Ouyang et al., 2016). L-Carnosine, also known as β-alanyl-L-histidine, is a natural imidazole-containing compound found abundantly in skeletal muscle and the olfactory bulb of mammals (Babizhayev et al., 1994; Boldyrev et al., 2013). L-Carnosine has a putative role in neuromodulation of glutamatergic sensory neurons (especially olfactory neurons) as well as antioxidant, metal chelating, and antiglycative properties in certain brain structures (Boldyrev et al., 2013). It also can play a protective role against brain ischemia (Shen et al., 2010). Due to neuroprotective functions of LCarnosine, it has been used in different disorders such as autism (Rossignol, 2009), schizophrenia (Chengappa et al., 2012), attention-deficit/hyperactivity disorder (Ghajar et al., 2018), obsessive compulsive disorder (Arabzadeh et al., 2017), Alzheimer’s and Parkinson’s disease (Boldyrev et al., 2008; Corona et al., 2011). Additionally, supplementation therapy with carnosine has been demonstrated to be effective for improving cognitive impairment in the elderly (Rokicki et al., 2015). Oxidative stress is thought to participate in the pathophysiology of major depressive disorder (Michel et al., 2012). Dietary supplementation with carnosine has been shown to suppress stress 13

and improve behavior and well-being. Hence, the therapeutic potential of carnosine supplementation for stress-related and depressive disorders is proposed to be studied (Hipkiss, 2015). Given its effect on glutamate, we hypothesized that L‐carnosine would reduce symptoms of major depressive disorder. To the best of our knowledge, the current study is the first doubleblind placebo-controlled clinical trial that investigated the efficacy and safety of L-carnosine in combination with citalopram, as a standard of care agent, in patients suffering from major depressive disorder. 5. Limitations Despite its several advantages, like the double-blind, placebo-controlled design and the careful adjustment for baseline clinical variables, this study had some limitations. Short follow-up period and small population size were two important limitations of this study. Although there was no significant gender difference between the two study groups, the male and female population was not equal. 6. Conclusion A 6-week treatment course with L-Carnosine in combination with citalopram showed favorable safety and efficacy profile in patients with major depressive disorder. Nevertheless, due to some limitations, further studies are indicated. Conflicts of Interest: All authors declare that they have no conflict of interest.

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Acknowledgments: This study was supported by a grant from Iran University of Medical Sciences. Role of funding source: The funding organization had no role in the design and conduct of this study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript and the decision to submit the paper for publication. The current study complies with contemporary laws and regulations in Iran.

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Author contributions Prof. Shahin Akhondzadeh, Dr. Mohammadreza Shalbafan, Dr. Elham Shirazi, Dr. Behnam Shariati and Dr. Mehrdad Eftekhar Ardebili designed the manuscript, provided the outlines for the presentation of the study, supervised the study process and edited the final manuscript. Dr Behin Araminia, Dr Amirhosein Mortezaei, Salomeh Ghaffari, Dr. Erfan Sahebolzamani, Dr. Seyyed Hosein Mortazavi, Dr. Ali Aqamolaei and Dr. Sina Naderi participated in data acquisition and the preparation of the manuscript. Acknowledgments This study was supported by a grant from Iran University of Medical Sciences. Role of funding source: The funding organization had no role in the design and conduct of this study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript and the decision to submit the paper for publication. The current study complies with contemporary laws and regulations in Iran. Conflicts of Interest: All authors declare that they have no conflict of interest.

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Figure 1 - Flow diagram representing case selection for the trial program

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Figure 2 - Repeated measure analysis for comparison of the two treatment effects on the Hamilton depression rating scale (HAM-D) scores. Values represent mean ± SEM (standard error of mean). P- values show the result of the independent sample t-test for comparison of the score change from the baseline between the two groups at each time point (*p ≤ 0.05).

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item

L-Carnosine Arms (n=26) 34.76 ± 6.00 13 (50%)

Age, mean ± SD Sex, Male (%) Marital status, n(%) Married 16 (61.5%) Single 10 (38.5%) Educational status, n(%) Under Diploma 8 (30.8%) Diploma 9 (34.6%) University Degree 9 (34.6%) Occupational status, n(%) Unemployed 2 (7.7%) Housewife 11 (42.3%) Student 8 (30.8%) Employed 5 (19.2%) Smoking, Yes (%) 9 (34.6%) Baseline HAM-D score, mean ± SD 26.38 ± 3.63 Table 1. Baseline characteristics of the participants

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Placebo Arms (n=26) 32.11 ± 7.17 16 (61.5%)

p-value 0.15 0.57 1.00

15 (57.7%) 11 (42.3%) 1.00 8 (30.8%) 9 (34.6%) 9 (34.6%) 0.89 2 (7.7%) 11 (42.3%) 6 (23.1%) 7 (26.9%) 10 (38.5%) 26.65 ± 3.76

1.00 0.79

Table 2. Comparison of score changes between the two groups

HAM-D score Change from baseline to week 2, mean ± SD Change from baseline to week 4, mean ± SD Change from baseline to week 6, mean ± SD

L-Carnosine group 7.69 ± 3.09

Placebo group 5.15 ± 3.96

Mean difference (95% CI) 2.53 (0.55-4.52)

t(50)

p-value

2.57

0.013

14.00 ± 4.25

10.96 ± 5.37

3.03 (0.34-5.73)

2.26

0.028

20.38 ± 3.57

17.23 ± 5.83

3.15 (0.45-5.84)

2.35

0.023

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Table 3. Comparison of response to treatment and remission rates at different study points between the two groups

Outcome Number (%) of responders, at week 2 Number (%) of responders, at week 4 Number (%) of responders, at week 6 Number (%) of remissions, at week 2 Number (%) of remissions, at week 4 Number (%) of remissions, at week 6

L-Carnosine group 1 (3.8%) 16 (61.5%) 26 (100%) 0 4 (15.4%) 19 (73.1%)

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Placebo group 0 9 (34.6%) 20 (76.9%) 0 0 9 (34.6%)

p-value

Odds Ratio (95%CI)

1.00 0.095 0.023 0.11 0.012

2.04 (1.54-2.69) 3.02 (0.97-9.35) 0.43 (0.31-0.60) 5.12 (1.56-16.76)

Table 4. Frequency of adverse events in the two study groups

Side effect

L-Carnosine

Placebo

(n = 26)

(n = 26)

Stiffness

2 (7.6%)

1(3.8%)

1.00

Day time drowsiness

1 (3.8%)

3 (11.53%)

0.61

Nausea

2 (7.6%)

1 (3.8%)

1.00

Constipation

2 (7.6%)

4 (15.38%)

0.66

Increased appetite

2(7.6%)

4(15.38%)

0.66

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p-value