Pregnenolone as an adjunct to risperidone for treatment of women with schizophrenia: A randomized double-blind placebo-controlled clinical trial

Accepted Manuscript Pregnenolone as an adjunct to risperidone for treatment of women with schizophrenia: A randomized double-blind placebo-controlled clinical trial Ladan Kashani, Nazila Shams, Ehsan Moazen-Zadeh, Mohammad-Ali KarkhanehYousefi, Gita Sadighi, Mohammad-Reza Khodaie-Ardakani, Farzin Rezaei, Fatemeh Rahiminejad, Shahin Akhondzadeh PII:

S0022-3956(17)30099-7

DOI:

10.1016/j.jpsychires.2017.06.011

Reference:

PIAT 3145

To appear in:

Journal of Psychiatric Research

Received Date: 21 January 2017 Revised Date:

21 June 2017

Accepted Date: 27 June 2017

Please cite this article as: Kashani L, Shams N, Moazen-Zadeh E, Karkhaneh-Yousefi M-A, Sadighi G, Khodaie-Ardakani M-R, Rezaei F, Rahiminejad F, Akhondzadeh S, Pregnenolone as an adjunct to risperidone for treatment of women with schizophrenia: A randomized double-blind placebo-controlled clinical trial, Journal of Psychiatric Research (2017), doi: 10.1016/j.jpsychires.2017.06.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1 Pregnenolone as an adjunct to risperidone for treatment of women with schizophrenia: A randomized double-blind placebo-controlled clinical trial

a*

MD., Nazila Shams

b*

Karkhaneh-Yousefi b* MD., Gita Sadighi

MD., Ehsan Moazen-Zadeh c

b*

MD., Mohammad-Ali

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Ladan Kashani

MD., Mohammad-Reza Khodaie-Ardakani

c

MD.,

a

b

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* The first four authors contributed equally in this study

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Farzin Rezaei d MD., Fatemeh Rahiminejad b MD., Shahin Akhondzadeh b# PhD.

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

Tehran, Iran

Razi Hospital, University of Social Welfare and Rehabilitation, Tehran, Iran

d

Qods Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran

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c

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# Correspondence to: Prof. Shahin Akhondzadeh Ph.D., Psychiatric Research Center, Roozbeh Psychiatric Hospital, Tehran University of Medical Sciences, South Kargar Street, Tehran

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13337, Iran. Tel: 98-21-55412222, Fax: +98-21-55419113, Email: [email protected]

ACCEPTED MANUSCRIPT 2 Abstract

There have been few studies of pregnenolone therapy in schizophrenia and those that exist have

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been subject to several critical limitations, thus yielding inconsistent results. We attempted to assess the therapeutic effect of pregnenolone in a patient sample as homogeneous as possible. In this randomized double-blind clinical trial, 82 female inpatients with chronic schizophrenia, who

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had discontinued their antipsychotic medications for at least one week in case of any oral antipsychotic medication or a month for any depot antipsychotic medication, received

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risperidone plus either pregnenolone (50mg/day) or placebo for 8 weeks. Inclusion criteria were acute illness with a baseline Positive and Negative Syndrome Scale (PANSS) negative subscale score of ≥20. Exclusion criteria were the presence of severe depression or other concomitant psychiatric disorders. Primary outcome was defined as the difference in the PANSS total score

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change from baseline to week 8 in the pregnenolone group compared to the placebo group. No significant difference was found in the PANSS total score changes between the two arms (mean difference (CI 95%) = -9.41 (-20.24 to 1.41); p= 0.087). Significant differences were initially

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found for PANSS negative change scores (mean difference (CI 95%) = -2.61 (-5.03 to -0.19); p=0.035) and general psychopathology change scores (mean difference (CI 95%) = -5.93 (-11.37

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to -0.48); p= 0.033). However, these findings did not survive Bonferroni correction for multiple testing. While this trial may suggest a potential effect of pregnenolone on schizophrenia symptoms, further studies are warranted. Keywords: Schizophrenia; Pregnenolone; Steroids; Clinical Trial

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1. Introduction

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As a chronic psychiatric disorder, schizophrenia affects the worldwide population with a lifetime prevalence of 1% and considerable long-term mortality, morbidity, and burden (Esan et al., 2012; Rössler et al., 2005; Saha et al., 2007; Switaj et al., 2012). Routine treatment regimens

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are accompanied by adverse effects, and a significant portion of patients remain symptomatic despite treatment, especially those with negative symptoms, which are the major disabling

and Stahl, 2007; Schooler et al., 2015).

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factors in these patients (Akhondzadeh and Moazen-Zadeh, 2017; Bobes et al., 2009; Buckley

Numerous lines of evidence indicated sex-specific characteristics of schizophrenia (da Silva and Ravindran, 2015; Melcangi and Garcia-Segura, 2010). Male patients are prone to

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higher risk, lower age of onset, and more severe course of the disorder (Aleman et al., 2003; McGrath et al., 2008). Compared to males, female patients are reported to respond better to treatment (Bloch et al., 2005; Grigoriadis and Seeman, 2002; Riecher-Rössler and Häfner, 2000),

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have lower remission and hospitalization rates (Desai et al., 2013), and express more concomitant affective symptoms such as depression, anxiety and irritability (Choi et al., 2001;

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Goldstein, 2006). Also, a second incidence peak of schizophrenia, around the age of 40, is reported in female patients (van der Werf et al., 2014). Further studies have suggested that neurosteroids, including pregnenolone and its metabolites such as estrogens, play an important role in pathophysiology of schizophrenia and can be potential therapeutic targets (Halbreich and Kahn, 2003; Heringa et al., 2015; Seeman, 2002).

ACCEPTED MANUSCRIPT 4 Among neurosteroids, pregnenolone is the single primary precursor for all others and has provoked less concern about adverse effects in case of long-term therapy compared to other neurosteroids including estrogens, progesterone, and dehydroepiandrosterone (DHEA)

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(Scheffers et al., 2015; Shulman et al., 2005; Wentzensen and Traberf, 2015). Furthermore, pregnenolone improved response to stress, mood regulation, and cognitive performance (Marx et al., 2011; Ritsner, 2010, 2011). Besides the direct effects of pregnenolone, such as modulation of

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neurotransmitters and hypothalamic-pituitary-adrenal (HPA) axis function (Zheng, 2009), further effects of pregnenolone are postulated to be mediated by its metabolites. For example,

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allopregnenolone decreases apoptosis and inflammation, increases neurogenesis, and markedly increases gamma-aminobutyric acid A (GABAA) receptor response. Pregnenolone sulfate, another metabolite, positively modulates N-methyl-D-aspartate (NMDA) receptors. Improving NMDA receptor function and GABA regulation have been proposed as two potential

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mechanisms through which pregnenolone may benefit patients with schizophrenia (MacKenzie et al., 2007; Marx et al., 2011). As other metabolites of pregnenolone, estrogens are widely studied in schizophrenia with promising effects according to some trials (Heringa et al., 2015).

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Interestingly, lower serum levels of pregnenolone was detected in schizophrenia patients compared to the controls (Ritsner et al., 2007), and oral administration of pregnenolone was

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associated with increased serum levels of pregnenolone as well as its metabolites by several folds (Marx et al., 2009, 2014). Furthermore, in animal models, pregnenolone serum levels were correlated with pregnenolone levels in the central nervous system (CNS) (Caruso et al. 2013). Considering the neural effects of pregnenolone, and its metabolites, and the fact that oral administration of pregnenolone potentially increases CNS levels of these neurosteroids, therapeutic effects of oral administration of pregnenolone in schizophrenia patients warranted

ACCEPTED MANUSCRIPT 5 further investigations. Meanwhile, the few available clinical trials on the pregnenolone adjunctive treatment in patients with schizophrenia had some limitations and yielded inconsistent results (Marx et al., 2009, 2014; Ritsner et al., 2010, 2014). A recent meta-analysis on these few

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trials found no significant difference between effects of pregnenolone adjunctive therapy and placebo on total, positive, or negative symptoms of schizophrenia measured by Positive and Negative Syndrome Scale (PANSS) (Heringa et al., 2015); however, the high heterogeneity of

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negative symptoms (Heringa et al., 2015) and inconsistent results of previous trials necessitated further scrutiny.

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We assessed the potential therapeutic effects of pregnenolone as an adjunctive therapy to risperidone in women with schizophrenia in an 8-week, randomized, double-blind, placebocontrolled clinical trial with a sample size of enough power for hypothesis testing. The primary outcome of interest was defined as the mean difference in change of PANSS total symptoms

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score from baseline to the end-point between the two treatment arms.

ACCEPTED MANUSCRIPT 6 2. Methods

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2.1. Trial Design

In this parallel-group, placebo-controlled, double-blind clinical trial, schizophrenia patients were equally randomized (1:1), treated, and followed for 8 weeks. The trial protocol was

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approved by the Institutional Review Board (IRB) of Tehran University of Medical Sciences (Approval number: 27746) in accordance with the World Medical Association (Declaration of

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Helsinki, as revised in Brazil 2013) code of ethics, and registered at the Iranian Registry of Clinical Trials (IRCT201504211556N76; http://www.irct.ir/). Written informed consent was obtained from patients and their legally authorized representatives in accordance with the procedures defined by the local IRB, and participants were informed that they were free to

healthcare provider.

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2.2. Participants

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withdraw from the study at any time without any negative effect on their relationship with the

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Participants were selected from female inpatients with chronic schizophrenia who were

18-50 years old. The diagnosis was based on the Structured Clinical Interview for Diagnostic and Statistical Manual-IV-TR Axis I Disorders (SCID) (First et al., 1997), and confirmed through an interview by a senior physician as well as a chart review. It was required that the participants be in the active phase of the illness, have a PANSS total score of ≥60, PANSS negative subscale score of ≥20, and illness duration of ≥2 years. Patients with any of the

ACCEPTED MANUSCRIPT 7 following conditions were excluded: history of comorbid DSM-IV Axis I disorders or alcohol/substance (other than nicotine) dependence based on DSM-IV-TR, a score of ≥14 on 17item Hamilton Rating Scale for Depression (HRSD) or ≥4 on depression item of PANSS, a score

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of ≥2 on the suicide item of HRSD or suicide ideation, mental retardation (intelligence quotient <70) based on clinical judgment, inability to communicate, severe extrapyramidal symptoms, serious neurological or medical conditions, pregnancy, lactation, women in child-bearing ages

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without a reliable method of contraception, and any history of prior thromboembolism, abnormal

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uterine bleeding, uterine or breast cancer, cerebrovascular accident, endocrine abnormality, hormone replacement therapy, or hypersensitivity to risperidone or pregnenolone. Patients were also excluded in case of any oral antipsychotic medication during the last week, any depot antipsychotic medication during the last month, or electroconvulsive therapy during the last 2 weeks prior to their enrollment. Patients’ prior medications were not changed or discontinued in

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this study, but rather the participants were selected from those who had discontinued their medication due to some other reason including lack of supportive care or incompliance with their treatment. It was also required that participants do not use mood stabilizers, antidepressants,

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sedating antihistamines, or a second antipsychotic, or receive behavior intervention therapy

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during the course of the trial. 2.3. Study settings

This multicenter clinical trial was conducted at Roozbeh Hospital (Tehran University of

Medical Sciences, Tehran, Iran), Razi Hospital (University of Social Welfare and Rehabilitation Sciences, Tehran, Iran), and Qods Hospital (Kurdistan University of Medical Sciences, Sanandaj, Iran) from May 2015 to November 2016. After the baseline visit, patients were evaluated in four

ACCEPTED MANUSCRIPT 8 consecutive visits with 2-week intervals. The trial was not subject to ethnical or regional restrictions as the patients were referred from different regions of Iran to these three large

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academic referral hospitals. 2.4. Interventions

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All the patients received risperidone (Risperdal®, Janssen Pharmaceuticals) in addition to the pregnenolone (Ray Kurzweil & Terry Grossman’s Health Products) or placebo from the

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beginning of the study. The starting dose for risperidone was 2 mg/day and was subsequently increased with 2 mg increment on a weekly basis to a maximum dose of 6 mg/day (2 mg TID) based on the clinical response. Pregnenolone was administered at a constant dose of 50 mg/day from baseline to the endpoint.

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2.5. Outcomes

In this trial, the PANSS was used for the assessment of treatment efficacy at baseline, and

for

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weeks 2, 4, 6, and 8. As a 30-item rating scale, the PANSS consists of three validated subscales measurement of

negative (seven

items),

positive (seven

items),

and

general

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psychopathological (16 items) symptoms of schizophrenia which are summed up in the PANSS total score (Kay et al., 1987). The PANSS was previously used in the Iranian population for clinical trials of schizophrenia (Iranpour et al., 2016; Nikbakhat et al., 2016; Tajik-Esmaeeli et al., 2017). Patients were rated on the PANSS scale by four trained raters who were third year residents of psychiatry and experienced in PANSS implementation in schizophrenia trials with an inter-rater reliability of >90% on PANSS total score. The 17-item HRSD (Hamilton, 1960; Jafarinia et al., 2016) was used for assessment of depressive symptoms at baseline and endpoint.

ACCEPTED MANUSCRIPT 9 The primary outcome of interest was defined as the mean difference in the PANSS total score change from baseline to week 8 between the pregnenolone and placebo arms. The secondary outcomes were the mean difference in the PANSS subscale score change between the

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two arms from baseline to the end-point, and the time × treatment interaction for PANSS total and subscale scores.

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2.6. Safety

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A broad range of adverse effects and warning symptoms was recorded at each visit through asking three open-ended questions followed by a 25-item comprehensive checklist (Akhondzadeh et al., 2008). In addition, the participants, their caregivers, and involved nurses were asked to report any unexpected signs or symptoms. The participants were also subject to a

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thorough physical examination at each visit. Extrapyramidal Symptom Rating Scale (ESRS) (part one: parkinsonism, dystonia, dyskinesia; sum of 11 items) (Chouinard and Margolese, 2005) was used for assessment of extrapyramidal symptoms at weeks 1, 2, 4, 6, and 8.

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Throughout the trial, independent raters were responsible for behavioral appraisal and assessment of side effects. In case of encountering side effects, a patient’s treatment would be

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discontinued, decreased, or continued based on the opinion of the appointed expert psychiatrist.

2.7. Sample size

Based on previous trials of schizophrenia, a final difference of 6.5 on the PANSS total score, a standard deviation of 10, a type I error of 0.05, and a power of 80% were assumed for

ACCEPTED MANUSCRIPT 10 sample size estimation. Primary estimated sample size was 74 (each group 37), and final sample

2.8. Randomization, allocation concealment, and blinding

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size was considered 90 assuming 20% dropout rate.

The patients were randomized to pregnenolone and placebo arms in a 1:1 ratio using

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computerized random number generation and random permuted blocks of four or six. Treatment allocation concealment from the physicians and patients was achieved using sequentially

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numbered, opaque, and sealed envelopes. Random allocation and rating of the patients were carried out by different entities. An investigational drug pharmacist was responsible to dispense the study medications in identical containers. Pregnenolone and placebo tablets were identical in size, shape, texture, color, taste, and odor. The physicians, nurses, patients, and the statistician

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2.9. Statistical methods

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were all blind to the treatment allocation.

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Data were analyzed and related plots were drawn using IBM SPSS Statistic 19.0.0 (IBM Corporations) and SigmaPlot 12.2.0 (SYSTAT Software, Incorporated) respectively. Continuous and categorical variables were reported in the respective formats of mean (± standard deviation, SD) and count (%).Chi-square test or Fisher’s exact test were used where appropriate. Mean differences in PANSS, HRSD, and ESRS scores between the two arms were reported in the format of mean (95% confidence intervals (95%CI)). Mean score changes from baseline to each time point were compared between the two groups using the independent sample’s t-test.

ACCEPTED MANUSCRIPT 11 Degrees of freedom and concordant p-values were corrected whenever the assumption of equality of variances was violated based on the Levene’s test results. Effect size was estimated for between-group differences in mean score change from baseline to each time-point using

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Cohen’s d (95% CI). Time × treatment interactions were assessed using two-way repeated measure analysis of variance (ANOVA) in which the treatment groups and PANSS measurements were considered as the between-subject and within-subject factors respectively.

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Greenhouse-Geisser’s correction was applied whenever the assumption of sphericity was violated based on the Mauchly’s test results. In order to adjust for multiple testing effect, p-

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values of <0.012 were considered significant in analysis of PANSS total and subscale score changes. P-value <0.05 was considered significant in analysis of baseline characteristics, time ×

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treatment interactions, and adverse events. Two-tailed p-values were reported.

ACCEPTED MANUSCRIPT 12 3. Results

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3.1. Participants

As shown in the flow diagram (Fig. 1), out of 122 screened patients, 90 were randomized to the pregnenolone and the placebo arms. Out of 45 patients in each group, 41 met the first post-

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baseline visit, continued to the end of the study, and were considered for analysis.

Analysis of baseline characteristics of patients did not show any significant difference between

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the two treatment arms for socio-demographics, illness duration, schizophrenia subtype, prior anti-psychotic medications, baseline HRSD score, or baseline ESRS score (Table 1). Also, there was no significant difference in baseline PANSS total or subscale scores between the two arms. The mean dose of risperidone administered during this study was 4.35 (0.65) mg/day and 4.40

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(0.70) mg/day in pregnenolone and placebo groups, respectively.

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3.2. Outcomes

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3.2.1. PANSS total score

As the primary outcome of interest, there was no significant difference in mean total

score changes between the pregnenolone and the placebo arms by the end of the study (Table 2, Fig. 2). During the study, there was no significant time × treatment interaction between the two

ACCEPTED MANUSCRIPT 13 arms [Two-way ANOVA with Greenhouse-Geisser correction: F (df, mean square) = 2.53 (1.17, 922.98), p = 0.110].

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3.2.2. PANSS negative subscale score

The pregnenolone arm experienced a significantly higher reduction in negative subscale

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scores compared to the placebo arm by the end of the study [mean difference (CI 95%) = -2.61 (5.03 to -0.19), t (df) = -2.15 (80), p = 0.035] (Table 2, Fig. 2) and by each time-point; however,

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the p-values moved out of significant range after Bonferroni correction. During the study, there was a significant time × treatment interaction between the pregnenolone and the placebo arms [Two-way ANOVA with Greenhouse-Geisser correction: F (df, mean square) = 4.08 (1.39,

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65.62), p = 0.033].

3.2.3. PANSS positive subscale score

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At study termination, there was no significant difference in mean positive subscale score change between the two arms (Table 2, Fig. 2). During the study, there was no significant time ×

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treatment interaction between the pregnenolone and the placebo arms [Two-way ANOVA with Greenhouse-Geisser correction: F (df, mean square) = 0.07 (1.33, 3.62), p = 0.860].

3.2.4. PANSS general psychopathology score

ACCEPTED MANUSCRIPT 14 By the end of the study, the pregnenolone arm experienced a significantly higher reduction in general psychopathology subscale scores compared to the placebo arm [mean difference (CI 95%)= -5.93 (-11.37 to -0.48), t (df) = -2.17 (80), p = 0.033] (Table 2, Fig. 2);

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however, the p-value dropped out of significance range after Bonferroni correction. During the study, there was a significant time × treatment interaction between the pregnenolone and the placebo arms [Two-way ANOVA with Greenhouse-Geisser correction: F (df, mean square) =

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4.00 (1.24, 365.47), p = 0.040].

3.3. HRSD score

At the end of the study, there was no significant difference in the mean HRSD score

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change between the pregnenolone and the placebo arms [mean difference (CI 95%) = -0.05(-0.38 to 0.28), t (df) = -0.29 (80), p = 0.772].

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3.4. ESRS score

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At the end of the study, there was no significant difference in the mean ESRS score

change between the pregnenolone and the placebo arms [mean difference (CI 95%) = -0.20(-0.64 to 0.24), t (df) = -0.88 (80), p = 0.380].

ACCEPTED MANUSCRIPT 15 3.5. Adverse Events

There was no significant difference between the pregnenolone and the placebo arms

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concerning adverse events other than extrapyramidal symptoms during the study course (Table 3). The reported adverse events included drowsiness, dizziness, headache, insomnia, acne, facial hair growth, constipation, diarrhea, nausea, vomiting, increased appetite, dry mouth, and

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increased blood pressure.

ACCEPTED MANUSCRIPT 16 4. Discussion

We found that the PANSS negative symptoms subscale scores decreased more

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prominently in the pregenolone arm compared to the placebo arm by each time-point. Also, we detected relatively higher reductions in the PANSS general psychopathology subscale scores in the pregenolone arm compared to the placebo arm by weeks 6 and 8. We also found a time ×

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treatment interaction effect for both negative symptoms and general psychopathology subscales. In case of PANSS total scores, mean differences in score change was considerable but not

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significant. For PANSS positive symptom subscale scores, there were no differences between the two arms. No time × treatment interaction was found for PANSS total or positive subscale scores by the end of the study at week 8.

Our findings are in line with a report by Ritsner et al. (2014) that patients with

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schizophrenia who received pregnenolone 50mg/day had significantly greater improvement in PANSS negative symptoms subscale scores compared to those who received placebo in a trial on 60 patients, out of whom 52 completed the study. Interestingly, the difference in reduction of

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PANSS negative symptoms subscale score was only significant for patients who were not treated with mood stabilizers (pregnenolone, n= 17; placebo, n=21). In another study, Marx et al. (2009)

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detected a significantly greater improvement in negative symptoms of patients treated with pregnenolone 500mg/day compared to the placebo in a trial on 21 patients, out of whom 18 completed the study; however, the difference in reduction was significant only for Scale for Assessment of Negative Symptoms (SANS) score but not for the PANSS negative subscale score. They reported a moderate correlation between SANS scores and PANSS negative subscale scores, and pointed out the low power of the study as the main cause of the non-significance in

ACCEPTED MANUSCRIPT 17 the results of comparison of PANSS negative symptoms score changes between the two treatment arms. In 2010, an interesting trial was carried out containing 4 arms: placebo (n=16), low dose

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pregnenolone (n=16), high dose pregnenolone (n=10), and DHEA (Ritsner et al., 2010). In contrast to our findings, they reported significantly higher reduction only in PANSS positive symptoms subscale scores of patients treated with low dose pregnenolone (30mg/day) compared

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to the placebo arm; however, this result was not replicated for the other subgroup of patients treated with high dose pregnenonlone (200mg/day). Recently, Marx et al. (2014) found no

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significant differences in SANS or PANSS subscale or total score changes between the pregnenolone and the placebo groups in a trial with a considerable sample size of 120 participants, out of whom 102 completed the study; however, the participants in that study had extremely low baseline SANS and PANSS scores on average which made it hard to detect

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difference in symptom improvement as the improvements are proportionately low when the baseline scores are low. Also, the antipsychotic dose received by 15 of patients was changed during the trial, and 62 patients were receiving two or more antipsychotics during that study,

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which might had confounded the effects of treatment with pregnenolone. The inconsistency observed in results of previous trials can be in part explained by the

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differences in methodology including sample size, schizophrenia subtypes of patients, pregnenolone dosage, and permitted concomitant psychotropic medications including mood stabilizers, antidepressants, and even second antipsychotics. In previous trials, patients were diagnosed with either paranoid schizophrenia and schizoaffective with suboptimal response to treatment (Ritsner et al., 2014), or schizophrenia and schizoaffective (Marx et al., 2009; Ritsner et al., 2010), or only schizophrenia (Marx et al., 2014). Patients were permitted to use mood-

ACCEPTED MANUSCRIPT 18 stabilizers and anti-depressants (Marx et al., 2009, 2014; Ritsner et al., 2014) except in one study in which the mood-stabilizer users were excluded (Ritsner et al., 2010). Finally, the patients were subject to a variety of antipsychotic regimens in all previous studies before the start of the trial;

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however, in analysis they founded no confounding effect for the type of antipsychotic used. In this trial, we included 82 patients to increase the power to more than 80%. We only included chronic schizophrenia patients and potential confounding effects of psychotropic drugs other

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than antipsychotics were removed by excluding all the patients who were using mood-stabilizers or anti-depressants. The fact that pregnenolone treatment was started alongside risperidone

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treatment is unique to this trial compared to previous trials.

A recent meta-analysis on previous trials found no significant difference between effects of pregnenolone adjunctive therapy and placebo on total, positive, or negative symptoms of schizophrenia measured by PANSS (Herniga et al., 2015); however, the few available trials with

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inconsistent results, diverse methodologies, and limitations makes it hard to come to a conclusion. We also did not find any significant difference in score changes between the two treatment arms after Bonferroni correction, but in case of PANSS negative symptom subscale

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scores, the p-values were <0.05 not only by the end-point but also the effect sizes were moderate (d>0.5) at each time-point. Also, it is noticeable that mean differences increased through

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subsequent time-points for PANSS total, negative symptoms, and general psychopathology scores, and the increase was prominent for total scores. No similar trend was detected for the effect sizes which was due to the increase in variance through subsequent measurements; however, this does not decrease the importance of the prominent increase in reduction of total scores through subsequent time-points.

ACCEPTED MANUSCRIPT 19 This trial was subject to some limitations. First, the fact that participants were selected based on restrictive criteria, and were treated with only risperidone as the single antipsychotic permitted, limits the generalizability of the results. Meanwhile, we hypothesize that potential

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therapeutic effects of pregnenolone is independent of the adjunctive antipsychotic, herein risperidone, and our restrictive inclusion criteria made our findings more reliable. Second, we only included patients who were not under treatment with any antipsychotics prior to the study,

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and the pregnenolone treatment began in our sample simultaneously with risperidone treatment. In fact, we were mainly interested in pregnenolone effects on patient’s overall symptoms

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measured by PANSS total score before the patient is stabilized on an antipsychotic. It is important to note that schizophrenia patients who are not stabilized on antipsychotics tend to express secondary rather than primary negative symptoms due to presence of higher depressive symptoms as well as higher positive symptoms. It is difficult to measure the true effects of

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pregnenolone on negative symptoms in such patients; however, we excluded patients with severe depressive symptoms. Also, baseline ESRS and PANSS positive symptom scores were similar in the two treatment groups which together make the comparison of pregnenolone effects on

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negative symptoms between the two arms reliable. Third, we used PANSS as the single measurement tool for assessment of schizophrenia symptoms. While we observed moderate

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effect of pregnenolone on negative symptoms, using SANS might show more prominent differences in change of negative symptoms between the two arms in the same way that was reported in a previous trial (Marx et al., 2009); however, our primary outcome of interest was the change in overall symptoms of schizophrenia rather than negative symptoms, and the PANSS has been used as a reliable and valid measurement tool whether for overall symptoms or negative symptoms in numerous previous trials. Fourth, we did not investigate cognitive effects of

ACCEPTED MANUSCRIPT 20 pregnenolone in schizophrenia patients. Fifth, we did not consider a long-term or high dose treatment with pregnenolone.

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4.1. Conclusions

Overall, in spite of the mentioned limitations, this trial may suggest a potential effect of

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pregnenolone as an adjunct to risperidone on symptom improvement in women with chronic schizophrenia, particularly in those with high negative and general psychopathology symptoms.

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While treatment with pregnenolone seems to benefit patients with schizophrenia independent of antipsychotics, it is a matter of further investigation to clarify this effect which would not be easy considering that monotherapy with pregnenolone may not reach ethical approval in most research settings. Further comprehensive trials are warranted with both high dose and low dose

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alongside other symptoms.

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pregnenolone for longer durations in conjunction with assessment of cognitive domains

ACCEPTED MANUSCRIPT 21 Role of the funding source This study complies with contemporary regulations in Iran and was supported by a grant to Prof. Shahin Akhondzadeh (Grant number 27746), from Tehran University of Medical Sciences

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(TUMS). The TUMS had no role in the design, conduct, data collection, analysis, data interpretation, manuscript preparation, review, final approval, or the decision to submit the paper

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for publication.

Acknowledgements

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This study was postgraduate thesis of Dr. Nazila Shams toward the Iranian Board of Psychiatry

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under supervision of Prof. Shahin Akhondzadeh.

ACCEPTED MANUSCRIPT 22 References Akhondzadeh, S., Malek-Hosseini, M., Ghoreishi, A., Raznahan, M., Rezazadeh, S.A., 2008. Effect of ritanserin, a 5HT2A/2C antagonist, on negative symptoms of schizophrenia: a double-

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Akhondzadeh, S., Moazen-Zadeh, E., 2017. More ACTIONS needed to reach a consensus on adjunctive antidepressant therapy for negative symptoms of schizophrenia. Evid. Based Ment.

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Health 20, e3.

Aleman, A., Kahn, R.S., Selten, J.-P., 2003. Sex differences in the risk of schizophrenia:

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ACCEPTED MANUSCRIPT 30 Fig. 1. Flow diagram of women with schizophrenia randomized to pregnenolone and placebo as adjunctive therapy to risperidone.

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Fig. 2. Positive and Negative Syndrome Scale (PANSS) total and subscales scores in women with schizophrenia randomized to pregnenolone and placebo as adjunctive therapy to risperidone. Values represent mean score ± SEM at each time point. P values show the result of independent-samples t test for

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comparison of the score change from baseline to each time point between the two groups. *p<0.05.

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Table 1. Baseline characteristics of the women with schizophrenia Variable

Risperidone + Pregnenolone

Risperidone + Placebo

(n=41)

(n=41)

34.85 (8.91)

36.61 (7.01)

Age, years, mean(SD) •

Single

25 (60.98)

•

Married

9 (21.95)

•

Divorced

7 (17.07)

24 (58.54)

6 (14.63)

•

Primary school

22 (53.66)

•

High school/diploma

10 (24.39)

•

University degree

6 (14.63)

2 (4.88)

0.950

23 (56.10) 9 (21.95)

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3 (7.32)

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Illiterate

0.862

11 (26.83)

Level of education, n, % •

0.324

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Marital status, n, %

P-Value*

7 (17.07)

37 (90.24)

35 (85.37)

0.738

19.56 (8.03)

20.46 (7.42)

0.598

20 (48.78)

21 (51.22)

0.967

8 (19.51)

7 (17.07)

4 (9.76)

5 (12.20)

9 (21.95)

8 (19.51)

25 (60.98)

27 (65.85)

0.819

15 (36.59)

16 (39.02)

1.000

10 (24.39)

11 (26.83)

1.000

8 (19.51)

7 (17.07)

1.000

4 (9.76)

5 (12.20)

1.000

HDRS score, mean(SD)

8.20 (1.49)

8.29 (1.50)

0.768

ESRS score, mean(SD)

2.27 (0.59)

2.15 (0.69)

0.394

Total

93.68 (23.57)

93.00 (22.17)

0.893

Negative symptoms

22.66 (7.27)

22.32 (5.71)

0.814

•

Positive symptoms

23.83 (8.36)

23.93 (8.91)

0.959

•

General psychopathology

47.12 (11.34)

46.76 (10.54)

0.880

Smoking, n, % Duration of illness, years, mean(SD) •

Paranoid

•

Residual

•

Disorganized

•

Undifferentiated

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Type of schizophrenia, n, %

Prior antipsychotic medications, n, % Risperidone

•

Halopridol

•

Fluphenazine

•

Olanzapine

•

Clozapine

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•

PANSS score, mean(SD) • •

*T-test, chi-square test, and Fisher’s exact test applied where appropriate PANSS, Positive and Negative Syndrome Scale; HDRS, Hamilton Depression Rating Scale; ESRS, Extrapyramidal Symptoms Rating Scale

ACCEPTED MANUSCRIPT Table 2. Change in Positive and Negative Syndrome Scale (PANSS) total and subscale scores from baseline in women with schizophrenia

Week 4

-17.70 (16.55)

Week 6

-25.70 (20.43)

Week 8

-33.76 (25.91)

-5.21 (5.69) -12.54 (13.12) -18.95 (18.66) -24.34 (23.30)

-0.83 (1.22)

Week 4

-4.44 (4.26)

-2.39 (2.57)

Week 6

-5.83 (4.73)

-3.68 (3.62)

Week 8

-7.73 (5.80)

-5.12 (5.19)

PANSS Positive symptoms -2.95 (4.86) Week 2

-2.61 (3.15)

Week 4

-5.63 (6.72)

-5.10 (5.89)

Week 6

-7.95 (8.01)

-7.76 (8.10)

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PANSS Negative symptoms -1.80 (2.24) Week 2

-10.22 (9.59)

-9.63 (9.94)

PANSS General psychopathology -3.15 (3.86) Week 2

-1.78 (2.41)

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Week 8

Week 4

-7.73 (7.78)

-5.05 (5.89)

Week 6

-11.88 (10.45)

-7.51 (8.33)

Week 8

-15.51 (13.84)

-9.59 (10.73)

p-value*

-2.68 (-5.97 to 0.60) -5.17 (-11.74 to 1.40) -6.76 (-15.36 to 1.84) -9.41 (-20.24 to 1.41)

-1.63 (68.12) -1.57 (76.04) -1.56 (80) -1.73 (80)

0.108

-0.98 (-1.77 to -0.18) -2.05 (-3.60 to -0.50) -2.15 (-4.00 to -0.3) -2.61 (-5.03 to -0.19)

-2.45 (61.92) -2.64 (65.67) -2.31 (80) -2.15 (80)

0.017

-0.34 (-2.14 to 1.46) -0.54 (-3.32 to 2.24) -0.20 (-3.74 to 3.34) -0.59 (-4.88 to 3.71)

-0.38 (80) -0.38 (80) -0.11 (80) -0.27 (80)

0.707

-1.37 (-2.79 to 0.05) -2.68 (-5.72 to 0.35) -4.37 (-8.52 to -0.21) -5.93 (-11.37 to -0.48)

-1.92 (67.09) -1.76 (74.56) -2.09 (80) -2.17 (80)

0.059

0.121

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PANSS Total score -7.90 (8.87) Week 2

Risperidone+ Placebo (n=41)

t (df*)

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Risperidone+ Pregnenolone (n=41)

Mean (CI 95%) difference in change

Cohen’s d (CI 95%)

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Mean (SD) change from baseline

0.122

0.087

0.010 0.024 0.035

0.702 0.913 0.787

0.082 0.040 0.033

0.36 (-0.08 to 0.80) 0.35 (-0.09 to 0.78) 0.35 (-0.09 to 0.78) 0.38 (-0.06 to 0.82) 0.54 (0.10.98) 0.58 (0.141.03) 0.51 (0.070.95) 0.47 (0.040.91) 0.08 (-0.35 to 0.52) 0.08 (-0.35 to 0.52) 0.02 (-0.41 to 0.46) 0.06 (-0.37 to 0.49) 0.43 (-0.01 to 0.86) 0.39 (-0.05 to 0.83) 0.46 (0.020.90) 0.48 (0.040.92)

*Degree of freedom and concordant p-value were calculated based on the results of Levene’s test for assessment of equality of variances

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Table 3. Incidence of the adverse events in women with schizophrenia Risperidone+ Placebo (n=41) 9 (21.95)

Pvalue*

Dizziness, n, %

8 (19.51)

7 (17.07)

1.000

Headache, n, %

10 (24.39)

7 (17.07)

0.587

Insomnia, n, %

5 (12.20)

3 (7.32)

0.712

Acne, n, %

6 (14.63)

3 (7.32)

0.482

Facial hair growth, n, %

4 (9.76)

2 (4.88)

0.676

Constipation, n, %

5 (12.20)

4 (9.76)

1.000

Diarrhea, n, %

4 (9.76)

4 (9.76)

1.000

Nausea, n, % Vomiting, n, % Increased appetite, n, % Dry mouth, n, % Increased blood pressure, n, %

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Drowsiness, n, %

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Risperidone+ Pregnenolone (n=41) 6 (14.63)

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Side effect

8 (19.51)

8 (19.51)

1.000

9 (21.95)

7 (17.07)

0.781

10 (24.39)

12 (29.27)

0.804

7 (17.07)

6 (14.63)

1.000

5 (12.20)

2 (4.88)

0.432

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*Fisher’s exact test is used, 2-tailed p-values presented

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0.569

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122 patients screened 32 excluded before run in:

45 assigned to risperidone+ placebo

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45 assigned to risperidone+ pregnenolone

4 discontinued at week 2 due to consent withdrawn

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41 completed the trial

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90 randomized

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20 Not meeting inclusion criteria 9 Meeting exclusion criteria 3 Refused to participate

4 discontinued at week 2 due to consent withdrawn

41 completed the trial

ACCEPTED MANUSCRIPT 1: The few previous clinical studies conducted on pregnenolone therapy on schizophrenia patients have yielded inconsistent results.

2: We found that the PANSS negative symptoms subscale scores decreased more prominently

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in the pregenolone group compared to the placebo group by each time-point.

3: While this trial may suggest a potential effect of pregnenolone on schizophrenia

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symptoms, further studies are warranted.