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Impact of alprazolam in allostatic load, neurocognition and quality of life of patients with neuroticism, anxiety

disorders and chronic stress (GEMA): Observational study protocol for a phase IV clinical trial.

Journal: BMJ Open

Manuscript ID: bmjopen-2014-007231

Article Type: Protocol

Date Submitted by the Author: 17-Nov-2014

Complete List of Authors: Soria, Carlos; Institute of Biosciences Henri Laborit, Córdoba Remedi, Carolina; Institute of Biosciences Henri Laborit, Córdoba Nuñez, Daniel; GADOR SA, Scientific Direction D'Alessio, Luciana; Cell Biology and Neuroscience Institute, CONICET Roldán, Emilio; GADOR SA, Scientific Direction

<b>Primary Subject Heading</b>:

Mental health

Secondary Subject Heading: Mental health

Keywords: CLINICAL PHARMACOLOGY, Adult psychiatry < PSYCHIATRY, Anxiety disorders < PSYCHIATRY

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Impact of alprazolam in allostatic load, neurocognition and quality of life of patients with

neuroticism, anxiety disorders and chronic stress (GEMA): Observational study protocol for a

phase IV clinical trial.

Authors

Carlos A. Soria, Carolina Remedi, Daniel A. Núñez, Luciana D’Alessio, Emilio J. A. Roldán.

Institution

Institute of Biosciences Henri Laborit, Córdoba, Argentina.

Scientific Direction Gador SA, Buenos Aires, Argentina.

Corresponding Author:

Luciana D’Alessio, Phd, MD

email: [email protected]

Darwin 429 (C1414CUI) CABA

Buenos Aires, Argentina

Phone 00541169518500

Background

Benzodiazepines are used as anxiolytics in interventions planned for a few weeks. However a

number of symptomatic patients with chronic stress and allostatic loads frequently demand to

continue their use because they cannot solve the underlying morbidity with two consequences:

poor results and risks due to prolonged consumption. Nevertheless, chronic stress and

exposure to allostatic load may induce riskier cognitive alterations. Hence, the use of

alprazolam in low doses might favor improvements in allostatic load and cognitive variables

beyond anxiolysis.

Methods/analysis: This study, GEMA (Gador study of Estres Modulation by Alprazolam), is an

ongoing prospective phase IV study, which aims to determine the impact of medication on

neurobiochemical variables related to chronic stress, metabolic syndrome, neurocognition and

quality of life in patients with anxiety, allostatic load and neuroticism. The study is an on demand

assignment of alprazolam doses in a wide range, in patients with anxiety disorders (six or more

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points in the Hamilton-A scale), neuroticism (a score of more than 18 points in the NEO-FFI

scale), neurocognitive performance (more than 25 points in the Folstein’s Mini-Mental State

Examination) and an allostatic load expressed by at least 3 positive clinical or biochemical items

(Crimmins and Seeman criteria).

Study participants will be assessed prior to treatment (week-1), on the day treatment started

baseline (day 0), and during treatment on weeks 1, 2, 4, 8 and 12. Clinical variables of anxiety,

neuroticism, allostatic load, neurobiochemical studies, neurocognition and quality of life will be

determine.

Ethics and dissemination: This study protocol was approved by an Independent Ethics

Committee of FEFyM (Fundación de Estudios Farmacológicos y Medicamentos/Foundation for

Pharmacological Studies and Drugs, Buenos Aires) and by regulatory authorities of Argentina

(ANMAT, Dossier # 61409-8 of April 20th, 2009), following the law of Habeas Data and

psychotherapeutic drug control. Trial Registration: GEMA - 20811

Limitations:

One of the important biases of the project is the lack of a parallel control group, which is not

regulatory allowed in our area, in a phase IV clinical trial with highly symptomatic patients with

inherent risks. As the project moves forward, however, the study itself can be justified in a more

advanced stage, for example, in a face-to-face comparison of different schemes.

Another difficulty is the existence of knowledge gaps among the clinical features, cognitive

observations and variables measured in laboratories. So, we assume that as we move forward

in the associations among them, other hypotheses on mechanism of action may arise that can

be formulated and tested by other investigators.

Key Words: Alprazolam, benzodiazepines, anxiety disorders, allostatic load, chronic stress,

cognition.

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1- Background

Stress related mental condition such as anxiety and neuroticism 123

are frequently

associated with higher allostatic loads and chronic stress, and overall all of these factors

increase the vulnerability to diseases and reduce resilience45678

.

Anxiety disorders have been linked to disturbances of hypotalamo- pituitary-adrenal

(HPA) axis with controversial results9 10

11

. Hormones such as cortisol and CRH (corticotrophin

releasing hormone) have been associated to states of fear and anxiety in experimental models

1213. Furthermore, increased basal levels of cortisol were found in patients with panic disorders

and in patients with GAD (general anxiety disorders) 14 15

. Nevertheless others authors did not

find any association in cortisol levels and anxiety scale measures 11

or as well found a reduction

in cortisol awakening response among older patients with anxiety disorders 16

. On the contrary

studies performed among posttraumatic stress disorder patients showed lower levels of cortisol

17. High levels of cortisol were also found in patiens with neuroticism (a predisposition to

respond with intense emotional reaction to psychosocial stressors) 12

.

In stress conditions both HPA axis and the noradrenergic (NA) system seem to function

in a synchronous fashion and these two systems are crucial for stress response in mammals 67

.

There is substancial evidence that anxiety in humans is associated with high levels of 3-

methoxy-4-hydroxyphenilglycol (MHPG), a principal NA metabolite, and salivary determination

of MHPG may be a useful marker for detecting changes in catecholamine metabolism 18

19

20

.

1.1 Does allostatic load and chronic stress affect cognition process?

The allostatic load model has been particularly informative in specifying the additive

effects of multiple biological processes that contribute to wear and tear on tissues and

accelerate pathophysiology related to stress6 57

21

. Stressful experiences have functionally-

relevant effects on dendritic arbor and spine/synapse number in many brain regions, including

the hippocampus, amygdala and the prefrontal cortex (PFC) with effects not only on cognitive

function but also on emotional regulation and other self-regulatory behaviors and upon

neuroendocrine and autonomic function 22

. By compromising hippocampal function, allostatic

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load and elevated levels of glucocorticoids impair normal hippocampal function as well memory

processes (declarative memory) 7 23

24

. Furthermore, stress-related effects upon the PFC has an

importance in working memory process (the ability to keep events in mind)25

and self regulatory

behaviors. The dorso lateral PFC is responsible for planning approaches and sequences of

behavior that are required for goal-directed behavior. This process is critical of executive

function 7 25

.

In experimental models, chronic restraint stress caused spine loss and debranching of dendrites

on PFC neurons and impaired working memory7 22

26

. In humans, adverse childhood

experiences were associated with smaller volume of the PFC greater activation of the HPA axis,

and elevation in inflammation levels 27

. Furthermore, it has been shown that stress and allostatic

load increases interference from irrelevant information and thus impairs selective attention 28

,

and high levels of cortisol in response to stressors showed lower performance in working

memory tasks in normal adults 29

. Also, it has been found that anxiety disorders affect cognitive

style of functioning, reversed by alprazolam treatment 30

.

1.2 How treatment anxiety with benzodiazepines impacts on allostatic load?

Benzodiazepines (BZD) are GABAergic substances with anxiolytic and sedative activity

available since the sixties. The therapeutic use to depress acute and subacute symptoms of

anxiety disorders has gained great popular acceptance up to the present 31

. Stress related

mental conditions such as anxiety disorders and neuroticism are treated with psychotherapy

and psychopharmacologic drugs, which are primarily prescribed to reduce pathological

symptoms but the impact on the underlying disorder is generally less understood. Furthermore,

many patients persist with symptoms and consequently require the use of BZD for longer

periods of time. Nevertheless, this indication is an off-label use is therefore regarded with

caution because of the possibility of favoring dependence on medication and other undesired

events such as cognitive impairment 31

32

33

.

In clinical practice prolonged prescription is described when there is recurrence of a

clinical situation and due to a positive perception of both patients and prescribers, there is a

favorable risk/benefit ratio because of the presence of a chronic condition which has not been

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completely resolved. This demand for medication could be significantly reduced if basic

phenomena that trigger anxiety, such as persistence of a harmful stress state, allostatic charge

accumulation and/or decrease of individual resilience level, could be efficiently modulated or

abolished. When it dosen’t happen and the stressors persist, cognitive variables could be

affected, making it difficult for the patient to take decisions that are favorable for QOL (quality of

life)33

34

35 36

Following the NICE (National Institute for Health and Clinical Excellence, UK)

guidelines, treatment of a person with GAD (general anxiety disorder) and chronic stress

consists of psychotherapy (cognitive behavioral therapy), but when symptoms induce a

significant functional impairment, it should be resorted with drugs, generally antidepressant and

benzodiazepines 36

. The objective of treatment is not only to suppress symptoms of anxiety, but

also to prevent a chronic outcome, and to guide patients to positive behaviors “decisions

making” for a better QOL. This involves strengthening native resilience mechanisms, balancing

the impact of individual allostatic loads and promoting an efficient use of cognitive resources

through supportive therapies. The result of this effort, however, is not always satisfactory or it

demands a long time, especially when the neurobiochemical function of the affected patient is

unbalanced by a chronic and unresolved morbid condition5 .

As BZD have anxioliytic and sedating properties, it is expected that they influence the

stress system. As well most studies showed a decrease in cortisol levels 37

38

39

40

41

, some

studies reported mixed results 41

42

43

, and these inconsistencies may be explained by

differences in patients groups, dosages, and BZD used among studies 41

. Some studies yield a

reduction of variations in noradrenaline (NA) metabolite levels (MHPG) "noradrenergic

volatility"44

, and in circulating and salivary cortisol levels using alprazolam in anxious patients 18

39 37

and in normal subjects 45

or as well an increase levels of dehydroepiandrostrerone (a anti-

cortisol mediator)46

. Nevertheless many controversial findings were found depending on age

and gender: younger anxious patients showed higher levels of salivary cortisol awakening

response 11

16

, but other authors found high salivary cortisol levels associated with severity of

late-life generalized anxiety disorders in older adults 15

. Furthermore women seem to yield a

different profile in the cortisol response to stress, in relation to men on response to alprazolam

treatment 40

. Previous data from our clinical practice showed that the administration of

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alprazolam results in a fast and significative decrease of MHPG concentrations and a decrease

of noradrenergic volatility accompanied by a slower but equally sustained decrease in salivary

cortisol levels 47

(Figure 1).

The direct action of alprazolam on GABAergic neurons, and secondarily, on

catecholaminergic and serotoninergic neurotransmitters may induce the indirect anti-stress

target reducing cortisol plasma levels. The reduction in "noradrenergic volatility" defined by the

sudden variations in the levels of NA (noradrenaline) and its metabolite MHPG observed in

patients with GAD, stress-related diseases and depressive states, may also be involved 44

48

49

.

In stress conditions both the NA system and the HPA axis seem to function in a synchronous

fashion. Increased activity in one system goes along with parallel variations in the other and

vice versa and the coordinated interaction of these two systems is a unique element for stress

response in mammals 50

51

52

. The noradrenergic function is hyperactive as it has been

documented in anxiety states, indeed the question is how the HPA axis responds, and if the

physiological synchronicity of both systems remains intact or not, during treatment.

1.3 Do benzodiazepines affect cognitive function positively in stressed patients?

Identification of drugs with adverse cognitive effects, requires dedicated monitoring of cognitive

function, which is often absent from routine hospital and ambulatory clinical care 53

. The short

term effects of BZD on cognition are well known and they are mediated through an agonist

action on receptors of γ aminobutyric acid (GABA A)54

53

. A systematic review of the association

between benzodiazepines and cognition found that cognitive impairment was presumed to be

transient and reversible in most studies 34

53

. Nevertheless the long term adverse effects of

BZD on cognition are still debated 32

53

53

and available data shows conflicting results. Some

authors found an improvement in memory and an increased risk of dementia in prospective

population based studies, performed in elderly patients with long term use of BZD32

55

56

57

58

.

Furthermore longer acting BZD were associated with greater risk of developing dementia

compared with shorter acting benzodiazepines 32

. Nevertheless, other concomitant factors could

be also responsible of dementia in such studies. Indeed, a higher degree of neuroticism in midlife

was associated with increased risk of dementia and long-standing distress, in a population-based study. 59

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Other studies showed not conclusive results 60

61

or even reported a potential protective effect

on behavioral disturbances associated to dementia: the role of GABA in dementia has yet to be

fully elucidated and behavioural symptoms associated with dementia may be a direct or indirect

result of GABAergic dysfunction 62

63

. Furthermore, in young adults, BZD may affects cognition

differentially depending on gender: cognitive alteration of long-term memory have been reported

in women, whereas there was no significant affectation in men 61

.

In this study we selected alprazolam (a triazolo-benzodiazepine) for different reasons: is

a high-potency benzodiazepines with an indirect activity through 5-HT1A serotonergic receptors,

which would produce a regulation of the hyperactive noradrenergic pathways and, through

them, a reduction of noradrenergic volatility. The reduction of noradrenergic volatility by high-

potency benzodiazepines would have a preventive effect via the noradrenergic system, beyond

the central GABAergic anxiolytic effects. The level of concentration and distribution of

alprazolam within the CNS is effective when it is administered in relatively low doses, with a

differential impact on GABAA receptors, upregulated in individuals under chronic stress, and

with less impact on cognitive functions because of the shorter acting 32

49

64

58

65

(Table 1)

1.4 General Hypothesis

In the present study we aim to investigate the intermediate term (12 weeks) biochemical and

neurocognitive effects of treatment with low doses of alprazolam in young adults with anxiety

and chronic stress. The question attempted to be solved is whether anxiolytic pharmacology

intervention with alprazolam, provided with secure schemes, modify allostatic load,

neurocognition performance and quality of life, beyond the known anxiolytic effect 49

66

67

. The

global hypothesis under investigation is if neurobiochemical indicators of allostatic load

(noradrenergic metabolites and cortisol levels), are modulated during treatment with alprazolam,

and if there are neurocognitive consequences when treatment maintains neurobiochemical

parameters within the normal range. A pharmacological modulation of such mediators may

contribute to reduce the impact of allostatic load, promoting resilience and better biochemical

and neurocognitive performance translated in a better quality of life.

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This study is based on the hypothesis that high potency benzodiazepines such as

alprazolam may serve as more than single anxiety suppressive agents reducing allostatic load

and facilitating adaptative responses. GEMA project proposes an intervention designed to

determine if minimum doses of alprazolam adjust neurobiochemical mediators, neurocognition

and affect quality of life, altered by stress (Figure 2).

1.5 Objectives (End-points): The project consists of three complementary stages (Figure 3).

1.5.1 To determine whether neurobiological and biochemical markers of allostatic load, tend to

normalize with 12 weeks of therapeutic intervention.

1.5.2 To determine if neurocognitive performance is modified after treatment and if there is a

positive correlation with biochemical variables of allostatic load and with therapeutic levels.

1.5.3 To assess QOL before and after treatment

Secondarily, the purpose of this study is to report conditions of failure due to lack of response or

emergence of undesired events, including adverse events, and possible situations of

habituation to or dependence on alprazolam.

2- Methods and analysis:

2.1 Protocol Design:

The GEMA project (Gador study of Estres Modulation by Alplax), is a prospective phase IV

clinical trial. The study began in 2010 and is still in advance. This is a prospective observational

study, controlled with on demand assignment of alprazolam doses in variable doses. It is a

phase IV study reported to regulatory authorities in compliance with the rules in force and

consequently enclosed for the use of experimental controls or interventions.

The presence of variable doses will allow us to establish a continuous range of dose-response.

2.2 Inclusion Criteria

Patients over than 21 years, who have at the time of admission anxiety symptoms (six or more

points in the Hamilton-A scale),68

neuroticism (18 or more points in the NEO-FFI scale),

69cognitive performance (25 or more points in the Folstein’s Mini-Mental State Examination)

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70and allostatic load expressed by at least 3 positive clinical or biochemical items (Crimmins and

Seeman criteria). 71

2.3 Exclusion Criteria

Other psychiatric disorders listed under Axis I of the DSM-IV (Diagnostic and Statistical Manual

of Mental Disorders, Fourth Edition)72

. Patients requiring other psychotropics added-on to

alprazolam, sympathicomimetics, corticosteroids or another medication that might kinetically or

dynamically interfere with alprazolam. Patients who had received fluoxetine in the previous 4

weeks or MAO (monoamine oxidase) inhibitors in the previous 2 weeks, or showing a history of

hypersensitivity to alprazolam. Women with confirmed or suspected pregnancy, or subjects

affected with concomitant conditions, severe or unstable, in the cardiovascular system,

gastrointestinal, hepatic, renal, neurological and other systems.

Patients will be withdrawn from this study if dose is prematurely modified, or if patient decides

not to continue with the administered drug, has a related adverse event that modifies his/her

daily performance, or if the investigator considers that the scheme is not safe or beneficial for

the patient according to his/her current clinical status.

2.4

Sample Size: Changes in variables higher than 25% (pre-treatment versus post-treatment or

significant correlations) with a 1-ß power of 0-80 are estimated to be detected. For the first

phase, an n = 29-55 cases, for the second phase an n = 112 and for the third an extension of

the sample to 182 assessable patients, according to a PC program (Statistica, Tulsa) will be

considered enough.

2. 5

Therapeutic Scheme and Dose Assignment

Patients will be extensively assessed by a team of medical doctors, clinicians and psychiatrists,

who decide the most appropriate approach for each case. The principal investigator will

determine the initial dose of alprazolam, usually from 0.75 mg/day to 3.0 mg/day, according to

the presence of anxiety symptoms and according to patient’s tolerance. The ongoing scheme of

alprazolam will be maintained for 3 months. Thereafter the investigator can decide to continue

or discontinue treatment gradually, by reducing 0.5 mg every 3 days.

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2.6 Patient Assessment.

Study participants will be evaluated prior to treatment (week-1), on the day treatment started

(day 0), and during treatment on weeks 1, 2, 4, 8 and 12. Clinical variables of anxiety,

neuroticism and allostatic load, neurocognitive and neurobiochemical studies will be

determined.

Anxiety assessment: Two scales will be rated: 1 - The Hamilton Anxiety Rating Scale (HARS) of

14 items. 68

The 0-5 score indicates absence of anxiety, 6-14 mild anxiety and more than 15

moderate to severe anxiety. 2 - The State-Trait Anxiety Inventory (STAI) of 40 items73

, which

assesses the current state and the anxiety trait as a stable tendency. The scale scores a

maximum of 60 points and percentiles are described for gender and age.

Neuroticism assessment: The NEO five factor inventory (NEO FFI) 69

is an abbreviated

measure of personality factors related to neuroticism, extraversion, openness, kindness and

responsibility.

Allostatic load determination: Clinical and biochemical evaluations will be determined: 1-

Systolic blood pressure will be measured in the supine and standing position, with a previous 2-

3 minutes rest A systolic pressure > 140mmHg and/or a diastolic pressure < 90 mmHg will be

considered a positive criteria.74

2- Heart frequency: a frequency exceeding 90 bpm (beats per

minute) in supine or standing position with a previous 2-3 minutes rest is considered a positive

pulse. 3- BMI (body mass index) and waist-hip ratio are measured while patient is standing and

breathing normally, horizontal over the edge of iliac crests, using a tight tape without

compressing the skin. 4- Laboratory biochemical tests in serum samples include total

cholesterol, and HDL and LDL fractions, glycosylated hemoglobin, fibrinogen, albumin,

andreactive protein. Furthermore, plasma noradrenaline and cortisol and salivary MHPG (3-

methoxy 4 hydroxyphenilglycol) and cortisol will be determined.

General condition and safety: 1-Clinical Global Impression Scale (National Institute of Mental

Health 1976) (CGI), which includes two subscales, 1a - clinical profile severity (CGI-S) and 1b-

clinical profile improvement (CGI-MJ) with 8 points each one. 2-The Mini-Mental State (MEC)

quantifies cognitive impairment in time-space orientation, immediate memory, concentration and

calculation, long-term memory, language and praxis.70

3- UKU scale (Udvalg fur klinische

undersogelser)75

to determine side effects; with 56 items: the first 9 are for psychic events, 8 for

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neurologic, 11 for autonomic and 25 for others. The last two items evaluate side events

interference with the patient's daily performance and their impact on medication intake (none,

dose reduction, withdrawal or change). In each one, the investigator assesses whether the

event was related to drug treatment or not. 4- Additional safety assessments include respiratory

rate and laboratory tests: complete blood count, creatinine, BUN (blood urea nitrogen), total

bilirrubin, alkaline phosphatase, liver enzymes. Human chorionic gonadotropin (hCG-B) will be

determined when appropriate. All patients will be informed about any eventual depressant effect

of alprazolam and risks in activities that require alert and the consumption of alcohol will be not

recommended. They will be also monitored to detect potential risks of suicide or overdose. All

adverse events are monitored, classified and reported according to GCP (good clinical practice)

standards of the local regulatory authority (ANMAT and Ministry of Health of the Province of

Córdoba, Argentina).

Cognitive assessment: To quantify the neurocognitive variables; attention, working memory,

executive functions, impulsiveness, verbal and nonverbal fluency the following tests will be

determined. 1- QPSS continuous performance test (CPT)76

; 2- Digit symbol test (DST) 77

; 3-

Digit span test (DST) 78

; 4- Verbal fluency test (VFT)79

; 5-Five point test (FPT)80

; 6-revised

Taylor complex test (RTCT)81

and 7- Stroop test (ST)82

. All of them will be applied after 6 hours

since the last administration of alprazolam (nadir plasma levels). Cognitive tests and laboratory

tests are blinded to the analyst, the remaining tests are open.

Quality of life (QOL) assessment: The health and quality of life scale, health questionnaire SF-

36 validated Spanish version will be determined. 83

Statistical assessment:

The mean, minimum and maximum values of quantitative variables will be determined. Pre- and

post-treatment values will be compared using t-test and/or non parametric test. Statistical

significance will be fixed at p<0.05 (2-sided; β-1 power≥0.80). Correlation analysis will be

performed by calculating the Pearson Test correlation coefficient for ungrouped data; with r>

0.80 and p<0.05 (2-sided) strong correlation and 0.80<r>0.50 and p<0.05 (2-sided), moderate

correlation.

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Treatment safety: Regarding patient safety, the use of alprazolam, a well known, low action,

nervous system depressant drug, at minimum doses, even when no symptoms palliation is

required, guarantees good tolerability, and no drawbacks are expected as a result of direct

effects. It is worth emphasizing that the proposed intervention tends to reduce long and/or very

frequently repeated demands for medication and with that improve the global safety rate and

QOL.

3. Ethics and dissemination:

The protocol was approved by an Independent Ethics Committee of FEFyM (Fundación de

Estudios Farmacológicos y Medicamentos/Foundation for Pharmacological Studies and Drugs,

Buenos Aires) and by regulatory authorities of Argentina (ANMAT, Dossier # 61409-8 of April

20th, 2009), following the law of Habeas Data and psychotherapeutic drug control. All

participants will agree voluntarily to participate, after having obtained the informed consent and

signed it, in accordance with local regulations and standards of bioethics. The study is

developed under Phase IV conditions, but the full therapeutic intervention has no cost for the

patient under the study protocol. The present study protocol is still ongoing and patient

recruitment is in process.

It is our expectation to see a decline within anxiety scores and allostatic load, reflected by

biochemical variables and an improvement in cognitive performance with a better quality of life,

even in patients treated with low doses of alprazolam. We expect to elucidate improvements in

cognitive variables, such as time-space orientation, immediate memory, concentration and

calculation, long-term memory, language and praxis, as well as a tendency to an overall

improvement of QOL scores.

If the GEMA project fulfills its basic purposes, an original road would open for the search of

specific modulators for the treatment of mental illnesses. It has been stated by health

economists, the “macro” conditions of the cost/health ratio are given more by classic or vintage

drugs rather than by innovations, many of which disappear after a promotional period. In this

sense alprazolam has persisted as a medical tool for several years, and regardless of what

formal pharmaceutical clinical trials and the data that investigators might justify their

permanence. The GEMA project is also an update in the knowledge of the impact of this

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compound for patients with anxiety and allostatic load, and is also a project designed to define a

new profile for the biomedical use of alprazolam.

4. Discussion

GEMA project proposes an intervention, designed to determine if minimum doses of alprazolam

treatment ameliorates additional neurobiochemical variables altered by stress as usually are

detected in anxiety patients, and if it contributes to prevent the impact of allostatic loads,

promoting resilience, better cognitive performance and making healthier decisions translated

into a better quality of life.

If the hypothesis is confirmed, a new paradigm would come into being, which would renew

interest in the clinical management of these compounds, beyond their typical use as anxiolytic

agents. In short, the proposal is to treat each episode more efficiently in order to reduce the

time required to achieve positive results (bene facere) while decreasing the tendency to

excessive consumption of drugs (non nocere).

5- List of abbreviations

HPA (hypotalamo pituitary adrenal)

CRH (corticotrophin releasing hormone)

GAD (general anxiety disorders)

PFC (prefrontal cortex)

MHPG (3-methoxy- 4- hydroxyphenilglycol)

BZD (benzodiazepines)

NICE (National Institute for Health and Clinical Excellence, UK)

NA (noradrenaline)

STH (somatotropic hormone)

GHRH (growth-hormone-releasing hormone)

MDD (major depressive disorder)

CRH (corticotropin releasing hormone)

MAO (monoamine oxidase)

STAI (state -trait anxiety inventory)

HARS (Hamilton anxiety rating scale)

NEO FFI (NEO five factor inventory for neuroticism)

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BMI (body mass index)

HDL (high density lipoprotein)

LDL (low density lipoprotein)

CGI (Clinical Global Impression Scale)

MEC (The Mini-Mental State)

UKU (Udvalg fur klinische undersogelser)

GCP (good clinical practice)

QOL (quality of life)

BUN (blood urea nitrogen)

CPT (continuous performance test

DST (digit symbol test)

DST (digit span test)

VFT (verbal fluency test

FPT (five point test)

RTCT (revised Taylor complex test)

ST (stoop test).

6- Competing interests

This work is carried out under a grant awarded by Gador SA, Buenos Aires (2010-2012) and

funds of the Instituto de Biociencias Henri Laborit, Córdoba. CS and LD are consultants at

Gador SA, and DN and EJAR are investigators employed by Gador SA, and CR does not

declare any conflict of interest. Gador SA, Darwin 429, Buenos Aires City, Argentina.

7- Authors’ contributions

CS, CR, DN and EJAR have made substantial contributions to conception and design. LD has

been involved in drafting and revising the manuscript for intellectual content.

EJAR has given final approval of the version to be published.

8- Author’ information

CS and CR: Institute of Biosciences Henri Laborit, Córdoba

DN and EJAR: Scientific Direction, Gador SA, Buenos Aires, Argentina.

LD: Institute of Biology and Neuroscience E de Robertis, CONICET, Argentina.

9- Acknowledgments

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Authors thank the participation and contributions of the work team involved in GEMA.

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12. Figure Legends

Table 1 Data come from different experimental models and do not represent human SNC condition, either in healthy or under allostatic loads. Gray boxes presume positive for the cognitive development under drug therapy. Adapted from references

4964

Figure 1 Percentage of reduction of salivary cortisol and MHPG levels among alprazolam (0.75 to 3.0 mg/day) responder patients. Modified from [47]

Figure 2 Upper panel: Repeated stress (S-blue) conditions plus accumulation of allostatic loads (AL-red) result in chronic stress, and with time loss of the individual resilience (R-green) levels. Anxiety (A-yellow) emerges as a reaction of an excessive stress applied to the body. Under chronic stress, cognitive resources are impaired and with it the possibilities of the affected subject to maintain its quality of life. Consequently, the affected live with a neurobiological environment poorly efficient to solve the daily challenges of life. Middle panel: With a short-term anxiolytic use of alprazolam, symptoms are reduced but demands of treatment persist, as well as the impact of the AL, and the decay of R, are not being significantly modified. Lower panel: With prolonged use of low-dose alprazolam anti-stress biochemical effects, plus a consequent increase of cognitive functions, may allow the progressive recovering of R, shorten S duration, and may further reduce the impact of AL. Under these new biological conditions the subject increased its chances to seek for an improved quality of life and may there of shorten the demand of palliative medication. Figure 3 Stage I: Short term evaluation of 0.75 to 3.0 mg/day alprazolam on biochemical variables related to chronic stress. Stage II: Short term evaluation of 0.75 to 3.0 mg/day alprazolam on cognitive and clinical variables related to chronic stress. Stage III: Intermediate term evaluation of low alprazolam doses in cognitive, clinical and quality of life indicators.

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Figure 1. MHPG and cortisol salivary levels

0 20 40 60 80 100

5% of reduction (salivary levels)

up to 5% of reduction (salivary levels)

58.33

41.67

95.65

4.35

Cortisol

MHPG

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Figure 2. Hypothesis of the therapeutic intervention postulated at the GEMA project.

No adequate therapy

Traditional use of

benzodiazepines

The GEMA project

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Figure 3. Development stages of the GEMA Project

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1

Table 1 Comparative affinity of different anxiolytics benzodiazepines to GABAA subtypes receptors. GABA receptor subtype

α1 α2 α3 α5 β3 γ2 SNC

depression

Motor

impairment

amnesia

Anxiolysis Disinhibition

Anticonvulsive Mio-relaxation

Anxiolysis

Learning memory

Anticonvulsive Muscle relaxation

sedation

Addiction respiratory

depression

Alprazolam Low High Moderate Low? Low High

Clonazepam Moderate High High Low? Moderate High

Diazepam Moderate High High Moderate? Moderate Moderate

Lorazepam Low High High Moderate? Low High

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SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and

related documents*

Section/item ItemNo

Description

Administrative information

Title 1 Descriptive title identifying the study design, population, interventions,

and, if applicable, trial acronym

Trial registration 2a Trial identifier and registry name. If not yet registered, name of

intended registry (page 1- abstract)

2b All items from the World Health Organization Trial Registration Data

Set (attached as additional information)

Protocol version 3 Date and version identifier( attached as additional information)

Funding 4 Sources and types of financial, material, and other support (pag 16 )

Roles and

responsibilities

5a Names, affiliations, and roles of protocol contributors (page 1 and 16)

5b Name and contact information for the trial sponsor (page 16)

5c Role of study sponsor and funders, if any, in study design; collection,

management, analysis, and interpretation of data; writing of the report;

and the decision to submit the report for publication, including whether

they will have ultimate authority over any of these activities (page 16)

5d Composition, roles, and responsibilities of the coordinating centre,

steering committee, endpoint adjudication committee, data

management team, and other individuals or groups overseeing the

trial, if applicable (see Item 21a for data monitoring committee)

(page 16)

Introduction

Background and

rationale

6a Description of research question and justification for undertaking the

trial, including summary of relevant studies (published and

unpublished) examining benefits and harms for each intervention

(point 1: pages 2-6)

6b Explanation for choice of comparators

Objectives 7 Specific objectives or hypotheses (point 1: page 7)

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Trial design 8 Description of trial design including type of trial (eg, parallel group,

crossover, factorial, single group), allocation ratio, and framework (eg,

superiority, equivalence, noninferiority, exploratory) (point 2 page 8)

Methods: Participants, interventions, and outcomes

Study setting 9 Description of study settings (eg, community clinic, academic hospital)

and list of countries where data will be collected. Reference to where

list of study sites can be obtained (point 2 page 8-9)

Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility

criteria for study centres and individuals who will perform the

interventions (eg, surgeons, psychotherapists) (point 2 page 8-9)

Interventions 11a Interventions for each group with sufficient detail to allow replication,

including how and when they will be administered (point 2.5 page 10)

11b Criteria for discontinuing or modifying allocated interventions for a

given trial participant (eg, drug dose change in response to harms,

participant request, or improving/worsening disease) (point 2.2 pag 9)

11c Strategies to improve adherence to intervention protocols, and any

procedures for monitoring adherence (eg, drug tablet return,

laboratory tests)

11d Relevant concomitant care and interventions that are permitted or

prohibited during the trial) (point 2.2 pag 9)

Outcomes 12 Primary, secondary, and other outcomes, including the specific

measurement variable (eg, systolic blood pressure), analysis metric

(eg, change from baseline, final value, time to event), method of

aggregation (eg, median, proportion), and time point for each

outcome. Explanation of the clinical relevance of chosen efficacy and

harm outcomes is strongly recommended (point 2.6 pages 10-12)

Participant

timeline

13 Time schedule of enrolment, interventions (including any run-ins and

washouts), assessments, and visits for participants. A schematic

diagram is highly recommended (see Figure) (point 2.5 page 10)

Sample size 14 Estimated number of participants needed to achieve study objectives

and how it was determined, including clinical and statistical

assumptions supporting any sample size calculations (point 2.4 page

10)

Recruitment 15 Strategies for achieving adequate participant enrolment to reach

target sample size

Methods: Assignment of interventions (for controlled trials)

Allocation:

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Sequence

generation

16a Method of generating the allocation sequence (eg, computer-

generated random numbers), and list of any factors for stratification.

To reduce predictability of a random sequence, details of any planned

restriction (eg, blocking) should be provided in a separate document

that is unavailable to those who enrol participants or assign

interventions

Allocation

concealment

mechanism

16b Mechanism of implementing the allocation sequence (eg, central

telephone; sequentially numbered, opaque, sealed envelopes),

describing any steps to conceal the sequence until interventions are

assigned

Implementation 16c Who will generate the allocation sequence, who will enrol participants,

and who will assign participants to interventions

Blinding

(masking)

17a Who will be blinded after assignment to interventions (eg, trial

participants, care providers, outcome assessors, data analysts), and

how

17b If blinded, circumstances under which unblinding is permissible, and

procedure for revealing a participant’s allocated intervention during

the trial

Methods: Data collection, management, and analysis

Data collection

methods

18a Plans for assessment and collection of outcome, baseline, and other

trial data, including any related processes to promote data quality (eg,

duplicate measurements, training of assessors) and a description of

study instruments (eg, questionnaires, laboratory tests) along with

their reliability and validity, if known. Reference to where data

collection forms can be found, if not in the protocol

18b Plans to promote participant retention and complete follow-up,

including list of any outcome data to be collected for participants who

discontinue or deviate from intervention protocols

Data

management

19 Plans for data entry, coding, security, and storage, including any

related processes to promote data quality (eg, double data entry;

range checks for data values). Reference to where details of data

management procedures can be found, if not in the protocol

Statistical

methods

20a Statistical methods for analysing primary and secondary outcomes.

Reference to where other details of the statistical analysis plan can be

found, if not in the protocol

20b Methods for any additional analyses (eg, subgroup and adjusted

analyses)

20c Definition of analysis population relating to protocol non-adherence

(eg, as randomised analysis), and any statistical methods to handle

missing data (eg, multiple imputation)

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Methods: Monitoring

Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role

and reporting structure; statement of whether it is independent from

the sponsor and competing interests; and reference to where further

details about its charter can be found, if not in the protocol.

Alternatively, an explanation of why a DMC is not needed

21b Description of any interim analyses and stopping guidelines, including

who will have access to these interim results and make the final

decision to terminate the trial

Harms 22 Plans for collecting, assessing, reporting, and managing solicited and

spontaneously reported adverse events and other unintended effects

of trial interventions or trial conduct

Auditing 23 Frequency and procedures for auditing trial conduct, if any, and

whether the process will be independent from investigators and the

sponsor

Ethics and dissemination

Research ethics

approval

24 Plans for seeking research ethics committee/institutional review board

(REC/IRB) approval (point 2.3 page 9)

Protocol

amendments

25 Plans for communicating important protocol modifications (eg,

changes to eligibility criteria, outcomes, analyses) to relevant parties

(eg, investigators, REC/IRBs, trial participants, trial registries, journals,

regulators)

Consent or assent 26a Who will obtain informed consent or assent from potential trial

participants or authorised surrogates, and how (see Item 32)

26b Additional consent provisions for collection and use of participant data

and biological specimens in ancillary studies, if applicable

Confidentiality 27 How personal information about potential and enrolled participants will

be collected, shared, and maintained in order to protect confidentiality

before, during, and after the trial

Declaration of

interests

28 Financial and other competing interests for principal investigators for

the overall trial and each study site

Access to data 29 Statement of who will have access to the final trial dataset, and

disclosure of contractual agreements that limit such access for

investigators

Ancillary and

post-trial care

30 Provisions, if any, for ancillary and post-trial care, and for

compensation to those who suffer harm from trial participation

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Dissemination

policy

31a Plans for investigators and sponsor to communicate trial results to

participants, healthcare professionals, the public, and other relevant

groups (eg, via publication, reporting in results databases, or other

data sharing arrangements), including any publication restrictions

31b Authorship eligibility guidelines and any intended use of professional

writers

31c Plans, if any, for granting public access to the full protocol, participant-

level dataset, and statistical code

Appendices

Informed consent

materials

32 Model consent form and other related documentation given to

participants and authorised surrogates

Biological

specimens

33 Plans for collection, laboratory evaluation, and storage of biological

specimens for genetic or molecular analysis in the current trial and for

future use in ancillary studies, if applicable

*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013

Explanation & Elaboration for important clarification on the items. Amendments to the

protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT

Group under the Creative Commons “Attribution-NonCommercial-NoDerivs 3.0 Unported”

license.

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Impact of alprazolam in allostatic load and neurocognition of patients with anxiety disorders and chronic stress

(GEMA): Observational study protocol

Journal: BMJ Open

Manuscript ID: bmjopen-2014-007231.R1

Article Type: Protocol

Date Submitted by the Author: 21-May-2015

Complete List of Authors: Soria, Carlos; Institute of Biosciences Henri Laborit, Córdoba Remedi, Carolina; Institute of Biosciences Henri Laborit, Córdoba Nuñez, Daniel; GADOR SA, Scientific Direction D'Alessio, Luciana; Cell Biology and Neuroscience Institute, CONICET Roldán, Emilio; GADOR SA, Scientific Direction

<b>Primary Subject Heading</b>:

Mental health

Secondary Subject Heading: Mental health

Keywords: CLINICAL PHARMACOLOGY, Adult psychiatry < PSYCHIATRY, Anxiety disorders < PSYCHIATRY


BMJ Open on 10 June 2019 by guest. P

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Impact of alprazolam in allostatic load and neurocognition of patients with anxiety

disorders and chronic stress (GEMA): Observational study protocol

Authors

Carlos A. Soria, Carolina Remedi, Daniel A. Núñez, Luciana D’Alessio, Emilio J. A.

Roldán.

Institutions

Institute of Biosciences Henri Laborit, Córdoba, Argentina.

Scientific Direction Gador SA, Buenos Aires, Argentina.

Corresponding Author:

Luciana D’Alessio, Phd, MD

email: [email protected] , [email protected]

Darwin 429 (C1414CUI) CABA

Buenos Aires, Argentina

Phone 00541169518500

Abstract

Introduction

The allostatic load model explains the additive effects of multiple biological

processes that accelerate pathophysiology related to stress, particularly in central

nervous system. Stress related mental condition such as anxiety disorders and

neuroticism (a well-known stress vulnerability factor), have been linked to

disturbances of hypotalamo-pituitary-adrenal (HPA) with cognitive implications.

Nevertheless there are controversial results in the literature and there is a need to

determine the impact of the psychopharmacological treatment on allostatic load

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parameters and in cognitive functions. GEMA (Gador study of Estres Modulation by

Alprazolam), aims to determine the impact of medication on neurobiochemical

variables related to chronic stress, metabolic syndrome, neurocognition and quality

of life in patients with anxiety, allostatic load and neuroticism.

Methods/analysis: In this observational prospective phase IV study, highly

sympthomatic patients with anxiety disorders (six or more points in the Hamilton-A

scale), neuroticism (more than 18 points in the NEO-FFI scale), an allostatic load

(three positive clinical or biochemical items at Crimmins and Seeman criteria) will

be included. Clinical variables of anxiety, neuroticism, allostatic load,

neurobiochemical studies, neurocognition and quality of life will be determine prior

and periodically (one, two, four, eight, and twelve weeks) after treatment (on

demand of alprazolam from 0.75 mg/day to 3.0 mg/day). A sample of n =55/182

patients will be consider enough to detect variables higher than 25% (pre-

treatment versus post-treatment or significant correlations) with a 1-ß power of 0-

80. T-test and/or non parametric test, and Pearson Test for correlation analysis will

be determine.

Ethics and dissemination: This study protocol was approved by an Independent

Ethics Committee of FEFyM (Foundation for Pharmacological Studies and Drugs,

Buenos Aires) and by regulatory authorities of Argentina (ANMAT, Dossier # 61409-

8 of April 20th, 2009), following the law of Habeas Data and psychotherapeutic drug

control.

Limitations of this study

One of the important biases of the project is the lack of a parallel control group,

which is not regulatory allowed in our area, in a phase IV trial with highly

symptomatic patients with inherent risks. As the project moves forward, however,

the project itself can be justified in a more advanced stage, for example, in a face-

to-face comparison of different schemes.

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Another difficulty is the existence of knowledge gaps among the clinical features,

cognitive observations and variables measured in laboratories. So, we assume that

as we move forward in the associations among them, other hypotheses on

mechanism of action may arise that can be formulated and tested by other

investigators.

Key Words: Alprazolam, benzodiazepines, anxiety disorders, allostatic load, chronic

stress, cognition.

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1- Introduction

Stress related mental condition such as anxiety disorders are frequently

associated with chronic stress [1,2,3]. If the stress response is excessive and

prolonged, the cost of reinstating homeostasis become too high, a condition that is

termed allostatic load (an indicator of the of adverse health outcomes because of

physiologic dysregulation) [4-10].

Anxiety disorders have been linked to disturbances of hypotalamo-pituitary-

adrenal (HPA) axis with controversial results[11-13]. Hormones such as cortisol and

CRH (corticotrophin releasing hormone) have been associated to states of fear and

anxiety in experimental models [14,15]. Furthermore, increased basal levels of

cortisol were found in patients with panic disorders and in patients with GAD

(general anxiety disorders) [16,17]. Nevertheless others authors did not find any

association in cortisol levels and anxiety scale measures[13] or as well found a

reduction in cortisol awakening response among older patients with anxiety

disorders[18]. On the contrary studies performed among posttraumatic stress

disorder patients showed lower levels of cortisol[19].

Similar pattern of cortisol levels were also found in patients with neuroticism

(a predisposition to respond with intense emotional reaction to psychosocial

stressors) [1,2]. Neuroticism is a well-known stress vulnerability factor that is

robustly associated with psychiatric disorders such as anxiety and depression [20].

Neuroticism and stress domains do not measure an identical construct

nevertheless, it has been reported as that cumulative stress was more likely to

precipitate psychiatric symptoms in vulnerable individuals with high levels of

neuroticism [20] and furthermore, these individuals perceive and have more

stressors, respond exaggeratedly to them, and require more time to recover from

them [1,2,21].

During stress activation both HPA axis and the noradrenergic (NA) system

seem to function in a synchronous fashion and these two systems are crucial for

stress response in mammals [6,7]. There is substancial evidence that anxiety in

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humans is associated with high levels of 3-methoxy-4-hydroxyphenilglycol (MHPG),

a principal NA metabolite, and salivary determination of MHPG may be a useful

marker for detecting changes in catecholamine metabolism [22-24], however there

is a need for determine by objective measures the impact of stress mediators on

allostatic load and cognitive functioning in these patient and the impact of the

psychopharmacological treatment on these parameters.

The allostatic load model has been particularly informative in specifying the

additive effects of multiple biological processes that contribute to wear and tear on

tissues and accelerate pathophysiology related to stress, particularly in central

nervous system [5,6,7,25,10]. These allodynamic processes can be adaptive in the

short term (allostasis) and maladaptive in the long term (allostatic load). The brain

is the central organ of stress processes and allodynamic adaptation and it is a key

target of allostasis, thus enable protection and adaptation. Consequently allostatic

load can affect brain plasticity since adjustments of biological systems [9].

It has been demonstrated that stressful experiences have functionally-

relevant effects on dendritic arbor and spine/synapse number in many brain

regions, including the hippocampus, amygdala and the prefrontal cortex (PFC) with

effects not only on cognitive function but also on emotional regulation and other

self-regulatory behaviors and upon neuroendocrine and autonomic function[9]. By

compromising hippocampal function, allostatic load and elevated levels of

glucocorticoids impair normal hippocampal function as well memory processes

(declarative memory)[7,26,27]. Furthermore, stress-related effects upon the PFC

has an importance in working memory process (the ability to keep events in

mind)[28] and self regulatory behaviors. The dorso lateral PFC is responsible for

planning approaches and sequences of behavior that are required for goal-directed

behavior. This process is critical of executive function[7,28].

In experimental models, chronic restraint stress caused spine loss and

debranching of dendrites on PFC neurons and impaired working memory[7,9,29]. In

humans, adverse childhood experiences were associated with smaller volume of the

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PFC greater activation of the HPA axis, and elevation in inflammation levels [30].

Furthermore, it has been shown that stress and allostatic load increases

interference from irrelevant information and thus impairs selective attention

[31],and high levels of cortisol in response to stressors showed lower performance

in working memory tasks in normal adults [32].Also, it has been found that anxiety

disorders affect cognitive style of functioning, reversed by alprazolam treatment

[33].

Following the NICE (National Institute for Health and Clinical Excellence, UK)

guidelines, treatment of a person with GAD (general anxiety disorder) and chronic

stress consists of psychotherapy (cognitive behavioral therapy), but when

symptoms induce a significant functional impairment, it should be resorted with

drugs, generally antidepressant and benzodiazepines [34]). Benzodiazepines (BZD)

are GABAergic substances with anxiolytic and sedative activity available since the

sixties. The therapeutic use to depress acute and subacute symptoms of anxiety

disorders has gained great popular acceptance up to the present[35].Stress related

mental conditions such as anxiety disorders and neuroticism are treated with

psychotherapy and psychopharmacologic drugs, which are primarily prescribed to

reduce pathological symptoms but the impact on the underlying disorder is

generally less understood. Furthermore, many patients persist with symptoms and

consequently require the use of BZD for longer periods of time. Nevertheless, this

indication is an off-label use is therefore regarded with caution because of the

possibility of favoring dependence on medication and other undesired events such

as cognitive impairment[35,36,37].However when the stressors persist, cognitive

variables could be affected, making it difficult for the patient to take decisions that

are favorable for QOL (quality of life)[34,37,38]

The objective of treatment is not only to suppress symptoms of anxiety, but

also to prevent a chronic outcome, and to guide patients to positive behaviors

“decisions making” for a better QOL[5].

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As BZD have anxioliytic and sedating properties, it is expected that they

influence the stress system. As well most studies showed a decrease in cortisol

levels [39-43], some studies reported mixed results [43-45], and these

inconsistencies may be explained by differences in patients groups, dosages, and

BZD used among studies [43]. Some studies yield a reduction of variations in

noradrenaline (NA) metabolite levels (MHPG) "noradrenergic volatility"[46], and in

circulating and salivary cortisol levels using alprazolam in anxious patients

[22,39,41] and in normal subjects [47] or as well an increase levels of

dehydroepiandrostrerone (an anti-cortisol mediator)[48]. Nevertheless many

controversial findings were found depending on age and gender: younger anxious

patients showed higher levels of salivary cortisol awakening response [13,18], but

other authors found high salivary cortisol levels associated with severity of late-life

generalized anxiety disorders in older adults [17]. Furthermore women seem to

yield a different profile in the cortisol response to stress, in relation to men on

response to alprazolam treatment [42]. Previous data from our clinical practice

showed that the administration of alprazolam results in a fast and significative

decrease of MHPG concentrations and a decrease of noradrenergic volatility

accompanied by a slower but equally sustained decrease in salivary cortisol

levels[49](Figure 1).The direct action of alprazolam on GABAergic neurons, and

secondarily, on catecholaminergic and serotoninergic neurotransmitters may induce

the indirect anti-stress target reducing cortisol plasma levels. The reduction in

"noradrenergic volatility" defined by the sudden variations in the levels of NA

(noradrenaline) and its metabolite MHPG observed in patients with GAD, stress-

related diseases and depressive states, may also be involved [46,50-54].

Nevertheless the other aspect of benzodiazepines treatment are cognitive

adverse effects, and identification of them requires dedicated monitoring of

cognitive function, which is often absent from routine hospital and ambulatory

clinical care [55].The short term effects of BZD on cognition are well known and

they are mediated through an agonist action on receptors of γ aminobutyric acid

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(GABA A)[55,56]. A systematic review of the association between benzodiazepines

and cognition found that cognitive impairment was presumed to be transient and

reversible in most studies [38,55]. Nevertheless the long term adverse effects of

BZD on cognition are still debated[36,55] and available data shows conflicting

results. Some authors found an improvement in memory and an increased risk of

dementia in prospective population based studies, performed in elderly patients

with long term use of BZD[36,57-60]. Furthermore longer acting BZD were

associated with greater risk of developing dementia compared with shorter acting

benzodiazepines [36]. Nevertheless, other concomitant factors could be also

responsible of dementia in such studies. Indeed, a higher degree of neuroticism in

midlife was associated with increased risk of dementia and long-standing distress,

in a population-based study [61]. Other studies showed not conclusive

results[62,63] or even reported a potential protective effect on behavioral

disturbances associated to dementia: the role of GABA in dementia has yet to be

fully elucidated and behavioral symptoms associated with dementia may be a direct

or indirect result of GABAergic dysfunction[64,65]. Furthermore, in young adults,

BZD may affects cognition differentially depending on gender: cognitive alteration

of long-term memory have been reported in women, whereas there was no

significant affectation in men [63].

In this study we selected alprazolam (a triazolo-benzodiazepine) for different

reasons: is a high-potency benzodiazepines with an indirect activity through 5-HT1A

serotonergic receptors, which would produce a regulation of the hyperactive

noradrenergic pathways and, through them, a reduction of noradrenergic volatility.

The reduction of noradrenergic volatility by high-potency benzodiazepines would

have a preventive effect via the noradrenergic system, beyond the central

GABAergic anxiolytic effects. The level of concentration and distribution of

alprazolam within the CNS is effective when it is administered in relatively low

doses, with a differential impact on GABAA receptors, upregulated in individuals

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under chronic stress, and with less impact on cognitive functions because of the

shorter acting [36,51,60,66,67](Table 1).

In the present study GEMA (Gador study of Estres Modulation by Alprazolam), the

following research questions were formulated: How treatment anxiety with

benzodiazepines impacts on allostatic load and neuroticism? Does allostatic load

and chronic stress affect cognition process? Do benzodiazepines affect cognitive

function positively in stressed patients?

1.4 General Hypothesis

In the present study we aim to investigate biochemical and neurocognitive

variables in patients with high anxiety levels associated to chronic stress, and

neuroticism (a clinical condition that indicate an exaggeratedly response to the

stressors that require more time to recover from them) [51,68,69] and the impact

of intermediate term treatment (12 weeks) with low doses of alprazolam.

In clinical practice high symptomatic anxiety disorders are frequently associated

with chronic stress and neuroticism (a well-known stress vulnerability factor) [20]

and overall all of these factors increase the vulnerability to diseases and reduce

resilience. In these high symptomatic patients the use of benzodiazepines is

required for longer periods of time. Nevertheless, this indication is an off-label use,

and is therefore regarded with caution because of the possibility of favoring

dependence on medication and other undesired events such as cognitive functions,

reflexes and alert states [35].

The global hypothesis under investigation is if neurobiochemical indicators of

allostatic load (noradrenergic metabolites and cortisol levels) are modulated during

treatment with alprazolam, and if there are neurocognitive consequences when

treatment maintains neurobiochemical parameters within the normal range. A

pharmacological modulation of such mediators may contribute to reduce the impact

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of allostatic load, promoting resilience and better biochemical and neurocognitive

performance translated in a better quality of life.

This study is based on the hypothesis that high potency benzodiazepines such as

alprazolam may serve as more than single anxiety suppressive agents reducing

allostatic load and facilitating adaptative responses. GEMA project proposes an

intervention designed to determine if minimum doses of alprazolam adjust

neurobiochemical mediators, neurocognition and affect quality of life, altered by

stress (Figure 2)

The question attempted to be solved is, whether anxiolytic pharmacology

intervention with alprazolam in these high symptomatic patients modify allostatic

load, neurocognition performance and quality of life, beyond the known anxiolytic

effect.

1.5 Objectives (End-points): The project consists of three complementary stages

(Figure 3).

1.5.1 To determine whether clinical and biochemical markers of allostatic load, tend

to normalize with 12 weeks of therapeutic intervention.

1.5.2 To determine if neurocognitive performance is modified after treatment and if

there is a positive correlation with biochemical variables of allostatic load and with

therapeutic levels.

1.5.3 To assess QOL before and after treatment

Secondarily, the purpose of this study is to report conditions of failure due to lack

of response or emergence of undesired events, including adverse events, and

possible situations of habituation to or dependence on alprazolam.

2- Methods and analysis:

2.1 Protocol Design:

The GEMA project (Gador study of Estres Modulation by Alplax), is a prospective

phase IV clinical trial. The study began in 2010 and is still in advance. This is a

prospective observational study, controlled with on demand assignment of

alprazolam doses in variable doses. It is a phase IV study reported to regulatory

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authorities in compliance with the rules in force and consequently enclosed for the

use of experimental controls or interventions.

The presence of variable doses will allow us to establish a continuous range of

dose-response.

2.2 Inclusion Criteria

In this study highly sympthomatic patients over than 21 years, who have at the

time of admission anxiety symptoms (six or more points in the Hamilton-A scale),

[70] neuroticism (18 or more points in the NEO-FFI scale), [71] allostatic load

expressed by at least 3 positive clinical or biochemical items (Crimmins and

Seeman criteria) with normal cognitive performance (25 or more points in the

Folstein’s Mini-Mental State Examination)[72] were included. Allostatic load

measurement included markers of hypothalmic pituitary axis, sympathetic nervous

system, cardio-vascular system, metabolic system, renal function, lung function,

and markers of inflammation [10].

2.3 Exclusion Criteria

Other psychiatric disorders listed under Axis I of the DSM-IV(Diagnostic and

Statistical Manual of Mental Disorders, Fourth Edition)[73]. Patients requiring other

psychotropics added-on to alprazolam, sympathicomimetics, corticosteroids or

another medication that might kinetically or dynamically interfere with alprazolam.

Patients who had received fluoxetine in the previous 4 weeks or MAO (monoamine

oxidase) inhibitors in the previous 2 weeks, or showing a history of hypersensitivity

to alprazolam. Women with confirmed or suspected pregnancy, or subjects affected

with concomitant conditions, severe or unstable, in the cardiovascular system,

gastrointestinal, hepatic, renal, neurological and other systems.

Patients will be withdrawn from this study if dose is prematurely modified, or if

patient decides not to continue with the administered drug, has a related adverse

event that modifies his/her daily performance, or if the investigator considers that

the scheme is not safe or beneficial for the patient according to his/her current

clinical status.

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2.4

Sample Size: Changes in variables higher than 25% (pre-treatment versus post-

treatment or significant correlations) with a 1-ß power of 0-80 are estimated to be

detected. For the first phase, an n = 29-55 cases, for the second phase an n = 112

and for the third an extension of the sample to 182 assessable patients, according

to a PC program (Statistica, Tulsa) will be considered enough.

2.5 Therapeutic Scheme and Dose Assignment

Patients will be extensively assessed by a team of medical doctors, clinicians and

psychiatrists, who decide the most appropriate approach for each case. The

principal investigator will determine the initial dose of alprazolam, usually from 0.75

mg/day to 3.0 mg/day, according to the presence of anxiety symptoms and

according to patient’s tolerance. The ongoing scheme of alprazolam will be

maintained for 3 months. Thereafter the investigator can decide to continue or

discontinue treatment gradually, by reducing 0.5 mg every 3 days.

2.6 Patient Assessment.

Study participants will be evaluated prior to treatment (week-1), on the day

treatment started (day 0), and during treatment on weeks 1, 2, 4, 8 and 12.

Clinical variables of anxiety, neuroticism and allostatic load, neurocognitive and

neurobiochemical studies will be determined.

Anxiety assessment: Two scales will be rated: 1 - The Hamilton Anxiety Rating

Scale (HARS) of 14 items. [70] The 0-5 score indicates absence of anxiety, 6-14

mild anxiety and more than 15 moderate to severe anxiety. 2 - The State-Trait

Anxiety Inventory (STAI) of 40 items[74], which assesses the current state and the

anxiety trait as a stable tendency. The scale scores a maximum of 60 points and

percentiles are described for gender and age.

Neuroticism assessment: The NEO five factor inventory (NEO FFI)[71] is an

abbreviated measure of personality factors related to neuroticism, extraversion,

openness, agreeableness, and conscientiousness. Allostatic load determination:

Clinical and biochemical evaluations will be determined: 1- Systolic blood pressure

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will be measured in the supine and standing position, with a previous 2-3 minutes

rest A systolic pressure > 140mmHg and/or a diastolic pressure < 90 mmHg will be

considered a positive criteria.[75]2- Heart frequency: a frequency exceeding 90

bpm (beats per minute) in supine or standing position with a previous 2-3 minutes

rest is considered a positive pulse. 3- BMI (body mass index) and waist-hip ratio

are measured while patient is standing and breathing normally, horizontal over the

edge of iliac crests, using a tight tape without compressing the skin. 4- Laboratory

biochemical tests in serum samples include total cholesterol, and HDL and LDL

fractions, glycosylated hemoglobin, fibrinogen, albumin, and C reactive protein.

Furthermore, plasma noradrenaline and cortisol and salivary MHPG (3-methoxy 4

hydroxyphenilglycol) and cortisol will be determined.

General condition and safety: 1-Clinical Global Impression Scale (National Institute

of Mental Health 1976) (CGI), which includes two subscales, 1a - clinical profile

severity (CGI-S) and 1b-clinical profile improvement (CGI-MJ) with 8 points each

one. 2-The Mini-Mental State (MEC) quantifies cognitive impairment in time-space

orientation, immediate memory, concentration and calculation, long-term memory,

language and praxis [72] 3- UKU scale (Udvalg fur klinische undersogelser)[76] to

determine side effects; with 56 items: the first 9 are for psychic events, 8 for

neurologic, 11 for autonomic and 25 for others. The last two items evaluate side

events interference with the patient's daily performance and their impact on

medication intake (none, dose reduction, withdrawal or change). In each one, the

investigator assesses whether the event was related to drug treatment or not. 4-

Additional safety assessments include respiratory rate and laboratory tests:

complete blood count, creatinine, BUN (blood urea nitrogen), total bilirrubin,

alkaline phosphatase, liver enzymes and human chorionic gonadotropin (hCG-B)

will be determined when appropriate. All patients will be informed about any

eventual depressant effect of alprazolam and risks in activities that require alert

and the consumption of alcohol will be not recommended. They will be also

monitored to detect potential risks of suicide or overdose. All adverse events are

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monitored, classified and reported according to GCP (good clinical practice)

standards of the local regulatory authority (ANMAT and Ministry of Health of the

Province of Córdoba, Argentina).

Cognitive assessment: To quantify the neurocognitive variables; attention, working

memory, executive functions, impulsiveness, verbal and nonverbal fluency the

following tests will be determined. 1- QPSS continuous performance test (CPT)[77];

2- Digit symbol test (DST)[78]; 3- Digit span test (DST)[79]; 4- Verbal fluency test

(VFT)[80]; 5-Five point test (FPT)[81]; 6-revised Taylor complex test (RTCT)[82]

and 7- Stroop test (ST)[83]. All of them will be applied after 6 hours since the last

administration of alprazolam (nadir plasma levels). Cognitive tests and laboratory

tests are blinded to the analyst, the remaining tests are open.

Quality of life (QOL) assessment: The health and quality of life scale, health

questionnaire SF-36 validated Spanish version will be determined. [84]

2.7 Statistical assessment:

The mean, minimum and maximum values of quantitative variables will be

determined. Pre- and post-treatment values will be compared using t-test and/or

non parametric test. Statistical significance will be fixed at p<0.05 (2-sided; β-1

power≥0.80). Correlation analysis will be performed by calculating the Pearson Test

correlation coefficient for ungrouped data; with r>0.80 and p<0.05 (2-sided) strong

correlation and 0.80<r>0.50 and p<0.05 (2-sided), moderate correlation.

Treatment safety: Regarding patient safety, the use of alprazolam, a well known,

low action, nervous system depressant drug, at minimum doses, even when no

symptoms palliation is required, guarantees good tolerability, and no drawbacks are

expected as a result of direct effects. It is worth emphasizing that the proposed

intervention tends to reduce long and/or very frequently repeated demands for

medication and with that improve the global safety rate and QOL.

3. Ethics and dissemination: Approval and Informed Consent

The protocol was approved by an Independent Ethics Committee of FEFyM

(Fundación de Estudios Farmacológicos y Medicamentos/Foundation for

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Pharmacological Studies and Drugs, Buenos Aires) and by regulatory authorities of

Argentina (ANMAT, Dossier # 61409-8 of April 20th, 2009), following the law of

Habeas Data and psychotherapeutic drug control. All participants will agree

voluntarily to participate, after having obtained the informed consent and signed it,

in accordance with local regulations and standards of bioethics. The study is

developed under Phase IV conditions, but the full therapeutic intervention has no

cost for the patient under the study protocol. The present study protocol is still

ongoing and patient recruitment is in process. Trial Registration: GEMA - 20811

4. Expected results

It is our expectation to see a decline within anxiety scores and allostatic load,

reflected by biochemical variables and an improvement in cognitive performance

with a better quality of life, even in patients treated with low doses of alprazolam.

We expect to elucidate improvements in cognitive variables, such as time-space

orientation, immediate memory, concentration and calculation, long-term memory,

language and praxis, as well as a tendency to an overall improvement of QOL

scores. If the GEMA project fulfills its basic purposes, an original road would open

for the search of specific modulators for the treatment of mental illnesses. It has

been stated by health economists, the “macro” conditions of the cost/health ratio

are given more by classic or vintage drugs rather than by innovations, many of

which disappear after a promotional period. In this sense alprazolam has persisted

as a medical tool for several years, and regardless of what formal pharmaceutical

clinical trials and the data that investigators might justify their permanence. The

GEMA project is also an update in the knowledge of the impact of this compound for

patients with anxiety and allostatic load, and is also a project designed to define a

new profile for the biomedical use of alprazolam.

5. Discussion

GEMA project constitute an observational clinical trial designed to determine

objective measures of stress pathophysiology in high symptomatic patients with

comorbid anxiety, allostatic load and neuroticism treated with minimum doses of

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alprazolam, with the aim to determine neurobiochemical and neuropsychological

variables altered by stress.

Treatment aims to contribute to prevent the impact of allostatic load, promoting

resilience, better cognitive performance and making healthier decisions translated

into a better quality of life. If the hypothesis is confirmed, a new paradigm would

come into being, which would renew interest in the clinical management of these

compounds, beyond their typical use as anxiolytic agents. In short, the proposal is

to treat each episode more efficiently in order to reduce the time required to

achieve positive results (bene facere) while decreasing the tendency to excessive

consumption of drugs (non nocere).

6- List of abbreviations

HPA (hypotalamo pituitary adrenal)

CRH (corticotrophin releasing hormone)

GAD (general anxiety disorders)

PFC (prefrontal cortex)

MHPG (3-methoxy- 4- hydroxyphenilglycol)

BZD (benzodiazepines)

NICE (National Institute for Health and Clinical Excellence, UK)

NA (noradrenaline)

STH (somatotropic hormone)

GHRH (growth-hormone-releasing hormone)

MDD (major depressive disorder)

CRH (corticotropin releasing hormone)

MAO (monoamine oxidase)

STAI (state -trait anxiety inventory)

HARS (Hamilton anxiety rating scale)

NEO FFI (NEO five factor inventory for neuroticism)

BMI (body mass index)

HDL (high density lipoprotein)

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LDL (low density lipoprotein)

CGI (Clinical Global Impression Scale)

MEC (The Mini-Mental State)

UKU (Udvalg fur klinische undersogelser)

GCP (good clinical practice)

QOL (quality of life)

BUN (blood urea nitrogen)

CPT (continuous performance test

DST (digit symbol test)

DST (digit span test)

VFT (verbal fluency test

FPT (five point test)

RTCT (revised Taylor complex test)

ST (stoop test).

7- Competing interests

This work is carried out under a grant awarded by Gador SA, Buenos Aires (2010-

2012) and funds of the Instituto de Biociencias Henri Laborit, Córdoba. CS and LD

are consultants at Gador SA, and DN and EJAR are investigators employed by

Gador SA, and CR does not declare any conflict of interest. Gador SA, Darwin 429,

Buenos Aires City, Argentina.

8- Authors’ contributions

CS, CR, DN and EJAR have made substantial contributions to conception and

design. LD has been involved in drafting and revising the manuscript for intellectual

content.EJAR has given final approval of the version to be published.

9- Author’ information

CS and CR: Institute of Biosciences Henri Laborit, Córdoba

DN and EJAR: Scientific Direction, Gador SA, Buenos Aires, Argentina.

LD: Institute of Biology and Neuroscience E de Robertis, CONICET, Argentina.

10- Acknowledgments

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Authors thank the participation and contributions of the work team involved in

GEMA.

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12. Figure Legends

Table 1 Data come from different experimental models and do not represent human SNC condition, either in healthy or under allostatic loads. Gray boxes presume positive for the cognitive development under drug therapy. Adapted from references [51][66] Figure 1 Percentage of reduction of salivary cortisol and MHPG levels among alprazolam(0.75 to 3.0 mg/day) responder patients.Modified from [47]

Figure 2

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Upper panel: Repeated stress (S-blue) conditions plus accumulation of allostatic loads (AL-red) result in chronic stress, and with time loss of the individual resilience (R-green) levels. Anxiety (A-yellow) emerges as a reaction of an excessive stress applied to the body. Under chronic stress, cognitive resources are impaired and with it the possibilities of the affected subject to maintain its quality of life. Consequently, the affected live with a neurobiological environment poorly efficient to solve the daily challenges of life. Middle panel: With a short-term anxiolytic use of alprazolam, symptoms are reduced but demands of treatment persist, as well as the impact of the AL, and the decay of R, are not being significantly modified. Lower panel: With prolonged use of low-dose alprazolam anti-stress biochemical effects, plus a consequent increase of cognitive functions, may allow the progressive recovering of R, shorten S duration, and may further reduce the impact of AL. Under these new biological conditions the subject increased its chances to seek for an improved quality of life and may thereof shorten the demand of palliative medication. Figure 3 Stage I: Short term evaluation of 0.75 to 3.0 mg/day alprazolam on biochemical variables related to chronic stress. Stage II: Short term evaluation of 0.75 to 3.0 mg/day alprazolam on cognitive and clinical variables related to chronic stress. Stage III: Intermediate term evaluation of low alprazolam doses in cognitive, clinical and quality of life indicators.

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140x90mm (300 x 300 DPI)

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127x64mm (300 x 300 DPI)

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1

SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and

related documents*

Section/item ItemNo

Description

Administrative information

Title 1 Descriptive title identifying the study design, population, interventions,

and, if applicable, trial acronym

Trial registration 2a Trial identifier and registry name. If not yet registered, name of

intended registry (page 1- abstract)

2b All items from the World Health Organization Trial Registration Data

Set (attached as additional information)

Protocol version 3 Date and version identifier( attached as additional information)

Funding 4 Sources and types of financial, material, and other support (pag 16 )

Roles and

responsibilities

5a Names, affiliations, and roles of protocol contributors (page 1 and 16)

5b Name and contact information for the trial sponsor (page 16)

5c Role of study sponsor and funders, if any, in study design; collection,

management, analysis, and interpretation of data; writing of the report;

and the decision to submit the report for publication, including whether

they will have ultimate authority over any of these activities (page 16)

5d Composition, roles, and responsibilities of the coordinating centre,

steering committee, endpoint adjudication committee, data

management team, and other individuals or groups overseeing the

trial, if applicable (see Item 21a for data monitoring committee)

(page 16)

Introduction

Background and

rationale

6a Description of research question and justification for undertaking the

trial, including summary of relevant studies (published and

unpublished) examining benefits and harms for each intervention

(point 1: pages 2-6)

6b Explanation for choice of comparators

Objectives 7 Specific objectives or hypotheses (point 1: page 7)

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Trial design 8 Description of trial design including type of trial (eg, parallel group,

crossover, factorial, single group), allocation ratio, and framework (eg,

superiority, equivalence, noninferiority, exploratory) (point 2 page 8)

Methods: Participants, interventions, and outcomes

Study setting 9 Description of study settings (eg, community clinic, academic hospital)

and list of countries where data will be collected. Reference to where

list of study sites can be obtained (point 2 page 8-9)

Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility

criteria for study centres and individuals who will perform the

interventions (eg, surgeons, psychotherapists) (point 2 page 8-9)

Interventions 11a Interventions for each group with sufficient detail to allow replication,

including how and when they will be administered (point 2.5 page 10)

11b Criteria for discontinuing or modifying allocated interventions for a

given trial participant (eg, drug dose change in response to harms,

participant request, or improving/worsening disease) (point 2.2 pag 9)

11c Strategies to improve adherence to intervention protocols, and any

procedures for monitoring adherence (eg, drug tablet return,

laboratory tests)

11d Relevant concomitant care and interventions that are permitted or

prohibited during the trial) (point 2.2 pag 9)

Outcomes 12 Primary, secondary, and other outcomes, including the specific

measurement variable (eg, systolic blood pressure), analysis metric

(eg, change from baseline, final value, time to event), method of

aggregation (eg, median, proportion), and time point for each

outcome. Explanation of the clinical relevance of chosen efficacy and

harm outcomes is strongly recommended (point 2.6 pages 10-12)

Participant

timeline

13 Time schedule of enrolment, interventions (including any run-ins and

washouts), assessments, and visits for participants. A schematic

diagram is highly recommended (see Figure) (point 2.5 page 10)

Sample size 14 Estimated number of participants needed to achieve study objectives

and how it was determined, including clinical and statistical

assumptions supporting any sample size calculations (point 2.4 page

10)

Recruitment 15 Strategies for achieving adequate participant enrolment to reach

target sample size

Methods: Assignment of interventions (for controlled trials)

Allocation:

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Sequence

generation

16a Method of generating the allocation sequence (eg, computer-

generated random numbers), and list of any factors for stratification.

To reduce predictability of a random sequence, details of any planned

restriction (eg, blocking) should be provided in a separate document

that is unavailable to those who enrol participants or assign

interventions

Allocation

concealment

mechanism

16b Mechanism of implementing the allocation sequence (eg, central

telephone; sequentially numbered, opaque, sealed envelopes),

describing any steps to conceal the sequence until interventions are

assigned

Implementation 16c Who will generate the allocation sequence, who will enrol participants,

and who will assign participants to interventions

Blinding

(masking)

17a Who will be blinded after assignment to interventions (eg, trial

participants, care providers, outcome assessors, data analysts), and

how

17b If blinded, circumstances under which unblinding is permissible, and

procedure for revealing a participant’s allocated intervention during

the trial

Methods: Data collection, management, and analysis

Data collection

methods

18a Plans for assessment and collection of outcome, baseline, and other

trial data, including any related processes to promote data quality (eg,

duplicate measurements, training of assessors) and a description of

study instruments (eg, questionnaires, laboratory tests) along with

their reliability and validity, if known. Reference to where data

collection forms can be found, if not in the protocol

18b Plans to promote participant retention and complete follow-up,

including list of any outcome data to be collected for participants who

discontinue or deviate from intervention protocols

Data

management

19 Plans for data entry, coding, security, and storage, including any

related processes to promote data quality (eg, double data entry;

range checks for data values). Reference to where details of data

management procedures can be found, if not in the protocol

Statistical

methods

20a Statistical methods for analysing primary and secondary outcomes.

Reference to where other details of the statistical analysis plan can be

found, if not in the protocol

20b Methods for any additional analyses (eg, subgroup and adjusted

analyses)

20c Definition of analysis population relating to protocol non-adherence

(eg, as randomised analysis), and any statistical methods to handle

missing data (eg, multiple imputation)

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Methods: Monitoring

Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role

and reporting structure; statement of whether it is independent from

the sponsor and competing interests; and reference to where further

details about its charter can be found, if not in the protocol.

Alternatively, an explanation of why a DMC is not needed

21b Description of any interim analyses and stopping guidelines, including

who will have access to these interim results and make the final

decision to terminate the trial

Harms 22 Plans for collecting, assessing, reporting, and managing solicited and

spontaneously reported adverse events and other unintended effects

of trial interventions or trial conduct

Auditing 23 Frequency and procedures for auditing trial conduct, if any, and

whether the process will be independent from investigators and the

sponsor

Ethics and dissemination

Research ethics

approval

24 Plans for seeking research ethics committee/institutional review board

(REC/IRB) approval (point 2.3 page 9)

Protocol

amendments

25 Plans for communicating important protocol modifications (eg,

changes to eligibility criteria, outcomes, analyses) to relevant parties

(eg, investigators, REC/IRBs, trial participants, trial registries, journals,

regulators)

Consent or assent 26a Who will obtain informed consent or assent from potential trial

participants or authorised surrogates, and how (see Item 32)

26b Additional consent provisions for collection and use of participant data

and biological specimens in ancillary studies, if applicable

Confidentiality 27 How personal information about potential and enrolled participants will

be collected, shared, and maintained in order to protect confidentiality

before, during, and after the trial

Declaration of

interests

28 Financial and other competing interests for principal investigators for

the overall trial and each study site

Access to data 29 Statement of who will have access to the final trial dataset, and

disclosure of contractual agreements that limit such access for

investigators

Ancillary and

post-trial care

30 Provisions, if any, for ancillary and post-trial care, and for

compensation to those who suffer harm from trial participation

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Dissemination

policy

31a Plans for investigators and sponsor to communicate trial results to

participants, healthcare professionals, the public, and other relevant

groups (eg, via publication, reporting in results databases, or other

data sharing arrangements), including any publication restrictions

31b Authorship eligibility guidelines and any intended use of professional

writers

31c Plans, if any, for granting public access to the full protocol, participant-

level dataset, and statistical code

Appendices

Informed consent

materials

32 Model consent form and other related documentation given to

participants and authorised surrogates

Biological

specimens

33 Plans for collection, laboratory evaluation, and storage of biological

specimens for genetic or molecular analysis in the current trial and for

future use in ancillary studies, if applicable

*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013

Explanation & Elaboration for important clarification on the items. Amendments to the

protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT

Group under the Creative Commons “Attribution-NonCommercial-NoDerivs 3.0 Unported”

license.

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