clinical study the effect of chronic alprazolam intake on ...clinical study the effect of chronic...

Clinical StudyThe Effect of Chronic Alprazolam Intake onMemory Attention and Psychomotor Performance inHealthy Human Male Volunteers

Zahid Sadek Chowdhury1 Mohammed Monzur Morshed2

Mohammad Shahriar1 Mohiuddin Ahmed Bhuiyan1

Sardar Mohd Ashraful Islam1 and Muhammad Shahdaat Bin Sayeed13

1Department of Pharmacy University of Asia Pacific Dhaka 1209 Bangladesh2Department of Biochemistry and Molecular Biology University of Dhaka Dhaka 1000 Bangladesh3Department of Clinical Pharmacy and Pharmacology University of Dhaka Dhaka 1000 Bangladesh

Correspondence should be addressed to Muhammad Shahdaat Bin Sayeed shahdaat2013duacbd

Received 3 March 2016 Revised 12 May 2016 Accepted 7 June 2016

Academic Editor Joao Quevedo

Copyright copy 2016 Zahid Sadek Chowdhury et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Alprazolam is used as an anxiolytic drug for generalized anxiety disorder and it has been reported to produce sedation andanterograde amnesia In the current study we randomly divided 26 healthy male volunteers into two groups one group takingalprazolam05mg and the other taking placebo daily for twoweeksWeutilized theCambridgeNeuropsychological Test AutomatedBattery (CANTAB) software to assess the chronic effect of alprazolam We selected Paired Associates Learning (PAL) and DelayedMatching to Sample (DMS) tests for memory Rapid Visual Information Processing (RVP) for attention and Choice Reaction Time(CRT) for psychomotor performance twice before starting the treatment and after the completion of the treatment We foundstatistically significant impairment of visual memory in one parameter of PAL and three parameters of DMS in alprazolam groupThe PAL mean trial to success and total correct matching in 0-second delay 4-second delay and all delay situation of DMS wereimpaired in alprazolam group RVP total hits after twoweeks of alprazolam treatment were improved in alprazolam group But suchdifferences were not observed in placebo group In our study we found that chronic administration of alprazolam affects memorybut attentive and psychomotor performance remained unaffected

1 Introduction

There is a steadily increased rate of alprazolam prescriptionsin Bangladesh which has a population of more than 168million (IMSQ4 2015 in-house data) [1] Generally alprazo-lam is the most frequently used benzodiazepine [2] primarilyindicated for the treatment of panic disorder and generalizedanxiety disorder (GAD) [3 4] The clinical dose for themanagement of anxiety can range from 05 to 4 milligrams(mg) per day and a daily dose of up to 10mg is indicated forthe management of panic disorder [5] Alprazolam has beenshown to be as equally effective in the treatment of GAD asother benzodiazepines [6 7] tricyclic antidepressants [8 9]and serotonin reuptake inhibitors [10] Alprazolam is also

effective in the treatment of severe anxiety in patients duringalcohol withdrawal [11] It is also affective in the treatment ofmajor depressive disorder when prescribed in double dosesthat are used for anxiety relief [12] However alprazolam isnot a preferred choice in the treatment of body dysmorphicdisorders Alprazolam is considered as a preferred anxiolyticbecause of its additional role as an antidepressant [13]Another benefit of this drug is the relatively faster onset ofanxiety relief compared to other anxiolytics [14]

After a single dose of alprazolam it takes about 18 hoursto reach peak plasma concentration (119862max) The sublingualdosage form takes relatively longer time [15] The amountof drug ingested and peak plasma concentration are propor-tional The elimination half-life (119905

12) is from 10 to 18 hours

Hindawi Publishing CorporationBehavioural NeurologyVolume 2016 Article ID 3730940 9 pageshttpdxdoiorg10115520163730940

2 Behavioural Neurology

after a single oral dose [16]Themean absolute bioavailabilityof oral alprazolam was found to be 92 compared to thatof intravenous alprazolam The onset of alprazolam-inducedsedation was reported to occur more rapidly than oraladministration after intravenous administration Howeverthe volume of distribution is estimated to be higher in oralform than in the intravenous form [16] Coadministrationwith food does not alter the rate or extent of absorption [15]There is no apparent difference in pharmacokinetics profilebetweenmen andwomen [17] In pregnant women use in thefirst trimester is associated with increased risk of congenitalabnormalities [18] and therefore it is considered as pregnancycategory D The clearance and the half-life for multipledoses are similar to those of single dose administration Inmultiple dose administration the steady state concentrationis proportional to daily dose and is similar to that foundby single dose [16] Peak plasma levels are higher in elderlypopulation and clearance is reduced [19 20]

Alprazolam or benzodiazepines in general mainly exerttheir function through gamma aminobutyric acid (GABAA)receptor which consists of three subunits 120572 (alpha) 120573 (beta)and 120574 (gamma) Mainly 120572

1subunit is associated with the

sedative and amnestic function of benzodiazepines whereas1205722is associated with the anxiolytic effect [21] 120572

1subunit is

present mainly in the cerebellum and 1205722can be found in the

hippocampus striatum and spinal cord (reviewed in [21])Alprazolam has long been studied to observe its role in

developing abuse potential Inhaled and oral dosage form ofalprazolam could increase the abuse potential of the drugin subjects with histories of drug abuse [22] Alprazolamis scheduled as a psychotropic substance by the WHO andis also reported to be the most used benzodiazepine withalcohol for abuse purposes in Drug Abuse Warning Network(DAWN) report [23]

Alprazolam has been reported to develop aggression-likebehavior upon chronic use (reviewed by [24]) but in case ofpatients with dementia alprazolam decreases agitation witha significant improvement in symptoms on clinical globalimpression scale [25] Alprazolam has also been reported tohave the lowest risk associated with postprescription nonver-tebral fractures in elderly patients [26] and the premedicationof alprazolam with melatonin has been reported to improveanxiolysis in addition to having an effect on sedation scoreand amnesia [27]

However the cognitive side effects of alprazolam havereceived the greatest attention in the majority of the studiesIn general benzodiazepines have been reported to producegeneral CNS side effects and cognitive impairment Sedationreduced alertness drowsiness sleepiness confusion andheadache constitute the general side effects whereas poorattention and anterograde amnesia are thought to be the cog-nitive impairment [28] Verster et al [29] have demonstratedthe acute effects of 1mg alprazolam which impairs psy-chomotor performance and special cognitive skills requiredfor daily activities like driving ability Furthermore Leufkenset al [30] have added on the subject of acute effect of immedi-ate and extended release formulation of alprazolam in healthyvolunteers on the abovementioned study parameters Andthus the majority of the studies conducted with alprazolam

focused on the acute challenge posed immediately uponalprazolam administration (reviewed in [21]) These studiesclearly show that acute challengewith alprazolamdeterioratesspecific aspects of cognitive function However it has beenreported that some of the benzodiazepine-induced sideeffects improve upon time if not completely eliminated [31] Itis still unresolved whether the cognitive domains impaired byimmediate administration of alprazolam remained the sameupon chronic use Those few studies conducted with chronicuptake of alprazolam have focused mainly on psychomotorperformance and sleep activity and have drawn differentconclusions (reviewed in [21]) Impairment of psychomotorperformance was found when alprazolam was administeredat a high dose for 3 weeks [32] or 025mg tid for one week[33] On the other hand other studies found no impairingeffects when administering a total of 4mg drug for 4 days[34] 05mg once daily [35] or 025mg once daily [36] forone week As for two-week period studies it was reportedthat alprazolam 025mg tid did not impair psychomotorperformance [14] however another study claimed it to beimproved upon repeated use of 0125mg twice daily [37]Tests used in these studies also vary in nature and none ofthese studies reported possible effect upon chronic adminis-tration of alprazolamon attention psychomotor analysis andmemory together on study subjects

Therefore we explored the possible effects on memoryattention and psychomotor performance in healthy malevolunteers who were kept on alprazolam for two weeks Theoutcomes weremeasured using Cambridge Neuropsycholog-ical TestAutomatedBattery (CANTAB) softwareAssessmentof cognitive functions with CANTAB has been proved to besuperior to other traditional psychometric tests because of itslanguage and culture independency higher subjective com-pliance and standardized tests The validity of the CANTABtests has also been assessed by various researchers making it apreferred choice to study cognitive functions One study rana preliminary validity test to assess the execution functionin patients with schizophrenia and bipolar disorder where itcompared the results with Computerized Neurological Test(CNT) [38] Another study conducted a comparison of theCANTAB tests with ldquotraditionalrdquo neuropsychological testinginstruments The authors in [39] reported a modest asso-ciation with traditional neuropsychological test measuresWe assumed that the battery of CANTAB tests selected issensitive enough to detect any impairment or improvementthat might occur over the treatment period

2 Methods and Assessment

21 Participants The present study was conducted on 26healthy male volunteers All the volunteers were recruitedfrom theUniversity of Asia Pacific Bangladesh All were con-versant with English to carry out the instructions given on thescreen during the testThe healthy volunteers were randomlyassigned to two groups Among them 13 were treated withalprazolam and the remaining 13 were considered as placebo(control) group The range of age varied between 20 and 23years Prior to the study written consent was obtained from

Behavioural Neurology 3

all the participating volunteers To determine the eligibilityof the study volunteers as healthy individuals they wereprovided with medical health questionnaires Participantswere asked to provide their medical and psychiatric historyfor the last six months before taking part in the study If theparticipants were not able to provide sufficient informationthey were not recruited in the study Intelligence quotient(IQ) was measured by National Adult Reading Scale asmentioned previously [40] None of the volunteers werehabitual smokers They did not have any history of alcoholintake It was also ensured that study subjects did notconsume any caffeine before 12 hours upon completion of thetest Institutional ethical approval was obtained (ZSC201401)The study strictly followed the International Conference ofHarmonization (ICH) for Good Clinical Practice (GCP) andit was conducted in compliance with the Declaration ofHelsinki and its further amendments

22 Treatment andDesign Theduration of the study was twoweeks Volunteers were divided into two groups randomly(Figure 1) A dose of 05mg was selected for this particularstudy Although the usual dose ranges from 025 to 05mgthree times daily a low dose was selected to avoid subsequentdose tapering after completion of the study All recruitedsubjects were students and multiple dosing was assumed tolead to potential dose missing One group took local brandof alprazolam (containing 05mg alprazolam) every nightbetween 9 pm and 10 pm for two weeks Nighttime dosingwas selected to avoid possible occurrence of drowsiness to beconsidered as a side effect of the treatment in only one groupwhich may occur if given at daytime The control groupwas assigned to take placebo following the same time lengthand pattern The different outlooks like texture shape sizeand color for both alprazolam and placebo were the sameBesides as mentioned above a nighttime dosing schedulewas selected to minimize participantrsquos own intuition aboutthe treatment The volunteers were briefed thoroughly sothat they have a clear idea about the tests before they startthem Before the initiation of the first dose administeringwith either alprazolam or placebo the condition of memoryattentiveness and psychomotor performance was measuredto determine the baseline data After two weeks the subjectsperformed the tests again just after 8 hours of their last doseThe sequence of selected tests was maintained to be the samefor all the volunteers It was also ensured that the volunteersdo not know whether they are taking alprazolam or placeboThe groups were revealed only after the last test was donefor the last study volunteer Volunteers had the option tocontact the study center in case of any emergency during thetest Constant communication was maintained with all theparticipants throughout the study to keep track of the intakeof the recommended product

23 Assessment CANTABwas implied to assess thememoryattention and psychomotor function of the volunteers Con-sidering instruction from the CANTAB developers (productmanual and web resource) and our previous investigations[41] and based on the study purpose a battery of four

different neuropsychological tests were selected for memoryattention and psychomotor performance Paired AssociatesLearning (PAL) and Delayed Matching to Sample (DMS)were selected to study effects on visual memory RapidVisual Information Processing (RVP) and Choice ReactionTime (CRT) were selected to observe the possible effectof alprazolam on attention and psychomotor performancerespectively This computerized platform is now widely usedfor assessing cognitive function memory and attentionOur group has demonstrated the validity of these subtestson healthy volunteers [41 42] Similarly other researchersworking on Alzheimerrsquos disease [43] and with ataxia patients[44] have also reported the validity of these subtests

Test of Visual Memory Test of visual memory was carried outusing the following

Paired Associates Learning (PAL) PAL test is developedto measure visual memory and new learning It measuresmemory in an episodicmanner which requires rememberinga particular location previously paired with an object

One or more boxes with different patterns inside weredisplayed to the participants There were eight differentpositions on the screen in which the boxes can appear Theboxes appeared in random orders on those positions butonly one box at a time The patterns were then displayedin the middle part of the screen one at a time Thenstudy subjects had to identify the exact position of the boxin which the pattern was present This test has gradualpattern of progress so that the number of boxes with patternsincreases as subjects complete the previous stages Clinicalmode was selected for this study which has eight stages Eachstage should be completed by a maximum of ten attemptsEvaluation is based on the following

(a) Total errors adjusted (total errors committed in allstages and adjustment for each stage not attemptedbecause of prior failure)

(b) Mean error to success (mean errors done beforesuccessful completion of a stage)

(c) Mean trial to success (total trials needed to locate allpatterns accurately)

(d) Memory score on the first trial

Delayed Matching to Sample (DMS) DMS is the test fordetermination of visual memory The memory process isexamined in a nonverbal manner in this test A decline inperception or attention may affect the outcome of the studyThe systemic time interval and sensitivity in precision ofpatterns make this test more robust to study visual memory

A complex visual pattern is presented to the subject for45 seconds which is considered as the sample With orwithout a brief delay four similar patterns are displayed tothe subjectThe perfect pattern-match had to be identified bythe participant Sample and choice making patterns may beshown simultaneously or after a delay of 0 4 or 12 secondsA single test consists of 43 trials among which the first threeare not evaluated A subject can make a maximum of four


Study volunteer participation

Follow-up

Analysis

Alprazolam group

Distribution

Control group

(ii) Did not follow assigned intervention (n = 0)(i) Followed assigned intervention (n = 13)

Assigned to alprazolam intervention (n = 13)

Discontinued intervention (n = 0)Lost to follow-up (n = 0)

(i) Excluded from statistical analysis (n = 0)

Considered for eligibility (n = 26)

(iii) Miscellaneous reasons (n = 0)(ii) Disinterested in participating (n = 0)(i) Failed to meet inclusion criteria (n = 0)

Excluded (n = 0)

Randomized (n = 26)

(ii) Did not follow allocated intervention (n = 0)(i) Followed allocated intervention (n = 13)

Assigned to placebo intervention (n = 13)


(i) Excluded from statistical analysis (n = 0)n = 13)Analyzed ( Analyzed (n = 13)

Figure 1 CONSORT 2010 Flow Diagram

choices to match the pattern in each trial More choices ledto an increase in choice latency Evaluation is based on thefollowing

(a) Probability of error following error(b) Probability of error following correct response(c) Correct total(d) Correct simultaneous(e) Correct for 0 s delay (delay between presentation of

sample pattern and choice pattern)(f) Correct for 4 s delay (delay between presentation of

sample pattern and choice pattern)(g) Correct for 12 s delay (delay between presentation of

sample pattern and choice pattern)(h) Correct for all delay(i) Mean latency (average time needed to respond with

accurate response)

Test of Attention Attention is assessed using the following

Rapid Visual Information Processing (RVP) RVP is morefocused towards the assessment of attention RVP examinesthe attention that is visual and sustained It also measurescontinuous performance In this test different single digits(ranging from 2 to 9) appear in a box placed in the middle ofthe screen The digits are shown in a pseudo random orderone at a time with a rate of 100 digits appearing per minuteDuring the test subjects had to identify a particular sequenceof numbers (2 4 6 3 5 7 or 4 6 8) displayed at the upperright side of the box from the randomly appearing singledigits in the box Whenever a subject identifies the targetsequence from the random presentation of single numbersheshe had to register the response by using the press padSuccessful registration was counted as hit Pressing the padirrespective of the target sequence was counted as miss Asingle test consisted of 7 attempts in total Among them thefirst four attempts were not evaluated The target sequencesappeared 27 times in the latter three attempts Evaluation isbased on the following

(a) RVP A1015840 probability for the identification of the targetsequence


(b) RVP B10158401015840 probability to depress the press pad irrespec-tive of the occurrence of target sequence

(c) RVP total hits the number of occasions upon whichthe target sequence was correctly identified

Test of Psychomotor Skills Psychomotor skills are assessedusing the following

Choice Reaction Time (CRT) CRT is a reaction time testThis test follows 2-Choice Reaction Time test where speedof response provides the evaluation This test measuresalertness and motor speed Two conceivable stimuli andresponses were introduced to the study subjects Stimuluswas displayed in an ldquoarrow shaperdquo which appeared eitheron the left or on the right side of the computer screen Thestudy subject was supposed to follow arrow direction to pressthe corresponding left or right ldquopress padrdquo The duration ofresponse limit was 31 seconds A single test consisted of threeattempts among which the first one was not evaluated Thelatter two attempts each had 50 trialsThe average prestimulusdelay in both attempts was around 11 seconds Evaluationwasbased on mean latency of response

24 Statistical Analysis Results were analyzed independentlyfor each test To find the difference between alprazolamand placebo group we checked normality assumption andemployed statistical tests that are appropriate For each mea-sure performance under the drug condition was comparedwith that at baseline using a 2 (time baseline after twoweeks)times 2 (treatment placebo alprazolam) mixed model ANOVAwith repeated measures by using IBM Statistics 21 to find theeffect of alprazolam over the period of two weeks Chi-squaretest was performed for demographic data between groups119901 lt 005 was considered statistically significant

3 Result

31 Demographic Data Randomly recruited volunteers ineither group did not vary significantly in their age andestimated IQ (119901 gt 005)The age (meanplusmn standard deviation)of the volunteers was 2092 plusmn 095 and 2100 plusmn 100 years foralprazolam and placebo group respectively with 119901 = 0891The IQ (mean plusmn standard deviation) of the volunteers was11346 plusmn 1125 and 11423 plusmn 997 for alprazolam and placebogroup respectively with 119901 = 0912

32 Test of Visual Memory

PAL Mixed model repeated measures ANOVA with 2 levelsof group and 2 levels of time shows that one out of four testsof PAL had significant main effect of treatment for two weekswith 119865(1 24) = 1445 119901 = 0001 1205782 = 0376 for PAL meantrial to success 119865(1 24) = 0629 119901 = 0435 1205782 = 0026for PAL total errors adjusted 119865(1 24) = 0740 119901 = 03981205782= 0030 for PAL mean error to success and 119865(1 24) =3396 119901 = 0078 1205782 = 0124 for PAL memory score on thefirst trialTherewas significant interaction between treatment

for two weeks and PAL mean trial to success with 119865(1 24) =3747 119901 lt 0001 1205782 = 0610 indicating that alprazolamimpaired PAL mean trial to success significantly over timeSince all the subjects had the value of 4 for ldquoPAL stagescompletedrdquo at both time points analysis of this parameterwas not possible and was therefore excluded from analysis(Supplementary Table 1 in SupplementaryMaterial availableonline at httpdxdoiorg10115520163730940)

DMS Mixed model repeated measures ANOVA with 2 levelsof group and 2 levels of time shows that three out of the ninetests of DMS had significant main effect of treatment for twoweeks with 119865(1 24) = 7708 119901 = 0010 1205782 = 0243 for DMScorrect 0 s delay 119865(1 24) = 7078 119901 = 0014 1205782 = 0228for DMS correct 4 s delay 119865(1 24) = 5237 119901 = 0031 1205782 =0179 for DMS correct all delay 119865(1 24) = 0125 119901 = 07271205782= 0005 for DMS probability of error following error119865(1 24) = 0323 119901 = 0575 1205782 = 0013 for DMS probabilityof error following correct response 119865(1 24) = 3743 119901 =0065 1205782 = 0135 for DMS correct total 119865(1 24) = 0000 119901 =1000 1205782 = 0000 for DMS correct simultaneous 119865(1 24) =0064 119901 = 0803 1205782 = 0003 for DMS correct 12 s delayand 119865(1 24) = 1721 119901 = 0202 1205782 = 0067 for DMS meanlatency There was significant interaction between treatmentfor two weeks and DMS correct 0 s delay with 119865(1 24) =1046 119901 = 0010 1205782 = 0243 between treatment for twoweeks and DMS correct 4 s delay with 119865(1 24) = 0020119901 = 0890 1205782 = 0001 and between treatment for two weeksand DMS correct all delay with 119865(1 24) = 1061 119901 = 03131205782= 0042 indicating that alprazolam impaired DMS correct

0 s delay DMS correct 4 s delay and DMS correct all delaysignificantly over time (Supplementary Table 1)

33 Test of Attention

RVP Mixed model repeated measures ANOVA with 2 levelsof group and 2 levels of time shows that one of the tests ofRVP RVP total hits had significant main effect of treatmentfor two weeks with 119865(1 24) = 21608 119901 = 0000 1205782 = 0474but there was no such effect (119901 gt 005) observed for RVP Aand RVP B with 119865(1 24) = 0990 119901 = 0330 1205782 = 0040and 119865(1 24) = 1600 119901 = 0218 1205782 = 0062 respectivelyHowever significant interaction between treatment for twoweeks and group was not found with 119865(1 24) = 0031 119901 =0861 1205782 = 0001 indicating that error terms are so high thatthe interaction for RVP total hits could not be calculated overtime (Supplementary Table 2)

34 Test of Psychomotor Performance

CRT Mixed model repeated measures ANOVA with 2 levelsof group and 2 levels of time shows that there is no maineffect of treatment for two weeks with 119865(1 24) = 1425119901 = 0244 1205782 = 0056 for CRT mean latency indicatingthat alprazolam treatment neither impaired nor improvedpsychomotor performance (Supplementary Table 3)


4 Discussion

In the present study we observed the effect of 05mgalprazolam daily on healthy volunteers for two weeks Afterobserving individual performance in PAL test it was revealedthat the subjects who performed poorly before initiation ofthe therapy made fewer errors after alprazolam treatmentwhereas subjects who made fewer errors took more attemptsto complete the test after the treatment This seeminglyopposing effect can be interpreted as increased focus andattention of the subjects who failedmore on the first occasionIt has been suggested that subjective performance will not beimpaired due to drugrsquos effect if the cognitive and performancetest duration is not considerably long (reviewed in [21]) Asthe Paired Associates Learning test required 10ndash15 minutesto complete in either group before and after treatment weassume that the subjects who previously failed more tendedto show more attention than others who performed wellbefore We also note that the amount of drug intake mightalso be low that it did not produce any effect on the testparameters after two weeks Higher doses might yield resultsshowing impairment in Paired Associates Learning in theseparameters

Analysis of DMS test showed that the probability ofmaking an error either following error or following correctresponse did not change significantly (119901 gt 005) over twoweeks in alprazolam group Total correct responses whenthe choice patterns were present simultaneously with thesample pattern were also found to be not different betweenalprazolam and placebo group (119901 gt 005) However astatistically significant difference (119901 lt 005) was observedin alprazolam group over two weeks of treatment whenthere is a delay (0 and 4 seconds) in presentation of thechoice pattern Similarly when all correct responses weresummed up for all delay situations correct all delay we alsofound a significant difference (119901 lt 005) in alprazolamgroup over two weeks of treatment However total correctresponses in overall test increased slightly in both alprazolamand placebo group but such increment was not significantlydifferent Given that the dose administeredwas low high dosemight result in significantly fewer total correct responsesPrevious studies have confirmed that immediate and delayedlearning are affected upon acute alprazolam challenge [45]and it is evident from our study that alprazolam groupdid not perform equally compared to placebo after twoweeks of treatment This implies that learning was impairedupon long-term alprazolam administration On the contrarysome studies with chronic administration of alprazolamhave found no significant defect on memory as reviewedelsewhere [21] These studies used immediate and delayedword recalls and picture recognition tests which are differentfromCANTABrsquos DMS test Since the outcomemeasurementsin CANTABwere collected through software and in the unitsof milliseconds the impairment with alprazolam intake wasobservable in our study Over two weeks of treatment themean latency of matching the sample decreases sharply butnot significantly (119901 gt 005) in alprazolam group Similarbut less prominent trend was also observed in placebo groupThis indicates that the subjects tended to match the sample

quickly but in the process make less correct matching due toalprazolam intake

Overall measurement of attention in RVP showed thatalprazolam has a significant effect Probability of hitting thetarget sequence RVP A1015840 and the probability of pressingthe touch pad irrespective of target sequence RVP B10158401015840 weredifferent in alprazolam group over two weeks of treatmentTotal targets successfully identified RVP total hits increasedsignificantly (119901 lt 005) in alprazolam group which indicatesthat chronic alprazolam ingestion at least at a dose of05mg daily does not affect attention After acute alprazolamchallenge attention is commonly affected Our study showsthat at a low dose attention is not affected when the drug isadministered chronicallyThis is in accordance with previousstudy reporting that small and repeated dosing of alprazolamproduced less pronounced behavioral and adverse side effects[34]

We did not find any significant difference in the meanlatency of reaction time (milliseconds) in alprazolam groupover two weeks of treatment Both alprazolam and placebogroups showed that the mean latency of reaction time wasdecreased but not significantly (119901 gt 005) This is inaccordance with the findings of previous studies [34ndash3646] where chronic ingestion of alprazolam did not affectpsychomotor performance

The fact that mean choice latency for DMS and RVPtest decreased after alprazolam treatment indicates incrementof fine motor controls which may result from decreasedactivity of GABAA mediated inhibition or increased excita-tory activity of glutamatergic system It has been proposedthat chronic increased GABA receptor mediated inhibitionby benzodiazepines may result in increased sensitivity ofglutamatergic system themain excitatory system of the brain[47] There are studies in animals to support this hypothesisIn a study by Steppuhn and Turski [48] it was demonstratedthat mice develop benzodiazepine withdrawal symptomsafter chronic treatment which consists of an initial silentphase and then an active phase characterized by increasedanxiety muscle rigidity and seizure activity Administra-tion of N-methyl-D-aspartate (NMDA) receptor antagonistprevented the development of withdrawal symptom in theactive phase and administration of 120572-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antago-nist in the silent phase prevented subsequent developmentof withdrawal symptoms of active phase So far there areno such studies reported for benzodiazepines withdrawal inhumans but it is conceivable that this system becomes moresensitive upon chronic benzodiazepines treatment

There have been no studies measuring glutamate concen-tration during benzodiazepine intake in human to the bestof our knowledge Acute alcohol withdrawal increases glu-tamate concentration and the glutamatergic system becomessensitized [49] Hyperactive glutamatergic system can causedamage to superior cortical activity [50] which may alsoresult in chronic benzodiazepine users Future studies couldbe directed to observe the occurrence of hyperactive behaviorupon chronic benzodiazepine intake along with glutamateconcentration to find a correlation between glutamate andhyperactivity


weeks

Baseline scores are collected for memory attention and psychomotor performance via

CANTABs PAL and DMS RVP and CRT testrespectively

CRT scores(ii) No significant change in RVP and(i) PAL and DMS scores decreased

Alprazolam 05mg for two

Figure 2 Effect of 05mg alprazolam daily on healthy male volunteers for two weeks Baseline data are collected for CANTABrsquos PAL DMSRVP and CRT testsThen subjects took 05mg of alprazolam daily for two weeks After two weeks PAL and DMS score decreased comparedto controls RVP and CRT scores were unaffected

Our study indicates that chronic administration of alpra-zolam intake does not affect psychomotor performanceand attention but affects memory performance of healthyvolunteers (Figure 2) In a meta-analysis with patients kepton long-term benzodiazepines it was reported that patientsdevelop certain kinds of cognitive impairment upon with-drawal and during follow-up those impairments remained[51]These include sensory processing verbal memory speedof processing motor performance working memory andverbal speed We failed to recapitulate motor performancedefect in the current study possibly because of the low doseused Overall long-term benzodiazepine users may not be intheir full cognitive state upon withdrawal The mechanismof benzodiazepine-induced cognitive effect upon withdrawaland during treatment is not clear Given that the mechanismof such effects is independent of whether patients have mooddisorder or not the cognitive impairment might be of thesame amplitude However sincemood disorder patients haveinherent alternation in brain function the outcome of theresult may vary quite dramatically from that of not havingany disorders

Utilization of CANTAB software to conduct the studyyielded more accurate and reproducible results However05mg once daily dose is relatively low compared to standardalprazolam requirement for anxiety relief and nighttimedosing schedule does not mimic actual practice of drugprescription and the sample size was also not large enoughBecause of the delay between last dose of the drug and testingof cognitive function the obtained cognitive scores mightnot represent the scenario where the drug was at its peakplasma concentration Inclusion of patients group who arekept on alprazolam treatment for future study is suggestedAlthough alprazolam has not been reported to have anyactive metabolite we also propose to measure alprazolamconcentration in subjects over the treatment period in future

studies to more appropriately fit the pharmacokinetic andpharmacodynamic profile of this drug

Competing Interests

The authors declare that they have no competing interestsregarding the publication of this paper

Acknowledgments

Muhammad Shahdaat Bin Sayeed received a grant from theUniversity of Asia Pacific Bangladesh as well as InnovationResearch amp Development Grant Ministry of Science andTechnology Bangladesh The authors are thankful to DrSadikKhuder andDr Caren L Steinmiller for assisting in sta-tistical analysis and critically reading the paper respectively

References

[1] CIA The World FactBook 2015 httpswwwciagovlibrarypublicationsthe-world-factbookgeosbghtml

[2] RxList ldquoTop 200 prescriptions for 2014 by number of US pre-scriptions dispensedrdquo 2014 httpsymphonyhealthcomwp-contentuploads201505Top-200-Drugs-of-2014pdf

[3] D J Greenblatt and C EWright ldquoClinical pharmacokinetics ofalprazolam therapeutic implicationsrdquo Clinical Pharmacokinet-ics vol 24 no 6 pp 453ndash471 1993

[4] B Bandelow D S Baldwin and P Zwanzger ldquoPharmacologicaltreatment of panic disorderrdquo Modern Trends in Pharmacopsy-chiatry vol 29 pp 128ndash143 2013

[5] U E Busto H L Kaplan C E Wright et al ldquoA compara-tive pharmacokinetic and dynamic evaluation of alprazolamsustained-release bromazepam and lorazepamrdquo Journal ofClinical Psychopharmacology vol 20 no 6 pp 628ndash635 2000


[6] R Rimon E-R Kultalahti A Kalli et al ldquoAlprazolam andoxazepam in the treatment of anxious out-patients with depres-sive symptoms a double-blind multicenter studyrdquo Pharma-copsychiatry vol 24 no 3 pp 81ndash84 1991

[7] J B Cohn and C S Wilcox ldquoLong-term comparison ofalprazolam lorazepam and placebo in patients with an anxietydisorderrdquo Pharmacotherapy vol 4 no 2 pp 93ndash98 1984

[8] R Hoehn-Saric D R McLeod andW D Zimmerli ldquoDifferen-tial effects of alprazolam and imipramine in generalized anxietydisorder somatic versus psychic symptomsrdquo Journal of ClinicalPsychiatry vol 49 no 8 pp 293ndash301 1988

[9] D R McLeod R Hoehn-Saric W D Zimmerli E B DeSouza and L K Oliver ldquoTreatment effects of alprazolam andimipramine physiological versus subjective changes in patientswith generalized anxiety disorderrdquo Biological Psychiatry vol 28no 10 pp 849ndash861 1990

[10] R Enkelmann ldquoAlprazolam versus buspirone in the treatmentof outpatients with generalized anxiety disorderrdquo Psychophar-macology vol 105 no 3 pp 428ndash432 1991

[11] I S Kolin and O I Linet ldquoDouble-blind comparison ofalprazolam and diazepam for subchronic withdrawal fromalcoholrdquo Journal of Clinical Psychiatry vol 42 no 4 pp 169ndash173 1981

[12] N Casacalenda and J-P Boulenger ldquoPharmacologic treatmentseffective in both generalized anxiety disorder andmajor depres-sive disorder clinical and theoretical implicationsrdquo CanadianJournal of Psychiatry vol 43 no 7 pp 722ndash730 1998

[13] F Petty M H Trivedi M Fulton and A John Rush ldquoBen-zodiazepines as antidepressants does GABA play a role indepressionrdquo Biological Psychiatry vol 38 no 9 pp 578ndash5911995

[14] R P Hart C C Colenda and R M Hamer ldquoEffects ofbuspirone and alprazolam on the cognitive performance ofnormal elderly subjectsrdquo The American Journal of Psychiatryvol 148 no 1 pp 73ndash77 1991

[15] J M Scavone D J Greenblatt J E Goddard H FriedmanJ S Harmatz and R I Shader ldquoThe pharmacokinetics andpharmacodynamics of sublingual and oral alprazolam in thepost-prandial staterdquo European Journal of Clinical Pharmacologyvol 42 no 4 pp 439ndash443 1992

[16] D J Greenblatt and C EWright ldquoClinical pharmacokinetics ofalprazolamrdquo Clinical Pharmacokinetics vol 24 no 6 pp 453ndash471 1993

[17] F Kristjansson ldquoDisposition of alprazolam in human vol-unteers Differences between gendersrdquo Acta PharmaceuticaNordica vol 3 no 4 pp 249ndash250 1991

[18] M M Iqbal T Sobhan and T Ryals ldquoEffects of commonlyused benzodiazepines on the fetus the neonate and the nursinginfantrdquo Psychiatric Services vol 53 no 1 pp 39ndash49 2002

[19] D J Greenblatt M Divoll D R Abernethy L J MoschittoR B Smith and R I Shader ldquoAlprazolam kinetics in theelderly Relation to antipyrine dispositionrdquo Archives of GeneralPsychiatry vol 40 no 3 pp 287ndash290 1983

[20] D J Greenblatt J S Harmatz and R I Shader ldquoClinicalpharmacokinetics of anxiolytics and hypnotics in the elderlyTherapeutic considerations (Part I)rdquo Clinical Pharmacokineticsvol 21 no 3 pp 165ndash177 1991

[21] J C Verster and E R Volkerts ldquoClinical pharmacology clinicalefficacy and behavioral toxicity of Alprazolam a review of theliteraturerdquo CNS Drug Reviews vol 10 no 1 pp 45ndash76 2004

[22] C J Reissig J A Harrison L P Carter and R R Grif-fiths ldquoInhaled vs oral alprazolam subjective behavioral andcognitive effects and modestly increased abuse potentialrdquoPsychopharmacology vol 232 no 5 pp 871ndash883 2015

[23] United States Department of Health and Human ServicesSubstance Abuse and Mental Health Services AdministrationCenter for Behavioral Health Statistics andQuality DrugAbuseWarning Network (DAWN) 2011

[24] B Albrecht P K Staiger K Hall P Miller D Best and DI Lubman ldquoBenzodiazepine use and aggressive behavioura systematic reviewrdquo Australian and New Zealand Journal ofPsychiatry vol 48 no 12 pp 1096ndash1114 2014

[25] R J Ancill W W Carlyle R A Liang and S G HollidayldquoAgitation in the demented elderly a role for benzodiazepinesrdquoInternational Clinical Psychopharmacology vol 6 no 3 pp 141ndash146 1991

[26] W D Finkle J S Der S Greenland et al ldquoRisk of frac-tures requiring hospitalization after an initial prescription forzolpidem alprazolam lorazepam or diazepam in older adultsrdquoJournal of the American Geriatrics Society vol 59 no 10 pp1883ndash1890 2011

[27] K PokharelM Tripathi P K Gupta B Bhattarai S Khatiwadaand A Subedi ldquoPremedication with oral alprazolam and mela-tonin combination a comparisonwith either alonemdasha random-ized controlled factorial trialrdquo BioMed Research Internationalvol 2014 Article ID 356964 7 pages 2014

[28] S E Buffett-Jerrott and S H Stewart ldquoCognitive and sedativeeffects of benzodiazepine userdquo Current Pharmaceutical Designvol 8 no 1 pp 45ndash58 2002

[29] J C Verster E R Volkerts and M N Verbaten ldquoEffectsof alprazolam on driving ability memory functioning andpsychomotor performance a randomized placebo-controlledstudyrdquo Neuropsychopharmacology vol 27 no 2 pp 260ndash2692002

[30] T R M Leufkens A Vermeeren B E Smink P van Ruiten-beek and J G Ramaekers ldquoCognitive psychomotor and actualdriving performance in healthy volunteers after immediate andextended release formulations of alprazolam 1 mgrdquo Psychophar-macology vol 191 no 4 pp 951ndash959 2007

[31] G H OrsquoSullivan H Noshirvani M Basoglu et al ldquoSafetyand side-effects of alprazolam Controlled study in agoraphobiawith panic disorderrdquo British Journal of Psychiatry vol 164 pp79ndash86 1994

[32] N Pomara H Tun D DaSilva R Hernando D Deptula andD J Greenblatt ldquoThe acute and chronic performance effects ofalprazolam and lorazepam in the elderly relationship to dura-tion of treatment and self-rated sedationrdquo PsychopharmacologyBulletin vol 34 no 2 pp 139ndash153 1998

[33] K Aranko M J Mattila and D Bordignon ldquoPsychomotoreffects of alprazolam and diazepam during acute and subacutetreatment and during the follow-up phaserdquo Acta Pharmacolog-ica et Toxicologica vol 56 no 5 pp 364ndash372 1985

[34] R B Smith and P D Kroboth ldquoInfluence of dosing regimen onalprazolam andmetabolite serum concentrations and toleranceto sedative and psychomotor effectsrdquo Psychopharmacology vol93 no 1 pp 105ndash112 1987

[35] R Kumar D S Mac W F Gabrielli Jr and D W GoodwinldquoAnxiolytics and memory a comparison of lorazepam andalprazolamrdquo Journal of Clinical Psychiatry vol 48 no 4 pp158ndash160 1987

[36] J L Jurado R Fernandez-Mas and A Fernandez-GuardiolaldquoEffects of 1 week administration of two benzodiazepines on


the sleep and early daytime performance of normal subjectsrdquoPsychopharmacology vol 99 no 1 pp 91ndash93 1989

[37] M Bourin M-C Colombel and B Guitton ldquoAlprazolam0125mg twice a day improves aspects of psychometric perfor-mance in healthy volunteersrdquo Journal of Clinical Psychopharma-cology vol 18 no 5 pp 364ndash372 1998

[38] H S Kim Y M An J S Kwon and M-S Shin ldquoA prelim-inary validity study of the cambridge neuropsychological testautomated battery for the assessment of executive function inschizophrenia and bipolar disorderrdquo Psychiatry Investigationvol 11 no 4 pp 394ndash401 2014

[39] P J Smith A C Need E T Cirulli O Chiba-Falek andD K Attix ldquoA comparison of the Cambridge AutomatedNeuropsychological Test Battery (CANTAB) with lsquotraditionalrsquoneuropsychological testing instrumentsrdquo Journal of Clinical andExperimental Neuropsychology vol 35 no 3 pp 319ndash328 2013

[40] M S Bin Sayeed M Asaduzzaman H Morshed M MHossain M F Kadir and M R Rahman ldquoThe effect of Nigellasativa Linn seed onmemory attention and cognition in healthyhuman volunteersrdquo Journal of Ethnopharmacology vol 148 no3 pp 780ndash786 2013

[41] S Tasnim P S Haque M S Bari et al ldquoAllium sativumL improves visual memory and attention in healthy humanvolunteersrdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 103416 8 pages 2015

[42] S Akter M R Hassan M Shahriar N Akter M G Abbas andM A Bhuiyan ldquoCognitive impact after short-term exposure todifferent proton pump inhibitors assessment using CANTABsoftwarerdquoAlzheimerrsquos ResearchampTherapy vol 7 article 79 2015

[43] J Kuzmickiene and G Kaubrys ldquoCognitive results of CANTABtests and their change due to the first dose of donepezil maypredict treatment efficacy in alzheimer diseaserdquoMedical ScienceMonitor vol 21 pp 3887ndash3899 2015

[44] I Vaca-Palomares R Dıaz R Rodrıguez-Labrada et al ldquoStrat-egy use planning and rule acquisition deficits in spinocere-bellar ataxia type 2 patientsrdquo Journal of the InternationalNeuropsychological Society vol 21 no 3 pp 214ndash220 2015

[45] R H Pietrzak J C Scott B T Harel Y Y Lim P J Snyderand P Maruff ldquoA process-based approach to characterizingthe effect of acute alprazolam challenge on visual pairedassociate learning andmemory in healthy older adultsrdquoHumanPsychopharmacology vol 27 no 6 pp 549ndash558 2012

[46] Z Subhan C Harrison and I Hindmarch ldquoAlprazolam andlorazepam single and multiple-dose effects on psychomotorskills and sleeprdquo European Journal of Clinical Pharmacology vol29 no 6 pp 709ndash712 1986

[47] D N Stephans ldquoA glutamatergic hypothesis of drug depen-dencerdquo Behavioral Pharmacology vol 6 pp 425ndash446 1995

[48] K G Steppuhn and L Turski ldquoDiazepam dependence pre-vented by glutamate antagonistsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 90 no14 pp 6889ndash6893 1993

[49] G Brousse B Arnaud F Vorspan et al ldquoAlteration of glu-tamateGABA balance during acute alcohol withdrawal inemergency department a prospective analysisrdquo Alcohol andAlcoholism vol 47 no 5 Article ID ags078 pp 501ndash508 2012

[50] P L Prior and J C F Galduroz ldquoGlutamatergic hyperfunction-ing during alcohol withdrawal syndrome therapeutic perspec-tive with zinc and magnesiumrdquoMedical Hypotheses vol 77 no3 pp 368ndash370 2011

[51] M J Barker K M Greenwood M Jackson and S F CroweldquoPersistence of cognitive effects after withdrawal from long-term benzodiazepine use a meta-analysisrdquo Archives of ClinicalNeuropsychology vol 19 no 3 pp 437ndash454 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


after a single oral dose [16]Themean absolute bioavailabilityof oral alprazolam was found to be 92 compared to thatof intravenous alprazolam The onset of alprazolam-inducedsedation was reported to occur more rapidly than oraladministration after intravenous administration Howeverthe volume of distribution is estimated to be higher in oralform than in the intravenous form [16] Coadministrationwith food does not alter the rate or extent of absorption [15]There is no apparent difference in pharmacokinetics profilebetweenmen andwomen [17] In pregnant women use in thefirst trimester is associated with increased risk of congenitalabnormalities [18] and therefore it is considered as pregnancycategory D The clearance and the half-life for multipledoses are similar to those of single dose administration Inmultiple dose administration the steady state concentrationis proportional to daily dose and is similar to that foundby single dose [16] Peak plasma levels are higher in elderlypopulation and clearance is reduced [19 20]

Alprazolam or benzodiazepines in general mainly exerttheir function through gamma aminobutyric acid (GABAA)receptor which consists of three subunits 120572 (alpha) 120573 (beta)and 120574 (gamma) Mainly 120572

1subunit is associated with the

sedative and amnestic function of benzodiazepines whereas1205722is associated with the anxiolytic effect [21] 120572

1subunit is

present mainly in the cerebellum and 1205722can be found in the

hippocampus striatum and spinal cord (reviewed in [21])Alprazolam has long been studied to observe its role in

developing abuse potential Inhaled and oral dosage form ofalprazolam could increase the abuse potential of the drugin subjects with histories of drug abuse [22] Alprazolamis scheduled as a psychotropic substance by the WHO andis also reported to be the most used benzodiazepine withalcohol for abuse purposes in Drug Abuse Warning Network(DAWN) report [23]

Alprazolam has been reported to develop aggression-likebehavior upon chronic use (reviewed by [24]) but in case ofpatients with dementia alprazolam decreases agitation witha significant improvement in symptoms on clinical globalimpression scale [25] Alprazolam has also been reported tohave the lowest risk associated with postprescription nonver-tebral fractures in elderly patients [26] and the premedicationof alprazolam with melatonin has been reported to improveanxiolysis in addition to having an effect on sedation scoreand amnesia [27]

However the cognitive side effects of alprazolam havereceived the greatest attention in the majority of the studiesIn general benzodiazepines have been reported to producegeneral CNS side effects and cognitive impairment Sedationreduced alertness drowsiness sleepiness confusion andheadache constitute the general side effects whereas poorattention and anterograde amnesia are thought to be the cog-nitive impairment [28] Verster et al [29] have demonstratedthe acute effects of 1mg alprazolam which impairs psy-chomotor performance and special cognitive skills requiredfor daily activities like driving ability Furthermore Leufkenset al [30] have added on the subject of acute effect of immedi-ate and extended release formulation of alprazolam in healthyvolunteers on the abovementioned study parameters Andthus the majority of the studies conducted with alprazolam

focused on the acute challenge posed immediately uponalprazolam administration (reviewed in [21]) These studiesclearly show that acute challengewith alprazolamdeterioratesspecific aspects of cognitive function However it has beenreported that some of the benzodiazepine-induced sideeffects improve upon time if not completely eliminated [31] Itis still unresolved whether the cognitive domains impaired byimmediate administration of alprazolam remained the sameupon chronic use Those few studies conducted with chronicuptake of alprazolam have focused mainly on psychomotorperformance and sleep activity and have drawn differentconclusions (reviewed in [21]) Impairment of psychomotorperformance was found when alprazolam was administeredat a high dose for 3 weeks [32] or 025mg tid for one week[33] On the other hand other studies found no impairingeffects when administering a total of 4mg drug for 4 days[34] 05mg once daily [35] or 025mg once daily [36] forone week As for two-week period studies it was reportedthat alprazolam 025mg tid did not impair psychomotorperformance [14] however another study claimed it to beimproved upon repeated use of 0125mg twice daily [37]Tests used in these studies also vary in nature and none ofthese studies reported possible effect upon chronic adminis-tration of alprazolamon attention psychomotor analysis andmemory together on study subjects

Therefore we explored the possible effects on memoryattention and psychomotor performance in healthy malevolunteers who were kept on alprazolam for two weeks Theoutcomes weremeasured using Cambridge Neuropsycholog-ical TestAutomatedBattery (CANTAB) softwareAssessmentof cognitive functions with CANTAB has been proved to besuperior to other traditional psychometric tests because of itslanguage and culture independency higher subjective com-pliance and standardized tests The validity of the CANTABtests has also been assessed by various researchers making it apreferred choice to study cognitive functions One study rana preliminary validity test to assess the execution functionin patients with schizophrenia and bipolar disorder where itcompared the results with Computerized Neurological Test(CNT) [38] Another study conducted a comparison of theCANTAB tests with ldquotraditionalrdquo neuropsychological testinginstruments The authors in [39] reported a modest asso-ciation with traditional neuropsychological test measuresWe assumed that the battery of CANTAB tests selected issensitive enough to detect any impairment or improvementthat might occur over the treatment period

2 Methods and Assessment

21 Participants The present study was conducted on 26healthy male volunteers All the volunteers were recruitedfrom theUniversity of Asia Pacific Bangladesh All were con-versant with English to carry out the instructions given on thescreen during the testThe healthy volunteers were randomlyassigned to two groups Among them 13 were treated withalprazolam and the remaining 13 were considered as placebo(control) group The range of age varied between 20 and 23years Prior to the study written consent was obtained from

















Follow-up

Analysis

Alprazolam group

Distribution

Control group







Excluded (n = 0)

Randomized (n = 26)











accurate response)










3 Result












4 Discussion









weeks









Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































Follow-up

Analysis

Alprazolam group

Distribution

Control group







Excluded (n = 0)

Randomized (n = 26)











accurate response)










3 Result












4 Discussion









weeks









Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Result












4 Discussion









weeks









Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4 Discussion









weeks









Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















weeks









Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















clinical study the effect of chronic alprazolam intake on ...clinical study the effect of chronic...

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