artigo 2015

Upload: caio-oliveira

Post on 16-Feb-2018

216 views

Category:

Documents


0 download

TRANSCRIPT

  • 7/23/2019 Artigo 2015

    1/7

    Pharmacological Research 99 (2015)370376

    Contents lists available at ScienceDirect

    Pharmacological Research

    journal homepage: www.elsevier .com/ locate /yphrs

    Aiming for allosterism: Evaluation ofallosteric modulators ofCB1in aneuronal model

    Alex Straiker,Jos Mitjavila, Danielle Yin, Anne Gibson, Ken Mackie

    Department of Psychological and Brain Sciences,Gill Center for Biomolecular Science, Indiana University, 1101 E, 10th St,Bloomington, IN 47405,

    United States

    a r t i c l e i n f o

    Article history:

    Received 10 June 2015Received in revised form 16 July 2015

    Accepted 17 July 2015

    Available online 23 July 2015

    Keywords:

    Allosterism

    Allosteric

    Orthosteric

    Cannabinoid

    Depolarization-induced suppressionof

    excitation

    Tetrahydrocannabinol

    Excitatorypostsynaptic current

    a b s t r a c t

    Cannabinoid pharmacology has proven nettlesome with issues ofpromiscuity a common theme among

    both agonists and antagonists. One recourse is to develop allosteric ligands to modulate cannabinoid

    receptor signaling. Cannabinoids have come late to the allosteric table. The first-generation negative

    and positive allosteric modulators (NAMs and PAMs) represent an important first effort. However, most

    studies have relied on synthetic agonists, often tested in over-expression systems rather than a defined

    neuronal model system that utilizes endogenously synthesized and released cannabinoids.

    We have systematically examined first-generation NAMs and a PAM on endocannabinoid modula-

    tion of synaptic transmission in cultured autaptic hippocampal neurons. These neurons exhibit CB1and 2-arachidonoyl glycerol (2-AG)-mediated depolarization induced suppression of excitation (DSE)

    and therefore serve as a model to test CB1 modulators in a neuronal model ofendogenous cannabinoid

    signaling.

    WefindORG27569,PSNCBAM-1,andPEPCAN12attenuateDSEanddonotdirectly inhibitCB1 receptors.

    Ofthese PSNCBAM-1 is themost efficacious while PEPCAN12 has the distinction ofbeing an endogenous

    NAM. The reported NAMs pregnenolone and hemopressin as well as the reported PAM lipoxin A4 are

    without effect in this model ofendocannabinoid signaling.

    In summary, three ofthe allostericmodulators evaluated function in amannerconsistentwith alloster-

    ism in a neuronal 2-AG-based model ofendogenous cannabinoid signaling. 2015 Elsevier Ltd. All rights reserved.

    1. Introduction

    Cannabinoid receptors were first identified in the early 1990s

    [1,2]. These G protein-coupled receptors mediate most of the

    salient effects of marijuana consumption and also are also key

    components of an endogenous signaling system that is both phy-

    logenetically highly conserved[3] and active throughout thebody.

    Thecannabinoid research field benefited from the early identifica-

    tionof potent andefficacious orthosteric agonists(i.e.WIN55212-2

    and CP55940) and antagonists (i.e. SR141716, AM251), all still in

    experimental use. However, due to the sometimes unfavorable

    therapeutic profiles of the orthosteric CB1 ligands, there has been

    a strong interest in the development of allosteric modulators at

    Abbreviations: DSE, depolarization-induced suppressionof excitation; 2-AG,2-

    arachidonoly glycerol;NAM, negative allostericmodulator; PAM,positive allosteric

    modulator; 9THC, tetrahydrocannabinol; EPSC, excitatory postsynaptic current;

    pERK,phospho extracellular signaling related kinase. Corresponding author.

    E-mail address: [email protected] (A. Straiker)

    CB1. Negativeallostericmodulators (NAMs) inhibit binding and/or

    signaling while positive allosteric modulators (PAMs) potentiate

    bindingand/or signaling by an orthosteric agonist [4]. NAMs/PAMs

    may act in a variety of ways but in its simplest form the concept

    is that a receptormay have one ormore additional allosteric sites

    that when engaged will modulate orthosteric signaling. This usu-

    ally occursby altering thebinding kineticsof the orthostericligand

    and/or by potentiating/inhibiting the receptors signaling via one

    or more signaling pathways. A major advantage of allosteric mod-

    ulators is the potential to modulate only activated receptors. This

    selectivity is especially relevant to the nearly ubiquitous cannabi-

    noidsignaling systemwithits highlevelofendogenousactivityand

    consequent risk of off-target action during a therapeutic interven-

    tion.

    Allosteric modulation is not a new idea. For instance several

    important classes of drugs are allosteric modulators at GABAAreceptors (e.g. benzodiazepines [5]). However the first two CB1NAMs,ORG27569 andPSNCBAM-1werecharacterized lessthan10

    years ago [69], while the first CB1 PAM has been described more

    recently [10]. These first generation allosteric modulators gener-

    atedexcitementbut additionalstudieshave foundthattheypossess

    http://dx.doi.org/10.1016/j.phrs.2015.07.017

    1043-6618/ 2015 Elsevier Ltd. All rights reserved.

    http://localhost/var/www/apps/conversion/tmp/scratch_3/dx.doi.org/10.1016/j.phrs.2015.07.017http://www.sciencedirect.com/science/journal/10436618http://www.elsevier.com/locate/yphrsmailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_3/dx.doi.org/10.1016/j.phrs.2015.07.017http://localhost/var/www/apps/conversion/tmp/scratch_3/dx.doi.org/10.1016/j.phrs.2015.07.017mailto:[email protected]://crossmark.crossref.org/dialog/?doi=10.1016/j.phrs.2015.07.017&domain=pdfhttp://www.elsevier.com/locate/yphrshttp://www.sciencedirect.com/science/journal/10436618http://localhost/var/www/apps/conversion/tmp/scratch_3/dx.doi.org/10.1016/j.phrs.2015.07.017
  • 7/23/2019 Artigo 2015

    2/7

    A. Straiker et al. / Pharmacological Research 99 (2015) 370376 371

    complex pharmacological profiles. For instance,ORG27569 lowers

    cAMP inhibition consistent with NAM activity [6]. But ORG27569

    also increases binding of CP55940 [9], stimulates CB1 internal-

    ization [6], and potentiates G-mediated pERK production [11].Similarly PSNCBAM-1 has been demonstrated to enhance binding

    affinity [8]. Adding to thecomplexity, a recentstudythatexamined

    ORG27569 and PSNCBAM-1 found that even the reported NAM-

    like reduced CB1 internalization [11], may in fact be a secondary

    consequence of enhanced orthosteric binding and consequent

    desensitization [12]. Therefore these compounds do notbehave as

    pure NAMs under the conditions that they have been tested thus

    far.

    PEPCAN12 is a member of a novel class of endogenous com-

    pounds [13]. Discovered in2012, PEPCAN12may beanendogenous

    CB1-modulating peptide acting as a NAM. More recently levels

    of the important steroid hormone precursor, pregnenolone, were

    reported to beupregulated in vivo by9THC; pregnenolone acted

    as a NAM for 9THC at CB1 receptors [14]. While this is a highly

    interestingfinding,the interactionofpregnenolonewithCB1 recep-

    torsactivated byendogenous cannabinoidswasnotevaluated.And

    lastly lipoxin A4was reportedas one of the firstCB1PAMs [10]. The

    identificationof aPAMis ofparticular interestchieflybecausemany

    of thetherapeuticapplicationsforcannabinoidsinvolveCB1activa-

    tion.PAMsofferthepossibilityof targetinga subsetof CB1 receptors(i.e. those currently active). Additionally, a pathway-specific PAM

    may further limit off-target action and may avoid undesired psy-

    choactiveeffects,particularlywhenCB1is activatedbyendogenous

    ligands.

    The pharmacology of allosterism can become quite com-

    plex, particularly because allosteric modulation can be probe-

    dependent, i.e. it may depend greatly on the orthosteric ligand

    being used [4]. Generally speaking, the therapeutic use of an

    allosteric modulator will involve the endogenous ligand, yet the

    characterizations of most CB1 allosteric modulators have used

    synthetic agonists such as CP55940 or WIN55212-2 or the phy-

    tocannabinoid, 9THC.Wehave examined the effect of a range of

    first-generationCB1 allostericmodulatorson synaptictransmission

    using autaptic hippocampal neurons. These cultures are a well-characterized model of endogenous cannabinoid signaling that

    expresses themachinery to produce andmetabolize endocannabi-

    noids aswell as presynaptic CB1receptors [1517]. Depolarization

    of these neurons induces depolarization induced suppression of

    excitation (DSE) a form of retrograde inhibition involving endo-

    cannabinoids and CB1 receptors found in many brain regions [18].

    Theautapticmodel is well-suited to an examination of allosterism

    with the endocannabinoids since we have established that DSE is

    mediatedby theendocannabinoid2-arachidonoylglycerol[15].We

    now report our study of first-generation allosteric modulators in a

    neuronal model of endogenous CB1signaling.

    2. Methods

    2.1. Hippocampal culture preparation

    All procedures used in this study were approved by the Ani-

    mal Care Committee of Indiana University and conform to the

    Guidelines of the National Institutes of Health on the Care and

    Use of Animals. Mouse (CD1 strain) hippocampal neurons iso-

    lated from the CA1-CA3 region were cultured on microislands as

    describedpreviously [19,20]. Neuronswereobtainedfromanimals

    (agepostnatal day02)andplatedontoa feederlayerof hippocam-

    pal astrocytes that had been laid down previously [21]. Cultures

    were grown in high-glucose (20mM)DMEM containing 10%horse

    serum, without mitotic inhibitors and used for recordings after 8

    days in culture and for nomore than3h after removal fromculture

    medium.

    2.2. Electrophysiology

    When a single neuron is grown on a small island of permis-

    sive substrate, it forms synapsesor autapsesonto itself. All

    experimentswere performedon isolated autaptic neurons.Whole

    cell voltage-clamp recordings from autaptic neurons were carried

    out at room temperature using an Axopatch 200A amplifier (Axon

    Instruments, Burlingame, CA).Theextracellular solutioncontained

    (in mM) 119 NaCl, 5 KCl, 2.5 CaCl2, 1.5 MgCl2, 30 glucose, and

    20 HEPES. Continuous flow of solution through the bath chamber

    (2ml/min) ensured rapid drug application and clearance. Drugs

    were typically prepared as stocks, and then diluted into extracel-

    lular solution at their final concentration and used on the same

    day.

    Recording pipettes of 1.83Mwere filled with (in mM) 121.5

    KGluconate,17.5KCl,9NaCl,1 MgCl2, 10HEPES,0.2EGTA, 2MgATP,

    and 0.5 LiGTP. Access resistance and holding current were moni-

    tored andonly cellswith both stable access resistance and holding

    currentwereincludedfor dataanalysis. Conventionalstimuluspro-

    tocol: the membrane potentialwas held at70mV and excitatory

    postsynapticcurrents(EPSCs)were evokedevery20s by triggeringan unclamped action current with a 1.0ms depolarizing step. The

    resultant evoked waveform consisted of a brief stimulus artifact

    anda largedownwardspike representing inward sodiumcurrents,

    followedby the slowerEPSC. Thesizeof therecordedEPSCswascal-

    culatedbyintegratingthe evokedcurrentto yielda charge value(in

    pC). Calculating the charge value in thismanner yields an indirect

    measure of the amount of neurotransmitter released while mini-

    mizing theeffectsof cabledistortiononcurrentsgenerated far from

    the site of the recording electrode (the soma). Data were acquired

    at a sampling rate of 5kHz.

    DSE stimuli: After establishing a 1020s 0.5Hz baseline, DSE

    was evoked by depolarizing to 0mV for 50ms, 100ms, 300ms,

    500ms, 1s, 3s and 10s, followed in each case by resumption of

    a 0.5Hz stimulus protocol for 2080+ seconds, allowing EPSCs torecover to baseline values. This approach allowed us to determine

    the sensitivity of the synapses to DSE induction. To allow compar-

    ison, baseline values (prior to the DSE stimulus) are normalized

    to one. DSE inhibition values are presented as fractions of 1, i.e. a

    50% inhibition from the baseline response is 0.50 standard error

    of the mean. Thex axis of DSE depolarizationresponse curves are

    log-scalesecondsof thedurationof thedepolarizationused toelicit

    DSE.

    Depolarization response curves are obtained to determine

    pharmacologicalpropertiesofendogenous2-AGsignalingbydepo-

    larizingneurons forprogressively longerdurations(50ms, 100ms,

    300ms, 500ms, 1s, 3s and 10s). The data are fitted with a nonlin-

    ear regression, allowing calculation of an ED50, the effective dose

    or durationof depolarization atwhicha 50% inhibition is achieved.Statistical significance in these curves is taken as 95% confidence

    interval.

    2.3. Drugs

    Concentrations of the drugs were tested at 1M consideredto be a concentration that was high enough to expect to see an

    effectbutnotso high as to introducean artifact or off-target effect.

    The exception was PEPCAN12 which has been found to be effec-

    tive at very low concentrations. Thus we tested this compound at

    the lower concentration of 200nM. PEPCAN12 was the generous

    gift ofDr. JuergGertsch (Universitt Bern). ORG27569was the gen-

    erous gift of Dr. Ruth Ross (University of Toronto). Hemopressin

    was the generous gift of Dr. Lakshmi Devi (Mt. Sinai University).

  • 7/23/2019 Artigo 2015

    3/7

    372 A. Straiker et al. / Pharmacological Research 99 (2015) 370376

    PSNCBAM-1, lipoxin A4 and pregnenolone were purchased from

    TocrisBioscience (Bristol, UK), CaymanChemicals (AnnArbor,MI),

    and Sigma Aldrich (St. Louis,MO), respectively.

    3. Results

    We examined the signaling properties of candidate CB1allosteric modulators in autaptic hippocampal neurons, a model

    of endogenous cannabinoid signaling. CB1activation inhibits neu-rotransmitter release in these neurons via subunit-dependentinhibition of calcium channels, resulting in a smaller excitatory

    postsynaptic current (EPSC) [15,22,23]. A negative allosteric mod-

    ulator (NAM) at CB1 should not inhibit EPSCs on its own but it

    should reduce the inhibition induced by CB1 activation. Because

    2-AG mediated depolarization induced suppression of excitation

    (DSE) can be elicited in these neurons, we can test the effect of a

    given candidate NAM on endogenous 2-AG signaling by depolar-

    izing neurons for progressively longer durations (50ms, 100ms,

    300ms, 500ms, 1 s, 3 s and 10s). A NAM should shift the DSE

    response curve up and/or to the right, while a PAM should shift

    the curve down and/or to the left.

    3.1. ORG27569 and PSNCBAM-1 act in a manner consistent with

    negative allostericmodulation

    We first tested the twooriginal negative allosteric modulators:

    ORG27569 [6,7,9] and PSNCBAM-1 [8]. ORG27569 did not directly

    inhibit EPSCs directly at 1M (Fig. 1C, relative EPSC charge (1MORG): 0.990.01, n=6). We also found that at 1M ORG27569did not shift the ED50 of the DSE depolarization response curve

    (Fig. 1A, ED50(baseline): 1.25(1.151.36) ED50(ORG27569):1.30s

    (1.121.51); n=5, overlapping 95% CIs). However, there was a

    statistically significant difference in inhibition for longer depo-

    larizations (Fig. 1A, relative EPSC charge (baseline, 10s depol):

    0.380.04; (ORG): 0.590.03, n=6; p0.05 1 way ANOVA with Bonferroni posthoc

    test).

  • 7/23/2019 Artigo 2015

    4/7

    A. Straiker et al. / Pharmacological Research 99 (2015) 370376 373

    Fig. 2. PSNCBAM-1 substantially inhibits DSE. (A) Depolarization response curve

    before (blacksquares)and after/during 1MPSNCBAM-1treatment(red triangles).

    (B) Averaged DSE (3s depolarization)time courses beforeand after drug. (C) Effect

    of1MPSNCBAM-1aloneon EPSCs inWTneurons. (For interpretation ofreference

    to colorin this figurelegend,the readeris referredto thewebversionof this article.)

    3.3. Pregnenolone does not attenuate 2-AG signaling in autaptic

    neurons

    Lastly among the NAMswe tested the steroid hormone precur-

    sor,pregnenolone. Ina recent report pregnenolonewas foundto actin a manner consistent with negative allosteric modulation of CB1[14] a potentially significant finding for this important hormone

    precursor. However, we found that pregnenolone at 1M waswithout effect on endocannabinoid signaling in autaptic neurons

    (Fig.5, ED50(pre): 2.7s (2.43.0); ED50(Preg1 M): 2.2s (2.02.4);Relative EPSC charge after preg: 1.010.01, n=5).

    3.4. Lipoxin A4 antagonizes cannabinoid inhibition of

    neurotransmission

    To finish on a positive note, we turned to the putative PAM

    lipoxin A4 [10]. In contrast to NAMs, wewould expect lipoxin A4

    to shift the DSE curve to the left. However we found that if any-

    thing 1M lipoxin A4 shifted the curve to the right, suggesting

    Fig. 3. PEPCAN12 inhibits DSE. (A) Depolarization response curve before (black

    squares)and after/during200nM PEPCAN12treatment(red triangles). (B)Averaged

    DSE(series of depolarizations 100ms to 1s) timecourses beforeand after drug.(C)

    Effectof 200nM PEPCAN12aloneon EPSCs.(For interpretation of referenceto color

    in this figure legend, thereader is referred to theweb version of this article.)

    antagonismofCB1signaling (Fig. 6, ED50(baseline): 2.2s (2.12.4);

    (drug): 3.3 s (3.03.6); n=5; non-overlapping 95% CIs). Because

    lipoxin A4 did not inhibit EPSCs when applied on its own (Fig. 6C,relative EPSC charge: 1.020.02, n=5), it is possible that lipoxin

    A4 serves as a NAM rather than a PAMwith2-AG as the ligand.

    4. Discussion

    Allostericmodulators of cannabinoid receptor signaling are rel-

    atively new arrivals on the pharmacotherapeutic stage. Since CB1receptors (and the associated therapeutic potential) are abundant

    throughout the CNS and since 2-AG is implicated in many of the

    known forms of CB1-mediated plasticity, we have tested several

    of the first-generation negative andpositive allosteric modulators

    in a neuronal model that uses 2-AG as the endocannabinoid in an

    endogenous retrograde signaling system. Our chief finding is that

    two of the six candidates act in a manner consistent with their

  • 7/23/2019 Artigo 2015

    5/7

    374 A. Straiker et al. / Pharmacological Research 99 (2015) 370376

    Fig. 4. Hemopressin does not inhibit neurotransmission via CB1. (A) DSE response

    beforeandafter1Mhemopressin. (B)Effectof 2Mhemopressin (pipette)on DSE

    at indicated time alone on EPSCs inWT neurons.

    Fig.5. Pregnenolonedoesnot inhibit neurotransmissionvia CB1. (A)Depolarization

    response curve before (black squares) and after/during 1M pregnenolone treat-ment (red triangles).(B) Effectof 1Mpregnenolonealone onEPSCs inWTneurons.

    (For interpretation of reference to color in this figure legend, the reader is referred

    to theweb version of this article.)

    predicted allosteric modulation while the remaining compounds

    have a diversity of actions, includingagonist effects.

    While our experiments can rule out an allosteric candidate

    (in regard to 2-AG modulation of synaptic transmission), they

    cannot rule one in: the definitive identification of a compound

    as an allosteric agent requires binding studies that character-

    ize the impact of that compound on the binding kinetics of an

    orthosteric ligand.We donot thereforeconcludethat a given com-

    pound is or is not an allosteric modulator but have determined

    whether a compound does/does not act in a manner consis-

    tent with allosteric modulation in the specific pathway of 2-AG

    inhibition of glutamatergic transmission. And importantly all is

    not lost for compounds that fail to act as negative allosteric

    Fig. 6. ThePAM LipoxinA4 (LPA4)inhibitsDSE in autaptic neurons. (A)Depolariza-

    tion response curve before (black squares) and after/during 1M LPA4 treatment

    (red triangles). (B) Averaged DSE (3s depolarization) time courses before and after

    drug. (C) Effect of 1M LPA4 alone on EPSCs inWT neurons. (For interpretation of

    reference to color in this figure legend, the reader is referred to the web version of

    this article.)

    modulators in a neuronalmodel of inhibition of synaptic transmis-

    sion. As has been shown for ORG27569, a given compound may

    serve as a negative allosteric modulator for one signaling path-

    way but not another. Indeed thismay be a preferredoutcome. The

    CB1antagonist SR141716(akaRimonabant/Accomplia) ranafoulofantihedonic properties during clinical use followingEMAapproval

    [25,26] that led to its failure to be approved in the US and even-

    tual withdrawal from the market in Europe. A NAM that avoids

    inhibition of thereceptors/pathwaysresponsible for thisantihedo-

    nic actionmayproduce the desired therapeutic/cosmetic outcome

    free of this side-effect. Conversely one of the allures of positive

    allosteric modulators would be the ability to induce a therapeutic

    effect without the undesired psychoactive responses, perhaps via

    signalingpathway-specific actions.

    Ourresultsadda notch infavorof ORG27569asaNAM.Asnoted

    above ORG27569 has a mixed profile that includes an enhance-

    ment of CP55940 binding as well as an unexpected enhancement

    of pERK signaling [11]. Most studies of ORG27569 action have

    examined its interaction with the synthetic CB1 agonist CP55940

  • 7/23/2019 Artigo 2015

    6/7

    http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0265http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0260http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0255http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0250http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0245http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0240http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0235http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0230http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0225http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0220http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0215http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0210http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0205http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0200http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0195http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0190http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0185http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0180http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0175http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0170http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0165http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0160http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0155http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0150http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0145
  • 7/23/2019 Artigo 2015

    7/7

    376 A. Straiker et al. / Pharmacological Research 99 (2015) 370376

    (crescendo): a randomised, multicentre, placebo-controlled trial, Lancet 376(2010) 517523.

    [27] S.C. Hofer,W.T. Ralvenius, M.S. Gachet, J.M. Fritschy,H.U. Zeilhofer, J. Gertsch,Localizationand production of peptide endocannabinoids in therodent cnsand adrenal medulla, Neuropharmacology (2015), Mar 31. pii:S0028-3908(15)00113-6.

    [28] D.M. Shore,G.L. Baillie, D.H. Hurst,F. Navas III, H.H. Seltzman, J.P. Marcu,M.E.Abood, R.A. Ross, P.H. Reggio, Allosteric modulation of a cannabinoid gprotein-coupled receptor:binding site elucidationand relationship to gprotein signaling, J. Biol. Chem. 289 (2014)58285845.

    http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0280http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0275http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270http://refhub.elsevier.com/S1043-6618(15)00150-4/sbref0270