In-depth neuropharmacokinetic analysis of antipsychotics
based on a novel approach to estimate unbound target-site
concentration in CNS regions: Link to spatial receptor
occupancy
Irena Loryan1*, MD, PhD, Erik Melander1, MSc, Marielle Svensson1, MSc, Maryam
Payan1,2, PhD, Frida König1, MSc, Britt Jansson1, BSc, and Margareta HammarlundUdenaes1, PhD
1 – Translational PKPD Group, Department of Pharmaceutical Biosciences, Associate member of
SciLifeLab, Uppsala University, Sweden
2
– Current affiliation: Food and Drug Control Laboratories Research Center (FDLRC); Ministry of Health
and Medical Education (MOH); Tehran, Iran
*Corresponding author
*Dr. Irena Loryan
Translational PKPD group, Department of Pharmaceutical Biosciences
Box 591, 751 24 Uppsala, Sweden
+46 18 471 4995; +46 73 8914982 (mobile)
Irena.loryan@farmbio.uu.se
Running title
Neuropharmacokinetic analysis of antipsychotics
1
Materials and Methods
Chemicals
Haloperidol, clozapine, paliperidone, risperidone, olanzapine, quetiapine, 2-hydroxypropyl-βcyclodextrin (HPβCD), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), human serum
albumin, sodium deoxycholate and sodium dodecyl sulfate (SDS) were obtained from Sigma-Aldrich
(Stockholm, Sweden). Acetonitrile, ammonium formate, trichloroacetic acid and formic acid were
purchased from Merck (Darmstadt, Germany). PierceTM BCA protein assay kit and rat serum albumin
ELISA kit were purchased from Thermo Scientific (MA, USA) and GenWay Biotech Inc. (San Diego, CA,
USA), respectively. Water used in all experiments was purified using a Milli-Q Academic system
(Millipore, Bedford, MA, USA).
Animals
All experiments were performed on drug-naïve male Sprague-Dawley rats (Taconic, Lille Skensved,
Denmark) in accordance with guidelines from Swedish National Board for Laboratory Animals, and were
approved by the Animal Ethics Committee of Uppsala, Sweden. In total, 40 drug-naïve rats weighing
282±34 g were included in in vivo neuroPK studies (ethical approval C16/12). In vitro brain tissue
binding studies and brain tissue uptake studies were conducted on 16 and 18 rats, respectively,
weighing 250-300 g (ethical approval C351/11). All rats were housed in groups at 18 to 22°C under a 12h light/dark cycle with ad libitum access to food and water.
The CMA-ROI toolbox
The methodological platform of CMA-ROI includes i) in vivo neuroPK studies performed in rodents
followed by measurement of brain discrete region/spinal cord partitioning coefficient (Kp,ROI); ii) in vitro
drug tissue binding studies for determination of fraction of unbound drug in CNS regions (fu,ROI) and
blood (fu,plasma); iii) in vitro brain slice studies for evaluation of unbound volume of distribution in
discrete brain regions (Vu,ROI). The extent of unbound drug transport across the BBB/BSCB in the various
2
CNS regions (Kp,uu,ROI) is thereafter assessed through a combination of the experimentally determined
Kp,ROI, fu,ROI and fu,plasma using the following equation:
𝐾𝑝,𝑢𝑢,𝑅𝑂𝐼 =
𝐾𝑝,𝑅𝑂𝐼
𝑉𝑢,𝑅𝑂𝐼 ∙ 𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎
≈
𝐾𝑝,𝑅𝑂𝐼
1
𝑓𝑢,𝑅𝑂𝐼
∙ 𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎
(1)
where Kp,ROI is the ratio of total CNS regional to total plasma drug concentrations measured at steady
state conditions.
In addition, the extent of unbound drug transport into the neural cells (Kp,uu,cell,ROI) in various brain
regions, below exemplified by cortex and striatum, was estimated as:
𝐾𝑝,𝑢𝑢,𝑐𝑒𝑙𝑙,𝑅𝑂𝐼 = 𝑉𝑢,𝑏𝑟𝑎𝑖𝑛,𝑅𝑂𝐼 ∙ 𝑓𝑢,𝑏𝑟𝑎𝑖𝑛,𝑅𝑂𝐼
(2)
Below CMA-ROI key methodological platforms are discussed in details.
In vivo neuropharmacokinetic study
The CNS exposure of antipsychotic drugs was assessed in vivo at steady state conditions. Animals were
not randomized and study was not blinded. The femoral vein and artery were surgically catheterized 24
h before the experiment. Rats were thereafter individually placed into the CMA120 system (CMA, Solna,
Sweden) for freely moving animals with ad libitum access to food and water. The dosing solutions of
antipsychotic drugs were prepared individually the day before experiment by diluting the respective
stock solution in a vehicle containing 10% HPβCD in 0.9% saline for parenteral use (Uppsala University
Hospital Pharmacy, Uppsala, Sweden). Rats were initially given an intravenous loading dose of
haloperidol, clozapine, paliperidone, risperidone, olanzapine or quetiapine during five minutes using a
fast-rate infusion followed by a four hour constant-rate infusion with a flow rate of 1 mL/kg · h-1
(Supplementary Table 1). The dosing regimen was chosen based on a simulation of the plasma profile of
the drug, targeting a total plasma concentration of 100 ng/mL, using the Berkeley Madonna software
package (version 8.3.18 for Windows, Berkeley, CA).
/Supplementary Table 1/
3
Blood (300 µL) was sampled from arteria femoralis and collected into heparin containing tubes at 0,
150, 210 and 240 min after the start of the infusion, to confirm attainment of plasma steady state
conditions. At the end of the experiment, the rats were anaesthetized by inhalation of 5% isoflurane
(Abbot Scandinavia, Solna, Sweden). CSF was collected from the cisterna magna using BD insulin syringe
(BD Biosciences, Plymouth, UK). Exclusion criterion for CSF samples contaminated with the blood was
pre-established. To reduce the residual cerebral blood volume, up to 10 ml of blood was taken
intracardially using a 10 ml EDTA containing vacutainer (BD Biosciences, Plymouth, UK) (1). After
decapitation, the brain was removed and the structures of interest, hypothalamus, frontal cortex,
cerebellum, striatum, hippocampus, and brainstem, were dissected according to Glowinski and Iversen
(2) and Chiu et al. (3). The spinal cord was removed from the column using a ventral approach. Blood
samples were centrifuged for 5 minutes at 1000 rpm and plasma was transferred to Eppendorf tubes.
Plasma, brain regions/spinal cord and CSF samples were immediately frozen on dry ice and thereafter
stored at -80°C pending bioanalysis. Prior to analysis the brain structures/spinal cords were individually
homogenized on ice in 1:4 (w:v) phosphate buffered saline (PBS) pH 7.4 using ultrasonic processor VCX130 (Sonics, Chemical Instruments AB, Sweden).
Validation using cerebral microdialysis technique would be very valuable for the current study and was
planned for clozapine and risperidone. For both drugs cerebral microdialysis data were previously
reported. However, an extensive nonspecific sticking of compounds to the plastic tubing (fluorinated
ethylene propylene) was observed for both drugs. The use of 0.5 % albumin in perfusate significantly
prevented the sticking to the tubing for risperidone; however albumin was affecting the “true” in vivo
recovery estimates. Therefore, the validation was not completed in our lab. In fact, often in published
literature experimental data on cerebral microdialysis presented without thorough assessment of
sticking of substance to the plastic tubing and probe, in vitro and in vivo recovery. Consequently,
conclusions made based on this kind of “quick and dirty” experiments may be misleading.
4
In vitro drug tissue binding study
Equilibrium dialysis was used to assess the fraction of unbound antipsychotic drugs in plasma (fu,plasma),
in whole brain homogenate (fu,brain) and in discrete brain regions and spinal cord (fu,ROI). A Teflon 96-well
plate (model HTD96b, HTDialysis LLC, Gales Ferry, CT, USA) fitted with a regenerated cellulose
membrane (molecular weight cutoff 12-14 kDa) was used in all tissue binding experiments. The
cellulose membrane was treated according to the recommendations from manufacturer before starting
the experiment (HTDialysis LLC, Gales Ferry, CT, USA).
For the plasma protein binding measurements, frozen blank plasma was thawed and pH was adjusted
to 7.4 using 1 M phosphoric acid or 1 M sodium hydroxide. Plasma (n=4), at volumes of 125 µl, were
individually spiked to a final drug concentration of 200 nM. The plasma samples were dialyzed against
equal volumes of PBS, pH 7.4, for 6 hours at 37°C at 200 rpm in an incubator with orbital shaking
(MaxQ4450 Thermo Fisher Scientific, NinoLab, Sweden). At the end of the experiment, 80 µl was
sampled from the spiked plasma (donor side) and from the buffer (receiver side), and subsequently
mixed with 80 µl of either PBS pH 7.4 or with undiluted plasma, respectively. Samples were stored at 20oC pending bioanalysis. The fraction of unbound drug in plasma (fu,plasma) was assessed using Equation
3:
𝐵𝑢𝑓𝑓𝑒𝑟 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎 = 𝑃𝑙𝑎𝑠𝑚𝑎 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
(3)
For the brain tissue binding assay, dilution factors 5 and 10 were initially tested using whole brain tissue
homogenate. The frozen blank whole brain tissue was homogenized with an ultrasonic processor VCX130 (Sonics, Chemical Instruments AB, Sweden) in PBS using 1:4 (w:v) or 1:9 (w:v) dilution. Whole brain
tissue homogenate was treated similarly as plasma. No significant differences were found in fraction of
unbound drug in whole brain homogenate diluted 5-fold or 10-fold (data not shown); moreover, the
results were comparable to literature fu,brain values. Therefore, the decision was made to use a 10-fold
diluted homogenate in the regional tissue binding study. The brain regions and spinal cord were
isolated as described above. Tissues from rats were pooled (2 different pools, n=8 biological replicates
in each pool) to study the fraction of unbound drug in brain regions and spinal cord with 4 technical
5
replicates in each experiment, except the experiments with homogenates of hypothalamus and frontal
cortex that were studied in duplicates and triplicates, respectively). Whole brain tissue homogenate was
used in each experiment as a reference. Eighty µl was taken from both sides of the membrane at 6 h,
assuming equilibrium. To minimize the occurrence of potential matrix effect interference during the
bioanalysis the samples from receiver side were mixed with 80 µl of 1:9 (w:v) homogenate of the
respective brain region or spinal cord. Due to the limited volume of homogenates of hypothalamus and
frontal cortex those samples were mixed with 1:9 (w:v) whole brain homogenate. The brain/spinal cord
tissue samples were mixed with 80 µl of PBS. Drug recovery and stability in the plasma and brain/spinal
cord homogenates were tested in each experiment. Samples were stored at -20oC pending bioanalysis.
Olanzapine showed low stability during the 6 hour incubation of the brain and spinal cord
homogenates. Equation 4 was used to assess the fraction of unbound drug in brain tissue, according to
𝑓𝑢,𝑅𝑂𝐼,𝐷 =
𝐵𝑢𝑓𝑓𝑒𝑟 𝑠𝑎𝑚𝑝𝑙𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝐵𝑟𝑎𝑖𝑛 𝑅𝑂𝐼 𝑠𝑎𝑚𝑝𝑙𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
(4)
Where fu,ROI,D is the fraction of unbound drug in diluted homogenate of brain regions or spinal cord. D is
the dilution factor (in these experiments, D=10). Equation 5 was used to correct fu,ROI,D for the dilution
(4):
𝑓𝑢,𝑅𝑂𝐼 =
1⁄
𝐷
(5)
)−1)+1⁄𝐷
((1⁄𝑓
𝑢,𝑅𝑂𝐼,𝐷
In vitro brain slice uptake study
The volume of distribution of unbound antipsychotic drug in brain (Vu,brain, mL · g brain-1) was estimated
using the brain slice method according to previously published protocols (5, 6). In order to measure
Vu,brain in cortex (Vu,cortex) and striatum (Vu,striatum), microdissection of cortex and striatum was performed
from the brain slices at the end of the incubation period (Figure 2).
/Manuscript Figure 2/
6
Briefly, six 300 µm brain slices were cut using a vibrating blade microtome Leica VT1200 (Leica
Microsystems AB, Sweden), and incubated in a HEPES-buffered artificial extracellular fluid (aECF)
containing the antipsychotic drug with an initial concentration of 200 nM. Two approaches were used.
First, the cassette (mixture of all drugs in aECF) incubation approach was used to study the Vu,brain in the
entire brain slice (n=3 brains per cassette, i.e., 18 slices). Second, the single drug incubation approach
was used to study the brain tissue uptake in cortex and striatum (n=2 rat brains per drug, i.e., 12 slices).
Incubation was performed during 5 h at 37°C in a shaker (MaxQ4450 Thermo Fisher Scientific, NinoLab,
Sweden) with a rotation speed of 45 rpm and constant oxygen flow of about 75-80 mL per minute
through a glass frit. Buffer and brain slices were sampled by the end of the incubation. The slices were
weighed and homogenized in 9 volumes (w:v) of aECF using ultrasonic processor VCX-130 (Sonics,
Chemical Instruments AB, Sweden). The viability of the brain slices was assessed by using one of the
slices per incubation to measure the activity of released lactate dehydrogenase using a cytotoxicity
detection kit (Roche Diagnostics GmbH, Germany). The other slices were analyzed for their drug
content. The brain slices were microdissected, cortex and striatum were isolated, dried on the filter
paper, individually weighted and homogenized. In each experiment one brain slice was not dissected
and was used for estimation of Vu,brain in the entire brain slice as a reference control.
Assuming that the concentration of the drug in virtually protein free aECF is equal to the interstitial fluid
concentration in the brain slice at equilibrium, Vu,ROI was estimated as the ratio of the amount of drug in
the entire brain slice or in cortex/striatum (AROI, nmole · g brain-1) to the measured final aECF
concentration (Cbuffer, µmole · mL-1) using Equation 4. The brain tissue density was assumed to be 1 g ·
mL-1.
Vu,ROI =
AROI −Vi ·Cbuffer
Cbuffer ·(1−Vi )
(6)
where Vi (mL · g brain-1) is the volume of the surrounding brain slice layer of aECF. A volume of 0.094 mL
∙g brain-1 was obtained using [14C]inulin (5).
7
Assessment of albumin content in the homogenates
The residual blood in the brain vasculature is known to affect the evaluation of total drug concentration
in brain tissue (28). Particularly, the influence of residual blood is more pronounced for the drugs with
extensive efflux at the BBB level and very high plasma protein binding. In order to minimize the residual
blood volume intracardial perfusion with PBS is often recommended before decapitation of rat. Due to
the fast systemic elimination of studied antipsychotics the perfusion of brain was not performed to
avoid the disruption of equilibria. Therefore, it might be assumed that observed differences are due to
the existence of spatial differences in vascularization of discrete brain regions and spinal cord.
A rat serum albumin double antibody sandwich ELISA kit (GenWay Biotech Inc., San Diego, CA, USA) was
used for the assessment of the amount of serum albumin in the samples. Briefly, 100 µL of 1:3600 PBS
diluted brain and spinal cord samples obtained from the neuroPK study were added to the polystyrene
microtitre wells covered by anti-rat albumin antibodies. After washing, the anti-albumin antibodies
conjugated with horseradish peroxidase were added to the wells. Subsequently, the excess of the antialbumin antibodies was removed. Horseradish peroxidase bound to the formed complex was visualized
by addition of chromogenic agent 3,3’,5,5’-tetramethylbenzidine. The absorbance of the complex at 450
nm was proportional to the amount of serum albumin within the sample, as measured with a
microplate reader (Fluostar omega, BMG Labtech, Ortenberg, Germany). The optimal detection range
was 12.5 – 400 ng/mL. The total protein content was measured in the same samples using PierceTM
bicinchoninic acid protein assay kit. The optimal detection range was 125 – 2000 µg/mL.
Bioanalysis
A liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze the samples from
both in vivo and in vitro studies. The liquid chromatography system was equipped with two LC-10AD
pumps, a SIL-HTc autosampler (Shimadzu, Kyoto, Japan) and a HyPurity C18 column (3 μm particle size,
50 x 4.6 mm from Thermo Scientific, MA, USA) with a guard column (HyPurity C18 column, 3 μm particle
size, 10 x 4.0 mm from Thermo Scientific, MA, USA). The analysis was carried out by multiple reaction
8
monitoring in a positive electrospray mode using a Quattro Ultima Pt (Waters, Milford, MA, USA). Data
quantification was performed using the Masslynx 4.1 software (Micromass, Manchester, UK).
The details on the conditions for the tandem mass spectrometry and liquid chromatography for each
analyte are presented in Supplementary Table 2 and 3, respectively. Independent and non-biased LCMS/MS analyst was involved in processing of samples and data quantification.
/Supplementary Table 2/
/Supplementary Table 3/
The mobile phase used for the detection of haloperidol, a mixture of 0.05 M ammonium formate
(adjusted to pH 4.0 with formic acid) as elution solvent A (MPA) and acetonitrile:MQ-water (90:10, v:v)
as elution solvent B (MPB), was delivered at 0.8 mL/min. The mobile phase used for the detection of
clozapine, quetiapine, olanzapine, risperidone and paliperidone (9-OH risperidone), a mixture of 0.1%
formic acid as elution solvent C (MPC) and MPB, was delivered at 0.8 mL/min. Gradient elution of
haloperidol was achieved using a mixture of 90% of MPA and 10% MPB over 4 min, after which a step
gradient was used with 10% of MPA and 90% MPB until 4.5 min followed by re-equilibration of the
column with 90% of MPA and 10% MPB until 5 min. Gradient elution of clozapine, quetiapine,
olanzapine, risperidone and paliperidone (9-OH risperidone) was attained using a mixture of 90% of
MPC and 10% MPB over 3.5 min, after which a step gradient was used with 40% of MPC and 60% MPB
until 4 min followed by re-equilibration of the column with 90% of MPC and 10% MPB until 4.5 min. The
eluent was split in the ratio of 1:3 in order to allow 0.2 mL/min into the mass spectrometer.
Acetonitrile mediated protein precipitation (1:3, v:v) was performed for all matrices, i.e., brain and
spinal cord 1:4 (in PBS or in aECF) and 1:19 (in PBS) homogenates, plasma and CSF. Standard curves
were prepared in the respective control matrices using the cassette approach, i.e., all six substances at
each standard level. The concentration range in the standard curves was 0.5 - 5000 ng/g for brain/spinal
cord homogenate neuromatrices and 0.04 - 1000 ng/mL in plasma and artificial CSF, respectively. A
suitable dynamic range was attained for all the bioanalytical assays. Briefly, 50 µL of samples, blanks
9
and standards was precipitated using ice-cold 0.2 % formic acid in acetonitrile. Due to the smaller
volume of cortex and striatum samples obtained from brain slice uptake study 25 µL was precipitated
with 75 µL of ice-cold 0.2 % formic acid in acetonitrile. The precipitated samples were vigorously
vortexed and centrifuged for 3 min at 10,000 rpm. Thereafter, 50 µL of the supernatant was diluted
with 400 µL of either MPA or MPC. Ten to forty µL was injected into the column.
Estimation of receptor occupancy based on neuroPK parameters
In vivo receptor occupancy (RO) can be described with the Hill equation (Eq. 7) (7).
𝐶𝛾
𝛾
𝛾
𝑑 +𝐶
𝑅𝑂 = 𝐾
(7)
where Kd is the equilibrium dissociation rate constant of the drug determined directly in a binding assay
using a labeled form of the ligand, C is the concentration of the drug available for interactions with the
receptor and γ is the Hill coefficient. Assumptions that the concentration of the ligand is much lower
than the equilibrium dissociation rate Kd and that the endogenous ligands (dopamine or serotonin) are
not affecting the ligand-receptor binding were made. According to the free drug hypothesis only
unbound drug is able to interact with the target. Therefore, C could be replaced by the unbound drug
concentration in the brain (Cu,brain). The latter can be obtained via correction of total brain concentration
(Ctot,brain) for drug brain tissue binding using the fraction of unbound drug (fu,brain), or overall brain tissue
uptake using the unbound volume of distribution of drug (Vu,brain). To obtain approximate estimates of
brain regional receptor occupancy the Cu,ROI, fu,ROI, Vu,ROI and KI,ROI in the respective region of interest can
be used as presented below:
𝐶𝑢,𝑅𝑂𝐼 = 𝐶𝑡𝑜𝑡,𝑅𝑂𝐼 ∙ 𝑓𝑢,𝑅𝑂𝐼 =
𝐶𝑡𝑜𝑡,𝑅𝑂𝐼
𝑉𝑢,𝑅𝑂𝐼
(8)
Therefore, Equation 7 can be rewritten as:
𝑅𝑂𝑅𝑂𝐼 = 𝐾
𝐶𝑢,𝑅𝑂𝐼 𝛾
𝑑,𝑅𝑂𝐼
𝛾
𝛾 +𝐶
𝑢,𝑅𝑂𝐼
(9)
10
In order to be able to estimate brain regional receptor occupancy based on the unbound plasma
concentration we propose to use the ratio of brain regional ISF to plasma unbound drug concentrations
Kp,uu,ROI as a novel parameter. Hence, the brain regional receptor occupancy could be assessed as:
𝑅𝑂𝑅𝑂𝐼 = (𝐾
𝐶𝑢,𝑝𝑙𝑎𝑠𝑚𝑎 𝛾
𝑖,𝑅𝑂𝐼 /𝐾𝑝,𝑢𝑢,𝑅𝑂𝐼 )
𝛾
𝛾 +𝐶
𝑢,𝑝𝑙𝑎𝑠𝑚𝑎
(10)
where Ki refers to the equilibrium dissociation constant of a ligand determined in inhibition studies.
Cu,plasma is obtained by correction of total plasma concentration Ctot,plasma for drug plasma protein binding
using fraction of unbound drug (fu,plasma). In case of direct estimation from Ctot,plasma the following
equation could be used:
𝑅𝑂𝑅𝑂𝐼 = (𝐾
𝐶𝑡𝑜𝑡,𝑝𝑙𝑎𝑠𝑚𝑎 𝛾
𝑖,𝑅𝑂𝐼 /(𝑓𝑢,𝑝𝑙𝑎𝑠𝑚𝑎 ∙𝐾𝑝,𝑢𝑢,𝑅𝑂𝐼 ))
𝛾
𝛾 +𝐶
𝑡𝑜𝑡,𝑝𝑙𝑎𝑠𝑚𝑎
(11)
In order to show the contribution of differences in the extent of BBB transport on assessment of the RO
of striatal D2 receptors and the RO of cortical 5-HT2A receptors, the RO was also estimated using
conventional method assuming uniform distribution across the BBB. The average of Kp,uu,ROI values for
haloperidol (1.1), clozapine (0.34), olanzapine (0.84 (8)), quetiapine (0.55), risperidone (0.11) and
paliperidone (0.07) was used in the Eq. 3. In the current study in vitro Ki values for D2 receptors in
caudate-putamen (striatum) and 5-HT2A receptors in frontal cortex as well as the Hill coefficient values
were obtained from Schotte et al (9).
Statistical analysis
Pilot studies for each compound were conducted for all experiments, i.e., neuroPK study, brain tissue
binding and brain uptake studies, and the effect size was estimated from the pilot data. In average,
minimally required per-group sample size for a two-tailed t-test study was 6, given the probability level
(α=0.05), the anticipated effect size (Cohen’s d=1.8), and the desired statistical power level (0.8).
Statistical analysis of data was performed using GraphPad Prism 6.04 for Windows, GraphPad Software,
San Diego California USA, www.graphpad.com (10). D'Agostino-Pearson normality test was performed,
where p>0.05 indicated that the data passed the normality test. Descriptive statistics on data is
11
presented as a mean and standard deviation (mean±s.d.) in the tables and as a mean and standard
error mean (mean±s.e.m.) in figures (else stated). The differences between the mean values of fu,ROI,
Kp,ROI, Kp,uu,ROI, Vu,ROI and Kp,uu,cell,ROI were assessed by ordinary one-way ANOVA. Brown-Forsythe test,
which is less affected by slight deviations from normality, as well as Bartlett’s test were used for
comparison of standard deviations between groups. In case of p<0.05 by one-way ANOVA the ANOVA
analysis was followed by multiple comparison test using the Tukey method, i.e., comparing every mean
with every other mean. Confidence interval for the difference between the two means was computed.
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