Diapositiva 1

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BIOPARTITIONING MICELLAR CHROMATOGRAPHY TO
PREDICT BLOOD TO LIVER, BLOOD TO LUNG AND BLOOD
TO FAT PARTITION COEFFICIENTS OF DRUGS
Y. Martín Biosca, S. Torres-Cartas, R.M.
Villanueva-Camañas, M.J. Medina Hernández and S.
Sagrado Dpto. Química Analítica, Universitat de
València, C/ Vicente Andrés Estellés s/n, E-46100
Burjassot (Valencia), Spain Dpto. Química,
Escuela Politécnica Superior de Gandia, Ctra.
Nazaret-Oliva, s/n, E- 46730 Gandia (Valencia)
Spain
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BIOPARTITIONING MICELLAR CHROMATOGRAPHY TO
PREDICT BLOOD TO LIVER, BLOOD TO LUNG AND BLOOD
TO FAT PARTITION COEFFICIENTS OF DRUGS
INTRODUCTION Distribution of organic compounds
between blood and tissues is of crucial
importance in the understanding of potential
toxic effects and in pharmacokinetic analysis.
Several procedures have been developed for
measuring drug partition coefficients from blood
to different tissues (liver, lung, brain,
muscle). Retention data obtained in
biopartitioning micelar chromatography (BMC) is
useful in constructing good models (attributed to
the fact that the characteristics of the BMC
system are similar to the characteristics of
biological barriers). The capability of BMC as
an in-vitro technique to describe distribution of
organic compounds between blood and tissues is
evaluated. Values of in vitro blood to liver,
blood to lung and blood to fat partition
coefficients of a heterogeneous set of compounds
have been collected from the literature. Adequate
correlations between the BMC retention data of
compounds, obtained using a solution of Brij35 as
micellar mobile phase, and their partition
coefficients in different tissues are achieved.
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BIOPARTITIONING MICELLAR CHROMATOGRAPHY TO
PREDICT BLOOD TO LIVER, BLOOD TO LUNG AND BLOOD
TO FAT PARTITION COEFFICIENTS OF DRUGS
EXPERIMENTAL
Stationary phase Kromasil octadecyl-silane C18
column (5 ?m, 150 x 4.6 mm i.d.) (Scharlab,
Barcelona, Spain). Mobile phase Brij35 0.04 M
at pH 7.4 0.05 M phosphate buffer. HPLC
conditions flow 1 mLmin-1 detection at 220 nm
loop 20 ?L T 35.5 ºC.
BMC method
EXPERIMENTAL
Drugs included in this study were chosen in
order to cover a broad range of physico-chemical
properties. All retention factor values (k) were
averages of a least triplicate determinations.

SOFTWARE, AND DATA PROCESSING
Microsoft Excel 2000 software were used to
perform the statistical analysis of the
regressions. The Unscrambler Version 7.6 by CAMO
was used to perform multivariate analysis. EPI
SuiteTM (ACD LabsTM, Advanced Chemistry
Development Inc. Demo version) was used for
parameters estimation octanol-water partition
coefficient (logP), molar refractivity (MR),
polarizability (Pol), molar volumen (MV),
parachor (Pr) and water solubility.
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BIOPARTITIONING MICELLAR CHROMATOGRAPHY TO
PREDICT BLOOD TO LIVER, BLOOD TO LUNG AND BLOOD
TO FAT PARTITION COEFFICIENTS OF DRUGS
RESULTS AND DISCUSSION
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BIOPARTITIONING MICELLAR CHROMATOGRAPHY TO
PREDICT BLOOD TO LIVER, BLOOD TO LUNG AND BLOOD
TO FAT PARTITION COEFFICIENTS OF DRUGS
Blood to lung partition coefficient-retention
relationship (an example)
In order to study the importance of some
physico-chemical variables in the construction of
a regression model for predicting blood to lung
partition coefficients for drugs (Table 1), a
partial least squares analysis (PLS) was
performed. The loading plot corresponding to the
first two latent variables is shown in Figure 1.
Figure 1. PLS loading plot corresponding to the
first two latent variables (y-block in pink and
X-block in blue).
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BIOPARTITIONING MICELLAR CHROMATOGRAPHY TO
PREDICT BLOOD TO LIVER, BLOOD TO LUNG AND BLOOD
TO FAT PARTITION COEFFICIENTS OF DRUGS
Blood to lung partition coefficient-retention
relationship
Non-significant variables were eliminated step
by step (Figure 2), re-analyzing each time the
PLS model. Finally a PLS model was obtained by
selecting the variables kBMC and molar volume
(MV). This model accounts for 82 and 80 of
variance in calibration and cross-validation,
respectively.
Figure 2.- The PLS-model regression coefficients
together their uncertainty limits for the two
latent variables model
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BIOPARTITIONING MICELLAR CHROMATOGRAPHY TO
PREDICT BLOOD TO LIVER, BLOOD TO LUNG AND BLOOD
TO FAT PARTITION COEFFICIENTS OF DRUGS
MLR
Blood to lung partition coefficient-retention
relationship
Figure 3.- Validation plot of the QRAR model
logPLung (-1.7 ? 0.6) (0.003 ? 0.002) k
(0.010 ? 0.002) MV N 30 r2 0.81 S.E.
0.33 As can be observed, in Figure 3 the
ability of the proposed model to describe and
predict logPLung was adequate.
References 1 M. H. Abraham and a. Ibrahim, Eur.
J. Med. Chem. 4 (2006) 1403-1438.. 2 M. H.
Abraham et. al. Eur. J. Med. Chem. (2007),
doi10.1016/j.ejmech.2006.12.01. 1 Michael H.
Abraham et. al. Eur. J. Med. Chem. (2007),
doi10.1016/j.ejmech.2006.12.011.
MLR vs. PLS coef.
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ENANTIOSELECTIVE BINDING OF ANTIHISTAMINES TO
HUMAN SERUM ALBUMIN BY AFFINITY ELECTROKINETIC
CHROMATOGRAPHYPARTIAL FILLIG TECHNIQUE Mª
Amparo Martínez Gómez, S. Sagrado, R.M.
Villanueva Camañas and M.J. Medina
Hernández Dpto. Química Analítica, Universitat de
València, C/ Vicente Andrés Estellés s/n, E-46100
Burjassot (Valencia), Spain.
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ENANTIOSELECTIVE BINDING OF ANTIHISTAMINES TO
HUMAN SERUM ALBUMIN BY AFFINITY ELECTROKINETIC
CHROMATOGRAPHYPARTIAL FILLIG TECHNIQUE
INTRODUCTION A new methodology to evaluate
the enantioselective binding to HSA of highly
protein-bound drugs was proposed. This
methodology consists in ultrafiltrating samples
containing HSA and racemic drug and analysing the
bound drug fraction using AEKC-partial filling
technique (PFT) and HSA as chiral selector. The
protein binding values, the affinity constants to
HSA and the binding sites of the enantiomers of
four antihistamines, brompheniramine,
chlorpheniramine, hydroxyzine and orphenadrine,
on the HSA molecule were evaluated.
REFERENCES 1 J.J.Martínez-Plà, M.A.
Martínez-Gómez, Y. Martín-Biosca, S. Sagrado,
R.M. Villanueva-Camañas, M.J. Medina-Hernández,
Electrophoresis 25 (2004) 3176-3185. 2 M.A.
Martínez-Gómez, S. Sagrado, R.M.
Villanueva-Camañas, M.J. Medina-Hernández,
Analytica Chimica Acta 592 (2007) 2029.
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ENANTIOSELECTIVE BINDING OF ANTIHISTAMINES TO
HUMAN SERUM ALBUMIN BY AFFINITY ELECTROKINETIC
CHROMATOGRAPHYPARTIAL FILLIG TECHNIQUE
EXPERIMENTAL AND METHODOLOGY
Incubation 30 min at 36.5ºC
Unbound drug fraction
Ultrafiltration
SAMPLE
Bound drug fraction

Precipitation of HSA with MeOH (centrif.)

• Drug enantiomer (65-270 µM)
• HSA (475 µM)
• (-)-Sulpiride (I.S.) (80 µM)
• Phosphate buffer 67 mM pH 7.4

CHIRAL ANALYSIS
Optimum experimental conditions for
enantioresolution of drugs and resolution values

• HP 3D CE system, diode array detector and HP
  3DCE Chemstation software
• Fused-silica capillary of 50 ?m i.d and 363 ?m
  o.d. with total and effective length of 65 and
  56.5 cm, respectively.
• Temperature 30º C, voltage 15 kV and detection
  wavelength, 225 nm

Drug pH HSA (µM) SPL (s) Rs
Brompheniramine 8.50 180 180 2.50
Chlorpheniramine 8.25 160 150 1.49
Hydroxyzine 7.00 180 150 1.41
Orphenadrine 7.80 160 150 1.12
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ENANTIOSELECTIVE BINDING OF ANTIHISTAMINES TO
HUMAN SERUM ALBUMIN BY AFFINITY ELECTROKINETIC
CHROMATOGRAPHYPARTIAL FILLIG TECHNIQUE
The binding sites of antihistamines in the HSA
molecule were identified using warfarin, diazepam
and digitoxin as marker ligands representatives
of sites I, II and III, respectively in the HSA
molecule.
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ENANTIOSELECTIVE BINDING OF ANTIHISTAMINES TO
HUMAN SERUM ALBUMIN BY AFFINITY ELECTROKINETIC
CHROMATOGRAPHYPARTIAL FILLIG TECHNIQUE
Compound Binding Model Enantiomer I Enantiomer I Enantiomer II Enantiomer II Enantioselectivity
Compound Binding Model KE1 (M-1) Binding Site KE2 (M-1) Binding site ES
Brompheniramine Competitive (9.390.10)102 Site II Diazepam (2.600.17)103 Site II Diazepam 2.8 0.2
Chlorpheniramine Competitive (9.200.20)102 Site II Diazepam (1.690.17)103 Site II Diazepam 1.8 0.3
Hydroxyzine Independent (5.30.5)103 Non defined (6.30.4)103 Site I Warfarine 1.2 0.6
Orphenadrine Independent (1.260.13)103 Site III Digitoxin (1.670.11)104 Non defined 13.3 0.1

• Table 1 shows the estimated affinity constants
  obtained for each drug enantiomer evaluated using
  the results obtained at five concentration
  levels. Both enantiomers of brompheniramine and
  chlorpheniramine bind to the site II in the HSA
  molecule so, enantiomers follow a competitive
  binding model. On the contrary, enantiomers of
  orphenadrine and trimeprazine bind to different
  binding sites, following an independent binding
  model.
• Enantioselectivity (ES) values were in all
  cases higher than 1 indicating that a certain
  degree of enantioselective binding of
  antihistamines to HSA exists. The results
  obtained represent the first evidence of the
  enantioselective binding of antihistamines to
  HSA, the major plasmatic protein.

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15
STEREOSELECTIVE PLASMA PROTEIN BINDING OF BASIC
DRUGS BY CAPILLARY ELECTROPHORESIS Mª Amparo
Martínez Gómez, S. Sagrado, R.M. Villanueva
Camañas and M.J. Medina Hernández Dpto. Química
Analítica, Universitat de València, C/ Vicente
Andrés Estellés s/n, E-46100 Burjassot
(Valencia), Spain.
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STEREOSELECTIVE PLASMA PROTEIN BINDING OF BASIC
DRUGS BY CAPILLARY ELECTROPHORESIS
INTRODUCTION The stereoselective binding of
antihistamines (brompheniramine,
chlorpheniramine, hydroxyzine, orphenadrine and
phenindamine), phenothiazines (promethazine and
trimeprazine) and a local anaesthetic
(bupivacaine) to human plasma proteins was
evaluated. The results obtained represent the
first evidence of the enantioselective binding of
brompheniramine, hydroxyzine, orphenadrine,
phenindamine, promethazine and trimeprazine to
human plasma proteins.
REFERENCES 1 J.J.Martínez-Plà, Y.
Martín-Biosca, S. Sagrado, R.M.
Villanueva-Camañas, M.J. Medina-Hernández,
J.Chromatogr. A 1048 (2004) 111-118. 2 M.A.
Martínez-Gómez, S. Sagrado, R.M.
Villanueva-Camañas, M.J. Medina-Hernández,
Analytica Chimica Acta 592 (2007) 202209. 3
M.A. Martínez-Gómez, S. Sagrado, R.M.
Villanueva-Camañas, M.J.
Medina-Hernández, Analytica Chimica Acta 582
(2007) 223228.
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STEREOSELECTIVE PLASMA PROTEIN BINDING OF BASIC
DRUGS BY CAPILLARY ELECTROPHORESIS
EXPERIMENTAL AND METHODOLOGY
Incubation 30 min at 36.5ºC
Unbound drug fraction
Ultrafiltration
SAMPLE
Bound drug fraction

Precipitation of HSA with ACN

• Drug enantiomer (92-347 µM)
• Plasma
• (-)-Sulpiride (I.S.) (80 µM)
• Phosphate buffer 67 mM pH 7.4

CHIRAL ANALYSIS

• HP 3D CE system, diode array detector and HP
  3DCE Chemstation software
• Fused-silica capillary of 50 ?m i.d and 363 ?m
  o.d. with total and effective length of 65 and
  56.5 cm, respectively.
• Temperature 30º C, voltage 15 kV and detection
  wavelength, 225 nm

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STEREOSELECTIVE PLASMA PROTEIN BINDING OF BASIC
DRUGS BY CAPILLARY ELECTROPHORESIS
Optimum experimental conditions for
enantioresolution of drugs and resolution
values
Drug pH HSA (µM) SPL (s) Rs
Brompheniramine 8.50 180 180 2.50
Chlorpheniramine 8.25 160 150 1.49
Hydroxyzine 7.00 180 150 1.41
Orphenadrine 7.80 160 150 1.12
Phenindamine 6.80 140 150 1.75
Promethazine 7.60 170 170 2.00
Trimeprazine 7.50 170 190 1.53
Bupivacaine 8.00 140 180 1.52
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STEREOSELECTIVE PLASMA PROTEIN BINDING OF BASIC
DRUGS BY CAPILLARY ELECTROPHORESIS
RESULTS AND DISCUSSION
Figure 1 shows the experimental
electropherograms corresponding to the analysis
of the bound fractions of (A) 242
µ?chlorpheniramine (B) 201µ? orphenadrine (C)
180µ? hydroxyzine (D) 100µ? promethazine (E)
312 µ? bupivacaine
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STEREOSELECTIVE PLASMA PROTEIN BINDING OF BASIC
DRUGS BY CAPILLARY ELECTROPHORESIS
Table 1
Table 1 shows the protein-binding (PB) values of
each drug enantiomer and the enantioselectivity
(ES) values obtained at 3 concentrations. In
general, the first eluted enantiomer (E1)
presented lower affinity towards plasma proteins
than the second enantiomer (E2) Saturation
of binding sites of proteins was observed for
brompheniramine and promethazine. The
different behaviour between the enantiomers of
orphenadrine and trimeprazine indicated that the
enantiomers follow an independent binding model.
Decreased order of ES was Phenindaminegttrimepra
zinegtpromethazineorphenadrinegt Bupivacainegtchlorp
heniraminehydroxyzinebrompheniramine
Compound C tot (µM) PB () PB () ES
Compound C tot (µM) E1 E2 ES
Chlorpheniramine 124 762 835 1.090.08
242 802 822 1.020.02
346 802 822 1.010.02
Hydroxyzine 92 995 1003 1.010.02
180 923 982 1.060.03
261 933 962 1.030.02
Phenindamine 120 293 716 2.490.02
243 293 713 2.510.12
347 322 722 2.250.09
Bupivacaine 125 843 983 1.170.08
218 783 973 1.240.02
312 852 962 1.140.02
Brompheniramine 120 895 924 1.010.05
240 652 732 1.150.03
345 432 502 1.160.04
Promethazine 100 713 1003 1.400.12
200 332 472 1.430.02
290 242 352 1.500.13
Orphenadrine 104 656 995 1.530.06
201 815 962 1.180.09
288 992 1002 1.000.02
Trimeprazine 97 486 842 1.80. 2
194 514 873 1.70.2
277 615 892 1.50.2
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22
SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
Y. Martín-Biosca, L. Asensi-Bernardi, R.M.
Villanueva-Camañas, S. Sagrado and M.J.
Medina-Hernández Dpto. Química Analítica,
Universitat de València, C/ Vicente Andrés
Estellés s/n, E-46100 Burjassot (Valencia), Spain
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SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
INTRODUCTION Alzheimers disease (AD) is an
age-related neurodegenerative disorder that
causes dementia characterized by a low level of
the neurotransmitter acetylcholine in the brain.
The current clinical treatment of AD is mainly
based on acetylcholinesterase (AChE) inhibitors,
such as tacrine, donepezil, rivastigmine, and
galantamine, which pharmacological effect is to
inhibit the activity of AChE, so as to keep a
normal level of acetylcholine in the nerve
system. Capillary electrophoretic systems have
been successfully applied for in-line enzymatic
reactions by a methodology known as
electrophoretically mediated microanalysis (EMMA)
1,3. In this methodology, all the different
steps (i.e. mixing, incubation, separation and
in-line quantitation) are combined in the
capillary, which is used as a microreactor for
the enzymatic reaction. The aim of the present
work is to develop a simple EMMA method for
screening of AChE inhibitors in the early stage
of drug discovery.
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SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
EXPERIMENTAL
Instrumentation
A 50 ?m i.d. (363 ?m o.d.) fused-silica capillary
with total and effective length of 56 and 47.5 cm
respectively was employed (Agilent Technologies,
Germany) CE conditions 15 kV detection at 230
nm T 37º C hydrodynamical injection Background
electrolyte 30 mM borate-phosphate buffer, pH
8.0
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SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
EMMA procedure
Substrate
Product
Enzyme
Inhibitor
Figure 1.- AChE catalyzed reaction
The enzyme activity was directly assayed by
measuring the peak area of produced thiocholine
(TCh) with UV detection at 230 nm
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SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
The enzyme solution and the substrate solution,
with or without inhibitor, were introduced into
the inlet part of the capillary by a sandwich
injection mode 1) Water, 20 mbar for 2 sec
2) AChE, 50 mbar for 2 sec 3) Substrate (with
or without inhibitor), 50 mbar for 2 sec 4)
AChE, 50 mbar for 2 sec 5) Water, 20 mbar for
2 sec 6) Waiting time (mixing and incubation
time) 1 min 7) A voltage of 15 kV was applied
to separate the product TCh from the unreacted
substrate

-
Figure 2.- Schematic illustration of EMMA
technique for AChE acitvity assay.
NOTE I.S. in all solutions
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SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
Blank-area
Product (TCh)
x-area
Substrate (AThCh)
Alprenolol (IS)
Figure 3.- Typical electropherogram obtained
after EMMA methodology applied with (red) and
without (blue) the inhibitor edrophonium (100 µM)
added to the substrate plug. Conditions
concentration of AChE, 0.4 mg/mL AThCh, 10 mM
MgSO4 in the substrate solution, 20 mM.
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SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
RESULTS AND DISCUSSION (an example)
A
B
Characterize mixed-mode inhibitor
To estimate Ki
Figure 4.- (A) Michaelis-Menten and (B)
corresponding Lineweaver-Burk plots obtained
using the inhibitor edrophonium .
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SCREENING OF ACETYLCHOLINESTERASE INHIBITORS BY
CAPILLARY ELECTROPHORESIS AFTER ENZYMATIC
REACTION AT CAPILLARY INLET
The percentage of inhibition was determined
according to the following equation
x peak area of the product (TCh) determined
at a given concentration of inhibitor blank
peak are of the product (TCh) whitout
inhibitor being present
The measured IC50 for edrophonium
(concentration of compound at which the reaction
was inhibited by 50) with the EMMA assay was
aproximately 100 ?M at a concentration of
substrate 10 mM. The proposed methodology is
rapid, simple, automatic and can be very useful
for screening of AChE inhibitors in the early
stage of drug discovery.
References
Figure 5.- Inhibition plot of edrophonium
obtained using the proposed EMMA methodology for
several substrate concentrations.

1. J. Zhang, J. Hoogmartens and A. Van Schepdael,
   Electrophoresis 2006, 27, 35-43.
2. M. Telnarová, S. Vytisková, R. Chaloupková and Z.
   Glatz, Electrophoresis 2004, 25, 290-296.
3. M. Telnarová, S. Vytisková, M. Monincová and Z.
   Glatz, Electrophoresis 2004, 25, 1028-1033.

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