Metabolómica in vivo y ex vivo en el control de la Actividad Farmacológica B. Celda1,2,3, D. Monleón2,3,4, MC. Martínez1,2,3, V. Esteve1,2,3, B. Martínez1,3, E. Piñeiro1, R. Ferrer1 1Dept. Química Física-SCSIE, Universitat de Valencia, 2Laboratorio Imagen Molecular (UCIM) 3CIBER Bioingeniería, Biomateriales y Nanomedicina, ISC III 4Fundación de Investigación del Hospital Clínico Universitario de Valencia

Metabolómica in vivo y ex vivo en
el control de la Actividad
Farmacológica
B. Celda1,2,3, D. Monleón2,3,4, MC. Martínez1,2,3,
V. Esteve1,2,3, B. Martínez1,3, E. Piñeiro1,
R. Ferrer1
1Dept.
Química Física-SCSIE, Universitat de Valencia,
2Laboratorio Imagen Molecular (UCIM)
3CIBER Bioingeniería, Biomateriales y Nanomedicina, ISC III
4Fundación de Investigación del Hospital Clínico
Universitario de Valencia
(FP6-2002-LIFESCIHEALTH 503094)
Esquema
Metabolómica y Actividad Farmacológica
1.- Introducción
2.- Precisión diagnóstico œ Selección Terapia
in vivo, ex vivo e in vitro
3.- Sistemas Modelo
in vivo, ex vivo e in vitro
4.- Desarrollo Bioinformático
BIONÓMICA
ADN
ARN
Genómica
Chips ADN
SCSIE+UCIM
40.000 genes
Conocimiento
Limitado
Transcriptómica 105 transcripciones
Chips ADN
SCSIE+UCIM
Proteínas
Proteómica
109 proteínas
MALDI-Tof
MALDI-Tof
RMN SCSIE
Metabolitos Metabolómica 3000 metabolitos
Metabonómica estructura conocida
(RMN)
RMN Alto Campo
SCSIE+UCIM
B.Celda
Conocimiento
Adecuado
Aplicaciones Moleculares al
Diagnóstico, Pronóstico y Selección Terapia
Medicina Translacional
Metabolómica (in vivo y ex vivo)
Genómica (Transcriptómica y Genotipado)
Proteómica (Funcional y Estructural)
B.Celda
INTRODUCCION
NMR Applications
1H( MHz)
Magnetic
Field
Intensity (T)
Size
(cm)
9,6 - 100
0,25 - 3,0
< 100
MRI +
41 - 300
MRS
(biomedicine)
1,0 - 7,1
Miniimaging
300 - 400
NMR
Microscopy
200 - 600
Type
resonance
frequency
MRI +
MRS
(clínical)
NMR
400 - 900
multidimensional
B.Celda
Spatial
resolution
(mm3)
Application
System
d5
human
body
< 30
d2
small animals
7,1 - 9,4
< 12
# 0,005
mouses
4,7 - 14,1
< 2,5
0,00005
insects,
materials,
embrions
9,4 - 21
< 40 Å
< 30
d2
small animals
Laboratorio
Imagen Molecular
(rabits, mouses…)
UCIM (UVEG)
proteíns, DNA,
tissues ...
PERFIL BIOQUÍMICO
Espacio Intra y extracelular,
Fluidos corporales
Metaboloma
Fluidos Corporales
RMN
Metabonoma
Metabolómica Plantas
Metabolómica Mamíferos
Metabolómica humanos:
Cerebro
Corazón
Hígado:
Hepatocito
Riñón
Próstata
Cáncer Mama
Tejido parenquimal
Tejido Médula
Líquido Cefalorráquideo
Sangre:
Plasma
Suero
Bilis
Fluido Seminal
Orina
Exposición por enfermedad, cambio micro-flora, fármacos, tóxicos ...
B.Celda
In vivo + ex vivo Metabolism
Genetic profile (transcriptomics)
Protein expression (Proteomics)
Metabolism (Metabolomic)
Preceds to tissue physiological changes
B.Celda
Brain Tissue Metabolite Profile by SV 1H MRS
B.Celda
Alzheimer
NAA
mI
Co
TE = 31 ms
Cr
NAAp ; mIn
NAA/mIp
NAA/mI d 2,32
ERM
1
0.9
Cr
NAA
Global
0.8
RMI
0.7
TE = 136 ms
Sensibilidad
Cr
0.6
0.5
0.4
Europ.J.Neurology
2004: 11; 89-98
0.3
NAA/Crp
0.2
0.1
0
0
0.2
0.4
0.6
0.8
1 - Espe cificidad (fals os positivos)
B.Celda
1
MRSI vs. PET
B
A
NAA
Co Cr
Lac
NA
Metastatic Cystic
MRSI vs. PET
B
A
3
1
6
4
2
7
5
C
High Grade Glioma
Brain tumour Diagnosis MRSI + SV
SV location of AII
according
Cho High level
previous 2D TSI
TE 272 ms
MMA/Cr
TR 2s 1.5 T
MMB/Cr
(FP6-2002-LIFESCIHEALTH 503094)
3
4
Gliomas grading by two
TE (31 + 136 ms) SV
quantitative analysis
previous SV location
by 2D TSI high level
Choline image
Grade
2
http:// www.etumour.net,
M.C. Martínez-Bisbal et al. ESMRMB (2005); # 114 and Rev.Neurol. (2002), 34(4); 309
C. Majós et al. AJNR (2004), 25; 1696; http://www.carbon.uab.es/INTERPRET,
B.Celda
Brain primary tumour vs metastasis MRSI
2D TSI at 272 ms 1.5 T
Brain secondary lesion
metastasis (MT)
(Cho/Cr)MT<(Cho/Cr)GBM
Brain brimary tumour
(GBM)
(FP6-2002-LIFESCIHEALTH 503094)
B.Celda http:// www.etumour.net,
Brain tumour patient outcome and follow-up
by Perfusion MRI and MRS
Kaplan-Meier survival curves
Cerebral Blood Volume (CBV) predicts
more accurately the Low Grade Gliomas (LGG)
progression than initial histopathology grading
Longer survival o CBV<1.75
Shorter survival o CBV>1.75
M. Law et al. Proc. Intl. Soc. Mag. Reson. Med. 13 (2005); 330
Follow-up of AII by 1H MRS SV at 1.5 T
TE 136 ms
Cho
Cr
NAA
MRS study number
B.Celda
Guided Therapy; Radiotherapy, Quimio
Tumour
tissue Infiltration
A
Normal
tissue
B
Necrosis
T. Laudatio, MC. Martínez-Bisbal et al., NMR Biomedicine, in press (2006)
B.Celda
Brain tumour radionecrosis vs. recurrence MRSI
Transverse
2D MRSI 1.5 T
TE 31 ms TR 2 s
Cho
Tumour Recurrence (TR)
(Cho/NAA)TR >(Cho/NAA)RI
Co
Saggital
2D TSI 1.5 T
TE 272 ms TR 2 s
Radiation injury (RI)
B.Celda
http:// www.etumour.net,
(FP6-2002-LIFESCIHEALTH 503094)
Breast cancer diagnosis (MRS/CSI)
2D PRESS MRSI (CSI) with k-space sampling
1.5 T TE = 270 ms 16 x 16 11:40 min
8 averages
Two independent CHESS optimized pulses
for water and lipid suppression
2D 1H MRSI study after 5 days no Cho
Gd-enhanced areas
absence of viable tumour
High Cho levels
active tumour by histology
J. Hu et al. Proc.Intl.Soc.Mag.Reson.Med. 13 (2005); 134
Liver treatment response by 19F MRSI at 3 T
of Capacitabine colorectal cancer drug
19F CSI of liver of a
patient treated with oral Capecitabine FABL imaging
Capecitabine
Capecitabine o 5’ deoxy-5’ fluorocytidine o 5’ deoxy-5’ fluorouridine o D-fluor-E-alanine
Oral drug
5’ DFUR
5’ DFCR
FBAL
D.W. Klomp.et al. Proc. Intl. Soc. Mag. Reson. Med. 13 (2005); 128
Ex Vivo 1H MRS
Lac
Leu
Ala
PCho
mI
Germinoma
Val
?? Ile
??
Leu
Gly
Tau??
Cho
Leu
PCr
Cr
Glx
NAA Ile
NAA
Asp
GBM
NAA
lip
Biopsy
study by
HR-MAS
(ex vivo)
HR-MAS
PCA
T=0ȱCȱ(4ȱCȱinner)
4500ȱHz
ca.ȱ20ȱmgȱofȱtissue
Cylindricalȱrotor
(50ȱPl)
(PCAȱca.ȱ2g)
D2Oȱfieldȱhomogeneity
andȱmobility
HR-MAS
PCA
NMR in Biomedicine, Martínez-Bisbal et al. 2004;17:191-205
B.Celda
129
Brain tumour characterization ex vivo (HR-MAS)
Met
Ast
Men
GBM
(FP6-2002-LIFESCIHEALTH 503094)
B.Celda
M.C. Martínez-Bisbal et al. NMR Biomed.(2005), 17; 1
http:// www.etumour.net;
D. Monleón et al., ESMRMB (2005) #325
Brain tumour biopsies ex vivo metabolic profile
NMR Biomed. 2004;17:191-205
Brain tumour characterization ex vivo (HR-MAS)
(FP6-2002-LIFESCIHEALTH 503094)
B.Celda
M.C. Martínez-Bisbal et al. NMR Biomed.(2005), 17; 1
http:// www.etumour.net;
D. Monleón et al., ESMRMB (2005) #325
Fle 158
Metastasis
In vivo ERM 30 ms a 1,5 T
ex vivo HR-MAS a 11,5 T
in vivo ERM 30 ms a 1,5 T
Fle 153
Ganglioglioma
ex vivo HR-MAS a 11,5 T
Perfil Metabólico ex vivo Biopsias Hígado
Glucosa E/LipI
Lip I/LipII
Alanina/Lisina
p < 0,001
p = 0,01
p = 0,026
NMR Biomed. 2006;19:90-100
Breast cancer diagnosis and invasion
High-Resolution 1H NMR
malignant
bening
1H NMR spectra at 8.5 T, 37 C of
breast fine needle aspiration biopsy
Accuracy
Accuracy
benign vs malignant
96% (Cho/Cr)
lymph node
vascular invasion
95%
94%
1H NMR spectra at 8.5 T, 37 C of
breast ductal carcinoma in situ (DCIS)
fine needle biopsy
C.E. Mountford et al. Chem. Rev. (2004), 104; 3677
C.E. Mountford et al. Br. J. Surg. (2001), 88; 1234
C. Lean et al., J. Women´s Imaging (2000), 2; 19
Breast cancer grading and lymph node status (HR-MAS)
1H HR-MAS CPMGPR
aliphatic region of a breast
tumour tissue at 14 T
•
Breast cancer grading for
invasive ductal carcinoma IDC
IDC I IDC II IDC III
Sensitivity 100% 84%
81%
Specificity 100% 83%
86%
B) Breast cancer lymph node status prediction
by HR-MAS
Actual negative
Sensitivity
92%
Specifity
97%
Actual Positive
97%
92%
B. Sitter et al. NMR in Biomedicine 2002; 15: 327
T.F. Bathen et al., Proc. Intl. Soc. Reson. Med., 13 (2005), 130
L.R. Jensen et al., Proc. Intl. Soc. Reson. Med., 13 (2005), 131
Cervical cancer diagnosis in vivo MRS + ex vivo (HR-MAS)
normal control
Invasive cervical
carcinoma
sagittal T2 TSE
endovaginal coil
1.5 T
TR 2 s TE 80 ms
normal control
10 cm3 squamous
cell carcinoma
-CH2
normal uterine tissue
Cho
Cr
squamous cell carcinoma
uterine cervix
Triglyceride
CH2
1H MRS
SV 3.4 cm3
PRESS spectra
TR 1600 ms
TE 135 ms
biomarker of
invasive cervical
tumour (74% of
tumours NOT in
control p<0.001)
1H presat HR-MAS alipahtic region at 11.7 T 25C
lipid and Choline levels markedly increased in
squamous cell carcinoma biopsy
M.M. Mahon et al. NMR Biomed. (2004), 17; 1
Pangenomic DNA microarrays eTUMOUR protocols
GBM, OA, AII, MET, MEN.
Not supervised analysis
Hierarchical cluster
Supervised LDA analysis
MEN
GBM
MET
A II
OLIGO
Lung Metastases vs Breast Metastases
CPMG
Gly
Cho Cr
Ala
PCA
Lung Metastases
Breast Metastases
Lac
GBM high lipids content vs GBM lower lipid amount
CPMG
GBM with high lipids
content
GBM with lower lipid
content and higher
intensity for mobile
metabolites
PCA
LipoMed, Inc.
3009 New Bern A venue
Raleigh, NC 27610
www. lipoprof ile.com
NMR LipoProfile
LipoMe d, Inc. 0 4/99
P rod uced un der p atent license s
to U.S. P aten t Nos. 4,93 3,844
a nd 5,34 3,389
LipoMed, Inc.
3009 New Bern A venue
Raleigh, NC 27610
www. lipoprof ile.com
NMR LipoProfile
LipoM ed, Inc. 0 4/99
P ag e 2 of 2
P age 1 of 2
Pat ien t Na m e
Pat ie nt Name
Pa tien t ID
Se x
Age
M
63
Bir th Dat e
Spe cimen ID
7-29 -36
L M018 82 0
Dat e Co llect ed
Date Re ceive d
Dat e Re po rte d
8-24 -99
8-25 -99
8-2 6-99
Spe cim en ID
Da te Rep or te d
LM01 882 0
08-2 6-99
Physicia n N ame & Ad dr ess
Phone: (
FA X: (
SUBCLASS LEVELS
)
)
Comments
VLDL Subclasses
LDL Subclasses
(mg/ dL Tr igly cer ide )
( mg /d L C hole ste ro l)
( mg/d L Cho les ter ol)
HDL
Subclas ses
(107)
LIPOPROTEIN PANEL
(21)
(35)
C oronary Heart Disease (C HD) Risk Categories
(18)
LDL Particles
nmol/L
Optimal*
D es irable
B orderline-High
High Risk
1654
les s t han 1100
1100 - 1399
1400 - 1799
great er than 1800
(0 )
Pat tern A (large LD L)
nm
LDL Size
Large HDL
Large VLDL
19.1
22.0 - 20.6
20.5 - 19. 0
Low er-R isk
Higher-Ri sk
mg/ dL
N egative R isk Fac tor
Intermedi ate
Posit ive Ris k Factor
16
greater than 40
40 - 21
less than 21
mg/ dL
Lower-Ris k
less than 7
Intermedi ate
7 - 27
Higher-R isk
greatert han 27
35
(16)
Pat tern B (small LDL)
(5)
(0)
Lar ge Medium
VLDL
VLDL
Sm al l
VLDL
(V5+ V6 )
(V1+V2)
(V3+ V4)
IDL
Large
LDL
Medium
LDL
Sm all
LDL
( L3)
(L2)
(L1 )
NMR LIPID PANEL
RISK ASSESSMENT PANEL
Elevated
LDL Particles
Atherogenic
Dyslipidemia
Total Chole sterol
>1400 nmol/L
2 traits
Smal l LD L
Pattern B
( 20.5 n m)
Re duce d
Large HDL
(< 21 mg/dL)
Ele vated
Large VLDL
(> 27 mg/dL)
Smal l
HDL
(H3+H4 +H5) (H1+H2)
LDL Cholesterol
D es ira b le
18 5
le ss th an 2 00
112
Triglyce rides
O pt im al *
le ss th an 1 00
Bo r de r line -H igh
H ig h
20 0 - 2 3 9
2 40 o r g r ea te r
De sir a ble
Bo rd e rlin e- Hig h
H igh R isk
1 00 - 1 29
1 30 - 15 9
g re at er th a n 1 6 0
Ne ga tiv e R isk F ac to r
I nt er m ed ia te
6 0 or g re at er
5 9 - 35
m g/d L
De sir a ble
Bo rd e rlin e- Hig h
18 1
le ss tha n 2 00
m g/d L
HDL Chole sterol
Current NCEP Risk Categories
m g/d L
m g/d L
Diagnostic Trait s of Atherogenic Dyslipidemia
Large
HDL
-
37
Relative NMR frequency (Hz)
Perfil metabólico Plasma
P rod uced und er pa tent licenses
to U. S. P aten t Nos. 4,933 ,844
an d 5,34 3,389
Lipoprotein diameter (nm)
20 0 - 4 0 0
P osi tive Ri sk Fa ct or
les s t ha n 35
H igh
4 00 - 1 ,0 00
Determination of drug-toxicity (metabonomics)
Rat urines after
dosage of
liver- and
kidney-toxins
600 MHz-1H
noesypr1d
64scans
Sprague Dawley +
Han-Wistar
are standardised
laboratory rat strains
Hexachlorobutadiene
Thioacetamid
Hydrazin
Sprague Dawley
control
Han Wistar
control
Determination of drug-toxicity (metabonomics)
Coomans
Plot
showing
95%
confidence
level
of
classificlassification
normal rat
urines
versus
toxin treated
urines
Determination of drug-toxicity (metabonomics)
Classification of hydrazine induced hepatotoxicity against
renal cortical
toxicity and
controls
based on
rat urine
1D-NMR
spectra
Determination of drug-toxicity (metabonomics)
Time course
in rat urines
after dosage
of
a-naphtylisothiocyanat
(ANIT,
liver toxin)
96-120h
lactate
72-96 h
acetate
48-72 h
32-48 h
bile acids
24-32 h
glucose
8-24 h
0-8 h
control
Metabolomic of gene alterations from DNA
reparation
1D 1H spectra
comparison of
breast cell
cultures with
BRCA gene
blocked and not
blocked
The most
significant
changes in the
metabolite profile
are marked
Colaboration with
Dra. Armengod
FVIB
Cell Culture differences CONTROL/AICAR
Sample 124
Choline
Glucose,
amino acids
betaine
Lactate
Lactate
Creatine?
Lysina?
Asparagine?
Glucose,
amino acids
Lípidos
Colaboración
Prof. JM. Esplugues
F. Medicina
Alanine
Lípids
Lípids
Breast Cancer HR-MAS
Visualización Apoptosis RMN
a) Evolución temporal de 1H-ERM
de un glioma transfectado HSV-tk
tratado in vivo con glanciclovir
b) RMI convencional de un glioma no
tratado.
c) Imagen de microscopía electrónica de
transmisión (x5000) de células
tumorales no tratadas
d) Gotas de lípidos flechas vacías;
cuerpos de apoptosis triángulos negros
en células tumorales no tratadas
e) En tumores tratados (4 días) gotas
ricas en ácidos grasos poliinsaturados
señaladas mediante flechas abiertas.
Hakumäki y Brindle, Trends in Pharmac. Sci. ,24,3 (2003)
Visualización Apoptosis RMN
Imágenes de 1H RMI de un tumor de ratón tratado con glanciclovir posteriores a la
inyección del agente de contraste C2-SPIO. (a) imagen previa a contraste; (b), (c), (d),
(e) a 11, 47, 77 y 107 min después. Las áreas de apoptósis, captan contraste, áreas de
pérdida de intensidad (flecha blanca). Imágenes inferiores sustracciones.
Hakumäki y Brindle, Trends in Pharmac. Sci. ,24,3 (2003
CLAS
clasificación module
Metabonomic of Folicular Fluid
Proteins vs Metabolites
(0.1-3KDa)
Contribución Clínica y Biomédica RMN Alto Campo
•
•
•
•
Diagnóstico Clínico Biofluidos
Diagnóstico Clínico Tejidos
Estudios Biomédicos Líneas Celulares
Estudios Farmacocinéticos:
Tejidos
Biofluidos
• Desarrollo Biomarcadores:
Diagnóstico
Diseño Fármacos
• Mejora Compresión Bioquímica Patologías
NMR Applications
1H( MHz)
Magnetic
Field
Intensity (T)
Size
(cm)
9,6 - 100
0,25 - 3,0
< 100
d5
MRI +
41 - 300
MRS
(biomedicine)
1,0 - 7,1
< 30
d2
Miniimaging
300 - 400
7,1 - 9,4
< 12
# 0,005
mouses
NMR
Microscopy
200 - 600
4,7 - 14,1
< 2,5
0,00005
insects,
materials,
embrions
9,4 - 21
< 40 Å
Type
resonance
frequency
MRI +
MRS
(clínical)
NMR
400 - 900
multidimensional
B.Celda
Spatial
resolution
(mm3)
Application
System
human
body
small animals
(rabits, mouses…)
proteíns, DNA,
tissues ...
FIHCV-06
Aknowledgments
Hospital La Ribera-Alzira
Dr. J. Piquer
Dr. E. Mollá
Dr. A. Revert
Dr. P. Ferrer
IVO- Valencia
Dr. J. Cervera
Dr. A. Marhuenda
Dr. A Menendez
F. Medicina (UVEG)-Valencia
Prof. F. Pallardó
Prof. J.M. Rodrigo
Prof. M. Cerdá
Dr. M. Mata
Hospital Clínico-Valencia
Dr. J. Del Olmo
Dr. J. Talamantes
Fundación Investigación H.Clínico
Clínica Quirón
Dr. L. Martí-Bonmatí
Bruker Biospin Lab of Applications
Dr. M. Piotto
Dr. O. Assemat
B.Celda
eTUMOUR Consortium
Prof. C. Arús (UAB-Barcelona)
Dr.F. Howe (SGHMS-London)
Prof. A. Heerschap (UMCN-Nijmengen)
Prof. L. Buydens (KUN-Nijmengen)
Dr. C. Segerbart (INSERM-Grenoble)
Mr. M Lluch (Microart-Barcelona)
Dr. A. Capdevilla (HSJD-Barcelona)
Dr. G. D’Incerti (PQE-Milan)
Dr. L. Visani (PQE-Milan)
Prof. S. Van Huffel (KUL-Leuven)
Dr. P. Kreisler (Siemens-Etlinger)
Dr. A. Klaasen (SCITO-Paris)
Dr. M. Robles (UPVLC-Valencia)
Dr. S. Wolfhard (DKFZ-Heidelberg)
Dr. C. Brevard (Bruker-Biospin-Wissembourg)
Dr. R. Grundy (BU-Birmimghan)
Dr. F. Berger (INSERM-Grenoble)
Dr. J. Calvar (FLENI-Buenos Aires)
Dr. W. Gajewicz (MUL-Lodz)
UVEG-Valencia
Dr. V. Esteve; MB Martínez-Granados
E. Piñero; R. Ferre
IIIGERMN