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Aplicaciones terapéuticas de las células madre
Terapia Celular y Medicina Regenerativa
� Curar con células
� Regenerar órganos y funciones
Las células como fármaco
Clinical Trial Process
2001-2002Hay Células madre pluripotenciales en la médula ósea del individuo adulto
• Médula ósea• Sangre• Cornea• Retina• Cerebro• Músculo• Hígado• Piel• Páncreas• Tracto GI• Pulpa Dental
Ensayos clínicos en terapia celular (clinicaltrials.gov)
Ensayos clínicos en terapia celularFebrero 2008
Tabla: nº de EECC en terapias celulares a nivel mundial en la actualidad.Fuente: ClinicalTrials.gov
Febrero de 2008
18461846100100
0,16
99,84
PORCENTAJE (%)PORCENTAJE (%)
3?
2?
4
57
936
846
SUBTOTALSUBTOTAL
10381038
?
?
2
34
537
465
FASE IIFASE II
Músculo
Tejido adiposo
Cordón umbilical
Sangre periférica
Médula ósea
FUENTE DE FUENTE DE OBTENCIOBTENCIÓÓN N
CELULARCELULAR
266266539539TOTALTOTAL
??
419 1843
131250
CÉLULAS MADRE
ADULTAS
3??CÉLULAS MADRE EMBRIONARIAS
11
130269
TOTALTOTALFASE IIIFASE IIIFASE IFASE I
Ensayos clínicos con “stem-cells”
Mapa: nº de EECC en terapias celulares con células madre a nivel mundial en la actualidad. Fuente: ClinicalTrials.gov
Febrero de 2008
9
Cell Therapy clinical trials
Type of cells
• > 1700 clinical trials with hematopoietic stem cells
61
6
19
6
18
0
10
20
30
40
50
60
70
Mes
ench
yma
l
Cho
ndro
cyte
sFi
brob
last
s/Ke
rat
inocy
tes
Myo
blas
ts
Oth
er
• ~ 110 clinical trials with other cell types
Source: Clinical trials.gov. Companies web pages
Fuentes celulares de uso clínicoCélulas para curar
Fuentes celulares de utilidad clínicaCélulas para curar
� BIOPSIA MUSCULAR
Extracción de Células Madre Mesenquimales de la grasa (lipoaspirado)
� Realización por un Cirujano Plástico.
� Proceso con anestésico local. (mepivacaina al 1%)
� Mínima incisión (1 cm.)
� Introducción de una cánula perforada.
� Disrupción del tejido por movimientos mecánicos.
Procesado de la grasa
� Lavado con PBS (v/v)del lipoaspirado.� Agitación y centrifugación
� Eliminación de: células hemáticas, solución salina y anestésico local.
� Obtención de la “fracción vasculo estromal” (SVF)
Procesado de la grasa � Digestión de la matriz
extracelular con Colagenasa 0,075%
� Condiciones: 30 min a 37ºC
� Inactivación de la enzima con igual volumen de medio de cultivo.� Centrifugación: 10 min a 250 g.� Resultado : Precipitado de alta densidad proveniente de las células del lipoaspirado
Expansión celular I
Medio: - DMEM (Dulbecco modified Eagle medium)
- 10 % SFB ( Suero Fetal Bovino)- 1 % Antibiótico/antimicótico.
Condiciones de cultivo: 37ºC y 5% de CO2
Expansión celular II� A las 24 h. se lava el cultivo con PBS� Las células fijadas se mantienen en el
incubador en las mismas condiciones.
Expansión celular III
Entre 5-7 días se obtiene la confluencia celular
� Se disgregan las células incubándolas a 37ºC con tripsina.� Lavado celular con PBS.� Recogida de las células
� Proceso de criopreservación
� Best performance than Bone Marrow 1:100� BM stimulation is not required (G-CSF)� Low Cost� Easy to obtain
Ensayos clínicos con Terapia Celular
� IAM� Insuficiencia hepática aguda� Enfermedad del injerto contra el
huesped� Incontinencia urinaria� Incontinencia fecal� Úlceras de decúbito� Enfermedad inflamatoria
intestinal� Lesión medular� Pioderma gangrenoso� Reparación de defectos óseos� Fístulas bronquiales� Fístulas digestivas� Reparación corneal� Pié diabético� Arteriopatías
20
Cell Therapy clinical trials
Therapeutic areas (Non hematopoietic cells)
• Taking into account cells different from hematopoietic cells
Cardiology 35%
Orthopedics 13%Dermatology 17%
NSC 7%
Haematology 7%
Gastroenterology 6%
Endocrinology 4%
Oncology 3%
Aesthetics 2%
Odontology 1%
Neumology 1%Ophthalmology 1%
Nephrology 1%
Metabolic disorders 1%
Rheumatology 1%
Source: Clinical trials.gov. Companies web pages
Terapia celular en IAM.
Estenosis
coronariaCorazón
normal
Ulceras cutáneas
Transplante de células troncales límbicas
Progenitores en la insuficiencia hepática aguda
ULTRASOUND GUIDED INJECTION
depot of fibroblastsin submucosa
tip of needlein submucosa
depot of myoblastsin rhabdosphincter
tip of needlein rhabdosphincter
SUBMUCOSA RHABDOSPHINCTER
Incontinence score
0
1
2
3
4
5
6
pre post
QoL
22
42
62
82
102
pre post
Experiencias clínicas en terapia celular en elServicio de Cirugía General
Cicatrización/Reparación
1846-1982
…Es un proceso “celular”
+Células
pluripotenciales
2003 heart
2001 Grasa
1999 brain
1993 Muscle
Hematopoetic (bone marrow) 1986
1990 Intestine
1992Liver
1993Skin
1999Mesenchymal cells (bone marrow)
2001MAPC (bone marrow)
2003Pancreas
Adipose tissue
Adipose derived mesenchymal stem cells:
� Higher yield (between 100 and 1000 times higher yield than bone marrow)� BM stimulation is not required (G-CSF)� Expendable and accessible: Simple liposuction� Biosafety : No chromosomal alterations/ tumorigenic behavior after long term ex vivo
cultures� Wide range of potential applications
Stem Cells from Adipose TissueAdvantages
2002 2003 2004 2005 2006 2007 2008
1 case1 center
8 cases1 center
Clinical Development
50 cases3 centers
207 cases
10 centers
OngoingCompleted
Preclinical
Clinical Proof of Concept
Phase I
Phase II
Phase III
Successful PLA-based treatment of a young woman with a recurrent recto-vaginal fistula that had been unresponsive to medical treatment
2002 2003 2004 2005 2006 2007 2008
1 case1 center
8 cases1 center
Clinical Development
50 cases3 centers
207 cases
10 centers
OngoingCompleted
Preclinical
Clinical Proof of Concept
Phase I
Phase II
Phase III
Phase I clinical trial
TRIAL SUMMARY
Trial Location La Paz Hospital, Madrid
Start April 2002
Enrollment 5 patients (total of 8 fistulas)
DesignOpen Label; Feasibility / Safety
Study
Administration Intralesional use
DurationFirst evaluation of endpoint: 8
weeks
Controlled No
EndpointComplete closure/healing of the
fistula clinically assessed
Results 75% success
Fistula closure
Phase I Patients Treatments Rejection Complete Partial
n 5 8 0 6 2
2002 2003 2004 2005 2006 2007 2008
1 case1 center
8 cases1 center
Clinical Development
50 cases3 centers
207 cases
10 centers
OngoingCompleted
Preclinical
Clinical Proof of Concept
Phase I
Phase II
Phase III
� Directivas comunitarias (BOE 12 de diciembre de 2003) sobre medicamentos de terapia celular somática de origen humano
� Nuevas normas para ensayos clínicos en los que ya se contemplan los ensayos clínicos con medicamentos de terapia celular somática (BOE 7 de febrero de 2004)
� Real Decreto 176/2004 (B.O.E. 31/01/04) en el que se aprueba elEstatuto del Centro Nacional de Transplantes y Medicina Regenerativa
Células = Medicamento
¿Cómo lo hicimos?
� LA PAZ realiza y dirige el desarrollo clínico desde el inicio
� PROMOTORProductor celular
Phase II clinical trialGeneral considerations
� Multi-center:� Three major hospitals in Madrid, Spain
� Randomized� Randomization performed by an
independent organization
� Controlled� Control arm: fibrin glue as fistula tract
sealant (one of the elective procedures to avoid conventional surgery)
� Add-on trial� Treatment arm: Cx401administered
intralesionally and fibrin glue as tract sealant
� Open-label, primary endpoint evaluated by a blinded committee
� Committee formed by three surgeons experts in coloproctology not recruiting patients for the study
� Analyzed clinical and photographic data
Patient Selection:
� Older than 18 years
� Both sexes
� Complex perianal fistula pathology fulfilling some of the following conditions:
� Associated faecal incontinence
� Risk factors of anal incontinence
� At least 1 previous operation for a fistulous
disorder
� Rectovaginal fistula
� Crohn´s disease
Route of Administration:� Intralesional use:
� ½ in the fistula wall (*)� ½ mixed with the fibrin glue
(*) 50% of total cell dose placed in the intersphincteric tracts and adjacent to the internal opening; 50% in the tract walls in the direction of the external opening. Superficial injection (< 2mm)
Phase II clinical trialDesign
Experimental Treatment GroupCx401 + Fibrin glue
Control GroupFibrin glue
Randomise
25 patients 25 patients
50 patients
Primary Outcome
Secondary Outcome
24 patients (ITT) 25 patients (ITT)
Experimental Treatment GroupCx401 + Fibrin glue
Control GroupFibrin glue
� Cell inyection: ½ cell dose in the fistula wall
�½ cell dose mixed with fibrin glue
� Tract identification
� Curetage
� Internal opening closure
� Tract sealant
Primary endpoint� Proportion of patients whose fistula was healed at week 8 after last dose of study drug
� Definition of Healing: no suppuration + complete re-epithelization of the external fistula opening assessed by an independent evaluation committee
Secondary endpoints� Maintenance of healing at 12 months� Time to healing� Quality of Life evolution (SF-12 score)� Non serious and serious adverse events incidence
Phase II clinical trialVariables
EfficacyPrimary endpoint
TREATMENT Relative risk
Cx401 Fibrin glue CI (95%) p-value
Healing (all cases)
(N=24) (N=25) 4.43
17 (71%) 4 (16%) (1.74, 11.27)
p-value <0.001
Healing in non-Crohn’s population
(N=17) (N=18) 4.23
12 (71%) 3 (17%) (1.44, 12.44)
p-value = 0.0013
Healing in Crohn’s population 1
(N=7) (N=7) 5.00
5 (71%) 1 (14%) (0.77, 32.57)
p-value = 0.10
Healing in cases of suprasphincteric fistulous tract
(N=14) (N=16) 5.71
10 (71%) 2 (12%) (1.49, 21.78)
p-value = 0.001
1 No statistical significance recognized (total sample size of Crohn´s patients = 14)
Quality of LifeAcute phase
LSMEAN1
CI 95%Absolute difference
CI 95% P-value
Physical functions
Cx401
49.48
(46.42, 52.55) 6.25
n=22* p=0.0095
Fibrin glue 43.23 (1.62, 10.88)
(39.83, 46.63)
n=18*
Emotional functions
Cx401 53.21
(46.01, 52.74) 3.84
n=22* p=0.09
Fibrin glue 49.38 (-0.70, 8.38)
(46.01, 52.74)
n=18*
1 Least squares Means; final average, considering basal average
SafetyAcute phase
� Primary evaluation (eight weeks after last treatment) revealed 17 adverse events with Cx401 and 11 with fibrin glue
� Only 2 serious adverse events (SAEs) were observed with fibrin glue and 2 with Cx401
� Not a single SAEs was related to Cx401
Group Crohn ´s disease Description Severity Results Causality
Fibrin glue Yes Crohn´s crisis and intrabdominal abscess
Yes In recovery Not related
Cx401 No Perianal abscess Yes Recovered Not related
Cx401 No Cholecystitis and cholelithiasis. Choledocholothiasis after cholecystomy
Yes In recovery Not related
Fibrin glue Yes Perianal abscess Yes Recovered Related
El tratamiento con células madre mesenquimales es seguro y eficaz en pacientes con fistulas perianales complejas.
2002 2003 2004 2005 2006 2007 2008
1 case1 center
8 cases1 center
Clinical Development
50 cases3 centers
207 cases10 centers
OngoingCompleted
Preclinical
Clinical Proof of Concept
Phase I
Phase II
Phase III
Phase III clinical trial� FATT I (Fistula Advanced Therapy Trial I)
� No Crohn fistula-in-ano
� FATT II (Fistula Advanced Therapy Trial II)� Crohn fistula-in-ano
Starting: March 2007Participants : Spain, UK, Germany, Holland etc.Leader : La Paz University Hospital
Recruitment finished
2002 2003 2004 2005 2006 2007 2008
1 case1 center
8 cases1 center
Clinical Development
50 cases3 centers
207 cases
10 centers
OngoingCompleted
Preclinical
Clinical Proof of Concept
Phase I
Phase II
Phase III
¿Cómo funcionan?
� Las células troncales podrían diferenciarse en células de la reparación y mejorar las condiciones locales
� …O bien secretar factores de crecimiento y señalizadores que podrían colaborar con eficacia en los procesos locales de cicatrización
� …O bien…
Jerónimo Blanco FernándezCentro de Investigación Cardiovascular (CSIC-ICCC)Hospital Sta. Cruz y San PabloBarcelona
Cx401 Mechanism of action � Cx401 is activated in an inflamed
environment
� Activated Cx401 suppresses the proliferation of lymphocytes and suppress the inflammation
� Local treatment of inflammatory diseases with tissue damage/ wounds: Cx401 acts at the source of the inflammation and establish an environment that will permit a healing
� Systemic treatment of diseases with acute inflammatory component: Cx401 migrates to the inflammatory environment and suppresses inflammation, avoiding tissue damage
Los retos
� Células con calidad clínica
� ¿Criopreservación?� ¿Autólogas?� ¿Alogénicas?
In vitro culture
Cell expansion
Cryopreservation
ASC Obtention
ASC manipulation
Seeding in abio-compatible scaffold
ASC Implant
Liposuction isolation
Master CellBank
Muchas gracias!