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!