Aplicaciones terapéuticas de las células madre

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

Médula ósea Sangre Cornea Retina Cerebro Músculo Hígado Piel Páncreas Tracto GI Pulpa Dental 2001-2002 Hay Células madre pluripotenciales en la médula ósea del individuo adulto

Ensayos clínicos en terapia celular (clinicaltrials.gov)

Ensayos clínicos en terapia celular Febrero 2008 CÉLULAS MADRE ADULTAS Tabla: nº de EECC en terapias celulares a nivel mundial en la actualidad. Fuente: ClinicalTrials.gov Febrero de 2008 FUENTE DE OBTENCIÓN CELULAR Médula ósea Sangre periférica Cordón umbilical Tejido adiposo Músculo CÉLULAS MADRE EMBRIONARIAS TOTAL FASE I 250 269 FASE II 465 537 SUBTOTAL 846 936 19 34 4 57 1843 1?? 539 2?? 1038 FASE III 131 130 1?? 266 4 2? 3? TOTAL 3 PORCENTAJE (%) 99,84 0,16 100 1846

Ensayos clínicos con stemcells Mapa: nº de EECC en terapias celulares con células madre a nivel mundial en la actualidad. Fuente: ClinicalTrials.gov Febrero de 2008

Cell Therapy clinical trials Type of cells > 1700 clinical trials with hematopoietic stem cells ~ 110 clinical trials with other cell types 70 60 61 50 40 30 20 19 18 10 6 6 9 0 Mesenchymal Chondrocytes Fibroblasts/Keratinocytes Myoblasts Other Source: Clinical trials.gov. Companies web pages

Fuentes celulares de uso clínico Células para curar

Fuentes celulares de utilidad clínica Células 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

IAM Ensayos clínicos con Terapia Celular 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

Cell Therapy clinical trials Therapeutic areas (Non hematopoietic cells) Taking into account cells different from hematopoietic cells Metabolic disorders 1% Odontology 1% Aes thetics 2% Oncology 3% Neum ology 1% Ophthalm ology 1% Ne phrology 1% Rheum atology 1% Endocrinology 4% Gas troenterology 6% Cardiology 35% Haem atology 7% NSC 7% 20 Derm atology 17% Orthopedics 13% Source: Clinical trials.gov. Companies web pages

Terapia celular en IAM. Corazón normal Estenosis coronaria

Ulceras cutáneas

Transplante de células troncales límbicas

Progenitores en la insuficiencia hepática aguda

ULTRASOUND GUIDED INJECTION SUBMUCOSA RHABDOSPHINCTER tip of needle in submucosa depot of fibroblasts in submucosa tip of needle in rhabdosphincter depot of myoblasts in rhabdosphincter

Incontinence score 6 5 4 3 2 1 0 pre post QoL 102 82 62 42 22 pre post

Experiencias clínicas en terapia celular en el Servicio de Cirugía General

1846-1982 Cicatrización/Reparación

Es un proceso celular + Células pluripotenciales

Stem Cells from Adipose Tissue Advantages Hematopoetic (bone marrow) 1986 1990 Intestine Liver Skin 1992 1993 1993 Muscle Mesenchymal cells (bone marrow) 1999 1999 brain MAPC (bone marrow) 2001 2001 Adipose Grasa tissue Pancreas 2003 Adipose derived mesenchymal stem cells: 2003 heart 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

Clinical Development 2002 2003 2004 2005 2006 2007 2008 Preclinical Clinical Proof of Concept 1 case 1 center Phase I 8 cases 1 center Phase II 50 cases 3 centers Phase III 207 cases 10 centers Completed Ongoing

Successful PLA-based treatment of a young woman with a recurrent recto-vaginal fistula that had been unresponsive to medical treatment

Clinical Development 2002 2003 2004 2005 2006 2007 2008 Preclinical Clinical Proof of Concept 1 case 1 center Phase I 8 cases 1 center Phase II 50 cases 3 centers Phase III 207 cases 10 centers Completed Ongoing

Phase I clinical trial Trial Location TRIAL SUMMARY La Paz Hospital, Madrid Fistula closure Phase I Patients Treatments Rejection Complete Partial Start April 2002 Enrollment 5 patients (total of 8 fistulas) n 5 8 0 6 2 Design Administration Duration Controlled Endpoint Results Open Label; Feasibility / Safety Study Intralesional use First evaluation of endpoint: 8 weeks No Complete closure/healing of the fistula clinically assessed 75% success

Clinical Development 2002 2003 2004 2005 2006 2007 2008 Preclinical Clinical Proof of Concept 1 case 1 center Phase I 8 cases 1 center Phase II 50 cases 3 centers Phase III 207 cases 10 centers Completed Ongoing

Células = Medicamento 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 el Estatuto del Centro Nacional de Transplantes y Medicina Regenerativa

Cómo lo hicimos? LA PAZ realiza y dirige el desarrollo clínico desde el inicio PROMOTOR Productor celular

Multi-center: Phase II clinical trial General considerations Three major hospitals in Madrid, Spain Patient Selection: Older than 18 years Randomized Controlled Add-on trial Randomization performed by an independent organization Control arm: fibrin glue as fistula tract sealant (one of the elective procedures to avoid conventional surgery) 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 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 trial Design 50 patients Experimental Treatment Group Cx401 + Fibrin glue Control Group Fibrin glue Randomise Tract identification Experimental Treatment Group Cx401 + Fibrin glue 25 patients 25 patients Control Group Fibrin glue Cell inyection: ½ cell dose in the fistula wall ½ cell dose mixed with fibrin glue Curetage 24 patients (ITT) 25 patients (ITT) Internal opening closure Primary Outcome Secondary Outcome Tract sealant

Phase II clinical trial Variables 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

Efficacy Primary endpoint Healing (all cases) Healing in non-crohn s population Cx401 TREATMENT Fibrin glue Relative risk CI (95%) p-value (N=24) (N=25) 4.43 17 (71%) 4 (16%) (1.74, 11.27) p-value <0.001 (N=17) (N=18) 4.23 12 (71%) 3 (17%) (1.44, 12.44) p-value = 0.0013 Healing in Crohn s population 1 5 (71%) 1 (14%) (0.77, 32.57) (N=7) (N=7) 5.00 Healing in cases of suprasphincteric fistulous tract p-value = 0.10 (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 Life Acute phase LSMEAN 1 CI 95% Absolute difference CI 95% P-value Physical functions 49.48 (46.42, 52.55) 6.25 Cx401 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

Safety Acute 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.

Clinical Development 2002 2003 2004 2005 2006 2007 2008 Preclinical Clinical Proof of Concept 1 case 1 center Phase I 8 cases 1 center Phase II 50 cases 3 centers Phase III 207 cases 10 centers Completed Ongoing

Phase III clinical trial FATT I (Fistula Advanced Therapy Trial I) No Crohn fistula-in-ano Recruitment finished FATT II (Fistula Advanced Therapy Trial II) Crohn fistula-in-ano Starting: March 2007 Participants: Spain, UK, Germany, Holland etc. Leader: La Paz University Hospital

Clinical Development 2002 2003 2004 2005 2006 2007 2008 Preclinical Clinical Proof of Concept 1 case 1 center Phase I 8 cases 1 center Phase II 50 cases 3 centers Phase III 207 cases 10 centers Completed Ongoing

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 Fernández Centro de Investigación Cardiovascular (CSIC-ICCC) Hospital Sta. Cruz y San Pablo Barcelona

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 ASC Obtention ASC manipulation Liposuction isolation In vitro culture Células con calidad clínica ASC Implant Seeding in a bio-compatible scaffold Cryopreservation Master Cell Bank Criopreservación? Cell expansion Autólogas? Alogénicas?

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