7
• Perianal discharge • Pain • Swelling • Bleeding • Diarrhoea • Skin excoriation • External opening
Looking for a Testing Bench Fistula in Crohn’s Disease: a real problem of wound healing
8
Looking for the best cells source Stem Cells from 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
2003 heart
2001 Grasa
1999 brain
1993 Muscle
Hematopoetic (bone marrow) 1986
1990 Intestine
1992 Liver
1993 Skin
1999 Mesenchymal cells (bone marrow)
2001 MAPC (bone marrow)
2003 Pancreas
Adipose tissue
9
2002 2003 2004 2005 2006 2007 2008 2009 2010
1 case 1 center
8 cases 1 center
ASC Clinical Development in fistula
50 cases 3 centers
Preclinical
Proof of Concept
Phase I
Phase II
Phase III 207 cases
10 centers FATT1
210 cases
22 centers FATT2
24 cases
3 centers CX601
10 cases
1 center FIS NC
Ongoing Completed
Autologous Allogenic
10
2002 2003 2004 2005 2006 2007 2008 2009 2010
1 case 1 center
8 cases 1 center
ASC Clinical Development in fistula
50 cases 3 centers
Preclinical
Proof of Concept
Phase I
Phase II
Phase III
207 cases
10 centers FATT1
210 cases
22 centers FATT2
24 cases
3 centers CX601
10 cases
1 center FIS NC
Ongoing Completed
Autologous Allogenic
11
Clinical Proof of Concept Successful cell treatment of a young woman with a recurrent
recto-vaginal Crohn’s fistula unresponsive to medical treatment
12
2002 2003 2004 2005 2006 2007 2008 2009 2010
1 case 1 center
8 cases 1 center
ASC Clinical Development in fistula
50 cases 3 centers
Preclinical
Proof of Concept
Phase I
Phase II
Phase III
207 cases
10 centers FATT1
210 cases
22 centers FATT2
24 cases
3 centers CX601
10 cases
1 center FIS NC
Ongoing Completed
Autologous Allogenic
13
Phase I clinical trial
TRIAL SUMMARY
Trial Location La Paz Hospital, Madrid
Start April 2002
Enrollment 5 patients (total of 8 fistulas)
Design Open Label; Feasibility / Safety Study
Administration Intralesional use
Duration First evaluation of endpoint: 8 weeks
Controlled No
Endpoint Complete 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
NO SEVERE ADVERSE EVENTS NO TUMOROGENIC EVENTS Garcia Olmo 2011
14
2002 2003 2004 2005 2006 2007 2008 2009 2010
1 case 1 center
8 cases 1 center
ASC Clinical Development in fistula
50 cases 3 centers
Preclinical
Proof of Concept
Phase I
Phase II
Phase III
207 cases
10 centers FATT1
210 cases
22 centers FATT2
24 cases
3 centers CX601
10 cases
1 center FIS NC
Ongoing Completed
Autologous Allogenic
15
Injection of ADSC cell suspension
Cell suspension is injected in the internal opening (the syringe enters through the anus) and through the fistula tract walls (the syringe enters
trough the external opening of the fistula)
Needle
Needle
ADSC
(*) 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)
Garcia Olmo 2011
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 16
Administration of Adipose Derived Stem Cells was effective in inducing healing in patients with complex
fistula-in-ano including Crohn’s disease, and this procedure can be considered safe
18
2002 2003 2004 2005 2006 2007 2008 2009 2010
1 case 1 center
8 cases 1 center
ASC Clinical Development in fistula
50 cases 3 centers
Preclinical
Proof of Concept
Phase I
Phase II
Phase III 207 cases
10 centers FATT1
210 cases
22 centers FATT2
24 cases
3 centers CX601
10 cases
1 center FIS NC
Ongoing Completed
Autologous Allogenic
19
Phase III (Autologuous ASCs):
Crohn: FATT I TRIAL DESIGN
Condition Fistula in non-Crohn´s patients
Study design ● Randomised ● Double blind ● Parallel Assignment
Recruitment: Completed: 200 patients Dosed: 135 patients
Controlled Yes (fibrin and placebo)
# of centres 23 centres in Spain
Primary endpoint
Complete healing assessed clinically and through MRI
Secondary endpoint
Long term healing, Quality of Life parameters, surgeries avoided, etc
Results Release anticipated 1 Q 2010
Non-Crohn:
Garcia Olmo 2011
Safety
Treatment received group ASCs (N=64)
ASCs+Fibrin tissue adhesive
(N=59) Fibrin tissue
adhesive (N=60)
Total (N=183)
N
% N
% N
% N
% p-value, general
association
p-value ASCs vs Fibrin tissue
adhesive
p-value ASCs and Fibrin tissue
adhesive vs Fibrin tissue adhesive
p-value ASCs vs ASCs
and Fibrin tissue adhesive
Related to study treatment
4
6.3 4
6.8 6
10.00 14
7.7 0.7026
0.4473 0.5291
0.9053
Related to study procedures
28
43.8 24
40.7 25
41.7 77
42.1 0.9397
0.8147 0.9128
0.7305
Adverse events related to either study procedures or treatment*
Overall, there were no significant differences among the three treatment groups in the proportion and nature of both non-serious and serious adverse events. In
addition, all treatments were safe as they had a low proportion adverse events either related to study treatment or related to study procedures, and a few severe/life-
threatening adverse events. In conclusion, the results of this trial indicate that the use of ASCs alone or with fibrin tissue adhesive does not increase the risk for the
patients at least during the period covered in this phase III clinical trial.
Garcia Olmo 2011
FATT1 Non-Crohn Population
Efficacy 24 weeks COMMITTEE All cases
0
10
20
30
40
50
A (Cells Alone) B (Cells+FibrinGlue) C (FibrinGlue)
No statistical significance
Garcia Olmo 2011
Looking for subgroups
• Male/Female • Age • Physical evaluation (Drainage, pain, excoriation,
swelling, bleeding, Num. external openings, • Previous surgery for fistulous disease • Previously diagnosed perianal abcesses • Incontinence (Wexner score) • Complexity score • Etc…
No statistical significance
Garcia Olmo 2011
Efficacy by centre
-0,6
-11,5
0,38
N % N % N % N %
CenterYesSubTotal 3 100.00 5 100.00 3 100.00 11 6.01
Yes 1 100.00 . . . . 1 100.00
SubTotal 1 100.00 . . . . 1 0.55
Yes
1 33.33 1 25.00 . . 2 20.00
SubTotal 3 100.00 4 100.00 3 100.00 10 5.46
Yes
. . . . 1 33.33 1 20.00
SubTotal . . 2 100.00 3 100.00 5 2.73
Yes 4 50.00 4 57.14 3 50.00 11 52.38
SubTotal 8 100.00 7 100.00 6 100.00 21 11.48
Yes 3 42.86 1 16.67 2 40.00 6 33.33
SubTotal 7 100.00 6 100.00 5 100.00 18 9.84
Yes 1 50.00 1 50.00 1 50.00 3 50.00
SubTotal 2 100.00 2 100.00 2 100.00 6 3.28
Yes 2 66.67 2 50.00 3 100.00 7 70.00
SubTotal 3 100.00 4 100.00 3 100.00 10 5.46
No data 1 11.11 . . . . 1 3.85
Yes 1 11.11 2 25.00 3 33.33 6 23.08
SubTotal 9 100.00 8 100.00 9 100.00 26 14.21
Yes 3 42.86 2 33.33 . . 5 26.32
SubTotal 7 100.00 6 100.00 6 100.00 19 10.38
Yes . . . . 1 100.00 1 100.00
SubTotal . . . . 1 100.00 1 0.55
Yes . . . . 1 50.00 1 25.00
SubTotal 2 100.00 . . 2 100.00 4 2.19
No1 100.00 1 100.00 2 100.00 4 100.00
SubTotal 1 100.00 1 100.00 2 100.00 4 2.19
Yes 6 54.55 10 83.33 2 18.18 18 52.94
SubTotal 11 100.00 12 100.00 11 100.00 34 18.58
Yes 1 100.00 . . 1 100.00 2 100.00
SubTotal 1 100.00 . . 1 100.00 2 1.09
Yes 1 25.00 . . 1 100.00 2 33.33
SubTotal 4 100.00 1 100.00 1 100.00 6 3.28
No 1 100.00 . . . . 1 100.00
SubTotal 1 100.00 . . . . 1 0.55
Yes . . . . 1 100.00 1 25.00
SubTotal 1 100.00 2 100.00 1 100.00 4 2.19
Hosp. Virgen del Camino
Hosp. de Fuenlabrada
Hosp. Univ. Girona Josep Trueta (ICO Girona)
Hosp. Univ. La Paz
Hosp. Univ. Tarragona Joan XXIII
Hosp. Univ. de Salamanca
Hosp. Gregorio Marañón
Hosp. Mutua de Terrassa
Hosp. Ntra. Sra. de Valme
Hosp. Sagunto
Hosp. Clínico San Carlos
Hosp. Doce de Octubre
Hosp. Donostia
Hosp. Gral Univ. de Valencia
Clínica U. de Navarra
Corporación Sanitaria Parc Taulí
Hosp. Clinic i Provincial de Barcelona
Hosp. Clinico Univ. Lozano Blesa
2 66.67 6 54.551 33.33 3 60.00
Fistula closed? Investigator criteria- V4
Treatment
TotalA B C
Garcia Olmo 2011
Efficacy 24 weeks COMITTEE
0102030405060708090
A (Cells Alone) B (Cells+FibrinGlue) C (FibrinGlue)
All CasesLa PazOthers
Why? P<0.0001
Garcia Olmo 2011
Looking for diferences
• Male/Female • Age • Physical evaluation (Drainage, pain, excoriation,
swelling, bleeding, Num. external openings, • Previous surgery for fistulous disease • Previously diagnosed perianal abcesses • Incontinence (Wexner score) • Complexity score • Etc…
Garcia Olmo 2011
Patient distribution by “Complexity Score” and center (Median)
4
5
6
7
8
9
La Paz Others
P<0.001
Garcia Olmo 2011
Efficacy 24 weeks COMITTEE
0102030405060708090
A (Cells Alone) B (Cells+FibrinGlue) C (FibrinGlue)
Toda La SerieLa PazOtros
Why?
Garcia Olmo 2011
Technical survey
We detect a large numbers of mistakes during the surgical procedure in “others hospitals”: use of
hydrogen peroxide (H2O2), cell shake, cells spilling..
Garcia Olmo 2011
What have we learned?
Clinical Trials Peculiarities in Cell Therapy
• Surgical procedure for Cell delivering : • Enviroment • Aggressiveness • Scrub • Needle • …
Garcia Olmo 2011
• This is a “Living medicament” and hence a careful management is a key point.
What have we learned?
Clinical Trials Peculiarities in Cell Therapy
Garcia Olmo 2011
31
2002 2003 2004 2005 2006 2007 2008 2009 2010
1 case 1 center
8 cases 1 center
ASC Clinical Development in fistula
50 cases 3 centers
Preclinical
Proof of Concept
Phase I
Phase II
Phase III 207 cases
10 centers FATT1
210 cases
22 centers FATT2
24 cases
3 centers CX601
10 cases
1 center FIS NC
Ongoing Completed
Autologous Allogenic X
32
2002 2003 2004 2005 2006 2007 2008 2009 2010
1 case 1 center
8 cases 1 center
ASC Clinical Development in fistula
50 cases 3 centers
Preclinical
Proof of Concept
Phase I
Phase II
Phase III 207 cases
10 centers FATT1
210 cases
22 centers FATT2
24 cases
3 centers CX601
10 cases
1 center FIS NC
Ongoing Completed
Autologous Allogenic X
Next step: Alogenic ASCs
• 2 PHASE I/IIb Allogenic • Crohn recto-vaginal fistula • Crohn perianal fistula
Starting: December 2009 Results: December 2011
CX601
How does it work? • An inmunomodulator effect of adipose derived stem cells has been recently described. • These immunosuppressive properties are the same as MSCs (mesenchymal stem cells derived from bone marrow)
Inflammation
Infiltration oflymphocytes (PBLs)in wound areaSecretion of pro-inflammatorycytokinesSensation of pain
A B CDelivery of eASCs
Activation of eASC by a cytokine called IFN-γExpression of an enzyme called IDO by eASCsSuppression of the proliferation of activatedPBLsSuppression of production of inflammatorysignals
Healing
Elimination of activatedPBLsAbrogation of pro-inflammatory cytokinesCessation of painRepair of tissue
Garcia Olmo 2011
Mechanism of Action of ASCs
● ASCs are activated in an inflamed environment
● Activated ASCs suppress the proliferation of lymphocytes and suppress the inflammation
● Local treatment of inflammatory diseases with tissue damage/ wounds: ASCs act at the source of the inflammation and establish an environment that will permit a healing
● Systemic treatment of diseases with acute inflammatory component: ASCs migrate to the inflammatory environment and suppress inflammation, avoiding tissue damage
36 Garcia Olmo 2011
Indoleamine 2,3 dioxygenase activity: a Trp catabolizing enzyme
37
% o
f PB
MC
inhi
bitio
n
eASC
-em
pty
eASC
-IDO
si
1:25 (hASC: PBMC)
0
25
50
75
100
*
IDO enzyme is involved in the antiproliferative property of eASCs
IDO synthesis
IFN-γ eASC
cytoplasm
Trp IDO
Kyn
Trp
Kyn
eASC
cytoplasm
HAA
( DelaRosa, Lombardo, Tissue Engineering 2009)
Where do we go?
• Safety Profile Excellent
§ DOSE INCREASE
• Cell Mortality
§ IMPROVING METHODS FOR DELIVERING
• Soft effects
§ CELL INGENIERING
§ CELL IMPROVING WITH STIMULATING FACTORS (BMPs,…)
40
Matrices, andamiajes,…
§ Hidrogeles de polifosfoester (Quiang Li. Biomaterials. 2006).
§ Microesferas (Sang-Soo Kim, et. al. Apli Biomaterials. 2005)
§ Coágulos de fibrina y trombina (Steve Cox, et. al. Tissue Engineering. 2004).
§ Silastic, condroitin 6 sulfato, colageno (piel
artificial) (M. Kremer, E, et. al. B J Plastic Surg 2000)