Download Endotoxin testing

Design of the
trial,
a phase II trial testing bone marrow
mononuclear cells in patients
with acute myocardial infarction
Disclosures
E. Marc Jolicoeur
reports no disclosures
Cranach’s Fontain of Youth (1546)
Staatliche Museen, Berlin
Pathological study comparing 27 hearts of patients
undergoing cardiac transplantation.
Mitotic figures
A proposed experiment
Bone marrow
mononuclear cells
isolated by Ficoll
gradient
Adapted from Dimmeler et al. JCI 2005;115:572
Cellular Therapy
The paracrine cytokines release by BMMNC is
postulated to induce neovascularization and
support cardioprotection
Adapted from Dimmeler et al. JCI 2005;115:572
Medical Therapy Has
Reached a Plateau
One out of four patient will develop an adverse outcome
in the year following their heart attack
Pfefer
Pfefer et
et al.
al. NEJM
NEJM 2003;349:20
2003;349:20
BMMNC trials
BMMNC – Meta-analysis
# cells
(106)
Age
(y)
Anterior
AMI
Time
to PCI
Days to
therapy
Strauer et al
50
38 %
12h
8d
46
Burtanek et al
54
94 %
10h
12 d
--
Jannsens et al
57
63 %
4h
1d
304
BOOST
56
77 %
9h
5d
2,460
Zhan-Quan et al
60
60 %
24h
6d
73
MAGIC 3
60
54 %
9h
4d
1,500
TCT-STAMI
58
70 %
8h
1d
39
ASTAMI
57
100 %
4h
6d
68
REPAIR-AMI
56
78 %
7h
4d
236
Meluzin et al
55
85 %
8h
7d
55
Study
Lipinski, JACC 2007;50:1761
Ejection fraction change
EF change % (SE)
ASTAMI
Burtanek et al
BOOST
Jannsens et al
MAGIC 3
Meluzin et al
REPAIR-AMI
Strauer et al.
TCT-STAMI
Zhan-Quan et al
- 1.4 (0.72)
- 3.1 (3.10)
- 2.8 (1.12)
- 1.1 (0.79)
- 5.2 (1.01)
- 2.0 (0.49)
- 2.5 (0.54)
- 1.0 (1.56)
- 6.7 (1.63)
- 5.5 (0.85)
Total (95% CI)
Test for heterogeneity Chi2 = 33.62, df=9, (p=0.0001), I2 = 73.2%
Test for overall effect: Z = 5.35 (p < 0.00001)
+ 3.0% [1.9% to 4.1%]
-10 -5
Favors cell therapy
0
5
10
Favors control
Lipinski, JACC 2007;50:1761
Clinical Events - Longest Follow-Up
Events Odds Ratio
OR = 0.52
1.5
OR = 0.32
OR = 0.22
1.0
0.5
58
58%
Control
patients
Death
Reinfarction
Heart Failure
Cell therapy patients
Lipinski, JACC 2007;50:1761
Controls
BMMNC
LVESV
-7.4 ml [-12.2 to -2.7]
Infarct size
-5.6% [-8.7% to -2.5%]
P= 0.002
REPAIR-AMI – 1 year
Cell therapy
Placebo
EHJ 2006;27:2775
The larger the better?
Absolute Change in Global LVEF (%)
p = 0.002
p = 0.81
20
10
0
-10
Placebo
Cell therapy
P interaction = 0.02
-20
No. of patients
52
41
Baseline LVEF Below Median
(≤ 49%)
40
54
Baseline LVEF Above Median
(>
> 49%)
NEJM 2006;355:1210
PRINCIPAL
PRINCIPAL INVESTIGATORS
INVESTIGATORS
Christopher
Christopher B
B Granger,
Granger, MD
MD
Nelson
Nelson Chao,
Chao, MD
MD
Co-PRINCIPAL
Co-PRINCIPAL INVESTIGATORS
INVESTIGATORS
E.
E. Marc
Marc Jolicoeur,
Jolicoeur, MD
MD MSc
MSc
Thomas
Thomas J.
J. Povsic,
Povsic, MD
MD PhD
PhD
James
James Mills,
Mills, MD
MD
STUDY
STUDY COORDINATOR
COORDINATOR
Bernadette
Bernadette Druken
Druken
REGULATORY
REGULATORY ADVISORS
ADVISORS
Clare
Clare A
A Matti
Matti
Rosemary
Rosemary Beci
Beci
Rebecca
Rebecca Haley,
Haley, MD
MD DTRI
DTRI
Phase
Phase II,
II, Randomized,
Randomized,
Placebo-Controlled,
Placebo-Controlled,
Double-Blinded
Double-Blinded Trial
Trial
Testing
Testing the
the Efficacy
Efficacy // Safety
Safety
Autologous
Autologous Bone
Bone Marrow
Marrow
Mononuclear
Mononuclear Cell
Cell Transfer
Transfer
for
for Myocardial
Myocardial Salvage
Salvage in
in Acute
Acute
Myocardial
Myocardial Infarction
Infarction
Bone Marrow mononuclear cells
Nature Review Cancer
Circulation
Circulation 2003;107:2294
2003;107:2294
ST
-Segment myocardial
ST-Segment
myocardial infarction
infarction
-- ≥≥ 2mm
2mm in
in ≥≥ 22 precordial
precordial leads
leads
-- ≥≥ 1mm
1mm in
in ≥≥ 22 limb
limb leads
leads
-- New
New LBB
LBB
Intracoronary
BM-MNC
Intracoronary
BM-MNC
Symptoms
8
5.0
5.0±±11xx10
108cells
cells
Successful
Successful stent
stent placement
placement with
with
onset to
-- patent
patent infarct
infarct artery
artery
enrollment
-- ≥≥ 22 mm
mm in
in diameter
diameter
Intracoronary
placebo
Intracoronary
placebo
≤
120
hours
-- TIMI
flow
grade
2
or
3
TIMI flow grade 2 or 3
LVEF
LVEF ≤≤ 40%
40%
by
by echo
echo
Infarct
Infarct size
size >> 15%
15%
MRI to
therapy
2-4
days
of
of left
left ventricle
ventricle (MRI)
(MRI)
Primary
Primary objective:
objective:
Determine
Determine whether
whether IC
IC delivery
delivery of
of autologous
autologous BM
BM
MNC
MNC to
to patients
patients with
with large
large acute
acute MI
MI improves
improves LV
LV
function,
function, as
as measured
measured by
by MRI
MRI ventriculography
ventriculography
Primary
Primary efficacy
efficacy endpoint:
endpoint:
Variation
Variation of
of LVEF
LVEF change
change from
from baseline
baseline to
to day
day
90
90 in
in BM
BM MNC
MNC vs
vs placebo
placebo treated
treated patients
patients
Safety
Safety endpoints:
endpoints:
1.
1. Death,
Death, reinfarction,
reinfarction, rehospitalization,
rehospitalization,
recurrent
recurrent angina
angina
2.
2. Procedural
Procedural complications
complications
3.
3. Serious
Serious ventricular
ventricular arrhythmias
arrhythmias
Follow
-up at
Follow-up
at 365
365 days,
days, (requested
(requested FDA)
FDA)
and
and
1.
1. Planned
Planned bypass
bypass surgery
surgery or
or
prior
prior CABG
CABG
2.
-vessel PCI
2. Multi
Multi-vessel
PCI
3.
3. Prior
Prior myocardial
myocardial infarction
infarction
4.
4. Active
Active cardiogenic
cardiogenic shock
shock
5.
5. Successful
Successful reperfusion
reperfusion << 33
hrs
hrs from
from symptom
symptom onset
onset
6.
6. Significant
Significant chronic
chronic heart
heart
failure
failure
7.
7. Contraindication
Contraindication to
to MRI
MRI
8.
8. Significant
Significant hepatic
hepatic
dysfunction
dysfunction or
or renal
renal
insufficiency
insufficiency
9.
9. Pregnancy,
Pregnancy, lactation
lactation within
within
the
the past
past 30
30 days
days
10.
10. Active
Active or
or planned
planned treatment
treatment
with
with chemotherapy
chemotherapy
11.
11. Evidence
Evidence of
of aa serious,
serious,
active
active infection
infection
12.
12. Any
Any known
known severe
severe
hematological
hematological disease,
disease,
malignancy,
malignancy, systemic
systemic or
or
life
life threatening
threatening disorder
disorder
Bone Marrow Processing
How you prepare cells matters
- Absolute improvement in LVEFREPAIR
AMI
REPAIR
AMI
trialtrial
10%
25.5 + p=0.01
13
8%
99 + 0
6%
6.8 + 4.8
4%
2% 5270 + 3919
230 + 123
ASTAMI
trial
ASTAMI
trial
p=0.77
10%
BMC recovery
8%
viability
6%
34+
CD cells
4%
CFU2%
3891 + 2425
MSC
161 + 160
19.1 + 7.6
99 + 0
4.4 + 3.6
1244
+ 1307Placebo
Basal invasion Cell448 +Placebo
384
Cell
NEJM 2006;355:1199
NEJM 2006;355:1210
*All comparison < 0.05
European
European Heart
Heart Journal
Journal 2007;28:766
2007;28:766
Automated Density Gradient Separation
1 h 15
+
Chamber
Bone
filling
Marrow
+
Sedimentation
Plasma
Density Gradient extraction
Washing
medium
Buffer
GMP-compliant
processing of small
volume bone marrow
samples for tissue
repair
Buffy coat
extraction
RBC
BMMNC
extraction
• Fully automated
• Closed environment
• High CD34 recovery
Adapted
Adapted from
from Atkas
Atkas et
et al.
al. Cytotherapy;10:203
Cytotherapy;10:203
Atkas
Atkas et
et al.
al. Cytotherapy
Cytotherapy 2008;10:203
2008;10:203
Validation of SePAX (Biosafe)
Automated system at Duke
Cell count (108 cells)
6
4
2
5.2
2.1
Manual
CD34+ cell (%)
0.86
0.8% 0.69
0.4%
SePAX
Viability (%)
100%
91%
97%
50%
Manual
SePAX
Manual
SePAX
Manual Ficoll vs automated Ficoll SePAX (Biosafe)
Validation performed on umbilical cord blood cells
(n = 3, p = NS)
Data
Data provided
provided by
by Dr
Dr Johan
Johan Kurtzberg
Kurtzberg
Timeline: Manual vs Automated Ficoll
7:00
7:00
7:00
7:00
9:00
9:00
9:00
9:00
Bone
Bone marrow
marrow
harvest
harvest
Bone
Bone marrow
marrow to
to
laboratory
laboratory
Manual
Manual Ficoll
Ficoll
separation
separation
Transplantation
Transplantation
to
to patient
patient
Automated
Automated
Ficoll
Ficoll
separation
separation
11:00
11:00
11:00
11:00
Stat
Stat Gram
Gram staining
staining
++ Endotoxin
Endotoxin testing
testing
Patient
Patient arrival
arrival
to
to cath
cath lab
lab
Transplantation
Transplantation
Stat
Stat Gram
Gram staining
staining Cells released for
to
to patient
patient
Cells released for
++ Endotoxin
Endotoxin testing
testingclinical use
clinical use
13:00
13:00
13:00
13:00
Patient
Patient arrival
arrival
Procedure
Procedure completed
completed
to
to cath
cath lab
lab
15:00
15:00
Cells
Cells released
released for
for
clinical
clinical use
use
Procedure
Procedure completed
completed
Cells Release Criteria
The final BM MNC product must meet the following
release criteria prior to infusion:
• Viability > 70% by Trypan blue exclusion
• Stat Gram stain negative for bacteria
• Product concentration equal or inferior to 5.0 ±
1.0 x 107 cells/ml
• Endotoxin testing < 5 EU per Kg of body weight)
(Limulus Amebocyte Lysate (LAL) PYROGENT®,
by Lonza)
• Sterility testing by detection of growth in a BAC/T
alert system for 5 days
Validation – Maverick Catheter
CD34+ cell (%)
Viability (%)
p=0.25
0.3%
0.28
p=0.22
0.30
100%
99%
97%
Pre
Post
0.2%
50%
0.1%
Pre
Post
Stability of cells after a passage through a Maverick
delivery catheter. Validation performed bone marrow
mononuclear cells (n = 10)
Acknowledgements
Duke Bone Marrow Transplantation Program
Barbara Waters-Pick
Linda Sledge
Duke Clinical Research Institute
Rose-Mary Beci
Clare A Matti
Diane Joseph
Duke Translational Research Institute
Dr Rebecca Haley
Adapted
Adapted from
from Atkas
Atkas et
et al.
al. Cytotherapy;10:203
Cytotherapy;10:203
Cellular therapy-comparisons
IC
Infusion
Volume
24 x 108
4-5
26
130 +/- 22 mL
3 x 108
3
10
ASTAMI3
49 +/- 9 mL
0.7 x 108
3
10
REPAIR-AMI4
50 mL
2.1 x 108
3
10
RENEW
160 mL
5.0 x 108
4
10
Study
BM aspirate
Total cells
BOOST1
128 +/- 33 mL
Janssens2
Sub-groups: timing
Outcome
LVEF(%)
BMMNC < 5
days after MI
BMMNC > 5
days after MI
P interaction
2.76
4.00
0.68
Infarct size
-2.44
-8.80
0.10
LVESV
-5.64
-6.51
0.86
LVEDV
-2.14
-5.34
0.58
Arch Intern Med 2007;167:989
Sub-groups: Dose of BMMNC
< 80 x 106
3.17
> 80 x 106
3.53
Infarct size
-4.58
-5.93
0.72
LVESV
-3.55
-4.58
0.79
LVEDV
-2.67
-0.89
0.74
Outcome
LVEF(%)
P interaction
0.84
Arch Intern Med 2007;167:989
LVEF improvment
Group BMMNC:
Group control:
+6.1±14.7%
+3.4±9.7%
BMMNC
p=0.26
control
Rando
18 mois
Rando
18 mois
LE (ml)
33±21
20±14
29±17
20±10
LVEDV
(ml)
84±17
96±17
79±12
82±14
Abstract
Abstract AHA
AHA 2004
2004
Repeated autologous BMMNC
further improves cardiac function
p = 0.001
p = 0.05
9.6% ± 2.8%
LVEF (MRI)
10%
6.3% ± 1.7%
5%
16%
2.8% ± 2.1%
58
58%
Placebo
Single dose
Repeated
dose
Yao et al. China Interventional Therapeutics 2007 (abstract)
Activity
Week 2
Full Physical Exam
Day 30
Day 90
Day 180
√
√
√
√
MRI
√
Echocardiogram
Holter monitoring
√
EKG
√
√
√
√
√
√
√
√
NYHA classification
Telephone contact
√
√
DASI questionnaire
SAE /Con med
documentation
Day 365
√
√
Based on the sub-group analysis in the
REPAIR-AMI trial, in patients with EF ≤ 40%
- Estimated an absolute change of +3.5%
in the global LVEF (by MRI)
+6% (+/-4%) change in treated patients
+2.5% (+/- 4%) change in the control group
- Assuming an α of .05, a sample size of
25 patients in each group will provide
85% power to detect this difference.
Adapted
Adapted from
from Atkas
Atkas et
et al.
al. Cytotherapy;10:203
Cytotherapy;10:203
How you prepare cells matters
REPAIR AMI trial
ASTAMI trial
25.5 + 13
BMC recovery
19.1 + 7.6
99 + 0
viability
99 + 0
6.8 + 4.8
CD34+ cells
4.4 + 3.6
5270 + 3919
CFU
3891 + 2425
230 + 123
MSC
161 + 160
1244 + 1307
Basal invasion
448 + 384
*All comparison < 0.05
European
European Heart
Heart Journal
Journal 2007;28:766
2007;28:766