Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT01595776 |
| Other study ID # |
CHVAS-01-08 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
Phase 1/Phase 2
|
| First received |
February 14, 2012 |
| Last updated |
June 13, 2012 |
| Start date |
January 2009 |
| Est. completion date |
December 2011 |
Study information
| Verified date |
June 2012 |
| Source |
IRCCS Policlinico S. Matteo |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
Italy: Ethics Committee |
| Study type |
Interventional
|
Clinical Trial Summary
Aim: to value the safety and efficacy of local intramuscular administration of
immunoselected autologous endothelial progenitor cells in the treatment of critical limb
ischemia in patients without revascularization options.
Primary goal: to value the feasibility of mobilization, harvesting, immunoselection and auto
transplantation of endothelial progenitor cells.
Secondary goal: to value the efficacy of local administration of autologous endothelial
progenitor cells in the treatment of critical limb ischemia
Description:
Type of the study. Prospective single centre not randomized. Aim of the study. To assess the
safety, feasibility and efficacy of local intramuscular administration of autologous
selected CD 133+ cells in patients suffering from CLI.
All the patients enrolled were suffering from CLI according to the TASC 2 definitions and
had no revascularization option, on the basis of contrast CT or angiography imaging. A
detailed informed consent, approved by our EC, had been required. An age lower than 18 years
and upper than 70 years because of a poor marrow responsiveness to drug simulation in
elderly people Clinical unsteadiness of the CLI, such as gangrene requiring major amputation
and a poor life expectancy were introduced as exclusion criteria for supposed latency of the
EPCs action. Severe systemic illness was judged to increase the risk of the marrow
stimulation. Patients with allergic diathesis, child-bearing age, previous EPCs muscular
implant, previous medical experimental protocol and any conflict of interest with the study
were excluded.
Patients: we enroll patients with a history of Rutherford stage 4 (rest pain) or 5 (small
ischemic lesions) PAD. All the patients have previous (contrast CT, MR or angiography)
vascular imaging excluding revascularization options, both endovascular and open and
encountered the enrolment and exclusion criteria. Every patient undergo routine physical and
instrumental examination including electrocardiogram, chest X-ray and blood sample analysis.
Younger patients met the Buerger disease criteria, whereas others had pure atherosclerotic
lesions.
CEUS Imaging Protocol. Two operators (F.C. and A.G., with 20 and 3 years experience,
respectively) who are blinded to treatment perform contrast-enhanced US for all patients.
Two US scanner (Philips iU22 (Bothell, WA, USA) with linear array L9-3 transducer and GE
Logiq e9 (GE Healthcare, Milwaukee, WI, USA) with linear array L6-9 transducer are used.
The imaging parameters are: reduced transmit power (mechanical index <0.06), at
approximately 7-10 frames per second and one focus well below the level of the target to
ensure a more uniform pressure field. Dual-mode presentation of a grayscale image
side-by-side with the contrast image facilitated real-time identification of anatomic
structures and ROI selection. Image loops of approximately 60 seconds were acquired. Effort
was made to have a uniform gain across the image and to avoid gain saturation. The TGC (time
gain compensation) was set such that before contrast arrival a uniform black image was
shown. A vial of contrast agent (SonoVue BR1; Bracco, Milan, Italy) is prepared at a
concentration of about 2x108 sulfur hexafluoride-filled microbubbles per milliliter,
according to the manufacturer's recommendations. Prior to each injection, microbubbles are
resuspended by shaking the vial. The position of the probe is recorded for each patient in
order to maintain the same position during follow up. The contrast injection consists of an
intravenous bolus of 4.8 ml of contrast agent injected in antecubital vein in 2 seconds
followed by a saline flush of 5 ml. The injection is made with the patient supine and after
10 minute of rest to avoid exercise related micro-vascular dilatation. The radiologist
maintains a constant image plane with the aid of the tissue (fundamental image) of the
"Contrast Side/Side" imaging mode.
Image analysis. The image loops are transferred to a personal computer for further analysis.
The main image analysis tasks are: a) identification of tibialis anterior artery (TAA) area,
b) selection of a representative region of normal tibialis anterior muscle (TAM) and c)
formulation of time-intensity curves. Two manually defined region of interest (ROI), 2 cm
and 4 cm sided-squares, are placed, respectively, over the tibialis anterior muscle with no
evidence of arterial branches, over tibialis anterior artery and over a small tibialis
arterial branch. The ROIs are placed in the same anatomical position for each patient to
avoid unwanted differences during follow up examinations. A time/intensity curve (TIC) is
obtained for each ROI. From an analysis of CEUS time-intensity curves, we compute regional
blood flow (RBF) and volume (RBV). Time-intensity curves are extracted using commercial
quantification software (QontraXt v.3.60, AMID, Rome, Italy). This software allows manual
region of interest (ROI) selection, measurement of the selected ROI area and provides linear
data for the time-intensity curves. For the ROI in the normal ATM effort is made to place
the region in an area without large vessels. The ROI of the ATA is a 2 cm square area and
the ATM ROI is a 4 cm square area. Time-intensity curves are obtained by computing the mean
intensity of pixels comprised within the ROI at each time point. For each image loop are
calculated:
- RBV which consists in the total amount of contrast media within the selected ROI, in a
period of time. Due to the characteristics of US contrast media, it reflects the
quantity of blood in a defined region. It is directly related to the area under the
curve (AUC).
- RBF that is in direct ratio to perfusion in a given ROI. It consist in the Contrast
Media flow (related to the Blood Flow) in a selected ROI. It is related to the Mean
Transit Time.
Marrow stimulation. Human recombinant granulocyte colony-stimulating factor (rhGCSF) is
administered subcutaneously for 4-5 consecutive days at a dosage of 10 µg/kg daily, split in
2 doses. Starting from the third day of mobilization, the CD133+ cell count is monitored
daily by cytofluorimetric analysis on a 2 ml sample obtained from the patients' peripheral
blood. The minimum CD133+ cell count acceptable for leukapheresis (LKF) collection was
10/µl. Patients are monitored for any G-CSF related side effects.
Leukapheresis collection. A single LKF collection is planned for each patient using a third
generation cell separator device (Spectra Cobe, Lakewood, CO), processing at least 2.5 blood
volumes according to our internal protocol for stem cell collection. Patients are monitored
for blood pressure and heart rate during the entire collection procedure. Immediately after
LKF a sample from patient's peripheral blood is taken for haemocytometric analysis to
evaluate platelet count and Hb levels. Each leukapheresis collection is diluted with 10%
acide citrate dextrose (ACD-A) and maintains overnight at 4°C before immunomagnetic cell
selection. A sample of 2 ml from each LKF bag is taken for cytofluorimetric analysis. Four
hours after LKF collection coagulation parameters, electrolyte levels and hemocytometric
analysis is evaluated.
Immunomagnetic cell selection. CD133 immunomagnetic cell selections are performed the day
after LKF collection using the Clini-MACS (Miltenyi Biotec) device according to the
manufacturer's standard protocol. Briefly, CliniMACS CD133 reagent (formed by super
paramagnetic particles composed of iron oxide and dextran conjugated to murine monoclonal
antibodies) is added to the cells for incubation. The product is subsequently washed by
dilution with buffer (CliniMACS PBS-EDTA buffer supplemented with 0.5% human serum albumin)
and the cell bag is hung on the device for the automated selection of CD133 labeled cells. A
sample of 2 ml from the positive fraction is taken for cytofluorimetric analysis.
Quality controls. Clonogenic assays. A sample taken from the CD133 cell positive fraction
after each immunomagnetic cell selection procedure is seeded for short term (14 days)
clonogenic assays. A standard mixture of methylcellulose plus recombinant human
erythropoietin, rh stem cell factor (SCF), rhGM-CSF, and rh interleukin-3 (IL-3) is employed
(Stem Cell Technologies, Vancouver, BC, Canada; MACS Media, Miltenyi Biotec GmbH, Bergisch
Gladbach,Germany). Microbial cultures. Microbial cultures on the immunoselection waste bag
containing the negative fraction are carried out to detect aerobic-anaerobic bacteria and
fungal contamination. A sample of 10ml is inoculated in the culture medium (Bact/Alert FA
and BacT/Alert FN, Organon Teknika Corp., Durham, NC) and incubated for 10 days at 37°C.
Cytofluorimetric analysis. Samples obtained from peripheral blood before mobilization with
G-CSF, at time of leukapheresis and after immunomagnetic cell selection are analyzed by flow
cytometry to evaluate the expression of specific stem cell and endothelial antigens. Becton
Dickinson FACSCanto was employed for all flow cytometric assays with a lyse no-wash
technique, using the following monoclonal antibodies: anti-CD45 fluorescein isothiocyanate
(FITC) (Becton Dickinson, San Jose, CA), anti-CD34 Peridinin-chlorophyll-protein complex
(PerCP) (8G12 clone, Becton Dickinson), anti-CD133 phycoerythrin (PE) (AC133 clone, Miltenyi
Biotec) and anti-VEGF-R2 allophycocyanin (APC) (R&D systems). Whole blood is processed
following the instructions for VEGF-R2 (KDR) determination. Each blood sample is transferred
to a 50 ml falcon tube and brought to a total volume of 30 ml by adding ammonium phosphate
containing lysis buffer (Becton Dickinson). After a lysis period of 5 minutes PB is
centrifuged at 500g for 5 minutes and washed two times with PBS containing 0,5% bovine serum
albumin (BSA). 100 µl of each sample are then transferred in a BD tube for cytometric
analysis and incubated for 15 minutes with 1µg/105 cells IgG in order to block all
nonspecific sites. 50 µl of the IgG blocked sample are incubated with 20 µl of anti-VEGF-R2
antibody for 30 minutes at 4°C in the dark and then washed twice with PBS. At the end of the
last wash step, 10 µl of each of the other antibodies (anti-CD45, anti-CD34, anti-CD133) are
added and incubated 10 minutes at room temperature in the dark. Each sample is acquired with
BD FACSCanto recording 100.000 events inside the lymphocyte plus monocyte gate. Data files
are analyzed with FACS Diva 6.1 software. Viability is assessed using 7-amino-actinomycin D
(7-AAD) (Molecular Probes, Eugene, OR). A sample for Hill and EBM2 clonogenic assays is
taken from patient's peripheral blood before and after mobilization (at time of LKF
collection).
Implant procedure. After loco-regional anesthesia and below the knee cutaneous disinfection,
45-48 ml of autologous CD133+ saline solution suspension is administered intramuscularly
with 1 ml deep injection through 18G needle. The injections were so allocated: 10 ml in the
anterior compartment of leg, 10 ml in the superficial posterior compartment, 10 ml in the
deep posterior compartment, 10 ml in the lateral compartment and the remaining part in the
foot.
Baseline assessment and follow up. Pain assessment is carried out with a personal scale of 3
degrees (mild, moderate and severe) and the pain killing drugs use monitoring. Ischemic
lesions are treated weekly by a wound management skilled nurse. Preoperative and at 3, 6 and
12 months after the implant are assessed CEUS, lesion evolution and pain management.
