Use Art-assist and Neupogen to Treat Chronic Limb Ischemia

This protocol is the next clinical step in our biochemical assessment of a cell therapy
approach to treat Chronic Limb Threatening Ischemia (CLI). Briefly, the capability to
enlarge collateral arteries ("arteriogenesis"), and to stimulate growth of new capillaries,
arterioles and venules at the tissue level ("angiogenesis") is innate. Investigators
attribute the failure for this to occur in CLI to the ischemic environment. In our first
protocol investigators used an external programmed pneumatic compression device (Art-Assist
Device) to improve hemodynamics in the limb and most importantly to provide the "on-switch"
to activate the endothelium so arteriogenesis can begin. This new protocol provides the
second step in improving this environment; improving the cellular deficit and protein
signaling. Investigators are seeking the proteomic, biochemical, and cytometry data needed
to refine the approach so a clinical trial can be performed. This project will use FDA
approved and CMS reimbursed products.

Rationale

Neovascularization in CLI fails for hemodynamic ("A") and cellular ("B") reasons:

A) The hemodynamic failure caused by multi-level arterial occlusive disease leads to:

1. Attenuation of infra-geniculate endothelial shear stress: Without this stimulus, the
endothelial cells lining small collateral arteries are not induced to attract and
capture circulating monocytes and progenitor cells that would orchestrate
arteriogenesis.

2. Impaired inflow of oxygenated nutritive blood flow into the ischemic tissue

3. Impaired clearance of waste products of metabolism from the ischemic tissue

4. Impaired dissemination of protein signals from the ischemic tissue that would recruit
pro-angiogenic reparative cells.

5. Impaired arrival of these pro-angiogenic cells to the ischemic tissue. B) Deficiency in
the circulating progenitor cell population: These cells participate in angiogenesis and
arteriogenesis. They are deficient in number and in function in CLI patients.

C) Impaired immunity. Even when blood flow is improved there is a population of patients
that still succumb to infection. Part of this relates to poor wound management, the other is
impaired immunity.

Our cell therapy approach is designed to address each of these. Specifically:

A) The hemodynamic failure caused by multi-level arterial occlusive disease will be
corrected by wearing the ACI device that

1. Restores the endothelial shear stress needed to activate vascular endothelium so as to
start the Arteriogenesis process.

2. Increases delivery of oxygenated nutritive blood flow into the ischemic tissue

3. Clears waste products of metabolism from the ischemic tissue

4. Increases dissemination of protein signals from the ischemic tissue that would recruit
pro-angiogenic reparative cells.

5. Enhances delivery of these pro-angiogenic cells back to the ischemic tissue.

B) The deficiency in the circulating progenitor cell population will be corrected:

G-CSF (Nuopogen, Amgen Inc.) is FDA approved for progenitor/stem cell mobilization. It has
been used clinically as stand-alone neovascularization therapy. Investigators will use it in
this protocol to correct the deficiency in this vital cell population in CLI patients,
enhancing arteriogenesis and angiogenesis. Investigators will measure its biochemical
impact.

C) The Impaired immunity will be addressed G-CSF (Nuopogen, Amgen Inc.) is FDA approved for
correcting the immunological deficit following cytotoxic chemotherapy. CLI occurs in
elderly, often diabetic, patients with impaired immunity. Amputations result when infections
arise in the forefoot and reach the mid-foot.

Our hypothesis: is that the combined use of a PPCD and G-CSF will promote arteriogenesis.
Investigators have limited but strong clinical evidence that this combined approach
dramatically improves blood flow. The recently completed IRB project (IRB#12-1198) focused
on the biochemistry of the PPCD. The present submission addresses the biochemical impact of
the PPCD and G-CSF together.

B.OBJECTIVES:

Specific Aim 1: is to measure the influence of progenitor cell mobilization on the PPCD
proteomic profile investigators derived during our recently completed protocol (IRB #
12-1198). Ten CLI patients will be enrolled. All will wear the PPCD as before. All will
receive 10 mcg/kg of Filgrastim (Nuopogen, Amgen Inc.) for a total of 5 doses. The FDA
approved indication is stem cell mobilization. The dosimetry is less aggressive than the
product label as the dosimetry is being spread out to once every 72 hours rather than once a
day. The FDA has granted the PI an IND waiver for this dosing interval. As with our first
protocol, cytometry will yield the distribution of progenitor CD34+ and VEGFR2+ cells and
monocytes (CD14+). The distribution of mature circulating endothelial cells (CD31+) cells
will be measured as a reference point. As with our first protocol investigators will do a
proteomic survey measuring levels of cytokines associated with arteriogenesis, as well as
serum nitrite (reflection of nitric oxide synthase activity).

Specific Aim 2: investigators will examine the association between these biochemical data,
hemodynamic testing, and clinical course (alleviation or progression of forefoot ischemic
rest pain and ischemic forefoot wounds) over a 6 month interval.

Study Design: Ten CLI patients with ischemic forefoot rest pain, non-healing forefoot
ulceration, or dry forefoot gangrene will be recruited. They will have already undergone
standard of care evaluation, including hemodynamic testing and duplex ultrasound delineation
of the arterial anatomy in the Non-invasive Vascular Laboratory. Those with tibial artery
occlusive disease, with normal or corrected proximal aorto-ilio-femoral arterial anatomy,
will be given the option of enrollment in lieu of surgery or catheter revascularization.
PPCD use will continue until the presenting symptoms resolve or traditional
revascularization becomes necessary to achieve limb salvage. The only three amendments to
our first IRB submission (IRB#12-1198) are:

1. All will receive 10 mcg/kg of Filgrastim (Nuopogen, Amgen Inc.) for a total of 5 doses.

2. The 30 day CRC visit will be replaced with a 14 day visit.

3. Cytometry will also be performed on the 14 day visit. As in our first IRB submission,
each patient serves as his/her own control. Two "pairs" of blood specimens will be
analyzed per patient. A "pair" includes phlebotomy prior to and immediately after two
hours of PPCD. The first pair is obtained on enrollment in the CRC. The second pair
will be done 2 weeks later 18-24 hours after the last dose of G-CSF. The patient will
return for standard of care clinical evaluation, with repeat hemodynamic testing 6
months after that 14 day CRC evaluation.

Baseline visit (Vascular Surgery Clinic):

1. Patients will undergo standard of care history and physical (H&P) examination.

2. Other standard of care tests will include: duplex ultrasound (DUS), Ankle and Toe
Pressures.

3. Upon verification that the patient is eligible (based on the inclusion/exclusion
criteria), an ICF (Informed consent form), will be explained to the patient to better
explain and the study and ask for authorization to participate.

Day1 (at the CRC; Clinical Research Center, University of Chicago): The following procedures
will be done in this order:

1. Initial blood draws (20 ml)

2. Each patient will be given Nuopogen shot (10 mcg/kg) subcutaneously.

3. Patients then will wear the PPCD in seated position for (2-hour session).

4. After the 2-hour use of the PPCD, another blood draw will be done (20 ml).

5. Patient will be given ample time to fill out the quality of life questionnaires and the
study coordinator will be available at this time to assist the patient.

6. Blood samples will be sent to the Dr. T.C. HE, PhD Lab for analysis.

Days 4, 7, 10, and 13 (at Home or in clinic): The following procedures will be done:

1. Nurse or P.I. will administer Nuopogen shot (10 mcg/kg) subcutaneously, every 3rd day,
for a total of 5 Nuopogen shots.

2. Patient will continue use of the PPCD every day, at least 3 hours a day, till symptoms
are resolved.

Day14 (at the CRC; Clinical Research Center, University of Chicago): The following
procedures will be done in this order:

1. Initial blood drawing (20 ml), 18-24 hours after the 5th shot of Nuopogen. Blood
samples will be sent to Dr. T.C. He, PhD Lab for biochemical analysis.

2. Patients will then wear PPCD for 2 hours in seated position.

3. Just prior to the end of the 2-hour use of the PPCD, another blood draw will be done
(20 ml).

4. QOL will be filled by the patient.

5. Patient will return the empty vials of Nuopogen.

Inclusion Criteria:

1. Male or female between the ages of 40 and 85.

2. Chronic limb ischemia Fontaine Class III (ischemic forefoot rest pain) and Class IV
(non-healing ischemic ulcers, gangrene) with confirmatory non-invasive vascular
testing.

Exclusion Criteria:

1. Acute limb ischemia requiring emergency treatment.

2. Non-salvageable foot (e.g. extensive gangrene, advanced infection, rigor mortis,
knee/hip flexion contracture, post-stroke paralysis, and hemiparesis).

3. Untreated hypercoagulability disorder, sickle cell anemia, myeloproliferative
disorder.

4. Dialysis, and sustained elevated Creatinine > 4 mg/dl.

5. Severe dementia; bed-ridden; non-compliance; unlikely to follow-up; unreliable.

6. Intolerance of PPCD compression

7. Morbid obesity (Body Mass Index > 34)

8. Severe venous insufficiency causing venous stasis ulceration and dermatitis.

9. Uncorrected significant aorto-iliac, common femoral, and profunda femoral arterial
disease

10. Ulceration precluding PPCD placement.

11. Active cancer.

12. Allergy to Nuopogen.

13. Uncorrected symptomatic coronary artery disease

14. History of lymphoma or leukemia