Cell suspension for use in the treatment of lower extremity peripheral artery disease

Abstract

The present invention refers to a cell suspension of autologous or allogeneic, preferably autologous, adult bone marrow-derived white blood cells, preferably derived from the crest of the ilium (or iliac crest), enriched for mononuclear cells, meaning more than 25% of the white blood cells (WBCs) present in the cell suspension are mononuclear cells, and comprising: A. Mononuclear cells (MNC) selected from the list comprising or consisting of: i. A population of Lymphocytes; ii. A population of Monocytes; and iii. A population of Hematopoietic stem cells expressing CD34; and wherein the cell suspension further comprises: B. Granulocytes; for use in the treatment or amelioration of lower extremity peripheral artery disease.

1 . A cell suspension comprising from 4×10 8  to 1.2×10 9  autologous or allogeneic white blood cells derived from the bone marrow of a human subject, wherein of the total number of from 4×10 8  to 1.2×10 9  white blood cells:
 i. 20% to 51% are lymphocytes, and 3.9% to 22.3% are monocytes; 
 ii. 1.4% to 10% are hematopoietic stem cells that express CD34; 
 iii. 25.3% to 83.3% of the total number of white blood cells are mononuclear cells; 
 iv. 16.7% to 74.7% are granulocytes; 
 v. 5.4% to 38.8% of the total number of white blood cells express CXCR4; 
 vi. 0.07% to 24.7% of total number of white blood cells express VEGFR2; 
 vii. Of the total number of hematopoietic stem cells that express CD34, 7.7% to 55.5% are early, non-committed hematopoietic stem cells that do not express CD38 and 0.7% to 10.3% are stem cells that express CD34 and CXCR4; and 
 viii. The maximum ratio of red blood cells to leucocyte cells is 6.7 and the maximum ratio of platelets to leucocyte cells is 32. 
 
       for use in the treatment or amelioration of lower extremity peripheral artery disease. 
     
     
         2 . The cell suspension for use according to  claim 1 , wherein of the total number of white blood cells 32.3% to 80.0% are mononuclear cells selected from the list consisting of lymphocytes, monocytes and hematopoietic stem cells that express CD34 cells and 20.0% to 67.7%, are granulocytes. 
     
     
         3 . The cell suspension for use according to  claim 1 , wherein the cell suspension comprises 5×10 8  to 1.2×10 9    
     
     
         4 . The cell suspension for use according to  claim 1 , wherein the cell suspension comprises 8×10 8  to 1.2×10 9    
     
     
         5 . The cell suspension for use according to  claim 1 , wherein the cell suspension comprises 9×10 8  to 1.1×10 9 . 
     
     
         6 . The cell suspension for use according to  claim 1 , wherein the cell suspension comprises 9.5×10 8  to 1.05×10 9 . 
     
     
         7 . The cell suspension for use according to  claim 1 , wherein the cell suspension comprises 9.8×10 8  to 1.02×10 9 . 
     
     
         8 . The cell suspension for use according to  claim 1 , wherein the lower extremity peripheral artery disease is critical limb ischemia. 
     
     
         9 . The cell suspension for use according to  claim 1 , wherein the cells of such cell suspension are suspended in a volume of from 5 to 30 ml of a heparinized saline solution or lactated Ringer solution, preferably comprising about 1% HSA and about 2.5% glucose. 
     
     
         10 . The cell suspension as defined in  claim 1 , for use in a method of treatment of lower extremity peripheral artery disease, preferably critical limb ischemia, via intra-arterial administration, wherein a low pressure blood flow of up to 4 atmospheres is obtained by positioning an inflatable balloon proximal to the occlusive vascular lesion at the distal femoral or popliteal artery and infusing said cell suspension intra-arterially. 
     
     
         11 . A syringe or a plurality of syringes comprising a cell suspension as defined in  claim 1 . 
     
     
         12 . The syringe(s) according to  claim 11  for use in the treatment or amelioration of lower extremity peripheral artery disease, preferably critical limb ischemia. 
     
     
         13 . The syringe(s) according to  claim 11 , for use in a method of treatment of lower extremity peripheral artery disease, preferably critical limb ischemia, via intra-arterial administration, wherein a low pressure blood flow of up to 4 atmospheres is obtained by positioning an inflatable balloon proximal to the occlusive vascular lesion at the distal femoral or popliteal artery and infusing said cell suspension intra-arterially. 
     
     
         14 . The cell suspension for use according to  claim 1  or the syringe(s) for use according to  claim 12 , wherein said suspension is provided as a single dose. 
     
     
         15 . The cell suspension or the syringe(s) for use according to  claim 10 , wherein the induction of low pressure flow is produced between 1 and 6 minutes and wherein the infusion of said cell suspension is carry-out between 2 and 10 minutes. 
     
     
         16 . A manufacturing process of a cell suspension, which comprises:
 a. Bone marrow (BM) collection from a human subject;   b. The BM is filtered, preferably by gravity, to remove any small bone fragments and to prevent clogging during later steps;   c. The initial BM volume is reduced, preferably to about 50-100 mL, by using the SmartRedux program on the Sepax 2.0 device and associated sterile disposable SmartRedux kit (CS-490.1);   d. The volume-reduced BM as obtained in step c), then goes through an automated density gradient centrifugation by performing a NeatCell density gradient centrifugation followed by two washes of the mononuclear cell (MNC) suspension using a washing solution, preferably composed of 2-4% HSA in saline solution (both pharmaceutical grade);   e. Collect the product of step d) and filter it, preferably through a 50 p.m filter, into a sterile falcon tube;   f. The BM product is then rinsed with wash solution and filtered to improve MNC (mononuclear cells) recovery; and   g. Optionally, the final volume of the BM product is adjusted to 40-60 mL.