Phase 2 Study of Intramyocardial Injection of Autologous CD34+ Cells to Treat Subjects with Refractory Chronic Myocardial Ischemia (CMI): Factors Influencing Mobilization and Apheresis.

CD34+ cells constitute a heterogeneous population of hematopoietic and endothelial progenitor cells, capable of producing mature blood cells and facilitating vascular repair, respectively. CD34+ cells predominantly reside in the bone marrow, circulate at low levels in the blood under steady state conditions, and can be mobilized to the periphery in response to acute tissue injury or exogenous stimuli such as granulocyte-colony-stimulating-factor (G-CSF). Although the ability to mobilize CD34+ cells is well established in a variety of clinical contexts, it has not been extensively studied in the CMI population. Accordingly, we examined the patterns and factors influencing mobilization of CD34+ cells in CMI subjects during a Phase 2 prospective, randomized, double blind, placebo controlled study to evaluate the safety and efficacy of two doses (1×10e5 or 5×10e5 (±10%) CD34+ cells per kg body weight, up to a maximum of 100 kg) of ISOLEX 300i Magnetic Cell Selection System (Baxter Healthcare Corporation, Deerfield, IL.) selected autologous-CD34+ cells administered via intramyocardial injection.

CMI subjects (n=167) who were no longer candidates for surgical or interventional revascularization procedures, were mobilized with subcutaneous injections of G-CSF at a dose of 5 ug/kg/day for 5 days. Peripheral blood CD34+ cell levels were measured using flow cytometry on Day 4 and Day 5. A single apheresis procedure, via a femoral or jugular placed catheter, was performed on Day 5. The number of total blood volumes (TBV) processed ranged from 2-5 and was based on each subject's Day 5 peripheral blood CD34+ cell level to minimize the collection time for good mobilizers and maximize the number of CD34+ cells collected from poor mobilizers. Simple and multiple linear regression analyses of demographic, clinical, and laboratory parameters were performed to identify characteristics associated with a CMI subject's ability to mobilize CD34+ cells.

The study group consisted of 87% males and 13% females ranging in age from 41-91, with an average age of 61 (±8.9) years and a mean body mass index (BMI) of 32.0 (±5.7). Ten percent were active smokers, 63% were former smokers and 27% had no history of smoking. Forty-seven percent of subjects had no history of diabetes and the remaining 53% were either insulin dependent (n=43) or non-insulin dependent (n=45) diabetics. Seventy-five percent of the subjects were able to mobilize ≥ 15 CD34+ cells/uL whole blood and required apheresis of ≤ 4 TBV resulting in a mean total of 197 × 10e6 CD34+ cells in the apheresis product. Subjects with < 15 CD34+ cells/uL whole blood apheresed 5 TBV resulting in a mean total of 76 × 10e6 CD34+ cells in the apheresis product. Increasing age (p=0.001) had the greatest negative impact on mobilization. Non-smokers mobilized better than smokers (p=0.024) and there was no statistically significant difference between current smokers and former smokers. Females tended to mobilize better than males (p=0.022) and there was a positive association between increasing BMI and mobilization (p=0.024). Higher platelet counts at baseline also correlated with better mobilization on Day 5 (p=0.005). Subjects with insulin dependent diabetes mobilized better than non-insulin dependent diabetics, who in turn mobilized better than non-diabetics (p=0.028). A history of hypertension, myocardial infarction, congestive heart failure, tachycardia, pericardial effusion or pacemaker had no significant impact on an individual's ability to mobilize CD34+ cells.

This is the first study providing insights into low dose G-CSF mobilization in a CMI population of older, sedentary individuals with multiple concomitant medical conditions. Female gender, increasing BMI, higher baseline platelet count and insulin-dependent diabetes positively correlated with mobilization. Increasing age and smoking, whether current or historical, negatively impacted mobilization. The TBV processed during a single apheresis collection can be adjusted to minimize collection time for good mobilizers and maximize the number of CD34+ cells collected from very poor mobilizers. Identifying these characteristics and recognizing their impact should be considered when designing future cell therapy based trials utilizing mobilization and apheresis, not only in a CMI population but other populations as well.

Off Label Use: Neupogen (G-CSF) is approved for stem cell mobilization in oncology patients whereas it was used in this study to mobilize stem cells in chronic myocardial ischemia patients.