Filgrastim, fibrinolysis, and neovascularization

Objective: Segmental recanalization of chronically occluded arteries was observed in patients with chronic limb-threatening ischemia (CLTI) treated with Filgrastim, a granulocyte colony stimulating factor, every 72 hours for up to a month, and an infra-geniculate programmed compression pump (PCP) for 3 hours daily. Molecular evidence for fibrinolysis and neovascularization was sought. Methods: CLTI patients were treated with PCP alone (N=19), or with Filgrastim and PCP (N=8 and N=6, at 2 institutions). ELISA was used to measure the plasma concentration of Plasmin and of Fibrin Degradation Products (FDP), and the serum concentration of proteins associated with neovascularization. In the PCP alone group blood was sampled on day 1 (baseline) and after 30 days of daily PCP. In the Filgrastim and PCP group blood was drawn on day 1, and one day after the 5th and the 10th Filgrastim doses. Each blood draw occurred before and after 2 hours of supervised PCP. Results: Significant (p<0.01) PCP independent increases in the plasma concentration of plasmin (>10 fold) and FDP (>5 fold) were observed one day after both the 5th and the 10th Filgrastim doses, compared to day 1. Significant (p<0.05) increases in the concentration of pro-angiogenic proteins (e.g., HGF, MMP-9, VEGF A) were also observed. Conclusion: Filgrastim at this novel dosimetry induced fibrinolysis without causing acute hemorrhage, in addition to inducing a pro-angiogenic milieu conducive to NV. Further clinical testing is warranted at this novel dosimetry in CLTI, as well as in other chronically ischemic tissue beds. Methods Population: CLTI patients who were not candidates for, or had failed, previous invasive revascularization procedures. Inclusion criteria: Patients complaining of ischemic forefoot rest pain, gangrene, and/or ischemic ulceration were enrolled if the ankle brachial arterial index was <0.45. Exclusion criteria: Acute limb ischemia, non-salvageable extremity, untreated hypercoagulable disorder, sickle cell disease, myeloproliferative disorder, dialysis, creatinine above 3.5 mg/dl, active cancer, dementia, non-compliance, intolerance of PCP, body mass index over 34, venous stasis ulcer, history of lymphoma or leukemia, uncorrected symptomatic coronary artery disease, severe carotid stenosis, sepsis proximal to the forefoot, allergy to Filgrastim. Patient groups: Table 2 lists the patient characteristics. Blood assays were performed on 2 patient groups. The first group was treated at a university-affiliated institution (UC, 2012-13) with PCP alone (N=19). The second group (N=14) was treated with PCP and Filgrastim: six at another independent university-affiliated institution (UIC, 2016-19) and eight at a hospital (2014-15). Filgrastim: Each patient was to receive Filgrastim subcutaneous injections, one every 72 hours at approximately 10 mcg/kg for up to ten doses. Filgrastim (Neupogen) was purchased from Amgen Inc (Thousand Oaks, CA) in 480 and 300 mcg vials. These were kept refrigerated between 2°C and 8°C and stored in the dark. Small losses of agent occurred during administration, which typically required two separate injections in the subcutaneous tissue in the lower abdomen. Fourteen patients received 5 doses. Ten of the 14 received all 10 doses. Mobilized progenitor cells circulated until either being engrafted or otherwise cleared from the circulation. The leukocyte count returned to normal approximately every 72 hours. The product label for Filgrastim lists progenitor cell mobilization as an indication (not CLTI). The FDA granted a waiver to use Filgrastim at the novel dosimetry in CLTI patients at both institutions. Filgrastim has been safely used in healthy patients for the purpose of bone marrow harvest of mobilized progenitor cells.[i] Oncologists have nearly 3 decades of experience with Filgrastim. Initial concerns over promoting tumor angiogenesis were not substantiated [ii]. While severe complications are uncommon, they can be significant (e.g., capillary leak syndrome, myocardial infarction, and splenic rupture). Hemorrhage is not a reported side effect; fibrinolysis and NV are not listed on the Filgrastim label. PCP: The ArtAssist device (ACI Medical LLC, San Marcos CA) applies sequential pressure to the calf, and foot. PCP was used at home on both legs in the seated position for 3 hours daily. Rapid inflation of pneumatic cuffs (0 to 120 mmHg in <0.3 sec) provides an endothelial shear stress stimulus, while at the same time driving in oxygenated nutritive blood flow and facilitating venous return (Table 1). The pressure is held in each of the cuffs for three seconds. Rapid deflation follows. Three cycles occur per minute. Phlebotomy: Group 1 patients were treated with PCP alone, and had blood drawn on Day 1 and Day 30. Group 2 patients were treated with Filgrastim and PCP and had three blood samples drawn: on Day 1, on the day after the 5th Filgrastim dose, and on the day after the 10th Filgrastim dose. In both Groups, on each day, blood was drawn using a 21- gauge butterfly inserted before and again after 2 hours of observed PCP. Blood was collected into serum separator and EDTA plasma separation tubes. Specimens were immediately transported on ice to the independent laboratories at the university-affiliated institutions. Serum separation: Whole blood was collected in serum separator tubes. Following clot formation (30-60 min) each tube was centrifuged for 15 min at 1000g. Serum aliquots were stored at -80°C in labeled cryovials (to avoid repetitive freeze/thaw) until batch analysis. Plasma separation was used for preparation of samples for ELISA, and for cytometry. Whole blood was collected in EDTA-treated tubes. Red cells and platelets were removed after centrifugation at 2,000g for 20 minutes at 4oC. Plasma aliquots were stored at -80°C. Enzyme-linked immunosorbent assay (ELISA): Protein concentrations were measured in duplicate in plasma or in serum using Human ELISA Kits according to each kit’s instructions (Table 7). Plasmin and Fibrin Degradation Products (FDP) were measured in plasma at UIC and in serum at UC to assess fibrinolysis. Proteins associated with NV included HGF[iii], VEGF-A[iv], MMP-9[v], Angipoietin-1 [vi],PDGF-AA, PDGF-BB, PDGF-AB [vii],TNF-a[viii], MCP-1[ix] and TGF-b[x], PLGF[xi], IL-6[xii], and IGF-1 [xiii]. All patient-specific samples were analyzed together using a single ELISA kit. Cytometry: Mature endothelial CD31+ (PECAM-1), progenitor CD34+, and endothelial progenitor CD309+ (VEGFR2+) cell populations were measured (Table 7). White Blood Cell (WBC) and differential counts were obtained on plasma in the hospital hematology laboratories. Serum nitrite: Serum nitrite, a breakdown product of nitric oxide (NO), reflects nitric oxide synthase (NOS) activity. It was measured before and after 2 hours of supervised PCP with a quantitative fluorometric assay based on the reaction of nitrite with 2,3- diaminonaphthalene under acidic conditions to form fluorescent 1-(H)-naphthotriazole (Table 7). Statistics: Each patient had 2 internal controls. The first internal control was obtained on Day 1, prior to PCP or Filgrastim. Changes resulting from daily PCP use for 30 days (Group 1) or caused by Filgrastim (Group 2) were referenced to data from Day 1. The second control was obtained on arrival (at T=0) to each 2 hour observed PCP session, and was used to ascertain the effect of PCP (at T=2HR) on Day 1 for all patients, on Day 30 for the PCP alone group, and on the day after the 5th and the 10th doses of Filgrastim in the Filgrastim+ PCP group. Percent changes are reported relative to each of these two controls (change from control divided by the control). The paired Student t-test was used to estimate a P value directly from the ELISA data (not from the percent changes). An unpaired T-Test was used when comparing the aggregated ELISA data in all patients obtained before any Filgrastim exposure to all data obtained after Filgrastim was given. P values <0.05 are considered significant. The significant findings in the UC study led to the confirmatory laboratory study at UIC in 6 CLTI patients. The UIC sample size was calculated from the UC data to provide 80% power to detect changes in protein levels of 0.66 standard deviations assuming a two-sided significance level of 0.05. [i] BE Shaw, DL Confer , W Hwang, MA Pulsipher. A review of the genetic and long-term effects of G-CSF injections in healthy donors: a reassuring lack of evidence for the development of haematological malignancies. 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