TRANSLATIONAL AND CLINICAL RESEARCH Improved Granulocyte Colony-Stimulating Factor Mobilization of Hemopoietic Progenitors Using Cytokine Combinations in Primates STEPHEN R. LARSEN,a,b,c KEEFE CHNG,a FIONA BATTAH,a ROSETTA MARTINIELLO-WILKS,a,c JOHN E.J. RASKOa,b,c a
Gene and Stem Cell Therapy Program, Centenary Institute, University of Sydney, Sydney, New South Wales, Australia; bInstitute of Haematology and cCell and Molecular Therapies, Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia Key Words. Peripheral blood stem cells • Mobilization • Cytokines • Thrombopoietin • Granulocyte colony-stimulating factor • Nonhuman primate
ABSTRACT Peripheral blood stem cells (PBSCs), usually mobilized with granulocyte colony-stimulating factor (G-CSF) alone or in combination with chemotherapy, are the preferred source of cells for hemopoietic stem cell transplantation. Up to 25% of otherwise eligible transplant recipients fail to harvest adequate PBSCs. Therefore it is important to investigate existing and novel reagents to improve PBSC mobilization. Because of marked interindividual variation in humans, we developed a robust nonhuman primate model that allows the direct comparison of the efficacy of two PBSC mobilization regimens within the same animal. Using this model, we compared pegylated G-CSF (pegG-CSF) with standard G-CSF and compared the combination of G-CSF and pegylated megakaryocyte growth and development factor (pegMGDF) with G-CSF plus
stem cell factor (SCF) by measuring the levels of CD34ⴙ cells, colony-forming cells (CFCs), and SCID repopulating cells (SRCs) before and after cytokine administration. Mobilization of CD34ⴙ cells, CFCs and SRCs using pegG-CSF achieved similar levels to those resulting from 5 days of standard G-CSF. The combination of G-CSFⴙpegMGDF mobilized progenitors to levels similar to G-CSFⴙSCF but greater than standard G-CSF for CD34ⴙ cells and CFC. This first direct comparison of PBSC mobilization in individual primates demonstrates that peg-G-CSF is equivalent to daily G-CSF and that the addition of pegMGDF to G-CSF improves mobilization. In light of the development of new thrombopoietin agonists, these data offer the potential for improved stem cell mobilization strategies. STEM CELLS 2008;26:2974 –2980
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION Mobilization of peripheral blood stem cells (PBSCs) is most commonly achieved using granulocyte colony-stimulating factor (G-CSF), with or without chemotherapy [1, 2]. However, marked variability in the degree of mobilization prevents safe transplantation of sufficient numbers of PBSCs in 5%–10% of allogeneic healthy donors [3] and in up to 50%–60% of patients for autologous transplantation who have previously received chemotherapy [4]. Stem cell factor (SCF) is approved for use in patients who have failed to achieve the minimal threshold of PBSC following mobilization. However, because of concerns related to allergic-like reactions mediated by mast cell degranulation, SCF is available only in Australia, Canada, and New Zealand. For this reason, it is important to investigate for novel reagents, such as AMD3100, to improve PBSC mobilization. Two existing reagents that warrant further investigation are pegylated (polyethylene glycol; peg) G-CSF (pegG-CSF) and thrombopoietin. Although both G-CSF and its pegylated counterpart have been used extensively in humans, surprisingly, they have not been carefully compared for their direct mobilizing abilities in primates. Non-pegylated G-CSF (Neupogen, Amgen, Thousand Oaks, CA,
http://www.amgen.com) has a half-life of approximately 4 – 8 hours in humans and requires daily administration to produce a sustained increase in circulating granulocytes and mobilization [5]. pegG-CSF was developed in the late 1990s and has been shown to have a half-life of approximately 42 hours [6]. Like standard G-CSF, it was initially approved for use in patients receiving chemotherapy to reduce the period of neutropenia [7, 8]. However, in 1999, pegG-CSF was first used to mobilize hemopoietic progenitors in normal humans. Molineux et al. [9] and subsequent investigators [10] demonstrated sustained duration of action in vivo in both mice and humans and showed that a single subcutaneous dose between 30 and 300 g/kg could mobilize CFU-GM peaking on day 5. Recent studies have explored the application of a single dose of pegG-CSF after chemotherapy to mobilize hemopoietic progenitors. Bruns et al. showed that a single dose of 6 or 12 mg of pegG-CSF mobilized CD34⫹ progenitors to a similar degree in patients with multiple myeloma [11]. In patients with lymphoma, 24 of 25 patients reached the target cell dose of 2 ⫻ 106 CD34⫹ cells after receiving a single dose of 6 mg after combination chemotherapy [12]. One case report of successful mobilization described a single 12-mg dose of pegG-CSF on day 5 after combination chemotherapy in a patient who had failed mobilization
Author contributions: S.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; K.C.: provision of study material or patients, collection and/or assembly of data; F.B.: conception and design, collection and/or assembly of data; R.M.-W.: statistical analysis; J.E.J.R.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript. Correspondence: John Rasko, Ph.D., Gene and Stem Cell Therapy Program, Centenary Institute, Amgen, Thousand Oaks, CA, http://www. amgen.com, Locked Bag No. 6, Newtown, New South Wales 2042, Australia. Telephone: 61-2-9565-6156; Fax: 61-2-9565-6101; e-mail:
[email protected] Received June 9, 2008; accepted for publication August 6, 2008; first published online in STEM CELLS EXPRESS August 21, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2008-0560
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Figure 1. Experimental design: protocol for the administration of cytokines for mobilization experiments. Day 0 indicates when leukapheresis was performed. Exp 1 was designed to compare pegG-CSF with G-CSF, and Exp 2 compared G-CSF⫹pegMGDF with G-CSF⫹ SCF. Each experiment comprised four animals. Abbreviations: Exp, experiment; G-CSF, granulocyte colony-stimulating factor; pegGCSF, pegylated granulocyte colony-stimulating factor; pegMGDF, pegylated megakaryocyte growth and development factor; SCF, stem cell factor.
twice with filgrastim [13]. Its use in normal, healthy donors for allogeneic transplantation has also been explored; 20 of 25 donors were successfully harvested after one leukapheresis, having receiving a single 12-mg dose of pegG-CSF [14]. Steidl et al. reported the successful use of pegG-CSF in patients with multiple myeloma; however, the median CD34⫹ cell peak was lower compared with filgrastim (78 vs. 111/l) [15]. Thrombopoietin is the most important physiological regulator of megakaryopoiesis and thrombopoiesis [16]. Pegylated megakaryocyte growth and development factor (pegMGDF) is a nonglycosylated, truncated form of human thrombopoietin conjugated with peg [17, 18]. Since its development in the mid1990s, pegMGDF has been shown to mobilize PBSCs in patients with advanced cancer [19]. Levels of progenitors on day 15 of chemotherapy were significantly greater in patients administered 0.3–5 g/kg pegMGDF and filgrastim compared with those receiving placebo and filgrastim. In patients with solid tumors pegMGDF alone induced a dose-dependent mobilization of progenitors of all lineages [20]. The kinetics of mobilization is different from that of G-CSF, with the peak of mobilization occurring on day 12. In patients receiving highdose chemotherapy and autologous PBSC transplantation, recombinant thrombopoietin increased CD34⫹ cell yield and reduced the number of apheresis procedures required [21, 22]. Despite the demonstration of PBSC mobilization with pegG-CSF and pegMGDF, there are no studies directly comparing their effectiveness to that of standard cytokines. Human studies are confounded by small numbers and variability in patient factors such as the underlying disease, previous chemotherapy exposure, and the known interindividual variability. For ethical reasons, studies comparing the mobilization of hemopoietic progenitors with two or more cytokines in the same person, thereby controlling for these variables, is not possible. Using a robust experimental design in a nonhuman primate model, we sought to directly compare the ability of pegG-CSF and pegMGDF to mobilize hemopoietic progenitors with current clinically available mobilization regimens. Two separate experiments were performed; the first experiment compared pegGCSF with standard G-CSF, and the second compared the combination of pegMGDF plus G-CSF with SCF plus G-CSF.
MATERIALS
AND
METHODS
Animals Baboons (Papio hamadryas) were housed and handled in accordance with the Australian National Health and Medical Research
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Council policy on the care and use of nonhuman primates for scientific purposes (National Health and Medical Research Council, 2003). The experiments were approved and conducted under the guidance of the Royal Prince Alfred Hospital Animal Welfare Committee. Five- to 8-week-old specific pathogen-free NOD/SCID (substrain designation: NOD.CB17-Prkdcscid/ARC) female mice were obtained from the Animal Resources Centre (Perth, Western Australia, Australia). Mice were housed in a barrier facility in static microisolator cages with sterilized paper pelleted bedding. Nutritional requirements were provided with Gordon’s Stockfeed Irradiated Rodent Diet (Gordon’s Speciality Stock Feed Pty Ltd, Yanderra, New South Wales, Australia) ad libitum and sterile water. Use of these animals was approved by the University of Sydney Animals Ethics Committee.
Cytokine Administration and Experimental Design The experimental design is summarized in Figure 1. Male baboons, ages 7–11 years, received two regimens of subcutaneously injected cytokines as follows: experiment 1, G-CSF (100 g/kg/day) for 5 days versus pegG-CSF, single dose (300 g/kg); experiment 2, G-CSF (100 g/kg/day) ⫹ SCF (50 g/kg/day) for 5 days versus pegMGDF (1 g/kg) every second day for 10 days ⫹ G-CSF (100 g/kg/day) for 5 days. A total of four animals were included in each of the two experiments, and mobilizations in each animal were separated by a minimum of 12 weeks to control for the possibility of a priming effect caused by the initial mobilization [23]. For each regimen, two baboons received two identical mobilizing regimens in one order, whereas the other two animals received them in the reverse order. The doses of G-CSF and SCF were selected on the basis of previous studies in baboons [24, 25]. The doses of pegGCSF and pegMGDF were based on preliminary data in nonhuman primates [26, 27]. G-CSF, pegG-CSF, and SCF were provided by Amgen, and pegMGDF was provided by Kirin Brewery Co. (Tokyo, http://www.kirin.co.jp). Peripheral blood mononuclear cells (PBMCs) were collected at baseline and on the day of peak mobilization to quantify CD34⫹ cells, colony-forming cells (CFCs), and SCID repopulating cells (SRC).
Baboon Leukapheresis To harvest PBMCs for the SRC assay, baboons underwent leukapheresis using a Gambro Cobe Spectra (Gambro BCT, Denver, http://www.gambro.com). Animals were initially sedated with intramuscular ketamine and then anesthetized using inhalational halothane after endotracheal intubation. Leukapheresis outflow access comprised an 18-gauge arterial catheter (RA-04018; Arrow International, Reading, PA, http://www.arrowintl.com) placed in a femoral artery. The return line comprised an 18gauge venous cannula placed in a cubital fossa vein. Anticoagulation was achieved using the combination of sodium heparin (50 U/kg bolus followed by a continuous infusion at 400 U/hour; Pfizer, La Jolla, CA, http://www.pfizer.com) and acid citrate dextrose-A (ACDA; 0.9 ml/minute; Baxter Healthcare Corpora-
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Figure 2. Kinetics of neutrophils and CD34⫹ cells after administration of pegylated granulocyte colony-stimulating factor (pegG-CSF). The neutrophil and CD34⫹ cell counts are shown for four animals after a single dose of pegG-CSF (300 g/kg subcutaneously). Day ⫺4 was defined as the day of administration, in accordance with the experimental design in Figure 1. The peak CD34⫹ cell count occurred 5 days after administration, whereas the peak neutrophil count occurred after 3 days.
tion, Deerfield, IL, http://www.baxter.com). Animals were monitored using ECG, blood pressure, CO2, and pulse oximetry throughout leukapheresis. To avoid citrate-induced hypocalcaemia due to ACDA, blood ionized calcium levels were measured on at least two occasions during leukapheresis. To reduce the risk of severe hypocalcaemia, an infusion of calcium gluconate (640 mg/hour; Hospira Australia Pty Ltd, Melbourne, Victoria, Australia, http://www.hospira.com.au) was administered throughout the procedure and titrated to the ionized calcium level. An AutoPBSC collection set (catalog no. 777-006-100; Gambro BCT) was used according to the manufacturer’s instructions. The AutoPBSC kit was primed using 180 –240 ml of autologous blood that had been collected in ACDA up to 2 weeks prior to leukapheresis. A maximum of 120 ml of blood was collected on each of two occasions separated by 1 week. Blood was processed at a rate of 28 –32 ml/minute, and PBMCs were collected into a bag containing 20 ml of ACDA. At the completion of the leukapheresis collection, protamine sulfate (5 mg) was administered, and firm pressure was placed on the femoral artery after removal of the arterial line. Prior to the administration of cytokines and on the day of leukapheresis, each animal was anesthetized with intramuscular ketamine for bone marrow aspiration.
Baboon CD34ⴙ Cell Quantitation A single-platform method using an anti-human CD34 antibody (clone 563; BD Biosciences, San Diego, http://www.bdbiosciences. com) was used to quantitate baboon CD34⫹ cells (Stemkit; Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Peripheral blood (40 l) collected in EDTA was incubated with 10 l of anti-CD34 antibody for 20 minutes on ice in triplicate. Samples with more than 30 ⫻ 109 white blood cells per liter were diluted 1:1 with phosphate-buffered saline (PBS). Two milliliters of NH4 lysing solution was then added to each tube, and the samples were incubated for 10 minutes at room temperature and then analyzed on a FACSCalibur flow cytometer (BD Biosciences). Immediately prior to analysis, 40 l of stem-count fluorospheres (Stem Kit IM3630; Beckman Coulter) of known concentration were added to each tube. The CD34⫹ cell count was calculated by taking the ratio of CD34positive events divided by the number of fluorosphere-positive events and multiplying by the known concentration of fluorospheres (taking into account any dilution).
CFC Quantitation CFC were enumerated using MethoCult H4434 methylcellulose medium (StemCell Technologies, Vancouver, BC, Canada, http:// www.stemcell.com) according to the manufacturer’s protocol.
Briefly, 4 l of whole peripheral blood was added to 4 ml of MethoCult containing cytokines (GM-CSF, interleukin [IL]-3, SCF) and plated in triplicate into 35-mm Petri dishes (1 ml per dish). The plates were incubated at 37°C in 5% CO2 for 14 days, and colonies were counted at ⫻10 magnification using an MZFLIII microscope (Leica, Wetzlar, Germany, http://www.leica.com).
Baboon SRC Quantitation To quantify primitive hemopoietic cells, the SRC frequency of baseline and mobilized PBMCs was assessed using limiting dilution analysis by transplantation into cohorts of NOD/SCID mice following nonmyeloablative total body irradiation (TBI). The TBI was administered using a cesium-137 gamma ray source via a Gammacell 40 Exactor (MDS Nordion, Ottawa, http://www.mds.nordion. com) at a dose of 92 centigray (cGy) per minute to a total of 300 cGy, according to our published protocol [28]. The antibiotic Enrofloxacin (Bayer Health Care, Monheim, Germany, http://www. bayer.com; 25 mg/ml; approximately 50 mg/kg body weight) was added to the drinking water for all mice commencing 24 hours before TBI and continuing for the duration of the study. For mobilized PBMCs, cells collected using leukapheresis were injected via the tail vein (1 ⫻ 106 to 100 ⫻ 106 cells per mouse). Each mobilized sample required six cell dose cohorts of five mice (total, 30 mice per mobilized sample). For baseline PBMCs, 60 ml of peripheral blood was collected in EDTA via venipuncture, followed by isolation using light density centrifugation in Ficoll Plus Hypaque (GE Healthcare, Little Chalfont, U.K., http://www.gehealthcare.com). In brief, 60 ml was diluted 1:1 with PBS, and 10 ml of diluted blood was layered over 9 ml of Ficoll in 50-ml Falcon tubes. Following centrifugation at 400g for 30 minutes, the mononuclear layer was collected, washed, and then resuspended in Hanks’ balanced saline solution for injection into NOD/SCID mice and for CFU-F analysis (described below). Up to 5 ⫻ 106 PBMCs obtained at baseline were injected into each of five mice. Engraftment of baboon hemopoietic cells was assessed using flow cytometry of murine bone marrow harvested 5– 6 weeks after transplantation using an antibody against CD11a (clone HI111; BD Biosciences), which is expressed on all leukocytes. This antibody has been shown to cross-react against baboon but not murine hemopoietic cells (data not shown). This antibody was chosen in preference to the anti-human CD45 antibody, as pilot studies showed suboptimal cross-reactivity of the latter on baboon hemopoietic cells (in contrast to the CD11a antibody). Engraftment was defined as donor cells being more than 0.5% of total bone marrow nucleated cells for all experiments, and the SRC frequency was determined by applying Poisson statistics to the single-hit model individually for each of the mobilized samples
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[29, 30]. Due to the extremely low frequency of SRC in the peripheral blood at baseline, data from all animals at baseline were pooled to generate the baseline SRC frequency.
Statistics Comparison of the mobilization of CD34⫹ cells, CFC, and SRC between two cytokine regimens in the same animals was analyzed using the Wilcoxon signed rank test. Comparison of CD34⫹, CFC, and SRC between cytokine regimens in different experiments was analyzed using the Kruskal-Wallis test. SCID repopulating frequency was assessed using limiting dilution analysis and applying Poisson statistics as described above.
RESULTS Kinetics of CD34ⴙ Cell Mobilization with pegG-CSF The CD34⫹ cell and neutrophil kinetics over 10 days after administration of 300 g/kg pegG-CSF in four animals are summarized in Figure 2. These data demonstrate that the peak CD34⫹ cell level occurred 5 days after administration in all four animals. The neutrophil count peaked on the 3rd day before reducing over the subsequent week. Notably, the CD34⫹ cell count had fallen sharply by the 7th day; however, it is important to emphasize that for animal welfare reasons, peripheral blood was not collected every day, and therefore the CD34⫹ cell peak may have occurred on either the 4th or 6th day.
Pegylated G-CSF Mobilized Hemopoietic Precursors to a Level Similar to That of Standard G-CSF The experimental design summarized in Figure 1 controlled for interindividual variation by comparing mobilization with two different cytokines in the same animal. The measurement of CD34 ⫹ cells, CFC, and SRC was conducted on the peak day of mobilization. Figure 3 shows the peak CD34⫹ cell (Fig. 3A), CFC (Fig. 3B), and SRC (Fig. 3C) mobilization in animals from experiment 1 after administration of G-CSF and pegG-CSF. The degree of mobilization was similar between the two cytokines in each animal for all of the three cell populations quantified. The fold increases compared with baseline after G-CSF of CD34⫹ cells, CFC, and SRC were 15.1, 17.6, and 11, respectively, whereas they were 20.7, 16.7, and 8.0 after pegG-CSF.
G-CSFⴙpegMGDF Mobilized Hemopoietic Precursors to a Level Similar to G-CSFⴙSCF, Which Is Superior to G-CSF or pegG-CSF Alone The degree of mobilization of CD34⫹ cells, CFC, and SRC in four additional animals receiving G-CSF⫹SCF and G-CSF⫹ pegMGDF is summarized in Figure 4. The degree of mobilization of the three cell types was similar between the two regimens and also between all four animals. The fold increases compared with baseline after G-CSF⫹SCF of CD34⫹ cells, CFC, and SRC were 49.2, 41.25, and 59.5, respectively, whereas they were 58.8, 42.2, and 60.2 after G-CSF⫹pegMGDF. A comparison of all four regimens is summarized in Figure 5. This graph includes the data on mobilizations in addition to the two experiments described (four additional animals for G-CSF and five for G-CSF⫹SCF). Of note, as a demonstration of interindividual variation, animal 1 in experiment 1 mobilized significantly more (up to 10 times) than the other three animals, although the level was similar for either cytokine. Results with G-CSF alone in four other animals were similar to those observed in animals 2, 3, and 4. For this reason, animal 1 was considered an outlier and excluded from the analysis reprewww.StemCells.com
Figure 3. Mobilization after G-CSF and pegG-CSF treatment in four animals. A summary of CD34⫹ cell (A), colony-forming cell (CFC) (B), and SRC (C) mobilization after G-CSF and pegG-CSF in four baboons in experiment 1 (n ⫽ 4). The measurement of CD34 ⫹ cells, CFC, and SRC was performed on the peak day of mobilization. The p value represents the comparison between mobilization with the two cytokines in the same animal. Abbreviations: G-CSF, granulocyte colony-stimulating factor; pegG-CSF, pegylated granulocyte colony-stimulating factor; SRC, SCID repopulating cell.
sented in Figure 5. The combination of G-CSF and pegMGDF mobilized CD34⫹ cells, CFC, and SRC to levels similar to G-CSF and SCF but greater than G-CSF alone. The result
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Figure 4. Mobilization after G-CSF⫹pegMGDF and G-CSF⫹SCF treatment in four animals. A summary of CD34⫹ cell (A), colonyforming cell (B), and SRC (C) mobilization after G-CSF⫹SCF and G-CSF⫹pegMGDF in experiment 2 (n ⫽ 4). The p value represents the comparison between mobilization with the two cytokines in the same animal. Abbreviations: G-CSF, granulocyte colony-stimulating factor; pegMGDF, pegylated megakaryocyte growth and development factor; SCF, stem cell factor; SRC, SCID repopulating cell.
for G-CSF⫹SCF compared with G-CSF mobilization was statistically significant for CD34⫹ cells (p ⫽ .002) and CFC (p ⫽ .005) but borderline for SRC (p ⫽ .057). The comparison of G-CSF⫹pegMGDF was statistically significant for CD34⫹ cells
PBSC Mobilization in a Nonhuman Primate Model
Figure 5. Mobilization of CD34⫹ (A), CFC (B), and SRC (C) from baseline using all cytokine regimens. Mobilization of primitive hemopoietic cell types following G, pegG, G⫹S, and G⫹pegM. This graph includes data on mobilizations performed in addition to the two experiments, including four G-alone and five G-CSF⫹SCF mobilizations. The number of animals is indicated below the cytokine or combination tested. Animal 1, which received G-CSF versus pegG-CSF, was excluded as an outlier. Abbreviations: CFC, colony-forming cell; G, granulocyte colony-stimulating factor; pegG, pegylated granulocyte colony-stimulating factor; pegM, pegylated megakaryocyte growth and development factor; S, stem cell factor; SRC, SCID repopulating cell.
(p ⫽ .006) and CFC (p ⫽ .012) but was also of borderline significance for SRC (p ⫽ .057).
DISCUSSION pegG-CSF is now routinely used in clinical practice to improve neutrophil recovery after chemotherapy. The convenience of administering a single injection is very attractive, and a single
Larsen, Chng, Battah et al. injection appears to be at least as effective as once-daily injections with standard G-CSF [31]. No previous study has directly compared whether pegG-CSF can mobilize PBSC as effectively as G-CSF in a given individual. Although it is clear that pegGCSF does mobilize hemopoietic progenitors in humans, it is difficult to perform studies that allow a direct comparison in a rigorous manner. The experimental design used in these studies allowed the direct comparison of pegG-CSF versus G-CSF in the same animals, controlling for interindividual variation. Analysis of the kinetics of pegG-CSF demonstrated that the peak neutrophil count occurred on day 3, whereas the peak CD34⫹ cell count occurred on day 5. Since the peripheral blood was collected on days 1, 3, 5, 7, and 10, the peaks may have occurred 1 day earlier or later. These results recapitulate the kinetics observed from human data, which suggested that the peak CD34⫹ cell count occurs on day 4 [9], and offer additional confidence in validation of the nonhuman primate model. The degree of CD34⫹, CFC, and SRC mobilization was similar in all four animals investigated, although one animal mobilized to a greater degree than the other three, emphasizing the known interindividual variation and justifying our experimental design. This finding suggests that pegG-CSF has the potential to be used instead of standard G-CSF to mobilize hemopoietic progenitors in the context of a patient in whom daily administration is difficult. In addition to convenience, work by Morris et al. suggests that the cellular content of pegG-CSF-mobilized harvests may provide a qualitative benefit from the point of view of minimizing graft-versus-host disease (GVHD) [32]. In a murine model, pegG-CSF was markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation due to the generation of IL-10-producing regulatory T-cells. In a subsequent phase 1/2 study, it was demonstrated that a single dose of pegG-GSF (12 mg) mobilized CD34⫹ cells in normal, healthy human donors, although a direct comparison with standard G-CSF was not made [33]. The peak CD34⫹ count was 99 ⫾ 11/l, and 12 of 13 donors collected sufficient stem cells for transplantation in a single apheresis. The benefit of adding SCF to G-CSF in mobilizing CD34⫹ cells is well established [34 –36], although the availability of SCF has been limited to a few countries because of concerns about mast cell activation. The risks of being denied a potentially life-saving PBSC transplant may need to be recalibrated against the known risks of SCF in light of our results. Compared with the combination of G-CSF plus SCF, the combination of G-CSF plus pegMGDF mobilized baboon CD34⫹ cells, CFC, and SRC to similar levels. Analysis comparing all four regimens showed that, compared with G-CSF, the increase in mobilization following G-CSF ⫹ pegMGDF was statistically significant for CD34⫹ cells and CFC and borderline for SRC. Despite early promise, the further investigation of recombinant thrombopoietins to enhance PBSC mobilization ceased
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because of the development of immune-mediated persistent thrombocytopenia (platelet count, ⬍100 ⫻ 109 per liter). Thrombocytopenia was observed in 13 of 325 healthy volunteers who received two or three doses of pegMGDF and in four of 650 oncology patients undergoing intensive nonmyeloablative chemotherapy who received multiple doses [37]. However, there is a renewed interest in megakaryopoiesis-stimulating molecules, such as AMG531, AKR-501, and eltrombopag, all of which are currently undergoing clinical development in thrombocytopenia [38, 39]. Phase 3 clinical trials have now been completed that demonstrate the efficacy and safety of AMG531 (Romiplostim, Amgen) and eltrombopag in patients with chronic idiopathic thrombocytopenia (ITP) [40, 41], and trials are proceeding in other disorders, such as myelodysplastic syndrome. At the time of writing, approval for Romiplostim is anticipated in Australia for patients with chronic ITP. On the basis of their mechanism of action through Mpl [42], these molecules may have an effect similar to that of thrombopoietin on mobilization, providing potential for an additional option for patients who are difficult to mobilize using standard approaches. The importance of this study is that using a robust nonhuman primate model of PBSC mobilization, (a) a single dose of pegG-CSF mobilized was as effective as daily doses of standard G-CSF, and (b) pegMGDF plus G-CSF was superior to standard G-CSF alone. To increase the available options in the patient who is difficult to mobilize, additional studies are warranted using the newer generation of thrombopoietin mimetics.
ACKNOWLEDGMENTS We thank Annemarie Hennessy, Sally Thomson, and Scott Heffernan for assistance with the baboon colony; Margaret Armstrong, Jillian Severs, Yok Eng Loh, Kate Walsh, and Louise Duke for leukapheresis operation; Doug Joshua and John Gibson for support; Louie Leung and Marcus Hayward for technical assistance; Lisa Barrow, Kon Zarkos, and Simon Cooper for assistance with cell processing; Jenny Kingham for advice and help with the NOD/SCID colony; Gambro BCT (Denver) for providing access to a Cobe Spectra Apheresis machine; and Amgen and Kirin Brewery for providing cytokines. S.R.L. was supported by the Anthony Rothe Trust and Cancer Institute New South Wales. This work was funded in part by the Leukemia Foundation of Australia, Cell and Gene Trust and an anonymous foundation.
DISCLOSURE
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CONFLICTS
The authors indicate no potential conflicts of interest.
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