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Hematopoietic Recovery Following Autologous Bone Marrow Transplantation in a Nonhuman Primate: Effect of Variation in Treatment Schedule with PEG-rHuMGDF ANN M. FARESE,a THOMAS J. MACVITTIE,a LORIN ROSKOS,b RICHARD B. STEADb a
University of Maryland Greenebaum Cancer Center, Baltimore, Maryland, USA; b Amgen, Inc., Thousand Oaks, California, USA Key Words. PEG-rHuMGDF · Nonhuman primate · Platelet · Hematopoietic · Bone marrow transplant
A BSTRACT Mathematical modeling of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) pharmacokinetics (PK) and pharmacodynamics (PD) suggest that variations in the PEGrHuMGDF treatment schedule could reduce the severity and duration of thrombocytopenia following myeloablation and bone marrow transplant (BMT). We tested this hypothesis in a rhesus monkey model of autologous (Au) bone marrow-derived mononuclear cell (BM-MNC) transplantation following lethal myeloablation. On day 0, animals were myeloablated by total body exposure to 920 cGy, 250 kVp x-irradiation (TBI). Four cohorts of animals were infused with 1 × 108 AuBMMNC/kg body weight within 2 hours of TBI. The AuBMT-alone cohort received no cytokine, the daily dosage cohort received PEG-rHuMGDF (2.5 µg/kg/day, s.c.) post TBI and AuBMT, and the pre/post-transplant cohort received PEG-rHuMGDF (2.5 µg/kg/day, s.c.)
pre (day -9 to day -5) and post TBI and AuBMT. The post-transplant PEG-rHuMGDF administration in the above cohorts was begun on day 1 post TBI and continued until platelet counts reached 200,000 µl (range, 1531 days). Another group received PEG-rHuMGDF (300 µg/kg/day, s.c.) on days 1 and 3 only following TBI and AuBMT. The TBI controls received neither AuBMT nor cytokine therapy. In this model of AuBMT, with regard to the PEG-rHuMGDF administration schedule, the daily dosage of the post-transplant cohort did not significantly improve platelet recovery; the pre/posttransplant schedule and an abbreviated high-dosage, post-transplant schedule (days 1 and 3) significantly improved the duration and nadir of thrombocytopenia and platelet recovery. These data confirm predictions from PK/PD modeling of PEG-rHuMGDF that thrombocytopenia is preventable following AuBMT. Stem Cells 2003;21:79-89
INTRODUCTION Bone marrow (BM) and/or mobilized peripheral bloodderived stem cell transplantation (PBSCT) are methods of choice for a variety of hematologic diseases and certainly may
allow for new generation treatment protocols involving doseintensive myeloablative regimens. While PBSCT has resulted in significantly improved engraftment parameters [1-4], hematopoietic reconstitution following myeloablative therapy
Correspondence: Ann M. Farese, M.S., University of Maryland Greenebaum Cancer Center, 655 West Baltimore Street, Baltimore, Maryland 21201, USA Telephone: 410-328-5347; Fax: 410-328-5488; e-mail:
[email protected] Received July 22, 2002; accepted for publication September 4, 2002. ©AlphaMed Press 1066-7159/2003/$5.00/0
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and autologous (Au) bone marrow transplant (BMT) is relatively prolonged and associated with considerable morbidity [5-10]. In clinical trials and preclinical studies, administration of myeloid growth factors (GFs) following BMT has improved the time to neutrophil recovery [11-16]. Results from pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or thrombopoietin (TPO) treatment protocols in rodent, canine, and nonhuman primate models of respective syngeneic BMT, allogeneic (allo)BMT, and AuBMT have been contradictory. Several studies using rodent models showed significant efficacy of PEG-rHuMGDF in stimulating platelet (PLT) recovery following syngeneic BMT [17-19]. Fibbe et al. [20], however, could not demonstrate such efficacy unless the marrow inoculum was derived from TPO-treated donor animals. Three additional studies in models using the nonhuman primate or canine demonstrated that standard post-transplant administration of TPO or PEG-rHuMGDF could not accelerate PLT reconstitution following AuBMT or alloBMT, respectively, in myeloablated hosts [21-23]. These contradictory results were surprising in light of the consistent and substantial preclinical data base demonstrating the significant therapeutic efficacy of the Mpl-ligands (Mpl-Ls) in accelerating PLT recovery in both modest and severe models of myelosuppression in rodents, canines, and nonhuman primates [23-35]. In fact, additional studies demonstrated that the administration schedule of the Mpl-L or promegapoietin, an engineered chimeric receptor agonist containing the Mpl-L, could be significantly abbreviated, such as in a single or two separate injections, and yet achieve PLT recovery equivalent to the conventional multiple daily dose protocols [24, 28, 31, 32, 36]. The successful transition of the Mpl-Ls through clinical trials has proved difficult. Early trials demonstrated that MplLs were potent stimulators of thrombopoiesis and enhanced PLT recovery from chemotherapy-induced myelosuppression consistent with the preclinical literature [37-41]. However, the demonstration of clinically meaningful benefit following more intensive cytotoxic therapies has remained elusive [42-44]. Additional studies using the full-length glycosylated molecule, rHuTPO, have demonstrated clinical efficacy in ameliorating chemotherapy-induced thrombocytopenia when administered via the intravenous route, and it remains in clinical trials for further evaluation of schedule and dose modification [40, 45]. However, several trials evaluating the treatment efficacy of rHuTPO after Au stem cell transplant (SCT) have shown no correlation between the dose of rHuTPO and recovery of PLT counts [46-49]. These results suggested that control of cytotoxic therapyassociated thrombocytopenia might be dependent on defining optimal dose, schedule, and route of administration of the respective Mpl-Ls. Roskos et al. approached this through the
PEG-rHuMGDF Enhances Hematopoietic Recovery Post AuBMT use of a cytokinetic model of PLT production and destruction following administration of PEG-rHuMGDF to normal and myeloablated rhesus macaques [50]. The pharmacodynamics (PD) of PEG-rHuMGDF are dependent on the pharmacokinetics (PK) of PEG-rHuMGDF, the cytokinetics of megakaryocytes (MKs) and PLTs, and the kinetics of induced myelosuppression. These investigators suggested an optimum dose and schedule based on model parameters derived from the PK/PD profiles of the normal and myeloablated rhesus macaques. Therefore, in this study we examined the relative efficacy of administering PEG-rHuMGDF in three different regimens: a daily dose post-transplant regimen, an abbreviated high-dose post-transplant regimen, and pretreatment plus post-treatment at the same dosage as the daily post-transplant cohort compared with controls receiving no cytokine support. MATERIALS AND METHODS Animals Domestic-born, male rhesus monkeys, Macaca mulatta, 3-5 kg, were housed in individual stainless steel cages in conventional holding rooms at the University of Maryland Veterinary Science Department in an animal facility accredited by the American Association for Accreditation of Laboratory Animal Care. Monkeys were provided 10 air changes/hour of 100% fresh air, conditioned to 72° ± 2°F with a relative humidity of 50% ± 20%, and maintained on a 12-hour light/dark fullspectrum light cycle, with no twilight. Monkeys were provided with commercial primate chow, and supplemented with fresh fruit and tap water ad libitum. Research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, National Research Council. Radiation Rhesus monkeys received myeloablative conditioning as total body exposure to a midline tissue dose of 920 cGy, 250 kVp x-irradiation at 13 cGy/minute. Ketamine-anesthetized animals (Ketaset® [10 mg/kg, i.m.], Fort Dodge Laboratories; Fort Dodge, Indiana) were placed in a Plexiglass restraint chair (to which they had been previously prehabituated), allowed to regain consciousness and were x-irradiated in the posterior-anterior direction, then rotated at mid-dose to the anterior-posterior direction to complete the exposure. Dosimetry was performed using paired 0.5-cm3 ionization chambers, with calibration factors traceable to the National Institute of Standards and Technology. Bone Marrow Harvest Rhesus monkeys were anesthetized with ketamine plus buprenorphine (Buprenex® Injectable [10 µg/kg, i.m.], Reckett
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& Coleman Pharmaceuticals; Richmond, Virginia). Following sedation, approximately 30-40 ml of heparinized BM were harvested from the humeri and/or iliac crest. Low-density (10 kg) was administered when the PLT count was 500/µl by day 16.7 ± 1.0 days (Fig. 2B, Table 1). Daily Post-Transplant PEG-rHuMGDF Administration The administration of PEG-rHuMGDF following AuBMT using a daily dose (2.5 µg/kg/day) treatment (range, 15-31 days until attainment of PLT = 200,000/µl), did not significantly improve the duration of thrombocytopenia (3.7 days ± 2.3; p = 0.305), the PLT nadir (21,333/µl ± 6,960), or day of recovery of PLT counts to ≥ 30,000/µl (day 9.7 ± 4.9; p = 0.182) relative to the AuBMTonly controls (5.6 days and 20,300/µl, respectively) (Fig. 2A, Table 1). Neither did the post-transplant administration of PEG-rHuMGDF significantly improve neutrophilrelated parameters compared with the AuBMT-only cohort (Fig. 2B, Table 1). Pre/Post-Transplant PEG-rHuMGDF Administration As expected from PK/PD modeling [50, 51], pretreatment of animals with PEG-rHuMGDF from days -9 to -5 prior to transplant increased the circulating PLT counts to 1,500,000/µl on day 0, immediately prior to marrow aspiration, myeloablation, and transplant. Daily PEG-rHuMGDF administration (2.5 µg/kg/day) was resumed on day 1 post transplant according to protocol. This schedule of pre/posttransplant administration of PEG-rHuMGDF eliminated the duration of thrombocytopenia relative to the control
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Figure 2. A) Mean peripheral PLT × 103/µl and B) mean peripheral ANC × 103/µl (± standard error [SE]) in rhesus monkeys after myeloablation. Animal received either no AuBMT (● Control, n = 2), AuBMT with no cytokine treatment (䡩 AuBMT, n = 9), or AuBMT treatment with daily therapeutic administration of PEG-rHuMGDF (2.5 µg/kg/day, from day 1 post AuBMT until PLT reached 200,000/µl, 䡲 PEG-rHuMGDF, n = 4). Treatment with PEG-rHuMGDF ranged from 15-31 days post AuBMT.
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Table 1. Hematopoietic recovery following AuBMT in nonhuman primates: effect of variation in PEG-rHuMGDF dose and schedule Platelet-related parameters Treatment
Duration (days) PLT
