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Stem Cells 2003;21:79-89 www.StemCells.com
© 2003 AlphaMed Press

Hematopoietic Recovery Following Autologous Bone Marrow Transplantation in a Nonhuman Primate: Effect of Variation in Treatment Schedule with PEG-rHuMGDF

Ann M. Faresea, Thomas J. MacVittiea, Lorin Roskosb, 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

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:
afarese{at}umaryland.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mathematical modeling of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) pharmacokinetics (PK) and pharmacodynamics (PD) suggest that variations in the PEG-rHuMGDF 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 x 108 AuBM-MNC/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, 15-31 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/post-transplant 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone marrow (BM) and/or mobilized peripheral blood-derived stem cell transplantation (PBSCT) are methods of choice for a variety of hematologic diseases and certainly may allow for new generation treatment protocols involving dose-intensive myeloablative regimens. While PBSCT has resulted in significantly improved engraftment parameters [1–4], hematopoietic reconstitution following myeloablative therapy 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 Mpl-Ls 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 therapy-associated 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 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 full-spectrum 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 & Coleman Pharmaceuticals; Richmond, Virginia). Following sedation, approximately 30-40 ml of heparinized BM were harvested from the humeri and/or iliac crest. Low-density (<1.077 g/cm3) mononuclear cells (MNCs) were separated using Histopaque (Sigma Aldrich; St. Louis, Missouri; http://www.sigmaaldrich.com) and resuspended in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO; Grand Island, New York; http://www.lifetech.com).

Study Design
For each experimental cohort, animals were irradiated on day 0 and thereafter were provided clinical support as required. The irradiation controls (n = 2) received neither AuBM-MNCs nor cytokine therapy prior to or following myeloablation. Four cohorts of animals were each infused with 1 x 108 AuBM-MNCs/kg body weight within 2 hours of irradiation exposure (Fig. 1Go). The control AuBMT cohort (n = 9) received autologous serum (0.1%, s.c.) for 18 consecutive days; the daily dose cohort (n = 3) received PEG-rHuMGDF (2.5 µg/kg/day, s.c., 18 days) (Amgen, Inc.; Thousand Oaks, California; http://www.amgen.com) beginning on day 1 and continuing until the animal had a PLT count of 200,000/µl (range, 26-30 days). The pre/post-treatment cohort (n = 4) received PEG-rHuMGDF (2.5 µg/kg/day, s.c.) prior to myeloablation from day -9 through day -5, and then again following total body irradiation (TBI) and AuBMT as per the daily dose-cohort postirradiation; the high-dose abbreviated post-transplant cohort (n = 5) received PEG-rHuMGDF (300 µg/kg/day, s.c.) on days 1 and 3 post TBI and AuBMT.



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  		Figure 1. Schedule and dose variations for PEG-rHuMGDF administration in a nonhuman primate model of AuBMT. A) Daily dose PEG-rHuMGDF administration post transplant (2.5 µg/kg/day, qd, day 1 to PLT of 200,000/µl); B) Pretransplant (day -9 to day -5) and post-transplant PEG-rHuMGDF administration (2.5 µg/kg/day, qd, as in A above), and C) Post-transplant high-dose PEG-rHuMGDF administration (300 µg/kg/day, days 1 and 3).

 
Clinical Support
All animals received clinical support that consisted of antibiotics, fresh irradiated whole blood, and fluids as needed. An antibiotic regimen was initiated prophylactically when the WBC was <1,000/µl and continued daily until the WBC was >1000/µl for 3 consecutive days. Gentamicin 10 mg/day (Abbott Laboratories; North Chicago, IL) and Rocephin 250 mg/day (Roche; Nutley, New Jersey; http://www.roche.com) were given s.c. Fresh, irradiated (1,500 cGy Co-60) whole blood (approximately 30 ml/transfusion) from a random donor pool (monkeys >10 kg) was administered when the PLT count was <20,000/µl and the hematocrit was <18%.

Hematologic Evaluations

Complete Blood Counts   Peripheral blood was obtained from the saphenous vein to assay complete blood (Sysmex K-4500, Long Grove, Illinois; http://www.sysmex.com/usa) and differential counts (Wright-Giemsa Stain, Ames Automated Slide Stainer, Elkhart, Indiana) at selected time points up to 50 days post TBI and AuBMT. The durations of thrombocytopenia (PLT <30,000/µl) and neutropenia (absolute neutrophil count <500/µl) and time to recovery to PLT counts >30,000/µl and neutrophil counts >500/µl were assessed.

BM-Derived Clonogenic Assay   Culture medium contained 0.9% methylcellulose (MethoCult H4230, Stem Cell Technologies; Vancouver, British Columbia; http://stemcell.com) in IMDM. In addition, a combination of recombinant human cytokines, G-CSF (5 ng/ml), stem cell factor (50 ng/ml), erythropoietin (2 U/ml), PEG-rHuMGDF (20 ng/ml), interleukin (IL)-3 (20 ng/ml), GM-CSF (5 ng/ml), and IL-6 (40 ng/ml) (Sandoz Pharmaceuticals; East Hanover, New Jersey) were added to each culture dish. MNCs were cultured at a plating density of 3-5 x 104 cells/ml (days 0, 21, 48 post TBI) or 1 x 105 cells/ml (days 7, 14 post TBI). Cells were incubated for 10 days at 37°C with 5% CO2 in air in a fully humidified incubator. Granulocyte macrophage-colony forming cell (GM-CFC)- and BFU-E-derived colonies (>50 cells) were expressed as the number of CFCs/105 MNCs. MK-CFCs were not distinguished from MK-BFUs in this assay. All MK colonies ranged from 10-50 cells.

Statistical Analysis
The Normal Scores test was used to make pairwise comparisons of the durations and nadirs of neutropenia and thrombocytopenia. The exact p values were obtained. The test was carried out using the software package StatXact (Cytel Software Corp.; Cambridge, Massachusetts; http://www.cytel.com). Mean values are presented ± the standard error. The clonogenic data were analyzed by applying a square root transform to all data. A repeated-measures analysis followed by Dunnett’s test was used to compare each day’s values with day 0. Significant differences among treatments were determined by applying a Least Significant Difference post hoc (two-sided) test to groups that had a significant one-way analysis of variance.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AuBMT-Associated Engraftment

Control Cohorts   The irradiation-only control animals succumbed by day 19 to the hematopoietic syndrome consequent to 920 cGy midline tissue (250 kVp x-irradiation) TBI. Each animal experienced severe thrombocytopenia and absolute neutropenia, and marked decrease of hematocrit despite aggressive clinical support consisting of antibiotics, fluids, and whole blood transfusions. Animals receiving TBI and transplanted with 1 x 108 AuBM-MNCs without cytokine support re-established hematopoiesis following a thrombocytopenic duration of 5.6 ± 1.5 days and a PLT nadir of 20,300/µl ± 2,700/µl. The PLT count recovered to >=30,000/µl by day14.8 ± 2.2 days (Fig. 2AGo, Table 1Go). Neutrophil engraftment was characterized by a neutropenic duration of 11.4 ± 0.9 days, a neutrophil nadir of 36/µl ± 9/µl, and a recovery to ANC >500/µl by day 16.7 ± 1.0 days (Fig. 2BGo, Table 1Go).




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  		Figure 2. A) Mean peripheral PLT x 103/µl and B) mean peripheral ANC x 103/µl (± standard error [SE]) in rhesus monkeys after myeloablation. Animal received either no AuBMT ({bullet} Control, n = 2), AuBMT with no cytokine treatment ({circ}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, {blacksquare} 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
 

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 AuBMT-only controls (5.6 days and 20,300/µl, respectively) (Fig. 2AGo, Table 1Go). Neither did the post-transplant administration of PEG-rHuMGDF significantly improve neutrophil-related parameters compared with the AuBMT-only cohort (Fig. 2BGo, Table 1Go).

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/post-transplant administration of PEG-rHuMGDF eliminated the duration of thrombocytopenia relative to the control AuBMT-only cohort (5.6 days) (p = 0.007) and was less than, although not significantly different from (p = 0.107), the PEG-rHuMGDF post-transplant-only cohort (3.7 days) (Fig. 3AGo, Table 1Go). The mean PLT nadir was 66,250/µl ± 12,809 relative to 20,300/µl and 21,333/µl for the AuBMT-only cohort (p = 0.001) and PEG-rHuMGDF post-transplant-only cohorts (p = 0.029), respectively. The cohort receiving the PEG-rHuMGDF treatment pre/post-AuBMT never experienced PLT counts below 30,000/µl as compared with the controls (recovery at day 14.8, p = 0.294) or the PEG-rHuMGDF treatment post-transplant-only cohort (recovery to PLT >=30,000/µl at day 9.7, p = 0.143).




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  		Figure 3. A) Mean peripheral PLT x103/µl and B) mean peripheral ANC x 103/µl (± standard error) in rhesus monkeys after myeloablation and AuBMT. Animals received either no cytokine treatment ({circ} AuBMT, n = 9), PEG-rHuMGDF administration pre- and post-transplant (2.5 µg/kg/day, from day -9 to day -5 and day 1 post AuBMT until PLT reached 200,000/µl, {bigtriangleup} PEG-rHuMGDF pre/post AuBMT, n = 4), or high-dose PEG-rHuMGDF administration (300 µg/kg/day) on days 1 and 3 only post AuBMT ({triangleup} high-dose PEG-rHuMGDF post AuBMT, n = 5).

 
The addition of the pretreatment schedule to the post-transplant administration of PEG-rHuMGDF did not significantly improve the duration of neutropenia (9.0 days ± 1.9, p = 0.123) nor the day of recovery of ANC to >500/µl (day 14.3 ± 2.1, p = 0.147) and did not improve the ANC nadir (30.5/µl ± 9.9, p = 0.355) relative to the control. When compared to the PEG-rHuMGDF post-transplant-only cohort, the pre/post-treatment schedule cohort experienced a significantly shorter duration of neutropenia (9.0 days versus 15.3 days, p = 0.029), an improved nadir (30.5 versus 19.0, p = 0.20), and earlier recovery of the ANC (day 14.3 versus 20.3, p = 0.057) (Fig. 3BGo, Table 1Go).

Abbreviated High-Dose Post-Transplant PEG-rHuMGDF Administration   PEG-rHuMGDF (300 µg/kg/day) administered on days 1 and 3 following AuBMT significantly improved the PLT nadir (28,600/µl ± 3,789 versus 20,300/µl, p = 0.053), the duration of thrombocytopenia (1.0 days ± 0.6 versus 5.6 days, p = 0.022), and shortened the day of PLT recovery to >=30,000/µl (day 4.4 ± 2.7 versus day 14.8, p = 0.008) as compared with the control AuBMT cohort (Fig. 3AGo, Table 1Go). The thrombocytopenic duration and time to PLT count recovery of >=30,000/µl following the high-dose abbreviated schedule were comparable (p = 0.278) to those observed for the pre- and post-PEG-rHuMGDF treatment cohort (1.0 and 4.4 days versus 3.7 and 9.7 days, respectively).

The high-dose abbreviated schedule of PEG-rHuMGDF administration significantly shortened the duration of neutropenia (7.0 days ± 1.3 versus 11.4 days [p = 0.022] and 15.3 days [p = 0.018]) and improved the neutrophil nadir (74.4/µl ± 15.8 versus 36.0/µl [p = 0.027] and 19/µl [p = 0.054]) relative to the control AuBMT and post-transplant-only PEG-rHuMGDF-treated AuBMT cohorts (Fig. 3BGo, Table 1Go). The day of recovery for ANC to >500/µl (day 12.0 ± 1.0) was also significantly shortened in this cohort as compared with the AuBMT controls (day 16.7, p = 0.01) and the post-treatment cohort (day 20.3, p = 0.018) but was comparable to the pre/post-treatment cohort (day 14.3, p = 0.222).

Bone Marrow-Derived Clonogenic Activity   Clonogenic frequency (CFC/105 MNCs), assessed at selected times pre- and post-AuBMT, suggested that the administration of PEG-rHuMGDF in the post-AuBMT or the high-dose days 1 and 3 post-AuBMT schedules did not influence overall recovery of BM-derived MK-CFC, GM-CFC, or BFU-E relative to the daily administration PEG-rHuMGDF cohort. However, PEG-HuMGDF administered in the pre/post-AuBMT schedule significantly (p = 0.01) increased the concentration of MK-CFCs in the day 0 (63/105 MNCs) bone marrow transplant inoculum relative to the control AuBMT-alone (18/105 MNCs) inoculum (Fig. 4Go).



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  		Figure 4. Bone marrow-derived concentration of (A) GM-CFC, (B) MK-CFC, (C) BFU-E, and (D) granulocyte-erythroid-macrophage-megakaryocyte (GEMM) after myeloablation and AuBMT. Animals received either no cytokine treatment (n = 9), PEG-rHuMGDF administration post transplant (2.5 µg/kg/day, day 1 post AuBMT until PLT reached 200,000/µl, n = 3), PEG-rHuMGDF administration pre/post-transplant (2.5 µg/kg/day, from day -9 to -5 and day 1 post AuBMT until PLT reached 200,000/µl, n = 4), or high-dose PEG-rHuMGDF administration (300 µg/kg/day) on days 1 and 3 only post AuBMT (n = 5). Clonogenic concentrations (per 105 MNC) are reported as mean values ± standard error. Abbreviation: BL = baseline; ND = none detected.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are consistent reports in the literature showing the rapid recovery of PLTs induced by administration of the Mpl-Ls in rodent, canine, and nonhuman primate models of moderate to severe radiation- or chemotherapy-induced myelosuppression [23–28, 30–32, 34, 35, 52, 53]. The use of various forms of the Mpl-L in a standard, daily treatment schedule consistently improved all PLT-related parameters and, in some instances, demonstrated enhanced multilineage recovery.

Additional preclinical studies evaluating the Mpl-Ls suggested that the efficacy of PEG-rHuMGDF or rHuTPO for enhancing PLT production following radiation-induced myelosuppression or SCT might be dependent on the treatment schedule and/or dose [20–22]. With regard to BMT, several studies in large animals demonstrated that daily administration of either rHuTPO or PEG-rHuMGDF at a standard dose (2.5-10 µg/kg/day) for several weeks post transplant did not increase PLT recovery relative to the control cohorts [21–23]. Neelis et al. and Farese et al., using primate models of AuBMT, and Nash et al., using a canine model of alloBMT, were consistent in demonstrating that a standard dose and schedule of the Mpl-Ls did not enhance early recovery of PLTs post transplant [21–23]. In this study, we demonstrated that thrombopoiesis following myeloablation and AuBMT can be enhanced by varying the schedule and/or dose of PEG-rHuMGDF administration.

The successful transition of the Mpl-Ls to clinical trials has proved difficult. Early trials demonstrated the ability to stimulate thrombopoiesis [37–40]. However, conventional doses (1.0-25 µg/kg/day) and schedule of daily administration of rHuTPO or Peg-rHuMGDF were ineffective in demonstrating meaningful benefit post SCT [46–49, 54]. Neither dose nor schedule of the Mpl-Ls appeared to have any effect on the time course of PLT recovery, duration of thrombocytopenia, or transfusion requirements. Also, the demonstration of clinically meaningful outcome in patient populations submitted to more intensive cytotoxic therapies remained elusive for PEG-rHuMGDF administered in standard dose and schedule [42, 43]. Several clinical trials, however, have shown the ability of rHuTPO to abrogate chemotherapy-induced thrombocytopenia using variations in the administration schedule [40, 45, 55]. These results suggested that schedule modification such as pre- and post-dosing via intravenous bolus would be effective in attenuating thrombocytopenia following dose-intensive chemotherapy.

Additional insights into the possible mechanism(s) of action and potential clinical efficacy of rHuTPO are provided by a number of studies in mice and nonhuman primates [24, 28, 31, 32, 53, 56–58]. These studies suggest that timing and dose of rHuTPO may have a significant impact on the viability and proliferation of hematopoietic stem cells (HSCs) subsequent to myelosuppression or lethal cytotoxic insult. A single administration of rHuTPO at pharmacologic doses, within 24 hours of myelosuppressive irradiation or chemotherapy, stimulated hematopoietic recovery in a manner equivalent to that noted with conventional, daily administration [24, 31, 32, 53, 58]. It is of further interest that a single administration of a relatively high dose of rHuTPO within an immediate to 24-hour time frame after supralethal doses of radiation or chemotherapy could stimulate early hematopoietic restoration and prevent lethality [28, 56, 57]. The evidence suggests that the early administration of rHuTPO promoted survival of HSCs and progenitor cells (HPCs), as well as stimulated their expansion and differentiation within the early, postirradiation microenvironment [57–60]. Other studies have clearly documented that delaying the start of Mpl-L administration after irradiation significantly lessened its therapeutic effects [32, 61, 62].

A more recent study substantiates the role of rHuTPO in stimulating expansion of HSCs after transplant [63]. It may also shed light on the role of rHuTPO dose and dose schedule relative to early HSC expansion and survival postirradiation. In this study, TPO–/– mice were found to require several fold more bone marrow cells to engraft and reconstitute hematopoiesis than the TPO+/+ cohort. The marked loss of stem cell expansion in the TPO–/– mice relative to the TPO+/+ controls would be reversed by administration of physiologic levels (80 ng) of rHuTPO. However, of additional interest, is that the administration of the low-dose rHuTPO to the transplanted TPO–/– cohort could not afford radioprotection through short-term hematopoietic reconstitution. In an earlier study in mice, Shibuya et al. administered a single high dose of PEG-rHuMGDF 1 hour after sublethal irradiation, then 48 hours later, BM cells were transplanted into lethally irradiated recipients. Transplantation of the cells from PEG-rHuMGDF-treated mice resulted in 70% survival, whereas all recipients that received cells from the vehicle-treated cohort died within 17 days post transplant [32]. The aforementioned studies and the data presented herein suggest that pharmacologic doses of rHuTPO administered within an immediate to 24-hour time frame post transplant may have induced early HSC expansion and hematopoietic rescue.

It is worth noting that endogenous TPO levels remain at normal, constitutive levels for several days post myelosuppression or transplant, as circulating TPO levels are regulated by PLT and MK mass [64–68]. Serum TPO levels begin to rise as PLT counts decrease after myeloablative conditioning. Enhanced survival and hematopoietic reconstitution therefore require pharmacologic doses early after cytotoxic insult to maintain viability and to promote early HSC/progenitor cell expansion, whereas this effect wanes with increasing interval post insult due to loss of HSC/HPC target cells and increasing levels of endogenous TPO.

There are also several differences between the myelosuppression and SCT models that may account for the unexpected difficulty in demonstrating the efficacy of rHuTPO or PEG-rHuMGDF post AuBMT. Although the myelosuppression models are characterized by a significant depletion of stem and progenitor cells, those surviving cells remain lodged within the inductive hematopoietic microenvironment. These surviving HSCs and HPCs are most likely in an accessible/responsive state to endogenous TPO and other thrombopoietic cytokines, as well as exogenous rHuTPO or PEG-rHuMGDF, whereas transplanted stem and progenitor cells must effectively seed within the microenvironment and become responsive to endogenous and exogenous GFs. The transplanted MK progenitor cells may do this with low efficiency and therefore must await replenishment from earlier noncommitted stem and progenitor cells. Neelis et al. and Wagemaker et al. have also suggested that the cellular composition of the respective grafts may have resulted in a less than optimal number of TPO-responsive progenitors post transplant [21, 58].

It is probable that the surviving, relatively radiation-resistant marrow MKs may represent a particularly interactive cell population within the myelosuppressed versus ablated marrow microenvironment [32, 69]. The MKs are capable of responding to both endogenous TPO and exogenous rHuTPO or PEG-rHuMGDF with enhanced endoreduplication and consequent increased marrow MK mass and production of PLTs [51, 70]. The surviving MKs may also respond to endogenous cytokines in the post-myelosuppressive marrow milieu by secreting additional GFs that are capable of acting in a paracrine/autocrine fashion and stimulating additional megakaryocytopoiesis [71–73]. The high doses of x-irradiation employed in this study (920 cGy) and those of Neelis et al. [21] and Nash et al. [23], while not limiting the seeding efficiency of transplanted stem and progenitor cells, may have further depleted the concentration of endogenous marrow MKs and immediate precursors and thus significantly diminished their contribution to post-transplant production of high-ploidy MKs and PLTs compared with effects noted in the myelosuppression models. As mentioned earlier, several rodent studies showed that single, high doses of Mpl-Ls, would significantly increase the early hematopoietic restoration required for survival following supralethal doses of radiation or chemotherapy [28, 56, 57]. This supports the contention that dose of the Mpl-L and time of administration are critical determinants of therapeutic efficacy in a model where Mpl-L target cells are minimal in number [28, 32, 56–58].

There are several possible explanations for the enhanced post-transplant PLT recovery in the two different schedule/dose variations that were selected based on PK/PD modeling. Pretreatment with low-dose PEG-rHuMGDF from day -9 to day -5 prior to AuBMT significantly increased the circulating PLT count to approximately 1,500 x 103/µl and the marrow concentration of MK-CFCs (threefold) in the autologous transplant inoculum. In addition, the pre/post treatment may provide a larger number of PEG-rHuMGDF-responsive MK-CFCs seeding the myeloablated marrow. Assuming no change in PLT half-life, compared with AuBMT controls, PK/PD modeling predicts that the elevated PLT count should account for a substantial elevation of the circulating PLT count above control values for several days. In this regard, the concentration of marrow-derived MK-CFCs at 7 days post transplant in the pretreatment cohort were significantly greater than both the AuBMT control and standard post-AuBMT PEG-rHuMGDF treatment cohorts. The relative contribution of these to the improved nadir, lack of thrombocytopenia, and earlier recovery of PLT counts to normal levels has not been assessed.

The Mpl-Ls remain as promising therapeutic agents. These results suggest that further definition of the schedule, dose, and route of administration of the Mpl-Ls may allow for optimal supportive care of the thrombocytopenic patient.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by a grant from Amgen, Inc., Thousand Oaks, California.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received July 22, 2002; accepted for publication September 4, 2002.




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