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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 837-845
HEMATOPOIESIS
From the Greenebaum Cancer Center, University of Maryland,
Baltimore; and Searle R & D, Monsanto Company, St. Louis, MO.
Myelopoietins (MPOs) constitute a family of engineered, chimeric
molecules that bind and activate the IL-3 and G-CSF receptors on
hematopoietic cells. This study investigated the in vivo hematopoietic response of rhesus monkeys administered MPO after radiation-induced myelosuppression. Animals were total body irradiated (TBI) in 2 series,
with biologically equivalent doses consisting of either a 700 cGy dose
of Cobalt-60 (60Co)
The major dose limiting sequelae, consequent to
radiation and/or chemotherapy are neutropenia and thrombocytopenia.
Protocols involving dose intensification and/or schedule compression
will most likely exacerbate the duration and degree of myelosuppression associated with standard dose therapy. Although G-CSF or GM-CSF are
used clinically to reduce the obligate neutropenic consequences of
cytotoxic therapy, the effective treatment of thrombocytopenia has
remained elusive.1-7 Recently, cytokines such as IL-11 and the cognate ligand (L) of the c-mpl receptor (Mpl-L) offer renewed potential for increasing platelet production after cytotoxic
therapy.8-16 The Mpl-L, the full length, glycosylated,
recombinant human (rHu) thrombopoietin (Tpo), and the nonpegylated or
pegylated (PEG) truncated form (encompassing the Epo-like domain) of
the Mpl-L, designated as megakaryocyte growth and development factor
(MGDF), significantly induced recovery of thrombopoiesis in
myelosuppressed nonhuman primates.8,11,12,17 The extension
of these models to combination studies showed that the Mpl-L in
combination with G-CSF augmented the G-CSF-induced recovery of
neutrophils without evidence of lineage competition after
radiation-induced myelosuppression.11,17 Furthermore, the
combination of daniplestim, an engineered IL-3 receptor agonist with a
truncated Mpl-L (Epo-like domain), enhanced hemopoietic regeneration
and reduced clinical support requirements noted with respective
daniplestim or truncated Mpl-L monotherapy in the irradiated nonhuman
primate.18
Another approach to inducing multilineage hematopoietic recovery,
subsequent to myelosuppressive therapy, has been to engineer new
molecules such as chimeric growth factor (GF) receptor agonists that
possess greater biologic activity than either GF agonist alone.
Furthermore, the ability to engineer each GF component of the chimeric
molecule offers the potential for enhancing the efficacy of GFs with
narrow therapeutic windows when used as monotherapy, such as IL-3.
These recently described families of chimeric proteins are composed of
combined GF agonists for either IL-3 and G-CSF receptors
(myelopoietin), IL-3 and Mpl-L receptors (promegapoietins), or Flt-3L
and G-CSF receptors (progenipoietins).19-24
The combination of engineered IL-3 and G-CSF receptor agonists to form
myelopoietin (MPO) was based on a consistent amount of in vitro and in
vivo evidence, suggesting the combination would enhance both neutrophil
and platelet regeneration within a favorable toxicity
profile.25-31 In fact, the chimeric IL-3/G-CSF receptor agonist, MPO, had a significantly higher potency for multilineage CFU
stimulatory activity on human CD34+ bone marrow cells compared with
mixtures of the IL-3 and G-CSF receptor agonists.20 The in
vivo priming effect of IL-3 for the action of other cytokines on early
hematopoietic progenitors suggested that maximum stimulation would be
achieved by the combination of IL-3 and G-CSF.32 In addition, the combined therapeutic administration of daniplestim (IL-3
receptor agonist) and G-CSF to high-dose, sublethally irradiated nonhuman primates further decreased the cytopenic durations and nadirs
for both platelets and neutrophils relative to the respective cytokine
monotherapy.33 Also, preliminary pharmacokinetic studies of
the MPO molecule in normal rhesus monkeys demonstrated an increased biologic half-life and suggested that a reduced administration schedule
would be biologically equivalent to the normally required twice daily
schedule used with native, or the engineered IL-3, daniplestim.31,33,34 This study evaluated the preclinical efficacy of myelopoietin, using twice daily or daily administration schedules on multilineage hematopoietic recovery in a nonhuman primate
model of high-dose, radiation-induced myelosuppression.
Conduct of experiments
Animals
Irradiation Monkeys, after a prehabituation period, were irradiated in plexiglass restraining chairs for both protocols. First series. In the AFRRI protocol, monkeys received bilateral, total body irradiation (TBI) with Cobalt-60 (60Co) -radiation to a total midline tissue dose of 700 cGy at a dose rate of 40 cGy/min. Second series. In the GCC
protocol, monkeys received unilateral TBI with 250 kVp x-radiation at
13 cGy/min in the posterior-anterior position, rotated 180° at the
mid-dose (300 cGy) to the anterior-posterior position for completion of the total 600 cGy midline tissue exposure. The 700 cGy 60Co
-radiation exposure is bioequivalent to the 600 cGy x-irradiation relative to hematopoietic myelosuppression (see Results, Table 1). Dosimetry was performed using paired
0.5 cm3 ionization chambers, with calibration factors
traceable to the National Institute of Standards and Technology.
Recombinant cytokines Myelopoietin (SC-68420) (MPO), an engineered, chimeric hematopoietic growth factor that binds and activates the IL-3 and G-CSF receptors was produced in Escherichia coli through the use of a plasmid-based expression vector. It was expressed in insoluble inclusion bodies within the E coli cells. Washed inclusion bodies were solubilized in urea buffer and disulfide bonds formed through air oxidation after lowering the urea concentration. MPO was purified by ion exhange chromatography and filtration. Endotoxin levels were undetectable (Limulus amebocyte assay [Associates of Cape Cod, Inc, Woods Hole, MA]). Purified protein was stored as a frozen solution in 10 mmol/L Tris buffer, pH 8.0. In the previous study daniplestin (SC-55494) was provided by Searle R&D (St Louis, MO). G-CSF was purchased as Neupogen (Filgrastin) (Amgen Inc, Thousand Oaks, CA).Study design In each experimental series, animals were irradiated at day 0 and randomly assigned to a treatment protocol. First series, 700 cGy-irradiation protocols. On day 1 after 700 cGy -irradiation, animals received MPO at either 200 µg/kg/d (n = 4) or 50 µg/kg/d (n = 2), or HSA (Plasbumin-5, Miles Inc, Cutter Biological, Elkhart, IN). MPO or control protein, 15 µg/kg/d, n = 10, was injected twice
daily (BID) as a divided dose (10-hour interval) as 1 mL subcutaneous
(SC) injection bolus for 18 to 23 days. Administration was stopped when
the white blood cell count reached 1 × 105/µL. Of
the 4 animals injected with 200 µg/kg of MPO, 1 animal each was
stopped after 18, 20, 22, and 23 days of administration. Both animals
administered MPO at 50 µg/kg/d received the full 23-day schedule.
Second series, 600 cGy x-irradiation protocols. On day 1 after
600 cGy x-irradiation, animals received MPO 200 µg/kg/d, n = 5 or
0.1% autologous serum (AS, n = 11) as control protein in 1 mL bolus
SC injections once a day (QD) for 18 days.
Rationale for cytokine dose and schedule The dose (50 µg/kg and 200 µg/kg) and administration schedule (BID vs QD) for MPO were based on several observations: (1) Native rhuIL-3 or engineered the IL-3 receptor agonist, daniplestim, have 20- to 50-fold less binding affinity for rhesus IL-3 receptor than the homologous, rhesus IL-3.36,37 (2) We previously demonstrated the enhanced therapeutic efficacy of daniplestim administered BID at 100 µg/kg/d relative to 25 µg/kg/d.31 (3) The MPO chimeric is constructed at approximately equimolar ratios of engineered IL-3 and G-CSF receptor agonists. Therefore, the 200 µg/kg dose of MPO provides approximately 100 µg/kg of engineered IL-3. (4) Monotherapy protocols that use native rhuIL-3 or daniplestim, required BID administration for optimal biologic response34 (Farese et al, unpublished observations, 1998). (5) The plasma pharmacokinetic parameters of daniplestim in irradiated monkeys31 and MPO in normal monkeys (Table 2, herein) suggested that an abbreviated schedule (QD vs BID) of MPO would induce an equivalent biologic response to the full BID schedule.
Clinical support Clinical support consisting of an antimicrobial regimen and whole blood transfusions were provided to all animals as required. An antibiotic regimen and transfusions of fresh, irradiated (1500 cGy, 60Co-irradiation) whole blood were administered as
previously described.11,33
Hematologic evaluations Peripheral blood. Peripheral blood was obtained from the saphenous vein to assay complete blood (Baker, System 9000, Serono-Baker, Allentown, PA or Sysmex K-4500, Long Grove, IL) and differential counts (Wright-Giemsa Stain, Ames Automated Slide Stainer, Elkhart, IN). Assessment of hematologic evaluations has been previously described.31 Bone marrow. Approximately 2 mL of heparinized-bone marrow (BM) was aspirated from the humerus and/or iliac crest of anesthetized primates (Ketaset, Fort Dodge, IA, 10 mg/kg intramuscular). Low density (< 1.077 g/cm3) mononuclear cells (MNC) were separated and cultured as previously described.31 GM-CFC and E-BFU derived colonies (> 50 cells) were expressed as the number of CFC/105 MNC. MK-CFC (10-50 cells/colony) are not distinguished from MK-BFC in this study, and are termed MK-CFC. Pharmacokinetic analysis The pharmacokinetics of MPO were determined in female rhesus monkeys (n = 3) after intravenous (IV) bolus (10 µg/kg) and SC (30 µg/kg) administration. MPO concentrations in plasma were determined by sandwich ELISA (enzyme-linked immunosorbent assay) procedures. The ELISA uses a murine monoclonal anti-G-CSF antibody, bound in microtiter plate wells, to bind MPO present in plasma samples. After removal of unbound plasma proteins by washing with buffer, MPO was quantified by the addition of goat polyclonal antidaniplestim antibody that is conjugated to horseradish peroxidase. The color produced by incubation with peroxidase substrate is read at 650 nm, and MPO concentrations are determined by reference to a standard curve prepared with MPO in plasma with a sensitivity limit of 0.150 ng/mL.Statistical analysis The Normal Scores Test was used to make pairwise comparisons of the durations of neutropenia and thrombocytopenia and evaluate the statistical significance between nadirs, the time to recovery and transfusion and antibiotic requirements. The tests were carried out using the software package StatXact (Cytel Software Corp, Cambridge, MA). The exact P values were obtained for all analysis.
Relative hematologic effects of total body exposure to 700 cGy
-irradiation is not significantly different from
that observed consequent to 600 cGy of 250 kVp x-irradiation (Table 1).
The key parameters, indicative of the relative degree of
myelosuppression, namely, the duration of neutropenia, absolute neutrophil count (ANC) < 500/µL, 14.8 and 16.2 days,
P = .065, and thrombocytopenia, platelet count (PLT) < 20 000/µL, 11.9 and 10.4 days, P = .207, absolute
neutrophil (0 and 8/µL, P = .139), and platelet nadirs,
5 000 and 3 000/µL, P = .197, are not statistically different after exposure to 700 cGy 60Co -irradiation or
600 cGy 250 kVp x-irradiation, respectively (Table 1). Inspection of
the BM-derived clonogenic activity noted in Figures 2 and 4 for the
respective control-treated cohorts also substantiates the equivocal
hematopoietic myelosuppressive effects of these different quality
radiation exposures.
Pharmacokinetics of MPO in normal animals Elimination of MPO from rhesus monkey plasma occurred in 2 phases after bolus IV administration of 10 µg/kg. The mean values of the first and second phase half-lives were 0.533 and 25.3 hours, respectively (Table 2). The total body clearance of MPO was 0.415 mL/min/kg. The central compartment (Vc) and steady state (VSS) volumes of distribution were 0.0220 and 0.0706 L/kg, respectively. These volume of distribution values are similar to the plasma volume and whole blood volume, respectively, and indicate little, if any, distribution from blood into other tissues.First series, 700 cGy Neutrophil recovery.
Neutrophil parameters were significantly improved by administration of
MPO at a total daily dose of 200 µg/kg/d in a BID protocol after 700 cGy total body
Platelet recovery.
The BID administration of MPO at a total daily dose of 200 µg/kg/d
significantly enhanced the recovery of platelets after 700 cGy total
body Production of red blood cells (RBC), nucleated red blood cells (NRBC) and transfusion requirements. The recovery of RBC in the MPO-treated cohort (200 µg/kg/d, BID) appears equivalent to the control cohort (Figure 1C). However, the appearance of NRBCs in the peripheral blood occurred earlier after MPO administration (17 to 23 days) than the HSA-treated controls (24 days) (Figure 1D). In addition, the HSA-treated cohort received an average of 1.7 transfusions per animal, whereas the MPO-treated cohort required 1.0 transfusion per animal (Table 3). Bone marrow-derived clonogenic activity.
Bone marrow-derived GM-CFC, E-BFU, MK-CFC, and GEMM-CFC activity was
evaluated at baseline and day 7, 14, 21, 48, and 100 after 700 cGy TBI.
In the HSA-treated controls, the concentration of MK-CFC was
significantly reduced through 21-day after exposure, whereas GM-CFC and
E-BFU were significantly decreased through 48-day after TBI (Figure
2). The number of recognizable GEMM-CFC were very low in baseline BM samples. An increase in GEMM-CFC per
105 BM cells was only observed at 48-day after TBI in the
control animals (Figure 2). The recovery of clonogenic activity
measured in the MPO-treated cohort (200 µg/kg/d, BID) was
significantly enhanced relative to that noted for time-matched controls
or respective baseline values (Figure 2A through D). All clonogenic
activity noted for GM-CFC, MK-CFC, E-BFU, and GEMM-CFC in MPO-treated
animals had returned to baseline values within 14 days after TBI.
Second series, 600 cGy x-irradiation, MPO QD administration protocol Neutrophil recovery.
Neutrophil regeneration was profoundly affected by the administration
of MPO at 200 µg/kg/d in a QD schedule after TBI. Significant reduction in the duration of neutropenia and improvement in neutrophil nadir were evident in the MPO-treated animals compared with AS-treated controls 7.2 days versus 16.2 days and 377/µL versus 8/µL,
P < .001 respectively (Table 3, Figure
3A). The recovery of ANC to
Platelet recovery.
Administration of MPO at 200 µg/kg/d, QD for 18 consecutive days
after TBI significantly reduced the duration of thrombocytopenia relative to AS-treated controls 3.2 days versus 10.4 days,
respectively, P < .001 (Table 3, Figure 3B). The depth of
platelet nadir was also significantly improved by MPO administration
from the control value of 3 000/µL to 28 000/µL (Table 3, Figure
3B). Recovery times of platelet count to Production of red blood cells (RBC), nucleated red blood cells (NRBC) and transfusion requirements. The MPO-induced recovery of RBCs was modestly improved relative to the control cohort (Figure 3C). However, the RBC recovery observed in the AS-treated animals reflects the effect of whole blood transfusions. The control cohort necessitated an average 2.6 whole blood transfusions per animal, whereas the MPO-treated animals were transfusion independent (P < .001) (Table 3). MPO administration after 600 cGy TBI induced an earlier appearance of NRBC in a fashion similar to that noted for the 700 cGy TBI protocol (Figure 3D). Bone marrow-derived clonogenic activity.
Bone marrow-derived GM-CFC, MK-CFC, and E-BFU activity
(CFC/105 MNCs) were evaluated before (baseline) and at days
7, 14, 21, and 46 after 600 cGy TBI. In the control-treated animals,
all clonogenic activity was significantly decreased at least through 21 days after x-irradiation. Administration of MPO-stimulated recovery
of GM-CFC and MK-CFC to within preirradiation values by 21 days after
irradiation (Figure 4). Interestingly,
E-BFU recovery was also enhanced by day 14 after QD administration of MPO.
Comparison of MPO-induced hematopoietic recovery to that of
coadministered daniplestim plus G-CSF.
We previously investigated the relative efficacy of coadministered
daniplestim (BID) plus G-CSF (QD) to monotherapy with daniplestim (BID)
or G-CSF (QD) in the equivalent 700 cGy
Myelopoietin, a novel, chimeric molecule that binds and activates
the IL-3 and G-CSF receptors had marked neutrophil and platelet restorative activity in a nonhuman primate model of radiation-induced myelosuppression. Furthermore, the favorable pharmacodynamic profile exhibited by MPO conferred an equivalent therapeutic efficacy between
BID or QD administration protocols. MPO administration in either
protocol significantly improved hematopoietic parameters indicative of
myelopoiesis, thrombocytopoiesis, and erythropoiesis subsequent to
high-dose sublethal radiation exposure. The neutrophil and platelet
nadirs were significantly improved, neutropenic and thrombocytopenic
durations were significantly reduced, and respective neutrophil and
platelet recovery time to
We wish to thank Michael Flynn, Nelson Fleming, Lisa B. Lind, Lorelei Dacquel Smith, Daniel Casey, and Heather Webster for their superb technical assistance and William Jackson for assistance with the statistical analysis.
Submitted October 14, 1998; accepted September 11, 1999.
Reprints: Thomas J. MacVittie, Greenebaum Cancer Center, S9D11, University of Maryland, 22 S Greene St, Baltimore, MD 21201.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Top
Abstract
Introduction
500/µL was significantly
improved from 19.7 days in the control-treated group to 12.3 days
(P = .007) in the 200 µg/kg/d, BID, MPO-treated group.
Consequently, the days on antibiotics were significantly reduced from
17.4 days for controls to 7.5 days (P = .005) for MPO-treated
animals. Treatment with a lower dose of MPO (50 µg/kg/d), although
less effective than the 200 µg/kg dose, improved the neutropenic
duration (12.0 days), neutrophil nadir (36/µL), recovery time to ANC 
denotes zero values; ND denotes not
done.
a multifunctional agonist which activates both IL3 and G-CSF receptors.
Blood.
1995;86:154a(abstract).