Pegylated Recombinant Human Megakaryocyte Growth And Development Factor Research Articles

Autonomous Megakaryocyte Growth in Essential Thrombocythemia and Idiopathic Myelofibrosis Is Not Related to a c-mpl Mutation or to an Autocrine Stimulation by Mpl-L

AbstractEssential thrombocythemia (ET) and idiopathic myelofibrosis (PMF) are two myeloproliferative diseases characterized by a marked megakaryocytic (MK) involvement. The pathogenesis of these two diseases is unknown. Recently it has been shown that overexpression of Mpl-ligand (Mpl-L) in mice induces thrombocytosis and myelofibrosis. In this study, we investigated whether Mpl-L was responsible for the pathogenesis of ET and PMF. Using in vitro cultures of blood or marrow CD34+ cells, we investigated whether MK growth was abnormal in these two diseases. Spontaneous MK growth involving only a fraction (20%) of the MK progenitors, as compared with growth in the presence of pegylated recombinant human megakaryocyte growth and development factor (PEG-rhuMGDF), was found in both diseases (21ET and 14PMF) using serum-free semisolid and liquid cultures, including cultures at one cell per well. We first searched for ac-mpl mutation/deletion by sequencing the entire coding region of the gene by polymerase chain reaction (PCR) in nine ET patients and five PMF patients, but no mutation was found. We subsequently investigated whether an autocrine stimulation by Mpl-L could explain the autonomous MK growth. Addition of different preparations of soluble Mpl receptor (sMpl) containing a Fc domain of IgG1 (sMpl-Fc) markedly inhibited MK spontaneous growth in both ET and PMF patients. This effect was specific for sMpl because a control soluble receptor (s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fc fragment had the same inhibitory efficacy as the sMpl-Fc. This inhibition was reversed by addition of PEG-rhuMGDF or a combination of cytokines. The sMpl-Fc markedly altered the entry into cell cycle of the CD34+ cells and increased the apoptosis that occurs in most patient CD34+ cells in the absence of exogenous cytokine, suggesting an autocrine stimulation. In contrast, a neutralizing antibody against Mpl-L did not alter the spontaneous MK growth, whereas it totally abolished the effects of 10 ng/mL PEG-rhuMGDF on patient or normal CD34+ cells. Mpl-L transcripts were detected at a very low level in the patient CD34+cells and MK and only when a highly sensitive fluorescent PCR technique was used. By quantitative reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per actin transcripts was lower than detected in human Mpl-L–dependent cell lines, suggesting that this synthesis of Mpl-L was not biologically significant. In favor of this hypothesis, the Mpl-L protein was not detected in culture supernatants using either an enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3huc-mpl) assay, except in one PMF patient. Investigation of Mpl-L signaling showed an absence of constitutive activation of STATs in spontaneously growing patient MKs. Addition of PEG-rhuMGDF to these MKs activated STATs 3 and 5. This result further suggests that spontaneous growth is neither related to a stimulation by Mpl-L nor to ac-mpl mutation. In conclusion, our results show that Mpl-L or Mpl are not directly implicated in the abnormal proliferation of MK cells from ET and PMF. The mechanisms by which the sMpl mediates a growth inhibition will require further experiments.

Autonomous Megakaryocyte Growth in Essential Thrombocythemia and Idiopathic Myelofibrosis Is Not Related to a c-mpl Mutation or to an Autocrine Stimulation by Mpl-L

Essential thrombocythemia (ET) and idiopathic myelofibrosis (PMF) are two myeloproliferative diseases characterized by a marked megakaryocytic (MK) involvement. The pathogenesis of these two diseases is unknown. Recently it has been shown that overexpression of Mpl-ligand (Mpl-L) in mice induces thrombocytosis and myelofibrosis. In this study, we investigated whether Mpl-L was responsible for the pathogenesis of ET and PMF. Using in vitro cultures of blood or marrow CD34+ cells, we investigated whether MK growth was abnormal in these two diseases. Spontaneous MK growth involving only a fraction (20%) of the MK progenitors, as compared with growth in the presence of pegylated recombinant human megakaryocyte growth and development factor (PEG-rhuMGDF), was found in both diseases (21ET and 14PMF) using serum-free semisolid and liquid cultures, including cultures at one cell per well. We first searched for ac-mpl mutation/deletion by sequencing the entire coding region of the gene by polymerase chain reaction (PCR) in nine ET patients and five PMF patients, but no mutation was found. We subsequently investigated whether an autocrine stimulation by Mpl-L could explain the autonomous MK growth. Addition of different preparations of soluble Mpl receptor (sMpl) containing a Fc domain of IgG1 (sMpl-Fc) markedly inhibited MK spontaneous growth in both ET and PMF patients. This effect was specific for sMpl because a control soluble receptor (s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fc fragment had the same inhibitory efficacy as the sMpl-Fc. This inhibition was reversed by addition of PEG-rhuMGDF or a combination of cytokines. The sMpl-Fc markedly altered the entry into cell cycle of the CD34+ cells and increased the apoptosis that occurs in most patient CD34+ cells in the absence of exogenous cytokine, suggesting an autocrine stimulation. In contrast, a neutralizing antibody against Mpl-L did not alter the spontaneous MK growth, whereas it totally abolished the effects of 10 ng/mL PEG-rhuMGDF on patient or normal CD34+ cells. Mpl-L transcripts were detected at a very low level in the patient CD34+cells and MK and only when a highly sensitive fluorescent PCR technique was used. By quantitative reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per actin transcripts was lower than detected in human Mpl-L–dependent cell lines, suggesting that this synthesis of Mpl-L was not biologically significant. In favor of this hypothesis, the Mpl-L protein was not detected in culture supernatants using either an enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3huc-mpl) assay, except in one PMF patient. Investigation of Mpl-L signaling showed an absence of constitutive activation of STATs in spontaneously growing patient MKs. Addition of PEG-rhuMGDF to these MKs activated STATs 3 and 5. This result further suggests that spontaneous growth is neither related to a stimulation by Mpl-L nor to ac-mpl mutation. In conclusion, our results show that Mpl-L or Mpl are not directly implicated in the abnormal proliferation of MK cells from ET and PMF. The mechanisms by which the sMpl mediates a growth inhibition will require further experiments.

Mobilization of primitive haemopoietic progenitor cells and stem cells with long-term repopulating ability into peripheral blood in mice by pegylated recombinant human megakaryocyte growth and development factor.

We investigated in detail the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization in male BDF1 mice. Treatment with PEG-rHuMGDF for 5 d stimulated a striking expansion of the circulating levels of multiple types of colony-forming units in culture (CFU-c), including CFU-granulocyte-macrophage, CFU-megakaryocyte, burst-forming units-erythroid, and multipotent CFU-c, and primitive day-12 CFU-spleen. All of these progenitors were mobilized into the peripheral blood (PB) with similar kinetics; their numbers peaked after the cessation of treatment and then declined earlier than platelet numbers peaked. The maximal increase in any of the four CFU-c in the PB was attained with at least 300 microg/kg/d of PEG-rHuMGDF, whereas peripheral platelet counts plateaued at 30 microg/kg/d. Adoptive transfer with PB from PEG-rHuMGDF-treated donor mice resulted in greater survival of lethally irradiated recipients. The majority of the recipients that survived at 187 d after transplantation with PEG-rHuMGDF-mobilized PB showed significant donor engraftment at the progenitor cell level. The combined administration of appropriate doses of PEG-rHuMGDF and recombinant human granulocyte colony-stimulating factor induced a synergistic increase in the circulating levels of the four CFU-c compared to either factor alone. These results indicate that PEG-rHuMGDF as a single agent can mobilize a full spectrum of PBPCs in mice.

Reactivity of Platelets from Healthy Individuals and from Patients with Hematologic Diseases following Incubation with Pegylated RecombinantHuman Megakaryocyte Gr owth and Development Factor in vitro

Platelets express the receptor for thrombopoietin. It is possible that thrombopoietin modulates platelet reactivity. We examined the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet activation in vitro using flow cytometry. We compared samples from healthy individuals and from patients with various hematologic diseases (AML, myeloma, postchemotherapy). Citrated whole blood was incubated with PEG-rHuMGDF (10 or 100 ng/ml), then a mild stimulus was added (0.1 U thrombin/ml). Blood from healthy individuals showed a significantly higher degree of platelet activation (CD62p expression), microparticle generation, and aggregate formation after incubation with PEG-rHuMGDF+thrombin versus thrombin alone (p < 0.05). However, this difference could not be shown for platelets from patients with thrombocytopenia or other hematologic diseases. The use of PEG-rHuMGDF should be safe and not cause an additional risk of thromboocclusive disease in these patients.

Recombinant human megakaryocyte growth and development factor attenuates postbypass thrombocytopenia

Background. Cardiopulmonary bypass contributes to platelet loss and dysfunction by exposure to shear stresses, foreign surfaces, and hypothermia. This study tested the hypothesis that pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) accelerates recovery of the platelet population after hypothermic extracorporeal circulation (HEC). Methods. In a blinded study, subcutaneous injections of drug or placebo were given to dogs daily for 3 days preoperatively (day 0, 1, and 2) with no drug on day 3. On day 4, the animal was prepared for arteriovenous HEC. After heparinization, HEC was initiated at 30 to 40 mL · kg −1 · min −1. Hypothermic extracorporeal circulation (25°C) was continued for 90 minutes. Results. Preoperative platelet count (×10 3 platelets/μL) did not differ from predrug count in placebo (256 ± 27 versus 255 ± 20) or PEG-rHuMGDF (271 ± 30 versus 291 ± 38). During 60 minutes of HEC, the platelet count decreased to ∼10% of baseline in placebo (29 ± 5) and PEG-rHuMGDF (46 ± 8), and recovered to ∼70% of baseline after rewarming (90 minutes of HEC: placebo, 185 ± 17, versus PEG-rHuMGDF, 169 ± 22). After HEC, platelet count was greater in PEG-rHuMGDF–treated animals ( p < 0.05) without altering function (aggregation responses). Within the first 6 hours after HEC, platelet count in PEG-rHuMGDF–treated animals was rising and increased to 260 ± 29 ( p < 0.01), but was unchanged in placebo animals (186 ± 21). Thereafter, platelet count in placebo animals declined to a nadir of 124 ± 15 (72 hours after HEC), whereas platelet count in PEG-rHuMGDF animals approximated the preoperative value (>200) at all times. Conclusions. Appropriately timed presurgical administration of PEG-rHuMGDF counteracts post-HEC relative thrombocytopenia without increasing platelet population and enhancing aggregation preoperatively or during extracorporeal circulation.

Treatment of Thrombocytopenia in Chimpanzees Infected With Human Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte Growth and Development Factor

AbstractThree chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 ± 19 × 103/μL (P = .004 compared with 228 ± 92 × 103/μL in 44 normal control animals), mean platelet volumes of 11.2 ± 1.8 fL (P > .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P < .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 × 103 RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 μg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 ± 19 to 599 ± 260 × 103 platelets/μL;P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells); and (4) serum levels of Mpl ligand from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/μL to 9.9 ± 5.0 × 103/μL (P= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P > .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P < .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P > .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.

Treatment of Thrombocytopenia in Chimpanzees Infected With Human Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte Growth and Development Factor

Three chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 ± 19 × 103/μL (P = .004 compared with 228 ± 92 × 103/μL in 44 normal control animals), mean platelet volumes of 11.2 ± 1.8 fL (P &gt; .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P &lt; .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 × 103 RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 μg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 ± 19 to 599 ± 260 × 103 platelets/μL;P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells); and (4) serum levels of Mpl ligand from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/μL to 9.9 ± 5.0 × 103/μL (P= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P &gt; .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P &lt; .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P &gt; .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.

Overview of the safety and biologic effects of PEG-rHuMGDF in clinical trials.

Completed randomized placebo-controlled phase I/II studies of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) have demonstrated that this recombinant Mpl ligand has potent and lineage-dominant effects on megakaryopoiesis and platelet production. Platelets produced after PEG-rHuMGDF administration display normal ultrastructure and functional attributes. In these early studies, PEG-rHuMGDF accelerated the recovery of baseline platelet counts after cytotoxic chemotherapy in cancer patients by six to seven days, indicating the potential for clinical benefit in this setting. PEG-rHuMGDF has been well-tolerated in clinical trials, with similar adverse events in placebo and PEG-rHuMGDF populations, and an observed adverse event profile consistent with the effects of underlying malignancy and chemotherapy. The lack of inflammatory cytokine effects in the clinic is consistent with results of animal studies, the narrow tissue distribution of Mpl and the lineage-dominant effect of PEG-rHuMGDF on megakaryopoiesis. Additional phase I/II studies have commenced in the fields of cancer chemotherapy and augmentation of platelet donation, and a phase III study is underway in patients undergoing bone marrow transplantation.

Multilineage Hematopoietic Recovery by a Single Injection of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor in Myelosuppressed Mice

Previous studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 μg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit–spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG-rHuMGDF (80 μg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG-rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG-rHuMGDF, a single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 μg/kg) treatment even when rhG-CSF doses were escalated to 1,000 μg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.

Multilineage Hematopoietic Recovery by a Single Injection of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor in Myelosuppressed Mice

AbstractPrevious studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 μg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit–spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG-rHuMGDF (80 μg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG-rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG-rHuMGDF, a single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 μg/kg) treatment even when rhG-CSF doses were escalated to 1,000 μg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.

The role of transforming growth factor-beta in PEG-rHuMGDF-induced reversible myelofibrosis in rats.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 microg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-beta1 (TGF-beta1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7- and 2-fold, respectively. The elevated levels of TGF-beta1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 microg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-beta1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

Erratum: Improvement of thrombocytopenia following bone marrow transplantation by pegylated recombinant human megakaryocyte growth and development factor in mice

We examined whether pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) is capable of improving thrombocytopenia and promoting thrombopoietic reconstitution following lethal irradiation and bone marrow transplantation (BMT) in mice. Immediately after receiving 10 Gy whole body irradiation (day 0), male C3H/HeN mice were inoculated with 10(6) bone marrow cells obtained from syngeneic mice. Circulating platelet counts decreased to below 4% of the normal counts with a nadir on day 10, and then returned to the normal level on day 28 in the control mice undergoing BMT. Subcutaneous consecutive treatment with PEG-rHuMGDF at doses from 10 to 300 micrograms/kg/day from day 1 for 13 days significantly improved the platelet nadir and promoted platelet recovery. The white blood cell counts and hemoglobin concentration following BMT were not influenced by the PEG-rHuMGDF. PEG-rHuMGDF-injection starting from day 5 did not improve the platelet nadir following BMT. Furthermore, administration with PEG-rHuMGDF on alternate days at 55.7 micrograms/kg/day for 7 days or at an interval of 3 days at 78 micrograms/kg/day for 4 days (twice a week for 2 weeks) had a significant efficacy, but these administration regimens had less efficacy than consecutive administration at 30 micrograms/kg/day for 13 days. The numbers of megakaryocytes and megakaryocyte progenitor cells decreased to 5 and 0.2% of normal level, respectively, in the control mice. Consecutive administration of PEG-rHuMGDF enhanced the recovery of the mean number of these cells compared to those in vehicle-treated mice, although such effects were not statistically significant except for the number of megakaryocyte progenitors on day 12. These results suggest that consecutive treatment with PEG-rHuMGDF beginning from the day after BMT may be effective in improving thrombocytopenia following BMT.

Prevention of Thrombocytopenia and Neutropenia in a Nonhuman Primate Model of Marrow Suppressive Chemotherapy by Combining Pegylated Recombinant Human Megakaryocyte Growth and Development Factor and Recombinant Human Granulocyte Colony-Stimulating Factor

This report examines the effects on hematopoietic regeneration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF ) (2.5 μg/kg/d) alone and in combination with recombinant human granulocyte colony stimulating factor (rHu-GCSF ) (10 μg/kg/d) for 21 days in rhesus macaques receiving intense marrow suppression produced by single bolus injections of hepsulfam (1.5 g/m2). In six hepsulfam-only control animals thrombocytopenia (platelet count &lt;100 × 109/L) was observed between days 12 and 25 (nadir 39 ± 20 × 109/L on day 17), and neutropenia (absolute neutrophil count &lt;1 × 109/L) occurred between days 8 and 30 (nadir 0.167 ± 0.120 × 109/L on day 15). PEG-rHuMGDF (2.5 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.9 ± 0.2 ng/mL and increased the platelet count twofold over basal prechemotherapy levels (856 ± 594 × 109/L v baseline of 416 ± 88 × 109/L; P = .01). PEG-rHuMGDF alone also shortened the period of posthepsulfam neutropenia from 22 days to 12 days (P = .01), although the neutropenic nadir was not significantly altered (neutrophil count 0.224 ± 0.112 × 109/L v 0.167 ± 0.120 × 109/L; P &lt; .3). rHu-GCSF (10 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.4 ± 1.1 ng/mL, and reduced the time for the postchemotherapy neutrophil count to attain 1 × 109/L from 22 days to 4 days (P = .005). The postchemotherapy neutropenic nadir was 0.554 ± 0.490 × 109neutrophils/L (P = .3 v hepsulfam-only control of 0.167 ± 0.120 × 109/L). However, thrombocytopenia of &lt;100 × 109 platelets/L was not shortened (persisted from day 12 to day 25), or less severe (nadir of 56 ± 32 × 109 platelets/L on day 14; P = .7 compared with untreated hepsulfam animals). The concurrent administration of rHu-GCSF (10 μg/kg/d) and PEG-rHuMGDF (2.5 μg/kg/d) in four animals resulted in postchemotherapy peripheral platelet counts of 127 ± 85 × 109/L (P = .03 compared with 39 ± 20 × 109/L for untreated hepsulfam alone, and P = .02 compared with 856 ± 594 × 109/L for PEG-rHuMGDF alone), and shortened the period of neutropenia &lt;1 × 109/L from 22 days to 4 days (P = .8 compared with rHu-GCSF alone). Increasing PEG-rHuMGDF to 10 μg/kg/d and maintaining the 21-day schedule of coadministration with rHu-GCSF (10 μg/kg/d) in another four animals produced postchemotherapy platelet counts of 509 ± 459 × 109/L (P &lt; 10−4compared with untreated hepsulfam alone, and P = .04 compared with 2.5 μg/kg/d PEG-rHuMGDF alone), and 4 days of neutropenia. Coadministration of rHu-GCSF and PEG-rHuMGDF did not significantly alter the pharmacokinetics of either agent. The administration of PEG-rHuMGDF (2.5 μg/kg/d) from day 1 through day 22 and rHu-GCSF (10 μg/kg/d) from day 8 through day 22 in six animals produced peak postchemotherapy platelet counts of 747 ± 317 × 109/L (P &lt; 10−4 compared with untreated hepsulfam alone, and P = .7 compared with PEG-rHuMGDF alone), and maintained the neutrophil count &lt; 3.5 × 109/L (P = .008 v rHu-GCSF therapy alone). Thus, both thrombocytopenia and neutropenia are eliminated by initiating daily PEG-rHuMGDF therapy on day 1 and subsequently adding daily rHu-GCSF after 1 week in the rhesus model of hepsulfam marrow suppression. This improvement in platelet and neutrophil responses by delaying the addition of rHu-GCSF to PEG-rHuMGDF therapy demonstrates the importance of optimizing the dose and schedule of cytokine combinations after severe myelosuppressive chemotherapy.

Prevention of Thrombocytopenia and Neutropenia in a Nonhuman Primate Model of Marrow Suppressive Chemotherapy by Combining Pegylated Recombinant Human Megakaryocyte Growth and Development Factor and Recombinant Human Granulocyte Colony-Stimulating Factor

AbstractThis report examines the effects on hematopoietic regeneration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF ) (2.5 μg/kg/d) alone and in combination with recombinant human granulocyte colony stimulating factor (rHu-GCSF ) (10 μg/kg/d) for 21 days in rhesus macaques receiving intense marrow suppression produced by single bolus injections of hepsulfam (1.5 g/m2). In six hepsulfam-only control animals thrombocytopenia (platelet count <100 × 109/L) was observed between days 12 and 25 (nadir 39 ± 20 × 109/L on day 17), and neutropenia (absolute neutrophil count <1 × 109/L) occurred between days 8 and 30 (nadir 0.167 ± 0.120 × 109/L on day 15). PEG-rHuMGDF (2.5 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.9 ± 0.2 ng/mL and increased the platelet count twofold over basal prechemotherapy levels (856 ± 594 × 109/L v baseline of 416 ± 88 × 109/L; P = .01). PEG-rHuMGDF alone also shortened the period of posthepsulfam neutropenia from 22 days to 12 days (P = .01), although the neutropenic nadir was not significantly altered (neutrophil count 0.224 ± 0.112 × 109/L v 0.167 ± 0.120 × 109/L; P < .3). rHu-GCSF (10 μg/kg/d) injected subcutaneously into four animals from day 1 to day 22 following hepsulfam administration produced trough serum concentrations of 1.4 ± 1.1 ng/mL, and reduced the time for the postchemotherapy neutrophil count to attain 1 × 109/L from 22 days to 4 days (P = .005). The postchemotherapy neutropenic nadir was 0.554 ± 0.490 × 109neutrophils/L (P = .3 v hepsulfam-only control of 0.167 ± 0.120 × 109/L). However, thrombocytopenia of <100 × 109 platelets/L was not shortened (persisted from day 12 to day 25), or less severe (nadir of 56 ± 32 × 109 platelets/L on day 14; P = .7 compared with untreated hepsulfam animals). The concurrent administration of rHu-GCSF (10 μg/kg/d) and PEG-rHuMGDF (2.5 μg/kg/d) in four animals resulted in postchemotherapy peripheral platelet counts of 127 ± 85 × 109/L (P = .03 compared with 39 ± 20 × 109/L for untreated hepsulfam alone, and P = .02 compared with 856 ± 594 × 109/L for PEG-rHuMGDF alone), and shortened the period of neutropenia <1 × 109/L from 22 days to 4 days (P = .8 compared with rHu-GCSF alone). Increasing PEG-rHuMGDF to 10 μg/kg/d and maintaining the 21-day schedule of coadministration with rHu-GCSF (10 μg/kg/d) in another four animals produced postchemotherapy platelet counts of 509 ± 459 × 109/L (P < 10−4compared with untreated hepsulfam alone, and P = .04 compared with 2.5 μg/kg/d PEG-rHuMGDF alone), and 4 days of neutropenia. Coadministration of rHu-GCSF and PEG-rHuMGDF did not significantly alter the pharmacokinetics of either agent. The administration of PEG-rHuMGDF (2.5 μg/kg/d) from day 1 through day 22 and rHu-GCSF (10 μg/kg/d) from day 8 through day 22 in six animals produced peak postchemotherapy platelet counts of 747 ± 317 × 109/L (P < 10−4 compared with untreated hepsulfam alone, and P = .7 compared with PEG-rHuMGDF alone), and maintained the neutrophil count < 3.5 × 109/L (P = .008 v rHu-GCSF therapy alone). Thus, both thrombocytopenia and neutropenia are eliminated by initiating daily PEG-rHuMGDF therapy on day 1 and subsequently adding daily rHu-GCSF after 1 week in the rhesus model of hepsulfam marrow suppression. This improvement in platelet and neutrophil responses by delaying the addition of rHu-GCSF to PEG-rHuMGDF therapy demonstrates the importance of optimizing the dose and schedule of cytokine combinations after severe myelosuppressive chemotherapy.

Thrombopoietic effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in patients with advanced cancer

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) is a potent stimulator of megakaryocyte colony formation and platelet production. It is likely to be useful in the management of severe thrombocytopenia. To determine its clinical activity and safety, we gave it to patients with advanced cancer before chemotherapy. Patients were randomly assigned to receive either PEG-rHuMGDF or placebo in a three to one ratio. PEG-rHuMGDF was given at a dose of 0.03, 0.1, 0.3, or 1.0 microgram/kg body weight. The study drug or placebo were administered daily by subcutaneous injection for up to 10 days or until a target platelet count was reached. 17 patients, median age 59 years, received either PEG-rHuMGDF (13 patients) or placebo (four patients). PEG-rHuMGDF produced a dose-dependent increase in platelet counts. Patients given placebo. 0.03, and 0.1 microgram/kg of PEG-rHuMGDF had median increases in platelet counts of 16%, 12%, and 39%. Those receiving 0.3 and 1.0 microgram/kg of PEG-rHuMGDF had an increase in blood platelets of between 51% and 584%. Platelets rose from day 6 of PEG-rHuMGDF administration and continued to rise after stopping the drug. The platelet count peaked between days 12 and 18 and remained above 450 x 10(9)/L for up to 21 days. There were no alterations in white-blood-cell count or haematocrit, and low toxicity. Platelets taken from patients during PEG-rHuMGDF administration and at the time of peak platelet count were morphologically and functionally normal. The potency with which PEG-rHuMGDF stimulates platelet production and its low toxicity indicate that this is likely to be a useful agent for the management of thrombocytopenia.

In vivo effects of pegylated recombinant human megakaryocyte growth and development factor on hematopoiesis in normal mice.

The in vivo effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human thrombopoietin modified with polyethylene glycol, were investigated in normal Balb/c mice. PEG-rHuMGDF was more potent in producing platelets and the dose-response curve was steeper compared with the case of the nonpegylated form of this molecule. Five consecutive injections with PEG-rHuMGDF caused a dose-dependent increase in peripheral platelet counts with a peak on day 8. There was a dose-dependent rise in platelet counts on day 8 at daily doses from 0.333 to 30 micrograms/kg. Intermediate doses of PEG-rHuMGDF (1.111 to 10 micrograms/kg/day) caused a significant decrease in mean platelet volume, and conversely, higher doses of PEG-rHuMGDF (30 to 270 micrograms/kg/day) induced a dose-dependent increase in mean platelet volume. There was a dose-dependent decrease in hemoglobin concentration with a minimum on day 8 but no significant reduction in reticulocyte counts following PEG-rHuMGDF administration. White blood cell counts were unchanged by PEG-rHuMGDF treatment. Marrow megakaryocyte size enlarged to 1.5-fold and the number of marrow megakaryocytes increased to sixfold by consecutive administration of PEG-rHuMGDF at 30 micrograms/kg/day. A twofold increase in the number of marrow megakaryocytic progenitor cells (colony-forming units-megakaryocyte) was also observed. Marrow erythroid progenitor (colony-forming units-erythroid) counts decreased but splenic colony-forming units-erythroid, marrow and splenic erythro/myeloid progenitor cell counts, and splenic granulocyte/macrophage progenitor cell counts increased with PEG-rHuMGDF treatment. Marrow and splenic erythroid burst-forming cells were unchanged. These results indicate that PEG-rHuMGDF, a truncated molecule of thrombopoietin, is a potent stimulator for megakaryopoiesis and thrombopoiesis, and also affects the development of other hematopoietic cells in normal mice.

Administration of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor to Humans Stimulates the Production of Functional Platelets That Show No Evidence of In Vivo Activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.

Effects of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia induced by a new myelosuppressive chemotherapy regimen in mice.

Thrombopoietin, the endogenous c-Mpl ligand, is a novel lineage-specific hematopoietic factor that plays a pivotal role in the regulation of megakaryocytopoiesis and thrombopoiesis. In this study, we examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human c-Mpl ligand derivatized with polyethylene glycol, on myelosuppressive chemotherapy-induced thrombocytopenia in mice. We developed a new murine model of thrombocytopenia induced by i.v. injections of mitomycin C (MMC) for two consecutive days. In control mice, platelet counts began to decrease on day 6, reached a nadir of less than 5% of basal level on day 14, and could not recover to basal level by day 26. Administration of PEG-rHuMGDF greatly enhanced recovery of the number of megakaryocyte progenitor cells and the megakaryocytes in bone marrow, and markedly reduced the severity of thrombocytopenia; it also accelerated platelet recovery in a dose-dependent manner in myelosuppressed mice. Mice receiving consecutive administration of higher doses of PEG-rHuMGDF showed no thrombocytopenia but rather had platelet counts being increased over basal level. Although absolute neutrophil counts and red cell counts also were decreased following MMC treatment, administration of PEG-rHuMGDF also improved neutropenia and anemia. Administration of PEG-rHuMGDF on alternate days or once a week after chemotherapy was almost as effective as consecutive administration in improving thrombocytopenia. Combined administration of PEG-rHuMGDF and rHuG-CSF had an additive effect on improvement of thrombocytopenia and neutropenia. These results suggest that PEG-rHuMGDF is a therapeutically effective agent in the treatment of thrombocytopenia associated with chemotherapy.

Megakaryocyte growth and development factor accelerates platelet recovery in peripheral blood progenitor cell transplant recipients

We have investigated the potential of PEGylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a molecule related to thrombopoietin (mpl ligand or TPO) in minimizing the thrombocytopenia associated with hematopoietic ablation and peripheral blood progenitor cell (PBPC) transplant. Irradiated mice that received PBPC mobilized by PEG-rHuMGDF or granulocyte colony-stimulating factor (G-CSF) had a reduced number of thrombocytopenic days with platelets below 100 x 10(6) per mL of blood. Recipients of unmobilized PBPC had a 9 day thrombocytopenic phase which was shortened to 7 days if they were given granulocyte-macrophage colony-stimulating factor (GM-CSF)-mobilized PBPC. This was further reduced to 2 or 3 days of thrombocytopenia in recipients of G-CSF- or PEG-MGDF-mobilized PBPC. Despite our observation that PEG-rHuMGDF is a relatively modest stimulator of the mobilization of myeloid progenitors to the blood, MGDF-mobilized PBPC do effect accelerated recovery of platelets after transplantation. However, the most effective use of PEG-rHuMGDF is when it is given during the recovery phase after PBPC transplantation to hematopoietically ablated mice. Posttransplant treatment with PEG-rHuMGDF reduces thrombocytopenia to a single day or less, in recipients of most types of PBPC. Mice that were treated during the first 2 weeks after PBPC transplant with PEG-rHuMGDF had 1 thrombocytopenic day compared to 9 days in carrier-treated recipients of unmobilized PBPC and 2 to 3 days in carrier-treated recipients of the optimally mobilized PBPC from G-CSF or G-CSF/PEG-rHuMGDF treated donors. In groups where PEG-rHuMGDF was included in the mobilization protocol and used to treat recipients as well thrombocytopenia was effectively eliminated. These data show that PEG-rHuMGDF is a highly effective agent in eliminating the thrombocytopenia associated with PBPC transplantation.

Megakaryocyte growth and development factor accelerates platelet recovery in peripheral blood progenitor cell transplant recipients

We have investigated the potential of PEGylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a molecule related to thrombopoietin (mpl ligand or TPO) in minimizing the thrombocytopenia associated with hematopoietic ablation and peripheral blood progenitor cell (PBPC) transplant. Irradiated mice that received PBPC mobilized by PEG-rHuMGDF or granulocyte colony-stimulating factor (G-CSF) had a reduced number of thrombocytopenic days with platelets below 100 x 10(6) per mL of blood. Recipients of unmobilized PBPC had a 9 day thrombocytopenic phase which was shortened to 7 days if they were given granulocyte-macrophage colony-stimulating factor (GM-CSF)- mobilized PBPC. This was further reduced to 2 or 3 days of thrombocytopenia in recipients of G-CSF- or PEG-MGDF-mobilized PBPC. Despite our observation that PEG-rHuMGDF is a relatively modest stimulator of the mobilization of myeloid progenitors to the blood, MGDF-mobilized PBPC do effect accelerated recovery of platelets after transplantation. However, the most effective use of PEG-rHuMGDF is when it is given during the recovery phase after PBPC transplantation to hematopoietically ablated mice. Posttransplant treatment with PEG- rHuMGDF reduces thrombocytopenia to a single day or less, in recipients of most types of PBPC. Mice that were treated during the first 2 weeks after PBPC transplant with PEG-rHuMGDF had 1 thrombocytopenic day compared to 9 days in carrier-treated recipients of unmobilized PBPC and 2 to 3 days in carrier-treated recipients of the optimally mobilized PBPC from G-CSF or G-CSF/PEG-rHuMGDF treated donors. In groups where PEG-rHuMGDF was included in the mobilization protocol and used to treat recipients as well thrombocytopenia was effectively eliminated. These data show that PEG-rHuMGDF is a highly effective agent in eliminating the thrombocytopenia associated with PBPC transplantation.