Effects of recombinant human megakaryocyte growth and development factor on platelet activation

The c-Mpl receptor for thrombopoietin and its recombinant related protein, the megakaryocyte growth and development factor (MGDF), is also expressed on circulating platelets. In the present study we evaluated the effect of MGDF on platelet aggregation in platelet-rich plasma (PRP) and in whole blood. The results obtained indicate that MGDF by itself did not affect platelet aggregation. However, when added before other agonists such as adenosine diphosphates (ADP), epinephrine (EPI), and thrombin (THR), it rendered platelets more sensitive. This "priming" effect of MGDF was dependent on the dose and on the time of platelet preincubation, and it occurred both in PRP and in whole-blood platelet aggregation. MGDF also "primed" the release of adenosine triphosphates and the production of thromboxane B2 by platelets stimulated with ADP, EPI, and THR. When added 15 seconds after the preincubation of platelets with subthreshold concentrations of ADP, EPI, and THR, MGDF exhibited a synergism with these agonists. Moreover, we observed a "priming" effect of MGDF on the phosphorylation of p-42 mitogen-activated protein kinase promoted by ADP, EPI, and THR. These observations suggest that thrombopoietin may play a physiologic role in modulating the response of platelets to several stimuli and thereby their hemostatic potential.     

Effects of pegylated recombinant human megakaryocyte growth and development factor in patients with idiopathic thrombocytopenic purpura

We conducted a phase 1-2 clinical trial to evaluate the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in patients with chronic idiopathic thrombocytopenic purpura (ITP) refractory to standard therapy who had platelet counts below 30 x 10(9)/L. Four patients received PEG-rHuMGDF (0.5 microg/kg of body weight per day) by daily intravenous injection for up to 7 days. Administration of PEG-rHuMGDF increased platelet counts in 3 patients. A striking thrombocytosis occurred in 2 patients, whose platelet counts were elevated to more than 700 x 10(9)/L a week after the last administration of PEG-rHuMGDF and returned to baseline levels within 4 to 6 weeks. Before the platelet peak, the percentage of reticulated platelets increased transiently in 3 patients tested, including one patient who had no response. Bleeding episodes decreased after the start of PEG-rHuMGDF therapy. These results suggest that PEG-rHuMGDF might have a clinical benefit in ameliorating thrombocytopenia associated with ITP.     

Pharmacokinetics of pegylated recombinant human megakaryocyte growth and development factor in healthy volunteers and patients with hematological disorders

OBJECTIVES: The purpose of this study is to examine the pharmacokinetics of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in healthy volunteers with normal hematopoiesis and patients with idiopathic thrombocytopenic purpura (ITP), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and aplastic anemia (AA). METHODS: PEG-rHuMGDF was intravenously administered to healthy volunteers and patients with ITP, AML, MDS, and AA. The serum concentration of PEG-rHuMGDF was measured and the pharmacokinetics was investigated using non-linear mixed-effects modeling technique. RESULTS: The systemic clearance (CL) and volume of distribution at steady-state (Vss) consistently decreased in the healthy subjects, when the dose increased. In AML patients, CL and Vss decreased when the dose increased, but the change of CL was not statistically significant. In contrast, no significant dose dependency of these parameter estimates was observed in MDS patients. In AA patients there was no significant change in Vss but the CL of the higher dose groups was slightly smaller than that of the lower dose groups. Relatively smaller CL and Vss in ITP patients than those of healthy volunteers at the same dose were observed. CONCLUSIONS: This saturable pathway of CL may involve the receptor-mediated endocytosis and degradation by megakaryocyte lineage cells and platelets. The saturable distribution space can be also explained by the receptors on hematopoietic cells. The non-saturable distribution space corresponds to the value of plasma and interstitial fluid volume.     

Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates

Thrombopoietin (TPO) is the physiologic Mpl-ligand regulating platelet production. Pegylated human recombinant megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated polypeptide Mpl-ligand derivitized with poly-(ethylene glycol), induces megakaryocyte endoreduplication and proliferation in vitro and in vivo. In the present study, the dose-response effects of PEG-rHuMGDF on pharmacokinetics, megakaryocytopoiesis, platelet production, and platelet function were characterized for dosing 0.05, 0.10, 0.50, or 2.5 micrograms/kg/d in 22 baboons for 28 days. Daily subcutaneous injections of PEG-rHuMGDF produced linear log-dose responses in (1) steady-state trough plasma levels of PEG-HuMGDF (P < 10(-3)); (2) marrow megakaryocyte volume (P < 10(-3)), ploidy (P < 10(-4)), and number (P < .01); and (3) peripheral platelet concentrations (P < 10(- 4)) and platelet mass turnover (P < 10(-3)). Platelet morphology, life span, and recovery were normal, and peripheral leukocyte, neutrophil, and erythrocyte counts were not significantly affected by PEG-rHuMGDF (P > .1 in all cases). PEG-rHuMGDF at 0.5 micrograms/kg/d produced similar blood concentrations of Mpl-ligand and platelets as 10 times the dose of rHu-MGDF (5.0 micrograms/kg/d), reflecting the extended plasma half-life achieved through pegylation. Whereas PEG-rHuMGDF did not induce platelet aggregation in vitro, platelet aggregatory responsiveness induced by thrombin receptor agonist peptide (TRAP1-6) and collagen was transiently enhanced ex vivo during the initial few days of PEG-rHuMGDF administration. However, adenosine diphosphate (ADP)-induced platelet aggregation was not enhanced ex vivo by PEG- rHuMGDF therapy. 111In-platelet deposition on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts increased in direct proportion to the circulating platelet concentration (P < 10(-4) for both EA and VG); 125l-fibrin accumulation was not affected by PEG-rHuMGDF-induced increases in peripheral platelet counts. Changes in platelet production and function produced by PEG-rHuMGDF returned to baseline within 2 weeks after discontinuing treatment. Thus, in nonhuman primates, PEG- rHuMGDF increases platelet production in a linear log-dose-dependent manner by stimulating megakaryocyte endoreduplication and new megakaryocyte formation from marrow hematopoietic progenitors. These findings suggest that appropriate dosing of PEG-rHuMGDF therapy during periods of chemotherapy-induced marrow suppression may maintain hemostatic concentrations of peripheral platelets without increasing the risk of thrombosis.     

Long-term administration of pegylated recombinant human megakaryocyte growth and development factor dramatically improved cytopenias in a patient with myelodysplastic syndrome

To date, there are no curative therapeutic options for patients with myelodysplastic syndromes (MDS) other than allogeneic stem cell transplantation. We treated an MDS patient with 10 microg/kg pegylated recombinant human megakaryocyte growth and development factor (rHuMGDF) for more than 450 d. The patient's platelet counts increased from <10 x 10(9)/l to 50 x 10(9)/l. Interestingly, haemoglobin levels increased dramatically and reached over 13 g/dl without additional transfusion. Adverse events and neutralizing antibodies were not observed during treatment, suggesting that the long-term administration of rHuMGDF might be of clinical benefit to patients with MDS.     

Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.     

Marked improvement of thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura by pegylated recombinant human megakaryocyte growth and development factor

OBJECTIVE: We examined the stimulatory effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production in male (NZW x BXSB) F(l) (W/B F(1)) mice, a murine model of idiopathic thrombocytopenic purpura. MATERIALS AND METHODS: A cohort of 19- to 25-week-old, severely thrombocytopenic male W/B F(1) mice were given PEG-rHuMGDF at different dosing schedules. Before and at various times after therapy, platelet counts, reticulated platelets, platelet lifespan, and levels of platelet-associated immunoglobulin G were measured. Analysis of megakaryocytic cells was performed. RESULTS: Treatment of male W/B F(1) mice with PEG-rHuMGDF (30 microg/kg/day) three times per week for several weeks resulted in sustained thrombocytosis, accompanied by increased megakaryocytopoiesis in both the bone marrow and spleen. The degree of the platelet response to PEG-rHuMGDF varied between individual mice, likely reflecting the heterogeneity of the disease. Production of new platelets in response to PEG-rHuMGDF was manifested by an increase in reticulated platelets. Levels of platelet-associated immunoglobulin G decreased inversely during periods of thrombocytosis. PEG-rHuMGDF therapy also improved thrombocytopenia in male W/B F(1) mice refractory to splenectomy. Platelet lifespan was not affected by PEG-rHuMGDF. Male W/B F(1) mice treated with pegylated murine MGDF, a homologue of PEG-rHuMGDF, had persistent thrombocytosis for at least 7 months, suggesting that antiplatelet antibody production was not enhanced. CONCLUSIONS: PEG-rHuMGDF therapy potently stimulated platelet production, effectively ameliorating thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura.     

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia

Recombinant human megakaryocyte growth and development factor (rHuMGDF), a truncated form of the Mpl ligand, stimulates megakaryopoiesis both in vitro and in vivo. We describe the in vitro effect of pegylated recombinant human MGDF (PEGrHuMGDF) alone and in combination with other haemopoietic growth factors (G-CSF, GM-CSF, IL3, IL6, erythropoietin, SCF) on megakaryopoiesis in bone marrow from 11 normal subjects and 19 patients with aplastic anaemia (AA). We used semi-solid cultures to assess megakaryocyte colony growth (CFU-Mk) and 7 d suspension cultures to assess production of platelet glycoprotein IIIa (CD61) positive cells. CFU-Mk growth from normal marrow increased 3-4-fold and CD61+ve cells in suspension culture increased 8-10-fold with the addition of 10 ng/ml PEGrHuMGDF. In normal subjects growth factor combinations further increased responses in suspension culture, PEGrHuMGDF + SCF, PEGrHuMGDF + IL3 and PEGrHuMGDF + SCF + IL3 + Epo (P<0.05). IL6, GM-CSF, G-CSF or Epo added with PEGrHuMGDF did not consistently give this increase. CFU-M. growth from AA marrow remained very low in the presence of PEGrHuMGDF, with or without the addition of other growth factors. CD61+ve cells in suspension culture were, however, increased in the presence of PEGrHuMGDF alone in 12/19 AA cases. Of the 12 patients responsive to PEGrHuMGDF, nine were tested with additional growth factors and further responses were seen in six. In the AA cases PEGrHuMGDF+SCP and PEGrHuMGDF+SCF+IL3+Epo gave the highest responses. These data suggest that PEGrHuMGDF, alone or in combination with SCF and/or IL3, can enhance megakaryocyte proliferation in some patients with aplastic anaemia and may therefore have a role in the treatment of thrombocytopenia in these cases.     

Megakaryopoiesis in vitro in myelodysplastic syndromes and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG-rHuMGDF combined with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61-positive cells in suspension cultures increased with PEG-rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL-3 and/or SCF were added to PEG-rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU-Mk growth was poor overall, but could be enhanced by PEG-rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG-rHuMGDF can be augmented by IL-3 and SCF in many MDS and AML patients.     

The effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet recovery in breast cancer patients undergoing autologous bone marrow transplantation

OBJECTIVE: To assess the safety and efficacy of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) administered after autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Two randomized, double-blind, placebo-controlled studies were done. In the phase 1/2 study, 75 breast cancer patients underwent a bone marrow harvest and myeloablative STAMP V chemotherapy and were randomized to receive placebo or one of three doses of PEG-rHuMGDF. In the phase 3 study, 64 patients were randomized to receive placebo or the minimally effective dose of PEG-rHuMGDF. The study drug was administered daily starting on the day of bone marrow infusion until the platelet count was greater than or equal to 50 x 10(9)/L (without transfusion) or for a maximum of 28 days. All patients received 10 microg/kg/day filgrastim starting on day 2 until neutrophil count recovery. RESULTS: PEG-rHuMGDF appeared to be safe and well tolerated. No significant differences were noted in mortality or disease progression rates. Antibodies to MGDF were not observed. In the phase 1/2 study, the time to platelet recovery to greater than or equal to 20 x 10(9)/L and platelet transfusion requirements were significantly reduced for patients treated with PEG-rHuMGDF compared with placebo (p < 0.05). In the phase 3 study, no significant differences in the kinetics of early thrombopoiesis or platelet transfusions after ABMT were observed. CONCLUSIONS: PEG-rHuMGDF was not consistently efficacious in reducing the duration of severe thrombocytopenia. The maximum platelet counts for PEG-rHuMGDF-treated patients occurred a median of 2 weeks after the last dose of drug, suggesting that the biologic effects of this hematopoietic cytokine are delayed compared with other hematopoietic cytokines.     

A double-blind, placebo-controlled trial of pegylated recombinant human megakaryocyte growth and development factor as an adjunct to induction and consolidation therapy for patients with acute myeloid leukemia

Newly diagnosed patients with acute myeloid leukemia (AML) were randomized to receive either 2.5 or 5 microg/kg/day of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or a placebo administered subcutaneously after completion of chemotherapy. The study evaluated the toxicity of PEG-rHuMGDF and any effect on the duration of thrombocytopenia. Each of 35 patients under 60 years of age received the following therapy: 45 mg/m(2) daunorubicin on days 1-3, 100 mg/m(2) cytarabine (ARA-C) for 7 days, and 2 gm/m(2) high-dose ARA-C (HIDAC) for 6 doses on days 8-10. The 22 patients 60 years or older received standard daunorubicin and ARA-C without HIDAC. PEG-rHuMGDF was well tolerated, and no specific toxicities could be attributed to its use. There was no difference in the time to achieve a platelet count of at least 20 x 10(9)/L among the 3 groups (median 28-30 days for patients less than 60 years old and 21-23 days for patients 60 years or older). Patients receiving PEG-rHuMGDF achieved higher platelet counts after remission. However there was no significant difference in the number of days on which platelet transfusions were administered among the 3 groups. The complete remission rate was 71% for patients less than 60 years and 64% for those 60 years or older, with no significant difference among the 3 groups. Postremission consolidation chemotherapy with either placebo or PEG-rHuMGDF was given to 28 patients beginning the day after completion of chemotherapy. There was no apparent difference in the time that was necessary to reach a platelet count of at least 20 or 50 x 10(9)/L or more platelets or in the number of platelet transfusions received. In summary, PEG-rHuMGDF was well tolerated by patients receiving induction and consolidation therapy for AML; however, there was no effect on the duration of severe thrombocytopenia or the platelet transfusion requirement. (Blood. 2000;95:2530-2535)     

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 μg/kg/d of PEG-rHuMGDF, whereas peripheral platelet counts plateaued at 30 μg/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.     

Synergistic effects of pegylated recombinant human megakaryocyte growth and development factor and granulocyte colony-stimulating factor on mobilization of hematopoietic progenitor and stem cells with long-term repopulating ability into peripheral blood in mice

We investigated the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization and the combined effect of PEG-rHuMGDF plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) in C57BL/6 mice. Treatment of mice with PEG-rHuMGDF increased the numbers of day 8 and day 12 spleen colony-forming units (CFU-S), and pre-CFU-S in the PB. Ten days administration of PEG-rHuMGDF could mobilize higher numbers of days 8 and day 12 CFU-S than 5 days administration. An optimal dose of PEG-rHuMGDF mobilized a higher number of committed progenitor cells (day 8 CFU-S) and a lower number of immature progenitor cells (pre-CFU-S) into PB than rhG-CSF. The combined administration of optimal or suboptimal doses of PEG-rHuMGDF and rhG-CSF induced synergistic effects on mobilization of CFU-S and pre-CFU-S into PB compared to either factor alone. Four months after sex-mismatched PBPC transplantation, long-term donor-derived engraftment was observed in all recipients that had been transplanted with PBPCs mobilized by rhG-CSF and/or PEG-rHuMGDF, as determined by Y-chromosome polymerase chain reaction (PCR) analysis. Our data suggest that cytokine-induced pathways for PBPC mobilization may be different between PEG-rHuMGDF and rhG-CSF and indicate that PEG-rHuMGDF alone or the combination with rhG-CSF may be useful in effective PBPC mobilization.     

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF) on megakaryopoiesis in normal subjects and patients with myelodysplasia and acute myeloid leukaemia

Mpl ligand is a recently cloned haemopoietic growth factor that stimulates megakaryopoiesis in vitro and in vivo . We describe the in vitro effect of a truncated form of Mpl ligand, recombinant human megakaryocyte growth and development factor (rHuMGDF), on megakaryopoiesis in bone marrow from normal subjects and patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We used both semi-solid and suspension culture techniques to assess the effect of pegylated (PEG) rHuMGDF on megakaryocyte colony growth (CFU-Mk) and on the production of CD61⁺ cells in 7 d suspension cultures. PEG rHuMGDF increased CFU-Mk growth and CD61⁺ cell production in a dose-dependent fashion in all normal marrows tested. Normal CFU-Mk growth was increased threefold with the addition of 10 ng/ml PEG rHuMGDF to cultures and CD61⁺ cells were increased 8–10-fold by the same dose. Although increased CFU-Mk growth was only seen in 1/10 AML and 6/16 MDS marrows, CD61⁺ cell numbers in suspension culture were increased in 9/13 AML and 12/15 MDS samples, responses ranged from very limited to normal magnitude. There was no correlation between platelet count and CFU-Mk number, CD61⁺ cell number or response to PEG rHuMGDF. We did not find any increased CFU-GM colony or cluster growth in response to PEG rHuMGDF and the CD61⁺ cells produced in suspension culture had features of megakaryocytic differentiation. These data suggest that PEG rHuMGDF can enhance megakaryocyte proliferation in some patients with MDS and AML, and may have a role in the treatment of thrombocytopenia in these patients.     

Phase 1 study of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in breast cancer patients after autologous peripheral blood progenitor cell (PBPC) transplantation

Forty-seven patients with stage II, III, or IV breast cancer undergoing autologous peripheral blood progenitor cell (PBPC) transplantation were randomized to placebo (n = 13) or to one of five sequential dose cohorts of pegylated (PEG) recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) (1.0, 2.5, 5.0, 7.5, or 10.0 microg/kg/day) (n= 34). Blinded study drug was started on the day of transplantation and was continued until the platelet count was > or =100 x 109/l or a maximum of 21 days. PBPCs were mobilized with filgrastim (r-metHuG-CSF) and all patients received filgrastim starting on day +2 after transplantation. The nadir platelet count was not affected by treatment. The median time to platelet recovery was 11 and 12 days for the placebo and combined PEG-rHuMGDF groups, respectively. No trends in adverse events suggested dose- or treatment-related toxicity. Two patients withdrew from the study because of an adverse event (allergic reaction in the 7.5 microg/kg group) probably related to study drug, and veno-occlusive disease (VOD) (in the 5 microg/kg group) which was felt not to be related to study drug by the investigator. No patients developed neutralizing antibodies to MGDF. Day +21 and day +28 platelet counts were higher in the group receiving PEG-rHuMGDF (246 vs 148 x 109/l and 299 vs 145 x 109/l, respectively; both P < 0. 05). PEG-rHuMGDF up to 10 microg/kg/day was well tolerated. In this study, there was no effect of study drug on initial platelet engraftment at the doses studied. However, the efficacy of other doses is unknown.     

Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.     

A randomized, double-blind, placebo-controlled study with pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) as an adjunct to chemotherapy for adults with de novo acute myeloid leukemia

To determine the safety, biologic, and clinical benefits of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF; Amgen, Thousand Oaks, CA) after myelosuppressive chemotherapy in acute myeloid leukemia (AML), 108 adult patients with de novo AML were randomized to receive either PEG-rHuMGDF (2.5 microg/kg/d or 5 microg/kg/d) for up to 21 doses (group A), a single dose of 2.5 microg/kg PEG-rHuMGDF, 7 daily doses of 2.5 microg/kg PEG-rHuMGDF (group B), or placebo. The greatest biologic activity was seen in group A with a median peak platelet count of 1,084 x 10(9)/L, occurring at a median 9 days after the last dose of study drug, compared with 517 x 10(9)/L and 390 x 10(9)/L in group B and placebo group, respectively. Thrombocytosis (platelets >1,000 x 10(9)/L) was seen at rates of 52%, 8%, and 9% in groups A, B, and placebo, respectively, but were not associated with any adverse event. There was no effect on median time to transfusion independent platelet recovery (> or = 20 x 10(9)/L). The median time to neutrophil recovery (> or = 500/microL) and red blood cell transfusion requirements were similar in all groups, and there was no apparent stimulation of leukemia. PEG-rHuMGDF was biologically active and well tolerated. Further investigation of dose and scheduling is required, specifically earlier dosing before and during chemotherapy.     

In vivo effects of recombinant human interleukin-6 in primates: stimulated production of platelets

In cynomolgus monkeys, twice daily subcutaneous injections of recombinant human interleukin-6 (rhIL-6) at doses of 5 to 80 micrograms/kg/d for 14 consecutive days caused dose-dependent increases in platelet count, usually continuing for more than 1 week after cessation of the injections. The count reached a level approximately twofold or more above the preinjection level even at 5 micrograms/kg/d, and at doses of more than 20 micrograms/kg/d, the increase became biphasic with a higher second peak 3 days after cessation of the injections. Morphologic analysis of the bone marrow after the 7 day-injections with 80 micrograms/kg/d revealed a marked increment in size of megakaryocytes compared with control, indicating the promotion of megakaryocyte maturation. Other changes attributable to the rhIL-6 treatment include dose-dependent loss of body weight, anemia, neutrophilia and monocytosis, elevation of serum C-reactive protein and alpha-1 acid glycoprotein levels, and decrease of serum albumin; all of which returned to normal within 1 week after cessation of the injections and were tolerable at doses of less than 10 micrograms/kg/d. These findings suggest that rhIL-6 may be an effective strategy for the treatment of thrombocytopenia.     

Leukemia inhibitory factor/human interleukin for DA cells: a growth factor that stimulates the in vitro development of multipotential human hematopoietic progenitors.

We investigated the in vitro hematopoietic stimulatory activity of leukemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) on bone marrow progenitor populations in 17 normal individuals. In serum-free cultures LIF/HILDA did not induce colony growth. In serum containing media, LIF/HILDA stimulated the growth of colony forming unit (CFU)-MIX and CFU-EO in a dose-dependent fashion and resulted in an increased CFU-MIX and burst forming unit-erythrocytes (BFU-E) colony size. Similar stimulatory effects were seen on a highly purified hematopoietic progenitor population obtained after immunomagnetic depletion of mature myeloid precursors and lymphoid cells. Addition of LIF/HILDA to cultures containing maximally stimulatory concentrations of recombinant human interleukin-3 (rhuIL3), rhuIL3 + rhuIL6, or rhu granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) in serum containing media significantly increased the number of CFU-MIX and eosinophil colonies and increased size and cluster number of CFU-MIX and BFU-E. Depletion of accessory T lymphocytes or monocytes from bone marrow progenitors did not alter the response of hematopoietic precursors to LIF/HILDA. A similar increased colony growth was seen when LIF/HILDA was added to cultures of positively selected CD34/HLA-DR+ or CD34+/HLA-DR- bone marrow hematopoietic progenitor cells stimulated with maximally stimulatory concentrations of rhuIL3 + rhuIL6. LIF/HILDA is a novel cytokine capable of stimulating growth and proliferation of multi-lineage, erythroid, and eosinophil colonies in the presence of serum. LIF/HILDA exerts its activity by direct interaction with highly purified immature bone marrow progenitor cells, has an additive effect when used with other cytokines known to stimulate primitive hematopoietic precursors, and does not require accessory cells.