Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice

The effects of recombinant human interleukin-11 (rhIL-11) on in vivo mouse megakaryocytopoeisis were examined. Normal C57Bl/6 mice and splenectomized C57Bl/6 mice were treated for 7 days with 150 micrograms/kg rhIL-11 administered subcutaneously. In normal mice, peripheral platelet counts were elevated compared with vehicle-treated controls after 3 days of rhIL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhIL-11 showed elevated peripheral platelet counts that were similar in magnitude to normal rhIL-11-treated mice. However, on day 10 the platelet counts in rhIL-11- treated, splenectomized mice were no longer elevated. Analysis of bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the percentage of 32N megakaryocytes in both normal and splenectomized animals treated with rhIL-11. In normal mice, the number of spleen megakaryocyte colony-forming cells (MEG-CFC) were increased twofold to threefold relative to controls after 3 and 7 days of rhIL-11 treatment, whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone marrow of rhIL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3 and 7 of the study. These data show that in vivo treatment of normal or splenectomized mice with rhIL-11 increased megakaryocyte progenitors, stimulated endoreplication of bone marrow megakaryocytes, and increased peripheral platelet counts. In addition, results in splenectomized mice showed that splenic hematopoiesis was not essential for the observed increases in peripheral platelets in response to rhIL-11 administration.     

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.     

The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis

Thrombopoietin (TPO), the ligand for the c-Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor-binding portion of c-Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 μg/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 μg/kg/d resulted in platelet counts 5-fold above normal. However, doses >60 μg/kg/d resulted in less-than-optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose-dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF-mediated inhibition was specific, since the addition of the truncated form of the c-Mpl receptor reversed the inhibition in a dose-dependent manner. Other recombinant factors, interleukin-6, interleukin-11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.     

Multilineage mobilization of peripheral blood progenitor cells in humans following administration of PEG-rHuMGDF

The most important physiological regulator of megakaryocytopoiesis is the ligand for the c-mpl receptor (thrombopoietin/megakaryocyte growth and development factor, MGDF). We examined the effect of pegylated-recombinant human MGDF (PEG-rHuMGDF): patients received PEG-rHuMGDF at doses of 0.03, 0.1, 0.3 or 1.0 μg/kg/d or placebo for 10 d maximum in a double-blinded randomized study. There was a dose-dependent elevation in circulating platelet counts but no alteration in erythrocyte or total leucocyte counts. The number of bone marrow megakaryocytes was increased approximately 2-fold. The frequency of bone marrow progenitor cells was not altered. In contrast, both to the bone marrow results and to published pre-clinical data, there was a dose-dependent mobilization into the blood of progenitor cells of multiple cell lineages. Increased levels of Meg-CFC (maximum increase 30-fold), day 7 and day 14 GM-CFC and BFU-E were demonstrated at doses of 0.3 and 1.0 μg/kg/d PEG-rHuMGDF. At 0.1 μg/kg/d, mobilization of Meg-CFC alone occurred in two-thirds of patients. Maximum blood levels of progenitor cells occurred at day 12. Thus, administration of PEG-rHuMGDF to humans resulted in mobilization of progenitor cells of multiple lineages despite its 'lineage-specific' activity on mature cell development.     

In vivo stimulation of platelet production in a primate model using IL-1 and IL-3

The in vivo administration of various cytokines for hematopoietic stimulation has led primarily to the enhancement of the myeloid response with an insignificant contribution toward stimulating any increase in platelet production. Current studies have suggested that interleukin 1 (IL-1) and interleukin 3 (IL-3) are two of several factors that have an effect on either megakaryocyte formation or platelet production. The objective of our research was to investigate how the in vivo administration of IL-1 or IL-3 or a combination could be used to regulate megakaryocytopoiesis and platelet production in nonhuman primates. A single dose of IL-1 was able to stimulate an increase in platelet production for 3 weeks. The response was shown to be biphasic, with increased platelet counts of 46% and 49% above baseline on days 8 and 17, respectively. In contrast, the administration of IL-3 for 6 days led to an increase of 29% above baseline on day 17. An interesting observation was that the increased platelet counts were accompanied by a transient increase in the peripheral blood of a highly proliferative megakaryocyte colony-forming cell (MK-CFC), which attained a maximum concentration on day 7. The administration of a sequential combination of IL-1, then IL-3, was further evaluated to elucidate a possible potentiation on platelet production. The result was a similar increase in platelets to that observed in IL-1-only-treated monkeys for the first 7 days. However, the most significant effect was observed on day 17, when the 85% increase in platelets was demonstrated to be additive of the single-agent effects on that day. A reversal in the order of cytokine administration did not affect platelet production in this manner. In IL-1, then IL-3-treated monkeys, the increased platelet counts were also accompanied by an increase in the concentration of the peripheral blood MK-CFC from days 7 through 14. These results demonstrate that a combination of factors may be required to enhance platelet production, stimulating not only the formation of megakaryocytes but also stimulating the production and release of platelets into the peripheral blood.     

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.     

Effects of recombinant human interleukin-11 on hematopoietic reconstitution in transplant mice: acceleration of recovery of peripheral blood neutrophils and platelets

We have examined the effects of recombinant human interleukin-11 (rhIL- 11) on the recovery of peripheral blood cell counts and proliferation of progenitors and hematopoietic stem cells (day 12 colony-forming units-spleen-CFU-S12) in vivo using a mouse bone marrow (BM) and spleen cell transplantation model. Recovery of leukocytes was accelerated in animals receiving daily administration of rhIL-11 (100 micrograms/kg/d) and reached normal levels by day 14 posttransplantation. This increased total leukocyte count reflected mainly an increase in neutrophils. Neutropenia (absolute neutrophil count [ANC] < 1,500) was present in control transplant mice for 14 to 15 days, while in the rhIL-11-treated group, neutrophils recovered to normal by days 8 to 10 and continued to increase until day 19. Animals treated with rhIL-11 had only 1 day with ANC demonstrated < 500. Correspondingly, rhIL-11 treatment increased granulocyte-macrophage progenitors (CFU-GM) derived from both spleen and BM cells. Higher doses of IL-11 increased CFU-GM nearly threefold and CFU-Mix fourfold to fivefold, while increasing burst-forming units- erythroid to a lesser degree. BM and spleen cellularity were both increased in IL-11-treated mice, but no increase in CFU-S12 was noted. In addition, in vivo daily administration of IL-11 increased peripheral platelet counts by threefold over control transplant mice at day 10 posttransplantation during the post-irradiation platelet nadir. Further treatment led to platelet counts higher than normal 18 days posttransplantation when control animals had just attained normal platelet counts. IL-11 can accelerate the recovery of the peripheral blood leukocytes, mainly neutrophils, and platelets in transplant mice, effects that may be clinically useful in future applications for BM transplantation and chemotherapy-related cytopenias.     

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.     

In vivo synergism of recombinant human interleukin-3 and recombinant human interleukin-6 on thrombopoiesis in primates.

Using a primate model, we examined the effect of recombinant human interleukin-3 (rhIL-3) and rhIL-6 on thrombopoiesis in vivo. Administration of 33 micrograms/kg/d of rhIL-3 for 11 to 14 days increased levels of circulating colony-forming units megakaryocyte (CFU-Mk) by approximately 15-fold in five rhesus monkeys without raising their platelet counts. In contrast, administration of 30 micrograms/kg/d of rhIL-6 for 10 days in four animals did not increase CFU-Mk levels but significantly raised platelet counts from a mean pretreatment value of 460 x 10(3)/microL (range 360 to 610) to a mean maximum of 746 x 10(3)/microL (665 to 790) on day 8. If monkeys were pretreated with rhIL-3 (33 or 100 micrograms/kg/d for 11 days) to expand their CFU-Mk compartment, the thrombopoietic effect of rhIL-6 was synergistically enhanced leading to platelet counts above 1,000 x 10(3)/microL (mean maximum value 1,247) in all three primates studied. The sequential administration of rhIL-3 and rhIL-6 might represent a powerful strategy to stimulate thrombopoiesis in vivo.     

Effects of human interleukin-6 on megakaryocyte development and thrombocytopoiesis in primates

Recombinant human interleukin-6 (IL-6) has previously been shown to increase platelet counts in mice and primates. To elucidate the mechanisms underlying this phenomenon, serial analyses were performed on megakaryocytes obtained from rhesus monkeys treated for 8 days with 30 micrograms/kg/d of recombinant human IL-6. Platelet counts increased to a maximum of 7.8 x 10(5)/microL with biphasic peaks on days 7 and 12 without significant changes in platelet volumes. Large increases in DNA content were seen by two-color flow cytometry and digital image analysis. Ploidy distribution underwent a significant shift between study days 3 and 11 (P less than .0001) with large increases in the frequency of 64N and 128N megakaryocytes. The modal ploidy increased from the normal 16N to 64N. Megakaryocyte size, as measured by area, was increased 2- to 2.7-fold. On day 3, multiple megakaryocytes were seen in endomitosis, along with an abundance of young cells with wide, organelle-free peripheral zones. The giant megakaryocytes seen on days 5 to 7 exhibited marked membrane hyperplasia that occupied much of the cell. Emperipolesis occurred frequently, as did megakaryocyte cell death. No giant platelets were seen. We conclude that IL-6 significantly alters the process of megakaryocyte maturation and thrombocytopoiesis, and that these effects, at least in the doses of IL-6 administered, should not be equated with the physiologic mechanisms operative during accelerated platelet production.     

The effect of oestrogen on megakaryocyte differentiation and platelet counts in vivo

BACKGROUND: Oestrogen alters megakaryocyte number in humans and mice. In mice, high-dose oestrogen stimulates an initial increase in megakaryocyte number followed by a decrease. However, the cellular action, effect of physiologically circulating and smaller supra-physiological oestrogen doses and whether changes in megakaryocyte number alter platelet counts have not been studied. METHODS: To further examine oestrogen's effect on megakaryocytes and platelets we administered intact or ovariectomised mice various doses of oestrogen and measured megakaryocyte and platelet counts. To determine the cellular mechanism by which oestrogen influences megakaryocytopoesis we also examined its effect on markers of megakaryocytic differentiation (CD41, CD61, CD34). RESULTS: We found that large doses of oestrogen (500 microg/kg) increased mature CD41+ megakaryocyte number within 2 days, and this was associated with an increase in circulating platelets. Smaller supra-physiological doses (100 microg/kg) lacked this anabolic effect, but still suppressed megakaryocyte and platelet number by day 10 in intact and ovariectomised mice. This was preceded by a reduction in the number of CD61+ megakaryoblasts and CD34+ precursors available to form mature megakaryocytes. In contrast, ovariectomy had no effect on megakaryocyte or platelet number, indicating that circulating oestrogen concentrations do not influence megakaryocyte differentiation or activity. CONCLUSIONS: Our data suggest that in mice at least platelet counts reflect changes in megakaryocyte number, and while both are independent of physiological hormone concentrations, they are sensitive to even small supra-physiological doses of oestrogen. Therefore, to ovoid disrupting platelet homeostasis the dose of oestrogen given should be no more than replacement.     

A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia

To evaluate the hematologic effects of recombinant human interleukin-6 (rhIL-6, Escherichia coli, SDZ ILS 969, IL-6), and determine its toxicity profile, we performed a phase I trial of IL-6 in 22 patients with various myelodysplastic syndromes (MDS), platelet counts < 100,000/microL, and < 5% bone marrow (BM) blasts. Patients received one of four doses of IL-6 (1.0, 2.5, 3.75, and 5.0 micrograms/kg/d) as a subcutaneous injection on day 1, followed by a 7-day wash-out period, and then 28 days of IL-6 therapy. Dose-limiting toxicities of fatigue, fever, and elevated alkaline phosphatase were seen at 5.0 micrograms/kg/d; the maximum tolerated dose was 3.75 micrograms/kg/d. All patients experienced at least grade II fever and all had an increase in acute phase proteins. Eight patients (36%) experienced at least a transient improvement in platelet counts; three fulfilled the criteria for response, whereas five others had clinically significant increases that failed to meet response criteria. Various IL-6-related toxicities prevented more than three patients from receiving maintenance therapy. Two of the three patients who received maintenance IL-6 therapy had a persistent increase in platelet counts, during 3 and 12 months of IL-6 therapy, respectively. Laboratory studies indicated that IL-6 increased the frequency of higher ploidy megakaryocytes but did not significantly increase the number of assayable megakaryocytic progenitor cells, suggesting that IL-6 acts as a maturational agent rather than a megakaryocyte colony-stimulating factor. Although IL-6 therapy can promote thrombopoiesis in some MDS patients, its limited activity and significant therapy-related toxicity preclude its use as a single agent in this patient population. Further studies, combining low doses of IL-6 with other hematopoietic growth factors, are underway.     

In vitro and in vivo activity of human recombinant granulocyte-macrophage colony-stimulating factor in dogs

Although it is well documented that human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production and functional activity of human and nonhuman primate granulocytes and macrophages, relatively little is known about its effects on cells obtained from other species. The molecular cloning of the complementary DNA for human GM-CSF has made it possible to determine the cross-reactivity of the purified recombinant human material (rhGM-CSF) on cells of other species. The results presented herein show that specific receptors for human GM-CSF exist on dog bone marrow cells and mature circulating dog granulocytes. The number of the receptors and the apparent binding affinity of the rhGM-CSF to its receptors on granulocytes were similar to those observed either on human or monkey cells. In cultures of dog bone marrow cells, rhGM-CSF was capable of promoting colony formation in a dose-dependent manner. Human GM-CSF also primed dog granulocytes for increased production of reactive oxygen metabolites in response to either phorbolmyristic acetate-or zymosan-activated dog serum. In vivo, s.c. administration to healthy dogs of rhGM-CSF in daily doses of 15, 50, or 150 micrograms/kg body weight over a period of 7-20 days induced a dose-dependent rise of up to a maximum of a fourfold increase in peripheral WBC counts. The rise in WBC counts was mainly due to elevated neutrophil levels, but an increase in the numbers of monocytes and eosinophils was also observed. However, the rhGM-CSF-induced leukocytosis in dogs was not as dramatic as that observed in nonhuman primates. In all rhGM-CSF-treated dogs, circulating platelet counts dropped to nadir levels of about 20%-30% of normal numbers. Dogs that were treated with 150 micrograms/kg rhGM-CSF developed specific antibodies after about 10-12 days of treatment. These antibodies were able to neutralize the effect of rhGM-CSF in in vitro assays. In vivo WBC counts began to decline when specific antibodies developed, but they never dropped below normal levels. Taken together, the results suggest that human GM-CSF does not appear to exhibit absolute species specificity.     

In vitro response of myelodysplastic megakaryocytopoiesis to megakaryocyte growth and development factor (MGDF)

To evaluate a potential therapeutic role for megakaryocyte growth and development factor (MGDF) in myelodysplastic syndromes (MDS) we compared the in vitro response of normal and myelodysplastic megakaryopoiesis to MGDF on bone marrow mononuclear cells from nine MDS patients and four healthy donors in a short term liquid culture system. The cells were incubated with MGDF alone (1 ng/ml, 10 ng/ml and 100 ng/ml) and in combination with 20 ng/ml stem cell factor, 2 U/ml erythropoietin (EPO) and 100 ng/ml interleukin-3 (IL-3). Cytospins were prepared after 4 days and 10 days for CD61 APAAP staining. In addition, the ploidy of propidium iodide stained CD61 positive cells were detected by flow cytometry. The CD61+ cell number significantly increased (13-fold after 4 days, 56-fold after 10 days of culture) when MGDF (100 ng/ml) was added to the normal bone marrow (NBM) cultures (P=0.0003; P=0.0001). In the MDS cultures the absolute number of endomitotic cells as well as the percentage of polyploid cells remained significantly lower than in NBM after 10 days (P=0.0001). The effect of MGDF on polyploidization of MDS cells was significantly dose dependent (P=0.0051). We found no correlation between peripheral platelet count, cellularity of the bone marrow, number of bone marrow megakaryocytes or FAB-subtype and response to MGDF. Additional administration of IL-3 resulted in a left-shift of megakaryopoiesis in both groups. Even though the response of myelodysplastic megakaryocytic progenitors to MGDF shows inter-individual variations, it is significantly impaired overall. Our data suggest that higher doses of MGDF may be able to ameliorate thrombocytopenia in a subgroup of MDS patients.     

Chronic exposure to retroviral vector encoded MGDF (mpl-ligand) induces lineage-specific growth and differentiation of megakaryocytes in mice

Megakaryocyte growth and development factor (MGDF) has recently been identified as a ligand for the c-mpl receptor. Using retroviral- mediated gene transfer, MGDF has been overexpressed in mice to evaluate the systematic effects due to chronic exposure to this growth factor. MGDF overexpressing mice had more rapid platelet recovery than control mice after transplantation. Following this recovery, the platelet levels continued increasing to fourfold to eightfold above normal baseline levels and remained elevated (five-fold above control mice) in these animals, which are alive and well at more than 4 months posttransplantation. Increased megakaryocyte numbers were detected in a number of organs in these mice including bone marrow, spleen, liver, and lymph nodes. Prolonged overexpression of MGDF led to decreased marrow hematopoiesis, especially erythropoiesis, with a shift to extramedullary hematopoiesis in the spleen and liver. All the MGDF overexpressing mice analyzed to date developed myelofibrosis and osteosclerosis, possibly induced by megakaryocyte and platelet produced cytokines. No significant effect on other hematopoietic lineages was seen in the MGDF overexpressing mice, showing that the stimulatory effect of MGDF in vivo is restricted to the megakaryocyte lineage.     

Systemic hematologic effects of PEG-rHuMGDF-induced megakaryocyte hyperplasia in mice

PEG-rHuMGDF injected daily in normal mice causes a rapid dose-dependent increase in megakaryocytes and platelets. At the same time that platelet numbers are increased, the mean platelet volume (MPV) and platelet distribution width (PDW) can be either decreased, normal, or increased depending on the dose and time after administration. Thus, PEG-rHuMGDF at a low dose causes decreases in MPV and PDW, MGDF at an intermediate dose causes an initial increase followed by a decrease in MPV and PDW, and PEG-rHuMGDF at higher doses causes an increase in MPV and PDW followed by a gradual normalization of these platelet indices. In addition to the expected thrombocytosis after 7 to 10 days of daily injection of high doses of PEG-rHuMGDF, a transient decrease in peripheral red blood cell numbers and hemoglobin is noted accompanied in the bone marrow by megakaryocytic hyperplasia, myeloid hyperplasia, erythroid and lymphoid hypoplasia, and deposition of a fine network of reticulin fibers. Splenomegaly, an increase in splenic megakaryocytes, and extramedullary hematopoiesis accompany the hematologic changes in the peripheral blood and marrow to complete a spectrum of pathologic features similar to those reported in patients with myelofibrosis and megakaryocyte hyperplasia. However, all the PEG-rHuMGDF-initiated hematopathology including the increase in marrow reticulin is completely and rapidly reversible upon the cessation of administration of PEG-rHuMGDF. Thus, transient hyperplastic proliferation of megakaryocytes does not cause irreversible tissue injury. Furthermore, PEG-rHuMGDF completely ameliorates carboplatin-induced thrombocytopenia at a low-dose that does not cause the hematopathology associated with myelofibrosis.     

Pharmacokinetics and adverse events following 5-day repeated administration of lenograstim, a recombinant human granulocyte colony-stimulating factor, in healthy subjects

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) (lenograstim) was administered to healthy subjects at doses of 2, 5 and 10 micrograms/kg/day for 5 days (twice a day subcutaneously) to examine the optimal dose and schedule of lenograstim in mobilizing peripheral blood progenitor cells (PBSC) for allogeneic transplantation. Lenograstim administration significantly increased CD34+ cells in a dose-related manner. A significant correlation was observed between the maximal post-dosing counts and the pre-dosing baseline counts of CD34+ cells. Peripheral neutrophils increased markedly by seven to 13 times from the baseline to a peak of approximately 40,000/microliter on day 5 for the 5 and 10 micrograms/kg/day doses. After peak serum concentration (Cmax) was attained 4 h following administration, serum G-CSF declined with time in a log-linear fashion. The Cmax and 12 h area-under-the-curve increased dose dependently, but minimum drug level increased up to day 2 and then decreased until day 5. Clearance decreased with increasing dosage at the first dose, and increased significantly at the last dose. We found a highly significant correlation between absolute neutrophil counts and clearance for each dose. Adverse events most frequently occurred on day 6, with increases of alkaline phosphatase and lactate dehydrogenase and onset of bone pain. Increases of aspartate aminotransferase and alanine aminotransferase occurred as delayed events. Platelet count gradually decreased after the end of drug administration to 57% of the pre-dosing count on day 10, but was still within the normal range. These preliminary results suggest that repeated doses of lenograstim induce mobilization of PBSC in a dose-dependent manner and the pre-dosing baseline count of PBSC may predict the post-dosing maximal mobilization. The drug treatment may cause delayed-onset moderate thrombocytopenia and increased transaminase, and the drug clearance changes in a complex manner during repeated dosing.     

PEG-rHuMGDF ameliorates thrombocytopenia in carboplatin-treated rats without inducing myelofibrosis.

We examined the effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on carboplatin-induced thrombocytopenia in rats. The focus was on whether myelofibrosis is associated with the PEG-rHuMGDF treatment in this chemotherapy model. After a single injection of carboplatin, rats received subcutaneous PEG-rHuMGDF at pharmacologic doses (1,3, or 30 micrograms/kg) or a vehicle daily for 7 days. PEG-rHuMGDF at more than 3 micrograms/kg ameliorated the thrombocytopenia at day 10. Histologically, no myelofibrosis was detected in the rats treated with PEG-rHuMGDF or vehicle. Subsequently, PEG-rHuMGDF at a suprapharmacologic dose (100 micrograms/kg) was subcutaneously administered to normal and to carboplatin-treated rats daily for 7 days. Histological analysis revealed that the treatment with PEG-rHuMGDF induced myelofibrosis in the normal rats but not in the carboplatin-treated rats. Additionally, the transforming growth factor-beta 1 (TGF-beta 1) levels in the extracellular fluid and the whole extract of the bone marrow were increased to a much lesser degree in the carboplatin-treated rats compared to the normal rats. These findings suggest that PEG-rHuMGDF is effective for carboplatin-induced thrombocytopenia. Proper control of platelet counts and TGF-beta 1 levels is essential so that myelofibrosis is not induced in clinical use.     

Thrombocytopoiesis in normal and sublethally irradiated dogs: response to human interleukin-6.

The response of megakaryocytes and platelets to the administration of recombinant human interleukin-6 (IL-6) was investigated in normal and sublethally irradiated dogs. IL-6 was administered for 2 weeks at doses of 10 to 160 micrograms/kg/d to normal animals to assess dose-response and toxicity. Subsequently, 40, 80, or 160 micrograms/kg/d for 2 weeks was administered to animals treated with 200 cG total body irradiation. Analysis of normal dogs showed a significant increment in the platelet count detectable approximately 11 days after initiation of IL-6 at all administered doses. Large platelets greater than 6.3 microns in diameter were observed 1 day after beginning IL-6, progressively increasing to as many as 19.1% of the total circulating platelets by day 10. The ploidy distribution of the marrow megakaryocytes did not differ from the normal at doses of less than or equal to 80 micrograms/kg/d, but at 160 micrograms/kg/d, a shift toward higher ploidy cells was noted. No change in total white count was noted; however, a decrease in hematocrit was seen at all doses. In the irradiated animals, the platelet count recovered earlier in the IL-6-treated dogs than in the controls, but no consistent change in the ploidy distribution was observed irrespective of dose. Large platelets were also noted in the treated animals, comprising up to 6.9% of the total platelet count. Fibrinogen levels were elevated to greater than 4 times normal. A significant decrease in hematocrit was seen in all animals, while no consistent change was noted in the white count. Elevations in serum cholesterol, triglycerides, and alkaline phosphatase, together with a decline in serum albumin were observed in all the treated animals (both normal and irradiated), but clinical symptoms were observed only in the dogs receiving greater than or equal to 80 micrograms/kg/d. The data show that IL-6 alone is capable of enhancing platelet recovery in dogs with bone marrow suppression.     

Physiology and clinical applications of platelet growth factors

Recombinant human thrombopoietin, or pegylated recombinant human megakaryocyte growth and development factor, produces log-linear increases in megakaryocytopiesis and platelet production. Recent clinical studies investigating the efficacy, safety, and cost benefit of administering platelet growth factors indicate that appropriate dosing of pegylated recombinant human megakaryocyte growth and development factor, or recombinant human thrombopoietin, during chemotherapeutic marrow suppression produces these increases in four ways. First, these factors improve ensuant thrombocytopenia without producing platelet-dependent thrombo-occlusive complications. Second, recombinant human thrombopoietin or pegylated recombinant human megakaryocyte growth and development factor mobilize hematopoietic progenitor cells into the peripheral blood. Third, chronic dosing of HIV-infected thrombocytopenic patients with pegylated recombinant human megakaryocyte growth and development factor normalizes peripheral platelet counts without antibody formation. Fourth, the administration of pegylated recombinant human megakaryocyte growth and development factor to normal human volunteer platelet donors increases platelet yields nearly fourfold, with corresponding increases in peripheral platelet counts when transfused into thrombocytopenic recipients. The clinical application of platelet growth factors is continuing to be defined.