Evaluation of bleeding and thrombotic events during long-term use of romiplostim in patients with chronic immune thrombocytopenia (ITP).

BACKGROUND: Romiplostim is a peptibody protein that raises platelet counts during long-term treatment of patients with chronic immune thrombocytopenia (ITP). Clinical outcomes related to increased platelet counts include a reduced risk of bleeding and a potential risk of thrombosis. OBJECTIVE: To evaluate bleeding and thrombotic events occurring in chronic ITP patients during two phase 3, randomized, placebo-controlled, 24-week studies of romiplostim and during subsequent treatment in an open-label extension study. PATIENTS/METHODS: In the phase 3 trials, 125 patients were randomized to romiplostim or placebo; romiplostim dose was adjusted to maintain platelet counts of 50-200 x 10(9) L(-1). Patients who completed the phase 3 trials could enroll in the extension study in which all patients received romiplostim. RESULTS: In the phase 3 trials, a significantly greater percentage of patients treated with placebo (34%) had bleeding adverse events of moderate or greater severity than did patients treated with romiplostim (15%, P = 0.018). In the extension study, the incidence of bleeding adverse events of moderate or greater severity decreased from 23% of patients in the first 24 weeks to 12% after 24-48 weeks, remaining < or = 6% thereafter. The exposure-adjusted incidence of thrombotic events was 0.1 per 100 patient-weeks in the phase 3 studies, and 0.08 per 100 patient-weeks in the extension study where patients received romiplostim for up to 144 additional weeks. CONCLUSIONS: The incidence and severity of bleeding was decreased in chronic ITP patients treated with romiplostim compared with placebo, and the incidence of thrombotic events was not different between the two groups.

Serum thrombopoietin levels and anti-thrombopoietin antibodies in systemic lupus erythematosus.

Thrombocytopenia is a common phenomenon in patients suffering from systemic lupus erythematosus (SLE). The cause of thrombocytopenia in SLE, however, is poorly understood. In this study, 100 patients with SLE were evaluated for serum thrombopoietin levels, anti-thrombopoietin antibodies and routine laboratory parameters such as peripheral blood counts, parameters of blood chemistry and immunologic parameters of SLE. The median platelet count of SLE patients was 230 g/l and 19 were thrombocytopenic (range 8-148 g/l). Thrombopoietin levels in SLE patients were found to be significantly higher than in healthy controls (n = 96; median, 117 pg/ml vs 64 pg/ml, P < 0.01). When excluding thrombocytopenic SLE patients, thrombopoietin levels in SLE were still above controls (111 pg/ml, P < 0.01). The thrombopoietin levels were correlated to erythrocyte sedimentation rate and ECLAM score of disease activity, and inversely correlated to complement factor C4, but not to the platelet count. Anti-thrombopoietin antibody reactivity was found in 23% of SLE patients. Interestingly, these patients had lower platelet counts than SLE patients without anti-thrombopoietin antibodies (median 174 g/l and 253 g/l, respectively, P < 0.01), but thrombopoietin levels were not significantly different. Taken together, thrombopoietin levels are significantly higher in the sera of SLE patients than in healthy controls and anti-thrombopoietin antibodies are frequently found.

Cyclic immune thrombocytopenia responsive to thrombopoietic growth factor therapy.

We report a patient with cyclic thrombocytopenia and antiplatelet antibodies, a variant of chronic immune thrombocytopenic purpura (ITP), with a several year history of periodic fluctuation of the platelet count, megakaryocytic hyperplasia and high-titer anti-GPIb-specific antiplatelet antibodies. The patient was resistant to multiple forms of therapy but has responded to the thrombopoietic growth factor, pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF). This case suggests that some patients with classic ITP may respond to thrombopoietic growth factors.

Indirect study of thrombopoiesis (TPO, reticulated platelets, glycocalicin) in patients with hereditary macrothrombocytopenia.

Chronic isolated hereditary macrothrombocytopenia (CHMT) is a congenital form of macrothrombocytopenia that seems to be due to defective production secondary to a disturbance in megakaryocyte fragmentation. To better understand the pathogenesis of thrombopoiesis in this hereditary thrombocytopenic disorder, we determined the percentage of reticulated platelets (RP), plasma glycocalicin (GC) and thrombopoietin (TPO) levels in 29 patients with CHMT, 23 patients with immune thrombocytopenic purpura (ITP), and 17 patients with thrombocytopenia secondary to decreased bone marrow megakaryocytes (hypoplasia). The % RP was similar in CHMT (2.27 +/- 1.33) and hypoplasia (1.98 +/- 1.35) patients and markedly lower than that in ITP patients (8.80 +/- 7.97; p <0.001), suggesting that the production of new platelets is reduced in CHMT. Plasma GC was within the normal range (0.84 +/- 0.16 microg/mL) both in patients with CHMT (0.63 +/- 0.20 microg/mL) and ITP (0.82 +/- 0.90 microg/mL), while it was significantly decreased in patients with hypoplasia (0.16 +/- 0.04 microg/mL; p < 0.001). When the GC value was normalized for platelet count, the GC index was normal in CHMT patients (2.05 +/- 1.1) and in patients with hypoplasia (0.85 +/- 0.10) while it was significantly increased in ITP patients (10.88 +/- 18.00; p<0.001); thus, patients with CHMT seem to have a normal platelet turnover. TPO was significantly increased in CHMT (195 +/- 72 pg/ml) as compared with normal (80 +/- 53 pg/ml; p < 0.002); however, the mean level was not as high as in ITP patients (345 +/- 167 pg/mL; p < 0.001). This finding suggests that CHMT syndrome is not secondary to a defective production of TPO and that megakaryocyte mass is nearly normal.

Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression.

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.

Haematological abnormalities in early abstinent alcoholics are closely associated with alterations in thrombopoietin and erythropoietin serum profiles.

Numerous reports exist on haematological pathology in alcoholism. However, no data are available regarding a potential involvement of haematopoietic growth factors in the recovery from alcohol-induced haematological abnormalities upon abstinence. Therefore, thrombopoietin (TPO) and erythropoietin (EPO) serum levels along with haematological and other routine laboratory parameters were closely followed in 14 thoroughly characterized male alcoholic patients over one to five months of controlled abstention from alcohol. Haematological changes in these early abstinent alcoholics consisted predominantly of (a) the well known rebound surge of platelets, (b) an early reticulocyte peak, and (c) persistently low haematocrit levels over months without signs of recovery. Observations on EPO and TPO during early abstinence can be summarized as follows: (1) Increased TPO levels precede the rebound thrombocytosis by several days, (2) both EPO and TPO concentrations are higher in anaemic than in nonanaemic alcoholics, with (3) nonanaemic subjects exhibiting levels of TPO in the range of healthy controls but levels of EPO below controls and (4) TPO concentrations show a stronger correlation with initial haematocrit values than with thrombocyte counts. To conclude, haematological recovery in early alcohol abstinence appears to be, at least in part, growth factor-driven, involving both TPO and EPO, and may reflect an intense interaction of erythro- and thrombopoiesis.

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice.

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

High levels of thrombopoietin in sera of patients with essential thrombocythemia: cause or consequence of abnormal platelet production?

Thrombopoietin (TPO) is the most important regulator of megakaryocyte development and platelet production. Platelet production is thought to be regulated by a negative regulatory feed back loop. In an attempt to evaluate the role of TPO in the pathobiology of essential thrombocythemia (ET), we have examined levels of TPO and other cytokines with thrombopoietic activity (interleukin-6 and interleukin-11) in sera obtained from 25 patients with ET (ten treated, 15 untreated) and 117 healthy control subjects. TPO serum levels were assessed using a sandwich-antibody ELISA that utilizes a polyclonal rabbit antiserum for both capture and signal. The mean serum TPO level in 25 ET patients was significantly elevated (545+/-853 pg/ml) as compared with that in healthy controls (95.3+/-54.0 pg/ml,p<0.001). The difference in TPO serum levels between ten treated (781+/-1229 pg/ ml) and 15 untreated ET patients (388+/-458 pg/ml) did not reach statistical significance (p = 0.09). We conclude that either consumption or production of TPO is altered in ET. Failure of appropriate feedback regulation and continued megakaryocyte stimulation by an elevated TPO may play an important role in the pathobiology of ET.

Thrombopoietin levels in HIV-associated thrombocytopenia in children.

OBJECTIVES: To determine the mechanism of human immunodeficiency virus (HIV)-associated thrombocytopenia by using thrombopoietin (TPO) levels. STUDY DESIGN: TPO levels were measured in 14 HIV+ children with thrombocytopenia (TCP+), 28 HIV+ children without thrombocytopenia (TCP-), and 15 matched control subjects. RESULTS: For the patients with moderate symptoms, TPO levels were similar for the TCP+ and TCP- groups (251 pg/mL vs 263 pg/mL; P =.98) and similar to those of control subjects. For the patients with severe symptoms, TPO levels were significantly higher for the TCP+ group versus the TCP- group (1172 pg/mL vs 222 pg/mL; P =.03). Patients with severe symptoms and thrombocytopenia had significantly higher TPO levels than those with moderate symptoms and thrombocytopenia (P <.005), were more likely to require growth factors, and did not respond to treatment with intravenous immunoglobulin. CONCLUSIONS: TPO levels can distinguish 2 groups of patients with HIV-associated thrombocytopenia. Patients with severe disease had elevated TPO levels, did not respond to treatment with intravenous immunoglobulin, and were more likely to be growth factor-dependent, suggesting marrow failure.

The Mpl ligand and platelet homeostasis.

The classification of Mpl as a cytokine receptor present on cells of the platelet lineage has led to the identification and cloning of its ligand. This has resulted in a rapid accumulation of data advancing the understanding of the processes of megakaryopoiesis and thrombopoiesis and the regulation of endogenous Mpl ligand (thrombopoietin, eTPO). Highlights of in vitro human and non-human primate data will be discussed, as well as preclinical (in vivo) non-human primate studies. Two recombinant forms of Mpl ligands (rTPO) are currently being tested in clinical trials and early results will be reviewed. The preclinical and clinical studies will be summarized with consideration of the observations which provide insights into the biology of the response to exogenous rTPO. Understanding the biology of platelet production and the condition of target cells in treatment populations will facilitate the appropriate use of this potential therapeutic agent.

Thrombopoietin levels after chemotherapy and in naturally occurring human diseases.

The identification of the primary regulator of thrombopoiesis, the Mpl ligand, has led to an explosion of information concerning the factors that control this process. The circulating endogenous form of this molecule, commonly termed thrombopoietin, was the source material used by several groups in the discovery efforts. In the past year, more information has become available regarding the circulating levels of endogenous thrombopoietin in chemotherapy-induced thrombocytopenia and naturally occurring diseases. This review discusses these new observations and how they contribute to our understanding of the regulation of thrombopoiesis.

Ineffective platelet production in thrombocytopenic human immunodeficiency virus-infected patients.

Thrombocytopenia has been characterized in six patients infected with human immunodeficiency virus (HIV) with respect to the delivery of viable platelets into the peripheral circulation (peripheral platelet mass turnover), marrow megakaryocyte mass (product of megakaryocyte number and volume), megakaryocyte progenitor cells, circulating levels of endogenous thrombopoietin (TPO) and platelet TPO receptor number, and serum antiplatelet glycoprotein (GP) IIIa49-66 antibody (GPIIIa49-66Ab), an antibody associated with thrombocytopenia in HIV-infected patients. Peripheral platelet counts in these patients averaged 46 +/- 43 x 10(3)/microL (P = . 0001 compared to normal controls of 250 +/- 40x 10(3)/microL), and the mean platelet volume (MPV) was 10.5 +/- 2.0 fL (P > 0.3 compared with normal control of 9.5 +/- 1.7 fL). The mean life span of autologous 111In-platelets was 87 +/- 39 hours (P = .0001 compared with 232 +/- 38 hours in 20 normal controls), and immediate mean recovery of 111In-platelets injected into the systemic circulation was 33% +/- 16% (P = .0001 compared with 65% +/- 5% in 20 normal controls). The resultant mean peripheral platelet mass turnover was 3.8 +/- 1.5 x 10(5) fL/microL/d versus 3.8 +/- 0.4 x 10(5) fL/microL/d in 20 normal controls (P > .5). The mean endogenous TPO level was 596 +/- 471 pg/mL (P = .0001 compared with 95 +/- 6 pg/mL in 98 normal control subjects), and mean platelet TPO receptor number was 461 +/- 259 receptors/platelet (P = .05 compared with 207 +/- 99 receptors/platelet in nine normal controls). Antiplatelet GPIIIa49-66Ab levels in sera were uniformly increased in HIV thrombocytopenic patients (P < .001). In this cohort of thrombocytopenic HIV patients, marrow megakaryocyte number was increased to 30 +/- 15 x 10(6)/kg (P = .02 compared with 11 +/- 2.1 x 10(6)/kg in 20 normal controls), and marrow megakaryocyte volume was 32 +/- 0.9 x 10(3) fL (P = .05 compared with 28 +/- 4.5 x 10(3) fL in normal controls). Marrow megakaryocyte mass was expanded to 93 +/- 47 x 10(10) fL/kg (P = .007 compared with normal control of 31 +/- 5.3 x 10(10) fL/kg). Marrow megakaryocyte progenitor cells averaged 3.3 (range, 0.4 to 7.3) CFU-Meg/1,000 CD34(+) cells compared with 27 (range, 0.1 to 84) CFU-Meg/1,000 CD34(+) cells in seven normal subjects (P = .02). Thus, thrombocytopenia in these HIV patients was caused by a combination of shortening of platelet life span by two thirds and doubling of splenic platelet sequestration, coupled with ineffective delivery of viable platelets to the peripheral blood, despite a threefold TPO-driven expansion in marrow megakaryocyte mass. We postulate that this disparity between circulating platelet product and marrow platelet substrate results from direct impairment in platelet formation by HIV-infected marrow megakaryocytes.

Thrombopoietin serum levels in patients with aplastic anaemia: correlation with platelet count and persistent elevation in remission.

In an attempt to evaluate the role of thrombopoietin (TPO) in the pathobiology of aplastic anaemia (AA), we have examined TPO levels in sera from 54 AA patients and 119 healthy controls. A total of 92 samples were collected from AA patients: 43 samples were harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples when patients were in partial (n=10) or complete remission (n=16) following immunosuppressive treatment. TPO serum levels were assessed by a sandwich-antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal. Serum samples from normal donors revealed a mean TPO level of 95.3 +/- 54.0 pg/ml (standard deviation). Mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728 +/- 1074 pg/ml (P<0.001 compared to normal controls: mean platelet count at that time: 27x10(9)/l). TPO serum levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P<0.001). However, despite normal platelet counts (mean 167x10(9)/l), TPO levels remained significantly elevated in complete remission (mean TPO 1009 +/- 590 pg/ml, P<0.001 compared to normal controls). There was a significant inverse correlation between serum TPO levels and platelet counts in AA patients who were not transfused for at least 2 weeks prior to sample collection (coefficient of correlation (r) = -0.70, P<0.0001). In summary, TPO levels were highly elevated in sera of patients with AA. Thus there is no evidence to suggest an impaired TPO response contributing to thrombocytopenia in AA. Thrombopoietin did not return to normal levels in remission, indicating a persisting haemopoietic defect in remission of AA. We hypothesize that elevated levels of TPO may be required to maintain normal or near normal platelet counts in remission of AA.

Endogenous TPO (eTPO) levels in health and disease: possible clues for therapeutic intervention.

The factor which is the primary regulator of megakaryocyte and platelet production has recently been identified as the ligand for the receptor Mpl. This discovery has resulted in substantial advances in our understanding of platelet homeostasis. The access to new experimental reagents has enabled studies of the endogenous circulating form of this ligand, endogenous thrombopoietin, in normal individuals and in patients with altered platelet numbers. The relationship of endogenous TPO in health and disease will be examined with consideration of the implications for successful therapeutic intervention with exogenous recombinant Mpl ligands in selected settings.

Chronic thrombocytopenia is induced in dogs by development of cross-reacting antibodies to the MpL ligand.

The MpL ligand (ML) is a potent stimulus for thrombocytopoiesis. To create an in vivo model of ML deficiency, we injected dogs with a recombinant human ML (rhML) to determine whether cross-reacting antibodies would develop and cause thrombocytopenia. RhML was administered subcutaneously for 8 weeks to three normal dogs (mean platelets, 197 +/- 5.5 x 10(3)/microL). Within 5 days their platelet counts were twice baseline and greater than 4 times baseline by day 21. Then, uniformly, chronic thrombocytopenia developed. At 1 week after terminating rhML, mean platelets were 0.5 times baseline and at 2 months 0.25 times baseline. Early in treatment, marrow biopsies showed increased megakaryocyte number and ploidy, which decreased as platelets declined. Paralleling these changes, high titer anti-rhML antibodies developed. Autologous 51Cr-labeled platelet recovery and survival measurements indicated that the thrombocytopenia was principally due to decreased production. Infusion of plasma from the thrombocytopenic dogs into two normal dogs and one dog previously made thrombocytopenic with rhML caused platelet counts to fall gradually. These studies show that dogs with anti-rhML antibodies develop thrombocytopenia, presumably because the cross-reacting antibodies neutralize endogenous canine ML. The results strongly suggest that ML plays an essential role in maintaining normal platelet levels.

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 micrograms/ kg/d) alone and in combination with recombinant human granulocyte colony stimulating factor (rHu-GCSF) (10 micrograms/ 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 x 10(9)/L) was observed between days 12 and 25 (nadir 39 +/- 20 x 10(9)/L on day 17), and neutropenia (absolute neutrophil count < 1 x 10(9)/L) occurred between days 8 and 30 (nadir 0.167 +/- 0.120 x 10(9)/L on day 15). PEG-rHuMGDF (2.5 micrograms/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 x 10(9)/L v baseline of 416 +/- 88 x 10(9)/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 x 10(9)/L v 0.167 +/- 0.120 x 10(9)/L; P > .3). rHu-GCSF (10 micrograms/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 x 10(9)/L from 22 days to 4 days (P = .005). The postchemotherapy neutropenic nadir was 0.554 +/- 0.490 x 10(9)neutrophils/L (P = .3 v hepsulfam-only control of 0.167 +/- 0.120 x 10(9)/L). However, thrombocytopenia of < 100 x 10(9) platelets/L was not shortened (persisted from day 12 to day 25), or less severe (nadir of 56 +/- 32 x 10(9) platelets/L on day 14; P = .7 compared with untreated hepsulfam animals). The concurrent administration of rHu-GCSF (10 micrograms/kg/d) and PEG-rHuMGDF (2.5 micrograms/kg/d) in four animals resulted in postchemotherapy peripheral platelet counts of 127 +/- 85 x 10(9)/L (P = .03 compared with 39 +/- 20 x 10(9)/L for untreated hepsulfam alone, and P = .02 compared with 856 +/- 594 x 10(9)/L for PEG-rHuMGDF alone), and shortened the period of neutropenia < 1 x 10(9)/L from 22 days to 4 days (P = .8 compared with rHu-GCSF alone). Increasing PEG-rHuMGDF to 10 micrograms/kg/d and maintaining the 21-day schedule of coadministration with rHu-GCSF (10 micrograms/kg/d) in another four animals produced postchemotherapy platelet counts of 509 +/- 459 x 10(9)/L (P < 10(-4) compared with untreated hepsulfam alone, and P = .04 compared with 2.5 micrograms/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 micrograms/kg/d) from day 1 through day 22 and rHu-GCSF (10 micrograms/kg/d) from day 8 through day 22 in six animals produced peak postchemotherapy platelet counts of 747 +/- 317 x 10(9)/L(P < 10(-4) compared with untreated hepsulfam alone, and P = .7 compared with PEG-rHuMGDF alone), and maintained the neutrophil count > 3.5 x 10(9)/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 chemotherap

Serum thrombopoietin levels in patients with aplastic anaemia.

Endogenous serum thrombopoietin (TPO) levels were measured in 31 patients with aplastic anaemia (AA) using an enzyme immunoassay with a sensitivity of 20 pg/ ml. The median platelet count for all AA patients was 30 +/- 29 x 10(9)/l (range 5-102) compared with a median of 284 +/- 59 x 10(9)/l (range 148-538) for normal controls. Serum TPO levels were significantly elevated in all patients compared with normals (1706 +/- 1114.2, range 375-5000 v 78 +/- 54, range 16.5-312.9, P < 0.0001). There was no correlation between serum TPO levels and the degree of thrombocytopenia in AA patients, but TPO levels were significantly higher in patients who were platelet transfusion dependent than in patients who were transfusion independent (P < 0.01). There was a trend for higher TPO levels in patients with severe AA compared with non-severe AA patients. Clinical trials of TPO and a related truncated, pegylated molecule, megakaryocyte growth and development factor (PEG-rHuMGDF), are awaited to determine whether treatment with these drugs will result in increased platelet counts in patients with AA.

Preclinical biology of megakaryocyte growth and development factor: a summary.

Since the discovery of the ligand for the cytokine receptor c-Mpl, much has transpired. The development of this protein has been rapid, and the amount of information available on the effects of this molecule in vitro and in vivo is vast. This paper will highlight some of the major studies and observations which are part of the ongoing pre-clinical development of the megakaryocyte growth and development factor encoding the erythropoietin-like domain of the c-Mpl ligand. A summary of in vitro effects on human cells, as well as the key in vivo observations, are included. This molecule is currently in clinical trials, and the initial results are promising.

Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice.

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.