Romiplostim promotes platelet recovery in a mouse model of multicycle chemotherapy-induced thrombocytopenia.

Chemotherapy-induced thrombocytopenia can lead to chemotherapy treatment delays or dose reductions. The ability of romiplostim, a thrombopoietin (TPO) mimetic, to promote platelet recovery in a mouse model of multicycle chemotherapy/radiation therapy (CRT)-induced thrombocytopenia was examined. In humans, an inverse relationship between platelet counts and endogenous TPO (eTPO) concentration exists. In a CRT mouse model, eTPO was not elevated during the first 5 days after CRT treatment (the "eTPO gap"), then increased to a peak 10 days after each CRT treatment in an inverse relationship to platelet counts seen in humans. To bridge the eTPO gap, mice were treated with 10-1,000 µg/kg of romiplostim on day 0, 1, or 2 after CRT. In some mice, the romiplostim dose was approximately divided over 3 days. Platelet recovery occurred faster with romiplostim in most conditions tested. Romiplostim doses of ≥100 µg/kg given on day 0 significantly lessened the platelet nadir. Fractionating the dose over 3 days did not appear to confer a large advantage. These data may provide a rationale for clinical studies of romiplostim in chemotherapy-induced thrombocytopenia.

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates.

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Molecular mechanism of hepcidin-mediated ferroportin internalization requires ferroportin lysines, not tyrosines or JAK-STAT.

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. In vitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. In vivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

The development of romiplostim for patients with immune thrombocytopenia.

Comprised of peptide and carrier components in an overall structure superficially resembling an antibody, romiplostim is the prototype of a new class of protein therapeutics called peptibodies. Romiplostim (AMG 531, Nplate) was designed to evade the immune response to recombinant thrombopoietin and offer a new method of treatment for patients with immune thrombocytopenia (ITP), an "orphan" disease. In contrast with agents designed to suppress immune function or hinder the processes of platelet destruction, romiplostim works by stimulating the production of new platelets. It was proven to increase platelet counts, reduce the need for other ITP therapies and emergency treatments, and demonstrate an acceptable safety profile. In addition, romiplostim improves patient-reported outcomes and quality of life in those suffering from this rare disease.

Serum hepcidin but not prohepcidin may be an effective marker for anemia of inflammation (AI).

Anemia in cancer patients can result from a complex interaction of numerous factors including iron deficiency, inflammation, toxicity related to therapy and effect of cancer on the marrow. Determining effective anemia treatment can therefore be complex, requiring a combination of diagnostic tests. Research on iron metabolism has highlighted the importance of hepcidin and its potential role in development of anemia of inflammation (AI). Hepcidin is a peptide that controls iron flow, is induced by inflammation and is speculated to cause the sequestration of iron in patients with inflammation. In the present study, serum hepcidin concentration determined by LC-MS/MS was shown to correlate with inflammatory markers in patients with anemia of cancer (AoC). In the absence of a widely-available serum hepcidin detection assay, detection of prohepcidin using a commercial assay has been used for several years as a surrogate for measuring serum hepcidin concentration. Analysis of prohepcidin concentration did not reveal any correlation with hepcidin or with inflammatory markers in patient samples and our data suggest that prohepcidin may not be stable in serum. Algorithms to sub-classify AoC patients showed that hepcidin was strongly associated with the population subset with inflammation and without iron deficiency. Serum hepcidin concentrations may therefore be a good predictor of AI, useful in diagnosis of anemia etiology and in treatment determination.

Development of romiplostim for the treatment of patients with chronic immune thrombocytopenia: from bench to bedside.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterised by abnormally low platelet counts (<100 x 10(9)/l), purpura, and bleeding episodes, and can be categorised in three phases: newly-diagnosed, persistent, and chronic. As many patients become refractory to standard treatments (corticosteroids, danazol, azathioprine, splenectomy), there is an urgent need for alternative treatments. The successful isolation and cloning of thrombopoietin (TPO) in the mid-1990s and identification of its key role in platelet production was a major breakthrough, rapidly followed by the development of the recombinant thrombopoietins, recombinant human TPO and a pegylated truncated product, PEG-rHuMGDF. Both agents increased platelet counts but development was halted because of the development of antibodies that cross-reacted with native TPO, resulting in prolonged treatment-refractory thrombocytopenia. Experimentation with novel platforms for extending the circulating half-life of therapeutic peptides by combining them with antibody fragment crystallisable (Fc) constructs led to the development of a new family of molecules termed 'peptibodies'. The 60Da recombinant peptibody romiplostim was finally produced by linking several copies of an active TPO-binding peptide sequence to a carrier Fc fragment. In clinical trials, romiplostim was effective in ameliorating thrombocytopenia in patients with chronic ITP, was well tolerated and did not elicit cross-reacting antibodies. Romiplostim has recently been approved for the treatment of adults with chronic ITP.

Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia.

Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

Investigation of the effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: Nonerythropoietin receptor-mediated pathways may play a major role.

Erythropoietin (EPO) receptor-mediated endocytosis and degradation in the bone marrow has been hypothesized to be the major clearance pathway of erythropoiesis-stimulating agents (ESA). We investigated the role of this pathway in ESA clearance by determining the pharmacokinetic profiles after intravenous (IV) dosing in rats and mice of recombinant human EPO (rHuEPO) and rHuEPO derivatives with different receptor binding activities and biochemical properties. These derivatives included NM385 (no detectable receptor binding activity), hyperglycosylated analogs with different carbohydrate contents and receptor binding activities; (NM294: +1 carbohydrate chain; darbepoetin alfa: +2 carbohydrate chains) and polyethylene glycol (PEG) derivatives (PEG-darbepoetin alfa, PEG-rHuEPO and PEG-NM385). After IV administration in rats, NM385 had a mean clearance (CL) similar to rHuEPO. Hyperglycosylated ESAs, compared with rHuEPO, had a progressively longer half-life (t(1/2)) and a progressively slower CL with increasing number of carbohydrates or amount of added PEG that correlated more closely with carbohydrate and/or PEG content than receptor binding activity. Taken together, these results suggest that (1) EPO receptor-independent pathway(s) play a substantial role in ESA clearance; (2) the longer half-life and reduced clearance of hyperglycosylated and/or PEGylated ESAs are primarily the result of decreased susceptibility to receptor-independent elimination mechanisms.

Repeated hematopoietic stem and progenitor cell mobilization without depletion of the bone marrow stem and progenitor cell pool in mice after repeated administration of recombinant murine G-CSF.

Administration of recombinant-human G-CSF (rhG-CSF) is highly efficient in mobilizing hematopoietic stem and progenitor cells (HSC/HPC) from the bone marrow (BM) toward the peripheral blood. This study was designed to investigate whether repeated G-CSF-induced HSC/HPC mobilization in mice could lead to a depletion of the bone marrow HSC/HPC pool with subsequent loss of mobilizing capacity. To test this hypothesis Balb/c mice were treated with a maximum of 12 repeated 5-day cycles of either 10 microg rhG-CSF/day or 0.25 microg rmG-CSF/day. Repeated administration of rhG-CSF lead to strong inhibition of HSC/HPC mobilization toward the peripheral blood and spleen after >4 cycles because of the induction of anti-rhG-CSF antibodies. In contrast, after repeated administration of rmG-CSF, HSC/HPC mobilizing capacity remained intact for up to 12 cycles. The number of CFU-GM per femur did not significantly change for up to 12 cycles. We conclude that repeated administration of G-CSF does not lead to depletion of the bone marrow HSC/HPC pool.

Comparison of epoetin alfa and darbepoetin alfa biological activity under different administration schedules in normal mice.

The unit of erythropoietic activity has long been the standard by which erythropoietic agents are judged, but the development of long-acting agents such as darbepoetin alfa has highlighted the shortcomings of this approach. To this point, we compared the in vivo activity of Epoetin alfa and darbepoetin alfa per microgram of protein core. Using the established mass-to-unit conversion for Epoetin alfa (1 microg congruent with 200 U), we then calculated darbepoetin alfa activity in units. Activity varied with treatment regimen (1 microg darbepoetin alfa congruent with 800 U for 3 times weekly dosing to 8,000 U for a single injection). This analysis reveals the inadequacy of evaluating darbepoetin alfa activity in terms of standard erythropoietic units. We therefore propose that for molecules with heightened biological activity, a more legitimate basis for comparison is the protein mass.

Darbepoietin alfa potentiates the efficacy of radiation therapy in mice with corrected or uncorrected anemia.

Darbepoietin alfa (DA) is a long-acting analogue of erythropoietin that has reduced receptor affinity and enhanced biological activity. Experiments were done to test the hypothesis that correction of anemia in tumor-bearing mice by DA would increase tumor oxygenation and potentiate radiation-induced tumor cell killing. A SCC VII tumor model was used to study tumor responses to fractionated radiation therapy in mice with anemia induced by total body irradiation. Administration of DA reduced the extent and duration of anemia and associated tumor hypoxia, protected the bone marrow cells and prevented the body weight loss from the effect of irradiation, and facilitated the recovery in a time-dependent manner, with the administration of DA prior to total body irradiation having the greatest protective effect. When combined with fractionated radiation therapy, DA increased the tumor growth delay time from 2.7 days for irradiation alone to 7.3 to 10.6 days for combination of DA and irradiation. The effect of DA on tumor responses to fractionated radiation therapy was observed when DA was given 18 to 4 days before starting radiation therapy, but DA was also equally effective as a radiosensitizer when given only 2 hours before fractionated irradiation therapy. Weekly dosing of DA was as efficacious for the enhancement of radiation responses of tumors as biweekly dosing. Similar results were obtained in the RIF-1 fibrosarcoma tumor model. These studies show that DA can effectively correct anemia in tumor-bearing mice and sensitize tumor cells to fractionated radiation therapy. Importantly, DA was also able to sensitize tumors to radiation in mice with uncorrected anemia and hypoxia, suggesting that the effect of DA on radiosensitivity was independent of these factors and a different mechanism of action may be responsible for this effect.

Drug-induced and antibody-mediated pure red cell aplasia: a review of literature and current knowledge.

Anti-erythropoietin (EPO)-induced pure red cell aplasia (PRCA) is an uncommon, potentially life-threatening condition in which the bone marrow stops manufacturing red blood cells. In the past few years, reports of drug-induced, anti-EPO antibody-mediated PRCA have increased substantially, with most cases attributed to the use of one erythropoiesis-stimulating protein, Eprex. A literature review was undertaken to document the reports of drug-induced PRCA, with all drugs and drug regimens. The sudden increase in reports of antibody-mediated PRCA is discussed.

Pegylation: engineering improved biopharmaceuticals for oncology.

Pegylation, the technology of polyethylene glycol (PEG) conjugation, holds significant promise in maintaining effective plasma concentrations of systemically administered drugs that might otherwise be hampered in vivo by a number of factors, such as rapid elimination by the kidneys. Mobile, nontoxic PEG chains can be conjugated to biotherapeutics, increasing their hydrodynamic volume, which can in turn prolong their plasma retention time, increase their solubility, and shield antigenic determinants on the drug from detection by the immune system. Attaching PEG molecules for optimal pharmacokinetics without obstructing the active sites that are essential for drug efficacy is a major challenge in pegylation. Current pegylation technology uses linkerless conjugation methods to produce coupling without added toxicity or immunogenicity, and may keep the innate surface charge of the pegylated molecule intact. In addition to controlling the size and complexity of PEG molecules, the attachment site can be manipulated to avoid steric hindrance of the drug's active receptor-recognition or substrate-interaction site. A few pegylated drugs have been engineered to have an improved pharmacokinetic profile with preserved bioactivity. They often have prolonged steady plasma concentrations in vivo, thereby making a reduced number of doses possible. Other interesting effects have also emerged, such as the self-regulating pharmacokinetics of pegfilgrastim, a pegylated version of the granulocyte colony-stimulating factor filgrastim that is administered for management of chemotherapy-induced neutropenia. The improved dosing schedule, with longer intervals between administrations of the pegylated agents, will improve compliance and quality of life in patients with chronic disease.

Kinetics of haematopoietic recovery after dose-intensive chemo/radiotherapy in mice: optimized erythroid support with darbepoetin alpha.

Despite its frequency and impact on clinical outcomes, anaemia in cancer patients remains poorly understood and suboptimally treated. The definition of optimum treatment schedules with erythropoietic agents requires a suitable model of chemotherapy-induced progressive anaemia. This study investigated novel strategies such as once-per-chemotherapy-cycle dosing, synchronization between erythroid supportive care and chemotherapy, and definition of the optimum timing of erythroid support. A murine model of carboplatin chemotherapy/radiotherapy (CRT)-induced anaemia was used, which caused progressive anaemia across multiple cycles. Weekly administration of recombinant human erythropoietin (rHuEPO) was effective, but the longer-acting darbepoetin alpha resulted in superior responses. In all animals, anaemia became progressive and more refractory across cycles because of accumulated bone marrow damage. Exploiting a specific enzyme-linked immunosorbent assay, which could distinguish between darbepoetin alpha and endogenous erythropoietin, the effect of CRT upon the pharmacokinetics of darbepoetin alpha showed that clearance of darbepoetin alpha, and presumably erythropoietin, was at least partially dependent on a chemotherapy-sensitive pathway. Scheduling data suggested that administration of erythropoietic agents prior to chemotherapy was more effective than administration after chemotherapy. There was no evidence that erythropoietic agents exacerbated anaemia, even when administered immediately prior to CRT in an attempt to "prime" erythroid cells for the effects of CRT.

Pegfilgrastim: using pegylation technology to improve neutropenia support in cancer patients.

Pegylation of a protein can improve not only its formulation properties, but also both its pharmacokinetic and pharmacodynamic performance. Pegfilgrastim was made by linking a 20-kDa polyethylene glycol molecule to filgrastim, producing a long-acting cytokine requiring less frequent dosing than its parent drug. This review describes the clinical development of pegfilgrastim, and discusses its potential benefits to patients and caregivers in the prophylaxis of chemotherapy-induced neutropenia. Pegfilgrastim has a longer half-life and slower elimination rate than filgrastim, resulting in an increased serum concentration over time. Serum levels of pegfilgrastim are maintained until after a chemotherapy-induced neutrophil nadir and then decline rapidly as the neutrophil count recovers, consistent with a neutrophil-mediated clearance mechanism. In two pivotal phase III studies of women receiving chemotherapy for breast cancer, a single injection of pegfilgrastim per chemotherapy cycle, dosed either by body weight (100 microg/kg) or as a fixed dose (6 mg), was comparable to daily filgrastim (5 microg/kg) for all efficacy parameters, including duration of severe neutropenia and depth of neutrophil nadir. Analysis of pooled data from these studies showed a significantly lower incidence of febrile neutropenia in patients receiving pegfilgrastim compared with filgrastim (11 versus 19%, p<0.05), and a trend towards a lower risk of hospitalization and use of i.v. anti-infectives. The safety profiles of pegfilgrastim and filgrastim were similar. Pegfilgrastim given once per chemotherapy cycle is as effective and well tolerated as daily injections of filgrastim. With its more convenient dosing regimen, pegfilgrastim has the potential to improve quality of life and compliance in patients, and to be more cost effective.

Mono-N-terminal poly(ethylene glycol)-protein conjugates.

A site-directed method of joining proteins to poly(ethylene glycol) is presented which allows for the preparation of essentially homogeneous PEG-protein derivatives with a single PEG chain conjugated to the amine terminus of the protein. This selectivity is achieved by conducting the reductive alkylation of proteins with PEG-aldehydes at lower pH. Working examples demonstrating the application of this method to improve the delivery characteristics and therapeutic value of several proteins are provided.

Physiologic and pathologic consequences of granulocyte colony-stimulating factor deficiency.

Published studies have extended our understanding of granulocyte colony-stimulating factor deficiency. In granulocyte colony-stimulating factor withdrawal in humans, apoptosis seems to account for the dramatic loss of neutrophils, but whether the apoptosis results from normal cellular aging processes or accelerated cell loss upon granulocyte colony-stimulating factor withdrawal is unclear. In granulocyte colony-stimulating factor(-/-) mice, the introduction of bcl-2, an antiapoptotic gene product, did not affect the number of neutrophils in the circulation. This finding suggests that apoptosis does not account for the lack of neutrophils in this setting and that the reduction in neutrophil numbers in these mice results from processes other than the loss of large numbers of granulocyte colony-stimulating factor-dependent cells after commitment to the neutrophil lineage. In an experimental model of the induction of antigranulocyte colony-stimulating factor antibodies, the animals appeared remarkably similar to granulocyte colony-stimulating factor(-/-) mice. This finding confirms that at least some neutrophils are produced by granulocyte colony-stimulating factor-independent mechanisms and suggests that the antigranulocyte colony-stimulating factor antibodies reported in clinical studies before and after granulocyte colony-stimulating factor administration do not lead to similar consequences. A gross loss of neutrophils as seen in these granulocyte colony-stimulating factor-immune mice has never been reported in patients, suggesting that such an occurrence is unlikely to have occurred.