Kinetics of neutrophil production in normal and neutropenic animals during the response to filgrastim (r-metHu G-CSF) or filgrastim SD/01 (PEG-r-metHu G-CSF).

Filgrastim G-CSF has a short, biologically active half-life, and its effective use depends on repeated inoculations. A major aim, therefore, has been to develop a once-per-chemotherapy cycle formulation. To this end, a polyethylene glycolylated form of Filgrastim, known as SD/01, has been developed. In this study, we compared the cellular kinetics of granulocyte production in mice stimulated with SD/01 and granulocyte colony-stimulating factor (G-CSF). Mice were injected with a single dose of SD/01 (1 mg/kg) or G-CSF (125 microg/kg) twice per day for 4 days. Mice rendered leukopenic with a single injection of cyclophosphamide (200 mg/kg) and temozolomide (90 mg/kg) were similarly treated at their 3-day neutrophil nadir. Tritiated thymidine was injected for autoradiographic labeling studies. Bone marrow labeling indices and the release of labeled neutrophils and monocytes into the peripheral blood were assessed. Granulocytopoiesis was stimulated similarly by both SD/01 and G-CSF in both normal and neutropenic animals, with counts rising to >20 x 10(9) polymorphonuclear neutrophils/l in both cases. Bone marrow thymidine labeling indices were increased, indicating a greater proportion of cells in DNA synthesis and an elevated proliferative activity. Compared with the normally slow release of neutrophils into the peripheral blood, labeled neutrophils (and monocytes) were rapidly released, increasing to peak levels at approximately 24 h. The peripheral half-life of neutrophils was not significantly different from normal, and the mitotic amplification factors for increase in granulocytopoiesis, accounted for by 3-3.9 extra cell divisions, were comparable for both factors. We conclude that neutrophil kinetics are stimulated in the same way and to the same extent by both SD/01 and G-CSF.

Development of spleen CFU-S colonies from day 8 to day 11: relationship to self-renewal capacity.

To investigate the persistence of spleen colonies from day 8 to day 11 of their development, we injected low numbers of marrow cells in order to obtain single colonies on the spleens of irradiated mice. Colonies were isolated on either half of the spleen on the eighth day. The position of day-11 colonies, determined relative to the ligature, indicated where novel colonies appear between those times. The results showed no evidence of the persistence of colonies from day 8 to day 11. The self-reproduction capacity of CFU-S that survive various cytotoxic drugs depends on the specific subpopulations that are affected by the drug. Using cyclophosphamide, busulphan, or BCNU, the self-renewal capacity of surviving CFU-S was manipulated. The results show that after cytotoxic treatments, a high day-11-day-8 ratio is not necessarily a reflection of a high self-renewal capacity of the CFU-S population that forms the day-11 colonies.

Effects of long-term in vivo treatment of mice with purified murine recombinant GM-CSF.

Normal adult BDF1 mice were injected s.c. with 1 or 10 micrograms/kg/day of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF; specific activity 3.9 x 10(7) U/mg) for periods up to 11 weeks. After 1, 3, 7, 9, or 11 weeks, groups of three mice were killed and various hematological parameters were evaluated. Such treatment induced a sustained increase in the number and phagocytic activity of peritoneal macrophages, increased weight and cellularity of the spleen, and increased numbers of lineage-restricted precursor cells and multipotent stem cells in the spleen. No significant differences in blood cell numbers or differential counts were observed during the experiment. Femoral granulocyte-macrophage colony-forming cells (GM-CFCs), mixed-lineage colony-forming cells (Mix-CFCs), and 7- and 12-day spleen colony-forming units (CFU-S) were increased in the first week of treatment, but returned to normal (or subnormal levels) later. Examination of histological preparations of liver, lung, skin, ileum, thymus, Peyer's patches, kidney, skin, and brain revealed no differences from control preparations. Mast cell numbers were not increased in any of these tissues. Despite continuing treatment, most hematological parameters normalized upon prolonged rmGM-CSF treatment. The reasons for this are unclear but are not apparently due to the presence of inhibitory factors in the blood of treated mice. The results indicate little evidence of side effects during prolonged therapy with doses of rGM-CSF commonly used in clinical situations.

Hemopoietic effects of short-term in vivo treatment of mice with various doses of rhG-CSF.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was injected s.c., twice daily, into normal adult male BDF1 mice for a period of 4-5 days in doses ranging from 10 to 2500 micrograms/kg/day. The number of peripheral blood cells and their morphology was examined daily from the beginning of the experiment for 6 days. On the 5th day of treatment parameters such as spleen weight and cellularity, bone marrow morphology and cellularity, content of granulocyte-macrophage colony-forming cells (GM-CFC) or multipotential stem cells (CFU-S) in spleen and marrow, and also histology of lung and liver were examined. We observed a dose-dependent increase in the number of blood neutrophils, and an increase in weight, cellularity, and numbers of GM-CFC and CFU-S in the spleen. In the bone marrow, cellularity decreased to 40% of normal. Numbers of GM-CFC and CFU-S in marrow were also decreased, and examination of marrow morphology revealed an inhibition of erythropoiesis.

Standardization of procedures for ectopic marrow grafting. II. Influence on recipients of radiation dose and field size.

The ectopic implantation of mouse marrow to the kidney capsule offers considerable scope as an assay of the hemopoietic microenvironment. Our previous work has shown that whole-body irradiation of the graft recipient prior to implantation results in superior ossicle formation in the kidney of the host. Here we report that a range of irradiation doses over a 4-Gy threshold are equivalent with respect to conditioning the graft recipient. We also show that two distinct and separable influences affect graft growth in the irradiated recipient, namely, a local effect brought about in the irradiated kidney (and restricted to it) and secondly, a systemic effect resulting from irradiation of sites other than the kidney, which nevertheless affects ossicle growth in the shielded renal capsule.

On the late seeding of CFU-S to the spleen: 8- vs 12-day CFU-S.

Marrow from 5-fluorouracil- or cyclophosphamide-treated mice, injected into lethally irradiated recipients, gives an increasing number of spleen colonies between days 7 and 14. It has been suggested that the later-forming colonies result from the more primitive spleen colony-forming units (CFU-S), which first seed into the marrow, only later to be recirculated and form colonies in the spleen. Strontium 89 (89Sr), a bone-seeking radionuclide, was injected into recipient mice to block such putative recirculation. A dose of 89Sr, which killed at least 99.8% of CFU-S in, or entering, the bone cavities, was incapable of preventing the increase in spleen colony numbers. Similarly, the splenic environment, modified by the presence of spleen colonies and able to provide a better bed for trapping CFU-S from the peripheral circulation, yielded the same number of further CFU-S, whether or not the animal had received 89Sr. Thus, it was concluded that the 12-day CFU-S does not seed initially into the marrow spaces. Direct observation of the quality of CFU-S initially seeding into the bone marrow and spleen showed, by retransplantation into secondary irradiated mice, that a similar spectrum of CFU-S types had seeded both organs.

PEG-rHuMGDF promotes multilineage hematopoietic recovery in myelosuppressed mice.

PEG-rHuMGDF administered to normal mice is a lineage-specific growth factor for megakaryocytes and platelets as judged by morphologic examination of hematologic cells in marrow and peripheral blood smears. The purpose of this study was to document that PEG-rHuMGDF in myelosuppressed mice promotes multilineage hematopoietic recovery. High-dose 5-fluorouracil (5-FU) in mice results in profound myelosuppression and 0-30% survival. Mice receiving a single dose of PEG-rHuMGDF (1000 microg/kg) 1 day after 5-FU (225 mg/kg) demonstrate an increased survival (76% vs 27% in control mice at 14 days). Compared to surviving controls, PEG-rHuMGDF-treated mice not only show the expected higher platelet counts, but also increased marrow colony-forming unit granulocyte-macrophage, increased multilineage marrow cellularity, and increased neutrophil, monocyte, and lymphocyte counts in peripheral blood. PEG-rHuMGDF- and vehicle-treated mice both develop hepatic abscesses after 5-FU treatment, but the abscesses in the PEG-rHuMGDF-treated mice contain more neutrophils, suggesting that myeloid reconstitution contributes to their survival. Furthermore, survival in 5-FU-treated mice is significantly improved by granulocyte colony-stimulating factor and antibiotics, suggesting that infection rather than thrombocytopenia is the predominant cause of death. PEG-rHuMGDF after 5-FU promotes survival accompanied by accelerated lymphohematopoietic repopulation, suggesting that PEG-rHuMGDF, a lineage-specific thrombopoietic factor in normal mice, promotes multilineage hematopoietic recovery in myelosuppressed mice.

Prolonged neutropenia in a novel mouse granulocyte colony-stimulating factor neutralizing auto-immunoglobulin G mouse model.

Therapeutic use of recombinant human cytokines in humans can result in the generation of drug-specific antibodies. To predetermine the maximum potential effects of a granulocyte colony-stimulating factor (G-CSF) neutralizing auto-immunoglobulin G (auto-IgG) response during recombinant human G-CSF therapy, we developed a mouse model of mouse G-CSF (mG-CSF) neutralizing auto-IgG response. Mice were immunized and boosted with mG-CSF chemically conjugated to either keyhole limpet hemocyanin or ovalbumin on an alternating schedule. Sera were analyzed for mG-CSF-specific titers and full blood counts were performed on a Technicon H-1E. On day 252, tissues were collected for histology. IgG was protein A affinity purified from pooled mG-CSF autoimmune sera. Mice immunized with mG-CSF conjugates produced mG-CSF-specific auto-IgG responses that lasted for the length of the study. Significant neutropenia (p(max) < 0.004) was concurrent with the rise in mG-CSF-specific IgG titers. However, neutrophil counts remained at approximately 20% of preimmunization levels through day 252. Endogenous mG-CSF neutralizing auto-IgG had no significant effect on hemoglobin, erythrocyte, lymphocyte, eosinophil, basophil, and platelet counts, and had minor, transient, or no effects on monocyte counts. Bone marrow colony assays from mG-CSF autoimmune mice demonstrated no significant effect of G-CSF neutralization on the numbers or proliferative capacity of preneutrophil lineage progenitors. Purified IgG from mG-CSF autoimmune mice neutralized mG-CSF in vitro. High-titer G-CSF neutralizing auto-IgG in adult mice partially inhibited steady-state granulopoiesis and had little or no effect on steady-state levels of other hematopoietic cells.

Novel erythropoiesis stimulating protein (darbepoetin alfa) alleviates anemia associated with chronic inflammatory disease in a rodent model.

OBJECTIVE: We developed a rodent model of noninfectious systemic inflammation to examine the pathogenesis of the associated anemia of chronic disorders (ACD), to evaluate the similarity of this ACD model to human ACD, and to evaluate the potential efficacy of novel erythropoiesis stimulating protein (darbepoetin alfa) as an ACD therapy. METHODS: Lewis rats were immunized with peptidoglycan-polysaccharide polymers (PG-APS), the chronic inflammation and associated ACD were characterized, and the effects of darbepoetin alfa treatment on complete blood counts (CBC), red blood cell (RBC) indices, and iron metabolism were analyzed weekly. RESULTS: Acutely inflamed rats had reduced peripheral blood (PB) RBC counts and hemoglobin (Hb) concentrations and increased reticulocyte counts. PB RBC numbers normalized during chronic inflammation, but RBC remained hypochromic and microcytic. Consequently, the rats remained chronically anemic. Anemic rats had fluctuating serum erythropoietin (EPO) concentrations, but mean EPO concentrations never varied significantly from baseline control levels. Histology of anemic rat spleen sections revealed reticuloendothelial siderosis. Total serum iron concentrations were chronically low. Peritoneal exudate cells (PEC) isolated from anemic rats and stimulated with PG-APS in vitro produced more interleukin (IL)-1alpha and interferon (IFN)-gamma, and significantly more tumor necrosis factor (TNF)-alpha and IL-10 than control cultures. Darbepoetin alfa restored Hb concentrations to baseline levels within 2 to 7 weeks, depending on dosage. A refined treatment strategy restored Hb to baseline and maintained those levels with reduced dosing. CONCLUSION: ACD in this rodent model closely replicates human ACD. Darbepoetin alfa treatment reversed ACD in this model by increasing RBC production and RBC hemoglobinization while reducing siderosis and hypoferremia.

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.

A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans.

Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.

Pegylation: engineering improved pharmaceuticals for enhanced therapy.

Conjugating biomolecules with polyethylene glycol (PEG), a process known as pegylation, is now an established method for increasing the circulating half-life of protein and liposomal pharmaceuticals. Polyethylene glycols are nontoxic water-soluble polymers that, owing to their large hydrodynamic volume, create a shield around the pegylated drug, thus protecting it from renal clearance, enzymatic degradation, and recognition by cells of the immune system. Agent-specific pegylation methods have been used in recent years to produce pegylated drugs that have biologic activity that is the same as, or greater than, that of the parent drug. These agents have distinct in vivo pharmacokinetic and pharmacodynamic properties, as exemplified by the self-regulated clearance of pegfilgrastim, the prolonged absorption half-life of pegylated interferon alpha-2a, and the altered tolerability profile of pegylated liposomal doxorubicin. Pegylated agents have dosing schedules that are more convenient and more acceptable to patients, and this can have a beneficial effect on the quality of life of patients with cancer.

The design and development of pegfilgrastim (PEG-rmetHuG-CSF, Neulasta).

Recombinant protein technology produces drugs for human therapy in unprecedented quantity and quality. Research is now focusing on the relationship between pharmacokinetic and pharmacodynamic properties of molecules, with the aim of engineering proteins that possess enhanced therapeutic characteristics in contrast to being used as simple replacements for the natural equivalent. The addition of a polyethylene glycol (PEG) moiety to filgrastim (rmetHu-G-CSF, Neupogen) resulted in the development of pegfilgrastim. Pegfilgrastim is a long-acting form of filgrastim that requires only once-per-cycle administration for the management of chemotherapy-induced neutropenia. The covalent attachment of PEG to the N-terminal amine group of the parent molecule was attained using site-directed reductive alkylation. Pegylation increases the size of filgrastim so that it becomes too large for renal clearance. Consequently, neutrophil-mediated clearance predominates in elimination of the drug. This extends the median serum half-life of pegfilgrastim to 42 hours, compared with between 3.5 and 3.8 hours for Filgrastim, though in fact the half-life is variable, depending on the absolute neutrophil count, which in turn reflects of the ability of pegfilgrastim to sustain production of those same cells. The clearance of the molecule is thus dominated by a self-regulating mechanism. Pegfilgrastim retains the same biological activity as filgrastim, and binds to the same G-CSF receptor, stimulating the proliferation, differentiation and activation of neutrophils. Once-per-chemotherapy cycle administration of pegfilgrastim reduces the duration of severe neutropenia as effectively as daily treatment with filgrastim. In clinical trials, patients receiving pegfilgrastim also had a lower observed incidence of febrile neutropenia than patients receiving filgrastim.

The radiation sensitivity of the haemopoietic microenvironment--effect of dose rate on ectopic ossicle formation.

The haemopoietic microenvironment (HM) consists of a complex mixture of cellular types and extra-cellular matrix. It is essential for prolonged haemopoiesis in both the normal situation and after bone marrow transplantation. The competence of the HM can be assessed by ectopic grafting of femoral marrow. A complete haemopoietic organ develops at the site of implantation. Stem cells (CFU-S) which inhabit the ossicle formed after ectopic implantation can be measured, to assess the function of the engrafted HM to support haemopoiesis. Using this functional endpoint we have examined the radiation sensitivity of the HM at both high and low dose rates, and conclude that high doses of gamma-irradiation delivered at 4 Gy/min or 0.016 Gy/min have widely different effects on the HM, the former proving much more damaging than the latter.

Residual haemopoietic damage in the mouse after fractionated gamma-irradiation, down to 0.1 Gy per fraction.

Residual damage in haemopoietic progenitor cell populations, spleen and granulocyte-macrophage colony-forming cells (CFU-S and GM-CFC) was detected in mice after 15 daily fractions where the dose per fraction was as low as 0.1 Gy. The injury was dose-dependent and after higher total fractionated doses of 7.5-10 Gy the CFU-S population recovered to about 50% of control between 2 and 12 months after irradiation. Residual damage was also detected in the stroma, in the form of reduced numbers of fibroblastoid colony-forming cells and of CFU-S in ossicles under the kidney capsule. The response to a second course of 15 fractions, given 3 weeks after the end of the first course, was similar and additive to the response to the first course in the short term. However, in the long term, recovery levels were similar after either one or two courses.

The effect of low dose rate on recovery of hemopoietic and stromal progenitor cells in gamma-irradiated mouse bone marrow.

Long-term recovery of mouse hemopoietic stem cells (CFU-S and CFU-S per colony), granulocyte-macrophage precursor cells (GM-CFC), and stromal colony-forming units (CFU-F) after doses up to 12.5 Gy was almost complete by 1 year when the dose rate was reduced to 0.0005 Gy/min compared to incomplete recovery after doses up to only 6.5 Gy given at greater than 0.7 Gy/min. This sparing effect of dose rate on long-term hemopoietic recovery is in contrast to the generally reported lack of dependence on dose rate for acute survival of hemopoietic progenitors after doses up to 5 Gy. The present results are compatible with the hypothesis that good recovery of the stroma should be reflected in the long-term recovery of hemopoiesis.

A cellular analysis of long-term haematopoietic damage in mice after repeated treatment with cyclophosphamide.

Following repeated treatment of mice with cyclophosphamide (5 X 200 mg/kg) it was found that slight, but significant, residual marrow damage persisted for at least half the lifespan of the animals. This long-term damage occurred despite preferential sparing of those multi-potential haematopoietic cells (CFU-S) that had a high self-renewal capacity after each step of the multistep regimen and despite a smaller CFU-S kill after each successive dose. The damage was characterized by low mean numbers of CFU-S and stromal colony-forming cells (CFU-F) which were around 70% of control values. Examination of individual animals revealed that the majority had slightly subnormal numbers of CFU-S and CFU-F, with only a few suffering a more severe injury, including 8% of mice with clinical hypoplasia or myelodysplasia.

Long-term bone marrow damage in experimental systems and in patients after radiation or chemotherapy.

Long-term bone marrow damage, characterised by stem, progenitor and stromal cell abnormalities is a frequent occurrence after cytotoxic treatments. The relative contributions of each of these components are difficult to analyse, especially in the case of patients who have received combined chemotherapy. The damage may be latent, and not manifested in low numbers of mature functional cells in the blood, but may become apparent as an hypoplastic syndrome at later times. Little tendency of recovery to normal parameters is seen in experimental animals and in patients.

Alpha-irradiation of haemopoietic tissue in pre- and postnatal mice: 2. Effects of mid-term contamination with 239Pu in utero.

The distribution of 239Pu in various tissues of foetal and postnatal offspring of pregnant mice, injected i.v. at 13 days gestation with 30 kBq 239Pu/kg (in some cases with 10 or 100 kBq/kg), together with the numbers of haemopoietic progenitors in the bone marrow, spleen and liver, were measured through to 1 year post-partum. The quality of the haemopoietic microenvironment in these mice was also measured using the renal-capsule implant method. The largest radiation dose received by any haemopoietic organ was that in the liver, amounting to 10-14 mGy, as reported previously. In spite of normal numbers of haemopoietic spleen colony-forming cells (CFC-S) in the liver and seeding, at birth, into the bone marrow where the level of plutonium was minimal, a long-term deficit in their number rapidly developed. The development of the stromal microenvironment, however, was also deficient, suggesting that the dose of alpha-irradiation to the foetal liver was sufficient to cause sublethal damage in those cells destined to become the precursors of the supportive haemopoietic microenvironment in bone marrow and spleen. The results of this study suggest that although the placenta affords significant shielding to the tissues of the developing foetus from maternal contamination, the long-term effects on haemopoiesis are comparable to those in mice contaminated as adults. This further implies that the developing haemopoietic tissues are exquisitely sensitive to 239Pu contamination.

Long-term effects of plutonium-239 and radium-224 on the distribution and performance of pluripotent haemopoietic progenitor cells and their regulatory microenvironment.

Experiments are described which investigate the long-term damage to haemopoietic progenitor cells (CFU-S) and their microenvironment in mouse marrow resulting from the administration of leukaemogenic amounts of plutonium-239 and radium-224. 239Pu (35 Bq g-1 body weight) and 224Ra (555 Bg g-1 body weight) were injected into 10-12-week-old mice, and numbers, proliferative activity and self-renewal capacity of CFU-S were measured at different locations in femoral marrow at intervals over the following 2 years. Parallel measurements were also made of the quality of the haemopoietic microenvironment by ectopic transplantation of bone marrow cells. There was some recovery from the initial effects of 239Pu on CFU-S numbers after 3-6 months, although the recovery was not maintained in all marrow fractions. Following 224Ra administration there was an initial transient increase in CFU-S numbers in the fraction of marrow furthest from bone surfaces but a considerable depression in numbers in other regions of marrow; there was no recovery between 3 and 6 months and subsequent recovery was not complete in all regions of marrow. The differential responses of CFU-S and the haemopoietic microenvironment following 224Ra or 239Pu administration seemed in some ways related to the metabolism of the radionuclides. There was a profound reduction in the ability of marrow to generate ossicles when transplanted under the kidney capsule as a result of the administration of either 224Ra or 239Pu, with only transient recoveries from the effects of 239Pu at 4 days and at 3 months after injection.