Toxicological Sciences 65, 246-255 (2002)
Copyright © 2002 by the Society of Toxicology
SAFETY EVALUATION |
Twenty-Six-Week Repeat-Dose Toxicity Study of a Recombinant Human Granulocyte Colony-Stimulating Factor Derivative (Nartograstim) in Cynomolgus Monkeys
,1
* SNBL USA, Ltd., 6605 Merrill Creek Parkway, Everett, Washington 98203;
Shin Nippon Biomedical Laboratories, Ltd., 2438 Miyanoura, Yoshida, Kagoshima 891-1374, Japan; and
Toxicological Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 2548 Fujimagari, Ube, Yamaguchi 755-8501, Japan
Received June 15, 2001; accepted October 26, 2001
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An rhG-CSF derivative, nartograstim (NTG), at dose levels of 0 (saline), 0.1, 1, 10, and 100 µg/kg, was administered subcutaneously to groups of 3 male and 3 female cynomolgus monkeys once daily for 26 weeks to investigate its toxicity. In Week 4 or later, an increase in leukocyte counts consisting mainly of neutrophils was noted in all NTG dose groups, and was considered to be attributable to the pharmacological action of NTG. The degree of this increase was reduced with repetition of dosing. Increases in granulocytic cells and granulocytic cells/erythrocytic cells (G/E) ratio in the bone marrow, increase in serum ALP activity, and enlarged spleens with increase of neutrophils in the red pulp were observed at 10 µg/kg and higher. Anemia was noted at 10 µg/kg and higher in Week 4 and was accompanied by an increase in reticulocytes and a decrease in total cholesterol level at 100 µg/kg. Anti-NTG antibody was detected in 1 female at 100 µg/kg, but neutralizing antibodies were not detected at any dose levels in Week 4. In Weeks 13 and 26, these antibodies were detected sporadically at all dose levels. However, there were considerable individual variations in antibody titer, and no definite correlation could be found between the dose levels and the antibody titer. Seven NTG-dosed animals including 3 high dose-group animals showed obvious increases in leukocyte counts until Week 26 but no obvious elevation of anti-NTG or neutralizing antibody. In these animals, changes including anemia became slighter but were still observed in Week 26. Under the conditions in this study, 1 µg/kg was concluded to be the no-observed-adverse-effect level (NOAEL) in cynomolgus monkeys.
Key Words: rhG-CSF derivative; nartograstim; cynomolgus monkey; granulocytosis; anemia; no observed adverse effect level.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors committed to the granulocyte lineage (Metcalf, 1984
). Human G-CSF (hG-CSF) is clinically useful for the therapy of neutropenia associated with cancer chemotherapy (Bronchud et al., 1987; Morstyn et al., 1988). Nartograstim (NTG) is an hG-CSF derivative produced by Escherichia coli and is a single-chain polypeptide with a molecular weight of about 19,000 composed of 175 amino acids (Kuga et al., 1989). Certain N-terminal amino acids are different in NTG as compared to hG-CSF. Specifically, Thr-1, Leu-3, Gly-4, Pro-5, and Cys-17 of human G-CSF are substituted by Ala, Thr, Tyr, Arg, and Ser, respectively. NTG was found to bind to G-CSF receptor (Asano et al., 1991) and in vitro possesses a 3-fold higher specific activity and more potent granulopoietic activity in mice than intact hG-CSF (Okabe et al., 1990). The present study was undertaken to assess the toxicity of repeated doses of NTG in cynomolgus monkeys.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NTG.
Prepared and provided by Kyowa Hakko Kogyo Co., Ltd. (Hofu, Japan), NTG was a clear, colorless, sterile protein solution, with a purity of 99% or more (SDS-PAGE method). Endotoxin contamination was less than 1 ng/mg of pure protein as determined by the Limulus amebocyte lysate assay. The homogenous protein had a concentration of 1.8 mg/ml in 10 mM phosphate buffer saline, pH 7.2. The NTG used in the present study was diluted to the appropriate concentrations with saline and formulated in vials as a sterile solution [Lot No. 9002-1G, 9002-3G, 9002-4G, 9002-5G, specific activity 2.93 (± 0.30) x 105 – 1.34 (± 0.07) x 108 U/ml, pH 6.20–7.05]. The vials were provided by Kyowa Hakko Kogyo Co., Ltd. and stored at –80°C. Each vial was removed from the freezer and allowed to thaw in water at room temperature just before use.
Animals and housing conditions.
Cynomolgus monkeys (3 to 9 years of age and with body weights ranging from 2.21–5.22 kg in males and 2.41–3.21 kg in females at the start of dosing) were obtained from Charles River Research Primate, Inc. (Wilmington, MA) and used for this study. Animals were housed in individual stainless steel cages conforming to USDA standards in a study room with a regulated temperature of 24 ± 2°C, relative humidity of 50 ± 10%, and a 12-h light cycle (0600–1800 h). Approximately 100 g of chow (Primate Chow No. 5048, Purina, Bethlehem, PA) was supplied to each animal daily (around 1500 h). Water, certified to the Japanese Tap Water Quality Standard, was available ad libitum throughout the study.
All animal experiments were conducted in compliance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science and according to the Law Concerning the Protection and Control of Animals (Law No. 105, October 1, 1973) and the Standards Relating to the Care and Management of Experimental Animals (Notification No. 6, March 27, 1980 of the Prime Minister's Office in Japan) throughout the study.
Grouping and identification of the animals.
Group number, treatment, dose levels, dose concentration, dose volume, and number of animals are given in Table 1. The study consisted of 4 treatment groups and a control group, and the number of animals per group was 3 males and 3 females. Animals were assigned to each group using a stratified randomization computer program to achieve approximately equal mean body weights among the groups.
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Dose levels and administration method.
Enlarged spleens and elevated serum ALP levels at the dose level of 150 µg/kg were reported in a previous 3-month sc dose study in monkeys (Saijo et al., unpublished report). Furthermore, considering the 6-month dosing duration in this study, a 100 µg/kg dose level was selected as the high dose, anticipated to produce toxic signs. The other dose levels were selected as log decrements of the high dose. The highest dose level was estimated to be 25 to 100 times the expected clinical dose level and the low dose level, 0.1 µg/kg, was expected to be the no observed adverse effect level (NOAEL). NTG was dosed into the subcutis of the dorsal skin once daily (1300–1500 h) for 26 weeks.
Observation and examinations.
The first day and week of dosing were designated as Day 0 and Week 0. The clinical observations, measurements, analyses, and examinations were done on all animals as described below.
Clinical signs, food consumption, body weight, and ophthalmology.
Clinical signs were observed 3 times per day, food consumptions were determined once weekly, body weights were taken once weekly, and an ophthalmological examination was conducted once prior to the start of dosing, and in Weeks 13 and 26 of dosing using a slit-lamp binoscope SL-2 and an RC-2 funduscope (Kowa, Tokyo, Japan).
Clinical pathology.
Urinalyses were conducted once prior to the start of dosing and in Weeks 13 and 26, and the parameters of color, pH, protein, glucose, ketone-body, bilirubin, occult blood, urobilinogen, sediment, lysozyme, N-acetyl-ß-D-glucosaminidase (NAG), volume, specific gravity, creatinine, Na+ and K+ ions were measured or analyzed. Hematological examinations were performed once prior to the initiation of dosing, and in Weeks 4, 13, and 26 of dosing. Only leukocyte and differential leukocyte counts were analyzed 3 times including before dosing, 8 and 24 h after dosing in each examination day. Venous blood samples treated with di-potassium-EDTA were used for the determinations of erythrocyte (RBC), leukocyte (WBC) and platelet counts, hematocrit value (Hct), hemoglobin concentration (Hgb), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration using an CC-800 (Toa Medical Electronics, Kobe, Japan), and reticulocyte and differential leucocyte counts using a MICROX HEG-70 (Omron, Kyoto, Japan). Coagulation tests were performed on 3.8% sodium citrate treated plasma once prior to the initiation of dosing and in Weeks 3, 12, and 25 of dosing. Prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen concentration were measured using a COAG-A-MATE -3881µX2 (Warner Lambert, Ann Arbor, MI), and plasminogen concentration and 
2-plasmin inhibitor were measured using a U-3200 (Hitachi, Tokyo, Japan). Bone marrow examination (total nucleated cell counts and myelogram) was performed at sacrifice. Blood chemistry examination was performed using a Clinalyzer RX-10 (Jeol, Akishima, Japan) once prior to the initiation of dosing and in Weeks 4, 13, and 26 of dosing. Venous serum was used for the determination of the following blood chemistry parameters: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), 
-glutamyl transpeptidase (-GTP), creatinine phosphokinase (CPK), total bilirubin, total protein, albumin, A/G, total cholesterol, free cholesterol, triglyceride, phospholipid, glucose, blood urea nitrogen (BUN), creatinine (CRN), uric acid, inorganic phosphate (IP), cholinesterase, Ca++, Na+, K+, and Cl-.
Gross pathology, organ weight, and histopathology.
After 26 weeks of dosing, animals were euthanized by iv injection of pentobarbital sodium and exanguination. Absolute and relative organ weights of brain including cerebellum, pituitary, thyroid, submandibular glands, thymus, heart, lung, liver, adrenal, kidneys, spleen, pancreas, testes, epididymis, seminal vesicle, prostate, ovaries, and uterus were determined, and a full histopathological examination was performed.
Anti-NTG antibody and neutralizing antibody assay.
Additional serum samples were collected at the same time points as for the blood chemistry examinations, and anti-NTG antibody titers were determined using the ELISA method (NTG coated well plates and peroxidase-linked antimonkey IgG antibody as a secondary antibody), and neutralizing antibody titers against NTG were determined using the growth of G-CSF dependent NFS-60 cells (Kato et al., 1991b; Saijo et al., 1991, unpublished report).
Statistical analyses.
Statistical analyses were performed according to the following procedures comparing the control and each NTG group, using a Micro VAX3600 (DEC, Maynard, MA). Data on food consumption, body weight, urine volume, urinary specific gravity, urinary electrolytes, urinary creatinine, NAG, lysozyme, hematology, bone marrow, blood chemistry, and organ weights (absolute and relative) were first analyzed for homogeneity of variance by Bartlett's test (Bartlett, 1937). In cases where homogeneity of variance was achieved, one-way ANOVA (Staples and Hasemen, 1974) was applied. If the result was significant, Dunnett's test (Dunnett, 1964) or Scheffe's test (Scheffe, 1959) was performed. When there was no homogeneity of variance by Bartlett's test, the data were analyzed by Kruskal-Wallis's H-test (Kruskal and Wallis, 1952). If the result was significant, Dunnett's test or Scheffe's test was performed. No statistical analyses were performed on other data collected.
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RESULTS |
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No deaths occurred in any group. No NTG-related abnormalities were observed in clinical sign observations, body weights or in food consumption measurement, ophthalmological examination or urinalyses in any group.
Hematological analyses revealed decreases in RBC count, Hct value, and Hgb concentration in both sexes in the 10 µg/kg dose group and higher in Week 4 (p < 0.01 at 100 µg/kg, Table 2). These changes were accompanied by a statistically significant increase in reticulocytes at 100 µg/kg (p < 0.01). The increase in leukocyte counts consisted mainly of neutrophils, with some monocytes and lymphocytes seen in both sexes in the 1 µg/kg dose group and higher in Week 4 (p < 0.01 in males and p < 0.05 in females at 100 µg/kg) and in the 10 µg/kg dose group and higher in Week 13 (p < 0.05 in males at 100 µg/kg) that persisted. Increases in mean leukocyte counts peaked 8 h after dosing and the counts subsequently returned to predose baseline values. The leukocyte count peak was noted in the 0.1 µg/kg dose group and higher in Week 4 (p < 0.05 at 10 µg/kg and p < 0.01 at 100 µg/kg) and in the 1 µg/kg group and higher in Week 13 (p < 0.05 in males at 10 µg/kg and p < 0.01 in males at 100 µg/kg) and afterwards. The extent of this change was reduced with continuation of dosing (Figs. 1, 2, 3 and 4).
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Bone marrow examination showed no abnormality in the nucleated cell counts in any group. However, group means for immature neutrophil counts and the granulytic cells/erythrocytic cells (G/E) ratios were slightly higher in males in the 100 µg/kg dose group and in females in the 10 µg/kg group and higher. Blood chemistry analysis showed high ALP values in males and females in the 10 µg/kg dose group and higher (p < 0.05 in females at 10 µg/kg and p < 0.01 in females at 100 µg/kg) and low total cholesterol values in females in the 100 µg/kg group (p < 0.05) in Week 4 (Table 3
). Low total cholesterol values were also sporadically noted in a few animals in the 100 µg/kg group in Weeks 4, 13, and 26.
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Gross pathological examination showed enlargement of the spleen in 2 males and 2 females in the 100 µg/kg dose group. Histopathological examination (Tables 4 and 5
) showed an increase in neutrophils in the red pulp of the spleen in a few animals in the 10 µg/kg group and higher. This was associated with high splenic weights in these animals and atrophy of the lymph follicle in the spleen in some of these animals. In the femoral bone marrow, hyperplasia of the granulocytic cells or erythrocytic cells was noted in a few animals in the 1 µg/kg group and higher, fibrosis and hemorrhage were also noted in 1 male and 1 female in the 100 µg/kg group, and only hemorrhage was observed in 1 female of the same dose group. In the sternal bone marrow, hyperplasia of granulocytic cells or erythrocytic cells was noted in a few animals in the 1 µg/kg group and higher. Neutrophil infiltration and extramedullary hematopoiesis were noted in the renal cortex of 1 male in the 100 µg/kg group. Additionally, neutrophils were increased in the pulmonary capillary bed in 1 female in the 100 µg/kg group. In the subcutis of injection site, there was leukocyte infiltration in a few animals in the 1 µg/kg group and higher, and hemorrhage in 2 females in the 1 µg/kg group and 1 male in the 10 µg/kg group.
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Anti-NTG antibody titers, neutralizing antibody titers, and leukocyte counts are shown in Tables 6 and 7
. A high titer of anti-NTG antibody was detected in 1 female in the 100 µg/kg group in Week 4, in a few male and female animals in the 0.1 µg/kg group and higher in Week 13, and in a few male and female animals in the 1 µg/kg group and higher in Week 26. Neutralizing antibody was not detected in any dose group in Week 4 but was seen in 1 male and 1 female each in the 0.1, 1, and 100 µg/kg groups, and 1 female in the 10 µg/kg group in Week 13, and also in 1 and 2 males and 1 to 3 females per group in the 0.1 µg/kg group and higher in Week 26. There were considerable individual variations in antibody titers, and no definite correlation could be found between the dose and the antibody titer (r = 0.2405 for anti-NTG antibody and r = 0.5160 for neutralizing antibody). Titers of both anti-NTG and neutralizing antibodies were generally elevated in animals with low leukocyte counts. On the other hand, anti-NTG and neutralizing antibody titers in animals showing a continuous increase in leukocytes were generally low or below the detection limit even at the end of the administration period. They were found in 1 male in the 1 µg/kg group, 1 male and 2 females in the 10 µg/kg group, and 1 male and 2 females in the 100 µg/kg group. In these animals, changes including anemia became slighter but were generally observed even in Week 26. However, in some animals there was no definite correlation between leukocyte count and the titers, or between anti-NTG antibody titers and neutralizing antibody titers.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several major NTG-related changes were observed in cynomolgus monkeys receiving repeated sc injections of an rhG-CSF derivative, NTG. These changes included an increase of peripheral neutrophils along with monocytes and lymphocytes, high serum ALP activity, enlarged spleens with an increase of neutrophils in the red pulp, retention of neutrophils in the pulmonary capillaries, and high ratios of granulocytic cells in the bone marrow. The increase in neutrophils was considered to be NTG-induced since NTG is a granulocyte colony-stimulating factor (Okabe et al., 1990
). NTG induced notable differentiation and proliferation of granulocytic cells in the hematopoietic organs. Furthermore, slight increases in monocyte and lymphocyte counts were noted that paralleled the increase in neutrophils and therefore were also thought to be NTG-related. High serum ALP activity was considered to be associated with neutrophil proliferation in the bone marrow since the relationship of high serum ALP activity with an increase of peripheral neutrophils has already been reported in repeated dose toxicity studies in rats and monkeys (Kato et al., 1991a; Saijo et al., 1991), and also since isozyme analysis by lectin-affinity electrophoresis on serum with high ALP activity showed that this ALP might be neutrophilic in origin (Yamamoto et al., 1989). Anemia, consisting of low erythrocyte count, hematocrit, and hemoglobin concentrations was noted at the 100 µg/kg dose level. This was attributed to the accelerated proliferation of granulocytic cells in bone marrow, and the resulting slight suppression of erythrocyte hematopoiesis.
An increase in peripheral reticulocyte counts, hyperplasia of erythroid cells in the bone marrow, and extramedullary hematopoiesis in the kidney were also observed in a few animals. There appeared to be individual differences between the animals in erythrocytic hematopoiesis. Erythroid cell hyperplasia in the bone marrow was noted in 7 animals. In 5 of these 7 animals, a decrease in leukocyte count was observed during the dosing period, suggesting that the pharmacological effect of NTG might be neutralized by antibody formation. In an iv repeated-dose study in rats (Kato et al., 1991a), decreased erythrocyte counts were also noted in males in the 100 µg/kg dose group and higher during the dosing period. After the completion of dosing, accelerated erythrocytic hematopoiesis was noted in these animals and was postulated to be a rebound phenomenon. For these reasons, the hyperplasia of erythroid cells observed in several animals in this study was thought to be related to antibody-mediated neutralization of NTG.
In animals with extramedullary hematopoiesis in the kidney, the erythroid hyperplasia was assumed to be a compensatory hematopoiesis since high G/E ratios in the bone marrow were noted and peripheral erythrocyte counts were low. Neutrophil infiltration was noted in the kidney of 1 male in the 100 µg/kg group. This was an independent of lymphocyte infiltration, which was observed in the control animals. Retention of peripheral neutrophils was observed in the blood vessel and was similar to changes seen in a 13-week sc toxicity study in monkeys (Saijo et al., unpublished report). For these reasons, it was hypothesized that neutrophil infiltration might be induced by administration of NTG. In the 100 µg/kg dose animals, leukocyte infiltration was noted in the subcutis at the injection site and it was thought that NTG might induce local irritation.
Changes in total blood cholesterol levels were noted in the NTG-dosed animals. It has been reported that reduced serum cholesterol is related to GM-CSF treatment (Goldstein et al., 1989; Nimer et al., 1988) and that GM-CSF might affect monocytes, macrophages, Kuppfer cells, etc. to produce factor(s) that act on the biosynthesis and elimination of cholesterol. This change might be related to the increased formation of cellular membranes in neutrophils stimulated by NTG. The change in total blood cholesterol levels was reported in the previously cited rat and monkey studies (Kato et al., 1991a; Saijo et al., 1991) and was thought to be toxicologically insignificant.
Anti-NTG and neutralizing antibodies were noted randomly in the NTG groups. There were considerable individual variations in antibody titer, and definite correlation could not be found between the dose and the antibody titer. There was no obvious immunotoxicity affecting the spleen or other lymphoid tissues in the anti-NTG antibody positive animals. Animals exhibiting successive increases in leukocyte counts generally had antibody levels at the limit of detection or at a low titer of anti-NTG and neutralizing antibodies. In these animals, changes including anemia became slighter but were still observed in Week 26. On the other hand, animals with a decrease of leukocytes showed an elevation in titer of anti-NTG and neutralizing antibodies. Therefore, changes in leukocyte count seemed to be correlated to antibody production in these animals. However, no correlation of any kind was found in some animals. It has been reported that production of neutrophils might be controlled by a feedback loop involving blood factors, such as lactoferrin, interferon, etc. (Broxmeyer et al., 1980; Shirafuji et al., 1990). In this study, there was a low correlation between anti-NTG antibody and neutralizing antibody, suggesting the titer of neutralizing antibodies might also include factors regulating neutrophil production.
Under these study conditions, several findings related to proliferation of granulocytic cells were thought to be NTG-related. The NOAEL in both sexes was shown to be 1 µg/kg, since adverse effects consisting mainly of anemia were observed at the 10 µg/kg dose level or higher.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (425) 407-8601. E-mail: kokasaki{at}snblusa.com
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REFERENCES |
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Asano, M., Gomi, K., and Okabe, M. (1991). Characterization of the receptor for granulocyte colony-stimulating factor (G-CSF) on murine myeloblastic leukemia cell, NFS-60 using NTG, a mutant G-CSF and analysis of the structure-activity relationship. Jpn. Pharmacol. Ther. 19, 2781–2787.
Bartlett, M. S. (1937). Some examples of statistical methods of research in agriculture and applied biology. J. Royal Stat. Soc. (Suppl. IV), 137–170.
Bronchud, M. H., Scarffe, J. H., Thatcher, N., Crowther, D., Souza, L. M., Alton, N. K., Testa, N. G., and Dexter, T. M. (1987). Phase I/II study of recombinant human granulocyte colony-stimulating factor in patients receiving intensive chemotherapy for small cell lung cancer. Br. J. Cancer 56, 809–813.[Web of Science][Medline]
Broxmeyer, H. E., DeSousa, M., Smithyman, A., Ralph, P., Hamilton, J., Kurland, J. I., and Bognacki, J. (1980): Specificity and modulation of the action of lactoferrin, a negative feedback regular of myelopoiesis. Blood 55, 324–333.
Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482–491.[Web of Science]
Goldstein, M. R., Mazur, E. M., Herbert, P. N., Nimer, S. D., Champlin, R. E., and Golde, D. W. (1989). Lowering cholesterol with granulocyte-macrophage colony-stimulating factor. JAMA 261, 2331–2332.
Kato, Y., Ikegami, J., Yamamoto, M., Tanaka, T., Shoukei, Y, Hara, T., and Deguchi, T. (1991a). Repeated dose toxicity of Marograstim (KW-2228). Intravenous administration to rats for 13 weeks. Pharmacometrics 41, 67–99.
Kato, Y., Yamamoto, M., Ikenaga, T., Shintome, T., Asano, M., Okabe, M., Hara, T., and Deguchi, T. (1991b). In vivo effect of human granulocyte colony-stimulating factor derivatives on hematopoiesis in primates. Acta Haematol. 86, 70–78.[Web of Science][Medline]
Kruskal, W. H., and Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. J. Am. Stat. Assoc. 47, 583–621.[Web of Science]
Kuga, T., Komatsu, Y., Yamasaki, M., Sekine, S., Miyaji, H., Nishi, T., Sato, M., Yokoo, Y., Asano, M., Okabe, M., Morimoto, M., and Itoh, S. (1989). Mutagenesis of human granulocyte colony stimulating factor. Biochem. Biophys. Res. Commun. 159, 103–111.[Web of Science][Medline]
Metcalf, D. (1984). The Hemopoietic Colony Stimulating Factors. Elsevier, Amsterdam.
Morstyn, G., Campbell, L., Souza, L. M., Alton, N. K., Keech, J., Green, M., Sheridan, W., Metcalf, D., and Fox, R. (1988). Effect of granulocyte colony stimulating factor on neutropenia induced by cytotoxic chemotherapy. Lancet 1, 667–672.[Web of Science][Medline]
Nimer, S. D., Champlin, R. E., and Golde D. W. (1988). Serum cholesterol-lowering activity of granulocyte-macrophage colony-stimulating factor. JAMA 260, 3297–3300.
Okabe, M., Asano, M., Kuga, T., Komatsu, Y., Yamasaki, M., Yokoo, Y., Itoh, S., Morimoto, M., and Oka, T. (1990). In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor. Blood 75, 1788–1793.
Saijo, T., Kato, Y., Ikenaga T., Yamamoto, M., Mataki, Y., Hara, T., and Deguchi T. (1991). Repeated dose toxicity of Marograstim (KW-2228). Intravenous administration to monkeys for 13 weeks. Pharmacometrics 42, 111–149.
Scheffe, H. (1959). The Analysis of Variance. Wiley, New York.
Shirafuji, N., Matsuda, S., Ogura, H., Tani, K., Kodo, H., Ozawa, K., Nagata, S., Asano, S., and Takaku, F. (1990). Granulocyte colony-stimulating factor stimulates human mature neutrophilic granulocytes to produce interferon-. Blood 75, 17–19.
Staples, R. E., and Hasemen, J. K. (1974). Commentary: Selection of appropriate experimental units in teratology. Teratology 9, 259–260.[Web of Science][Medline]
Yamamoto, M., Sano, J., Kato, Y., and Takahira, H. (1989). A possible mechanism of elevation of serum alkaline phosphatase activity in the monkeys treated with human G-CSF. Acta Haematol. Jpn. 52, 385 (Abstract).
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