* 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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: rhG-CSF derivative; nartograstim; cynomolgus monkey; granulocytosis; anemia; no observed adverse effect level.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study was undertaken to assess the toxicity of repeated doses of NTG in cynomolgus monkeys.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and housing conditions.
Cynomolgus monkeys (3 to 9 years of age and with body weights ranging from 2.215.22 kg in males and 2.413.21 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 (06001800 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.
|
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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
).
|
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartlett, M. S. (1937). Some examples of statistical methods of research in agriculture and applied biology. J. Royal Stat. Soc. (Suppl. IV), 137170.
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, 809813.[ISI][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, 324333.[ISI][Medline]
Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482491.[ISI]
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, 23312332.
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, 6799.
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, 7078.[ISI][Medline]
Kruskal, W. H., and Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. J. Am. Stat. Assoc. 47, 583621.[ISI]
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, 103111.[ISI][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, 667672.[ISI][Medline]
Nimer, S. D., Champlin, R. E., and Golde D. W. (1988). Serum cholesterol-lowering activity of granulocyte-macrophage colony-stimulating factor. JAMA 260, 32973300.[Abstract]
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, 17881793.[Abstract]
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, 111149.
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, 1719.[Abstract]
Staples, R. E., and Hasemen, J. K. (1974). Commentary: Selection of appropriate experimental units in teratology. Teratology 9, 259260.[ISI][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).