Reproductive Toxicology 46 (2014) 12–19
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Developmental toxicity study of CBLB502 in Wistar rats C. Paul Chow a , Ali S. Faqi b,∗ a b
Cleveland BioLabs, Inc., Buffalo, NY 14203, United States MPI Research, Mattawan, MI 49071, United States
a r t i c l e
i n f o
Article history: Received 29 July 2013 Received in revised form 18 February 2014 Accepted 22 February 2014 Available online 3 March 2014 Keywords: Developmental toxicity CBLB502 Wistar rats
a b s t r a c t CBLB502 is a derivative of a microbial protein that binds to Toll-like receptor 5. It is demonstrated to reduce inflammatory response from acute stresses, such as radiation in animal models. We determined the potential developmental toxicity of CBLB502 in rats. Four groups of 25 time-mated female Wistar rats/group received subcutaneously 0, 30, 100, or 300 g/kg/day of CBLB502 from Gestation Days (GD) 6 to 17 at a dose volume of 1.0 mL/kg. Toxicokinetic evaluation was performed on GD 6 and 17. On GD 20 C-section was performed for uterine evaluation and blood samples collected from each dam for immunogenicity assay. Significant decrease in gestation body weight, weight changes and food consumption indicative of maternal toxicity were observed in all dose groups. Also adjusted body weight and weight changes were seen at 300 g/kg/day. No external, visceral and skeletal abnormalities were observed. The NOAEL for developmental toxicity was estimated to be ≥300 g/kg/day. © 2014 Published by Elsevier Inc.
1. Introduction CBLB502, is a polypeptide drug derived from Salmonella flagellin that binds to Toll-like receptor 5 (TLR5) and activates nuclear factor-kappa B (NF-b) signaling [1]. The activation of NF-B is a double-edged sword. While needed for proper immune system function, inappropriate NF-B activation can mediate inflammation and tumorigenesis. This duality is particularly remarkable in relation to cancer, a proinflammatory disease [2]. CBLB502 is a rationally designed derivative of Salmonella flagelin that lacks the highly immunogenic central globular domain and contains N- and C-terminal domains of the parental protein connected by a flexible linker. It is less immunogenic than the full flagella length but retains the TLR5-dependent NF-B inducing activity and the capability to counteract damage induced by radiation [1]. CBLB502 has demonstrated the capacity to reduce inflammatory response from acute stresses, such as radiation in animal models. It also mobilizes several cell protective mechanisms, including inhibition of programmed cell death (apoptosis), reduction of oxidative damage and induction of regeneration-promoting cytokines [1]. Toll-like receptors (TLRs) play a critical role in the detection of pathogens invading the body and the subsequent immune response
∗ Corresponding author. Tel.: +1 269 668 3336; fax: +1 269 668 4151. E-mail address: [email protected] (A.S. Faqi). http://dx.doi.org/10.1016/j.reprotox.2014.02.007 0890-6238/© 2014 Published by Elsevier Inc.
[3,4]. Upon recognition of a microbial product, TLRs initiate a signaling pathway to activate innate immunity. There are two major TLR pathways; one is mediated by MyD88 adaptor proteins, and the other is independent of MyD88. With exception of TLR3, all other TLRs commonly use MyD88 as the downstream adopter protein. Generally, upon activation with their individual ligands, activated TLRs recruit MyD88, leading to subsequent activation of the downstream targets, including NF-B (a nuclear factor of kappa light polypeptide gene enhancer in B-cells), mitogen-associated protein (MAP) kinase and interferon regulatory factors (IRFs) [5–7]. CBLB502 is currently being developed by Cleveland BioLabs under the FDA’s animal efficacy rule to treat acute radiation syndrome (ARS) or radiation poisoning from any exposure to radiation such as a nuclear or radiological weapon/dirty bomb, or from a nuclear accident. A single injection of CBLB502 before lethal total-body irradiation protected mice from both gastrointestinal and hematopoietic ARS and resulted in improved survival [1]. It also showed radioprotective activity in lethally irradiated rhesus monkeys. In contrast to normal tissues, this TLR5 ligand didn’t change the radio sensitivity of mouse tumor, suggesting that TLR5 ligand may be valuable adjuvants for cancer radiotherapy and relatively safe protector for normal cells against high dose-radiation during irradiation [1]. As a drug the CBLB502 is intended to be used in women of child bearing potential for the treatment of ARS; its safety must be assessed. CBLB502 is pharmacologically active in rats; therefore, rats were selected for use as animal species in nonclinical safety
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testing. The purpose of the present study was to determine the embryo-fetal developmental toxicity of CBLB502 when administered during the period of organogenesis. 2. Materials and methods 2.1. Animals and animal care Care and use of the animals was in conformity with the American Association for Laboratory Animal Science Policy on the Humane Care and Use of Laboratory Animals [8]. All procedures performed on the animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at MPI Research. Timemated female Wistar (Crl: WI) rats 8–10 weeks of age were obtained from Charles River Laboratories (Portage, MI). The animals were acclimated from the time of arrival on Gestation Day (GD 0 = sperm positive) until the day of dosing on GD 6. All animals were identified by a microchip implant. Each dam was individually housed in suspended, stainless steel, wire-mesh type cages in environmentally controlled rooms with an approximate 12-hour light/12-hour dark cycle. The animal rooms were maintained at 22 ± 2 ◦ C and 30–70% relative humidity. Rats were provided with certified meal lab diet® (# 5002) and city of Mattawan municipal tap water ad libitum. 2.2. Experimental design Four groups (a vehicle and three treatment groups) of 25 time-mated animals/group were used in the study. The treatment groups received CBLB502 at respective dose levels of 30, 100 or 300 g/kg/day. The vehicle group received Phosphate Buffered Saline (PBS, pH 7.4) containing 0.1% Tween® 80. CBLB502 or vehicle was administered to all groups via subcutaneous (sc) injection once per day from GD 6–17, at a dose volume of 1.0 mL/kg. Additionally, one group of three animals and three groups of nine animals/group served as toxicokinetic (TK) animals and received the vehicle or CBLB502 in the same manner as the main study groups at respective dose levels of 0, 30, 100 and 300 g/kg/day. The dose levels were selected on the basis of available data from previous studies in Wistar rats. Doses above and/or 1 mg/kg/day showed substantial effects on body weight and food consumption. The projected clinical efficacious dose of CBLB502 is approximately 0.5 g/kg. 2.3. In-life and postmortem parental evaluation The animals were observed for morbidity, mortality and the availability of food and water twice daily throughout the study. Cage and detailed clinical observations were performed daily from GD 6 to GD 20 (60–90 min post dose on treatment days). Body weights for all animals were measured and recorded on GD 0, 6, 9, 12, 15, 18 and 20. Gestation body weight changes, adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to GD 20) were calculated. Food consumption was measured and recorded on the corresponding body weight days and calculated for the same intervals. Dams were euthanized by CO2 asphyxiation on GD 20, and cesarean sections performed. The uterus was removed from the dam, trimmed free of excess adherent tissue and weighed, with the ovaries, prior to removal of the fetuses. After weighing, the corpora lutea were counted and recorded for the left and right ovaries. Each uterine horn was inspected for resorption and fetal deaths. Resorptions were counted and classified as an early resorption (placenta only), late resorptions (placenta and attached fetal parts), early death (fetus weighing less than 0.8 g), or late death (fetus weighing more than 0.8 g).
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Each live fetus was counted and received a gross external morphologic examination. Live fetuses from each litter were weighed, sexed, and tagged. Approximately one-half of the fetuses in each litter were placed in Bouin’s solution, preserved and processed for visceral examination using a freehand razor-blade sectioning technique [9,10]. The remaining fetuses designated for skeletal examinations were eviscerated, skinned, fixed in ethyl alcohol, and stained with alizarin red.
2.4. Toxicokinetic evaluation Blood samples (approximately 0.3 mL) were collected from TK animals via the orbital sinus following anesthesia with carbon dioxide/oxygen inhalation for determination of the serum concentrations of CBLB502. Samples were collected from cohorts of three animals per group (control group had only one cohort) at rotating time points of 0, 1, 3, 6, 12 and 24 h post dose on GD’s 6 and 17. Following final blood collections, TK animals were euthanized, pregnancy status was determined and animals were discarded without further evaluation.
2.5. Anti-CBLB502 antibody analysis Blood samples (approximately 0.2 mL) were collected from all main study animals prior to c-section on GD 20 for determination of anti-CBLB502 neutralizing antibodies.
2.5.1. Statistical procedures Means and standard deviations were calculated for all measured parameters. Levene’s test was used to assess homogeneity of group variances for each of the following endpoints (see below) and for all collection intervals. Parental in-life data: Gestation body weights, gestation body weight changes, gestation food consumption, adjusted body weights and adjusted body weight changes (days 0–20). Uterine examination: Gravid uterine weights, corpora lutea/dam, total implantations/dam, litter size/dam, viable fetuses/dam, total number resorptions/dam, number early resorptions/dam and number late resorptions/dam. If Levene’s test was not significant (p ≥ 0.01), a pooled estimate of the variance (Mean Square Error or MSE) was computed from a one-way analysis of variance (ANOVA) and utilized by a Dunnett’s comparison of each treatment group with the control group. If Levene’s test was significant (p < 0.01), comparisons with the control group was performed using Welch’s t-test with a Bonferroni correction. Mean fetal body weight was analyzed using analysis of covariance. Litter size was the covariate. Each treatment satisfying the sample size assumption was compared to control using Dunnett’s test under the analysis of covariance model. The LSMEANS (Least Square Means), which are the means adjusted for values of the covariate are presented. Pregnancy index, and litter incidence of fetal abnormal findings were analyzed using Fisher’s exact test. If this overall test is significant (p < 0.05) a follow up analysis (pair-wise test) was done where each treatment group was compared to the control group. Results will be reported at the 0.05 and 0.01 significance levels. All endpoints will be analyzed using two-tailed tests unless indicated otherwise. Fetal sex ratio (% males/litter), % preimplantation loss and % postimplantation loss were transformed using arcsin of the square root. The data was then assessed for homogeneity using Levene’s test and appropriate evaluation (as described above) was conducted depending whether the test was significant or not.
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Table 1 Summary of maternal body weight during pregnancy. Study interval (day)
Dose level 0 g/kg/day Mean ± SD
0 6 9 12 15 18 20
210.0 246.0 262.3 283.5 307.8 347.5 381.3
± ± ± ± ± ± ±
13.92 14.92 16.0 17.43 18.15 24.04 28.67
30 g/kg/day Mean ± SD 210.6 250.3 253.9 273.9 297.2 339.1 373.6
± ± ± ± ± ± ±
12.57 13.33 13.60 17.42 18.46 22.39 24.82
100 g/kg/day Mean ± SD 210.7 249.0 248.7** 269.5* 291.8** 336.6 369.2
± ± ± ± ± ± ±
13.76 11.94 11.76 14.03 13.49 19.86 24.52
300 g/kg/day Mean ± SD 210.4 246.6 240.9** 259.0** 281.0** 326.9** 359.7*
± ± ± ± ± ± ±
12.26 11.71 12.31 15.57 17.77 26.27 31.97
SD, standard deviation. * Significantly different from control (p ≤ 0.05). ** Significantly different from control (p ≤ 0.01).
A minimum significance level of p ≤ 0.05 was used for all comparisons.
Subcutaneous treatment with CBLB502 to a dose level up 300 g/kg/day during the period of organogenesis did not affect these parameters.
3. Results 3.2. Fetal effects 3.1. Maternal effects In the dams, there was no CBLB502 treatment- related mortality or clinical signs of toxicity. One animal at 300 g/kg/day died on GD 20 following blood collection. The death of this animal was related to the blood collection complications. Maternal toxicity including effects on gestation body weight, body weight changes and food consumption were observed in all the treatment groups. Gestation body weight (Table 1) were significantly decreased at 100 or 300 g/kg/day in comparison to the control group. Gestation body weight changes were significantly reduced on study interval days (6–9 and 6–18) at 30, 100 or 300ug/kg/day and study interval days 6–9, 6–18, 6–20 and 0–20 at 300 g/kg/day (Table 2). Gestation food consumption was significantly decreased on study interval 6–9, 9–12, 12–15, 15–18, 18–20, 6–18, 6–20 and 0–20 at 300 g/kg/day. Gestation food consumption was also significantly reduced at 100 g/kg/day on five occasions and on three occasions at 30 g/kg/day in comparison to the control (Table 3). In addition adjusted final body weight (final body weight minus uterine weight) and adjusted body weight change (final body weight minus uterine weight minus Gestation Day 0 weight) were significantly reduced at 300 g/kg/day. This was considered to be treatment-related. Adjusted weight changes were also significantly decreased at 30 g/kg/day, but not at 100 g/kg/day when compared to the control animals; therefore, was not considered to be adverse. Gestational data, including, pregnancy, corpora lutea, implantation sites, pre- and post-implantation loss, viable and nonviable fetuses, and early and late resorptions are presented in Table 4.
There was no effect on the fetal sex ratio and fetal body weight (Table 4). The mean male and female fetal body weights and combined fetal body weights were comparable among the groups. No fetal external malformations were observed in any of the CBLB502 dose groups. Likewise, no CBLB502 related fetal visceral abnormalities were seen. Slightly increased, but not statistically significant incidence of incomplete ossification of the parietal (number of litters affected; control: 6; 100 g/kg/day: 13; 300 g/kg/day: 15), squamosal (control: 6; 100 g/kg/day: 11; 300 g/kg/day: 13) and supro-occipital bones (control: 9; 100 g/kg/day: 13; 300 g/kg/day: 15) was observed at 100 and 300 g/kg/day (Table 5). These findings were not considered to be CBLB502 treatment-related because of the lack of statistical significance. 3.3. Toxicokinetic evaluation The pharmacokinetics of CBLB502 was evaluated following the first dose (GD 6) and the 12th dose (GD 17) of repeated once daily s.c. injections in female rats. Assay results for many of the collected serum samples were not reportable because quality control samples on some of the assay runs failed to meet the acceptance criteria, and sample quantities were not sufficient to repeat the assays. Consequently no results were available for the intermediate dose (100 g/kg/day) due to this difficulty (Table 6 and Fig. 1). On the first day of dosing (GD 6) serum CBLB502 concentrations exceeded the assay low limit of quantification (LLOQ) by the time the first post-dose sample was collected at 1 h. Concentrations declined
Table 2 Summary of maternal body weight gain/loss during pregnancy. Study interval (day)
Dose level 0 g/kg/day Mean ± SD
0–6 6–9 9–12 12–15 15–18 18–20 6–18 6–20 0–20
36.0 16.3 21.2 24.3 39.7 33.8 101.5 135.3 171.3
SD, standard deviation. * Significantly different from control (p ≤ 0.05).
± ± ± ± ± ± ± ± ±
7.18 3.53 4.98 5.42 10.53 6.50 18.65 23.85 29.44
30 g/kg/day Mean ± SD 39.7 3.7 20.0 23.3 41.9 34.4 88.9 123.3 163.0
± ± ± ± ± ± ± ± ±
5.90 6.21* 5.85 6.12 8.07 7.23 14.94* 18.28 20.88
100 g/kg/day Mean ± SD 38.3 −0.3 20.8 22.3 44.9 32.5 87.6 120.2 158.4
± ± ± ± ± ± ± ± ±
6.90 5.46* 6.19 5.86 10.23 7.24 15.29* 19.50 22.58
300 g/kg/day Mean ± SD 36.2 −5.7 18.1 22.0 45.9 31.6 80.3 112.8 148.4
± ± ± ± ± ± ± ± ±
5.7 5.75* 6.68 7.12 11.59 8.19 18.60* 24.32* 25.52*
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Table 3 Summary of maternal food consumption during pregnancy. Study interval (day)
0–6 6–9 9–12 12–15 15–18 18–20 6–18 6–20 0–20
Dose levels 0 g/kg/day Mean ± SD
30 g/kg/day Mean ± SD
20.0 ± 2.02 23.5 ± 2.36 25.9 ± 2.49 27.7 ± 2.76 30.2 ± 3.07 29.4 3 ± .23 26.8 ± 2.56 27.2 ± 2.58 25.0 ± 2.32
20.4 16.9 23.4 26.1 29.8 30.2 24.0 24.9 23.6
± ± ± ± ± ± ± ± ±
100 g/kg/day Mean ± SD
1.89 2.84* 4.30 4.39 4.36 2.79 3.58* 3.31* 2.80
20.5 14.0 21.3 25.8 31.3 30.2 23.1 24.1 23.0
± ± ± ± ± ± ± ± ±
1.69 2.12* 2.56* 2.79 3.24 2.47 2.29* 2.26* 2.04*
300 g/kg/day Mean ± SD 19.8 12.1 19.3 23.0 28.3 28.9 20.7 21.8 21.2
± ± ± ± ± ± ± ± ±
1.66 2.51* 3.15* 2.94* 4.20 2.87 2.51* 2.50* 2.06*
SD, standard deviation. * Significantly different from control (p ≤ 0.05). Table 4 Uterine parameters, fetal weight, sex ratio and adjust uterine weight and weight changes. Endpoints
Dose levels
Number of females on study Number not pregnant Number pregnant Number died pregnant Viable litters Corpora lutea per litter (M ± SD) Implantation sites per litter (M ± SD) Pre-implantation loss per litter (%) (M ± SD) Viable fetuses per litter (M ± SD) Postimplantation loss per litter (M ± SD) Resorptions Early + late (M ± SD) Early (M ± SD) Late (M ± SD) Fetal weight (g) Males (M ± SD) Females (M ± SD) Combined (M ± SD) Fetal sex ratio mean % males per animal (SD) Gravid uterine weight (g) (M ± SD) Final body weight (g) (M ± SD) Adjusted final body weight (g) (M ± SD) Adjusted weight change from day 0 (g) (M ± SD)
0 g/kg/day
30 g/kg/day
100 g/kg/day
300 g/kg/day
25 3 22 0 22 13.5 ± 1.65 13.0 ± 2.06 4.53 ± 8.35 11.7 ± 3.34 10.67 ± 19.16
25 2 23 0 23 13.8 ± 1.40 13.0 ± 1.11 5.32 ± 6.26 12.0 ± 1.78 8.03 ± 10.82
25 1 24 0 24 13.2 ± 1.69 12.3 ± 2.11 7.48 ± 10.99 11.0 ± 2.77 11.95 ± 15.76
25 0 25 1a 24 13.8 ± 2.04 12.5 ± 2.25 9.43 ± 13.16 11.0 ± 3.41 12.95 ± 20.42
1.3 ± 2.19 1.3 ± 2.19 0.0 ± 0.00
1.0 ± 1.43 1.0 ± 1.41 0.0 ± 0.21
1.3 ± 1.43 1.2 ± 1.38 0.1 ± 0.41
1.4 ± 2.04 1.1 ± 1.68 0.3 ± 1.43
4.30 ± 0.307 4.10 ± 0.197 4.19 ± 0.21 50.1 ± 16.08 74.2 ± 19.27 381.3 ± 28.67 307.1 ± 19.88 97.1 ± 18.20
4.42 ± 0.22 4.20 ± 0.229 4.31 ± 0.21 52.7 ± 11.36 78.5 ± 10.89 373.6 ± 24.82 295.1 ± 22.13 84.5* ± 17.36
4.42 ± 0.33 4.25 ± 0.257 4.34 ± 0.27 45.5 ± 15.07 72.5 ± 16.54 369.2 ± 24.52 296.6 ± 14.98 85.9 ± 17.50
4.38 ± 0.32 4.26 ± 0.336 4.31 ± 0.28 52.5 ± 16.49 75.2 ± 19.49 359.7 ± 31.97* 284.5 ± 21.11* 73.2 ± 15.26*
Postimplantation loss = No. implantations − No. viable fetuses/No. implantations × 100. Preimplantation loss; No. corpora lutea − No. implantations/No. corpora lutea × 100. Adjusted final body weight = final body weight (g) minus uterus weight (g). Adjusted body weight change = final body weight (g) minus uterus weight (g) minus Gestation Day 0 body weight (g). a Animal died due to bleeding complications on GD 20. * Significantly different from control (p ≤ 0.05).
Fig. 1. Mean (±SD) serum CBLB502 concentrations in female rats with subcutaneous injections of 300 g/kg/day (semilog coordinates).
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Table 5 Summary of individual fetal skeletal observations. Study interval (Day)
Dose level 0 g/kg/day Mean ± SD
30 g/kg/day Mean ± SD
100 g/kg/day Mean ± SD
300 g/kg/day Mean ± SD
Neural arch(es), additional ossification center 2 (9.1) No. litters (%) 3 (2.3) No. fetuses (%)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
2 (8.3) 3 (2.3)
Neural arch(es), incompletely ossified No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
1 (4.2) 1 (0.8)
Scapula, bent No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
1 (4.2) 1 (0.8)
Ischium, incompletely ossified No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
2 (8.7) 2 (1.4)
1 (4.2) 1 (0.8)
2 (8.3) 3 (2.3)
Rib(s), bent No. litters (%) No. fetuses (%)
5 (22.7) 6 (4.7)
5 (21.7) 10 (7.2)
9 (37.5) 16 (12.2)
7 (29.2) 13 (9.9)
16 (72.7) 44 (34.1)
20 (87.0) 61 (43.9)
21 (87.5) 54 (41.2)
15 (62.5) 34 (26.0)
Rib(s), rudimentary No. litters (%) No. fetuses (%) Rib(s), unilateral full rib No. litters (%) No. fetuses (%)
2 (9.1) 2 (1.6)
5 (21.7) 5 (3.6)
2 (8.3) 2 (1.5)
0 (0.0) 0 (0.0)
Frontal bone, incompletely ossified No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
1 (4.3) 2 (1.4)
1 (4.2) 1 (0.8)
1 (4.2) 1 (0.8)
Hyoid, not ossified No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
3 (13.0) 4 (2.9)
7 (29.2) 11 (8.4)
5 (20.8) 11 (8.4)
Interparietal bone: incompletely ossified No. litters (%) No. fetuses (%)
4 (18.2) 6 (4.7)
2 (8.7) 2 (1.4)
5 (20.8) 6 (4.6)
6 (25.0) 10 (7.6)
Jugal (Zygoma), fused No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
1 (4.3) 1 (0.7)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
Jugal (Zygoma), incompletely ossified No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
6 (26.1) 8 (5.8)
5 (20.8) 5 (3.8)
6 (25.0) 8 (6.1)
1 (4.3) 1 (0.7)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
9 (40.9) 14 (10.9)
11 (47.8) 24 (17.3)
13 (54.2) 34 (26.0)
16 (66.7) 46 (35.1)
6 (27.3) 8 (6.2)
6 (26.1) 11 (7.9)
11 (45.8) 23 (17.6)
13 (54.2) 37 (28.2)
Supra occipital bone, incompletely ossified 9 (40.9) No. litters (%) 14 (10.9) No. fetuses (%)
8 (34.8) 12 (8.6)
13 (54.2) 22 (16.8)
15 (62.5) 35 (26.7)
Sternebra(e), misaligned No. litters (%) No. fetuses (%)
6 (27.3) 7 (5.4)
2 (8.7) 2 (1.4)
1 (4.2) 1 (0.8)
2 (8.3) 2 (1.5)
Sternebra(e), not ossified No. litters (%) No. fetuses (%)1
7 (31.8) 10 (7.8)
Parietal bone, additional ossification center No. litters (%) 0 (0.0) 0 (0.0) No. fetuses (%) Parietal bone, incompletely ossified No. litters (%) No. fetuses (%) Squamosal, incompletely ossified No. litters (%) No. fetuses (%)
Sternebra(e), sternoschisis No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
4 (17.4) 4 (2.9)
4 (16.7) 4 (3.1)
8 (33.3) 9 (6.9)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
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Table 6 Toxicokinetic parameters of CBLB502 in pregnant rats following subcutaneous injections. Parameter
Unit
Dose levels 30 g/kg/day
Dose Cmax Cmax /dose Tmax AUCt AUCt /dose AUC∞ z T1/2 CL/F Vz /F
g/kg/day ng/mL (ng/mL)/(g/kg) h ng/h/mL (ng/h/mL)(g/kg) ng/h/mL 1/h h L/h/kg L/kg
100 g/kg/day
300 g/kg/day
GD 6
GD 17
GD 6
GD 17
GD 6
GD 17
30 3.79 0.126 1 13.16 0.439 NE NE NE 2.28 NE
30 1.14 0.038 1 3.89 0.13 NE NE NE 7.72 NE
100 NE NE NE NE NE NE NE NE NE NE
83 NE NE NE NE NE NE NE NE NE NE
300 98.22 0.327 1 187.24 0.624 190.78 0.257 2.69 1.57 6.11
259 10.82 0.0417 1 38.66 0.149 39.89 0.298 2.32 6.5 21.8
NE = not evaluated. Table 7 Summary of immunogenicity results. Group number
Screening result
Immunodepletion result
Neutralizing antibody % inhibition (cutpoint 14.68%)
Neutralizing antibody result
1
2 PP 23 Negative 19 PP 2 IN 4 Negative 24 PP 1 Negative 22 PP 3 Negative
2 Positive
30.46%
1 Positive
16 Positive
15.43–85.25%
17 Positive
20 Positive
18.5–67.98%
16 Positive
20 Positive
22.28–69.64%
17 Positive
2
3 4
PP = presumed positive; IN = inconclusive. CBLB502 is a derivative of a microbial protein that binds to Toll-like receptor 5. It is demonstrated to reduce inflammation from acute stresses, such as radiation in animal models. We determined the potential developmental toxicity of CBLB502 in rats. Four groups of 25 time-mated female Wistar rats/group received subcutaneously 0, 30, 100, or 300 g/kg/day of CBLB502 from Gestation Days (GD) 6–17 at a dose volume of 1.0 mL/kg. Toxicokinetic evaluation was performed on GD 6 and 17. On GD 20 C-section was performed for uterine evaluation and blood samples collected from each dam for immunogenicity assay. Significant decrease in gestation body weight, weight changes and food consumption indicative of maternal toxicity were observed in all dose groups. Also adjusted body weight and weight changes were seen at 300 g/kg/day. No external, visceral and skeletal abnormalities were observed. The NOAEL for developmental toxicity was estimated to be ≥300 g/kg/day.
following the 1-hour sample such that they were below the LLOQ by 24 hours post-dose after even the highest dose (300 g/kg/day) on both GD 6 and GD 17. The Cmax and AUCt increased at a slightly greater than dose proportional manner on GD 6, but their increases were approximately dose proportional on GD 17. The terminal elimination half-life was approximately 2.5 h after the high dose (the only dose for which it was calculable) on both observation days. Apparent clearance of CBLB502 on GD 6 ranged from 1.6 to 2.3 L/h/kg, with a trend to decrease with increasing dose; on GD 17 the mean apparent clearance was 3–4 times the clearance on GD 6 (6.5–7.7 L/hr/kg), also with a trend to decrease with increasing dose. The systemic exposures to CBLB502 on GD 17 in these rats were 1/4 to 1/3 the peak and total exposures observed following the first dose on GD 6, indicating that CBLB502 clearance from the rat serum was substantially increased with daily repeated dosing. 3.4. Anti-CBLB502 antibody analysis The presence of anti-CBLB502 neutralizing antibodies in Wister rat serum was assessed. A total of 100 samples (25 samples/group, all collected on GD 20) were screened for the presence of antiCBLB502 antibodies in Wistar rat serum. Of these, 67 samples (2 from control, 19 at 30 g/kg/day, 24 at 100 g/kg/day, and 22 at 300 g/kg/day) yielded optical densities (OD) above their corresponding plate-specific cutpoint and were presumed positive for anti-CBLB502 antibodies. Nine of the presumptive positive samples were deemed QNS (Quantity Not Sufficient) for confirmatory immune-depletion analysis and titering, and were reserved for
testing for the presence anti-CBLB502 neutralizing antibodies. The remaining 58 presumptive positive samples were subjected to confirmatory immune-depletion analysis, and all the 58 samples were confirmed positive for the presence of anti-CBLB502 antibodies. All of these samples plus the 9 presumptive positive samples were then subjected for titration assay. In all, 51 samples were positive for anti-CBLB502 neutralizing antibodies: (control: 1; 30 g/kg/day: 17; 100 g/kg/day: 16, and 300 g/kg/day: 17 – see Table 7). For an unknown reason, one control animal was found to be positive for neutralizing antibodies. Overall, the frequency of neutralizing antibody-positive animals did not appear to be dose dependent. 4. Discussion and conclusions The objective of this study was to determine the developmental toxicity including of the potential teratogenicity of ‘CBLB502 in rats. CBLB502 is a derivative of a microbial protein that reduces inflammatory response from acute stresses by mobilizing several natural cell protecting mechanisms, including inhibition of programmed cell death (apoptosis), reduction of oxidative damage and induction of regeneration promoting cytokines [1]. It is currently being developed by Cleveland BioLabs under the FDA’s Animal Efficacy Rule to treat ARS or radiation poisoning from any exposure to radiation such as a nuclear or radiological weapon/dirty bomb, or from a nuclear accident. No mortality or CBLB502-related clinical signs of toxicity were observed at a dose level up to 300 g/kg/day. Significantly decreased gestation body weight, gestation body weight changes
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and gestation food consumption were observed at 30, 100 or 300 g/kg/day dose when compared to the controls. Gestation body weight and gestation body weight changes are viewed as indicators of maternal toxicity for rodent species. Indeed body weight changes may provide more information than a daily body weight measured during treatment or during gestation as changes in weight gain during treatment could occur that would not be reflected in the total weight change throughout gestation, because of compensatory weight gain that may occur following post-treatment [11]. Additional endpoints affected include adjusted body weight and adjusted body weight changes that were significantly reduced at 300 g/kg/day. Effects on adjusted body weight and weight gain with no effect in gravid uterine weight suggest maternal toxicity. The current practice for the assessment of embryo-fetal developmental toxicity (EFD) necessitates administration of high, maternally toxic doses to pregnant laboratory animals [12–14] and avoid marked maternal toxicity leading to mortality or decreased body weight gains of greater than 20% for prolonged periods [15]. In EFD studies it is very common to see that maternal effects are accompanied by developmental toxicity. However, in the present study no fetal external, visceral and skeletal malformations were observed. In addition uterine data including implantation sites, preand post-implantation loss, viable/nonviable fetuses and resorptions were not adversely affected. Likewise, fetal body weights were comparable among the groups; although adjusted final body weight and adjusted weight changes were significantly reduced at 300 mg/kg/day. Slightly increased, but statistically not significant incidence of incomplete ossification of parietal, squamosal bones, were observed at 100 and 300 g/kg/day. Delayed ossification may pose some challenges in the interpretation because these abnormalities occur at a relatively high background incidence in control animals and are observed frequently in a dose-related manner [16]. Incomplete/delay in ossification is often related to maternal toxicity [17]. Carney and Kimmel (2007) [17] proposed a weight-of-evidence approach for the interpretation of delayed ossifications and other variations. They have classified the findings into three different categories, namely insignificant findings, low significance findings and findings that warrant increased attention. Findings that warrant increased attention include patterns of ossification that do not follow normal sequence, specific delays involving bones that normally are well ossified in term fetuses (e.g., ribs, clavicle, long bones of the limbs, lumbar vertebral centra), abnormally-shaped cartilage template, significantly increased delayed ossification without decreases in fetal body weight, delayed ossification in the absence of maternal toxicity, and delayed ossification associated with teratogenesis. The delayed ossifications observed in our study do not fall into the category of the findings that warrant increased attention and nevertheless such minor variations would not generally be considered adverse in and of themselves but should be interpreted in the context of other maternal and fetal finding. In our study, there were no fetal findings and the maternal toxicity was limited to reduced body weight, body weight changes and food consumptions as well as adjusted final body weight and weight changes. We therefore, conclude that these findings were not CBLB502 treatment-related because of the lack of statistical significance. We have also examined the presence of anti-CBLB502 neutralizing antibodies in Wister rat serum. It is common for the animals to develop anti-drug antibodies (ADA) following administration of foreign proteins (such as CBLB502). This may lead to increased clearance of the biopharmaceuticals, yielding exposure reduction and over estimating safety [18]. The immunogenicity assay conducted revealed that not all animals had anti-CBLB502 neutralizing antibodies. The ADA was not detected in all the animals and the frequency of neutralizing antibody-positive animals did not
manifest in a dose-dependent manner. However, anti-CBLB502 neutralizing antibodies may have induced some impact to the toxicokinetic behavior of CBLB502. The Cmax and AUCt increased at a slightly greater than dose proportional manner on GD 6, but their increases were approximately dose proportional on GD 17. Apparent clearance of CBLB502 on GD 6 ranged from 1.6 to 2.3 L/h/kg, with a trend to decrease with increasing dose; on GD 17 the mean apparent clearance was 3–4 times the clearance on GD 6 (6.5–7.7 L/h/kg), also with a trend to decrease with increasing dose. Having said that we can conclude that anti-CBLB502 neutralizing antibodies had little or no impact and/or does not disqualify the overall conduct of the study as the doses of CBLB502 used were able to elicit and maintain persistence maternal effects throughout the study. For example the effect on maternal body weight at 300 g/kg/day emerged on GD 9 and continued throughout the study. Likewise, the effects on body weight changes and food consumption at the high dose followed similar trend. Nevertheless, there are some unknown variables including how many dosing days was necessary to elicit anti-CBLB502 neutralizing antibody as the immunogenicity assay was conducted only on GD 20, three days after completion of treatment. But, the toxicokinetic data of GD 17 at 300 g/kg/day and the persistence maternal effects observed in this study may suggest that the anti-CBLB502 neutralizing antibodies may have been induced late during the treatment. Based on the results of this study, the NOAEL (No-ObservedAdverse-Effect-Level) for developmental toxicity was estimated to be to be ≥300 g/kg/day after administration to Wistar rats for 12 days. Since the high dose did not cause developmental effects; therefore, a dose greater or equal than the high dose is estimated to be the NOAEL. It is unknown exactly what dose, but for sure is greater or equal than the high dose. Preclinical safety results available to date support the continued development of CBLB502 in human. Conflict of interest Paul Chow works for Cleveland Biolabs that is developing CBLB502. Ali S. Faqi has no conflict of interest. Transparency document The Transparency document associated to this article can be found, in the online version. References [1] Burdelya LG, Krivokrysenko V, Tallant TC, Strom E, Gleibeman AS, Gupta D, et al. An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models. Science 2008;320(5873):226–30. [2] Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet 2001;357:539–45. [3] Akira S. Toll-like receptor signaling. J Biol Chem 2003;278(40):38105–8. [4] Means TK, Hayashi F, Smith KD, Aderem A, Luster AD. The toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J Immunol 2003;170(10):5165–75. [5] Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004;16:3–9. [6] Moynagh PN. TLR signalling and activation of IRFs: revisiting old friends from the NF-kappaB pathway. Trends Immunol 2005;26:469–76. [7] Yamamoto M, Akira S. TIR domain-containing adaptors regulate TLR signaling pathways. Adv Exp Med Biol 2005;560:1–9. [8] AAALAC. American Association for Laboratory Animal Science policy on the humane care and use of laboratory animals. Lab Anim Sci 1991;41:91. [9] Wilson JG. Methods for administering agents and detecting malformations in experimental animals. In: Teratology, Principles and Techniques. Chicago: University of Chicago Press; 1965. p. 62. [10] Staples RE. Detection of visceral abnormalities in mammalian fetuses. Teratology 1974;9(3):A37–8. [11] Kimmel CA, Price CJ. Developmental toxicity studies. In: Arnold DL, Grice HC, Krewski DR, editors. Handbook of In Vivo Toxicity Testing. San Diego, CA: Academic Press; 1990. p. 271–301.
C.P. Chow, A.S. Faqi / Reproductive Toxicology 46 (2014) 12–19 [12] Rogers JM, Chernoff N, Keen CL, Daston GP. Evaluation and interpretation of maternal toxicity in Segment II studies: issues, some answers, and data needs. Toxicol Appl Pharmacol 2005;207(2 Suppl.):367–74. [13] Carney E. Maternally mediated developmental toxicity. In: Charlene A, McQueen, editors. Comprehensive Toxicology, vol. 12. Oxford: Academic Press; 2010. p. 163–76. [14] Faqi AS. Methods for detection of developmental toxicity. In: Charlene A, McQueen, editors. Comprehensive Toxicology, vol. 12. Oxford: Academic Press; 2010. p. 259–78. [15] Beyer BK, Chernoff N, Danielsson BR, Davis-Bruno K, Harrouk W, Hood RD, et al. ILSI/HESI maternal toxicity workshop summary: maternal toxicity and its
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impact on study design and data interpretation. Birth Defects Res B Dev Reprod Toxicol 2011;92(1):36–51. [16] Daston GP, Seed J. Skeletal malformations and variations in developmental toxicity studies: interspecies issues for human risk assessment. Birth Defects Res B Dev Reprod Toxicol 2007;80(6):421–4. [17] Carney EW, Kimmel CA. Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res B Dev Reprod Toxicol 2007;80(6):473–96. [18] Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin-Dupont P, et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 2002;346:469–75.
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Developmental toxicity study of CBLB502 in Wistar rats C. Paul Chow a , Ali S. Faqi b,∗ a b
Cleveland BioLabs, Inc., Buffalo, NY 14203, United States MPI Research, Mattawan, MI 49071, United States
a r t i c l e
i n f o
Article history: Received 29 July 2013 Received in revised form 18 February 2014 Accepted 22 February 2014 Available online 3 March 2014 Keywords: Developmental toxicity CBLB502 Wistar rats
a b s t r a c t CBLB502 is a derivative of a microbial protein that binds to Toll-like receptor 5. It is demonstrated to reduce inflammatory response from acute stresses, such as radiation in animal models. We determined the potential developmental toxicity of CBLB502 in rats. Four groups of 25 time-mated female Wistar rats/group received subcutaneously 0, 30, 100, or 300 g/kg/day of CBLB502 from Gestation Days (GD) 6 to 17 at a dose volume of 1.0 mL/kg. Toxicokinetic evaluation was performed on GD 6 and 17. On GD 20 C-section was performed for uterine evaluation and blood samples collected from each dam for immunogenicity assay. Significant decrease in gestation body weight, weight changes and food consumption indicative of maternal toxicity were observed in all dose groups. Also adjusted body weight and weight changes were seen at 300 g/kg/day. No external, visceral and skeletal abnormalities were observed. The NOAEL for developmental toxicity was estimated to be ≥300 g/kg/day. © 2014 Published by Elsevier Inc.
1. Introduction CBLB502, is a polypeptide drug derived from Salmonella flagellin that binds to Toll-like receptor 5 (TLR5) and activates nuclear factor-kappa B (NF-b) signaling [1]. The activation of NF-B is a double-edged sword. While needed for proper immune system function, inappropriate NF-B activation can mediate inflammation and tumorigenesis. This duality is particularly remarkable in relation to cancer, a proinflammatory disease [2]. CBLB502 is a rationally designed derivative of Salmonella flagelin that lacks the highly immunogenic central globular domain and contains N- and C-terminal domains of the parental protein connected by a flexible linker. It is less immunogenic than the full flagella length but retains the TLR5-dependent NF-B inducing activity and the capability to counteract damage induced by radiation [1]. CBLB502 has demonstrated the capacity to reduce inflammatory response from acute stresses, such as radiation in animal models. It also mobilizes several cell protective mechanisms, including inhibition of programmed cell death (apoptosis), reduction of oxidative damage and induction of regeneration-promoting cytokines [1]. Toll-like receptors (TLRs) play a critical role in the detection of pathogens invading the body and the subsequent immune response
∗ Corresponding author. Tel.: +1 269 668 3336; fax: +1 269 668 4151. E-mail address: [email protected] (A.S. Faqi). http://dx.doi.org/10.1016/j.reprotox.2014.02.007 0890-6238/© 2014 Published by Elsevier Inc.
[3,4]. Upon recognition of a microbial product, TLRs initiate a signaling pathway to activate innate immunity. There are two major TLR pathways; one is mediated by MyD88 adaptor proteins, and the other is independent of MyD88. With exception of TLR3, all other TLRs commonly use MyD88 as the downstream adopter protein. Generally, upon activation with their individual ligands, activated TLRs recruit MyD88, leading to subsequent activation of the downstream targets, including NF-B (a nuclear factor of kappa light polypeptide gene enhancer in B-cells), mitogen-associated protein (MAP) kinase and interferon regulatory factors (IRFs) [5–7]. CBLB502 is currently being developed by Cleveland BioLabs under the FDA’s animal efficacy rule to treat acute radiation syndrome (ARS) or radiation poisoning from any exposure to radiation such as a nuclear or radiological weapon/dirty bomb, or from a nuclear accident. A single injection of CBLB502 before lethal total-body irradiation protected mice from both gastrointestinal and hematopoietic ARS and resulted in improved survival [1]. It also showed radioprotective activity in lethally irradiated rhesus monkeys. In contrast to normal tissues, this TLR5 ligand didn’t change the radio sensitivity of mouse tumor, suggesting that TLR5 ligand may be valuable adjuvants for cancer radiotherapy and relatively safe protector for normal cells against high dose-radiation during irradiation [1]. As a drug the CBLB502 is intended to be used in women of child bearing potential for the treatment of ARS; its safety must be assessed. CBLB502 is pharmacologically active in rats; therefore, rats were selected for use as animal species in nonclinical safety
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testing. The purpose of the present study was to determine the embryo-fetal developmental toxicity of CBLB502 when administered during the period of organogenesis. 2. Materials and methods 2.1. Animals and animal care Care and use of the animals was in conformity with the American Association for Laboratory Animal Science Policy on the Humane Care and Use of Laboratory Animals [8]. All procedures performed on the animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at MPI Research. Timemated female Wistar (Crl: WI) rats 8–10 weeks of age were obtained from Charles River Laboratories (Portage, MI). The animals were acclimated from the time of arrival on Gestation Day (GD 0 = sperm positive) until the day of dosing on GD 6. All animals were identified by a microchip implant. Each dam was individually housed in suspended, stainless steel, wire-mesh type cages in environmentally controlled rooms with an approximate 12-hour light/12-hour dark cycle. The animal rooms were maintained at 22 ± 2 ◦ C and 30–70% relative humidity. Rats were provided with certified meal lab diet® (# 5002) and city of Mattawan municipal tap water ad libitum. 2.2. Experimental design Four groups (a vehicle and three treatment groups) of 25 time-mated animals/group were used in the study. The treatment groups received CBLB502 at respective dose levels of 30, 100 or 300 g/kg/day. The vehicle group received Phosphate Buffered Saline (PBS, pH 7.4) containing 0.1% Tween® 80. CBLB502 or vehicle was administered to all groups via subcutaneous (sc) injection once per day from GD 6–17, at a dose volume of 1.0 mL/kg. Additionally, one group of three animals and three groups of nine animals/group served as toxicokinetic (TK) animals and received the vehicle or CBLB502 in the same manner as the main study groups at respective dose levels of 0, 30, 100 and 300 g/kg/day. The dose levels were selected on the basis of available data from previous studies in Wistar rats. Doses above and/or 1 mg/kg/day showed substantial effects on body weight and food consumption. The projected clinical efficacious dose of CBLB502 is approximately 0.5 g/kg. 2.3. In-life and postmortem parental evaluation The animals were observed for morbidity, mortality and the availability of food and water twice daily throughout the study. Cage and detailed clinical observations were performed daily from GD 6 to GD 20 (60–90 min post dose on treatment days). Body weights for all animals were measured and recorded on GD 0, 6, 9, 12, 15, 18 and 20. Gestation body weight changes, adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to GD 20) were calculated. Food consumption was measured and recorded on the corresponding body weight days and calculated for the same intervals. Dams were euthanized by CO2 asphyxiation on GD 20, and cesarean sections performed. The uterus was removed from the dam, trimmed free of excess adherent tissue and weighed, with the ovaries, prior to removal of the fetuses. After weighing, the corpora lutea were counted and recorded for the left and right ovaries. Each uterine horn was inspected for resorption and fetal deaths. Resorptions were counted and classified as an early resorption (placenta only), late resorptions (placenta and attached fetal parts), early death (fetus weighing less than 0.8 g), or late death (fetus weighing more than 0.8 g).
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Each live fetus was counted and received a gross external morphologic examination. Live fetuses from each litter were weighed, sexed, and tagged. Approximately one-half of the fetuses in each litter were placed in Bouin’s solution, preserved and processed for visceral examination using a freehand razor-blade sectioning technique [9,10]. The remaining fetuses designated for skeletal examinations were eviscerated, skinned, fixed in ethyl alcohol, and stained with alizarin red.
2.4. Toxicokinetic evaluation Blood samples (approximately 0.3 mL) were collected from TK animals via the orbital sinus following anesthesia with carbon dioxide/oxygen inhalation for determination of the serum concentrations of CBLB502. Samples were collected from cohorts of three animals per group (control group had only one cohort) at rotating time points of 0, 1, 3, 6, 12 and 24 h post dose on GD’s 6 and 17. Following final blood collections, TK animals were euthanized, pregnancy status was determined and animals were discarded without further evaluation.
2.5. Anti-CBLB502 antibody analysis Blood samples (approximately 0.2 mL) were collected from all main study animals prior to c-section on GD 20 for determination of anti-CBLB502 neutralizing antibodies.
2.5.1. Statistical procedures Means and standard deviations were calculated for all measured parameters. Levene’s test was used to assess homogeneity of group variances for each of the following endpoints (see below) and for all collection intervals. Parental in-life data: Gestation body weights, gestation body weight changes, gestation food consumption, adjusted body weights and adjusted body weight changes (days 0–20). Uterine examination: Gravid uterine weights, corpora lutea/dam, total implantations/dam, litter size/dam, viable fetuses/dam, total number resorptions/dam, number early resorptions/dam and number late resorptions/dam. If Levene’s test was not significant (p ≥ 0.01), a pooled estimate of the variance (Mean Square Error or MSE) was computed from a one-way analysis of variance (ANOVA) and utilized by a Dunnett’s comparison of each treatment group with the control group. If Levene’s test was significant (p < 0.01), comparisons with the control group was performed using Welch’s t-test with a Bonferroni correction. Mean fetal body weight was analyzed using analysis of covariance. Litter size was the covariate. Each treatment satisfying the sample size assumption was compared to control using Dunnett’s test under the analysis of covariance model. The LSMEANS (Least Square Means), which are the means adjusted for values of the covariate are presented. Pregnancy index, and litter incidence of fetal abnormal findings were analyzed using Fisher’s exact test. If this overall test is significant (p < 0.05) a follow up analysis (pair-wise test) was done where each treatment group was compared to the control group. Results will be reported at the 0.05 and 0.01 significance levels. All endpoints will be analyzed using two-tailed tests unless indicated otherwise. Fetal sex ratio (% males/litter), % preimplantation loss and % postimplantation loss were transformed using arcsin of the square root. The data was then assessed for homogeneity using Levene’s test and appropriate evaluation (as described above) was conducted depending whether the test was significant or not.
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Table 1 Summary of maternal body weight during pregnancy. Study interval (day)
Dose level 0 g/kg/day Mean ± SD
0 6 9 12 15 18 20
210.0 246.0 262.3 283.5 307.8 347.5 381.3
± ± ± ± ± ± ±
13.92 14.92 16.0 17.43 18.15 24.04 28.67
30 g/kg/day Mean ± SD 210.6 250.3 253.9 273.9 297.2 339.1 373.6
± ± ± ± ± ± ±
12.57 13.33 13.60 17.42 18.46 22.39 24.82
100 g/kg/day Mean ± SD 210.7 249.0 248.7** 269.5* 291.8** 336.6 369.2
± ± ± ± ± ± ±
13.76 11.94 11.76 14.03 13.49 19.86 24.52
300 g/kg/day Mean ± SD 210.4 246.6 240.9** 259.0** 281.0** 326.9** 359.7*
± ± ± ± ± ± ±
12.26 11.71 12.31 15.57 17.77 26.27 31.97
SD, standard deviation. * Significantly different from control (p ≤ 0.05). ** Significantly different from control (p ≤ 0.01).
A minimum significance level of p ≤ 0.05 was used for all comparisons.
Subcutaneous treatment with CBLB502 to a dose level up 300 g/kg/day during the period of organogenesis did not affect these parameters.
3. Results 3.2. Fetal effects 3.1. Maternal effects In the dams, there was no CBLB502 treatment- related mortality or clinical signs of toxicity. One animal at 300 g/kg/day died on GD 20 following blood collection. The death of this animal was related to the blood collection complications. Maternal toxicity including effects on gestation body weight, body weight changes and food consumption were observed in all the treatment groups. Gestation body weight (Table 1) were significantly decreased at 100 or 300 g/kg/day in comparison to the control group. Gestation body weight changes were significantly reduced on study interval days (6–9 and 6–18) at 30, 100 or 300ug/kg/day and study interval days 6–9, 6–18, 6–20 and 0–20 at 300 g/kg/day (Table 2). Gestation food consumption was significantly decreased on study interval 6–9, 9–12, 12–15, 15–18, 18–20, 6–18, 6–20 and 0–20 at 300 g/kg/day. Gestation food consumption was also significantly reduced at 100 g/kg/day on five occasions and on three occasions at 30 g/kg/day in comparison to the control (Table 3). In addition adjusted final body weight (final body weight minus uterine weight) and adjusted body weight change (final body weight minus uterine weight minus Gestation Day 0 weight) were significantly reduced at 300 g/kg/day. This was considered to be treatment-related. Adjusted weight changes were also significantly decreased at 30 g/kg/day, but not at 100 g/kg/day when compared to the control animals; therefore, was not considered to be adverse. Gestational data, including, pregnancy, corpora lutea, implantation sites, pre- and post-implantation loss, viable and nonviable fetuses, and early and late resorptions are presented in Table 4.
There was no effect on the fetal sex ratio and fetal body weight (Table 4). The mean male and female fetal body weights and combined fetal body weights were comparable among the groups. No fetal external malformations were observed in any of the CBLB502 dose groups. Likewise, no CBLB502 related fetal visceral abnormalities were seen. Slightly increased, but not statistically significant incidence of incomplete ossification of the parietal (number of litters affected; control: 6; 100 g/kg/day: 13; 300 g/kg/day: 15), squamosal (control: 6; 100 g/kg/day: 11; 300 g/kg/day: 13) and supro-occipital bones (control: 9; 100 g/kg/day: 13; 300 g/kg/day: 15) was observed at 100 and 300 g/kg/day (Table 5). These findings were not considered to be CBLB502 treatment-related because of the lack of statistical significance. 3.3. Toxicokinetic evaluation The pharmacokinetics of CBLB502 was evaluated following the first dose (GD 6) and the 12th dose (GD 17) of repeated once daily s.c. injections in female rats. Assay results for many of the collected serum samples were not reportable because quality control samples on some of the assay runs failed to meet the acceptance criteria, and sample quantities were not sufficient to repeat the assays. Consequently no results were available for the intermediate dose (100 g/kg/day) due to this difficulty (Table 6 and Fig. 1). On the first day of dosing (GD 6) serum CBLB502 concentrations exceeded the assay low limit of quantification (LLOQ) by the time the first post-dose sample was collected at 1 h. Concentrations declined
Table 2 Summary of maternal body weight gain/loss during pregnancy. Study interval (day)
Dose level 0 g/kg/day Mean ± SD
0–6 6–9 9–12 12–15 15–18 18–20 6–18 6–20 0–20
36.0 16.3 21.2 24.3 39.7 33.8 101.5 135.3 171.3
SD, standard deviation. * Significantly different from control (p ≤ 0.05).
± ± ± ± ± ± ± ± ±
7.18 3.53 4.98 5.42 10.53 6.50 18.65 23.85 29.44
30 g/kg/day Mean ± SD 39.7 3.7 20.0 23.3 41.9 34.4 88.9 123.3 163.0
± ± ± ± ± ± ± ± ±
5.90 6.21* 5.85 6.12 8.07 7.23 14.94* 18.28 20.88
100 g/kg/day Mean ± SD 38.3 −0.3 20.8 22.3 44.9 32.5 87.6 120.2 158.4
± ± ± ± ± ± ± ± ±
6.90 5.46* 6.19 5.86 10.23 7.24 15.29* 19.50 22.58
300 g/kg/day Mean ± SD 36.2 −5.7 18.1 22.0 45.9 31.6 80.3 112.8 148.4
± ± ± ± ± ± ± ± ±
5.7 5.75* 6.68 7.12 11.59 8.19 18.60* 24.32* 25.52*
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Table 3 Summary of maternal food consumption during pregnancy. Study interval (day)
0–6 6–9 9–12 12–15 15–18 18–20 6–18 6–20 0–20
Dose levels 0 g/kg/day Mean ± SD
30 g/kg/day Mean ± SD
20.0 ± 2.02 23.5 ± 2.36 25.9 ± 2.49 27.7 ± 2.76 30.2 ± 3.07 29.4 3 ± .23 26.8 ± 2.56 27.2 ± 2.58 25.0 ± 2.32
20.4 16.9 23.4 26.1 29.8 30.2 24.0 24.9 23.6
± ± ± ± ± ± ± ± ±
100 g/kg/day Mean ± SD
1.89 2.84* 4.30 4.39 4.36 2.79 3.58* 3.31* 2.80
20.5 14.0 21.3 25.8 31.3 30.2 23.1 24.1 23.0
± ± ± ± ± ± ± ± ±
1.69 2.12* 2.56* 2.79 3.24 2.47 2.29* 2.26* 2.04*
300 g/kg/day Mean ± SD 19.8 12.1 19.3 23.0 28.3 28.9 20.7 21.8 21.2
± ± ± ± ± ± ± ± ±
1.66 2.51* 3.15* 2.94* 4.20 2.87 2.51* 2.50* 2.06*
SD, standard deviation. * Significantly different from control (p ≤ 0.05). Table 4 Uterine parameters, fetal weight, sex ratio and adjust uterine weight and weight changes. Endpoints
Dose levels
Number of females on study Number not pregnant Number pregnant Number died pregnant Viable litters Corpora lutea per litter (M ± SD) Implantation sites per litter (M ± SD) Pre-implantation loss per litter (%) (M ± SD) Viable fetuses per litter (M ± SD) Postimplantation loss per litter (M ± SD) Resorptions Early + late (M ± SD) Early (M ± SD) Late (M ± SD) Fetal weight (g) Males (M ± SD) Females (M ± SD) Combined (M ± SD) Fetal sex ratio mean % males per animal (SD) Gravid uterine weight (g) (M ± SD) Final body weight (g) (M ± SD) Adjusted final body weight (g) (M ± SD) Adjusted weight change from day 0 (g) (M ± SD)
0 g/kg/day
30 g/kg/day
100 g/kg/day
300 g/kg/day
25 3 22 0 22 13.5 ± 1.65 13.0 ± 2.06 4.53 ± 8.35 11.7 ± 3.34 10.67 ± 19.16
25 2 23 0 23 13.8 ± 1.40 13.0 ± 1.11 5.32 ± 6.26 12.0 ± 1.78 8.03 ± 10.82
25 1 24 0 24 13.2 ± 1.69 12.3 ± 2.11 7.48 ± 10.99 11.0 ± 2.77 11.95 ± 15.76
25 0 25 1a 24 13.8 ± 2.04 12.5 ± 2.25 9.43 ± 13.16 11.0 ± 3.41 12.95 ± 20.42
1.3 ± 2.19 1.3 ± 2.19 0.0 ± 0.00
1.0 ± 1.43 1.0 ± 1.41 0.0 ± 0.21
1.3 ± 1.43 1.2 ± 1.38 0.1 ± 0.41
1.4 ± 2.04 1.1 ± 1.68 0.3 ± 1.43
4.30 ± 0.307 4.10 ± 0.197 4.19 ± 0.21 50.1 ± 16.08 74.2 ± 19.27 381.3 ± 28.67 307.1 ± 19.88 97.1 ± 18.20
4.42 ± 0.22 4.20 ± 0.229 4.31 ± 0.21 52.7 ± 11.36 78.5 ± 10.89 373.6 ± 24.82 295.1 ± 22.13 84.5* ± 17.36
4.42 ± 0.33 4.25 ± 0.257 4.34 ± 0.27 45.5 ± 15.07 72.5 ± 16.54 369.2 ± 24.52 296.6 ± 14.98 85.9 ± 17.50
4.38 ± 0.32 4.26 ± 0.336 4.31 ± 0.28 52.5 ± 16.49 75.2 ± 19.49 359.7 ± 31.97* 284.5 ± 21.11* 73.2 ± 15.26*
Postimplantation loss = No. implantations − No. viable fetuses/No. implantations × 100. Preimplantation loss; No. corpora lutea − No. implantations/No. corpora lutea × 100. Adjusted final body weight = final body weight (g) minus uterus weight (g). Adjusted body weight change = final body weight (g) minus uterus weight (g) minus Gestation Day 0 body weight (g). a Animal died due to bleeding complications on GD 20. * Significantly different from control (p ≤ 0.05).
Fig. 1. Mean (±SD) serum CBLB502 concentrations in female rats with subcutaneous injections of 300 g/kg/day (semilog coordinates).
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Table 5 Summary of individual fetal skeletal observations. Study interval (Day)
Dose level 0 g/kg/day Mean ± SD
30 g/kg/day Mean ± SD
100 g/kg/day Mean ± SD
300 g/kg/day Mean ± SD
Neural arch(es), additional ossification center 2 (9.1) No. litters (%) 3 (2.3) No. fetuses (%)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
2 (8.3) 3 (2.3)
Neural arch(es), incompletely ossified No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
1 (4.2) 1 (0.8)
Scapula, bent No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
1 (4.2) 1 (0.8)
Ischium, incompletely ossified No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
2 (8.7) 2 (1.4)
1 (4.2) 1 (0.8)
2 (8.3) 3 (2.3)
Rib(s), bent No. litters (%) No. fetuses (%)
5 (22.7) 6 (4.7)
5 (21.7) 10 (7.2)
9 (37.5) 16 (12.2)
7 (29.2) 13 (9.9)
16 (72.7) 44 (34.1)
20 (87.0) 61 (43.9)
21 (87.5) 54 (41.2)
15 (62.5) 34 (26.0)
Rib(s), rudimentary No. litters (%) No. fetuses (%) Rib(s), unilateral full rib No. litters (%) No. fetuses (%)
2 (9.1) 2 (1.6)
5 (21.7) 5 (3.6)
2 (8.3) 2 (1.5)
0 (0.0) 0 (0.0)
Frontal bone, incompletely ossified No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
1 (4.3) 2 (1.4)
1 (4.2) 1 (0.8)
1 (4.2) 1 (0.8)
Hyoid, not ossified No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
3 (13.0) 4 (2.9)
7 (29.2) 11 (8.4)
5 (20.8) 11 (8.4)
Interparietal bone: incompletely ossified No. litters (%) No. fetuses (%)
4 (18.2) 6 (4.7)
2 (8.7) 2 (1.4)
5 (20.8) 6 (4.6)
6 (25.0) 10 (7.6)
Jugal (Zygoma), fused No. litters (%) No. fetuses (%)
0 (0.0) 0 (0.0)
1 (4.3) 1 (0.7)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
Jugal (Zygoma), incompletely ossified No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
6 (26.1) 8 (5.8)
5 (20.8) 5 (3.8)
6 (25.0) 8 (6.1)
1 (4.3) 1 (0.7)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
9 (40.9) 14 (10.9)
11 (47.8) 24 (17.3)
13 (54.2) 34 (26.0)
16 (66.7) 46 (35.1)
6 (27.3) 8 (6.2)
6 (26.1) 11 (7.9)
11 (45.8) 23 (17.6)
13 (54.2) 37 (28.2)
Supra occipital bone, incompletely ossified 9 (40.9) No. litters (%) 14 (10.9) No. fetuses (%)
8 (34.8) 12 (8.6)
13 (54.2) 22 (16.8)
15 (62.5) 35 (26.7)
Sternebra(e), misaligned No. litters (%) No. fetuses (%)
6 (27.3) 7 (5.4)
2 (8.7) 2 (1.4)
1 (4.2) 1 (0.8)
2 (8.3) 2 (1.5)
Sternebra(e), not ossified No. litters (%) No. fetuses (%)1
7 (31.8) 10 (7.8)
Parietal bone, additional ossification center No. litters (%) 0 (0.0) 0 (0.0) No. fetuses (%) Parietal bone, incompletely ossified No. litters (%) No. fetuses (%) Squamosal, incompletely ossified No. litters (%) No. fetuses (%)
Sternebra(e), sternoschisis No. litters (%) No. fetuses (%)
1 (4.5) 1 (0.8)
4 (17.4) 4 (2.9)
4 (16.7) 4 (3.1)
8 (33.3) 9 (6.9)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0)
C.P. Chow, A.S. Faqi / Reproductive Toxicology 46 (2014) 12–19
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Table 6 Toxicokinetic parameters of CBLB502 in pregnant rats following subcutaneous injections. Parameter
Unit
Dose levels 30 g/kg/day
Dose Cmax Cmax /dose Tmax AUCt AUCt /dose AUC∞ z T1/2 CL/F Vz /F
g/kg/day ng/mL (ng/mL)/(g/kg) h ng/h/mL (ng/h/mL)(g/kg) ng/h/mL 1/h h L/h/kg L/kg
100 g/kg/day
300 g/kg/day
GD 6
GD 17
GD 6
GD 17
GD 6
GD 17
30 3.79 0.126 1 13.16 0.439 NE NE NE 2.28 NE
30 1.14 0.038 1 3.89 0.13 NE NE NE 7.72 NE
100 NE NE NE NE NE NE NE NE NE NE
83 NE NE NE NE NE NE NE NE NE NE
300 98.22 0.327 1 187.24 0.624 190.78 0.257 2.69 1.57 6.11
259 10.82 0.0417 1 38.66 0.149 39.89 0.298 2.32 6.5 21.8
NE = not evaluated. Table 7 Summary of immunogenicity results. Group number
Screening result
Immunodepletion result
Neutralizing antibody % inhibition (cutpoint 14.68%)
Neutralizing antibody result
1
2 PP 23 Negative 19 PP 2 IN 4 Negative 24 PP 1 Negative 22 PP 3 Negative
2 Positive
30.46%
1 Positive
16 Positive
15.43–85.25%
17 Positive
20 Positive
18.5–67.98%
16 Positive
20 Positive
22.28–69.64%
17 Positive
2
3 4
PP = presumed positive; IN = inconclusive. CBLB502 is a derivative of a microbial protein that binds to Toll-like receptor 5. It is demonstrated to reduce inflammation from acute stresses, such as radiation in animal models. We determined the potential developmental toxicity of CBLB502 in rats. Four groups of 25 time-mated female Wistar rats/group received subcutaneously 0, 30, 100, or 300 g/kg/day of CBLB502 from Gestation Days (GD) 6–17 at a dose volume of 1.0 mL/kg. Toxicokinetic evaluation was performed on GD 6 and 17. On GD 20 C-section was performed for uterine evaluation and blood samples collected from each dam for immunogenicity assay. Significant decrease in gestation body weight, weight changes and food consumption indicative of maternal toxicity were observed in all dose groups. Also adjusted body weight and weight changes were seen at 300 g/kg/day. No external, visceral and skeletal abnormalities were observed. The NOAEL for developmental toxicity was estimated to be ≥300 g/kg/day.
following the 1-hour sample such that they were below the LLOQ by 24 hours post-dose after even the highest dose (300 g/kg/day) on both GD 6 and GD 17. The Cmax and AUCt increased at a slightly greater than dose proportional manner on GD 6, but their increases were approximately dose proportional on GD 17. The terminal elimination half-life was approximately 2.5 h after the high dose (the only dose for which it was calculable) on both observation days. Apparent clearance of CBLB502 on GD 6 ranged from 1.6 to 2.3 L/h/kg, with a trend to decrease with increasing dose; on GD 17 the mean apparent clearance was 3–4 times the clearance on GD 6 (6.5–7.7 L/hr/kg), also with a trend to decrease with increasing dose. The systemic exposures to CBLB502 on GD 17 in these rats were 1/4 to 1/3 the peak and total exposures observed following the first dose on GD 6, indicating that CBLB502 clearance from the rat serum was substantially increased with daily repeated dosing. 3.4. Anti-CBLB502 antibody analysis The presence of anti-CBLB502 neutralizing antibodies in Wister rat serum was assessed. A total of 100 samples (25 samples/group, all collected on GD 20) were screened for the presence of antiCBLB502 antibodies in Wistar rat serum. Of these, 67 samples (2 from control, 19 at 30 g/kg/day, 24 at 100 g/kg/day, and 22 at 300 g/kg/day) yielded optical densities (OD) above their corresponding plate-specific cutpoint and were presumed positive for anti-CBLB502 antibodies. Nine of the presumptive positive samples were deemed QNS (Quantity Not Sufficient) for confirmatory immune-depletion analysis and titering, and were reserved for
testing for the presence anti-CBLB502 neutralizing antibodies. The remaining 58 presumptive positive samples were subjected to confirmatory immune-depletion analysis, and all the 58 samples were confirmed positive for the presence of anti-CBLB502 antibodies. All of these samples plus the 9 presumptive positive samples were then subjected for titration assay. In all, 51 samples were positive for anti-CBLB502 neutralizing antibodies: (control: 1; 30 g/kg/day: 17; 100 g/kg/day: 16, and 300 g/kg/day: 17 – see Table 7). For an unknown reason, one control animal was found to be positive for neutralizing antibodies. Overall, the frequency of neutralizing antibody-positive animals did not appear to be dose dependent. 4. Discussion and conclusions The objective of this study was to determine the developmental toxicity including of the potential teratogenicity of ‘CBLB502 in rats. CBLB502 is a derivative of a microbial protein that reduces inflammatory response from acute stresses by mobilizing several natural cell protecting mechanisms, including inhibition of programmed cell death (apoptosis), reduction of oxidative damage and induction of regeneration promoting cytokines [1]. It is currently being developed by Cleveland BioLabs under the FDA’s Animal Efficacy Rule to treat ARS or radiation poisoning from any exposure to radiation such as a nuclear or radiological weapon/dirty bomb, or from a nuclear accident. No mortality or CBLB502-related clinical signs of toxicity were observed at a dose level up to 300 g/kg/day. Significantly decreased gestation body weight, gestation body weight changes
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and gestation food consumption were observed at 30, 100 or 300 g/kg/day dose when compared to the controls. Gestation body weight and gestation body weight changes are viewed as indicators of maternal toxicity for rodent species. Indeed body weight changes may provide more information than a daily body weight measured during treatment or during gestation as changes in weight gain during treatment could occur that would not be reflected in the total weight change throughout gestation, because of compensatory weight gain that may occur following post-treatment [11]. Additional endpoints affected include adjusted body weight and adjusted body weight changes that were significantly reduced at 300 g/kg/day. Effects on adjusted body weight and weight gain with no effect in gravid uterine weight suggest maternal toxicity. The current practice for the assessment of embryo-fetal developmental toxicity (EFD) necessitates administration of high, maternally toxic doses to pregnant laboratory animals [12–14] and avoid marked maternal toxicity leading to mortality or decreased body weight gains of greater than 20% for prolonged periods [15]. In EFD studies it is very common to see that maternal effects are accompanied by developmental toxicity. However, in the present study no fetal external, visceral and skeletal malformations were observed. In addition uterine data including implantation sites, preand post-implantation loss, viable/nonviable fetuses and resorptions were not adversely affected. Likewise, fetal body weights were comparable among the groups; although adjusted final body weight and adjusted weight changes were significantly reduced at 300 mg/kg/day. Slightly increased, but statistically not significant incidence of incomplete ossification of parietal, squamosal bones, were observed at 100 and 300 g/kg/day. Delayed ossification may pose some challenges in the interpretation because these abnormalities occur at a relatively high background incidence in control animals and are observed frequently in a dose-related manner [16]. Incomplete/delay in ossification is often related to maternal toxicity [17]. Carney and Kimmel (2007) [17] proposed a weight-of-evidence approach for the interpretation of delayed ossifications and other variations. They have classified the findings into three different categories, namely insignificant findings, low significance findings and findings that warrant increased attention. Findings that warrant increased attention include patterns of ossification that do not follow normal sequence, specific delays involving bones that normally are well ossified in term fetuses (e.g., ribs, clavicle, long bones of the limbs, lumbar vertebral centra), abnormally-shaped cartilage template, significantly increased delayed ossification without decreases in fetal body weight, delayed ossification in the absence of maternal toxicity, and delayed ossification associated with teratogenesis. The delayed ossifications observed in our study do not fall into the category of the findings that warrant increased attention and nevertheless such minor variations would not generally be considered adverse in and of themselves but should be interpreted in the context of other maternal and fetal finding. In our study, there were no fetal findings and the maternal toxicity was limited to reduced body weight, body weight changes and food consumptions as well as adjusted final body weight and weight changes. We therefore, conclude that these findings were not CBLB502 treatment-related because of the lack of statistical significance. We have also examined the presence of anti-CBLB502 neutralizing antibodies in Wister rat serum. It is common for the animals to develop anti-drug antibodies (ADA) following administration of foreign proteins (such as CBLB502). This may lead to increased clearance of the biopharmaceuticals, yielding exposure reduction and over estimating safety [18]. The immunogenicity assay conducted revealed that not all animals had anti-CBLB502 neutralizing antibodies. The ADA was not detected in all the animals and the frequency of neutralizing antibody-positive animals did not
manifest in a dose-dependent manner. However, anti-CBLB502 neutralizing antibodies may have induced some impact to the toxicokinetic behavior of CBLB502. The Cmax and AUCt increased at a slightly greater than dose proportional manner on GD 6, but their increases were approximately dose proportional on GD 17. Apparent clearance of CBLB502 on GD 6 ranged from 1.6 to 2.3 L/h/kg, with a trend to decrease with increasing dose; on GD 17 the mean apparent clearance was 3–4 times the clearance on GD 6 (6.5–7.7 L/h/kg), also with a trend to decrease with increasing dose. Having said that we can conclude that anti-CBLB502 neutralizing antibodies had little or no impact and/or does not disqualify the overall conduct of the study as the doses of CBLB502 used were able to elicit and maintain persistence maternal effects throughout the study. For example the effect on maternal body weight at 300 g/kg/day emerged on GD 9 and continued throughout the study. Likewise, the effects on body weight changes and food consumption at the high dose followed similar trend. Nevertheless, there are some unknown variables including how many dosing days was necessary to elicit anti-CBLB502 neutralizing antibody as the immunogenicity assay was conducted only on GD 20, three days after completion of treatment. But, the toxicokinetic data of GD 17 at 300 g/kg/day and the persistence maternal effects observed in this study may suggest that the anti-CBLB502 neutralizing antibodies may have been induced late during the treatment. Based on the results of this study, the NOAEL (No-ObservedAdverse-Effect-Level) for developmental toxicity was estimated to be to be ≥300 g/kg/day after administration to Wistar rats for 12 days. Since the high dose did not cause developmental effects; therefore, a dose greater or equal than the high dose is estimated to be the NOAEL. It is unknown exactly what dose, but for sure is greater or equal than the high dose. Preclinical safety results available to date support the continued development of CBLB502 in human. Conflict of interest Paul Chow works for Cleveland Biolabs that is developing CBLB502. Ali S. Faqi has no conflict of interest. Transparency document The Transparency document associated to this article can be found, in the online version. References [1] Burdelya LG, Krivokrysenko V, Tallant TC, Strom E, Gleibeman AS, Gupta D, et al. An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models. Science 2008;320(5873):226–30. [2] Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet 2001;357:539–45. [3] Akira S. Toll-like receptor signaling. J Biol Chem 2003;278(40):38105–8. [4] Means TK, Hayashi F, Smith KD, Aderem A, Luster AD. The toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J Immunol 2003;170(10):5165–75. [5] Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004;16:3–9. [6] Moynagh PN. TLR signalling and activation of IRFs: revisiting old friends from the NF-kappaB pathway. Trends Immunol 2005;26:469–76. [7] Yamamoto M, Akira S. TIR domain-containing adaptors regulate TLR signaling pathways. Adv Exp Med Biol 2005;560:1–9. [8] AAALAC. American Association for Laboratory Animal Science policy on the humane care and use of laboratory animals. Lab Anim Sci 1991;41:91. [9] Wilson JG. Methods for administering agents and detecting malformations in experimental animals. In: Teratology, Principles and Techniques. Chicago: University of Chicago Press; 1965. p. 62. [10] Staples RE. Detection of visceral abnormalities in mammalian fetuses. Teratology 1974;9(3):A37–8. [11] Kimmel CA, Price CJ. Developmental toxicity studies. In: Arnold DL, Grice HC, Krewski DR, editors. Handbook of In Vivo Toxicity Testing. San Diego, CA: Academic Press; 1990. p. 271–301.
C.P. Chow, A.S. Faqi / Reproductive Toxicology 46 (2014) 12–19 [12] Rogers JM, Chernoff N, Keen CL, Daston GP. Evaluation and interpretation of maternal toxicity in Segment II studies: issues, some answers, and data needs. Toxicol Appl Pharmacol 2005;207(2 Suppl.):367–74. [13] Carney E. Maternally mediated developmental toxicity. In: Charlene A, McQueen, editors. Comprehensive Toxicology, vol. 12. Oxford: Academic Press; 2010. p. 163–76. [14] Faqi AS. Methods for detection of developmental toxicity. In: Charlene A, McQueen, editors. Comprehensive Toxicology, vol. 12. Oxford: Academic Press; 2010. p. 259–78. [15] Beyer BK, Chernoff N, Danielsson BR, Davis-Bruno K, Harrouk W, Hood RD, et al. ILSI/HESI maternal toxicity workshop summary: maternal toxicity and its
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impact on study design and data interpretation. Birth Defects Res B Dev Reprod Toxicol 2011;92(1):36–51. [16] Daston GP, Seed J. Skeletal malformations and variations in developmental toxicity studies: interspecies issues for human risk assessment. Birth Defects Res B Dev Reprod Toxicol 2007;80(6):421–4. [17] Carney EW, Kimmel CA. Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res B Dev Reprod Toxicol 2007;80(6):473–96. [18] Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin-Dupont P, et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 2002;346:469–75.
