Reproductive Toxicology 52 (2015) 101–107
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Absence of developmental toxicity in a canine model after infusion of a hemoglobin-based oxygen carrier: Implications for risk assessment J.F. Holson a , D.G. Stump b , L.B. Pearce c , R.E. Watson d , J.M. DeSesso e,f,∗ a
Ashland, OH, United States WIL Research Laboratories, Ashland, OH, United States Biologics Consulting Group, Alexandria, VA, United States d SNBL USA, Everett, WA, United States e Exponent, Inc., Alexandria, VA, United States f Georgetown University School of Medicine, Washington, DC, United States b c
a r t i c l e
i n f o
Article history: Received 29 May 2014 Received in revised form 24 December 2014 Accepted 20 January 2015 Available online 16 February 2015 Keywords: Hemoglobin-based oxygen carriers Inverted yolk sac Dog
a b s t r a c t Bovine-derived hemoglobin-based oxygen carriers (HBOCs) have been investigated for use in humans (HBOC-201) and approved for veterinary medicine (HBOC-301). We infused pregnant beagles with HBOC201 to test whether HBOC-induced developmental toxicity previously observed in rats would occur in a species devoid of an inverted visceral yolk sac (invVYS). Phase 1 assessed developmental toxicity of 6 g/kg HBOC-201 on gestational day (GD) 21. Phase 2 investigated single infusions of 6 g/kg HBOC-201 on one of GDs 21, 25, 29 or 33. Phase 3 studied multiple sequential infusions on GDs 21, 23,25,27,29, 31, and 33 at 0.52 g/kg/day (3.6 g/kg total dose). Mild to moderate maternal toxicity occurred in all phases. There was an unequivocal absence of developmental toxicity in all phases. Overall, our hypothesis that HBOC, which interferes with the function of the invVYS, would not affect the offspring in dogs was supported. The implications relative to human risk are discussed. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Hemoglobin-based oxygen carriers (HBOCs) are under development to expand blood volume and deliver oxygen in cases where there has been a precipitous loss of blood and compatible blood is not available for transfusion. HBOC-201 is produced from tetrameric purified bovine hemoglobin (PBH) molecules that have been bound together in a 250 kDa glutaraldehyde-stabilized polymer. Safety assessment studies in pregnant rats infused intravenously with HBOC-201 resulted in significant developmental toxicity, as demonstrated by a decrease in live litter size and an increase in external fetal malformations [1]. Subsequent investigatory studies that controlled for the colloidal properties, osmolarity, protein content, and hemoglobin content of HBOC-201 have shown that the developmental toxicity observed is directly related to the hemoglobin content of HBOC-201 [1]. The appearance of these effects correlated with infusion of HBOC-201 only during gestational days (GDs) 7–9, a period during rat organogenesis when the inverted visceral yolk sac (invVYS) supplies the rat embryo with its
∗ Corresponding author at: Exponent, Inc., 1800 Diagonal Road, Suite 500, Alexandria, VA 22314, United States. Tel.: +1 571 227 7261; fax: +1 571 227 7299. E-mail address: [email protected] (J.M. DeSesso). http://dx.doi.org/10.1016/j.reprotox.2015.01.006 0890-6238/© 2015 Elsevier Inc. All rights reserved.
primary source of nutrition through a histiotrophic process. This period precedes the establishment of the chorioallantoic placental circulation, which starts on GD 11.5 [2]. In rat whole embryo culture experiments, it has been demonstrated that HBOC inhibits the transport function of the inVYS [1]. The developmental toxicity observed in the rat may not accurately predict human developmental toxicity because the human yolk sac does not function as a placenta. Consequently, to more fully understand whether HBOCs might cause potential developmental toxicity in humans, the use of an alternative animal model that does not rely on an invVYS during organogenesis was investigated. The dog is an attractive animal model because, as in humans, the canine yolk sac membrane does not invert nor does it serve as a primary nutritive organ during organogenesis. Additional advantages of the canine model include previous knowledge of critical periods of organogenesis and approval of a similar hemoglobin-based oxygen carrying therapeutic agent (HBOC-301) for veterinary use in the dog. Non-human primates were not deemed to be a more predictive model because the stress of compound delivery juxtaposed with superficial implantation predisposes non-human primates to early abortion [3], which might confound interpretation. The aim of these studies was to assess potential developmental toxicity related to infusion of pregnant dogs with HBOC-201 during organogenesis. A dose of 6 g/kg HBOC-201 was an estimate of a dose
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that would exceed the approximate clinically relevant single dose in the human without causing significant maternal toxicity that could complicate analysis of developmental toxicity. A preliminary experiment was performed to verify that 6 g/kg HBOC-201 is an appropriate dose in the dog. Based on the success of the preliminary experiment at that dose level, two larger scale definitive studies were performed. In the first definitive study, single-day infusions of 6 g/kg HBOC-201 were given to groups of pregnant dogs on one of GDs 21, 25, 29, or 33. In the second definitive study, sequential infusions in the same dogs were administered on seven occasions throughout organogenesis (GDs 21, 23, 25, 27, 29, 31, and 33) for a total dose of 3.6 g/kg. This dose mimics the repeat administration proposed for the human clinical setting.
2. Materials and methods 2.1. Animals Time-mated multigravid female beagle dogs were obtained from Covance Research Products, Inc. (Cumberland, VA). Dogs had been mated naturally at Covance on two separate days; the second breeding was conducted 2–6 days following the first breeding. The first day on which mating was observed was designated as GD 0. The dogs were approximately 2–5 years old and ranged from GD 7 to 10 on the day of receipt. Each dog was allowed 8 days to acclimate to the laboratory environment, and during this period animals were observed twice daily for mortality and changes in general appearance and behavior. Animals were fed PMI Nutrition International, Inc. (Richmond, IN) Certified Canine LabDiet® 5007 ad libitum throughout acclimation and the study. Drinking water was delivered by an automatic watering system and was provided ad libitum throughout the acclimation period and during the study. Dogs were housed individually in whelping pens that were controlled for temperature and light/dark conditions in accordance with the “Guide for the Care and Use of Laboratory Animals” [4]. At the conclusion of the acclimation period, the animals were assigned to groups using a computerized randomization procedure. The protocols for these studies were submitted to and approved by the WIL Research Institutional Animal Care and Use Committee. 2.2. Test articles The infusate vehicle consisted of a 0.9% sodium chloride solution (saline; Baxter Healthcare Corporation; Deerfield, IL) stored at room temperature. Solutions of HBOC-201 (13 g/dL) in vehicle were prepared by Biopure® (Cambridge, MA). HBOC-201 was kept at room temperature and was considered stable under this condition. To control for the concentrated protein nature of HBOC-201, a 13% solution of human serum albumin (HSA; equivalent protein concentration) was used. 2.3. Surgical placement of jugular vein catheters All animals were acclimated to wearing jackets and collars prior to surgery. Surgeries for the implantation of indwelling jugular vein catheters were performed on or before GD 18. The dogs were anesthetized by isoflurane inhalation prior to catheter implantation. Each implanted catheter was attached to a validated CADD-Legacy® pump (Sims Deltec, St. Paul, MN) with a remote reservoir and an extension set placed in the vest pouch closest to the vein to be catheterized. Following surgery, collars protecting the catheters were placed on the animals. Reservoirs were replaced, as necessary, throughout the acclimation period. To maintain the patency of access, saline was administered at an
infusion rate of approximately 0.5 mL/h following surgery until test article infusion was initiated on GD 21. 2.4. Infusion of test article A preliminary study established that the infusion procedures did not result in significant maternal toxicity. Dogs were infused with 6 g/kg HBOC-201 (N = 4) or saline (N = 5) at a rate of 0.1 mL/kg/min for 8 h on GD 21. These dogs tolerated the HBOC-201 well, and thus, this dose and infusion procedure was deemed appropriate for use in larger scale studies. Single-day exposures to groups of dogs (N = 20) were performed at 6 g/kg HBOC-201 on GDs 21, 25, 29, or 33. These dates were selected to provide significant plasma levels of HBOC-201 at intervals that collectively spanned the period of major organogenesis. This dosing regimen was based on the long plasma half-life of HBOC in dogs at this dose1 (∼55 h) and when all four single-day regimens are combined results in exposure over the period of major organogenesis. The 13% HSA control (46 mL/kg) was administered on GD 21. The saline control group was infused on all four days that HBOC-201 was administered. Twenty pregnant dogs were assigned to each of the six groups used in this study. All infusions were delivered at a rate of 0.1 mL/kg/min for an 8-h period. In the sequential infusion study, repeat administration of HBOC201 or saline (control) was performed on groups of 20 pregnant dogs over a 13-day period during organogenesis. On GDs 21, 23, 25, 27, 29, 31 and 33, saline or HBOC-201 was infused at a flow rate of 0.1 mL/kg/min for approximately 40 min resulting in a total infusate volume of 4 mL/kg/day. Each infusion delivered 0.52 g HBOC-201/kg resulting in a total dose of 3.6 g/kg. 2.5. Maternal observations during gestation and lactation Dogs were observed twice daily for moribundity and mortality. Detailed clinical observations were recorded daily for each female. Observations for signs of toxicity included, but were not limited to, evaluations for changes in the skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems, somatomotor activity and behavior. On the days of treatment, animals were observed for signs of toxicity approximately 1 h following completion of the 8 h infusion. All significant clinical findings were recorded at this observation period. On GD 30 ± 5, each dog was palpated by the attending veterinarian in an attempt to determine pregnancy status. Maternal body weights were recorded on several occasions throughout gestation and mean body weight changes were calculated for GD 21–60. Dogs that were non-gravid and control dogs which aborted were not included in the statistical analysis of maternal parameters. 2.6. Plasma hemoglobin assessment In order to monitor the elimination of HBOC-201 from the plasma, assessments for plasma hemoglobin were performed on the first six dogs infused with HBOC-201 on GD 33 prior to infusion, immediately following infusion and 1, 4, 16, 24, 36, 48, 60, 72, 84, 96, 144 and 192 h after the end of infusion. EDTA was used as the anticoagulant and the analyses were conducted using the HemoCue® B-Hemoglobin System (HemoCue AB, Angelholm, Sweden). In the sequential infusion study, assessments for plasma hemoglobin were performed on the first six dogs infused with
1 The plasma half-life of HBOC-201 is dose-dependent and ranged from ∼22 h at low doses to ∼55 h.
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HBOC-201 immediately prior to infusion and 30 min following infusion on GDs 21, 23, 25, 27, 29 and 31 and GD 33 prior to infusion, 30 min, 1, 4, 8, 12, 16, 20, 26, 32, 38, 46, 56 and 72 h after the end of infusion.
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puppy body weight parameters. The Kruskal–Wallis test [9] with the Mann–Whitney U test [9] was performed for non-parametric parameters including puppy sexes at birth, postnatal survival, litter proportions of puppy malformations and developmental variations.
2.7. Whelping Females were allowed to deliver naturally. When necessary, 0.2–0.5 mL of oxytocin was administered intramuscularly to aid uterine contraction. For each litter, the numbers of stillborn and live puppies as well as their sex were recorded. The duration of gestation was calculated using the interval between the date on which whelping was initiated and the date of first mating provided by the animal supplier. Animals that failed to deliver were considered to be nongravid and were removed from the study. The number of nongravid animals was small and their occurrence did not appear to be related to HBOC-201 administration. 2.8. Offspring morphological examination Puppies were sexed on postnatal day (PND) 0. On PND 1 they were weighed and euthanized by an intraperitoneal injection of sodium pentobarbital. Each puppy (whether viable or nonviable) received a detailed external examination that included, but was not limited to, an examination of the eyes, palate and external orifices. All findings were recorded uniquely for each puppy. Radiographs were taken of the dorsal to ventral, ventral to dorsal, right lateral and left lateral positions. Each puppy then was examined viscerally by a modified Stuckhardt and Poppe [5] fresh dissection technique. The examination included the heart and major vessels as well as confirmation of gender. The heads were inspected by a mid-coronal slice. All carcasses were then eviscerated, fixed in 100% ethyl alcohol, macerated in potassium hydroxide, and stained with Alizarin Red S by a method similar to that described by Dawson [6]. Skeletal examinations were performed through a combination of reviewing the radiographs and evaluating the stained skeletal preparations. External, visceral and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effects on animal health or body conformity, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function or may be incompatible with life). The percentage of pups with malformations, the mean percentage of malformed pups/litter, and the percentage of pups surviving to PND 1 were calculated. 2.9. Statistical analyses All analyses were performed on the litter as the experimental unit, using two-tailed tests for minimum significance levels of 5% and 1% that compared the test article-treated group to the control group. A one-way ANOVA [7] with Dunnett’s post hoc test [8] was performed with parametric parameters: maternal body weight changes, number of puppies born, live litter size, and
3. Results Table 1 presents the data from the preliminary study. Compared to saline controls, infusion of 6 g/kg HBOC-201 at 46 mL/kg did not lead to significant changes in live litter size, male or female pup weight, survival from birth to PND 1, or the incidence of external malformations. A numerical decrease in maternal weight gain during gestation (not statistically significant, likely due to the large standard deviation) and overt clinical signs of maternal toxicity, primarily yellow sclerae were associated with HBOC-201 infusion. Excreta-related effects (e.g., soft stool, decreased defecation, mucoid feces and/or diarrhea) were observed at a similar incidence in the saline control and 6 g/kg HBOC-201 group. Because the 6 g/kg dose elicited clinical signs without severe effects that might induce developmental toxicity secondary to maternal toxicity, this dose was deemed an appropriate amount to administer for daily infusions in the definitive studies. Maternal and developmental toxicity assessments were next performed on pregnant dogs given a single-day infusion of 6 g/kg HBOC-201 on GDs 21, 25, 29, or 33 or hetastarch on GD 21 (Table 2). Numerical decreases in maternal weight gain (not statistically significant, likely due to the large standard deviation) and overt signs of maternal toxicity, primarily yellow sclerae, were observed in dogs infused with 6 g/kg HBOC-201 on each GD examined. Excretarelated effects were noted in the dogs infused with HBOC-201 and saline on each GD examined as well as for pregnant dogs infused with 13% HSA on GD 21. However, infusion with neither HSA nor HBOC-201 at any dose level led to any significant changes in litter size, survival rate from birth to PND 1, male or female pup weight, or the rate of external, visceral or skeletal malformations when the results are compared to those of the saline and HSA controls. Table 3 summarizes the results of repeated infusions of 0.52 g/kg/day HBOC-201 or saline into pregnant dogs every other day during GDs 21–33. Infusion of HBOC-201 led to decreases in maternal weight gain during gestation and overt signs of maternal toxicity, primarily yellow sclera; however, no signs of developmental toxicity were detected in offspring. Taken together, the results of these robust studies found no indications of developmental toxicity in pups after maternal infusions of HBOC-201 at doses that caused clinical signs of toxicity in pregnant dogs. The mean plasma hemoglobin concentration immediately following infusion of 6 g/kg of HBOC-201 on GD 33 was approximately 6.0 g/dL demonstrating that marked plasma concentrations of HBOC-201 were achieved (Table 4). The mean plasma hemoglobin concentration 72 h following infusion (2.6 g/dL) was slightly less than one-half the concentration immediately following infusion,
Table 1 Developmental toxicity results after infusion of HBOC-201 into pregnant dogs on gestational day 21. Parameter
Saline (46 mL/kg)
HBOC-201 (6.0 g/kg = 46 mL/kg)
Number of pregnant dogs Maternal weight gain from GDs 21 to 28 (g ± S.D.) Clinical findings Live litter size ± S.D. Postnatal survival birth to PND 1 (% per litter ± S.D.) Mean weight for males (g ± S.D.) Mean weight for females (g ± S.D.) External malformations (no. affected/no. examined)
5 194 ± 1158 Excreta-related effects 6.2 ± 2.4 86.7 ± 29.8 297.3 ± 22.4 285.9 ± 24.1 0/29
4 −831 ± 115 Excreta-related effects 3.8 ± 3.2 100 ± 0.0 326.9 ± 76.4 308.6 ± 83.5 0/15
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Table 2 Maternal and developmental toxicity findings after single-day infusions of HBOC-201 on gestation days 21, 25, 29, and 33. Parameter
Saline (46 mL/kg) GD 25
13% HSA (6 g/kg = 46 mL/kg) GD 21
Number of pregnant dogs examined Clinical findings
18 Excreta-related effectsa
16 Excreta-related effects
Mean maternal body weight gain GDs 21–34 (g ± S.D.) Mean number of live fetuses/litter Postnatal survival from birth to PND 1 (% per litter) Mean pup weight males (g ± S.D.) Mean pup weight females (g ± S.D.) Number of pups (litters) examined Pups (litters) with external Malformations Cleft palate Bent tail w/vertebral anomaly Pups (litters) with visceral malformations Hydrocephaly Kidney and ureter absent Pups (litters) with skeletal malformations Rib anomalies Total malformations (mean % per litter ± S.D.)
−4 ± 222
a
HBOC-201 (6 g/kg = 46 mL/kg) GD 21
GD 25
GD 29
GD 33
−95 ± 178
19 Excreta-related effects, yellow sclera −334 ± 157
18 Excreta-related effects, yellow sclera −296 ± 204
18 Excreta-related effects, yellow sclera −442 ± 201
17 Excreta-related effects, yellow sclera −118 ± 224
5.4 ± 2.8 84.3 ± 32.8
4.9 ± 2.1 80.3 ± 27.06
4.9 ± 2.0 80.3 ± 27.1
6.1 ± 1.6 80.4 ± 28.2
5.4 ± 2.3 95.9 ± 8.2
4.7 ± 2.2 84.7 ± 21.3
260.9 ± 61.3 244.9 ± 52.3 92 (16) 4 (2)
249.1 ± 58.6 264.1 ± 42.7 75 (16) 0 (0)
272.7 ± 50.6 261.7 ± 43.4 88 (18) 0 (0)
267.1 ± 43.1 259.9 ± 32.9 100 (17) 3 (3)
254.9 ± 52.4 229.2 ± 42.0 97 (18) 0 (0)
265.5 ± 54.6 257.5 ± 52.6 76 (17) 0 (0)
3 (2) 1(1) 2 (2)
0 (0) 0 (0) 2 (2)
0 (0) 0 (0) 2 (2)
1 (1) 2 (2) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
2 (2) 0 (0) 0 (0)
1 (1) 2 (2) 1 (1)
2 (2) 1 (1) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 5.7 ± 13.9
1 (1) 5.3 ± 13.3
0 (0) 2.1 ± 6.4
0 (0) 4.5 ± 12.5
0 (0) 0.0 ± 0.0
0 (0) 0.0 ± 0.0
Soft stool, decreased defecation, mucoid feces and/or diarrhea.
Table 3 Maternal and development toxicity results after multiple infusions of HBOC-201 during gestational days 21–33. Parameter
Saline (4 mL/kg/day)
HBOC-201 (0.52 g/kg/day = 4 mL/kg/day)
Number of pregnant dogs examined Clinical findings Mean maternal body weight gain GDs 21–34 (g ± S.D.) Mean live litter number Postnatal survival birth PND 1 (% per litter) Mean pup weight males (g ± S.D.) Mean pup weight females (g ± S.D.) Number of pups (litters) examined Pups (litters) with external malformations Pups (litters) with visceral malformations Pups (litters) with skeletal malformations Rib anomalies Total malformations (mean % per litter ± S.D.)
19 Excreta-related effects 22 ± 586 5.2 ± 1.8 93.0 ± 12.7 293.2 ± 47.6 281.7 ± 42.6 91 (18) 0 (0) 0 (0) 1 (1) 1 (1) 0.9 ± 3.9
17 Excreta-related effects, yellow sclera −522 ± 484 5.5 ± 1.5 92.4 ± 11.8 278.1 ± 39.6 264.6 ± 60.0 91 (17) 0 (0) 0 (0) 0 (0) 0 (0) 0.0 + 0.0
and measurable plasma hemoglobin concentrations were still present 144 h following infusion. These results demonstrate that significant exposure to HBOC-201 occurred for 6 days following infusion.
Table 4 Plasma hemoglobin concentrations after a single-day infusion of 6 g/kg HBOC-201 on GD 33. Time after infusion
Plasma hemoglobin (g/dL)
Prior to infusion Immediately following infusion 1h 4h 16 h 24 h 36 h 48 h 60 h 72 h 84 h 96 h 144 h 192 h
0.0 6.0 5.9 5.8 5.2 5.0 4.3 3.8 3.2 2.6 2.0 1.6 0.4 0.1
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.33 0.38 0.38 0.33 0.37 0.34 0.34 0.58 0.37 0.50 0.44 0.17 0.08
The mean plasma hemoglobin concentrations 30 min following infusion of 0.52 g/kg of HBOC-201 on GDs 21, 23, 25,27, 29, 31 and 33 were approximately 1 g/dL demonstrating that consistent plasma concentrations of HBOC-201 were achieved and that no indication of accumulation (increased AUC) was observed following repeated administration of HBOC-201 (Table 5). The plasma hemoglobin concentration 16 h following infusion on GD 33 was approximately one-half the concentration 30 min following infusion. 4. Discussion In contrast to rats [1], no developmental toxicity was observed in puppies whose mothers were exposed during organogenesis to either single high dose exposures or repeated lower-dose exposures of HBOC-201. This supports the concept that the mode of action whereby HBOC-201 causes developmental toxicity in rats is by impeding the function of the inverted yolk sac. Although humans and dogs do have yolk sacs, they do not invert, nor do they function as a major conduit for nutrition during development. Therefore, substances that interfere solely with invYS function should not signal a concordant risk to development of canine or human embryos.
J.F. Holson et al. / Reproductive Toxicology 52 (2015) 101–107 Table 5 Plasma hemoglobin concentrations after multiple-day infusions of 0.52 g/kg HBOC201. Time after infusion
Plasma hemoglobin (g/dL)
GD 21 prior to infusion GD 21–30 min following infusion GD 23 prior to infusion GDs 23–30 min following infusion GD 25 prior to infusion GDs 25–30 min following infusion GD 27 prior to infusion GDs 27–30 min following infusion GD 29 prior to infusion GDs 29–30 min following infusion GD 31 prior to infusion GDs 31–30 min following infusion GD 33 prior to infusion GDs 33–30 min following infusion GDs 33–1 h GDs 33–4 h GDs 33–8 h GDs 33–12 h GDs 33–16 h GDs 33–20 h GDs 33–26 h GDs 33–32 h GDs 33–38 h GDs 33–46 h GDs 33–56 h GDs 33–72 h
0.0 1.0 0.1 1.1 0.1 1.1 0.4 1.2 0.2 1.1 0.2 1.0 0.1 1.0 1.0 0.9 0.7 0.6 0.5 0.4 0.3 0.3 0.2 0.1 0.1 0.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.58 0.04 0.16 0.05 0.10 0.49 0.15 0.08 0.13 0.18 0.18 0.14 0.21 0.23 0.24 0.23 0.19 0.18 0.19 0.19 0.11 0.12 0.16 0.07 0.04
HBOC-201 is a large molecule that is unlikely to cross biological membranes rapidly, or to any large extent [10,11]. If the material is not able to readily cross membranes, gain access to, or accumulate in nontrivial amounts within the embryo, it is unlikely that it could exert an embryonic site of action. Nevertheless, HBOC-201 caused developmental toxicity in rats, including malformations and decreased fetal weights, when it was infused continuously over gestational days 6–18. The malformations that occurred (mainly ocular defects and craniofacial dysmorphologies) are consistent with findings of agents that act early in gestation, while the observations of decreased fetal weights are consistent with agents that interfere with embryonal/fetal growth throughout gestation. These types of findings in rat offspring are similar to those reported for agents such as trypan blue [12] and concanavalin A [13], both molecules that do not cross into rodent embryos, but exert their influences through interference with the function of the inverted yolk sac [14,15]. When HBOC-201 was administered over 24-h periods during organogenesis, malformations occurred only when exposure occurred on gestational days 7–8 and 8–9, but not when administered at one-day intervals during gestational days 9–10; 10–11; 11–12; 12–13 [1], which is the period when the rat relies on the inverted yolk sac as its main source of nutrition. This is an important finding because these adverse events occur prior to establishment of the underpinnings of the chorioallantoic placenta which becomes functional at about gestational day 10.5–11.5 in rats [2]. The inverted yolk sac and chorioallantoic placenta differ dramatically in their modes of transport. Physiological transport via the inverted yolk sac comprises a multi-step process that includes (1) endocytosis or pinocytosis of uterine secretions from the luminal surface of the visceral yolk sac epithelium, (2) traversing of the endocytotic vacuoles into the cytoplasm to merge with lysosomes and subsequent enzymatic attack, and (3) diffusion of breakdown products across the epithelium toward embryonic vitelline blood vessels in the mesenchyme on the abluminal surface. This multistep process is termed histiotrophic nutrition, which supplies the embryo with nutrients from sources other than from the maternal blood. The histiotrophic mechanism can transfer nutrients and
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large molecular weight substances, but it is a time-consuming process that soon becomes inefficient for the needs of a rapidly growing embryo. The invVYS is soon superseded by the chorioallantoic placenta which depends on direct exchange between the separate maternal and embryonal blood circulations, termed hemotrophic nutrition. Hemotrophic nutrition enables the prompt transfer of relatively small molecules between the maternal and embryonic blood circulations; the transfer efficiency declines when the size of the molecules exceeds 600 Daltons and, typically, the largest molecules transferred by diffusion across non-fenestrated epithelia of the chorioallantoic placenta are less than 1200 Daltons [10,11]. The invVYS of the rat remains functional after the establishment of the chorioallantoic placenta, and transports nutrients and proteins into the embryo until at least GD 17 [16]. In the rapidly developing rat embryo, the yolk sac abuts the uterine wall and then collapses (inverts) such that lumen disappears and the somatic wall (the original outer yolk sac wall that touched the uterine epithelium) deteriorates, leaving behind a basal lamina (Reichert’s membrane) that interposes between the visceral epithelium of the invVYS and the wall of the maternal uterine lumen [2,17]. The visceral epithelium of the invVYS functions as the primary means for physiological exchange during the span between approximately GDs ∼8–11.5 [2,18]. During this period of development the rat embryo completes several major milestones including closure of the neural tube and establishment of the anlage for many organ systems [19]. Throughout this critical time period, transport of materials to the embryo must occur across the invVYS. Therefore, interference with this process early in gestation would be expected to cause adverse effects on embryo-fetal development. However, this mechanism of action is not applicable to the human or the dog. While disruption of the processes of endocytosis, lysosomal degradation, and subsequent transport of breakdown products across the cell could be expected to occur if HBOC-201 were presented to absorptive epithelia, the impact is of fundamental concern to developmental toxicity only when this process is the exclusive means available for transporting nutrients to the embryo. Humans and dogs have a much longer gestational period (266 days and ∼60 days, respectively) than rats (22 days). As a consequence, human and dog embryo/fetuses develop at a less hurried rate than rats, as illustrated in Table 6 summarizing the timing of gestational milestones in the rat, dog and human. Importantly, the milestones of rat embryogenesis that occur during the interval beginning when the rat is dependent on the inverted yolk sac placenta and continuing through the establishment of the rat chorioallantoic placenta are the same ones that occur in the dog during the infusion schedule used. The human and dog establish only one definitive placenta: the chorioallantoic placenta that begins functioning at the end of the fourth week of gestation. Because humans do not rely on histiotrophic nutrition as a means of obtaining nourishment, starvation of their embryos by interference with this process is irrelevant as a mode of action for developmental toxicity [20]. While the yolk sac does not contribute to definitive placenta formation in either the human or dog embryo it does play an important role in their development. It is the site of early hematopoiesis, and produces albumin, pre-albumin, alpha-fetoproteins, alphaantitrypsins, transferrin, and apolipoproteins. The yolk sac is also the site of origin of gonial cells. During early human development, the yolk sac enlarges considerably (such that it is larger than the embryo by about 22 days of gestation), but its wall never touches the chorion and, thus, it never functions as a placenta [18]. In dogs, a small area of the yolk sac wall briefly abuts the chorion from about gestational days 21 to 24, but the yolk sac does not invert [21,22]. Though the underpinnings of the chorioallantoic placenta in the human are present at the beginning of gestational week 4
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Table 6 Comparison of developmental milestones in the rat, dog and human.a Developmental milestones
Rat Age in daysb
Dog Age in daysc
Human Age in days
Fertilization Morula Blastocyst Implantation into uterine epithelium Yolk sac inverts Histiotrophic nutrition via inverted visceral yolk sac begins First somite Aortic arches I–III Anterior limb bud appears Lens placode Chorioallantoic placenta established Neural tube closure Herniation of intestines into umbilical cord Digital rays in anterior limb bud Palatal shelves begin to rotate Hard palate closure Parturition
0 3 5 5.5–6 6.5–7 7–9 9.5 10.5–11 11 11.5 11–11 .5 11.5 12.5 13.5 16.5 17 22
0 8 12–16 12–18 Does not invertd N/A 19 23 23 23 22 21–22 30 31 33 35 ∼60
0 3 4–5 7–8 Does not invert N/A 19–20 28–31 26 27 ∼28 28 36 35–36 54 ∼56 ∼266
N/A, not applicable. a Data assembled from [19,22,29,30]. b Gestational day 0 is the day of confirmed mating. c Gestational day 0 is the day of the LH surge. Yolk sac of the dog briefly abuts the chorion ∼19.5 day of gestation, but does not invert.
[18,21], some researchers believe that hemotrophic nutrition (from maternal blood circulating among the villi of the chorioallantoic placenta) is not fully functional before gestational weeks 8–10. This claim is based on Doppler ultrasound studies that are interpreted to indicate that the endometrial spiral arteries are blocked by plugs of cytotrophoblast cells prior to this time [23,24]. It has been suggested that the yolk sac plays an important role in transferring maternal nutrients from the mother to the fetus to address the nutritional needs of the fetus prior to gestational weeks 8–10 [25,26]. Transfer to the fetus of nutrients from uterine gland secretions is proposed to occur by a route that involves the pinocytotic uptake of nutrients by trophoblast, movement along the stromal channels in the chorion, diffusion into the coelomic fluid, and ultimate absorption into the yolk sac where the nutrients gain access to the fetus [25]. This process has been termed “histiotrophic” but it differs markedly from the more direct histiotrophic pathway in the rat involving the uptake of compounds through the visceral wall of the sac between gestational days 7 and 9. Research supporting the human histiotrophic pathway includes studies examining the transfer of human chorionic gonadotrophin (HCG) to the yolk sac lumen, as well as the movement of proteins secreted by the uterine glands. HCG mRNA was detected in cytotrophoblast and syncytiotrophoblast, but not in yolk sac tissue [27]. However, HCG is present in the lumen of the yolk sac, suggesting that HCG can be absorbed by the yolk sac wall [27]. This study suggests that although a high molecular weight protein (HCG at 27.5 kDa; [28]) can enter the yolk sac, the concentration of HCG in the yolk sac was 2.8 times less than that reported in the coelomic fluid [27]. Furthermore, there is no conclusive evidence that a compound from the maternal circulation gained access to the fetus via the proposed histiotrophic pathway in the human, and it is particularly unlikely that a much larger compound such as HBOC-201 (250 kDa) would be available to the fetus via this pathway. In conclusion, HBOC-201 was not developmentally toxic to dogs. It produced developmental toxicity in rats through impeded functioning of the rat inverted yolk sac, an organ that does not exist in dogs or humans and which functions by a physiological mechanism that differs from the hemotrophic placentae of dogs and humans. Thus, the mechanism of teratogenic action for HBOC-201 is not relevant to canines or humans.
Transparency document The Transparency document associated with this article can be found in the online version.
Conflict of interest Funding for the experiments was supplied by Biopure. Dr. Pearce had been an employee of Biopure; Drs. Stump and Holson were employees of WIL Research; Drs. Watson and DeSesso were consultants to Biopure.
Acknowledgements The authors are indebted to Drs. M. S. Gawryl and V. T. Rentko, formerly of Biopure Corporation, for early discussions concerning the design of studies.
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J.F. Holson et al. / Reproductive Toxicology 52 (2015) 101–107 [11] Miller RK, Levin AA, Ng WW. The placenta: relevance to toxicology. In: Clarkson T, Nordberg G, Sager P, editors. Reproductive and Developmental Toxicity of Metals. New York: Plenum Press; 1983. p. 569–605. [12] Beck F, Lloyd JB, Griffiths A. Lysosomal enzyme inhibition by Trypan Blue: a theory of teratogenesis. Science 1967;157:1180–2. [13] Hayasaka I, Hoshino K, Kameyama Y. Pathogenesis of ochratoxin Aand concanavalin A-induced exencephalies in mice. Cong Anom 1986;26: 11–24. [14] Beck F, Lloyd JB, Griffiths A. A histochemical and biochemical study of some aspects of placental function in the rat using maternal injection of horseradish peroxidase. J Anat 1967;101:461–78. [15] DeSesso JM, Niewenhuis RJ, Goeringer GC. Lectin teratogenesis II. Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the sensitive period. Teratology 1989;39:395–407. [16] Beckman DA, Brent RL, Lloyd JB. Pinocytosis in the rat visceral yolk sac: potential role in amino acid nutrition during the fetal period. Placenta 1994;15:171–6. [17] Smith KK, Strickland S. Structural components and characteristic of Reichert’s membrane, an extra-embryonic basement membrane. J Biol Chem 1981;256:4654–61. [18] Carney EW, Scialli AR, Watson RE, DeSesso JM. Mechanisms regulating toxicant disposition to the embryo during early pregnancy: an interspecies comparison. Birth Defects Res (C): Embryol Today 2004;72:345–60. [19] DeSesso JM. Comparative gestational milestones in vertebrate development. In: Hood RD, editor. Developmental and reproductive toxicology: a practical approach. 3rd ed. New York: Informa Healthcare; 2012. p. 93–138.
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[20] Holson JF, Stump DG, Pearce LB, Watson RE, DeSesso JM. Mode of action: yolk sac poisoning and impeded histiotrophic nutrition. HBOC-related congenital malformations. Crit Rev Toxicol 2005;35:739–45. [21] Ramsey EM. The Placenta: Human and Animal. New York: Praeger; 1982. [22] Evans HE. Prenatal development in: Miller’s Anatomy of the Dog. 3rd ed. Philadelphia, PA: W.B. Saunders Company; 1993. p. 32–97. [23] Jauniaux E, Jurkovic D, Campbell S. Current topic; in vivo investigation of the placental circulations by Doppler echography. Placenta 1995;16:323–31. [24] Jaffe R, Jauniaux E, Hustin J. Maternal circulation in the first-trimester human placenta-Myth or reality? Am J Obstet Gynecol 1997;176:695–705. [25] Burton GJ, Hempstock J, Jauniaux E. Nutrition of the human fetus during the first trimester – a review. Placenta 2001;22(Suppl. A(15)):S70–6. [26] Burton GJ, Watson AL, Hempstock J, Skepper JN, Jauniaux E. Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy. J Clin Endocrinol Metab 2002;97(6):2954–9. [27] Gulbis B, Jauniaux E, Cotton F, Stordeur P. Protein and enzyme patters in the fluid cavities of the first trimester gestational sac: relevance to the absorptive role of secondary yolk sac. Mol Hum Reprod 1998;4(9):857–62. [28] Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW. Crystal structure of human chorionic gonadotropin. Nature 1994;369:455–61. [29] Hill, MA. Embryology: Dog Development. https://php.med.unsw.edu. au/embryology/index.php?title=Dog Development [retrieved 31.08.14]. [30] Pretzer SD. Canine embryonic and fetal development: a review. Theriogenology 2008;70:300–3.
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Absence of developmental toxicity in a canine model after infusion of a hemoglobin-based oxygen carrier: Implications for risk assessment J.F. Holson a , D.G. Stump b , L.B. Pearce c , R.E. Watson d , J.M. DeSesso e,f,∗ a
Ashland, OH, United States WIL Research Laboratories, Ashland, OH, United States Biologics Consulting Group, Alexandria, VA, United States d SNBL USA, Everett, WA, United States e Exponent, Inc., Alexandria, VA, United States f Georgetown University School of Medicine, Washington, DC, United States b c
a r t i c l e
i n f o
Article history: Received 29 May 2014 Received in revised form 24 December 2014 Accepted 20 January 2015 Available online 16 February 2015 Keywords: Hemoglobin-based oxygen carriers Inverted yolk sac Dog
a b s t r a c t Bovine-derived hemoglobin-based oxygen carriers (HBOCs) have been investigated for use in humans (HBOC-201) and approved for veterinary medicine (HBOC-301). We infused pregnant beagles with HBOC201 to test whether HBOC-induced developmental toxicity previously observed in rats would occur in a species devoid of an inverted visceral yolk sac (invVYS). Phase 1 assessed developmental toxicity of 6 g/kg HBOC-201 on gestational day (GD) 21. Phase 2 investigated single infusions of 6 g/kg HBOC-201 on one of GDs 21, 25, 29 or 33. Phase 3 studied multiple sequential infusions on GDs 21, 23,25,27,29, 31, and 33 at 0.52 g/kg/day (3.6 g/kg total dose). Mild to moderate maternal toxicity occurred in all phases. There was an unequivocal absence of developmental toxicity in all phases. Overall, our hypothesis that HBOC, which interferes with the function of the invVYS, would not affect the offspring in dogs was supported. The implications relative to human risk are discussed. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Hemoglobin-based oxygen carriers (HBOCs) are under development to expand blood volume and deliver oxygen in cases where there has been a precipitous loss of blood and compatible blood is not available for transfusion. HBOC-201 is produced from tetrameric purified bovine hemoglobin (PBH) molecules that have been bound together in a 250 kDa glutaraldehyde-stabilized polymer. Safety assessment studies in pregnant rats infused intravenously with HBOC-201 resulted in significant developmental toxicity, as demonstrated by a decrease in live litter size and an increase in external fetal malformations [1]. Subsequent investigatory studies that controlled for the colloidal properties, osmolarity, protein content, and hemoglobin content of HBOC-201 have shown that the developmental toxicity observed is directly related to the hemoglobin content of HBOC-201 [1]. The appearance of these effects correlated with infusion of HBOC-201 only during gestational days (GDs) 7–9, a period during rat organogenesis when the inverted visceral yolk sac (invVYS) supplies the rat embryo with its
∗ Corresponding author at: Exponent, Inc., 1800 Diagonal Road, Suite 500, Alexandria, VA 22314, United States. Tel.: +1 571 227 7261; fax: +1 571 227 7299. E-mail address: [email protected] (J.M. DeSesso). http://dx.doi.org/10.1016/j.reprotox.2015.01.006 0890-6238/© 2015 Elsevier Inc. All rights reserved.
primary source of nutrition through a histiotrophic process. This period precedes the establishment of the chorioallantoic placental circulation, which starts on GD 11.5 [2]. In rat whole embryo culture experiments, it has been demonstrated that HBOC inhibits the transport function of the inVYS [1]. The developmental toxicity observed in the rat may not accurately predict human developmental toxicity because the human yolk sac does not function as a placenta. Consequently, to more fully understand whether HBOCs might cause potential developmental toxicity in humans, the use of an alternative animal model that does not rely on an invVYS during organogenesis was investigated. The dog is an attractive animal model because, as in humans, the canine yolk sac membrane does not invert nor does it serve as a primary nutritive organ during organogenesis. Additional advantages of the canine model include previous knowledge of critical periods of organogenesis and approval of a similar hemoglobin-based oxygen carrying therapeutic agent (HBOC-301) for veterinary use in the dog. Non-human primates were not deemed to be a more predictive model because the stress of compound delivery juxtaposed with superficial implantation predisposes non-human primates to early abortion [3], which might confound interpretation. The aim of these studies was to assess potential developmental toxicity related to infusion of pregnant dogs with HBOC-201 during organogenesis. A dose of 6 g/kg HBOC-201 was an estimate of a dose
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that would exceed the approximate clinically relevant single dose in the human without causing significant maternal toxicity that could complicate analysis of developmental toxicity. A preliminary experiment was performed to verify that 6 g/kg HBOC-201 is an appropriate dose in the dog. Based on the success of the preliminary experiment at that dose level, two larger scale definitive studies were performed. In the first definitive study, single-day infusions of 6 g/kg HBOC-201 were given to groups of pregnant dogs on one of GDs 21, 25, 29, or 33. In the second definitive study, sequential infusions in the same dogs were administered on seven occasions throughout organogenesis (GDs 21, 23, 25, 27, 29, 31, and 33) for a total dose of 3.6 g/kg. This dose mimics the repeat administration proposed for the human clinical setting.
2. Materials and methods 2.1. Animals Time-mated multigravid female beagle dogs were obtained from Covance Research Products, Inc. (Cumberland, VA). Dogs had been mated naturally at Covance on two separate days; the second breeding was conducted 2–6 days following the first breeding. The first day on which mating was observed was designated as GD 0. The dogs were approximately 2–5 years old and ranged from GD 7 to 10 on the day of receipt. Each dog was allowed 8 days to acclimate to the laboratory environment, and during this period animals were observed twice daily for mortality and changes in general appearance and behavior. Animals were fed PMI Nutrition International, Inc. (Richmond, IN) Certified Canine LabDiet® 5007 ad libitum throughout acclimation and the study. Drinking water was delivered by an automatic watering system and was provided ad libitum throughout the acclimation period and during the study. Dogs were housed individually in whelping pens that were controlled for temperature and light/dark conditions in accordance with the “Guide for the Care and Use of Laboratory Animals” [4]. At the conclusion of the acclimation period, the animals were assigned to groups using a computerized randomization procedure. The protocols for these studies were submitted to and approved by the WIL Research Institutional Animal Care and Use Committee. 2.2. Test articles The infusate vehicle consisted of a 0.9% sodium chloride solution (saline; Baxter Healthcare Corporation; Deerfield, IL) stored at room temperature. Solutions of HBOC-201 (13 g/dL) in vehicle were prepared by Biopure® (Cambridge, MA). HBOC-201 was kept at room temperature and was considered stable under this condition. To control for the concentrated protein nature of HBOC-201, a 13% solution of human serum albumin (HSA; equivalent protein concentration) was used. 2.3. Surgical placement of jugular vein catheters All animals were acclimated to wearing jackets and collars prior to surgery. Surgeries for the implantation of indwelling jugular vein catheters were performed on or before GD 18. The dogs were anesthetized by isoflurane inhalation prior to catheter implantation. Each implanted catheter was attached to a validated CADD-Legacy® pump (Sims Deltec, St. Paul, MN) with a remote reservoir and an extension set placed in the vest pouch closest to the vein to be catheterized. Following surgery, collars protecting the catheters were placed on the animals. Reservoirs were replaced, as necessary, throughout the acclimation period. To maintain the patency of access, saline was administered at an
infusion rate of approximately 0.5 mL/h following surgery until test article infusion was initiated on GD 21. 2.4. Infusion of test article A preliminary study established that the infusion procedures did not result in significant maternal toxicity. Dogs were infused with 6 g/kg HBOC-201 (N = 4) or saline (N = 5) at a rate of 0.1 mL/kg/min for 8 h on GD 21. These dogs tolerated the HBOC-201 well, and thus, this dose and infusion procedure was deemed appropriate for use in larger scale studies. Single-day exposures to groups of dogs (N = 20) were performed at 6 g/kg HBOC-201 on GDs 21, 25, 29, or 33. These dates were selected to provide significant plasma levels of HBOC-201 at intervals that collectively spanned the period of major organogenesis. This dosing regimen was based on the long plasma half-life of HBOC in dogs at this dose1 (∼55 h) and when all four single-day regimens are combined results in exposure over the period of major organogenesis. The 13% HSA control (46 mL/kg) was administered on GD 21. The saline control group was infused on all four days that HBOC-201 was administered. Twenty pregnant dogs were assigned to each of the six groups used in this study. All infusions were delivered at a rate of 0.1 mL/kg/min for an 8-h period. In the sequential infusion study, repeat administration of HBOC201 or saline (control) was performed on groups of 20 pregnant dogs over a 13-day period during organogenesis. On GDs 21, 23, 25, 27, 29, 31 and 33, saline or HBOC-201 was infused at a flow rate of 0.1 mL/kg/min for approximately 40 min resulting in a total infusate volume of 4 mL/kg/day. Each infusion delivered 0.52 g HBOC-201/kg resulting in a total dose of 3.6 g/kg. 2.5. Maternal observations during gestation and lactation Dogs were observed twice daily for moribundity and mortality. Detailed clinical observations were recorded daily for each female. Observations for signs of toxicity included, but were not limited to, evaluations for changes in the skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems, somatomotor activity and behavior. On the days of treatment, animals were observed for signs of toxicity approximately 1 h following completion of the 8 h infusion. All significant clinical findings were recorded at this observation period. On GD 30 ± 5, each dog was palpated by the attending veterinarian in an attempt to determine pregnancy status. Maternal body weights were recorded on several occasions throughout gestation and mean body weight changes were calculated for GD 21–60. Dogs that were non-gravid and control dogs which aborted were not included in the statistical analysis of maternal parameters. 2.6. Plasma hemoglobin assessment In order to monitor the elimination of HBOC-201 from the plasma, assessments for plasma hemoglobin were performed on the first six dogs infused with HBOC-201 on GD 33 prior to infusion, immediately following infusion and 1, 4, 16, 24, 36, 48, 60, 72, 84, 96, 144 and 192 h after the end of infusion. EDTA was used as the anticoagulant and the analyses were conducted using the HemoCue® B-Hemoglobin System (HemoCue AB, Angelholm, Sweden). In the sequential infusion study, assessments for plasma hemoglobin were performed on the first six dogs infused with
1 The plasma half-life of HBOC-201 is dose-dependent and ranged from ∼22 h at low doses to ∼55 h.
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HBOC-201 immediately prior to infusion and 30 min following infusion on GDs 21, 23, 25, 27, 29 and 31 and GD 33 prior to infusion, 30 min, 1, 4, 8, 12, 16, 20, 26, 32, 38, 46, 56 and 72 h after the end of infusion.
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puppy body weight parameters. The Kruskal–Wallis test [9] with the Mann–Whitney U test [9] was performed for non-parametric parameters including puppy sexes at birth, postnatal survival, litter proportions of puppy malformations and developmental variations.
2.7. Whelping Females were allowed to deliver naturally. When necessary, 0.2–0.5 mL of oxytocin was administered intramuscularly to aid uterine contraction. For each litter, the numbers of stillborn and live puppies as well as their sex were recorded. The duration of gestation was calculated using the interval between the date on which whelping was initiated and the date of first mating provided by the animal supplier. Animals that failed to deliver were considered to be nongravid and were removed from the study. The number of nongravid animals was small and their occurrence did not appear to be related to HBOC-201 administration. 2.8. Offspring morphological examination Puppies were sexed on postnatal day (PND) 0. On PND 1 they were weighed and euthanized by an intraperitoneal injection of sodium pentobarbital. Each puppy (whether viable or nonviable) received a detailed external examination that included, but was not limited to, an examination of the eyes, palate and external orifices. All findings were recorded uniquely for each puppy. Radiographs were taken of the dorsal to ventral, ventral to dorsal, right lateral and left lateral positions. Each puppy then was examined viscerally by a modified Stuckhardt and Poppe [5] fresh dissection technique. The examination included the heart and major vessels as well as confirmation of gender. The heads were inspected by a mid-coronal slice. All carcasses were then eviscerated, fixed in 100% ethyl alcohol, macerated in potassium hydroxide, and stained with Alizarin Red S by a method similar to that described by Dawson [6]. Skeletal examinations were performed through a combination of reviewing the radiographs and evaluating the stained skeletal preparations. External, visceral and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effects on animal health or body conformity, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function or may be incompatible with life). The percentage of pups with malformations, the mean percentage of malformed pups/litter, and the percentage of pups surviving to PND 1 were calculated. 2.9. Statistical analyses All analyses were performed on the litter as the experimental unit, using two-tailed tests for minimum significance levels of 5% and 1% that compared the test article-treated group to the control group. A one-way ANOVA [7] with Dunnett’s post hoc test [8] was performed with parametric parameters: maternal body weight changes, number of puppies born, live litter size, and
3. Results Table 1 presents the data from the preliminary study. Compared to saline controls, infusion of 6 g/kg HBOC-201 at 46 mL/kg did not lead to significant changes in live litter size, male or female pup weight, survival from birth to PND 1, or the incidence of external malformations. A numerical decrease in maternal weight gain during gestation (not statistically significant, likely due to the large standard deviation) and overt clinical signs of maternal toxicity, primarily yellow sclerae were associated with HBOC-201 infusion. Excreta-related effects (e.g., soft stool, decreased defecation, mucoid feces and/or diarrhea) were observed at a similar incidence in the saline control and 6 g/kg HBOC-201 group. Because the 6 g/kg dose elicited clinical signs without severe effects that might induce developmental toxicity secondary to maternal toxicity, this dose was deemed an appropriate amount to administer for daily infusions in the definitive studies. Maternal and developmental toxicity assessments were next performed on pregnant dogs given a single-day infusion of 6 g/kg HBOC-201 on GDs 21, 25, 29, or 33 or hetastarch on GD 21 (Table 2). Numerical decreases in maternal weight gain (not statistically significant, likely due to the large standard deviation) and overt signs of maternal toxicity, primarily yellow sclerae, were observed in dogs infused with 6 g/kg HBOC-201 on each GD examined. Excretarelated effects were noted in the dogs infused with HBOC-201 and saline on each GD examined as well as for pregnant dogs infused with 13% HSA on GD 21. However, infusion with neither HSA nor HBOC-201 at any dose level led to any significant changes in litter size, survival rate from birth to PND 1, male or female pup weight, or the rate of external, visceral or skeletal malformations when the results are compared to those of the saline and HSA controls. Table 3 summarizes the results of repeated infusions of 0.52 g/kg/day HBOC-201 or saline into pregnant dogs every other day during GDs 21–33. Infusion of HBOC-201 led to decreases in maternal weight gain during gestation and overt signs of maternal toxicity, primarily yellow sclera; however, no signs of developmental toxicity were detected in offspring. Taken together, the results of these robust studies found no indications of developmental toxicity in pups after maternal infusions of HBOC-201 at doses that caused clinical signs of toxicity in pregnant dogs. The mean plasma hemoglobin concentration immediately following infusion of 6 g/kg of HBOC-201 on GD 33 was approximately 6.0 g/dL demonstrating that marked plasma concentrations of HBOC-201 were achieved (Table 4). The mean plasma hemoglobin concentration 72 h following infusion (2.6 g/dL) was slightly less than one-half the concentration immediately following infusion,
Table 1 Developmental toxicity results after infusion of HBOC-201 into pregnant dogs on gestational day 21. Parameter
Saline (46 mL/kg)
HBOC-201 (6.0 g/kg = 46 mL/kg)
Number of pregnant dogs Maternal weight gain from GDs 21 to 28 (g ± S.D.) Clinical findings Live litter size ± S.D. Postnatal survival birth to PND 1 (% per litter ± S.D.) Mean weight for males (g ± S.D.) Mean weight for females (g ± S.D.) External malformations (no. affected/no. examined)
5 194 ± 1158 Excreta-related effects 6.2 ± 2.4 86.7 ± 29.8 297.3 ± 22.4 285.9 ± 24.1 0/29
4 −831 ± 115 Excreta-related effects 3.8 ± 3.2 100 ± 0.0 326.9 ± 76.4 308.6 ± 83.5 0/15
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Table 2 Maternal and developmental toxicity findings after single-day infusions of HBOC-201 on gestation days 21, 25, 29, and 33. Parameter
Saline (46 mL/kg) GD 25
13% HSA (6 g/kg = 46 mL/kg) GD 21
Number of pregnant dogs examined Clinical findings
18 Excreta-related effectsa
16 Excreta-related effects
Mean maternal body weight gain GDs 21–34 (g ± S.D.) Mean number of live fetuses/litter Postnatal survival from birth to PND 1 (% per litter) Mean pup weight males (g ± S.D.) Mean pup weight females (g ± S.D.) Number of pups (litters) examined Pups (litters) with external Malformations Cleft palate Bent tail w/vertebral anomaly Pups (litters) with visceral malformations Hydrocephaly Kidney and ureter absent Pups (litters) with skeletal malformations Rib anomalies Total malformations (mean % per litter ± S.D.)
−4 ± 222
a
HBOC-201 (6 g/kg = 46 mL/kg) GD 21
GD 25
GD 29
GD 33
−95 ± 178
19 Excreta-related effects, yellow sclera −334 ± 157
18 Excreta-related effects, yellow sclera −296 ± 204
18 Excreta-related effects, yellow sclera −442 ± 201
17 Excreta-related effects, yellow sclera −118 ± 224
5.4 ± 2.8 84.3 ± 32.8
4.9 ± 2.1 80.3 ± 27.06
4.9 ± 2.0 80.3 ± 27.1
6.1 ± 1.6 80.4 ± 28.2
5.4 ± 2.3 95.9 ± 8.2
4.7 ± 2.2 84.7 ± 21.3
260.9 ± 61.3 244.9 ± 52.3 92 (16) 4 (2)
249.1 ± 58.6 264.1 ± 42.7 75 (16) 0 (0)
272.7 ± 50.6 261.7 ± 43.4 88 (18) 0 (0)
267.1 ± 43.1 259.9 ± 32.9 100 (17) 3 (3)
254.9 ± 52.4 229.2 ± 42.0 97 (18) 0 (0)
265.5 ± 54.6 257.5 ± 52.6 76 (17) 0 (0)
3 (2) 1(1) 2 (2)
0 (0) 0 (0) 2 (2)
0 (0) 0 (0) 2 (2)
1 (1) 2 (2) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
2 (2) 0 (0) 0 (0)
1 (1) 2 (2) 1 (1)
2 (2) 1 (1) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
0 (0) 5.7 ± 13.9
1 (1) 5.3 ± 13.3
0 (0) 2.1 ± 6.4
0 (0) 4.5 ± 12.5
0 (0) 0.0 ± 0.0
0 (0) 0.0 ± 0.0
Soft stool, decreased defecation, mucoid feces and/or diarrhea.
Table 3 Maternal and development toxicity results after multiple infusions of HBOC-201 during gestational days 21–33. Parameter
Saline (4 mL/kg/day)
HBOC-201 (0.52 g/kg/day = 4 mL/kg/day)
Number of pregnant dogs examined Clinical findings Mean maternal body weight gain GDs 21–34 (g ± S.D.) Mean live litter number Postnatal survival birth PND 1 (% per litter) Mean pup weight males (g ± S.D.) Mean pup weight females (g ± S.D.) Number of pups (litters) examined Pups (litters) with external malformations Pups (litters) with visceral malformations Pups (litters) with skeletal malformations Rib anomalies Total malformations (mean % per litter ± S.D.)
19 Excreta-related effects 22 ± 586 5.2 ± 1.8 93.0 ± 12.7 293.2 ± 47.6 281.7 ± 42.6 91 (18) 0 (0) 0 (0) 1 (1) 1 (1) 0.9 ± 3.9
17 Excreta-related effects, yellow sclera −522 ± 484 5.5 ± 1.5 92.4 ± 11.8 278.1 ± 39.6 264.6 ± 60.0 91 (17) 0 (0) 0 (0) 0 (0) 0 (0) 0.0 + 0.0
and measurable plasma hemoglobin concentrations were still present 144 h following infusion. These results demonstrate that significant exposure to HBOC-201 occurred for 6 days following infusion.
Table 4 Plasma hemoglobin concentrations after a single-day infusion of 6 g/kg HBOC-201 on GD 33. Time after infusion
Plasma hemoglobin (g/dL)
Prior to infusion Immediately following infusion 1h 4h 16 h 24 h 36 h 48 h 60 h 72 h 84 h 96 h 144 h 192 h
0.0 6.0 5.9 5.8 5.2 5.0 4.3 3.8 3.2 2.6 2.0 1.6 0.4 0.1
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.33 0.38 0.38 0.33 0.37 0.34 0.34 0.58 0.37 0.50 0.44 0.17 0.08
The mean plasma hemoglobin concentrations 30 min following infusion of 0.52 g/kg of HBOC-201 on GDs 21, 23, 25,27, 29, 31 and 33 were approximately 1 g/dL demonstrating that consistent plasma concentrations of HBOC-201 were achieved and that no indication of accumulation (increased AUC) was observed following repeated administration of HBOC-201 (Table 5). The plasma hemoglobin concentration 16 h following infusion on GD 33 was approximately one-half the concentration 30 min following infusion. 4. Discussion In contrast to rats [1], no developmental toxicity was observed in puppies whose mothers were exposed during organogenesis to either single high dose exposures or repeated lower-dose exposures of HBOC-201. This supports the concept that the mode of action whereby HBOC-201 causes developmental toxicity in rats is by impeding the function of the inverted yolk sac. Although humans and dogs do have yolk sacs, they do not invert, nor do they function as a major conduit for nutrition during development. Therefore, substances that interfere solely with invYS function should not signal a concordant risk to development of canine or human embryos.
J.F. Holson et al. / Reproductive Toxicology 52 (2015) 101–107 Table 5 Plasma hemoglobin concentrations after multiple-day infusions of 0.52 g/kg HBOC201. Time after infusion
Plasma hemoglobin (g/dL)
GD 21 prior to infusion GD 21–30 min following infusion GD 23 prior to infusion GDs 23–30 min following infusion GD 25 prior to infusion GDs 25–30 min following infusion GD 27 prior to infusion GDs 27–30 min following infusion GD 29 prior to infusion GDs 29–30 min following infusion GD 31 prior to infusion GDs 31–30 min following infusion GD 33 prior to infusion GDs 33–30 min following infusion GDs 33–1 h GDs 33–4 h GDs 33–8 h GDs 33–12 h GDs 33–16 h GDs 33–20 h GDs 33–26 h GDs 33–32 h GDs 33–38 h GDs 33–46 h GDs 33–56 h GDs 33–72 h
0.0 1.0 0.1 1.1 0.1 1.1 0.4 1.2 0.2 1.1 0.2 1.0 0.1 1.0 1.0 0.9 0.7 0.6 0.5 0.4 0.3 0.3 0.2 0.1 0.1 0.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.58 0.04 0.16 0.05 0.10 0.49 0.15 0.08 0.13 0.18 0.18 0.14 0.21 0.23 0.24 0.23 0.19 0.18 0.19 0.19 0.11 0.12 0.16 0.07 0.04
HBOC-201 is a large molecule that is unlikely to cross biological membranes rapidly, or to any large extent [10,11]. If the material is not able to readily cross membranes, gain access to, or accumulate in nontrivial amounts within the embryo, it is unlikely that it could exert an embryonic site of action. Nevertheless, HBOC-201 caused developmental toxicity in rats, including malformations and decreased fetal weights, when it was infused continuously over gestational days 6–18. The malformations that occurred (mainly ocular defects and craniofacial dysmorphologies) are consistent with findings of agents that act early in gestation, while the observations of decreased fetal weights are consistent with agents that interfere with embryonal/fetal growth throughout gestation. These types of findings in rat offspring are similar to those reported for agents such as trypan blue [12] and concanavalin A [13], both molecules that do not cross into rodent embryos, but exert their influences through interference with the function of the inverted yolk sac [14,15]. When HBOC-201 was administered over 24-h periods during organogenesis, malformations occurred only when exposure occurred on gestational days 7–8 and 8–9, but not when administered at one-day intervals during gestational days 9–10; 10–11; 11–12; 12–13 [1], which is the period when the rat relies on the inverted yolk sac as its main source of nutrition. This is an important finding because these adverse events occur prior to establishment of the underpinnings of the chorioallantoic placenta which becomes functional at about gestational day 10.5–11.5 in rats [2]. The inverted yolk sac and chorioallantoic placenta differ dramatically in their modes of transport. Physiological transport via the inverted yolk sac comprises a multi-step process that includes (1) endocytosis or pinocytosis of uterine secretions from the luminal surface of the visceral yolk sac epithelium, (2) traversing of the endocytotic vacuoles into the cytoplasm to merge with lysosomes and subsequent enzymatic attack, and (3) diffusion of breakdown products across the epithelium toward embryonic vitelline blood vessels in the mesenchyme on the abluminal surface. This multistep process is termed histiotrophic nutrition, which supplies the embryo with nutrients from sources other than from the maternal blood. The histiotrophic mechanism can transfer nutrients and
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large molecular weight substances, but it is a time-consuming process that soon becomes inefficient for the needs of a rapidly growing embryo. The invVYS is soon superseded by the chorioallantoic placenta which depends on direct exchange between the separate maternal and embryonal blood circulations, termed hemotrophic nutrition. Hemotrophic nutrition enables the prompt transfer of relatively small molecules between the maternal and embryonic blood circulations; the transfer efficiency declines when the size of the molecules exceeds 600 Daltons and, typically, the largest molecules transferred by diffusion across non-fenestrated epithelia of the chorioallantoic placenta are less than 1200 Daltons [10,11]. The invVYS of the rat remains functional after the establishment of the chorioallantoic placenta, and transports nutrients and proteins into the embryo until at least GD 17 [16]. In the rapidly developing rat embryo, the yolk sac abuts the uterine wall and then collapses (inverts) such that lumen disappears and the somatic wall (the original outer yolk sac wall that touched the uterine epithelium) deteriorates, leaving behind a basal lamina (Reichert’s membrane) that interposes between the visceral epithelium of the invVYS and the wall of the maternal uterine lumen [2,17]. The visceral epithelium of the invVYS functions as the primary means for physiological exchange during the span between approximately GDs ∼8–11.5 [2,18]. During this period of development the rat embryo completes several major milestones including closure of the neural tube and establishment of the anlage for many organ systems [19]. Throughout this critical time period, transport of materials to the embryo must occur across the invVYS. Therefore, interference with this process early in gestation would be expected to cause adverse effects on embryo-fetal development. However, this mechanism of action is not applicable to the human or the dog. While disruption of the processes of endocytosis, lysosomal degradation, and subsequent transport of breakdown products across the cell could be expected to occur if HBOC-201 were presented to absorptive epithelia, the impact is of fundamental concern to developmental toxicity only when this process is the exclusive means available for transporting nutrients to the embryo. Humans and dogs have a much longer gestational period (266 days and ∼60 days, respectively) than rats (22 days). As a consequence, human and dog embryo/fetuses develop at a less hurried rate than rats, as illustrated in Table 6 summarizing the timing of gestational milestones in the rat, dog and human. Importantly, the milestones of rat embryogenesis that occur during the interval beginning when the rat is dependent on the inverted yolk sac placenta and continuing through the establishment of the rat chorioallantoic placenta are the same ones that occur in the dog during the infusion schedule used. The human and dog establish only one definitive placenta: the chorioallantoic placenta that begins functioning at the end of the fourth week of gestation. Because humans do not rely on histiotrophic nutrition as a means of obtaining nourishment, starvation of their embryos by interference with this process is irrelevant as a mode of action for developmental toxicity [20]. While the yolk sac does not contribute to definitive placenta formation in either the human or dog embryo it does play an important role in their development. It is the site of early hematopoiesis, and produces albumin, pre-albumin, alpha-fetoproteins, alphaantitrypsins, transferrin, and apolipoproteins. The yolk sac is also the site of origin of gonial cells. During early human development, the yolk sac enlarges considerably (such that it is larger than the embryo by about 22 days of gestation), but its wall never touches the chorion and, thus, it never functions as a placenta [18]. In dogs, a small area of the yolk sac wall briefly abuts the chorion from about gestational days 21 to 24, but the yolk sac does not invert [21,22]. Though the underpinnings of the chorioallantoic placenta in the human are present at the beginning of gestational week 4
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Table 6 Comparison of developmental milestones in the rat, dog and human.a Developmental milestones
Rat Age in daysb
Dog Age in daysc
Human Age in days
Fertilization Morula Blastocyst Implantation into uterine epithelium Yolk sac inverts Histiotrophic nutrition via inverted visceral yolk sac begins First somite Aortic arches I–III Anterior limb bud appears Lens placode Chorioallantoic placenta established Neural tube closure Herniation of intestines into umbilical cord Digital rays in anterior limb bud Palatal shelves begin to rotate Hard palate closure Parturition
0 3 5 5.5–6 6.5–7 7–9 9.5 10.5–11 11 11.5 11–11 .5 11.5 12.5 13.5 16.5 17 22
0 8 12–16 12–18 Does not invertd N/A 19 23 23 23 22 21–22 30 31 33 35 ∼60
0 3 4–5 7–8 Does not invert N/A 19–20 28–31 26 27 ∼28 28 36 35–36 54 ∼56 ∼266
N/A, not applicable. a Data assembled from [19,22,29,30]. b Gestational day 0 is the day of confirmed mating. c Gestational day 0 is the day of the LH surge. Yolk sac of the dog briefly abuts the chorion ∼19.5 day of gestation, but does not invert.
[18,21], some researchers believe that hemotrophic nutrition (from maternal blood circulating among the villi of the chorioallantoic placenta) is not fully functional before gestational weeks 8–10. This claim is based on Doppler ultrasound studies that are interpreted to indicate that the endometrial spiral arteries are blocked by plugs of cytotrophoblast cells prior to this time [23,24]. It has been suggested that the yolk sac plays an important role in transferring maternal nutrients from the mother to the fetus to address the nutritional needs of the fetus prior to gestational weeks 8–10 [25,26]. Transfer to the fetus of nutrients from uterine gland secretions is proposed to occur by a route that involves the pinocytotic uptake of nutrients by trophoblast, movement along the stromal channels in the chorion, diffusion into the coelomic fluid, and ultimate absorption into the yolk sac where the nutrients gain access to the fetus [25]. This process has been termed “histiotrophic” but it differs markedly from the more direct histiotrophic pathway in the rat involving the uptake of compounds through the visceral wall of the sac between gestational days 7 and 9. Research supporting the human histiotrophic pathway includes studies examining the transfer of human chorionic gonadotrophin (HCG) to the yolk sac lumen, as well as the movement of proteins secreted by the uterine glands. HCG mRNA was detected in cytotrophoblast and syncytiotrophoblast, but not in yolk sac tissue [27]. However, HCG is present in the lumen of the yolk sac, suggesting that HCG can be absorbed by the yolk sac wall [27]. This study suggests that although a high molecular weight protein (HCG at 27.5 kDa; [28]) can enter the yolk sac, the concentration of HCG in the yolk sac was 2.8 times less than that reported in the coelomic fluid [27]. Furthermore, there is no conclusive evidence that a compound from the maternal circulation gained access to the fetus via the proposed histiotrophic pathway in the human, and it is particularly unlikely that a much larger compound such as HBOC-201 (250 kDa) would be available to the fetus via this pathway. In conclusion, HBOC-201 was not developmentally toxic to dogs. It produced developmental toxicity in rats through impeded functioning of the rat inverted yolk sac, an organ that does not exist in dogs or humans and which functions by a physiological mechanism that differs from the hemotrophic placentae of dogs and humans. Thus, the mechanism of teratogenic action for HBOC-201 is not relevant to canines or humans.
Transparency document The Transparency document associated with this article can be found in the online version.
Conflict of interest Funding for the experiments was supplied by Biopure. Dr. Pearce had been an employee of Biopure; Drs. Stump and Holson were employees of WIL Research; Drs. Watson and DeSesso were consultants to Biopure.
Acknowledgements The authors are indebted to Drs. M. S. Gawryl and V. T. Rentko, formerly of Biopure Corporation, for early discussions concerning the design of studies.
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