Presented by Donald G. Stump and Joseph F. Holson in Symposium I ("A Detective Story: Is the Prenatal Toxicity of a Therapeutic in Rats Relevant to Human Risk?", Joseph F. Holson and L. B. Pearce, co-chairpersons) at the Forty-Third Annual Meeting of the Teratology Society, Philadelphia, PA, June 26, 2003.

1. Findings of Developmental Toxicity Studies of
HBOC-201 in Rodent and Canine Models
Donald G. Stump, Ph.D., D.A.B.T.
Joseph F. Holson, Ph.D.

2. Preliminary Rat Developmental
Toxicity Studies
1) Continuous 24-hour infusion throughout
organogenesis – Top loading
 A.M. of GD 6 – A.M. of GD 18
1) Single-day infusion during gestation
A. Intermittent 24-hour infusion - Top loading
 GD 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, or 12-13
A. 50% hemodilution and single-day infusion on GD 9
 HBOC-201
 Purified bovine hemoglobin (PBH)

3. GD 0
• Continuous 24-hour infusion
• 8 females/group
• Dose levels - 1.95, 3.9 and 5.85 g/kg/day + saline control
+ HSA control
GD 6 GD 18
I.V. Infusion
Pilot Continuous Infusional Developmental
Toxicity Study in Rats
GD 20
Uterine and Fetal
External Exam

4. Results of Pilot Continuous Infusional
Developmental Toxicity Study in Rats (GD 6 -18)
Biopure Study Report/ CTBR
• Dose-related decrease in fetal viability and weight
• Dose-related increase in external malformations
Parameter Saline
(45 mL/kg/day)
13% HSA
(45 mL/kg/day)
HBOC 201
1.95 g/kg/day 3.9 g/kg/day 5.85 g/kg/day
Clinical findings - -
Petechial
hemorrhages
on tails,
darkened eyes
and/or skin
Petechial
hemorrhages
on tails,
darkened eyes
and/or skin
Petechial
hemorrhages
on tails,
darkened eyes
and/or skin
Mean maternal
body weight gain
(GD 6-18; g)
101.5 76.3 48.0 27.4 19.2
Mean no. viable
fetuses
12.8 10.8 10.2 2.0 0
Mean fetal weight
(g)
4.27 4.25 3.77 1.82 N/A
% External
malformations
fetuses
0 0 25.9 100 100

5. Multiple Single-Day Infusional
Exposures in Rats - Top-Loading
• 24-hour infusion on GD 6-7, 7-8, 8-9, 9-10, 10-11, 11-12 or 12-13
• 6 females/group
• Dose levels - 1.95 and 5.9 g/kg + saline + Hetastarch controls
(22-group study)
GD 0 GD 6
6-7
GD 137-8
8-9
9-10
10-11
11-12
12-13
6-7
7-8
8-9
9-10
10-11
11-12
12-13
Biopure Study Report/ CTBR
GD 20
Uterine and Fetal
External Exam

6. • No effect on fetal weight after GD 6-7 exposure
• Fetal weight decreased after GD 7-8 through GD 12-13 exposure,
most pronounced on GD 8-9, least pronounced on GD 12-13
• Malformations seen after GD 7-8 and 8-9 exposures
Summary of Multiple Single-Day Infusional
Exposures in Rats - Top-Loading
Biopure Study Report/ CTBR

7. Hemodilution and Single-Day
Infusion Study in Rats
• Hemodilute (withdraw 10-15 mL of blood while administering
approximately two times blood withdrawal volume of saline)
on GD 9 to produce 50% decrease in hemoglobin
• 8 females/group
• Dose levels - 6 g/kg + saline control
1-Hour
Infusion
GD 0 GD 20
Uterine and Fetal
External Exam
GD 9

8. Results of Rat Hemodilution
Study - GD 9
• The adverse fetal effects observed in previous top-loading studies
were not a result of hyperosmotic changes

9. Hemodilution and Single-Day Infusional Study
with Purified Bovine Hemoglobin (PBH) in Rats
• Hemodilute (withdraw 10-15 mL of blood while administering
approximately two times blood withdrawal volume of saline)
on GD 9 to produce 50% decrease in hemoglobin
• 8 females/group
• Dose levels - 6 g/kg PBH + saline control + Hetastarch
1-Hour
Infusion
GD 0 GD 20
Uterine and Fetal
External Exam
GD 9

10. Results of Rat Hemodilution
Study with PBH - GD 9
• The adverse fetal effects observed in rat studies with HBOC-201 were due to
the hemoglobin component of HBOC-201

11. Why the Dog Model?
 Absence of inverted yolk sac (placenta), to test
hypothesis
 Extensive toxicologic and pharmacologic database for
HBOC-201 in dogs
 Approved for veterinary use in dogs
 Ease of delivery without requiring restraint
 Half-life >40 hours in dog
 Stress of compound delivery, timing of rodent effect
juxtaposed with susceptibility to early abortion thought to
be confounders in non-human primate

12. Dog Estrous Cycle
Feldman and Nelson, 1996
(INDIVIDUAL EGGS: FERTILE FOR 12 to 24
HOURS
FERTILIZATION PERIOD
PRIMARY OOCYTES REQUIRE 24-48 hr.
CAPACITATION
OVULATION OCCURS: Thru~24-96 hr.

13. Comparison of Rodent and
Canine Development Schedule
Reproductive
Parameter
Rat
(GD)
Dog
(GD)
Ovulation 0 2-3
Fertilization 0 3-6
Implantation 5.5 16-18
Neural Tube Closure 10.5-11 21-22
Hard Palate Closure 17 33-35
Parturition 22 60-65
GD 0 = Day of Mating

14. Reasons for Segmented Study Design
in the Dog
 Biologics often use single or limited treatments in these types of
studies.
 Relative to potential exposure of embryo, not really different than
average drug because of PK profile.
 More appropriate for intended clinical use.
 Sensitive period of effect in rat has been identified and delimited.
 This design has been proposed to be more sensitive than repeated
daily treatments (Neubert et al., 1990). This design allows
assessment of higher Cmax and greater AUC during sensitive phases
of development.
 For testing the rodent yolk-sac hypothesis, it better mimics the
previous rat studies

15. Pilot Single-Day Dog Infusion Study
Top-Loading
GD 0
Natural Mating
(Observed Tie)
GD 18
Catheter
Placement
GD 21
Infuse
5 Females/Group
0.1 ml/kg/min
(8 hours)
Saline
Hetastarch
HBOC-201 – 6 g/kg
GD 63-67
Dams
allowed to
deliver
naturally
Viability
Body Weights
External Exam
Live Litter Size
PND 1

16. Canine Infusion

17. Canine Infusion

18. Results of Pilot Single-Day Dog
Infusion Study - Mean (N)
Parameter
Saline
(46 mL/kg)
Hetastarch
(2.7 g/kg)
HBOC-201
(6.0 g/kg)
Live Litter Size 6.2 (5) 8.0 (1) 3.8 (4)
Postnatal Survival Birth to
PND 1
(% per litter)
86.7 (5) 37.5 (1) 100 (4)
Mean Litter Weight for
Males (Grams)
297.3 (4) 267.6 (1) 326.9 (3)
Mean Litter Weight for
Females (Grams)
285.9 (5) 255.3 (1) 308.6 (3)
External Malformations
(No. Affected/No.
Examined)
0/29 1/3 0/15

19. Definitive Dog Developmental Toxicity Study -
Single-Day Infusional Exposures - Top-Loading
GD 0
Natural Mating
(Observed Tie)
GD 18
Catheter
Placement
GD 21 25 29 33
GD 63-67
Dams
allowed to
deliver
naturally
Viability
Body Weights
External, Visceral
and Skeletal Exam
Live Litter Size
PND 1
• 8-hour infusion (0.1 mL/kg/hour) on GD 21, 25, 29 or 33
• 20 females/group
• Dose levels - 6 g/kg HBOC-201 + saline control + 13% HSA control

20. Radiograph of PND 1 Dog Pup

21. Alizarin Red S Stained PND 1
Dog Pup

22. Results of Definitive Dog Developmental Toxicity Study
- Single-Day Infusional Exposures - Mean ± S.D.

23. Results of Definitive Dog Developmental Toxicity Study
- Single-Day Infusional Exposures

24. Repeated Infusional Study in Dogs Top-Loading –
7 Exposures throughout Organogenesis
GD 0
Natural Mating
(Observed Tie)
GD 18
Catheter
Placement
GD 21 33
GD 63-67
Dams
allowed to
deliver
naturally
Viability
Body Weights
External, Visceral
and Skeletal Exam
Live Litter Size
PND 1
• Repeated 8-hour infusions (0.1 mL/kg/hour) on GD 21, 23, 25, 27, 29, 31 and 33
• 20 females/group
• Dose levels - 0.52 g/kg/day + saline control
Repeated
Exposure
Period

25. Results of Repeated Infusional Study
in Dogs – Mean ± S.D.

26. Results of Repeated Infusional Study
in Dogs

27. Conclusions
1) HBOC-201 produces dose-dependent developmental
toxicity (embryolethality and malformations) in the rat
2) The dysmorphogenesis in rats is a result of exposure
in the pre-chorioallantoic period only
3) Administration after chorioallantoic placenta formation
resulted in reduced fetal weights explainable by the
ongoing absorptive activity of the InvYSP (Morgan,
1973; Padykula, 1966)
4) No developmental toxicity was observed in the dog
after either single, high exposures or repeated
exposures with HBOC-201 during organogenesis