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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.
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 gestationA. 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)
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
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
fetuses0 0 25.9 100 100
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 13
7-8
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
• 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
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-HourInfusion
GD 0 GD 20
Uterine and Fetal External Exam
GD 9
Results of Rat Hemodilution Study - GD 9
• The adverse fetal effects observed in previous top-loading studies were not a result of hyperosmotic changes
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-HourInfusion
GD 0 GD 20
Uterine and Fetal External Exam
GD 9
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
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
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.
Comparison of Rodent and Canine Development Schedule
Reproductive Parameter
Rat (GD)
Dog (GD)
Ovulation 0 2-3Fertilization 0 3-6Implantation 5.5 16-18
Neural Tube Closure 10.5-11 21-22Hard Palate Closure 17 33-35
Parturition 22 60-65
GD 0 = Day of Mating
Reasons for Segmented Study Designin 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
Pilot Single-Day Dog Infusion Study Top-Loading
GD 0Natural Mating (Observed Tie)
GD 18Catheter
Placement
GD 21Infuse 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
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
Definitive Dog Developmental Toxicity Study - Single-Day Infusional Exposures - Top-Loading
GD 0Natural Mating (Observed Tie)
GD 18Catheter
Placement
GD 21 25 29 33
GD 63-67Dams
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
Results of Definitive Dog Developmental Toxicity Study - Single-Day Infusional Exposures - Mean ± S.D.
Repeated Infusional Study in Dogs Top-Loading – 7 Exposures throughout Organogenesis
GD 0Natural Mating (Observed Tie)
GD 18Catheter
Placement
GD 21 33
GD 63-67Dams
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
RepeatedExposure
Period
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