findings of developmental toxicity studies of hboc-201 in rodent and canine models

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



and/or skin



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


GD 20


• 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


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


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


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

Canine Infusion

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 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

Radiograph of PND 1 Dog Pup

Alizarin Red S Stained PND 1 Dog Pup

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

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

Repeated Infusional Study in Dogs Top-Loading – 7 Exposures throughout Organogenesis


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

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

Results of Repeated Infusional Study in Dogs

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

findings of developmental toxicity studies of hboc-201 in rodent and canine models

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