WIL Research Laboratories

Joseph F. HolsonWIL Research Laboratories

A probable false positive finding of prenatal toxicity in the rodent model with a high molecular weight protein

oxygen therapeutic: Evidence and Implications

Acknowledgements

Virginia Rentko BioPure

Maria Gawryl BioPure

Stephen Harris SBH Group

Anthony Scialli Georgetown UMC

Bennett Varsho Lisa Snyder Mark Nemec Teresa Gleason Carol Kopp Saundra Coe

Symposium Objectives

Role in Safety Assessment of: Whole-embryo culture Non-rodent, non-primate species model

Extensive Review and Analysis of Pertinent Placentology Clarifications

Evaluation of New Information Regarding Early Human Extraembryonic Fluids and Membranes Possible misconceptions or a new and important understanding

Weight of Evidence Strongly Suggests: Large molecules with active moieties likely to disrupt inverted yolk sac

placenta (InvYSP) Most potential developmental toxicants would be small molecules that

would have access to the InvYSP, but without effect For this candidate therapeutic, HBOC-201, effects in the rat are specious

findings

Comparison of Anamniotes’ and Amniotes’ Idealized Conceptuses

Modified from Beck, 1976

Chorio-allantois

Chorion

Chorion

YS

EEC

AC

AL

Coelom

Yolk

Ectoderm

Mesoderm

Periblast

Vitelline Vessels

Yolk Sac

Colleagues (Past & Present) Contributing to the Visceral Yolk Sac Literature (Incomplete)

John Everett Robert Brent Thomas Koszalka Marcella Jensen David Beckman Felix Beck Craig Harris Marshall Johnson James Wilson Tom Sadler Nigel Brown George Daston A.E. Beaudoin

John Lloyd William Jollie Ronald Jensh C.K. Leung Stuart Freeman L.E. Kobrin M. Kernis Ed Carney Dennis New Joseph Holson G. Schlüter E.S. Hunter Tom Shepard

Types of Embryonal/Fetal Nutrition

1) Histiotrophic Extracellular material including cellular debris

secreted/deposited in space between maternal/embryonal surfaces in direct contact with trophectoderm Phagocytosis of histiotroph is considered to be a

characteristic of both cellular and syncytial trophoblast.

1) Hemotrophic Materials (O2, electrolytes, amino acids, etc.)

carried in the maternal blood

Wooding and Flint, in Marshall’s Physiology of Reproduction, 1994

Transport Processes in the Early Embryonal Adnexae

Diffusion Concentration dependent (Ficke’s Law)

Pinocytosis Invagination of cell membrane

Phagocytosis Cell engulfs target

Endocytosis Invagination of receptor-bearing membrane

Functions of the Yolk Sac

Human Embryos Hematopoiesis Produces albumin, pre-albumin, alpha-fetoproteins,

alpha-antitrypsin, transferrin, apolipoproteins Origin of gonial cells

Rat Embryos All of the above PLUS role as placenta Regulates volume of other extraembryonic studies

(Payne & Deuchar, 1972) Makes possible survival of explanted whole embryos

in culture

Comparative Early Placentation: Human and Rat Conceptuses

Amniotic Cavity

Extra-Embryonic Coelom

Decidua

Yolk Sac

Uterine Lumen

Uterine Artery

Decidua

Ectoplacenta

Allantois

Visceral Yolk Sac

Vascular Lacuna

Human Conceptus (Pre-Chorioallantoic Placental Stage) Day 10 Rat Conceptus

The inverted yolk sac surrounds rodent embryo but not human

Mark Hill, UNSW

Ida Smoak, UNC - Winner of Clarke Fraser Award

Comparative Definitive Placentation: Human and Rat Conceptuses

Amniotic Cavity

Extra-Embryonic Coelom

DeciduaYolk Sac

Uterine Artery

Decidua

Re-EstablishedUterine Lumen

Amniotic Cavity

Visceral Yolk Sac

VascularLacuna

Human Conceptus at the Time of Chorioallantoic Placental Establishment Day 12 Rat Conceptus

ChorioallantoicPlacenta

ChorioallantoicPlacenta

InvYSP may be site of effect in rodents, not existent in human conceptus

Mark Hill, UNSW

Mark Hill, UNSW

Chronology of Selected Earlier Reports on InvYSP and Trypan Blue

1909 Goldman Trypan Blue accumulates in vitelline epithelium

1927 Brunschwig proposed yolk sac is functional placenta

1935 Everett Predicted yolk sac as important as chorioallantoic placenta

1947 Noer & Mossman Said yolk sac was complementary to chorioallantoic placenta

1948 Gillman, Gilbert, Gillman & Spence Trypan Blue a teratogen

1967 Beck, Lloyd & Griffiths Trypan Blue site most likely yolk sac

1968 Davis & Gunberg Trypan Blue in GD 11, 12, 13 rat; Trypan Blue very small amounts

in gut epithelium 1976 Williams, Roberts, Kidson, F. Beck & Lloyd

Reported decreased pinocytocis in yolk sac by Trypan Blue

In the Rat Beginning on Gestational Day 14:

Most potential developmental toxicants, small molecules, will reach InvYSP, but as gestation progresses, redistribution of blood flow to uterus will result in diminished access/presentation to antimesometrial margins of conceptus.

Day 13 Rat Conceptus Treated w/Trypan Blue (GD 11) 6/2003

Day 12 Rat Conceptus Treated w/Trypan Blue (GD 11) 6/2003

On the Human Yolk Sac:

Yolk sac development in the human is unusual in that most of the cells of the primary yolk sac form extraembryonic mesoderm, and only a small secondary yolk sac is formed. Although this sac enlarges considerably such that it is larger than the embryo by about 22 days of gestation, the definitive yolk sac never contacts the trophoblast to form a yolk sac placenta. Nevertheless, the yolk sac is the site of blood cell formation, and some of the primordial germ cells arise near the base of the structure.

Enders & Blankenship, 1999

The yolk sac is probably nonfunctional with respect to “histotrophic” absorption in the primate. It atrophies early and is not usually visible at term in the umbilical cord.

Noden and de Lahunta, 1985

On the Human Yolk Sac:

CAP

CAP

Temporal Comparison of Early Development: Rat and Human

Rat

Human

Conception

Day 0

Day 0

5.5 - 7

6-13

Implantation

Primitive Streak

Appears

13.5

8.5 9

18

Neural Folds

To reach equivalent lengths – 3 mm – Human: 25 days vs. Rat: 9 days (From: O’Rahilly & Muller, 1987)

27

11.511

26

InvYSP InvYSP

First Somite Formed

First Heartbeat

9.5

19 23

Circulation Begins

10

Forelimb Bud

Chorioallantoic Placenta

Rodent Visceral Yolk Sac Toxicants

TABLE 1. Agents which inhibit rodent visceral yolksac function1

*Anti-rat visceral yolk sac sera

*Chloroquine *Trypan Blue

*Hyperglycemia *Leupeptin *Cadmium

Ammonium ions *Suramin *Aurothiomalate

Methylammonium Elastatinal 2,4-dinitrophenol

Ethylammonium Antipain Chymostatin

Rotenone Iron Dextran Thorium dioxide1Asterisks indicate having reported to be developmentally toxic in the rodent; each is an inhibitor of pinocytosis or lysosomal proteinases/hydrolases.

Modified from Beckman, et al., 1990, reported by G. Schlüter

Comparative Molecular Masses

Agent Molecular Weight

Water 0.018 kDa

Glucose 0.18 kDa

Leupeptin 0.48 kDa

Suramin 1.4 kDa

Inulin 5 kDa

hCG 38 kDa

Purified Bovine Hemoglobin 60 kDa

Trypan Blue + Albumin 1 kDa + 66 kDa = 67 kDa

Alpha-fetoprotein 70 kDa

IgG 150 kDa

Iron Dextran 165 kDa

HBOC-201 250 kDa (each hemoglobin ~ 60 kDa)

Profile of HBOC-201 Developmental Toxicity in Rat(Single-Day Infusions w/5.9 g/kg)

6 7 8 9 1110 12 13 14 15 16 17 18 19 20

100%

50%

Colored days indicate single treatments with HBOC-201

De

velo

pme

nta

l To

xici

ty

Gestational Day of Infusion

Dose-related inhibition of embryonal growth but not somite development.

Interference of proteolysis by 30-50% in visceral yolk sac placenta.

Inhibition of endocytosis by 70% at concentrations of 1-10% v/v.

No indication of oxidative stress.

Study of HBOC-201 on InvYSP Function in Whole-Embryo Culture: Summary of Findings

Rationale for Canine Model

Infusion of HBOC-201 prior to day 12 in the rat was incompatible with development (resulting in malformations or death).

Infusion studies in rats demonstrated that developmental disruption was attributable to prechorioallantoic phase of gestation.

Spectrum of malformations in rats resulting from infusion of HBOC-201 similar to other known inverted yolk sac placenta (InvYSP) “teratogens.”

Direct study of InvYSP in explanted whole-embryo culture confirmed interference with endocytosis/proteolysis which are essential for histiotrophic nutrition in rat embryo.

Infusion of 6.0 g/kg in pregnant dogs at stage of development (and developmental sensitivity) comparable to rodent studies were without effect.

Infusion of 6.0 g/kg in pregnant dogs at stage of development (and developmental sensitivity) comparable to rodent studies were without effect.

Size of the molecule too large for chorioallantoic placental transport.

Late-gestation sheep placental perfusion study indicated no transport of HBOC-201.

Maternal toxicity equal to or greater in dog than rat at 6.0 g/kg. AUC and Cmax in nonpregnant dogs comparable or greater

than rat. No evidence for a qualitative difference in elimination of

hemoglobin between the species.

Rationale for Canine Model (cont’d)

Generalized Implications from our Studies and Analysis

There should be no doubt that the InvYSP can be a target for toxicity leading to serious developmental disruption. To the contrary, it has not been demonstrated that the noninverted yolk sac is a similar target.

Caution should be exercised In generalizing too broadly the findings of studies of this product, which by design, was given at high doses (mass) of hemoglobin protein, 6 g/kg.

Large and/or proteinaceous agents 1) with no pharmacologic action on the biochemical modalities of the InvYSP or 2) which do not contain a moiety with toxic properties would not be expected to exert similar effects.

The former types of agents would appear to represent a small number of the universe of xenobiotics and no broad sense lessens the value of current models.