Upload
joseph-holson
View
312
Download
0
Tags:
Embed Size (px)
Citation preview
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.