Prenatal Drug

Exposure:

Kinetics and

Dynamics

U.S. DEPARATMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Menial Health Administration
Prenatal Drug Exposure:

Kinetics and Dynamics

Editors:

C. Nora Chiang, Ph.D.

Charles C. Lee, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

NIDA Research Monograph 60

1985

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857
NIDA Research Monographs are prepared by the research divisions of the

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Temple University School of Medicine

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NB Associates. Health Sciences

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Gilbert J. Botvin, Ph.D.

Cornell University Medical College

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Joseph V. Brady, Ph.D.

The Johns Hopkins University School of Medicine

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Theodore J. Cicero, Ph.D.

Washington University School of Medicine

St. Louis, Missouri

Sidney Cohen, M.D.

Los Angeles, California

Reese T. Jones, M.D.

Langley Porter Neuropsychiatric institute

San Francisco, California

Denise Kandel, Ph.D.

College of Physicians and Surgeons of Columbia University

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Yale University School of Medicine

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NIDA Research Monograph Series

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Prenatal Drug Exposure:

Kinetics and Dynamics
ACKNOWLEDGMENT

Dr. Charles C. Lee, coeditor of this monograph, was on sabbatical

leave from the University of Houston during 1984 and serving as a

member of the professional staff with the Division of Preclinical

Research, National Institute on Drug Abuse, by an agreement under

the Intergovernmental Personnel Act of 1970.

This monograph is based upon papers presented at a technical review

on prenatal drug exposure and consequences of maternal drug use

which took place on September 24-25, 1984, at Bethesda, Maryland.

The meeting was sponsored by the Division of Clinical Research and

Division of Preclinical Research, National Institute on Drug Abuse.

The clinical papers have been published separately as NIOA Research

Monograph 59, Current Research on the Consequences of Maternal Drug

Abuse.

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The U.S. Government does not endorse or favor any specific commercial product or company. Trade or company names appearing in this

publication are used only because they are considered essential in

the context of the studies reported herein.

DHHS publication number (ADM)85-1413

Printed 1985

NIDA Research Monographs are indexed in the Index Medicus.

selectively included in the coverage of American Statistics

Biosciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology

Abstracts.

They

Index

are

iv
Contents

Introduction and Overview

C. Nora Chiang and Charles C. Lee 1

Animal Models for Study of Fetal Drug Exposure

Abraham M. Rudolph 5

Biotransformation of Drugs and Foreign Chemicals in the Human

Fetal-Placental Unit

M.R.Juchau 17

Pharmacokinetics of Drugs and Metabolites in the MaternalPlacental-Fetal Unit: General Principles

Laurene H. Wang, Abraham M. Rudolph, and Leslie Z. Benet 2 5

The Pharmacodynamics of Prenatal Chemical Exposure

Richard K. Miller and Carol K. Kellogg 39

Opioids and Development: New Lessons From Old Problems

Ian S. Zagon 58

Pharmacodynamics of Fetal Exposure to Narcotics

Hazel H. Szeto and Jason G. Umans 7 8

Positron-Emission Tomography: A New Approach to Feto-Maternal

Pharmacokinetics

Bo S. Lindberg, Per Hartvig, Anders Lilja, Hans Lundqvist.

Bengt Langstrom, Petter Malmborg, Anders Rane, Annika Rimland,

and Hans Svard 88

Placental Transfer of Drugs, Alcohol, and Components of

Cigarette Smoke and Their Effects on the Human Fetus

Betty R. Kuhnert and Paul M. Kuhnert 9 8

Appendix--Maternal-Fetal Transfer of Abused Substances:

Pharmacokinetic and Pharmacodynamic Data

Charles C. Lee and C. Nora Chiang 110

List of NIDA Research Monographs 148

v
Introduction and Overview

C. Nora Chiang and Charles C. Lee

It has become obvious from both animal experiments and clinical

observations that the placenta does not act as a barrier to protect the

fetus from exposure to xenobiotics taken by the mother. Nearly all drugs

or foreign chemicals, following maternal exposure, enter the fetus and

may exert adverse effects, depending on the reactivity (dynamics) of the

drug in the fetus and the extent of fetal drug exposure, which may be

assessed from the kinetics of the drug in the maternal-fetal unit. The

fetus may be affected through the direct action of a drug as well as

indirectly due to drug effects on the mother. Hence, it is a primary

health concern to understand the adverse effects of a drug, taken by the

mother, on the fetus. In addition, it is of paramount importance to

determine the kinetics and the dynamics of a drug in the maternal-fetal

unit in order to elucidate the mechanisms of drug action.

Studies in humans regarding drug effects due to prenatal exposure are

usually limited because of ethical and technical considerations. Such

studies are usually restricted to clinical observations of pregnancy

outcomes or the determination of drug concentrations in maternal and

fetal biological fluid samples collected at the time of delivery. Much of

the research assessing the potentially harmful effects of prenatal drug

exposure, and particularly the mechanisms of such effects, is performed

in animals. Because of the continuous morphological and physiological

alterations during pregnancy in the mother, placenta, and fetus, both the

kinetics and dynamics of a drug in the maternal-fetal unit may change

progressively. Studies on the effects and mechanisms of drug action in

the fetus are therefore very complicated and involve a diverse range of

disciplines. Furthermore, there are questions concerning the extrapolation of animal data to humans since there are anatomical differences

in the structure of placenta and species differences in the pharmacologic

action (dynamics) and the disposition (kinetics) of a drug.

The experimental complexity and broad spectrum of prenatal pharmacology and toxicology require the exchange of information as well as

integrated efforts among scientists from different disciplines, including

clinical research, chemistry, biochemistry, pharmacokinetics, pharmacology, and physiology. To coordinate an effort for information exchange

1
and interdisciplinary interaction, the National Institute on Drug Abuse

sponsored a technical review on September 24 and 25, 1984. This volume

presents the proceedings of the preclinical portion of the technical

review. It focuses primarily on the current research findings of prenatal

drug effects and current approaches for the investigation of mechanisms

of prenatal drug effects through applications of state-of-the-art

technology. In addition, an appendix containing a comprehensive review

of literature data regarding the dynamics and kinetics of drugs in the

maternal-fetal unit has been added as a supplement. Research articles

presented of the clinical portion of the meeting are collected in a

separate volume entitled “Current Research on the Consequences of

Maternal Drug Abuse.”

Various animal preparations have been used for the investigation of

prenatal drug effects. An overview of animal models was presented by

Dr. Rudolph. The presentation highlighted the pregnant ewe as an animal

model for quantitative assessment of the extent of drug exposure and the

intensity of pharmacological/physiological responses of the fetus.

Techniques for the preparation of the pregnant ewe for such investigations were illustrated. This preparation is highly praised and indeed

provides a useful model for studies of drug effects in the fetus.

Nevertheless, an extrapolation to the human situation must be made with

caution.

Drug disposition plays an important role in the elicitation or termination

of a pharmacologic effect. The biotransformation of drugs or other

exogenous chemicals in the placental-fetal unit was presented by Dr.

Juchau. Recent findings indicate that fetal liver and placenta of human

and nonhuman primates develop drug-metabolizing enzyme activities

during gestational maturation, while other subprimate animals in general

lack enzyme activities at this stage. One set of drug-metabolizing

enzymes (monooxygenases) in the placenta has been found to exhibit

increased activity in tobacco smokers. Preliminary evidence suggests

that these enzymes may catalyze the generation of highly reactive

metabolic intermediates capable of eliciting deleterious effects in the

fetus and newborn. Since research in this area is still in a developmental

stage, continuing efforts are required to establish the toxicological or

pharmacological significance of these drug-metabolizing enzymes.

The application of pharmacokinetics, a quantitative description of the

time course of a drug in the body, to studies of drug transport mechanisms

between the mother and fetus and the elicited pharmacological effects

was discussed by Dr. Benet. Using the ewe model, pharmacokinetic

principles were demonstrated with acetaminophen as a model drug for

studies of the transfer of drug and metabolites between the mother and

fetus as well as fetal disposition of all chemical entities. Pharmacokinetics, as applied in drug abuse research, will certainly help to provide

insight into the extent of fetal exposure and toxic consequences.

Prenatal effects of diazepam on rodents were discussed by Dr. Miller.

Reports indicate that persistent and selective alteration of the central

nervous system is induced in rats following low-dose diazepam exposure

during the last week of gestation and that coadministration of a diazepam antagonist effectively prevents these alterations. The data suggest

2
that administration of diazepam during pregnancy may result in permanent, selective, receptor-mediated alterations in behavioral and

transmitter functions in the neonate.

The overall opiate effects on the somatic and neurobiological development of the fetus and/or the infant were summarized by Dr. Zagon.

Laboratory investigations revealed that the actions of opiate agonists

were stereospecific and subject to blockade by coadministration of opiate

antagonists. This suggests that the locus of opiate action is at the level

of the opiate receptor. Recent research findings, which suggest that

opioid antagonists affect fetal development, led to the speculation that

endogenous opioid peptides may serve to regulate growth through

interaction with opiate receptors. The role of endogenous opioids on fetal

development clearly needs further investigation.

The adverse effects of opioids on the offspring due to maternal drug

exposure have been well documented, although the mechanisms of action

are not clear. In utero monitoring, utilizing the pregnant ewe model, of

the direct effects of morphine exposure on fetal behavioral, respiratory,

and cardiovascular functions was presented by Dr. Szeto. Results showed

that morphine exhibited a biphasic dose-response relationship, excitation

at low dose and suppression at high dose, and the fetal narcotic abstinence

symptom was precipitated in utero after a short 2-hour low-dose

morphine exposure. These findings suggest that the fetus may undergo a

cycle of acute physical dependence and withdrawal when opiates are

administered to normal clinical patients. Further research in this area is

needed to provide further insight into mechanisms for the neurobehavioral

disturbances observed in children born to pregnant narcotic users.

Noninvasive methods are clearly preferable for the study of drug distribution in maternal and fetal tissues. The use of the new and noninvasive

technique, Positron Emission Tomography (PET) for the study of morphine

and heroin distribution in pregnant monkeys was illustrated by Dr.

Lindberg. Following drug administration, both drugs readily distribute to

the fetus, with heroin reaching the fetus more rapidly than morphine. In

combination with pharmacokinetic modeling, PET may prove to be a

powerful tool for the evaluation of rapid distribution and disposition of

drugs in the maternal-fetal unit. It is also a potentially useful means for

the determination of specific sites of drug action in the fetus.

Clinical reports on infants born to tobacco-smoking mothers suggest

several adverse effects. According to Dr. Kuhnert, three components of

cigarette smoke--cadmium, lead, and thiocyanate--were present at

substantially higher than normal concentrations in the newborn of

cigarette smokers. These components were shown to exhibit some

deleterious biological activities. For example, cadmium was shown to be

placental-toxic in both animal studies and in the in vitro human placenta

preparation, as presented by Dr. Miller. The factors that clinically affect

the infant or fetus are not yet identified. Dr. Kuhnert also reported

adverse effects for infants born to mothers who used phencyclidine,

meperidine, or alcohol during pregnancy. Quantitative analysis of these

drugs in the biological fluids of infants at birth demonstrated their

presence.

3
All data gathered to date support the notion that nearly all drugs taken by

the pregnant mother readily cross the placenta to the fetus. Research

efforts to determine the concentrations of drugs/chemicals in body fluids

of the newborn of maternal drug users have provided valuable information

for the assessment of potential adverse effects to the infants. These

efforts should be continued with particular emphasis on the application of

new analytical technologies which enable drug determination in microliter

volumes of sample fluid.

It is well recognized that information on perinatal effect of drugs of

abuse is very limited. In many instances, the available data are confusing

or contradictory. Discrepancies may be attributed to different methodologies employed, different animal species studied, and varying routes of

drug administration, doses, and dosage forms. Despite the limitations of

the animal model, it is generally agreed that animal studies do provide

valuable information on prenatal drug effects. It is recommended,

however, that researchers be cautious about the selection of the animal

model as well as the route, dose, and dosage form of the drug to be

administered. It is further emphasized that a dose-response curve should

be generated for the selection of a proper dose for the animal studies, as

some drugs may exert multiphasic pharmacological effects.

In summary, it is clear that further research efforts are needed in this

critical area to further contribute to the substantial base of knowledge on

prenatal drug effects due to maternal drug exposure which has been

generated over years of research. As new technologies advance, more

sophisticated means for the investigation of the mechanisms of drug

action can be implemented. Of particular importance is integrated

research involving interdisciplinary collaboration which will certainly

contribute to the overall understanding of the preclinical and clinical

facets of the subject. Combining clinical and laboratory findings can be

anticipated to yield an improved basis for the prediction and minimization

of toxicities in humans through more effective treatment and prevention

efforts on the part of both physicians and the lay public.

AUTHORS

C. Nora Chiang, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857

Charles C. Lee, Ph.D.

University of Houston

Texas Medical Center

1441 Moursund Ave.

Houston. Texas 77030

4
Animal Models for Study of Fetal

Drug Exposure

Abraham M. Rudolph

Increasing concerns have developed regarding the potential harmful effects

on fetal developrnent of drugs injected into or ingested by the mother, and

from her exposure to chemicals encountered in occupations or the environment. Much of the earlier work regarding these potential hazards was

done in humans, and involved the detection of drugs or their metabolites in

umbilical cord blood at the time of delivery, or in urine in the newborn

infant soon after birth. Although detection of the parent drug reflected

passage across the placenta, the presence of metabolites did not confirm

that the drug itself was transferred, because the placenta may have been

permeable to the metabolite but not to the drug itself. Additional earlier

studies in the human were concerned with the effects that drugs taken by

the mother had on fetal function. Moya and Thorndike (1962) reviewed the

work done on detection of drugs in umbilical cord blood, and on observation

of behavior and certain physiological parameters of the human infant after

birth.

Another major interest in drug effects on the fetus concerned the

examination of their teratogenic effects. Considerable attention has been

directed toward studying the potential of drugs administered to the mother

at different stages of embryonic and fetal development to induce

congenital defects or even embryonic or fetal death. Most of this work has

been done in small rodents (Warkany and Takacs 1959). In addition, much

knowledge has been derived from examination of the coincidental

occurrence of specific congenital defects and administration of certain

drugs to the human mother.

More recently, interest has been directed toward obtaining more specific

information about factors that influence drug transfer across the placenta

and drug distribution in the fetus, and about the effects of drugs on fetal

physiological and biochemical functions. The perfused human placenta in

vitro has been used extensively to examine transfer of metabolites and

drugs (Schneider et al. 1972). There are several major concerns regarding

these studies. First, the perfusion medium has usually been an electrolyte

solution, sometimes with low molecular weight dextran; the flow rates are

usually quite low compared with what might be expected in vivo raising

questions about the degree of vasoconstriction present. Also, the duration

of viability of the preparation has not been established.

5
Physiological responses of the human fetus to drugs have been difficult to

assess. Heart rate responses can be monitored, and Schifferli and

Caldeyro-Barcia (1973) studied effects on fetal heart rate after administering atropine or beta-adrenergic drugs to the mother. Recently, it has

also become possible to monitor human fetal respiratory movements by

ultrasound techniques, and it will be possible to examine the effects of

drugs on this activity.

In this presentation, I will not address the use of animal models for

assessing the teratogenic effects of drugs. I plan to discuss some models

that have been used, and some that have the potential to be used for

studying pharmacokirretics and pharmacodynamics in the fetal placental

unit, and to address some of the limitations of these studies. The following

issues will be considered, along with models available for their study:

1. Placental transfer of drugs and their metabolites;

2. Fetal distribution;

3. Intrauterine distribution and excretion of drugs and metabolites;

4. Fetal drug metabolism;

5. Pharmacological effects of drugs and metabolites.

PLACENTAL TRANSFER

The influence of physical properties on the diffusibility of substances

across the placenta has been reviewed by Faber (1973). Lipid soluble

materials diffuse readily across the placenta. Rate of movement is largely

affected by umbilical and uterine blood flow rates rather than by

permeability, because this determines the rate of delivery and removal of

the substance at the exchange site. However, transfer of lipid insoluble

substances is dependent on molecular size and on the morphology of the

placental membrane. The rate of transfer of lipid insoluble substances is

proportional to their coefficient of free diffusion in water. If molecular

size is very large, there may be no diffusion. Since placental morphology is

very different in different species, pore size will be quite variable, and

diffusibility is markedly affected. Thus, the rabbit and guinea pig have a

hemoendothelial placenta: the fetal and maternal blood streams are

separated only by fetal endothelium. Because of this, molecules larger

than plasma albumin can diffuse across the placenta. The sheep placenta is

syndesmochorial; it has five layers separating fetal and maternal blood:

maternal endothelium and connective tissue, and fetal trophoblast,

connective tissue, and endothelium. The placentae of the human and the

primate are hemochorial, consisting of three fetal layers. Thus, because of

the histology of the membrane, the sheep placenta presents a considerably

greater diffusion barrier than the human placenta, which in turn is less

permeable than the rabbit or guinea pig placenta. These potentially

enormous differences in placental diffusing capacity must be appreciated

in experimental studies of drug transfer.

Diffusion rate is dependent on the mean concentration difference between

maternal and fetal blood. As Faber points out, the relevant concentrations

are those of physically dissolved material, not the total content. Thus,

protein binding of drugs. will have a marked effect on placental transfer,

either from mother to fetus, or fetus to mother. Potentially, fetal and

maternal protein-binding capacities may be different, apart from the fact

that, at least in some speceies, albumin concentrations are lower in fetal

blood.

6
Several animal models have been used to study drug transfer across the

placenta. Mirkin (1973) described a technique for examining transfer and

tissue distribution of a drug in the fetus by injection of an isotopically

labeled drug into the mother. After killing the animals at various intervals

after injection, whole fetuses were examined by radioautography.

Although this technique does demonstrate which fetal tissues are labeled,

there are several drawbacks. First, only small rodents can be used; second,

caution would have to be exercised that the tissue labelling was not caused

by passage of labeled metabolites rather than the parent drug; and third,

the technique does not provide quantitative data regarding drug transfer.

Several investigators have used techniques for studying drug transfer to

and from the fetus by placing intravascular catheters in the fetus and

mother, and administering drugs either to the fetus or mother. Maternal

and fetal plasma concentrations were measured either after a bolus

injection or after infusion. Many of these studies were done acutely in

anesthetized animals; this raises serious concerns regarding the reliability

of any quantitative measurements of transfer rates because anesthesia or

acute stress could greatly modify circulatory dynamics in both mother and

fetus and also influence hormonal and metabolic activity. Fouron (1973)

studied placental transfer of digoxin in the dog acutely under anesthesia

after either acute or chronic administration of the drug. Cosmi (1976)

studied transfer of anesthetic agents and other drugs in sheep; and

Ruckebusch et al. (1976) reported on the transfer of thiopental,

meperidine, and acepromazine (a phenothiazine) in sheep, as well as

monitoring some fetal responses to these drugs. Also, Szeto et al. (1978)

studied some aspects of pharmacokinetics of meperidine in the fetus and

mother in a chronically catheterized pregnant sheep model.

Numerous studies of similar type have been performed in chronically

catheterized fetal and maternal sheep, in which a fetal hindlimb artery and

vein and maternal artery and vein have been catheterized. This model is

useful for determining the rapidity of transfer of drugs in either direction

across, the placenta and the relative fetal and maternal plasma concentrations. Thus, Morishima et al. (1972, 1975) showed that lidocaine, when

injected into the mother, appears in fetal plasma very quickly. With this

model, it is also possible, after a bolus of the drug is injected into the

fetal or maternal circulation, to determine its biological half-life in the

fetus or mother, as well as the presence of, and rates of disappearance of,

metabolites. However, the information derived does not differentiate

between actual placental transfer and removal by metabolism or excretion

by other routes.

Transfer rates of drugs across the placenta can be measured more specifically by application of the Fick principle. Meschia et al. (1966) first

applied this approach to measure umbilical and uterine blood flows in

pregnant sheep by infusing antipyrine into the fetus. The rate of removal

of drug to the uterus and its contents is measured by the equation

A= Qut x (Cuta - Cutv)

where A is amount removed Qut is uterine blood flow, and Cuta and Cutv

are concentrations of drug in uterine artery and vein at steady state. This

technique has limitations because the sheep uterus has several draining

veins, and sampling from a single vein may not be representative of mixed

7
uterine venous blood. Also, uterine venous blood is a mixture of blood

derived from placental site perfusion as well as uterine musculature.

Myometrial blood flow in the sheep in mid- to late-gestation accounts for

only about 15% of total uterine blood flow, but this must be considered in

estimates of placental transfer.

The amount of a drug that is removed or taken up at the placenta on the

fetal side can be calculated similarly from the equation

A = Qun x (Cumv - Cuma)

where Qum is umbilical blood flow and Cumv and Cuma are concentrations

of drug in umbilical vein and artery, respectively, at steady state. Problems with umbilical venous sampling may present difficulties in making

accurate measurements. A technique used commonly to catheterize the

umbilical vein is to pass a catheter from a peripheral cotyledonary vein

centrally into one of the two main veins (Rudolph and Heymaun 1980). It is

possible that the sample obtained may not reflect mixed umbilical venous

blood. To overcome this, we developed a method for chronically catheterizing the common intra-abdominal segment of the umbilical vein (Young

et al. 1974). but recent experience has shown that there is no significant

difference in concentrations in the common vein as compared with the two

main veins.

Uterine and umbilical blood flow can be measured by several techniques.

The antipyrine steady-state diffusion method mentioned above suffers

from the inability to make repeated measurements after any manipulation

without waiting a considerable period for a new steady state to be

achieved. Techniques for applying electromagnetic flow transducers to the

vessels have been described for both the mother and fetus. Flow

transducers can be applied to the branches of the uterine artery, but to

measure total flow, the transducer can be applied to the distal aorta

beyond the origins of the common iliac arteries. In the sheep, this segment

of the aorta gives rise to the two uterine arteries, from which also arise

some small branches to the pelvis. Thus, measurement of flow at this site

slightly overestimates the uterine blood flow. In the fetal lamb, we

demonstrated that the umbilical arteries arise from a short distal segment

beyond the common iliac artery origin. We have described the technique

for applying a flow transducer to this segment to measure total umbilical

blood flow (Berman et al. 1975). Because some small arteries arising from

the umbilical arteries supply pelvic tissue, the measured flow slightly

overestimates actual umbilical blood flow. The great advantage of this

technique is that blood flow can be measured instantaneously and rapid

alterations can be monitored.

The radionuclide-labeled microsphere technique is a useful method for

measuring both umbilical and uterine blood flows (Rudolph and Heymann

1967; Heymann et al. 1977). In the mother, a catheter must be introduced

retrograde through a peripheral artery and advanced to the left ventricle.

If the placental portion of the uterus is separated from the surrounding

myometrium at autopsy, it is possible to measure uterine blood flow to

each portion separately. In the fetus, the microspheres can be injected

into a peripheral vein while a reference sample is collected from the

descending aorta or one of its branches. It is also feasible to measure

umbilical blood flow to the placental cotyledons separate from the

amniotic and chorionic membranes.

8
The placenta has high metabolic activity and a fairly large volume. Drugs

delivered to the placental site either from the maternal or fetal

circulations will be distributed in the placental volume. Also, some of the

drug may be metabolized by the placenta. Thus, even at steady state, the

calculated uptake of drug by the placenta on the fetal side may not equal

the calculated amount leaving the uterine circulation at the placental site.

The difference is a reflection of placental removal of drug.

It should also be appreciated that the uptake or release calculated from the

Fick equations above represents net transfer. Some of the drug in the

umbilical artery may cross to the maternal circulation, whereas some in

the uterine artery may enter the fetal circulation.

FETAL DISTRIBUTION AND EXCRETION OF DRUGS AND METABOLITES

The fetal circulation is characterized by the presence of shunts which

provide preferential distribution of oxygen and substrates to upper body

organs and also function as a partial bypass of the lungs. The

radioautography studies developed by Mirkin (1973) are not quantitative

enough to define distribution patterns of drugs in fetal tissues. The blood

flow patterns in the fetus are important to our understanding of how drugs

and chemicals delivered to the fetus across the placenta into umbilical

venous blood are distributed in the fetus. It is also important in

experimental studies in which drugs are infused directly into the fetus for

pharmacokinetic or pharmacodynamic studies. Furthermore, because of

the shunting, arterial concentrations of drugs may be very different in

upper and lower body arteries.

The patterns of blood flow in major venous channels have been studied in

fetal lambs by applying the radionuclide-labeled microsphere technique.

Simultaneous injection of microspheres into various peripheral veins has

defined the flow patterns shown in figure 1. Umbilical venous blood

contributes about 40% of total venous return; about 55% normally passes

through the ductus venosus, thus bypassing the liver, while the remainder is

distributed to the left and right lobes of the liver. Portal venous blood is

distributed exclusively to the right lobe of the liver (Edelstone et al.

1978). Distal inferior vena caval blood is joined by blood from the left and

right hepatic veins and the ductus venosus just below the diaphragm.

However, blood does not mix completely in the thoracic portion of the

inferior vena cava. We have shown that ductus venosus blood streams

preferentially through the foramen ovale to the left atrium, whereas distal

inferior vena caval blood preferentially flows through the tricuspid valve

into the right ventricle (Reuss and Rudolph 1980; Edelstone and Rudolph

1979). Superior vena caval blood passes almost exclusively through the

tricuspid valve into the right ventricle. Right ventricular blood largely

flows through the ductus arteriosus to the descending aorta, while left

ventricular blood is ejected into the ascending aorta. These flow patterns

account for the higher oxygen saturation in ascending aortic blood as

compared with descending aortic blood in the fetus, and also could result in

higher concentrations of drugs in ascending aortic blood when they enter

the fetus through the placenta.

We have also shown that left hepatic venous blood follows the preferential

streaming pattern of ductus venosus blood, whereas right hepatic venous

blood is distributed in a similar manner to distal inferior vena caval blood.

9
VENOUS FLOW PATTERNS IN THE FETAL LAMB

Figure 1. The course and distribution of venous blood entering the heart

in the fetus is depicted. Umbilical venous blood enters the hepatic hilum

and gives off branches to the left lobe. The ductua venous carries

predomanantley umbilical venous blood driecly to the inferior vena cava.

The umbilical vein then the arches, to the right to join the portal vein. Almost

all portal vnous blood enters the right lobe of the liver. The right hepatic

venous, blood, joins the abdominal inferior vena caval stream to be

preferentilly distributed through the tricuspid valve. Left hepatic venous

blood joins the ductus Venous stream and these two well-oxygenated

bloods are preferentially directed through the foramen orale. The superior

vena caval blood is almost completely directed through the tricuspid calve

(from Rudolph 1983, copyright 1983, American Association for the Study

of Liver Disease)

10
These distribution patterns could greatly influence concentrations of drugs

reaching different organs, depending on their route of administration and

on the rate of metabolism in various organs. Thus, if a drug enters the

umbilical circulation it first reaches the fetal liver, but about half passes

directly through the ductus venosus. If the drug is rapidly metabolized by

the liver, up to half of that entering the fetus may be removed on the first

pass. If the drug is administered through the portal vein, it will reach the

right lobe of the liver in highest concentration, with much lower

concentrations reaching the left lobe of the liver. When a drug is infused

into a forelimb vein, it will not be distributed to the brain or heart on the

first pass, and if the drug is readily removed by the placenta, large

amounts may be necessary to have an effect on the heart or brain. In the

same fetus, drugs infused at the same ratio could thus exert very different

effects, depending on the route of administration.

INTRAUTERINE DISTRIBUTION AND EXCRETION

In addition to distribution of drug administered to the mother into the fetal

body, some drugs may enter the chorioallantoic membranes and amniotic

and allantoic sacs. Exchanges between allantoic and amniotic fluids and

the fetal or maternal circulations are not clearly established. The

amniotic fluid surrounds the fetus, but the allantoic sac occupies a small

segment of the uterine space. In the latter trimester of the sheep, the

allantoic space is well defined in the horn of the uterus and in the cervical

region, but in the body of the uterus the allantoic and amniotic membranes

are closely applied and there is little allantoic cavity in this region (Mellor

1980). Because of this, there could well be poor mixing within the allantoic

cavity. Similarly, although the fetus is surrounded by amniotic fluid, the

degree of mixing of substances that enter the amniotic fluid is not well

established. In early gestation in the sheep, the fluid is a clear liquid and

mixing may be good, but beyond about 120 days gestation the fluid becomes

increasingly viscid and considerable mucus is present, and distribution of

materials added to it may be quite poor.

It is possible to place catheters into the amniotic and allantoic sacs and to

maintain them chronically. The amniotic catheter can be readily inserted

adjacent to the fetal body. To catheterize the allantoic sac, the muscle of

the horn of the uterus is carefully incised and the allantoic cavity is

entered; it is recognized by the presence of clear yellowish fluid, in

contrast with amniotic fluid, which is often turbid and, in later gestation,

viscid. Also, the cotyledonary branches of the umbilical arteries and veins

are clearly visible on the chorionic membrane.

The allantoic and amniotic spaces may have a prominent role in kinetics of

the parent drug or its metabolites. if the drug or metabolite enters these

spaces, it could be reabsorbed into the maternal or fetal circulation. The

drug or metabolite may enter the amniotic sac from fetal urinary output

via the urethra, or the allantoic sac from urine via the urachus. In

addition, drugs may enter the amniotic fluid in tracheal fluid, secreted by

the lung, or possibly via the gastrointestinal tract, but the latter source is

not likely.

The excretion of drug or metabolites by the fetal kidney can be measured

readily in the lamb fetus in which a catheter is implanted chronically into

the bladder. This can be done directly through a suprapubic abdominal

11
incision (Iwamoto and Rudolph 1983), but can also be readily accomplished

by passing a catheter through the urachus either intra-abdominally or as it

enters the umbilical cord, external to the umbilical ring. I now prefer the

urachus approach because, since the urachus is ligated, urine will not enter

the allantoic sac. Also, to be sure that no urine enters the amniotic sac,

the urethra can be ligated at the skin surface. If this is done, the urinary

catheter must be allowed to drain, so that retention does not occur. When

catheters are placed in the bladder and in the allantoic and amniotic sacs,

it is possible to measure the urinary excretion rates of drugs and their

metabolites, and also examine their rates of entry and removal from the

allantoic and amniotic fluid. As mentioned above, because there may be

problems of even distribution of substances in the allantoic and amniotic

fluid, the information regarding drug and metabolite concentrations and

kinetics in the allantoic and amniotic sacs may not be precise.

FETAL DRUG METABOLISM

A great deal of work has been done on developmental aspects of enzymes

involved in drug metabolism, but most studies have concentrated on

detection of enzymatic activity in vitro in microsomal fractions or

homogenates of fetal organs, or in fetal cell cultures. This work has been

reviewed extensively by Dutton (1978). However, the techniques used may

possibly interfere with activity of some enzymes and, furthermore, may

activate a11 enzyme present in inactive form, but which may not be active

in vivo. Study of metabolism of drugs by the fetus in vivo have taken two

directions. First, analysis of metabolites of the drug in fetal blood provide

an indication that the fetus has the capacity to metabolize the drug,

assuming that the metabolites do not cross the placenta. If they do cross

the placenta, it is difficult to determine whether they are derived from

maternal metabolism. Also, measurement of metabolite concentration

does not indicate where the drug is being metabolized.

We have developed a preperation ion in fetal lambs which allows the direct

measurement of rates of metabolism by the liver. Catheters are implanted

into a lower limb artery and vein, an umbilical vein, a mesenteric venous

tributary of the portal vein, and a left or right hepatic vein. These

techniques have been reported previously (Bristow et al. 1981). Blood flow

to each lobe of the liver from the umbilical veins, to the portal vein, and to

the hepatic artery can be measured using the radionuclide-labeled

microsphere method. By measuring concentrations of drugs or metabolites

in the various vessels and using the blood flow data, rates of hepatic uptake

or release can be calculated by the Fick method. We have shown that, in

the fetus, only a very small percentage of liver blood supply is derived

from the hepatic artery, and it can readily be discounted in these

calculations. Thus, for the left lobe

A = QLuv x Cuv - QLuv x CLhv

and for the right lobe

A = (QRuv x Cuv + QRpv x Cpv) - (QRuv + QRpv) x CRhv

Where A is the amount taken up or released, QLuv is blood flow to the left

liver lobe from umbilical vein, and Cuv and CLhv are concentrations of

drug or metabolites in umbilical venous and left hepatie venous blood.

12
Q Ruv and QRpv are blood flows to the right liver lobe from the umbilical

and portal veins. CUV, CPv and CRhv are concentrations in umbilical vein,

portal vein, and right hepatic vein.

These calculations depend for reliability on adequate concentration

differences across the liver; if this does not occur, it may be necessary to

use radioisotopically labeled drugs, with measurement of specific activity

of metabolites in hepatic venous blood.

Recently, we have also developed a preparation in fetal lambs in which the

renal vein is catheterized chronically (Iwamoto and Rudolph 1983). In

association with lower limb artery catheterization and measurement of

renal blood flow either by the microsphere method or by clearance

techniques, the rate of uptake of drugs or release of metabolites from the

kidney can be calculated.

PHARMACOLOGICAL EFFECTS OF DRUGS AND METABOLITES

Numerous fetal physiological and biochemical functions can now be

monitored in the chronically instrumented fetal lamb. Heart rate and

arterial pressure can be monitored from intraarterial catheters, or heart

rate and electrocardiogram can be recorded from chronically implanted

ECG leads sewn to fetal skin. Venous pressures or atrial and ventricular

pressures can be recorded from catheters inserted directly, or via

peripheral vessels.

Continuous measurement of blood flow in several fetal vessels has been

achieved by chronic implantation of electromagnetic flow transducers

around the vessel. Thus, umbilical blood flow can be measured by placing a

flow transducer around the common umbilical artery (Berman et al. 1975)

or the common umbilical vein (Oakes et al. 1976). Transducers around the

pulmonary trunk measure right ventricular output and around the ascending

aorta measure left ventricular output minus coronary flow (Rudolph and

Heymann 1973). The fetal circulatory effects of drugs administered to the

mother or fetus can be examined in these preparations. However, when

drugs are administered to the mother, fetal responses may not be a direct

effect, but may result from interference with uterine blood flow, causing

fetal hypoxemia and possibly aeidemia. Also, drugs that may depress the

fetal circulation or constrict the umbilical-placental vessels could cause

hypoxemia as a result of inadequate oxygen delivery. It is therefore

advisable to exarnine the effects of drugs administered directly to the

fetus as well as to the mother. Also, maternal cardiovascular variables

such as blood pressure and heart rate, and particularly uterine blood flow,

should be measured to assess maternal responses.

The radionuclide-labeled microsphere technique has greatly advanced the

opportunity to exarnine fetal cardiovascular responses to drugs (Rudolph

and Heymann 1967). Injection of microspheres with two different labels

into an upper and lower limb vein with simultaneous sampling from upper

and lower limb arteries allows measurement of cardiac output and blood

flow to every organ and tissue and to the placenta. Currently, we can

discriminate nine different gamma emitters, so that it is possible to obtain

a control measurement and the effects of a drug at three different

concentrations, if desired. The changes in vascular resistance in each

organ can be assessed.

13
Some of the effects of drugs on the central nervous system can be

examined by implanting leads to measure electrocortical activity or

electroocular activity and limb or nuchal muscle movement (Ruckebusch et

al. 1976).

The effects of drugs on renal function can readily be examined in the

preparation we reported (lwamoto and Rudolph 1983). Renal blood flow

and urinary output, as well as urinary electrolyte composition, can be

examined. Biochemical and endocrine effects of drugs can be studied by

measuring plasma concentrations of metabolic substrates or hormones.

This review indicates that there have been considerable advances in the

ability to study pharmacokinetics and pharmacodynamics of drugs in the

fetus and mother during pregnancy. At present, the pregnant sheep

preparation provides a useful model for drug studies. It must be

appreciated, however, that there are important species differences in

placental function. Furthermore, the state of maturation of the developing

fetus varies greatly in different species at the time of birth. It is

therefore not appropriate to assume that drug effects can be compared at

similar proportions of the total gestational period.

REFERENCES

Berman, W., Jr.; Goodlin, R.C.; Heymann, M.A.; and Rudolph, A.M. The

measurement of umbilical blood flow in fetal lambs in utero. J Appl

Physiol 39:1056-1059, 1975.

Bristow, J.; Rudolph, A.M.; and Itskovitz, J. A preparation for studying

liver blood flow, oxygen consumption, and metabolism in the fetal lamb

in utero. J Dev Physiol 3:255-266, 1981.

Cosmi, E.V. Drugs, anesthetics and the fetus. In: Scarpelli, E.M., ed.

Reviews in Perinatal Medicine. Vol. 1. Baltimore: University Park

Press, 1976;. pp. 191-253.

Dutton, G.J. Developmental aspects of drug conjugations with special

reference to glucuronidation. Am Rev Pharmacol Toxicol 18:17, 1978.

Edelstone. D.I., and Rudolph, A.M. Preferential streaming ductus

venosus blood to the brain and heart in fetal lambs. Am J Physiol

237:H724-H729, 1979.

Edelstone, D.I.; Rudolph, A.M.; and Heymann, M.A. Liver and ductus

venosus blood flows in fetal lambs in utero. Circ Res 42:426-433, 1978.

Faber, J.J. Diffusional exchange between foetus and mother as a function

of the physical properties of the diffusing materials. In: Proceedings of

the Sir Joseph Barcroft Centenary Symposium: Foetal and Neonatal

Physiology. Cambridge: Cambridge University Press, 1973. pp. 306-327.

Fouron, J.C. Dynamics of the placental transfer of digoxin in the dog.

Biol Neonate 23:116-122, 1973.

Heymm; M.A.; Payne, B.D.; Hoffman, J.I.E.; and Rudolph, A.M. Blood

flow measurements with radionuclide-labeled particles. Prog Cardiovasc

Dis 20:55-79, 1977.

Iwamoto, H.S., and Rudolph, A.M. Chronic renal venous catheterization in

fetal sheep. Am J Physiol 245:H524-H527, 1983.

14
Mellor, D.J. Investigations of fluid spaces of the sheep conceptus. In:

Nathanielsz, P.W., ed. Animal Models in Fetal Medicine. Vol. I.

Amsterdam: Elsevier/North-Holland Biomedical Press, 1980. pp.

59-106.

Meschia, G.; Cotter, J.R.; Makowski, E.L.; and Barron, D. Simultaneous

measurement of uterine and umbilical blood flows and oxygen uptakes.

Q J Exp Physiol 52:1-18, 1966.

Mirkin, B.L. Drug distribution in pregnancy. In: Boreus, L.O., ed; Fetal

Pharmacology. New York: Raven Press, 1973. pp. 1-28.

Morishima, H.O.; Heymann, M.A.; Rudolph, A.M.; and Barrett, C.T.

Toxicity of lidocaine in the fetal and newborn lamb and its relationship

to asphyxia. Am J Obstet Cynecol 112:72-79, 1972.

Morishima, H.O.; Heymann, M.A.; Rudolph, A.M.; Barrett, C.T.; and James,

L.S. Transfer of lidocaine across the sheep placenta to the fetus. Am J

Obstet Gynecol 122:58l-588, 1975.

Moya, F., and Thorndike, V. Passage of drugs across the placenta. Am J

Obstet Cynecol 84:1778-1797, 1962.

Oakes, G.K.; Walker, A.M.; Ehrenkranz, R.A.; Cafalo, R.C.; and Chez, R.A.

Uteroplacental blood flow during hyperthermia with and without

respiratory alkalosis. J Appl Physiol 41:197-201, 1976.

Reuss, M.L., and Rudolph, A.M. Distribution and recirculation of umbilical

and systemic venous blood flow in fetal lambs during hypoxia. J Dev

Physiol 2:71-84, 1980.

Ruckebusch, Y.; Graujoux, M.; and Eghbali, B. Placental transfer of central

nervous systein depressants in sheep. Eur J Pharmacol 37:193-196, 1976.

Rudolph, A. M. Hepatie and ductus venosus blood flows during fetal life.

Hepatology 3:254-258, 1983.

Rudolph, A.M., and Heymann, M.A. The circulation of the fetus in utero.

Circ Res 21:163-184, 1967.

Rudolph, A.M., and Heymann, M.A. Control of the foetal circulation. In:

Proceedings of the Sir Joseph Barcroft Centenary Symposium: Foetal

and Neonatal Physiology. Cambridge: Cambridge University Press,

1973. pp. 89-111.

Rudolph, A.M., and Heymann, M.A. Methods for studying the circulation of

the fetus in utero. In: Nathanielsz, P.W., ed. Animal Models in Fetal

Medicine. Vol. I. Amsterdam: Elsevier/North-Holland Biomedical

Press, 1980. pp. 1-57.

Schifferli, P.Y., and Caldeyro-Barcia, R. Effects of atropine and betaadrenergic drugs on the heart rate of the human fetus. In: Boreus, L.O.,

ed. Fetal Pharmacology. New York: Raven Press, 1973. pp. 259-279.

Schneider,.; H.; Panigel, M.; and Dancis, J. Transfer across the perfused

human placenta of antipyrine, sodium and leucine. Am J Obstet Gynecol

114:822-828, 1972.

Szeto, H.H.; Mann, L.I.; Bhaktharathsalan, A.; Liu, M.; and Inturrisi, C.E.

Meperidine pharmacokinetics in the maternal-fetal unit. J Pharmacol

Exp Ther 206:448-459, 1978.

Warkany, J., and Takacs, E. Experimental production of congenital

malformations in rats by salicylate poisoning. Am J Pathol 35:315-331,

1959.

Young, W.P.; Creasy, R.K.; and Rudolph, A.M. Catheterization of the

common umbilical vein for chronic fetal lamb studies. J Appl Physiol

37:620-621, 1974.

15
AUTHOR

Abraham M. Rudolph, M.D.

Professor of Pediatrics, Physiology, and

Obstetrics, Gynecology, and Reproductive Sciences

Neider Professor of Pediatric Cardiology

School of Medicine

University of California

San Francisco, California 94 143

16
Biotransformations of Drugs and

Foreign Chemicals in the Human

Fetal-Placental Unit

M.R. Juchau

Prenatal drug biotransformation has attracted investigative attention

since the late 1950s (Fouts and Adamson 1959; Jondorf et al. 1959) when it

was recognized that differences between mature and developing organisms, in terms of their responses to the pharmacologic/toxicologic actions

of drugs, could be due (at least in part) to developmental differences in

biotransformation. The common biotransformation pathways for drugs

and foreign chemicals are presented in table I. Early studies on fetal

drug metabolism primarily focused on hepatic tissues because the liver

was recognized as the principal site of drug biotransformation reactions in

adult animals. When compared in vitro with hepatic tissues from mature

experimental animals, the corresponding tissues from fetuses and

neonates exhibited extremely low or, in many cases, undetectable drug

biotransforming activities. Based primarily on studies in rats, mice,

rabbits, and guinea pigs, the concept arose that the drug biotransforming

capacity of prenatal animals was virtually negligible. Early studies of

fetal and neonatal drug biotransformation were reviewed by Brodie and

Maickel (1962) and Fouts and Hart (1965). This concept (absence of

prenatal drug biotransformation) persisted until the early 1970s when it

was discovered independently, in three separate laboratories (Pelkonen et

al. l971a, 1971b; Rane and Ackerman 1972; Rane and Sjöqvist 1972; Juchau

1971; Juchau et al. 1972; Juchau and Pedersen 1973), that the human fetal

liver and adrenal gland contained enzymes capable of catalyzing various

monooxygenation reactions--reactions for one of the common biotransformation pathways of drugs and foreign chemicals (table 1), at

reasonable respectable rates and at fairly early stages of gestation.

Several reviews of these findings also have appeared in the literature

(Pelkonen 1973, 1979, 1980d, 1980b, 1982; Yaffe and Juchau 1974; Rane et

al. 1973; Juchau et al. 1980; Juchau 1980a).

Almost simultaneously, it was discovered that human placental tissues

exhibited the capacity to biotransform drugs via each of the major

metabolic pathways. Possibly the most striking aspect of placental drug

biotransformation was the remarkably increased monooxygenase activities

observed in placentas as of tobacco smokers. Such increases were observed

primarily in placentas delivered near term and contrasted with a relative

17
TABLE 1. Common biotransformation pathways and enzymes involved in

biotransgermotaion

PATHWAY

I. Oxidation

a) Monooxygenation

b) Dioxygenation

c) Dehydrogenation

d) Peroxidation

e) Monoamine oxidation

Il. Reduction

a) Nitro groups

b) Azo linkages

c) Epoxides

d) Carbonyl groups

e) Unsaturated carboncarbon bonds

Ill. Hydrolysis

a) Carboxylic acid esters

b) Amides/peptides

c) Sulphate esters

d) Glucuronic acid esters

e) Epoxides

IV. Conjugation

a) Glycosylation

1. Glucuronidation

2. Riboside formation

3. Glucoside formation

b) Sulphation

c) Acylation

1. Acetylation

2. Clycine/glutamine

conjugation

d) Glutathione conjugation

e) Methylation

ENZYMES

I. Oxidoreductases

a1) Cytochrome P-450-

dependent systems

a2) Flavin-dependent

monoxygenases

b) Dioxygenases

c) Dehydrogenases

d) Peroxidases

e) Monoamine oxidases

II. Oxidoreductases

a) Nitro reductases

b) AZO reductases

c) Epoxide reductases

d) Dehydrogenases

e) Dehydrogenases

III.Hydrolases

a) Esterases

b) Amidases

c) Sulphatases

d) Glucuronidases

e) Epoxide hydrolases

IV. Transferases

a) Glycosyl transferases

1. Glucuronosyl

transferases

2. Ribosyl transferases

3. G1ucosyst transfeases

b) Sulphokinases

c) Acyl transferases

1. Acetyl transferases

2. Amino acid

transferases

d) Glutathione transferases

e) Methyl transferases

18
lack of response during the first third of gestation. Fetal livers and

adrenal glands also appeared not to respond significantly to inducing

agents present in tobacco smoke between weeks 7 to 20 of gestation.

Aspects of these researh findings have been reviewed on numerous

occasions. (For recent reviews, see Juchau l980b, 1982, 1984; Chao and

Juchau 1983; Juchau and Rettie, in press).

PHARMACOLOGIC SIGNIFICANCE

A somewhat disturbing aspect of the finding of comparitively high

monooxygenase activities in human placental and fetal tissues is the

indication provided by somewhat preliminary research that various

enzymes deemed extremely important for bioinactivation of drugs and

chemicals are present in low to negligible levels in each of the three

aforementioned. tissues (but particularly the placenta). They also appear

to remain at low levels following esposure to inducing agents. Of

principal cocern is the low to negligible complement of glucuronyl

transferases (in all fetal and placental tissues), enzymes which play a

prime role in terminiatirng the biologic effects of drugs and other

chemicals. Activities of other conjugating enzymes may also be low,

although no generalizations should be made at this time because of the

fact that relatively high activities can be observed depending upon the

substrate under investigation, the tissue preparation being studied, the

stage of gestation, exposure to inducing agents, and a multitude of other

genetic, physiological, and environmental factors known to affect rates of

conjugation.

Under conditions in which the tissue monooxygenase activities are

comparatively high, it can be shown that enzymes from each of these

three organs will catalyze the conversion of promutagens to mutagenic

intermediary metabolites (Jones et al. 1977; Juchau et al. 1978, 1980;

Juchau l980a). Other bioactivation reactions also have been documented

(Rollins et al. 1979; Pelkonen 1979, 1982). The toxicologic significance of

these observations is not fully understood at present but would suggest

that after exposure of pregnant women to drugs or chemicals,

steady-state levels of reactive, toxic intermediates would tend to be high

in fetal and placental tissues with high ratios of monooxygenase to

conjugating activities. Considerable research must be performed in order

to understand the implications of regulatory increases in xenobiotic

monooxygenation in the fetal-placental unit. From the viewpoint of

toxicologic effects of drugs and chemicals, the influence of such changes

will undoubtedly vary depending on the toxic chemical under

consideration. It is important to emphasize that we are now in possession

of only the barest framework of knowledge of xenobiotic biotransformation in human fetal and placental tissues and that it is entirely premature

to make predictions concerning the metabolic fate of a given chemical

within the human fetal-placental unit based on currently available

information. Recently, we discovered (Juchau et al. 1982; Namkung et al.

1983a, 1983b; Omiccinski et al. 1980) that additions of micromolar

quantities of hematin to reaction mixtures containing fetal or placental

tissues as enzyme source would increase rates of monooxygenation by as

much or more than a hundredfold, depending upon reaction conditions and

the tissue under investigation. The mechanism for elicitation of these

remarkable increases has not yet been elucidated, but evidence

accumulated to date suggests that the phenomenon may reflect a highly

19
important, short-term mode of regulation of P-450-dependent monooxygenation reactions. In view of the implications for generation of toxic

reactive intermediates, it would seem important to gain a much firmer

understanding of the phenomenon. We are currently pursuing studies of

“hematin-mediated” regulation of P-450-dependent monooxygenase

activities in fetal and placental tissues with the following working

hypothesis:

Extrahepatic tissues of mature animals and both

hepatic and extrahepatic tissues of immature (prenatal and neonatal) animals (including human and

nonhuman primates) contain significant pools of

apocytochrome P-450 isozymes which, under normal

circumstances, exhibit a relatively low affinity for

the corresponding hematin prosthetic group. Following contact with certain small organic chemicals

(not necessarily xenobiotics, as hydroxylation of

estradiol 17B also appears subject to this regulatory

mode), interaction of the apocytochrome with the

chemical and/or its metabolite(s) results in a conformational change in the apocytochrome(s) which

increases the affinity for its hematin prosthetic

group. The increased affinity, in turn, results in an

increased steady-state level of active holocytochrome(s) and thus, a marked increase in the corresponding P-450-dependent monooxygenase activities.

Although the hypothesis remains unproven, we have provided substantial

evidence to rule out a large number of possible alternative hypotheses and

also have found that all data obtained to date are consistent with the

stated hypothesis. In addition, we have been able to show (Giachelli et al.

1984) that embryonic tissues contain unusually high quantities of mRNA

which serve as template for P-450 synthesis, suggesting the possibility

that correspondingly high levels of inacative apocytochrome may also he

present prenatally. In view of the importance of the role of P-450-

dependent monooxygenation in the generation of reactive intermediates

and in the potential serious toxic effects of such intermediates, it is felt

that pursuit of these questions could provide considerable light on

mechanisms of chemically initiated toxicity in the fetal-placental unit.

It should he emphasized, however, that our current understanding of drug

biotransformation in the human fetal-placental unit is very primitive. It

is not possible to predict the metabolic fate of drugs and foreign

chemicals within that unit--even for chemicals that have received some

investigative attention. For obvious reasons, investigators have utilized

model substrates (primarily) for the study of the metabolic capabilities of

the pertinent human fetal and placental tissues. Thus, specific information on most drugs of abuse is not available at present, nor is it

possible to predict their biotransformational fate within the fetalplacental unit. Understandably, in view of the ethical questions involved,

progress in the area has been rather slow, with only a few published

artickes appearing each year. For these and other reasons, our

understanding of the effects of drugs of abuse on the prenatal organism

(particularly in terms of the more subtle, nonmorphologic and/or

Long-term effects) is incomplete at best. Future research, however, will

undoubadly help to clarify many of the currently existing questions.

20
SUMMARY

Several communications pertaining to research on human fetal drug

biotransformation have appeared in the literature in the past 3 to 4 years

(Aranda et al. 1979; Cresteil et al. 1982; Pacifici et al. 1981; Pacifici and

Rane 1982, 1983; Schroeter and Amon 1983). In general, research in this

area has produced no major recent surprises, but it has confirmed,

extended, and supported the earliest findings through those of the

mid-1970s and has served to expand our understanding of these important

systems. The important aspects of our current knowledge may be

summarized very briefly as follows:

1 . Each of the major drug metabolic reactions (oxidations,

reductions, hydrolyses, and conjugations) can he catalyzed at

generally low rates by enzymes present in human fetal and

placental tissues.

2. Reaction rates appear, in general, to increase with advancing

gestational age.

3. Placental xenobiotic-biotransforming monooxygenases display a

high sensitivity to the effects of MC-type (methylcholanthrene)

but not PB-type (phenobarbital) inducing agents during the later

stages of gestation. Responsivity is minimal during early

gestation.

4. Monooxygenases in other fetal tissues (liver, adrenal gland,

etc.) appear to respond minimally to inducing agents, at least

during the earlier stages of gestation.

5. Glucuronyl transferase activities appear to be very low in all

fetal and placental tissues.

6. Epoxide hydrolase, glutathione S-transferase, and sulfotransferase (and perhaps others) activities can be relatively

high in prenatal primate hepatic tissues but, at present, are

difficult to predict. Some of these may respond to environmental inducers in situ, but solid evidence for this is still

lacking.

7. Monooxygenases in all fetal and placental tissues (with the

exception of the liver in certain species) appear subject to a

hematin-Jnediated regulation of activity. Extremely large

increases have been observed.

8. Enzymes/enzyme systems present in human fetal and placental

tissues can catalyze the generation of reactive intermediary

metabolites capable of covalently binding to biomacromolecules

and inducing bacterial mutations.

9. In terms of substrates attacked by human tissue monooxygenases, the fetal hepatic enzyme exhibits a rather broad

specificity, the fetal adrenal gland exhibits a narrower specificity, and the human placenta exhibits an even narrower

specificity.

21
10. In comparison with hepatica systems from adult male rodents,

human fetal and placental tissues are virtually unexplored with

respect to knowledge of drug biotransformation.

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Chao, S.T., and Juchau, M.R. Placental drug metabolism. In: Johnson,

E.M., and Kochhar, D.M., eds. Teratogenesis and Reproductive

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York: Springer Verlag, 1983. pp. 31-49.

Cresteil, T.; Beaune, P.; Kremers, P.; Flinois, J.; and Leroux, J. Drug

metabolizing enzymes in human fetal liver: Partial resolution of

multiple cytochromes P-450. Pediatr Pharmacol 2:119-207, 1982.

Fouts, J.R., and Adamson, R.H. Drug metabolism in the newborn rabbit.

Science 129:807-898 1959.

Fouts, J.R., and Hart, L.G. Hepatir drug metabolism during the perinatal

period. Ann NY Acad Sci 123:245-251, 1965.

Giachelli, C.M.; Juchau, M.R.; and Omiecinski, C.J. Expression of cytochrome P-450 and epoxide hydrolase messenger RNA in developing rat

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Jondorf, W.R.; Maickel, R.P.; and Brodie, B.B. Inability of newborn mice

and guinea pigs to metabolize drugs. Biochem Pharmacol 1:352-354,

1959.

Jones, A.H.; Fantel, A.C.; Kocan, R.A.; and Juchau, M.R. Bioactivation

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Life Sci 21:1831-1836, 1977.

Juchau, M.R. Drug biotransformation in the human fetus: Nitro group

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Juchau, M.R. Enzymatic bioactivation and inactivation of chemical

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M.R., ed. The Biochemical Basis of Chemical Teratogenesis. New

York: Elsevier Press. 1980a. pp. 63-95.

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Snell, K., ed. Developmental Toxicology. London: Croom-Helm, 1982.

pp. 189-209.

Juchau, M.R. Placental drug metabolism with respect to transplacental

carcinogenesis. In: Reznik-Schuller, H.M., ed. Comparative Perinatal

Carcinogenesis. New York: CRC Press, 1984. pp. 117-137.

Juchau, M.R., and Pedersen, M.G;. Drug biotransformation reacations in the

human fetal adrenal gland. Life Sci 12:193-204, 1973.

Juchau, M.R., and Rettie, A.E. The metabolic role of the placenta. In:

Fabro, S.E., and Scialli, A.R., eds. Principles of Drug and Chemical

Action in Pregnancy. New York: Marcel-Dekker Inc., in press.

22
Juchau, M.R.; Pedersen, M.G.; and Symms, K.G. Hydroxylation of 3,4-

benzpyrene in human fetal tissue homogenates. Biochem Pharmacol

21:2269-2272, 1972.

Juchau, M.R.; Jones, A.H.; Namkung, M.J.; and DiGiovanni, J. Extrahepatic bioactivation of 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene man fetal tissues. In: Jones, P.W., and Freudenthal, R.I., eds.

Carcinogenesis Vol III: Polynuclear Aromatic Hydrocarbons. New

York: Raven Press, 1978. pp. 361-370.

Juchau. M.R.; Chao. S.T.; and Omiecinshi, C.J. Drug metabolism bv the

human fetus. Clin Pharmacokinet 5:320-339, 1980

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increases in P-450-dependent monooxygenation reactions: An apparent

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Microsomes, Drug Oxidations and Drug Toxicities. New York:

Wiley-Interscience, 1982. pp. 371-381.

Namkung, M.J.; Chao, S.T.; and Juchau, M.R. Placental monooxygenation:

characteristics and partial purification of a hematin-activated human

placental monooxygenase. Drug Metab Dispos 11:10-15, 1983a.

Namkung, M.J.; Faustman-Watts. E.M.; and Juchau. M.R. Hematinmediated increases in benzo(a)pyrene monooxygenation in maternal,

fetal and placental tissues of inducible and non-inducible mouse

strains. Dev Pharmacol Ther 6:199-207, 1983b.

Omiecinski. C.J.; Chao. S.T.; and Juchau. M.R. Modulation of monooxvgenases activity by hematin and 7,8-benzoflavone in fetal tissues of

rats, rabbits and humans. Dev Pharmacol Ther 1:90-100, 1980.

Pacifici, G.M., and Kane, A. Renal glucuronidation of morphine in the

human fetus. Acta Pharmacol Toxicol 50:155-160, 1982.

Pacifici, G.M., and Rane, A. Epoxide hydrolase in human fetal liver.

Pharmacol 26:241-248, 1983.

Pacifici, G.M.; Norlin, A.; and Rane, A. Glutathione S-transferase in

human fetal liver. Biochem Pharmacol 30:3367-3371, 1981 .

Pelkonen, O. Drug metabolism in human fetal tissues. Life Sci 13:1163-

1180, 1973.

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metabolism. In: Estabrook, R., and Lindelaub, J., eds. The Induction

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507-516.

Pelkonen, O. Environmental influences on human fetal and placental

xenobiotic metabolism. Eur J Clin Pharmacol 18:17-24, 1980a.

Pelkonen, O. Biotransformation of xenobiotics in the fetus. Pharmacol

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developmental toxicity. In: Snell, K., ed. Developmental Toxicology.

London: Croom-Helm, 1982. pp. 167-188.

Pelkonen, O.; Verne, M.; Jouppila, P.; and Karki, N.T. Metabolism of

chlorpromazine and p-nitrobenzoic acid in the liver, intestine and

kidney of the human fetus. Acta Pharmacol Toxicol 29:284-294, 197la.

Pelkonen, O.; Arvela, P.; and Karki, N.T. 3,4-Benzpyrene and N-melhylaniline metabolizing enzymes in immature human fetus and placenta.

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human fetal liver. Clin Pharmacol Ther 13:663-670, 1972.

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newborn infant. Pediatr Clin North Am 19:37-49, 1972.

23
Kane, A.; Sjöqvist, F.; and Orrenius, S. Drugs and fetal metabolism. Clin

Pharmacol Ther l4:666-67‘2, 1973.

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Acetaminophen: Potentially toxic metabolite formed by human fetal

and adult liver microsomes and isolated fetal liver cells. Science

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Pharmacol Toxicol 14:219-238, 1974.

AUTHOR

M.R. Juchau, Ph.D.

Department of Pharmacology

University of Washington SJ-30

Seattle, WA 98195

24
Pharmacokinetics of Drugs and Metabolites

in the Maternal-Placental-Fetal Unit:

General Principles

Laurene H. Wang, Abraham M. Rudolph, and Leslie Z. Benet

Drug use during pregnancy may result in both pharmacologic and toxicologic drug effects in the fetus. Prenatal pharmacology defines the

therapeutic use of drugs to treat fetal diseases in utero; while prenatal

toxicology evaluates the risks of drug exposure to the developing fetus.

To correctly assess the pharmacologic and toxicologic effects of a drug,

one must examine hoth the pharmacodynamics and pharmacokinetics of

the drug and its metaholites. Pharmacodynamic parameters which specify

the interaction of a drug or its metabolites with certain specific receptors

and the elucidation of their modes of action determine whether the drug

or its metabolites possess pharmacological or toxicological potential in

the fetus. On the other hand, pharmacokinetic parameters which

characterize the disposition of the drug and its metabolites in the

maternal-placental-fetal unit are of utmost importance in determining

whether an active form of the drug can reach the fetus, after maternal

administration, at a sufficient concentration and for a11 adequate period

to induce pharmacological or toxicological effects during the prenatal

period. Therefore, pharmacokinetic studies in the maternal-placentalfetal unit which evaluate the rate and extent of drug exposure to the

fetus play an important role in developmental pharmacology and

toxicology.

An index of relative exposure of the fetus to the drug taken by the mother

has been defined as the ratio of the total area under the drug concentration versus time curve for the fetus to that for the mother after a

single-dose administration (Levy and Hayton 1973). If multiple doses of a

drug are administered, the index of relative exposure of the fetus to the

drug can be determined by the ratio of the steady-state drug concentration in the fetus to that in the mother. Since the metabolite of a drug is,

in many cases, the active species responsible for a pharmacologic or

toxicologic effect, the concentrations of the metabolite should then be

measured to determine the fetal metabolite exposure. In addition, plasma

protein binding measurements should also be determined because the

unbound drug is generally believed to be the moiety that elicits a

pharmacologic or toxicologic action. The factors affecting the rate,

extent, and duration of exposure of a drug or its metabolites to the fetus

include the absorption, distribution,. and elimination of the drug in the

mother; the distribution and elimination of the drug metabolites in the

mother; the transfer of the drug and its metabolites across the placenta;

25
the metabolism of the drug or its metabolites in the placenta; and the

distribution and elimination of the drug and its metabolites in the fetus.

Theoretical considerations of all the above processes of drug disposition in

the maternal-placental-fetal unit are presented in this chapter. Pharmacokinetic experiments which have been utilized lo assess appropriate

pharmacokinetic parameters for characterizing the kinetic processes are

also discussed.

PHARMACOKINETIC STUDIES DURING PREGNANCY

Pharmacokinetic studies in humans during pregnancy are usually limited

to a single point determination of drug concentrations in the mother and

the fetus (umbilical cord blood) immediately following delivery. These

determinations are carried out in different subjects, at different times

after drug administration, and usually under different conditions. Consequently, pharmacokinetic chararterization of drug disposition in the

maternal-placental-fetal unit derived from these studies is extremely

difficult and by no means is definitive in reflecting in utero rondition.

Based on theoretical considerations, computer simultations various

pharmacokinelic models have been undertaken to predict the time course

of drug concentrations in the mother and fetus (Levy and Hayton 1973;

Gillette 1977). Nonetheless, the most useful information can only be

obtained from properly conducted experiments in which the time course

of maternal plasma concentrations of drug and metabolites is followed

after drug administration; and maternal, cord arterial, and venous blood

samples and amniotic fluid are collected at the time of delivery (Rajchgot

and MacLeod 1983). In addition, the plasma protein binding of the drug

and its metabolites should also be determined.

Since drug studies in pregnant woman are ethically and medically prohibited, most multiple sample pharmacokinetic investigations of drugs and

metabolites have been carried out in animals. Rats, mice, and rabbits

have been frequently utilized with a single time sample obtained in both

the mother and fetus at sacrifice. Pharmacokinetic analyses are then

undertaken with combined data from many animals sacrificed at different

times after dosing. A few multiple time sample studies have been carried

out in monkeys, but the pregnant sheep has evolved as the most frequently

used animal model.

The establishment of a chronically catheterized pregnant sheep model has

allowed complete pharmacokinetic studies in the maternal-placentalfetal unit in utero. Surgical procedures are used to implant chronic

indwelling catheters, enabling repeated sampling from various maternal

and fetal blood vessels, from maternal and fetal bladders, and from

amniotic and allantoic fluid cavities at predetermined time intervals after

drug administration. Chronic studies with multiple dosing of a drug to the

mother and kinetic studies with direct introduction of the drugs into the

fetal circulation can also be readily carried out. Additionally, the pregnant ewe preparation permits pharmacokinetic studies on the developmental aspects of drug disposition in the maternal-placental-fetal unit.

Therefore, the following discussion of the experimental methods utilized

in prenatal pharmacokinetic studies will concentrate on the use of

pregnant sheep as the animal model. However, comparative studies using

different animal models are essential to gain a full understanding of the

overall scheme of drug disposition in the maternal-placental-fetal unit.

26
Pharmacokinetic Models

Various compartmental models have been suggested for characterizing the

kinetics of drug disposition in the maternal-placental-fetal unit during

pregnancy (Levy and Hayton 1973; Krauer and Krauer 1977; Szeto 1982;

Szeto et al. 1982; Kajchgot and MacLeod 1983; Cabrielsson and Paalzow

1983). To obtain the pharmacokinetic parameters which best describe the

rate, extent, and duration of drug exposure to the fetus, all accountable

kinetic processes must be considered when constructing the pharmacokinetic model. The compartmental model can be as complicated as that

shown in figure I in which all possible physiological conditions of the

system are considered. However, the practicality of a kinetic model is

usually limited by the availability of sampling sites, and, thus the

experimental data. Therefore, preliminary in vivo and in vitro experiments should always be performed to provide the basis for a particular

model. Furthermore, model independent or noncompartmental methods

should be utilized to substantiate the validity of the derived compartmental parameters using the proposed model. If the experimental results

of both methods are not consistent, a model describing more complicated

processes should then be proposed and tested.

At present, it appears that the simplest kinetic model would be a twocompartment (maternal and fetal) model with transplacental transfer and

nontransplacental elimination occurring in both compartments (Szeto

1982). As depicted in figure 1, complicated situations arise when the drug

is metabolized in the placenta, which constitutes a third compartment, or

when the drug is transferred via nonplacental routes between the fetus

and the mother. This is exemplified by the reversible diffusion of the

drug between the amniotic fluid and the mother across the chorioallantoic

membranes (Mellor 1980). the reversible diffusion of the drug between the

amniotic fluid and the fetus across the fetal skin, chorioamnion, or

respiratory epithelium (Seeds 1981; Carson et al. 1979; Dawes 1973). or the

oral-renal-amniotic fluid recycling (Amon and Amon 1976). The pharmacokinetic scheme is further complicated when metabotite kinetics is

considered. Overall, the significance of each process in a kinetic model

depends upon its contribution to the overall estimation of the

pharmacokinetic parameters which govern the rate, extent, and duration

of drug exposure to the fetus.

Experimental Methods

Once a compartmental model is proposed for the disposition of a drug in

the maternal-placental-fetal unit, the rate equations describing the

change of drug concentration in each compartment can be written. When

steady-state infusions of the drug into each compartment are carried out

on separate occasions, the rate of change of drug concentration at steady

state in each compartment is equal to zero for every infusion experiment. The drug concentration in each compartment at steady state and

the infusion rate for each experiment allow one to determine the

clearance of the drug for each process describing the disposition of the

drug in the maternal-placental-fetal unit. A two-compartment open

model with elimination from both the maternal and fetal compartments

has been utilized by Szeto (1982) and Szeto et al. (1982) to determine the

transplacental clearances from both sides of the placental membranes and

the nontransplacental clearance from the mother and the fetus, as

expressed in the following equations:

27

483-942 0 - 85 - 2 : QL 3
FIGURE 1. Schematic Representation of the Drug Transfer and

Elimination Processes in the Mother, Placenta, and Fetus.

Mc = maternal central compartment; Mp = maternal peripheral

compartment; D = drug; and M = metabolite.
Where CLMF and CLFM are the respective transplacental clearances of drug

from the mother to the fetus and from the fetus to the mother; CLMO and

CLFO are the respective nontransplacental elimination clearances of drug

from the mother and the fetus; J and J’ are the respective infusion rates of

the drug into the maternal and the fetal compartments; CM and CF are the

respective steady-state drug concentrations in the mother and the fetus after

maternal infusion; and CM and CF are the respective steady-state drug

concentrations in the mother and the fetus after the fetal infusion.

When experimental techniques permit, direct measurement of these clearance terms should be undertaken independently in the mother, placenta, and

fetus to verify the credibility of the clearance terms obtained with the

compartmental model method. With catheterization of uterine and umbilical

veins in addition to the maternal and fetal femoral arteries, it is possible to

determine clearances across the placenta from either side using the extraction

ratio method by measuring the arterial and venous drug concentrations across

the placenta and the uterine and umbilical venous blood flow rates with

radiolabeled microspheres (Rudolph and Heymann 1980). The placental

clearance determined by the extraction ratio method consists of both the

transfer clearance across the placenta and the metabolic clearance, if any, in

the placenta; while the transplacental clearances (CLMF and CLFM) obtained

from the two-compartment model method solely represent the transfer

clearances. (For the two-compartment model method, placental metabolic

clearance would be included in the CLMO and CLFO terms.) If placental

metabolism does not exist, clearance values obtained from both methods

should be equal. In addition to the in vitro placental incubation experiments,

the extraction ratio method provides justification for incorporating placental

metabolism in the pharmacokinetic model.

29
The validity of the nontransplacental clearances in the mother and the

fetus, calculated from the compartmental model method at steady state,

can be verified by determining the metabolic and renal clearances of the

drug in the mother and the fetus following single-dose administration of

drug or metabolite to both the mother and fetus. If the drug metabolite(s) is (are) not transferred across the placenta, metabolic clearance(s)

of the drug can be determined using the general methods described by

Benet (1984). Thus, the disposition of the drug metabolite(s) in the

maternal-placental-fetal unit should be characterized prior to the

assessment of metabolic clearances of the drug in the mother and the

fetus. A valid nontransplacental clearance obtained from the model

dependent method should be equal to the sum of the clearances of the

drug via all elimination routes (metabolism and excretion) other than

placental transfer.

Preliminary experiments to provide evidence of drug transfer through

nonplacental routes can be performed by administering single bolus doses

of the drug respectively into the mother, fetus, amniotic fluid cavity, and

allantoic fluid cavity on separate occasions. Samples are collected from

the amniotic fluid and allantoic fluid cavities, the maternal femoral

artery and uterine vein, and the fetal femoral artery and umbilical vein.

The rate and extent of drug appearance at each sampling site, e.g., the

lag time, the peak time, peak concentration, and total area under the

concentration-time curve, can then be compared between the sampling

sites and between the studies. These comparisons allow one to better

understand the mechanism of drug transfer via nonplacental routes, and

they serve as a basis for upgrading a kinetic model to incorporate all the

necessary transfer routes for describing drug disposition in the

maternal-placental-fetal unit.

Metabolite Kinetics in the Fetus

Drug metabolism in the fetus has been extensively studied using in vitro

enzyme preparations (Petkonen 1980; Dutton and Leakey 1982). Very

limited literature information is available regarding the in utero drug

metabolism in the fetus. Since an understanding of the metabolic

activation and deactivation pathways in the fetus is essential to determine drug effects and toxicity during the prenatal period (Lucier et al.

1979). studies of in utero drug metabolism in the fetus are indispensable in

prenatal pharmacology and toxicology. Pharmacokinetics along with

appropriate animal models provide us with a very useful tool to

investigate fetal drug metabolism in utero.

A drug metabolite detected in the fetal circulation after drug administration to the mother may not reflect the ability of the fetus to

metabolize the drug. Further investigation is required to determine

whether the metabolite found in the fetus originates from fetal metabolic

activity or results from transfer of the metabolite from the mother across

the placenta. In addition, if a metabolite is formed in the fetus, the

distribution and elimination processes of the metabolite in the fetus must

be examined in order to quantitate the fetal metabolic activity in utero.

To this end, a single dose of the metabolite can be administered to the

mother to determine whether placental transfer of the metabolite

occurs. The metabolite should then be given to the fetus to examine the

30
fetal disposition of the metabolite. Upon the identification of all

elimination processes of the metabolite in the maternal-placental-fetal

unit, rate equations describing the change of the metabolite concentration

in the fetus can be written. As stated earlier, if the metabolite is not

transferable across the placenta, the formation clearance for the

metabolite (or metabolic clearance of the drug) can be determined using

general pharmacokinetic methods (Benet 1984). An example of these

methods will be demonstrated in the following section of this paper. If

the metabolite is formed in the fetus and is transferred across the

placenta from both sides, computer simulations and data fitting may be

helpful in obtaining the formation clearance for the metabolite in the

fetus. Studies of in utero metabolite kinetics in the fetus at different

gestational ages can then be carried out to investigate the prenatal

development of various metabolic pathways.

EXAMPLE OF PHARMACOKINETIC STUDIES DURING PREGNANCY

We have chosen acetaminophen as a model compound to carry out

pharmacokinetic studies of drug disposition in the sheep maternalplacental-fetal unit. These studies are presented here to illustrate the

concepts and methods discussed above. The primary reasons for choosing

acetaminophen are: (1) acetaminophen is the most widely administered

nonprescription drug during pregnancy and clinicians frequently question

the safety of its use in pregnant women; (2) acetaminophen is mostly

glucuronidated and sulfated in various animal species and can thus serve

as a model substrate to study the prenatal development of the two

conjugation pathways; and (3) pure acetaminophen conjugates can be

chemically synthesized in sufficient quantities for metabolite kinetic

studies in the mother and the fetus.

Single bolus doses of acetaminophen were first administered intravenously

(i.v.) into the mother and the fetus on two separate occasions (Wang et al.

1983). Acetaminophen was found to be transferred across the placenta

from both sides of the membrane and its half-life was about 1 hour in

both the mother and fetus. Both acetaminophen glucuronide and sulfate

were measurable in the fetus and their concentrations gradually increased

until the end of study. Both conjugates stayed in the fetal circulation for

a much longer period than in the maternal circulation. Compared to

acetaminophen glucuronide, acetaminophen sulfate had a higher concentration in the fetus but a lower concentration in the mother. This

preliminary study revealed the bidirectional placental transfer of

acetaminophen and the presence of its two conjugates in the fetus.

Half-Lives of acetaminophen and its conjugates in both the mother and the

fetus were also determined. Acetaminophen glucuronide and sulfate were

then administered i.v. simultaneously in single bolus doses into the mother

and the fetus on separate occasions to examine the possibility of placental

transfer of the two conjugates and to study the disposition of the two

conjugates in the mother and the fetus (Wang et al. 1985a). Samples were

collected from the maternal femoral artery and the uterine vein and

bladder catheters; and from the fetal femoral artery and the umbilical

vein and bladder catheters. It was found that acetaminophen glucuronide

and sulfate were not transferred across the placenta from either side and

that renal excretion is the predominant route of elimination for both

conjugates in the mother and the fetus. Considering the glomerular

filtration rate and the extent of plasma protein binding, we concluded

31
that renal elimination of both conjugates was primarily through

glomerular filtration in the fetus, but through renal tubular secretion in

the mother. The increasing glomerular filtration rate with gestational

age resulted in an increase of total (renal) clearances of both conjugates

with gestational age. Steady-state volumes of distribution were

determined for both conjugates in the fetus and were also found to be

linearly related to the age of the fetus. This study demonstrated that the

two conjugates detected in the fetal circulation following acetaminophen

dosing originated from metabolic conjugation activities in the fetus, but

not from placental transfer. Following the characterization of conjugates

disposition in the fetus, it was then possible to quantitate conjugation

activities in fetal lambs in utero using acetaminophen as the model

substrate.

Since preliminary in vitro and in vivo data suggested negligible placental

metabolism of acetaminophen, we proposed a two-compartment open

model (Szeto 1982). with elimination from both the mother and fetus, to

investigate the bidirectional placental transfer of acetaminophen and the

total nontransplacental elimination of acetaminophen in the ewe and the

fetal lamb (Wang et al. 1985b). Based on the data from steady-state i.v.

infusions of acetaminophen into the mother and the fetus on separate

occasions, the transplacental clearances from both sides of the membrane

and the total nontransplacental clearances in the mother and the fetus

were calculated using the equations stated above (1 to 4). The ratio of

steady-state acetaminophen concentration, fetus to mother, after

maternal infusion was found to be 0.77 and remained unchanged

throughout the gestational age of the fetus. Transplacental clearances

from mother to fetus and vice versa did not differ, indicating that

acetaminophen is transferred across the placenta exclusively through

passive diffusion since the plasma protein binding of acetaminophen is

insignificant and comparable in both the mother and the fetus. The

placental clearance was also determined using the extraction ratio

method and found to approximate the transplacent clearances obtained

from the compartmental model method, indicating that the model

dependent method yields credible results for transplacental clearances,

and that the placental metabolism is either nonexistent or negligible. The

model independent total nontransplacental clearance of acetaminophen in

the mother and the fetus were determined using the plasma and urine

data. Since acetaminophen conjugates are not transferred across the

placenta from either direction and renal excretion is the only elimination

route for both conjugates (Wang et al. 1985a), the metabolic clearance of

acetaminophen in the mother with respect to glucuronidation

or sulfation was determined at steady state as the amount of

the glucuronide or sulfate excreted in the urine divided by the area under

the plasma acetaminophen concentration-time curve at steady state. The

metabolic clearance in the fetus with regard to glucuronidation or

sulfation was estimated based on the following equation:

32
Where is the metabolic clearance of acetaminophen to a

particular conjugate; VSSF, C is the steady-state volume of distribution of

the conjugate in the fetus and is obtained from the previous conjugate

dose experiments; CF,C and CF,A are the respective plasma conjugate

and acetaminophen concentration in the fetus; and UF,C is the cumulative

amount of the conjugate excreted in the fetal urine.

The sum of glucuronidation and sulfation clearances and the renal clearance of acetaminophen in the mother accounted for 98% of the nontransplacental clearance in the mother obtained from the compartmental

model method. This provides further evidence of the validity of the two

compartment model used for describing acetaminophen disposition in the

sheep maternal-placental-fetal unit. Glucuronidation and sulfation

activities in the fetus in utero were found to be 15% and 50% of the

respective activities in the mother on a body weight basis. However, the

sum of glucuronidation and sulfation clearances and renal clearance of

acetaminophen cannot account for the overall nontransplacental

clearance in the fetus obtained from the model dependent method. We

speculate that diffusion of aretaminophen into the slow equilibrating

amniotic fluid or metabolism of acetaminophen across the amnion, fetal

intestine or lungs may partly explain this discrepancy. We now plan to

collect both amniotic and allantoic fluids in addition to blood samples and

to administer acetaminophen intra-amniotically and intra-allantoically to

study the kinetics of acetaminophen disposition in the two fetal fluids and

the possible drug transfer in relation to the maternal and fetal

circulations. By these means, we hope to be ahle to propose a more

sophisticated model to characterize the disposition of acetaminophen in

the sheep maternal-placental-fetal unit.

FACTORS AFFECTING FETAL DRUG EXPOSURE

The factors which may influence the rate, extent, and duration of drug

exposure to the fetus are essentially the factors that modify the

pharmacokinetic parameters characterizing the absorption, distribution,

metabolism, and elimination of the drug or/and its metabolites in the

pregnant mother, the placenta, and the developing fetus. The

determinants of fetal drug exposure during pregnancy are therefore

categorized as the maternal, placental, and fetal factors. Several review

articles on drug disposition in the maternal-placental-fetal unit have been

published (Mirkin and Singh 1976; Krauer et al. 1980); Juchau and

Faustman-Watts 1983; Bogaert and Thiery 1983), in which the maternal,

placental, and fetal factors affecting fetal drug exposure have been

discussed. Extensive and specific discussion of the maternal factors

which modify the absorption, distribution, and elimination of the drug

or/and its metaholites in pregnant women arc presented in the articles by

Krauer and Krauer (1977). Juchau and Faustman-Watts (1983), and Bogaert

and Thiery (1983). Articles by Mirkin and Singh (1976), Juchau (1976), Nau

and Liddiard (1978), and Goodman et al. (1982) specifically discuss

placental metabolism of drugs and factors affecting placental transfer of

drug or/and its metaholites. The distribution and elimination of drug

or/and its metabolites in the fetus are specifically discussed in the

articles by Mirkin and Singh (1976), Pelkonen (1980), Aranda and Stern

(1983). Juchau and Faustman-Watts (1983), and Rajchgot and MacLeod

(1983). Similar topics are addressed by Drs. Juchau and Miller in this

33
volume. The following section summarizes the literature discussions and

presents a brief review of the maternal, placental, and fetal factors

affecting fetal drug exposure.

Maternal Factors

Absorption. The gastrointestinal absorption of drugs in pregnant women

will be influenced by pregnancy-related physiological changes, such as

decreased gastrointestinal motility, delayed gastric and intestinal

emptying, reduction of gastric acid secretion, and increased mucous

secretion. During late pregnancy, an increase in alveolar ventilation as

well as tidal volume is observed (Alaily and Caroll 1978), which may

enhance the pulmonary absorption of drugs given by inhalation. In

addition, the increase in venous pressure in the lower limbs could alter the

absorption rate of drugs administered intramuscularly.

Distribution. The physical changes related to pregnancy which may

modify the distribution of a drug and/or its metabolites include the

increase in total body water (both intra- and extra-vascular), large

accumulation of body fat, the decrease in drug binding to plasma proteins

(albumin), and profound hemodynamic changes.

Metabolism. Maternal drug biotransformation could be a major

determinant of the therapeutic and/or fetotoxic effect(s) of a drug.

Increased hepatic microsomal production of a diol (via epoxide)

metabolite has been found for phenytoin during early pregnancy (Blake et

al. 1978). Increased plasma free fraction due to decreased albumin binding

may increase the rate of metabolism. Pseudocholinesterase concentrations fall during pregnancy and may result in an impaired ability to

hydrolyze the muscle relaxant succinylcholine. However, the rates of

maternal drug biotransformation may vary as a function of gestational

age and may be affected differently in various organs of the same

animal. The relative rates of bioactivating versus inactivating reactions

for any given substrate may also be affected by pregnancy. Most studies

on the effects of pregnancy on rates of drug biotransformation have been

performed using in vitro enzyme preparations. Therefore, further studies

need to be carried out various species of whole animal to demonstrate

the pregnancy-dependent differences in drug biotransforming enzyme

systems.

Excretion. Pregnancy-dependent increases in renal plasma flow and

glomerular filtration rate would increase the kidney excretion rates of

drug and/or its metabolites. Decreases in plasma albumin binding may

also increase the rate of renal excretion of the unbound drug or/and its

metabolites. Biliary excretion of the injected dye sulfobromophthalein

was found to decrease during pregnancy (Tindall 1975). Whether the

biliary excretion of other foreign chemicals would be similarly affected is

unknown. Pulmonary excretion of gases and volatile substances would be

increased due to increased respiratory rate, tidal volume, and minute

volume.

Placental Factors

Placental Transfer. The placental transfer of a compound may be

influenced by the following factors: blood flow rates of the placental

circulation--uterine and umbilical; lipid solubility of the compound and

34
its degree of ionization in the maternal and fetal circulations; molecular

weight of the compound; protein binding of the compound in the maternal

and fetal plasma and the stage of developement of the placenta. Mirkin

and Singh (1976) have provided a thorough review of this subject.

Placental Metabolism. Placental metabolism of drugs and foreign

chemicals has been thoroughly reviewed by Juchau (1976). The

therapeutic significance of drug biotransformation in in the placenta

remains to be defined under in vivo conditions. However, fetotoxic

effects of drugs or foreign chemicals may be induced by placental

metabolism and this area of research requires further investigation

(Goodman et al 1982) . . .

Fetal Factors

Distribution. Nearly all drugs distribute to various tissues after entry into

the fetus. The distribution is influenced by the altered permeability of

specific membranes, or by the content of total body water and lipids in

the fetus, and the altered distribution of fetal circulation. A variable

proportion of umbilical venous hlood will bypass the liver and an

alteration in hepatic hlood flow would occur. Some drugs accumulate in

the amniotic fluid via urinary excretion or by exchange across the

nonkeratinized fetal skin or across the chorioamnion or respiratory

epithelium. Recycling of drugs between the fetus and amniotic fluid may

occur through fetal swallowing of amniotic fluid.

Metabolism. Fetal drug metabolism has been extensively investigated

using in vitro enzyme preperations. Experiments under in vivo conditions

are essential to determine the role of fetal metabolism in eliciting the

therapeutic and fetotoxic effects of drugs in the fetus. The

development aspects of drug metabolizing enzymes at different

gestational ages should also be investigated in utero.

Excretion. Renal function in the fetus is underdeveloped. Both

glomerular filtration rate and renal plasma flow rate are lower on a body

weight basis; tubular secretion of organic compounds is restricted. This

results in prolonged elimination half-life for polar, water soluble

compounds in the fetal circulation.

CONCLUSION

The therapeutic and toxicologic effects of drug exposure to the fetus are

governed by a combination of factors which modify the pharmacokinetics

of drugs and metabolites in the mother, placenta, and fetus. The role of

pharmacokinetic studies in prenatal pharmacology and toxicology is,

therefore, to characterize the essential pharmacokinetic parameters that

determine the therapeutic benefits or/and fetotoxic risks of drug use

during pregnancy. Well-designed and properly conducted pharmacokinetic

experiments will provide valuable information on drug disposition in the

maternal-plac,fetal-fetal unit.

A systematic pharmacokinetic study of drug and metaholite disposition in

the maternal-placental-fetal unit was described. The model drug,

acetaminophen, was found to be transferred across the placenta via

passive diffusion into the fetal circulation and metabolized in the fetus to

35
the glucuronide and sulfate conjugates. Transplacental clearances of

acetaminophen from the maternal and fetal circulation were found to be

equivalent. No placental metabolism of acetaminophen was observed. No

transplacental transfer of acetaminophen conjugates was detected. Both

acetaminophen glucuronide and sulfate are predominantly eliminated from

the plasma through renal excretion via glomerular Filtration in the fetal

kidney and by renal tubular secretion and glomerular Filtration in the

maternal kidney. Knowing the disposition of the conjugates, we could

then determine the conjugation activities in the ewes and in the fetal

lambs in utero. Glucuronidation and sulfation activities are much lower in

the fetal lambs than those in the ewes. Therefore, both conjugation

pathways could possibly be saturated in the fetus should a large dose of

acetaminophen be taken by the mother. If a reactive metabolite of

acetaminophen could be formed in the fetus, saturation of the conjugation

pathways could lead to toxic effects in the Fetus. Further studies are

required to characterize the oxidative metabolism and glutathione

conjugation pathways in the fetus in utero before the potential toxicity of

acetaminophen to the fetus can be assessed.

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Obstet Cynaecol 85:318-524, 1978.

Amon, I., and Amon, K. Zum Ubertritt von Arzneimitteln in das Fruchtwasser. Zentralb Gynakol 98:961-969, 1976.

Aranda, J.V., and Stern, L. Clinical aspects of developmental pharmacology and toxicology. Pharmacol Ther 20:1-51, 1983.

Benet, L.Z. Pharmacokinetics: Basic principles and its use as a tool in

drug metabolism. In: Mitchell, J.R., and Horning, M.G., eds. Drug

Metabolism and Drug Toxicity. New York: Raven Press, 1984. pp.

199-211.

Blake, D.A.; Collins, J.M.; Mayasaki, B.C.; and Cohen, F. Influence of

pregnancy and folic acid on phenytoin metabolism by rat liver

microsomes. Drug Metab Dispos 6:246-250, 1978.

Bogaert, M.C., and Thiery, M. Pharmacokinetics and pregnancy. Eur J

Obstet Gynecol Reprod Biol 16:229-235, 1983.

Carson, G.D.; Bolla, J.D.; and Challis, J.R.G. The availability of cortisol

in amniotic fluid to the fetus and chorionic and amniotic membranes.

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Dawes, G.S. Breathing and rapid-eye movement sleep before birth. In:

Comline, R.S.; Cross, K.W.; Dawes, G.S.; and Nathanielsz, P.W., eds.

Fetal and Neonatal Physiology. Cambridge: Cambridge Press, 1973. pp.

49-62.

Dutton, G.J., and Leakey, J.E.A. The perinatal development of drug metabolizing enzymes. Prog Drug Res 25:189-273, 1982.

Gabrielsson, J.L., and Paalzow, L.K. A physiological pharmacokinetic

model for morphine disposition in the pregnant rat. J Pharmacokinet

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Gillette, J.R. Factors that affect drug concentrations in maternal plasma. In: Wilson, J.G., and Fraser, F.C., eds. Handbook of Teratology.

New York: Plenum Press, 1977. pp. 35-78.

Goodman, D.R.; James, R.C.; and Harson, R.D. Placental toxicology.

Food Chem Toxicol 20:123-128, 1982.

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Juchau, M.R. Drug biotransformation reactions in the placenta. In:

Mirkin, B.L., ed. Perinatal Pharmacology and Therapeutics. New York:

Academic Press, 1976. pp.71-118.

Juchau, M.R., and Faustman-Watts, E. Pharmacokinetic considerations

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1983.

Krauer, B., and Krauer, F. Drug kinetics in pregnancy. Clin Pharmacokinet 2:167-181, 1977.

Krauer, B.; Krauer, F.; and Hytten, F.E. Drug disposition and pharmacokinetics in the maternal-placental-fetal unit. Pharmacol Ther

10:301-328, 1980.

Levy, G., and Hayton, W.L. Pharmacokinetic aspects of placental drug

transfer. In: Boreus, L.O., ed. __ Fetal Pharmacology. New York:

Raven Press, 1973. pp.29-40.

Lucier, G.W.; Lui, E.M.K.; and Lamartiniere, C.A. Metabolic activation/

deactivation reactions during perinatal development. Environ Health

Perspect 29:7-16, 1979.

Mellor, D.J. Investigations of fluid spaces of the sheep conceptus. In:

Nathanielsz, P.W., ed. Animal Models in Fetal Medicine. Amsterdam:

Elsevier/North Holland Biomedical Press, 1980. pp.59-106.

Murkin, B.L., and Singh, S. Placental transfer of pharmacologically active

molecules. In: Mirkin, B.L., ed. Perinatal Pharmacology and

Therapeutics. New York: Academic Press, 1976. pp. l-69.

Nau, H., and Liddiard, C. Placental transfer of drugs during early human

pregnancy. In: Merker, H.J.; Nau, H.; and Langman, J., eds. Role of

Pharmacokinetics in Prenatal Pharmacology and Prenatal Toxicology.

Stuttgart: Georg Thieme Publishers, 1978. pp. 465-481.

Pelkonen, O. Biotransformation of xenobiotics in the fetus. Pharmacol

Ther 10:261-281, 1980.

Rajchgot, P., and MacLeod, S. Perinatal pharmacology. Prog Clin Biol

Res 135:3-23, 1983.

Rumph, A.M., and Heymann, M.A. Methods for studying the circulation

of the fetus in utero. In: Nathanielsz, P. W., ed. Animal Models in

Fetal Medicine. Amsterdam: Elsevier/North Holland Biomedical

1980. pp. 1-57.

Seeds, A.E. Basic concepts of maternal-fetal amniotic fluid exchange.

Pediatr Clin North Am 28:231-240, 1981.

Szeto, H.H. Pharmacokinetics in the ovine maternal-fetal unit. Annu

Rev Pharmacol Toxicol 2:221-243, 1982.

Szeto, H.H.; Umans, J.C.; and Rubinow, S.I. The contribution of transplacental clearances and Fetal clearance to drug disposition in the ovine

maternal-fetal unit. Drug Metab Dispos 10:382-386, 1982.

Tindall, V.R. The liver in pregnancy. Clin Obstet Gynaecol 2:441-462,

1975.

Wang, L.H.; Rudolph, A.M.; and Benet, L.Z. Acetaminophen disposition in

adult, neonatal and fetal sheep (abstr.) APHA Acad Pharm Sci 13(1):94,

1983.

Wang, L.H.; Rudolph, A.M.; and Benet, L.Z. The distribution and Fate

of acetaminophen conjugates in fetal lambs in utero. Submitted to J

Pharmacol Exp Ther, 1985a.

Wang, L. H.; Rudolph, A.M.; and Benet, L.Z. Pharmacokinetic studies of

the disposition of acetaminophen in the sheep maternal-placental-Fetal

unit. Submitted to J Pharmacol Exp Ther, 1985b.

37
ACKNOWLEDGMENTS

This work was supported in part by National Institute of General Medical

Sciences center grant CM26691. During the course of this work, L.H.

Wang was the recipient of a Chancellor’s Graduate Research Fellowship

at the University of California, San Francisco.

AUTHORS

Laurene H. Wang, B.S., M.S.

Graduate Research Fellow

Department of Pharmacy

School of Pharmacy

University of California

San Francisco, California 94143.

Abraham M. Rudolph, M.D.

Professor of Pediatrics, Physiology, and Obstetrics,

Gynecology and Reproduction Sciences

School of Medicine

University of California

San Francisco, California 94143

Leslie Z. Benet, Ph.D.

Professor and Chairman

Department of Pharmacy

School of Pharmacy

University of California

San Francisco, California 94143

38
The Pharmacodynamics of Prenatal

Chemical Exposure

Richard K. Miller and Carol K. Kellogg

Pregnancy represents a unique time in the life cycle of a species. It is a

collage of interacting and interdependent variables in the mother, conceptus, and placenta--modifying all three during the course of gestation in

mammals. If one superimposes the introduction of a chemical in this complex milieu, the number of interactions relating to the distribution,

metabolism, and effects of such an agent in mother and conceptus can be

considerable.

Substantial interest has recently been focused in three major areas of

developmental investigations which relate to: (1) the ability of chemicals to

be metabolized to toxic products which may then produce their effects in

either mother and/or conceptus; (2) specific interactions with developing

processes, e.g., cell-cell interactions necessary for appropriate organ

development, or receptor-specific responses; and (3) organ-specific responses leading to lethality, or reduced functional or structural capacity,

e.g., the central nervous system (CNS), reproductive system, and placenta.

No single set of experimental tools can be applied to explore such questions. Most often a comprehensive evaluation of the distribution, metabolism, and interactions of a compound, whether drug or environmental

chemical, must be completed for both mother and conceptus before any

understanding of mechanistic actions can be established. These are

common recommendations for or from a pharmacologist or toxicologist;

however, these principles are often not considered critical or widely appreciated by teratologists, obstetricians, or pediatricians. Other clinicians not devoted to studies in developmental pharmacology or toxicology

do not appreciate the fact that the conceptus can be a separately

functioning organism from a pharmacological point of view and, therefore,

these investigators are content with extrapolating observations of drug

distribution, metabolism, and interaction from the nonpregnant adult, and

applying them directly to the pregnant patient. This fallacy must be

eliminated forever and in its place must be imprinted the following:

1. There are dramatic and continuing changes in the physiology and

biochemistry of pregnancy, which include both mother and

conceptus and do persist throughout the entire course of gestation.

39
2. There are two entirely separate and distinct genomes existing in

the same organism (mother).

3. There are two separate and distinct blood supplies with a unique

interface, the trophoblast.

4. There is rapid and selective growth of specific cell types in the

conceptus at particular stages of gestation.

5. There are direct and indirect interactions among mother,

embryo/fetus, and placenta.

With such sensitivity to basic principles of pharmacology and toxicology, it

is possible to begin to assess the reasons by which certain responses in the

pregnant female may differ from that of the nonpregnant female, and

equally important why the conceptus (whether embryo, fetus, or placenta)

may respond differently at one time from another time in its fife cycle

(figure 1). Yet, to evaluate the effects of chemical exposure during pregnancy, one must consider not only the immediate impact of such exposure

on those cells, organs, or systems, but rather the impact on the total

development and life of the mother and conceptus. How persistent or

transient are the effects? Are these effects due to the presence of the

chemical at the time of testing or are these effects the result of permanent alterations in system function without the persistence of the agent

at the time of testing? Does the conceptus have mechanisms to guard

itself against such chemical invasions or is it at the mercy and benevolence

of the mother to protect and sustain the growth and development of the

conceptus?

FIGURE 1. The continuum of perinatal pharmacology and toxicology

40
These questions of pharmacodynamics will be addressed by evaluating two

classes of compounds and organ systems: (1) Heavy metal toxicity and

placental function; and (2) ataratics and CNS function. Through these

examples, specificity and selectivity of chemical distribution in mother and

conceptus as well as organs/cells will be explored with an emphasis on the

selectivity of chemical response in mother and conceptus either in utero or

as an adult.

Before investigating specific examples, please refer to figure 2 which

presents a general review of the principal variables for consideration

during pregnancy for the mother, placenta, and conceptus. These factors

will be discussed in the following pages only as they relate to the specific

examples presented. For a detailed discussion of these factors, the reader

is referred to Miller et al. (1976) and Miller (1983).

FIGURE 2. Biochemical and pysiological consideration in prenatal

exposure to chemicals (from Miller 1983, Cpoyright 1983, Alan R. Liss,

Inc.)

HEAVY METAL TOXICITY AND PLACENTAL FUNCTION

Heavy metals, in particular lead, mercury, and cadmium, are associated

with birth defects and alterations in growth (cf. Clarkson et al. 1983). The

usual exposure to such metals is considered to be occupational or environmental (in air, water, or food); however, it is becoming apparent that

substance abuse in the form of cigarette smoking can result in the accumulation of cadmium within the body. One organ which appears to

concentrate these metals is the placenta (cf. Miller and Shaikh 1983). The

concentration of these metals in the human placenta has been correlated

with such environmental and occupational exposures (Clark 1977;

Hubermont et al. 1978; Lauwerys et al. 1978; Khera et al. 1980).

Placentae from many regions of the world have been evaluated for the

concentration of cadmium (cf. Miller and Shaikh 1983). Unfortunately,

41
there is wide variation within any given locale for the actual levels of

cadmium when compared with lead or mercury. As noted above, the reason

for such variation is attributed to the personal habits of the individuals as

correlated with the amount of cigarette smoking. Studies in multiple

laboratories and geographical regions have confirmed this association

between cigarette smoking and placental concentration of cadmium (table

1). It has been proposed that the placenta be an exposure index for selected

environmental chemicals. Yet, such questions as level of risk, doseresponse relationships, and actual form of the metals have not been

investigated extensively.

Cadmium rapidly appears in the blood when a person inhales it via cigarette smoke. In our laboratory, we have noted cadmium levels hetween 4

and 8 ng/ml in blood after smoking a single cigarette. Thus, besides the

carbon monoxide, nicotine, cyanide, polycyclic aromatic hydrocarbons, and

other metals, cadmium is present in cigarette smoke and found in higher

concentrations in the placenta, whereas other metals are not significantly

altered, e.g., copper and zinc (table 1).

Such interest in cadmium and the placenta was first reported by Parizek

(1964) when he noted that a single injection of cadmium into a near-term

rat resulted in the death of the fetus and the appearance of placental

necrosis. This fetal lethality is now known to result from direct placental

toxicity and reduced placental blood flow rather than a direct effect on the

fetus (Levin and Miller 1980; Levin et al. 1981).

The sequence of toxic response (figure 3) is apparently an initial rapid

uptake of cadmium by the placenta at levels greater than that noted by the

kidney, an index for adult toxicity. The maternal blood levels of cadmium

peak at 18 to 20 nmoles/ml within 5 minutes, while the placental concentrations of cadmium reach 60 to 90 nmoles/gm by 1 hour. Interestingly,

only small quantities of cadmium (<0.1 nmoles/gm) actually enter the fetus

(Ahokas and Dilts 1979; Sonawane et al. 1975; Levin and Miller 1980; Levin

et al., in press). By 4 to 6 hours, there is a rise in mitochondrial calcium,

an index of cellular toxicity and ultrastructural damage (Levin et al. 1981,

1983; di Sant’Agnese et al. 1983). By 10 to 12 hours, a 5 percent incidence

of fetal death, a low incidence of histologic placental necrosis, and a 25

percent reduction in utero placental blood flow were all observed (Levin

and Miller 1981). By 18 hours, all of these observations increased

substantially. Recently, cadmium administration during late gestation in

rodents reduced the placental transfer of cobalamin while not significantly

altering neutral amino acid transfer (Danielsson and Dencker 1984). Such

dramatic changes indicate the potential for a single dose of cadmium

administered near term in the rat to produce placental toxicity with

resultant fetal lethality.

Wolkowski (1974) hypothesized that it is not cadmium which produces the

placental toxicity, but rather metallothionein, a 10,000 dalton protein

which binds cadmium and other metals. Metallothionein is a known

nephrotoxin. However, metallothionein injected intravenously into the

mother did not produce fetal death, but did produce nephrotoxicity. While

blood levels of cadmium dropped from 18 nmoles/ml to 1 nmole/ml, the

content of cadmium in the placenta did not increase comparably (Plautz et

al. 1980; Levin et al. 1981).

42
TABLE 1. Placental Levels of Cadmium, Copper, and Zinc in Women Who Do or Do Not Smoke

Location

Belgium

Nonsmoker

Smoker

Netherlands

Nonsmoker

Smoker

No333

109

31

30

Cadmium Copper Zinc

ng/g ug/g ug/g

12.5 ± 8.6

15.7 ± 9.2*

8.2 ± 3.2

10.6 ± 5.2*

Reference

Roels et al. 1978

Van Hattum et al. 1981

Rochester, NY

Nonsmoker 18

Smoker 24

4.3 ± 2.8 11.76 ± 0.65 1.24 ± 0.10 Miller and Gardner 1981

19.2 ± 4.6* 11.94 ± 0.64 1.36 ± 0.08

Cleveland, Ohio

Nonsmoker

Smoker

31

44

13.7 ± 6.4 Kuhnert et al. 1982

18.1 ± 7.3*

*Significantly different at least at (P < 0.05).
The placenta levels of cadmium in the human study were similar to levels

found in the rodent (Wier et al. 1983b), and the resultant functions of the

placenta during prolonged perfusion with complete transfusions every 4

hours were notably compromised. The fetal circuit began to lose volume

>5 ml/hr) between 8 to 10 hours of perfusion; the production, tissue

content, and perfusate levels of human chorionic gonadotropin were

depressed by 85 percent. Further, the stroma became edematous,

Hofbauer cells were vacuolated, and subsynctiotrophoblastic vacuolization

became widely apparent, as has also been noted for cycloheximide exposure

(Wier et al., in press).

Therefore, besides studying the distribution, metabolism, and transfer of

chemicals between the maternal and fetal circulations, the tissue content

and alterations in both structure and function over periods between 12 to

16 hours can be examined to answer the question: Does a compound appear

in the fetal circulation? In addition, the human question of how chemicals

interact with the placenta and its circulation and function now can be

studied without risk to mother or baby.

MATERNAL CIRCUIT FETAL CIRCUIT

FIGURE 4. Diagram of a dual perfused recirculating human placenta

lobule perfusion

The placenta, whether human or rodent, does result in chemical-induced

pathology (Panigel 1981; Miller and Thiede 1981, 1984). It is proposed that

prolonged perfusion of the human placenta under in vitro conditions may

offer an opportunity for assessing not only transfer and metabolism by

human fetal tissue-trophoblast but also the potential for determining the

toxicity of both drugs and environmental chemicals. The transfer and

metabolism of diazepam has been studied under short-term perfusion

conditions for the human placenta (Guerre-Milo et al. 1982). It was found

that the degree of plasma binding by diazepam and other benzodiazepines

did alter the placental transfer of these ataratics.

44
It is apparent that this dose of cadmium, which results in placental toxicity

in the rat, is greater than the levels in placentae from cigarette smokers

(table 1). A dose-response relationship for different species, including

man, presents multiple difficulties in gathering these epidemiologic data

following acute exposure.

et al. (1983).

FIGURE 3. Sequence of observed responses in the near-term pregnant rat

following single subcutaneous administration of 40 umoles/kg of cadmium

chloride. The lines represent a relative change in structure, concentration

or function without direct comparison to absolute values among the

variables measured. This figure summarizes the observation reported by

Levin and Miller (1980, 1981) Levin et al. (1981, 1983, in press), and Miller

To compare the species similarities and differences, an in vitro human

placental system that allows for 12-hour perfusion was developed (Miller et

al. 1985) to explore the direct toxicity of cadmium on the human placenta

(figure 4). This perfusion system is based on the model developed by

Panigel (1962) and Schneider et al. (1972). In the rat, following a subcutaneous dose of cadmium chloride of 40 nmoles/kg, the plasma levels of

cadmium were 18 nmoles/ml at 5 minutes and 1 nmole/ml after 1 to 2

hours (Levin et al., in press). These kinetics for cadmium were reproduced

in the in vitro human placental perfusion where initial doses were either 10

or 100 nmoles/ml. In particular, little cadmium passed to the fetal

circulation in either the rat or human study, while the placenta

concentrated cadmium in excess of 100 nmoles/gm.

45
PRENATAL DIAZEPAM AND POSTNATAL FUNCTION

The developing brain can be permanently altered by drugs which have only

acute or transient effects on the adult nervous system. Such permanent

alterations in CNS function following exposure of chemicals to the

developing brain may be different from those effects noted following adult

therapy. The underlying mechanisms for such differences in responses

between the adult and developing brains are reflected in the sequence of

events, e.g., appearance of specific receptors, neurotransmitters, cell

types, and cell-cell interactions, which are spatially related to particular

stages of in utero and neonatal development (figure 5). In the adult, these

relationships are all established and, therefore, drugs affect a complete

system; however, depending upon the species and the growth stage, the

development of neurotransmitters, receptors, cell types, and cell-cell

interactions are different.

FIGURE 5. Prenatal exposure of diazepam to rats during the last week of

gestation and the persistnce of the drug in the brains of the progeny as

related to developmental events in the fetal and neonatal brains. Prenatal

and postnatal age are indicated on the horizontal line. Late gestation in

the rat is characterized by rapid neuronal proliferation and differentation

as well as by the begining of synaptic formation. Specific receptor for

drugs and transmitters begin appearing during this period. Abbreviations:

CS = catecholamine. BZ = benzodiazepine. ACH = acetylcholine. GABBA =

gomma aminobutyric acid.

Even though organogenesis (embryogenesis) in both man and rat can be

compared, the rapid development of the rat suggests that even single

exposure to a drug may be equivalent to multiple exposures in man.

4 6
Malformations of the CNS, such as spina bifida and anecephaly, are

developmental anomalies due to the effects of an agent directly on the

neural tube during embryogenesis. However, during this time of gestation,

the specific receptors and neurotransmitters apparently are not present or

functional. Therefore, the specificity of CNS drug action on the developing CNS would be predominant when the appearance of these specific

neurochemical processes are present. Such development is seen from

approximately the 10th week in the human and day 10 or 11 in the rat.

Therefore, for a number of functional defects the fetal period may be the

most susceptible time in the life cycle.

Is there though a comparison between the human and the rodent when one

is assessing behavior, function, and the effects of drugs? The answer must

be an emphatic yes with the caveat that one is examining mechanisms of

action and, therefore, the potential species differences are adequately

considered before conclusions are drawn. Such examinations rnust include

pharmacokinetics/dynamics, developmental sequences, intercurrent disease, and the exact timing of the drug exposures.

The question of whether drugs or environmental chemicals do alter postnatal function has been reviewed extensively by other investigators (Rodier

1980; Vorhees and Butcher 1982; Hutchings, in press). Many psychoactive

agents act on specific mechanisms of neurotransmitter function, e.g.,

transmitter receptors, reuptake sites, synthesizing enzymes, or release

mechanisms. However, how specifically, selectively, and extensively can a

drug modify postnatal function following in utero drug exposure has not

been resolved.

Diazepam, a minor tranquilizer, has been investigated in animals to explore

these questions because of the variety of regulatory dysfunctions noted in

the human neonate whose mother had taken diazepam during pregnancy

(Scher et al. 1972; Cree et al. 1973; Safra and Oakley 1978; Rementeria

and Bhatt 1982). Even maternal stress itself induces functional impairments in the progeny (Ward 1972). Such functional alterations due to

maternal stress have been prevented by concurrent diazepam therapy

during pregnancy (Barlow et al. 1979). However, does such therapy represent a specific and permanent interaction within the developing brain?

Diazepam, when administered between days 13 to 20 of gestation in the rat

(where gestation is 21 to 22 days), can modify a number of behavioral endpoints during postnatal development and in adult life, i.e., startle response,

auditory temporal resolution, and coping with stress, without significantly

altering more general behaviors (Kellogg et al. 1980, 1983b; Simmons et al.

1984b). These specific behavioral changes are related to maternal doses of

diazepam (1 to 10 mg/kg). Such selectivity in response indicates a specific

mechanistic interaction. Previous investigations have characterized a

benzodiazepine receptor in the adult which interacts with both the

GABAminergic and catecholaminergic neurons (cf. Gee et al. 1984).

These alterations in adult behavior following prenatal exposure to diazepam are not related to the continued presence of diazepam into adult life

since the pups were fostered to control mothers at birth and diazepam was

undetectable in the neonatal brain or other tissues by day 20 (table 2).

47
TABLE 2. The Distribution of Diazepam and its Metabolites in Selected Tissues of the Mother and Neonates

Following In Utero Administration of 2.5 mg/kg of 14C-Diazepam Between Days 13 to 20 of Gestation (Simmons

et al. 1983).

Total Diazepam/Metabolites Proportion of Diazepam and

in Selected Tissues

pmoles/1OOmg

Plasma Liver Heart

Maternal 3.7±0.5 55.8±7.7 2O.1±1.4

Neonatal 0 ND 7.8±0.9 6.3±0.5

Neonatal 10 N D ND ND

Neonatal 20 N D ND ND

Metabolites in Brain

Brain

3.4±0.3

3.2±0.3

3.4±0.3

ND

N-desmethylDiazepam diazepam

24±5% 28±4%

24±9% 25±9%

19±8% 39±9%

ND ND

Oxazepam Glucuronide

0% 49±2%

0% 52±4%

12±6% 32±5%

ND ND

ND - not detectable
Thus, persistent functional alterations are present in the adult progeny

following only prenatal exposure to diazepam, even though the drug is no

longer present.

A mechanistic evaluation of these aberrant behaviors was initiated to

determine: (1) the relationship to stress, (2) the neurochemical impairments (GABA, GAD, norepinephrine), (3) regional specificity of such

changes, and (4) receptor specificity. Since sensory perception was altered

by prenatal exposure to diazepam, does diazepam permanently alter the

ability of the offspring to respond to stress? Following restraint stress, the

adult control progeny demonstrated the typical response for plasma corticosterone changes to such stress (figure 6). However, prenatal exposure to

diazepam prevents the surges in this hormone following such stress. Thus,

the ability of these animals treated in utero with diazeparn to respond

normally to stress was impaired.

FIGURE 6. Plasma levels of corticosterone in 90-day-old rat progeny

under basal conditions and following 30 or 120 minutes of restraint stress.

The stressor was restraint stress, which was induced by placing the animal

in a small plastic restraint cage located in a bright lighted room for

specific time intervals. Asterish denotes a significant differnce (p < 0.05)

from conrols at each individual time period as determined by Student's -ttest. Corticosterone was measured by a specific radioimmunoassay using

dog corticosterone binding globulin as the antibody (from Simmos et al.

1984b, Copyright 1984, Elsevier Science Publishers, B.V).

49
Such hormonal regulation would implicate the hypothalamus in the control

of these processes. Regional neurochemical determinations of GABA

receptors, norepinephrine, and glutamic acid-decarboxylase demonstrated

that only norepinephrinre content and turnover were decreased in a doserelated manner by diazepam in the hypothalamus of the adult progeny

(table 3). These alterations in norepinephrine neurons occured when no

significant changes in the number of binding sites, dissociation constant, or

maximal stimulation by GABA were noted for benzodiazepine binding in

newborns or adults prenatally exposed to 2.5 or 10 mg/kg/day of diazepam

(Kellogg et al. 1983b).

Such selectivity of neurochemical and functional responses would be

consistent with a specific activat ion mechanism. Fortunately, a benzodiazepine antagonist for the central binding sites (RO15-1788) is available.

KO15-1788 does not appear to have agonist activity but rat her blocks

diazepam at the receptor (Gee et al. 1984). To determine whether the

benzodiazepine recepot is involved in the product ion of these functional

impairments, RO15-1788 was coadministered to the pregnant dam with

diazepam. Prenatal RO15-1788 blocked the inhibitory effects of prenatal

diazepam on these stress-induced corticosterone surge (figure 6) and the

content and turnover of norcpinephrine in the hypothalamus of the adult

progeny (table 4).

Thus, it is becoming apparent that specific CNS functions may be permanently altered by prenatal exposures to drugs which have selective interactions with the CNS while many other functions may appear normal. Such

selectivity and specificity of interactions between drug and regions of the

brain demonstrate that what may be a transient yet spcific effect in the

adult brain may be just as selective and specific--receptor or mediated--in

the developing brain with a resultant permanent alteration.

CONCLUSION

Cigarette smoking has been associated with small for gestation age infants,

postnatal growth retardation, induction of mixed function oxidases, and

other delays in development. In particular, cadmium has been documented

to be at higher levels in placaentae of (cigarette smokers than in nonsmokers. In both animal studies and in vitro human placenta perfusion

studies, cadmium has been found to be placental toxic. Such specificity for

the placenta appears to be greater than for the organ commonly thought to

represent cadmium toxicity, the kidney. Yet, many other constituents of

cigarette smoke do modify the capacity of the conceptus, e.g., nicotine

affects uterine blood flow, placental transport of nutrients, and fetal

breathing; polycyclic aromatic hydrocarbons affect fetal and placental

metabolism of xenobiotics (cf. Sastry et al. 1983; Juchau 1980, 1982, this

volume; Pelkonen, 1980; Miller et al., 1983).

The metabolism of drugs and environmental chemicals has been implicated

in their developmental toxicity, e.g., benzo(a)pyrene (Nebert 1981),

phenytoin (Martz et al. 1977), cyclophosphamide (Manson and Smith 1977;

Kitchhin et al. 1981), diethylstilbestrol (cf. Metzler 1984), and thalidomide

(Gordon et al. 1981). Such formation of reactive intermediates may mean

that the parent compound has different therapeutic effects compared with

50
TABLE 3. Kinetics of Norepinephrine Turnover in Adult Hypothalamus in Rats Exposed In Utero to Diazepam

Between Days 13 to 20 of Gestation (Simmons et al. 1984a).

Prenatal exposure No. NE levels Turnover rate Turnover rate Turnover Linear regression

days 13-20 (ng/g) (ng/g/h) constant(h-1) time (h) coefficient

Uninjected 23 1895 ± 221 322 0.17 ± 0.01 5.9 0.89

Vehicle 21 1688 ± 108 388 0.23 ± 0.02* 4.4 0.88

Diazepam 1.0 mg/kg 20 1304 ± 130* 241 0.18 ± 0.02 5.6 0.81

Diazepam 2.5 mg/kg 21 1238 ± llO* 112 0.09 ± 0.02 11.1 0.92

Diazepam 10 mg/kg 17 657 ± 108* 46 0.07 v 0.02' 14.3 0.90

*Significantly different from control value, P < 0.05.

Mean + S.E.M.

No. = number of animals
TABLE 4. Kinetics of Norepinephrlne Turnover in the Adult Hypothalamus: Effect of Prenatal Exposure to a

Benzodiazepine Antagonist (Simmons et al. 1984a).

Prenatal exposure No. NE levels Turnover rate Turnover rate Turnover Linear regression

days 13-20 (ng/g) (ng/g/h) constant (h-') time (h) coefficient

Uninjected 15 1719 ± 138 292 0.17 ± 0.01 5.9 0.88

RO-1788 10 mg/kg 15 1695 ± 152 278 0.16 ± 0.02 6.1 0.82

RO-1788 10 mg/kg & 15 1662 ± 133 288 0.17 ± 0.01 5.8 0.92

Diazepam 2.5 mg/kg

Diazepam 2.5 mg/kg 15 1298 ± 112* 117 0.09 ± 0.01* 11.1 0.90

*Significantly different from control value, P < 0.05.

No. = number of animals

Mean ± S.E.M.
the toxicity of their reactive metabolites (arene oxides, epoxides, phenoxyradicals). In fact, it may be the metabolic capability of the conceptus

which determines the developmental toxicity of a compound and not just

the maternal capabilities as demonstrated for benzo(a)pyrene metabolism

by embryos in the same litter (Shum et al. 1979). Thus, the pharmacogenetics of the conceptus may partially account for why only 1 out of 10

babies have the fetal phenytoin syndrome, especially since electrophilic

intermediates of phenytoin have been implicated in producing birth defects

in mice (Martz et al. 1977). Thus, for some drugs, it may not be the effect

of the parent compound but other effects of its metabolites.

Besides metabolism, protein binding of drugs in both mother and conceptus

may be critical to the expressed toxicity. In the rodent, synthetic estrogens such as diethylstilbestrol (DES) are both carcinogenic and teratogenie at doses lower than those noted for natural estrogens, such as

estradiol. Such selectivity in response, especially in the rat, relates to: (1)

specific plasma binding protein for the natural estrogen, estradiol, which

circulates in the fetus; (2) the rapid metabolism of estradiol; and (3) the

responsiveness of the developing reproductive tract, which is substantially

different from that of the adult. In the fetus, one of the primary reasons

estradiol is less teratogenic than DES is that the affinity of alpha-fetoprotein for estradiol is much greater than for DES. Therefore, less

estradiol is available for entry into the tissue (cf. Miller et al. 1982, in

press).

Persistent and selective alterations in the CNS can be induced following

exposure to low doses of diazepam during the last week of gestation in the

rat. Aberrant responses to stress have been observed in the adult progeny

when no trace of the drug is detectable. Yet, of greater interest is the

selectivity of this prenatal diazepam exposure for reducing hypothalamic

norepinephrine levels and turnover in the adult progeny and the prevention

of these aberrant behavioral responses and neurochemical alterations in the

hypothalamus following coadministration of a diazepam antagonist with

diazepam during gestation. Thus, these examples of prenatal exposure to

cadmium, DES, and diazepam demonstrate the important differences in

their individual pharmacokinetics, which include distribution, metabolism,

protein binding, specific tissues, and even receptor interactions.

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ACKNOWLEDGMENTS

The original work presented was supported in part by National Institutes of

Health grants ES 02774, CA 22335, and MH 31850, and a research award

from Hoffmann-LaRoche.

AUTHORS

Richard K. Miller, Ph.D.

Associate Professor of Obstetrics/Gynecology, and

Pharmacology/ Toxicology

Director, Division of Research

University of Rochester

School of Medicine and Dentistry, Box 668

601 Elmwood Avenue

Rochester, New York 14642,

Carol K. Kellogg, Ph.D.

Professor of Psychology and Pharmacology

University of Rochester

Rochester, New York 14620

57
Opioids and Development: New

Lessons From Old Problems

Ian S. Zagon

OLD PROBLEMS

Human consumption of opium for religious, medicinal, social, and/or

personal reasons probably dates back 6,000 years (Blum 1969; Terry and

Pellens 1970; Musto 1973). An important corollary to opioid’ utilization by

pregnant women relates to the passive dependence of the fetus. Exactly

how long ago this relationship was perceived is unclear. However,

Hippocrates (ate Martin 1893) mentions opium in connection with “uterine

suffocation”; this may be a reference to opium’s toxicity on the embryo

and/or fetus. Clearly, by the end of the 19th century, clinical reports

evidenced the unusual behavior of the fetus and the neonate passively

addicted to opium. Infants with “chronic opium intoxication” were

reported to exhibit excessive nervousness, rapid breathing, and convulsive

movements right after birth, with death occurring within the first week of

life (Terry and Pellens 1970). A review of the early literature reveals an

impressive amount of information and insight into the field of perinatal

opioid dependence. For example, in 1895, Bureau found that morphine

could pass through the human placenta. Other workers recognized the

possibilities that maternal drug addict ion might influence the fetus

(Sainsbury 1909), and that enough opium passed into breast milk to

alleviate withdrawal (Laase ISIS; Langstein 1930; Petty 1912) or to even

establish drug dependency in a normal infant breast-fed by a woman who

became dependent on opioids after parturition (Lichenstein 1915). The

importance of therapeutic intervention by administration of morphine,

heroin, or paregoric to prevent withdrawal was also well recognized

(Menninger-Lerchenthal 1934) by the beginning of the 20th century. Even

the long-term implications of perinatal opioid exposure were noted by

these early investigators. For example, in a discussion following the

presentation of a paper by Earle (1888) concerning the adverse effects of

fetal opioid exposure, a number of particpants reported the association of

neurobiological abnormalities in offspring (including individuals reacahing

adulthood) maternally exposed to opium. The incidence of perinatal

exposure to opioids is difficult to gauge. Perlstein (1947) and Petty (1913)

refer to the “rare” occurrence of such situations in earlier periods (up to

the 1940s). However, Graham-Mulhall (1926) recorded over 800 pregnant

5 8
women consuming opioids in New York during a 1-year period in the early

1900s. The type of opioid abused has changed over the years. Until the

1950s. morphine appeared to be the drug of choice, with the 1956 report by

Goodfriend et al. signaling a change to heroin usage. Following Dole and

Nyswander’s (1965) advocation of the methadone maintenance treatment

program as an alternative to heroin dependency, numerous reports have

documented methadone-dependent offspring. Paralleling the change from

morphine to utilization of heroin and methadone in the 1950s, a marked

increase in the number of births to opioid-dependent women has been

recorded. In one municipal hospital in New York City, for example, only

42 infants were born to heroin-dependent mothers frorn 1955 to 1959, but

26 infants were delivered during 1960 (Zelson 1975). In this same hospital,

over a sixfold increase in drug-dependent babies was recorded between

1960 (1 in 164 births) and 1972 (1 in 27 births). Recent estirnates (Carr 1975)

place the birth rate of heroin and methadone-addicted mothers at

3,000/year in New York City alone; given that New York City has

one-third to one-half of the total number of chronic heroin and

methadone users in the United States (Carr 1975; Salerno 1977), one could

extrapolate 6,000 to 9,000 births/year to opioid-consuming women. Carr

also reports that, as of 1975, 115,000 children of mothers dependent on

illegal opiates and rnethadone maintenance were in the New York City

area. Assuming again that this number for New York City is

representative of one-third to one-half of the entire United States

population, one can estimate that at least 250,000 infants, children, and

young adults have already been born to females consuming opioids such as

methadone or heroin. Placed within the context of the average number of

births in the U.S. every year (roughly 3.3 million), one can calculate that

at least 1 in 1,000 births is by a mother using heroin or methadone. The

incidence for this “fetal opiate syndrome” or “fetal narcotic syndrome,” as

it may be termed, rivals estimates for many well-known and highly

publicized problems of early life, including the fetal alcohol syndrome,

Down’s syndrome, and neural tube defects, and it far exceeds the

incidence of cancer in children aged 1 to 15 years. Assuming the

population of the U.S. is 230 million, and given a population of 230,000

children already exposed perinatally to opioids, 1 in 1,000 people in the

U.S. have been subjected to opioids in early life. By thernselves, these

numbers are significant. However, unreported use of opioids by pregnant

women and the possible influence exerted by paternal opiate consumption

may indicate an error of underestimation.

Clinical Observation

Even before birth the offspring of opioid-dependent women are subjected

to a host of potential problems. During the course of pregnancy, the

mothers of these children often encounter medical complications,

including infectious diseases, nutritional deficits, and an abnormal

incidence of venereal disease. Obstetrical complications include toxernia

of pregnancy and intrauterine growth retardation. Additionally,

spontaneous miscarriage, abortion, and stillbirths have been suspected of

being higher than normal among opioid-dependent women (Salerno 1977).

Many of these coassociated medical and obstetrical complications appear

to be secondary to the lifestyle and habits of the pregnant addict

(Perlmutter 1974), with prenatal care often being neglected. The very

59

483-942 0 - 85 - 3 : QL 3
fact that enrollment in the methadone program places women in touch

with health professionals who encourage prenatal care has been an

extremely positive feature of this program.

A decrease in birthweight of infants born to heroin-dependent mothers

has been recorded (Finnegan et al. 1972; Wilson et al. 1979), with low

birthweight infants often being small for gestational age. These growth

delays associated with prenatal heroin do not appear to be related to

inadequate maternal nutrition or prenatal care (e.g., Naeye et al. 1973).

In contrast to heroin-exposed offspring, methadone-dependent neonates

have higher birthweights and are of greater gestational ages, although

mean weight is often less than among nondrug dependent controls (Kandall

et al. 1974).

Conclusive evidence that maternal opioid dependency is related to

congenital malformations has not been presented, although some

incidences have been reported (e.g., Ostrea and Chavez 1979). Abrahms

(1975) recorded increased chromosomal aberrations in infants exposed to

heroin, but not necessarily methadone, while Chavez et al. (1979) noted an

increased incidence of sudden infant death syndrome (SlDS) in opioidexposed infants. A lower incidence and severity of neonatal jaundice is

known to occur for heroin-exposed infants (Zelson et al. 1973), and

premature infants of heroin-dependent mothers often demonstrate a

lower incidence of respiratory distress syndrome (Glass et al. 1971).

The proportion of stillbirths and mortality in the opioid-exposed

population generally appears to be increased in comparison to control data

(Ostrea and Chavez 1979; Perlmutter 1967), but the higher mortality rate

may not be necessarily related to the neonatal withdrawal syndrome

(Ostrea and Chavez 1979). Few studies have pursued details of postmortem examination of drug-dependent infants, although evidence from

neuropathological studies (e.g., Rorke et al. 1977) suggests that, in

addition to a host of nonspecific secondary gestational complications,

primary and specific effects of addictive drugs on the developing nervous

system occur.

Perhaps the most well-recognized sign of fetal exposure to opioids is the

neonatal withdrawal syndrome, with 60 to 90% of opioid-exposed infants

undergoing some degree of abstinence (Perlmutter 1974). Symptoms of

withdrawal include irritability, tremors, high-pitched cry, hyperactivity,

wakefulness, diarrhea, disorganized sucking reflex, respiratory alkalosis,

and lacrirnation, as well as hiccups, sneezing, twitching, myoclonic jerks,

or seizures. Opioids reported to cause these symptoms in the neonate are

heroin, methadone, meperidine, morphine, codeine, and pentazocine. The

onset of symptorns may be present at birth or may begin up to 14 to 28

days after birth. This depends upon the drug the infant was exposed to in

utero and the profile of pharmacokinetic excretion. Subacute syrnptoms

may last for 4 to 6 months after birth (Desmond and Wilson 1975).

Depending on the severity of the withdrawal syndrome, treatment with

such agents as paregoric, phenobarbital, diazepam, and chlorpromazine

may be required. The period of treatment may last frorn a few days to

several rnonths (Zelson et al. 1970). The question of advisability of breast

60
feeding by women on opioids has been raised. Opioids are known to be

present in breast milk (Blinick et al. 1975) and numerous reports describe

“addictive” effects in infants consuming opioid-containing milk

(Menninger-Lerchenthal 1934). Given the permeability of the blood-brain

barrier during perinatal life, and the fact that only nanogram and

picogram quantities of drug are required to occupy opiate receptors, it

could easily be envisioned that opioids in milk could be capable of

influencing the infant.

The neurobehavioral sequelae associated with perinatal opioid exposure

have been extensively reviewed by Zagon and McLaughlin (1984a). In

summary, a number of highlights should be mentioned. “Classical”

neurological tests (Davis and Shanks 1975) have shown opioid-exposed

infants to exhibit central nervous system hyperactivity, irritability,

tremors and hypertonicity, impaired nutritive sucking, vomiting, severe

sleep deficit, autonomic dysfunction (vasomotor lability and diarrhea), and

a lability of “states” manifested by frequent shifts between sleep and

wakefulness. Hyperactivity was a persistent finding that endured through

the early school age (Davis and Shanks 1975). Utilizing the Brazelton

Neonatal Behavioral Assessment Scale (BNBAS), a number of investigators

have tried to quantitate infant response to external stimuli, motor

organization, and ability to regulate state of consciousness. These studies

show an impaired ability of opioid-dependent babies to adequately

organize their response to environment, with a lessened capacity to

attend and react to noxious stimuli and to habituate to disturbing events

(Davis and Shanks 1975). Opioid-exposed infants exhibited a depressed

response decrement to light, with problems in visual and auditory

orientation evident (Chasnoff et al. 1982). Using the BNBAS to decipher

the specific effects of methadone on the neonate, Marcus et al. (1982)

have suggested that behavioral problems in methadone infants may not be

due to generalized central system irritability, but actually to

neuromuscular dysfunction. Other investigators have suggested that the

orientation responsiveness and excitability recorded in the BNBAS have

substantial impact on caregivers’ perceptions of infants, which rnay lead

to long-term consequences for caregiver-infant interactions.

During the period of infancy, a significant decrease in quiet sleep

(Schulman 1969) along with an increase (Dinges et al. 1980) or decrease

(Sisson et al. 1974) in rapid eye movement has been noted. Kron and

colleagues (1976) have reported an uncoordinated and ineffective sucking

reflex as a manifestation of the opioid abstinence syndrome, whereas

Lodge and colleagues (1975) found that the withdrawal period was

characterized by heightened auditory responsiveness and orientation,

lowered overall alertness, and poor attentiveness to visual stimuli.

Differences in neurological status between opioid-dependent and control

groups have not been noted by some workers (Kaltenback et al. 1979).

However, Rosen and Johnson (1982a) reported tone discrepancies,

developmental delays, and eye problems in children up to 18 months of age

delivered by methadone-dependent mothers. Between 6 and 12 months, no

real language development was detected.

Beyond the withdrawal period, Wilson et al. (1973) have found adaptive

behavior, language performance, and personal-social developrnent of

61
heroin-exposed children to be normal, but gross motor coordination was

more advanced than fine motor coordination. Disturbances of activity

levels and/or attention span, along with sleep disturbances, temper

tantrums, and low tolerance to frustration were recorded by Wilson at 1

year, while the onset of hyperactivity began at 12 to 18 months. Using the

Bayley Scale of Infant Behavior as a monitor for mental and motor

development, a number of investigators have found children that

subjected perinatally to opioids are within the normal range (Blatman and

Lipsitz 1972; Chasnoff et al. 1980). However, Wilson et al. (1981) have

noted the less attentive nature of both methadone- and heroin-exposed

infants, while Kaltenbach et al. (1979) recorded a higher failure rate on

three specific items: naming of two objects, pointing to five pictures, and

naming of three objects. Kaltenbach suggested that subtle differences in

cognitive behavior occur in opioid-exposed children that may be occluded

by summary scores. Rosen and Johnson (1982a) reported a lower than

normal score for methadone-exposed infants on both the mental and

motor development indices at 12 and 18 rnonths of age, and Strauss et al.

(1976) also noted delays in the physical development index.

Opioid-subjected infants tested on the Cesell scales (Finnegan et al. 1977;

Wilson et al. 1973), Merrill-Palmer test, and Peabody Picture Vocabulary

test (Blatman and Lipsitz 1972). as well as the Object Permanence Scales

(Rosen and Johnson 1982), appeared normal. A number of workers have

reported disorders in speech and/or language development (Blatman and

Lipsitz 1972) in children subjected to opioids, although Lodge et al. (1975)

reported strength in the realm of language for methadone-exposed

offspring.

Few studies that extend beyond the first few years of childhood have been

conducted on children maternally subjected to opioids. Nichtern (1973),

examining children up to 15 years of age born of heroin-dependent

mothers, recorded a number of problems related to their capacity for

human relationships, dernonstration of excessive adult-peer interactions,

poor socialization, and the use of withdrawal to deal with difficult

situations. Rosen and Johnson (1982a), in a study of children exposed in

utero to methadone, recorded signs of impaired development, including

smaller head circumferences, strabismus and/or nystagmus, and

abnormalities in muscle tone, coordination, and language. Strauss et al.

(1979) found a cluster of behavioral differences related to greater

task-irrelevant activity in methadone-exposed children during testing

situations. These authors suggest that in structured and demanding

situations, qualities of attentiveness and motor inhibition may be a

domain in which opioid-exposed children might reveal particular

vulnerabilities.

The neurobehavioral implications of exposure to heroin during early life

have been reported by Wilson and colleagues (1979). Wilson did not find

problems with speech and language function, hut on a battery of

perceptual measures, visual, tactile, and auditory perception were less

than normal. Moreover, the parents of these children noted difficulties

for their heroin-exposed children in the areas of self adjustment, social

adjustment, and physical adjustment. Items included were: uncontrolled

6 2
temper, impulsiveness, poor self-confidence, aggressiveness, and difficulty in making and keeping friends. These children were very active,

although ratings of attention, cooperation, and alertness were not

abnormal. Wilson concludes that heroin-exposed children may have a

problem common to the general process of perception rather than a

specific sensory deficit, and feels that behavioral problems in these

children may be manifestations of “impaired attention and organizational

abilities.”

In addition to the neurobehavioral sequelae described for children born of

opioid-dependent women, a number of studies have rnonitored growth.

Some studies (Blatman and Lipsitz 1972) reveal no abnormalities, while

others (Strauss et al. 1976; Wilson et al. 1981; Finnegan 1981) have recorded

lower (but not significantly reliable) values for offspring exposed to

opioids. A substantial number of reports (Wilson et al. 1973; Wilson 1975;

Ting et al. 1974, 1978; Rosen and Johnson 1982a, 1982b; Chasnoff et al.

1982) document delayed growth properties associated with perinatal opioid

exposure. These delays may take the form of noticeably lower body

weights, shorter body lengths (height), and/or smaller head circumferences.

As in most areas of human research, data collection and interpretation

are often fraught with difficulties. In the study of perinatal opioid

exposure and human development, the potential for problems is further

compounded. The licit and illicit use of opioids so alters psychological,

behavioral, and physiological processes that it elevates the drug to a

paramount position in the life of any user. These individuals consume

opioids during pregnancy despite the ramifications of such consumption on

the health of their children. Moreover, the study of opioid exposure in

developing humans really involves two individuals: mother and offspring;

in some cases, other individuals (e.g., caregivers) may also be involved.

Thus, the interaction of these forces only serves to potentially confuse.

essential issues further. Fortunately, in the case of clinical studies on

opioids and development, recognition of and discussion about problems in

experirnental design and interpretation have not been overlooked (e.g.,

Householder et al. 1982; Strauss et al. 1979; Wilson et al. 1981; Aylward

1982). Some of these confounding variables include: polydrug abuse, poor

prenatal and/or postnatal care, demographics, socioeconomic states,

length of hospitalization, neonatal withdrawal, breast feeding, motherinfant/child interpretations, paternal influences, sample selection, type of

tests used for investigation, structuring of appropriate comparison groups,

“dropout” rate of patients in the study, and statistical analysis.

Rather than being overwhelmed by these confounding variables and simply

dismissing reports cited in this review, one needs to be aware that

although the goal of clinical research may be to determine a relationship

between cause and effects, this type of distinction may be unattainable.

Searching for the etiology of opioid-related problems (much less defining

all specific abnormalities) could be unrewarding. Given all of the possible

confounding variables enumerated above, it is therefore no wonder that

defining pathognomonic features of perinatal opioid exposure is a very

difficult task. A fine example of the problems in attempting to ascribe

specific characteristics to perinatal opioid exposure concerns the neonatal

abstinence syndrome. Neonatal withdrawal has been considered to be a

hallmark sign of maternal opioid consumption; yet, a variety of nonopioid

63
drugs consumed by the mother can elicit somewhat similar effects. Some

of these agents include alcohol, barbiturates, chlordiazepoxide, clomipramine, ethchlorvynol, glutethimide, hydroxyzine, and meprobamate

(Committee on Drugs 1983. Thus, at least in the clinical realm, we may

have to be satisfied with the knowledge that opioids are part of a

cumulative effect from an overall potentially dangerous milieu that

contributes to any damaging sequelae encountered.

Laboratory Observations

Laboratory studies offer another perspective as to the influences of

opioids on development. Through laboratory investigations, the goal of

achieving information in regard to opioids and biological development can

be realized. Laboratory models also allow us to address these questions in

a wide variety of methodologies that include, in addition to maternal

models of opioid consumption, studies in postnatally developing animals,

regenerating tissues, tissues and cells in culture, and in other in vitro

preparations. Thus, the laboratory has allowed workers to be unrestricted

in their designs, yet to be able to cast light on the essential biological

question being asked: Do opioids influence developrnent and, if so, how?

Just as in the earlier synopsis of the clinical literature, it simply is beyond

the scope of this paper to review all of the laboratory literature. Once

again, the reader should be aware of a series of bibliographies (Zagon et

al. 1982, 1984) that can serve as a guide to any laboratory (and clinical)

topic desired. Moreover, the reader can consult a recent review of the

neurobehavioral sequelae associated with perinatal opioid exposure by

Zagon and McLaughlin (1984a). In the following discussion, some of the

highlights of the laboratory literature will be mentioned.

In regard to studies that have attempted to create a “model” of maternal

opioid consumption to determine the effects on offspring, it should be

clearly recognized that a variety of routes of administration, drug

dosages, and schedules of treatment have been employed. Maternal

exposure to opioids does appear to have little effect on estrous cycle,

fertility, length of gestation, and parturition (e.g., Zagon and McLaughlin

1977a, 1977b, 1977c), although difficulties with conception and a

protraction of the gestational period (Buchenauer et al. 1974), as well as

positional malformations of the fetus (Chandler et al. 1975) have been

recorded. A distinct effect of maternal opioid consumption appears to be

a reduction in maternal body weight during pregnancy (McCinty and Ford

1976; White et al. 1978; Seidler et al. 1982; Zagon and McLaughlin 1977a),

but these weight deficits do not appear to be caused by insufficient

nutritional status (Ford and Rhines 1979; White et al. 1978). In general,

maternal exposure to morphine and methadone (Davis and Lin 1972; Zagon

and McLaughlin 1977b, 19778 does not have a detrimental influence on

titter size, although some decreases in litter size with higher doses of

methadone have been noted (Middaugh and Simpson 1980). Teratogenicity

appears to be associated with high drug dosages administered acutely or

over a short time period (Geber and Schramm 1975), but not with lower

dosages administered chronically. Some increase in stillborns has been

observed with high drug dosages (Freeman 1980; Sobrian 1977). while other

studies reveal little problem in this area (Davis and Lin 1972).

64
The effect of transplacental exposure to opioids on infant viability is

determined by drug dosage and whether or not the neonates continue to

receive opioids postnatally (e.g., breast milk, direct injection). Neonates

that do not continue to receive opioids often may be hypersensitive to

stimuli and experience tremors at birth, with notable infant mortality

found in the first few days of life for those pups exposed to morphine,

heroin, methadone, and levo-alpha-acetylmethadol (LAAM) (e.g., Davis

and Lin 1972; Zagon and McLaughlin 1977d; Freeman 1980).

Since the timetable of behavioral maturation in rodents is so well known,

evaluation of the developmental pattern in opioid-exposed offspring can

be determined (Zagon and McLaughlin 1978b). Delays in physical characteristics (e.g., eye opening), spontaneous motor physical characteristics

(e.g., walking), and reflexive tests (e.g., visual orientation) have ail been

noted. Interestingly, animals exposed only prenatally to opioids (i.e.,

allowed to go through withdrawal at birth and not receiving drug postnatally) exhibit the greatest number of delays in attaining behavioral

capacities and physical characteristics. Those animals receiving drug

prenatally and postnatally (a situation somewhat comparable to that in

humans when therapeutic intervention prevents withdrawal) are often

closest to the normal timetable of maturation.

In the rat, behavior in the period shortly after weaning (postnatal days 21

to 44) has generally been characterized by a reduction in activity and a

decreased emotionality relative to control offspring (Freeman 1980; Grove

et al. 1979; Zagon et al. 1979a). Zagon and McLaughlin (1981b) have also

found an abnormally high incidence of wet-dog-shake and bead-shake

behaviors during this period that resembled drug withdrawal. In contrast

to the reduced activity levels in opioid-treated pups at weaning, young

adults (postnatal days 45 to 89) generally were hyperactive and more

emotional (Davis and Lin 1972; Grove et al. 1979; Zagon et al. 1979a).

Once again, methadone-exposed pups often exhibited head-shake and

wet-dog-shake behaviors, suggesting a protracted phase of withdrawal.

Peters (1978) has also accumulated preliminary evidence of learning

disabilities in methadone- and morphine-exposed rats at this age.

Adult rats that were perinatally exposed to opioids exhibit a lasting

impairment in the ability to acquire fear (Sonderegger and Zimmerman

1976), a reduction in social dominance (Thompson and Zagon 1982), problems in learning (Middaugh and Simpson 1980; Zagon et al. 1979b), and a

facilitation toward self-administrative behavior (Rech et al. 1980).

In regard to other anatomical, physiological, and biochemical correlates

to opioid exposure in early life, a number of important observations have

been made. Somatic growth retardation (e.g., McLaughlin and Zagon 1980;

McLaughlin et al. 1978; Slotkin et al. 1976, 1980), smaller brain dimensions

(Zagon and McLaughlin 1977b, 1978a), and deficits in organ weights

(McLaughlin and Zagon 1980; McLaughlin et al. 1978) have been reported.

Physiological dysfunction in regard to thermoregulation (Thompson and

Zagon 1981; Thompson et al. 1979), nociceptive thresholds (Zagon and

McLaughlin 1980, 1981a, 1982b), and aberrant response to opioids and

nonopioid drugs (Zagon and McLaughlin 1981a, 1984b) have also been

recorded. Opioid-exposed offspring, particularly those subjected to drugs

only prenatally, often exhibit deficits in brain cell number, as well

65
as alterations in brain RNA and protein concentrations and content (Zagon

and McLaughlin 1978a). Changes in polyamine metabolism (Slotkin et al.

1976, 1979) and in the ontogeny of catecholaminergic systems, as well as a

retardation in the synaptic development of 5-hydroxytryptamine, dopamine, and epinephrine neurons in the nervous system have been cited

(McGinty and Ford 1980; Rech et al. 1980; Slotkin et al. 1982). Morphological investigations have shown that the timetable of neurogenesis is

dependent on the schedule of drug exposure (Zagon and McLaughlin 1982b),

and that prenatal exposure to opioids is related to reductions in cortical

thickness and number of cells in the neocortex during the first 2 weeks of

rat development, in addition to neuronal density changes in the hippocampus up to postnatal day 28 (Ford and Rhines 1979). It does not appear

that either maternal undernutrition or inadequate nutrition of the offspring form the etiologic basis for opioid-related problems (e.g., Ford and

Rhines 1979; Zagon and McLaughlin 1982a; McLaughlin and Zagon 1980,

Seidler et al. 1982; White et al. 1978; Raye et al. 1977; Smith et al. 1977).

Moreover, hypoxia due to opioid consumption does not appear to be

responsible for the sequelae recorded White and Zagon 1979).

Comparisons of Clinical and Laboratory Findings

Information discussed in this review and others (Zagon and McLaughlin

1983a, 1983b, 1983c, 1983d, 1984a), as well as inspection of individual

papers on the subject (see Zagon et al. 1982, 1984). reveals a number of

striking parallels between clinical and laboratory findings. In summary

fashion, these include: passive dependence of the fetus on opioids,

occurrence of the neonatal abstinence syndrome, high rate of morbidity

and mortality of the neonate when not given supportive therapy for

withdrawal, sleep disturbances in the neonate, protracted/subacute

withdrawal, delays in sensorimotor development, retardation in somatic

growth, smaller head circumferences in humans/smaller brain sizes in

laboratory animals, delays in walking, problems in visual and/or auditory

systems, abnormalities in neuropathological studies indicating alterations

in neuro-ontogeny, diminished alertness, poor attention spans in early

phases of development, hyperactivity in later phases of development,

learning disabilities, and social maladjustments.

Etiological Considerations of the Perinatal Opioid Syndrome

Although many studies to date have dealt more with the phenomenological

aspects of opioids and development, they all have, to some degree or

another, helped formulate a picture contributing to the uncovering of the

etiology and pathogenesis associated with the perinatal opioid syndrome.

Information garnered from other biological studies on opioids and early

life also should be mentioned before attempting to place this syndrome in

perspective. First, opioids are known to selectively accumulate in the

brains and nervous tissues of fetal rats (Peters et al. 1972) and preweaning

rata (Shah and Donald 19791, presumably because developing organisms

have an increased permeability of the blood-brain barrier. Moreover,

opioids have been reported to exert a stereospecific effect on growth in

animals (Smith et al. 1977; Crofford and Smith 1973), indicating that opioid

action is quite specific. The effects of opioids also have been shown to be

blocked by concomitant administration of opioid antagonists such as

naloxone (Crofford and Smith), suggesting that opioid action resides at the

66
level of the opiate receptor. In this regard, it should be noted that opiate

receptors have been found in body and brain tissues of developing

organisms (e.g., Gibson and Vernadakis 1982; Clendeninn et al. 1976).

Tissue culture studies in our laboratory (Zagon and McLaughlin 1984c;

McLaughlin and Zagon 1984) have verified stereospecific and naloxonereversible effects of opioid agonists on cell growth, and also reveal that

tolerance can develop to these influences. Additionally, cells that are

physically dependent on opioid agonists and withdrawn from drugs go

through a withdrawal. A principle manifestation of cellular withdrawal is

a diminution in mitotic activity.

Thus, it could be conjectured that opioid agonists serve to inhibit growth

at the locus of the opioid receptor. The relationship of the receptor to

growth remains an exciting new frontier for exploration. In the case of

maternal opioid agonist consumption, it can be envisioned that these

agonists depress developmental events until the cells are able to become

tolerant to drug exposure. The magnitude of prenatal effects on growth

depends on the drug dosage and the length of time needed to establish

tolerance in the developing organism. Withdrawal from opioid agonist

exposure at birth (or even during fetal life) would force developing cells

to readjust to an absence of drug, which would result in a delayed growth

profile; the extent of these delays would correlate with the magnitude of

withdrawal. Amelioration of withdrawal at birth by administration

(breast feeding or direct administration) of opioid agonistlike agents

would circumvent the need for readjustment and allow the cells and tissue

to continue developing in an established (albeit, containing opioid agonist)

milieu. This hypothesis fits very nicely with laboratory findings that

prenatal exposure followed by postnatal withdrawal is most detrimental to

development, whereas prenatal exposure along with administration of

opioids in the postnatal life usually exerts the Least short- and long-term

damage. In the clinical setting, elimination or at least minimization of

the withdrawal syndrome probably attenuates many detrimental

influences. Therefore, it is hardly any wonder that difficulties might

exist in establishing firm evidence separating the effects of perinatal

opioid exposure from other potentially damaging influences.

NEW LESSONS

If indeed exogenous opioids such as methadone, heroin, and morphine can

inhibit growth by acting stereospecifically and in a naloxone-reversible

fashion at the level of the opiate receptor, then the question arises as to

whether the body’s own morphine--the endorphins--could also regulate

growth by interaction with opiate receptors known to be present on

developing cells. To investigate this possibility further, we administered

naltrexone--a potent opioid antagonist--to preweaning rats. Preweaning

development in the rat (birth to day 21) is characterized by explosive body

and brain growth, and serves as a good model to examine the influences of

various agents on developmental events. Daily injections of a relatively

high dosage of naltrexone (50 mg/kg) stimulated development in preweaning rats, while daily injections of a low dosage (1 mg/kg) inhibited

growth (Zagon and McLaughlin 1983a, 1983b, 1984d, 1984e). For example,

the body weight of 21-day-old 50 mg/kg naltrexone-treated rats was 18%

more than controls and the body weights of 1 mg/kg naltrexone-treated

67
rats was 11% less than controls. Not only were body weights affected,

but examination of the wet weights of brain, heart, kidneys, liver, and

skeletal muscle were 11 to 32% more than controls for rats in the 50

mg/kg group, and 5 to 24% below normal for animals given 1 mg/kg

naltrexone (Zagon and McLaughlin 1983b).

The timing of the appearance of certain physical characteristics that

were examined (hair covering, incisor eruption, ear opening, and eye

opening) often revealed that many of these traits occurred significantly

earlier in the 50 mg/kg naltrexone group, and markedly later in rats

receiving 1 mg/kg naltrexone (Zagon and McLaughlin 1983b, 1985). The

appearance of spontaneous motor and sensorimotor behaviors was also

altered in a similar manner. For example, the initial appearance of

walking, a milestone in behavioral ontogeny, appeared 2 days earlier in

the 50 mg/kg naltrexone group relative to controls. Furthermore, at the

age when only SO% of the controls were able to walk, no rat in the 1

mg/kg naltrexone group could walk, and all (100%) of the animals in the 30

mg/kg naltrexone group could walk (Zagon and McLaughlin 1983b, 1985).

Macroscopic dimensions of the brains and the cerebellum at 21 days of age

showed marked increases for the 50 mg/kg naltrexone group and

decreases for the 1 mg/kg naltrexone group (Zagon and McLaughlin 1983a,

1984e). Morphornetric analysis of the cerebrum revealed a somatosensory

cortex in the 50 mg/kg naltrexone group that was 12% thicker than in

controls, but 9% thinner in rats of the 1 mg/kg group. In regard to the

cerebellum of 21-day-old rats, the 50 mg/kg group had areal

measurements of total, molecular layer, internal granule layer, and

medullary layer that were 27%, 24%. 28%, and 41%, respectively, greater

than controls; animals in the 1 mg/kg group had measurements that were

27%, 30%, 24%, and 27%, respectively, less than those of the control

group. The number of neural cells in the molecular layer of rats in the 50

mg/kg group was 76% greater than control levels, and the number of glial

cells in the medullary layer was 62% greater in these animals than in

control rat cerebella.

Dose-response experiments (Zagon and McLaughlin 1984d) have shown that

dosages from 0.1 to 10 mg/kg naltrexone inhibit body and brain growth,

whereas dosages of 20 to 100 mg/kg stimulate development. Utilizing

opiate challenge experiments (nociceptive response is recorded 30 minutes

following an injection of 0.2 mg/kg morphine) to examine the

pharmacological efficacy of various daily dosages of naltrexone, dosages

of 20 mg/kg or greater were found to block the opioid receptor from

interaction with a dose of morphine for 24 hr/day, and dosages of 10

mg/kg naltrexone and less blocked the opioid receptor for only 12 hr/day

or less. To further investigate whether the relationship between growth

processes, drug dosage, and the pharmacological properties of naltrexone

were more than coincidental, some preweaning pups received low dosages

(3 mg/kg) of naltroxone given three times daily, which in effect blocked

the opiate receptor for 24 hr/day. These rats demonstrated notable

increases in body and brain development. A cumulative dosage of 9 mg/kg

naltrexone once daily, which blocked the opioid receptor for less than 12

hr/day, retarded growth. Thus, developmental events appear to be

governed by the duration of opioid receptor blockade.

68
These findings, taken together with evidence that endorphins are present

in the plasma and brain of developing organisms (Kramer and

Teschemacher 1978; Bayon et al. 1979), that irnmunoreactive analogs of

endorphins have been detected in fetal and neonatal cells (Knodel and

Richelson 1980; Neale et al. 1978), and that opioid receptors have been

identified in brain and body tissues during ontogeny (Coyle and Pert 1976;

(Gibson and Vernadakis 1982, led us to the exciting and novel proposition

that endogenous opioids and their opioid receptors regulate developmental

events. These effects on growth do not appear to be due to nutritional or

hormonal events (Zagon and McLaughlin 1984d). In fact, we are not aware

of any agent (e.g., drug or hormone) or condition that makes a postnatally

developing animal larger than “normal” and increases brain development

such as we have demonstrated.

The specific mechanisms as to how endogenous opioid systems control

growth are unclear. Our studies do show that complete prevention of the

interaction between endorphins and opioid receptors by administration of

relatively high dosages of naltrexone once daily, or low dosages of

naltrexone given repeatedly each day, allows growth to proceed in an

“unrestricted fashion” that results in the larger animals. Thus, just as

with exogenous opioids such as heroin and methadone, endogenous opioids

serve to inhibit growth. Enhancement of this interaction, such as that

recorded with ternporary opioid receptor blockade by naltrexone

administration, results in smaller animals. Although the precise pathways

for this diminution in growth are unknown, naltrexone administration has

been reported to elevate plasma beta-endorphin levels (Recant et al. 1980)

and to increase opioid receptor number and supersensitivity to opioids

(Lahti and Collins 1978.; Schulz et al. 1979; Tang and Collins 1978; Zukin et

al. 1982). In distinction to a total blockade of this interaction, a

temporary blockade provides an interval each day when the inhibitory

action of basal or elevated levels of cndorphins can act on cells possessing

a greater complement of opioid receptors. Fascinatingly, naltrexone’s

effects on infant rat development and the establishment of endogenous

opioid control of growth is consistent with other studies in which

endogenous opioid systems have been demonstrated to control

tumorigenesis (Zagon and McLaughlin 1981c, 1981d, 1983c, 1983d, 1984f).

FOOTNOTE

‘The term opioid is used in the generic sense and refers to exogenous and

endogenous substrates, natural or synthetic in origin, that possess opiumor morphine-like properties.

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ACKNOWLEDGMENTS

The technical and intellectual contributions of P.J. McLaughlin, M.S. to

all studies mentioned in this review which originated from our laboratory

is gratefully acknowledged. This work was supported in part by grants

NS-20500, NS-20693, and NS-21246 from the National Institutes of Health.

76
AUTHOR

Ian S. Zagon, Ph.D.

Associate Professor of Anatomy

Department of Anatomy

The Milton S. Hershey Medical Center

The Pennsylvania State University

Hershey, PA 17033

77
Pharmacodynamics of Fetal

Exposure to Narcotics

Hazel H. Szeto and Jason G. Umans

INTRODUCTION

Infants born to narcotic addicts are known to be at risk of undergoing an

opiate-abstinence syndrome in the neonatal period. The most common

manifestations of this syndrome include irritability, hyperactivity, increased eye movements, diarrhea, tachypnea, tremors, exaggerated reflexes, and occasionally generalized seizures. in addition, these infants

exhibit abnormal sleep patterns, with an increase in wakefulness and

rapid-eye-movement sleep, at the expense of quiet sleep (Glass et al.

1972; Schulman 1969; Dinges et al. 1980).

Although most of the clinical signs of withdrawal subside in a matter of

days, some behavioral and neurological disturbances appear to persist into

early childhood. Many children have been found to be hyperactive, with

shorter attention spans and decreased fine motor coordination (Wilson et

al. 1981). These persistent neurological and behavioral effects are often

considered consequences of the withdrawal insult. However, it is also

possible that prolonged intrauterine exposure to methadone may have

direct effects on the development of the nervous system. In both clinical

and animal studies, it has been rather difficult to differentiate between

direct narcotic effects on fetal development and the consequences of the

unavoidable withdrawal syndrome at birth.

Furthermore, it has recently been suggested that the fetus may undergo

intermittent intrauterine withdrawal throughout gestation (Lichtblau and

Sparber 1981). and this may result in long-term behavioral and neurological

disturbances. It is known that detoxification of narcotic addicts during

pregnancy results in fetal distress, as evidenced by meconium staining

(Hill and Desmond 1963; Harper et al. 1974), increased amniotic fluid

catecholamines, and occasional fetal demise (Zuspan et al. 1975). Changes

in fetal hemodynamics and meconium staining have also been reported

following precipitated morphine withdrawal in pregnant ewes (Cohen et

al. 1980). These studies, however, did not examine the behavioral

responses which might allow comparison with the abstinence syndrome

described in the human neonate. Therefore, although intrauterine opiate

withdrawal presents a severe physiologic stress to the fetus, it remains to

be determined whether behavioral disturbances are a significant

component of the syndrome.

78
Our objectives were, therefore, to develop an animal model that would

permit us to examine the direct effects of morphine exposure in the fetus

and to fully characterize the fetal opiate-abstinence syndrome. An

understanding of these fundamental pharmacodynamic properties of a

narcotic drug in the fetus may provide a better understanding of the

long-term effects of narcotics exposure on the developing fetus and

neonate.

EXPERIMENTAL METHODS--GENERAL CONSIDERATIONS

The Animal Model

When considering the effects of acute morphine exposure, we are most

concerned about its effects on behavioral, respiratory, and cardiovascular

functions. This type of information obviously cannot be obtained from the

human fetus or the fetus of small laboratory animals. The pregnant

sheep, on the other hand, offers a unique opportunity for examining these

functional parameters in the fetus under physiologic conditions. The

relatively large size of the fetus allows chronic implantation of catheters

and electrodes necessary for intrauterine monitoring of behavioral,

respiratory, and cardiovascular functions.

The effects of morphine exposure on fetal cardiovascular function can be

easily determined with an indwelling catheter in the fetal aorta for

continuous monitoring of blood pressure and heart rate.

Respiratory function can be monitored with a pair of electrodes implanted

in the diaphragm and a catheter implanted in the trachea. Breathing

movements are indicated by a burst of activity in the diaphragmatic

electromyogram and a decrease in tracheal pressure.

Effects of drugs on fetal behavioral activity are much more difficult to

ascertain since it is not possible to directly observe the fetus. We have

recently developed techniques for intrauterine recording of electrocortical activity (ECoG), electromyographic activity (EMG), and eye

movements (EOG) in the fetal lamb, making it possible to interpret

indirectly from these electrophysiologic measurements the behavioral

activity of the fetus (Szeto 1983; Umans and Szeto 1983). Employing the

classical criteria for sleep-wake scoring (table 1), we are able to

differentiate three distinct states of behavior in the fetal lamb in the

third trimester.

TABLE 1

Behavioral State ECoG

Arousal desynchronized

Quiet Sleep synchronized

Active Sleep desynchronized

Nuchal EMG EOG

tone, bursts random, slow

tone, bursts random, slow

atonia REMs

Thus, with the development of sophisticated techniques for intrauterine

recording of physiologic functions in the fetal lamb, the pregnant sheep

has become an ideal animal model for studying both pharmacokinetics and

79
pharmacodynamics of drugs in the maternal-fetal unit. We have previously demonstrated that this model can be used for comparing the extent

of fetal exposure to different narcotic drugs (Szeto 1982; Szeto et al. 1978,

1981, 1982).

Experimental Design

In designing any pharmacodynamic study in the fetus, we should ideally

have some information regarding the pharmacokinetics of the drug in the

maternal-fetal unit. In addition, there are a few points that should be

considered in the design of the route and dose of drug administration.

Firstly, since the extent of fetal exposure following maternal drug

administration may vary considerably from animal to animal, it is

preferable to administer the drug directly to the fetus rather than to the

mother. This can be easily accomplished with an indwelling catheter in

the fetal vena cava (Szeto et al. 1978). Secondly, if we wish to correlate

pharmacodynamic effects with the dose of drug administered, the drug

should be administered by constant rate infusion to steady state. This is

particularly important when studying periodic events such as sleep-wake

behavior (Szeto 1983, Umans and Szeto 1983). Thirdly, a full range of

doses should be examined to document dose-related effects and biphasic

effects relationships. Single-dose studies may be very misleading,

especially if the dose is not within the linear dose range. Finally, for the

opiate withdrawal studies, naloxone will be used to precipitate withdrawal

in morphine-exposed animals. Naloxone should also be administered

directly to the fetus at doses that do not alter any of the measured

parameters, and the study should be carried out following different doses

of agonist exposure.

EXPERIMENTAL PROCEDURES

Animal Preparation

Thirteen pregnant ewes and their fetuses (gestational ages ranging from

121 to 138 days at the time of study, term being approximately 145 days)

were surgically instrumented for monitoring of fetal hemodynamics and

behavioral activity at least 5 days prior to study. Details of the surgical

procedure have been described previously (Szeto 1983; Umans and Szeto

1983, 1985). Briefly, chronic indwelling catheters were placed in the fetal

inferior vena cava for drug infusion and in the distal aorta for continuous

monitoring of blood pressure and heart rate. For continuous monitoring of

fetal behavioral activity, a pair of stainless steel screws were implanted

extradurally over the parietal cortex to record electrocortical activity, a

pair of stainless steel screws was implanted over the superior orbital ridge

to record eye movements, and stainless steel electrodes were implanted in

the dorsal neck muscles and quadriceps femoris muscle to assess nuchal

tone and body movements. In addition, fetal breathing movements were

monitored via a pair of stainless steel electrodes implanted in the

diaphragm and a catheter in the trachea.

Recording Procedures

All recordings of fetal blood pressure (FBP), heart rate (FHR), tracheal

pressure (TP), electrocorticogram (ECoC), electromyogram (EMG), and

80
electrooculogram (EOC) were carried out with the ewe standing or lying

quietly in an experimental cart with free access to food and water. A

companion sheep was kept in the same room to minimize restlessness.

All recordings were obtained with a Gould model 2800 analog recorder and

appropriate amplifiers. The ECoG, EMG, and EOG were recorded via

Universal amplifiers set at the following filter band widths: ECoC, 0.3 to

30 Hz; EMG, 30 to 300 Hz; and EOC, 1 to 10 Hz.

Drug Protocols

Control recordings of FBP, FHR, and all neurobehavioral parameters were

obtained for at least 2 hours prior to drug administration.

In a preliminary study, naloxone was administered intravenously at doses

of 0.75, 1.5, 3.0, 6.0, and 12.0 mg to opiate-naive fetuses, without any

significant effect on the monitored physiologic variables. We chose a

naloxone dose of 6.0 mg for use in all subsequent studies. In previous

studies of specific opiate effects, compararable doses precipitated opiate

abstinence in rodents (Wei et al. 1973; Umans and Inturrisi 1982).

Morphine was infused to the fetus at a constant rate, ranging from 0.075

to 80.0 mg/hr for 2 to 6 hours. Naloxone was then administered to the

fetus at the end of the morphine infusion. All hemodynamic and activity

parameters were monitored continuously throughout the control period,

during the morphine infusion, and for 2 hours following naloxone administration.

RESULTS

All predrug control recordings showed cyclic variation in electrocortical

activity, eye movements, total body movements, and breathing movements (figure I). Three behavioral states could be clearly distinguished in

all recordings according to classic sleep-wake scoring (table 1). During

the control period, the fetus spent 85% to 95% of its time alternating

between quiet sleep and active sleep. Very short episodes of arousal

would sometimes occur during the quiet sleep-active sleep transition or

during prolonged periods of quiet sleep.

The administration of morphine to the fetus resulted in two distinct

response clusters as a function of dose. At doses lower than 0.30 mg/hr,

morphine did not significantly alter any of the measured physiologic

parameters. Doses up to 2.5 mg/hr resulted in desynchronization of the

ECoG; increased nuchal tone and body and eye movements; continuous

fetal breathing movements; and increased heart rate and heart rate

variability (figure 2). Higher doses (through 80.0 mg/hr resulted in a

return of synchronized ECoC, accompanied by decreases in body, eye, and

breathing movements and heart rate variability (figure 3). These signs of

central nervous system depression were evident towards the end of the 2.5

mg/hr infusion in 30% of the animals. Fetal breathing movements were

completely suppressed at doses greater than 10 mg/hr. Upon termination

of the high-dose infusion, a prolonged period of arousal and enhanced

breathing activity was observed before return to control values.

81
FIGURE 1. Control recording demonstrating cyclic activity in ECoG.

Nuchal EMG (EMGN), EOG, diaphragmatic, EMG (EMGD), and tracheal

pressure (TP) prior to morphine exposure.

FIGURE 2. Effects of low-dose morphine infusion (2.5 mg/hr) on fetal

hemodynamic, behavioral and respiratory activity. Note continous fetal

arousal and breathing movements.

82
FIGURE 3. Effects of high-dose morphine infusion (25.0 mg/hr) on fetal

hemodynamic, behavioral, and respirtatory activity. Note prolonged quiet

sleep and apnea

The administration of 6 mg naloxone to opiate-naive fetuses did not result

in any alteration in the physiologic parameters which were monitored, or

in their pattern of cyclic variation (figure 4).

Following low-dose morphine infusion, naloxone resulted in further

desynchronization of the ECoG an immediate bradycardia, an increase in

both the rate and depth of breathing movements, and an increase in total

body movements (figure 5). Following high-dose morphine infusion,

naloxone provoked a qualitatively similar, but more intense, abstinence

syndrome (figure 6).

In fetuses which were subjected to an episode of precipitated abstinence

following morphine infusion of 25.0 mg/hr or greater, a second abstinence

could be precipitated by naloxone administration 24 hours following the

initial withdrawal. lmmediately prior to the second dose of naloxone, all

fetuses exhibited normal control traces, without evidence of acute opiate

effect. The second episode of abstinence was, in each case, similar to,

but less intense than, the first.

83
FIGURE 4. The effects of nalorone opiate-naive fetus.

FIGURE 5. The effects of nalorone on fetal hemodynamics and behavioral

activity parameters when administered subsequent to a 6-hour infusion of

morphine at 2.5 mg/hr.

8 4
FIGURE 6. The effects of nalorone on fetal hemodynamics and behavioral

activity parameters when administered subsequent to a 6-hour infusion of

morphine at 25.0 mg/hr.

DISCUSSION

It is generally assumed that neonatal morbidity and developmental abnormalities following intrauterine narcotic exposure result from repeated

episodes of fetal withdrawal (Lichtblau and Sparber 1981). Data which

suggest that both direct opiate pharmacodynamic effects and fetal withdrawal may contribute significantly to neonatal outcome following intrauterine narcotic exposure is presented below.

Using the chronically instrumented fetal lamb preparation, we have

defined the full dose-response relationship of morphine on fetal behavioral, respiratory, and cardiovascular functions. We have previously

shown that doses in the range of 2 mg/hr caused complete suppression of

quiet sleep in the fetus and induced prolonged episodes of arousal (Umans

and Szeto 1983). These arousal periods were associated with continuous

breathing movements and increased heart rate and heart rate variability.

We have now demonstrated that the morphine dose-response curve is

biphasic, with higher doses resulting in quiet sleep and apnea. The

biphasic nature of morphine’s dose-response curve in the fetus may

account for some of the confusion in the current literature, with some

investigators reporting a suppression of fetal breathing movements

(Rigatto et al. 1984), and others reporting both suppression and stimulation

of breathing movements in the fetal lamb (Olsen and Dawes 1984).

We have recently demonstrated a similar biphasic response to a stable

enkephalin analog, D-Ala2-MePhe4-Met(0)5-enkephalinol (FK 33-824), in

85
the fetal lamb (Umans et al., in press). At high plasma concentrations,

FK 33-824 resulted in apnea and quiet sleep. As plasma concentrations

declined, there was prolonged period of arousal and enhanced respiratory

activity. These studies suggest that the pharmacodynamic effects of

morphine in the developing fetus are similar to that of the opioid

peptides, and that these peptides may play a role in the physiologic

regulation of sleep, breathing, and cardiovascular function in the

developing fetus. In addition, these studies demonstrate that direct

effects of opiates may account for some of the effects observed in the

neonate following intrauterine opiate exposure, and that the nature of the

effect would be dependent on the extent of fetal drug exposure.

In these experiments, we have also characterized a syndrome of naloxoneprecipitated opiate abstinence following short-term infusion of morphine

to the fetus. The syndrome consists of desynchronization of electrocortical activity; increased total body movements, nuchal tone, and eye

movements; continuous rapid, deep breathing movements; an immediate

bradycardia associated with transient increases in systolic, diastolic, and

pulse pressure; and meconiwn staining of the amniotic fluid. This syndrome resembles that observed in the human neonate (Glass et al. 1972;

Schulman 1969; Dinges et al. 1980). The syndromes differ, however, in

that we observed no cortical seizure activity in the fetal lamb. There are

no human data available describing the cardiovascular manifestations of

neonatal withdrawal.

Based on the present observations, we agree with Zuspan and coworkers

(1975) in suggesting that intrauterine opiate withdrawal presents a severe

physiologic stress to the developing fetus. Our data, showing that a

significant abstinence syndrome may be precipitated following short (2

hours), low-dose fetal exposure to morphine, further suggest that the

fetus may undergo a cycle of acute physical dependence and withdrawal

following the administration of opiates in normal clinical patients.

In conclusion, we have presented evidence which suggests that both direct

opiate pharmacodynamic effects and fetal withdrawal may contribute significantly to neonatal outcome following intrauterine narcotic exposure.

REFERENCES

Cohen, M.S.; Rudolph, A.M.; and Melmon, K.L. Antagonism of morphine

by naloxone in pregnant ewes and fetal lambs. Dev Pharmacol Ther

1:58-69, 1980.

Dinges, D.F.; Davis, M.M.; and Glass, P. Fetal exposure to narcotics:

neonatal sleep as a measure of nervous system disturbance. Science

209:619-621, 1980.

Glass, L.; Rajegowda, B.K.; and Evans, H.E. Effects of heroin withdrawal

on respiratory rate and acid-base status in the newborn. N Engl J Med

186:746-748, 1972.

Harper, R.G.; Solish, G.I.; Purrow, H.M.; Sang, E.; and Panepinto, W.C.

The effects of a methadone treatment program upon pregnant heroin

addicts and their newborn infants. Pediatrics 54:300-305, 1974.

Hill, R.M., and Desmond, M.M.; Management of the narcotic withdrawal

syndrome in the neonate. Pediatr Clin North Am 10:67-86, 1963.

86
Lichtblau L., and Sparber, S.B. Opiate withdrawal in utero increases

neonatal morbidity in the rat. Science 212:943-945, 1981.

Olsen, G.D., and Dawes, G.S. Morphine-induced depression and stimulation of breathing movements in the fetal lamb. In: Jones, C.T., and

Nathanielsz, P.W. eds. Physiological Development of Fetus and

Newborn. New York: Academic Press, 1984.

Rigatto, H.; Lee, D.; Caces, R.; Albersheim, S; and Moore, M.: The

effect of morphine on fetal breathing and behavior. In: Jones, C.T., and

Nathanielsz, P.W., eds. Physiological Development of Fetus and

Newborn. New York: Academic Press, 1984.

Schulman, C.A. Alterations in the sleep cycle in heroin-addicted and

“suspect” newborns. Neuropaediatrie 1:89-108, 1969.

Szeto, H.H. Pharmacokineties in the ovine maternal-fetal unit. Annu

Rev Pharmacol Toxicol 22:221-243, 1982.

Szeto. H.H. Effects of narcotic drugs on fetal behavioral activity: Acute

methadone exposure. Am J Obste Gynecol 146:211-217, 1983.

Szeto. H.H.; Mann. L.I.; Bhakthavathsalan, A.; Liu. M.; and Inturrisi. C.E.

Meperidine pharmacokinetics in the maternal-fetal unit. J Pharmacol

Exp Ther 206:448-459, 1978.

Szeto, H.H.; Clapp, J.F.; Larrow, R.W.; Hewitt, J.; Inturrisi, C.E.; and

Mann, L.I. Disposition of methadone in the ovine maternal-fetal unit.

Life Sci 28:2111-2117, 1981.

Szeto, H.H.; Umans, J.G.; and McFarland, J.W. A comparison of morphine

and methadone disposition in the maternal-fetal unit. Am J Obstet

Gynecol 143:700-706, 1982.

Umans, J.G., and Inturrisi, C.E. Antinociceptive activity and toxicity of

meperidine and normeperidine in mice. J Pharmacol Exp Ther

223:203-206, 1982.

Umans, J.G., and Szeto, H.H. Effects of opiates on fetal behavioral

activity in utero. Life Sci 33(1):639-642, 1983.

Umans, J.G., and Szeto, H.H. Precipitated opiate abstinence in utero.

Am J Obstet Gynecol, 151:441-444, 1985.

Umans, J.G.; Umans, H.R.; and Szeto, H.H. Neurobehavioral activity

of a stable enkephalin analogue in the chronically-instrumented fetal

lamb. Neurosci Lett, in press.

Wei. E.; Loh. H.H.; and Way. E.L. Quantitative aspects of precipitated

abstinence in morphine-dependent rats. J Pharmacol Exp Ther

184:398-403, 1973.

Wilson, G.S.; Desmond, M.M.; and Wait, R.B. Follow-up of methadoneand untreated narcotic-dependent women and their infants: health,

developmental and social implications. J Pediatr 98:716-722, 1981.

Zuspan, F.P.; Gumpel, J.A.; Mejia-Zelaya, A.; Madden, J.; and Davis, R.

Fetal stress from methadone withdrawal. Am J Obstet Gynecol

122:43-46, 1975.

AUTHORS

Hazel H. Szeto, M.D., Ph.D.

Jason G. Umans, M.D., Ph.D.

Department of Pharmacology

Cornell University Medical College

1300 York Avenue

New York, New York 10021

87
Positron-Emission Tomography:

A New Approach to Feto-Maternal

Pharmacokinetics

Bo S. Lindberg, Per Hartvi , Anders Lilja, Hans Lundqvist,

Bengt Långström, Petter Malmborg, Anders Rane,

Annika Rimland, and Hans Svärd

INTRODUCTION

Positron emission tomography (PET) is a noninvasive technique for studies

of the regional kinetics of positron-emitting radiotracers. A drug or other

molecule is labeled with short-lived radionuclides such as 11C, 13N, 15O,

or

18F. After administration to animal or man, the distribution in the body

is visualized by external detectors and the radioactivity concentration in

various organs is quantitated as a function of time. With a high specific

radioactivity, PET is a true tracer technique and pharmacologic effects of

the radiolabeled compound will not occur.

PET has been widely used in studies of brain metabolism where 11C-labeled glucose, glucose analogs, and amino acids are commonly used

radiotracers (Ell and Holman 1982). Previously, PET has also been shown to

be a valuable tool in studies of tissue distribution of drugs (Hartvig et al.

1984) and studies on receptor-binding kinetics (Wagner et al. 1983).

Studies on the pharmacokinetics and on the metabolism of drugs in the

feto-maternal unit in any animal are hampered by the difficulties of

obtaining access to the fetal compartments without disturbing the normal

function. The close relationship between the placenta and fetal development of humans and some nonhuman primates, such as the rhesus monkey,

makes this species an attractive animal model for pharmacokinetic studies

during pregnancy. However, the uterus of rhesus monkeys is sensitive to

manipulation such as insertion of catheters for fetal blood sampling. In

addition, the blood volume of the fetus does not permit withdrawal of more

than 5 to 10 ml of blood during an experiment if normal physiologic

conditions are to be maintained. A noninvasive technique such as PET

would therefore be suitable for the study of rapid transfer of 11C-labeled

drugs from the mother to the fetus.

However, PET registers only the distribution of the total radioactivity. If

the radiolabeled molecule is metabolized, knowledge of the metabolic

pathways is necessary for interpretation of the results. More information

can be obtained either by labeling the molecule itself or its metabolites at

88
different sites in different experiments or by the use of additional

analytical techniques. Such techniques include chemical and radiochemical analyses (e.g., high performance liquid chromatography [HPLC]

and gel chromatography) of blood and other tissue fluids.

The objective of this study was to evaluate the PET technique in combination with other methods in studies of the kinetics of morphine and heroin

in pregnant rhesus monkeys. The possibilities and shortcomings of the PET

technique in such studies, as well as some preliminary results, are discussed

below.

MATERIALS AND METHODS

Radiopharmaceutical Preparation

11C was obtained as [11C]carbon dioxide by using the nuclear reaction

14N(p,a

)

11C and a gas target at the van de Graaff accelerator (11MeV),

University of Uppsala. [

11C]methyl iodide was prepared as described by

Langstrom et al. (1982). The corresponding N-demethylated analogs of

morphine and heroin were alkylated and then purified by liquid chromatography. Samples of the purified 11C-labeled compound were analyzed

for identity and radiochemical purity by use of reversed-phase liquid

chromatography. Radiochemical yields, when corrected for decay, were in

the range of 15 to 25%. The time of synthesis, including purification by

liquid chromatography, starting with the release of [

11C]carbon dioxide

was about 50 minutes. Before administration, the samples were filtered

through a 0.22 M membrane filter to ensure a sterile preparation. The

radioactivity dose was measured immediately before administration.

Heroin was used in this experiment with the permission of the Swedish

National Board of Health and Welfare.

Animals

Three healthy, well-nourished pregnant rhesus monkeys (Macaca mulatta)

from the Primate Laboratory for Reproductive Research in Uppsala were

used in the experiments. The age of the animals varied between 7 and 15

years and their weights ranged from 7.3 to 9.1 kg. Gestational age at the

time of experiment was 120 to 150 days (pregnancy duration in rhesus

monkeys is 167 days). After an overnight fast, the animals were anesthetized with 100 mg ketamine intramuscularly, and the anesthesia was

maintained with repeated doses of 50-108 mg ketamine. Two indwelling

catheters were placed in different leg veins, one for administration of the

drugs and the other for blood sampling. Catheters were also inserted in the

bladder and in the amniotic cavity. Ultrasonography was used for visualization of the internal organs of the mother and the fetus. Fetal blood

was obtained by ultrasonographic-guided puncture of the umbilical vein

(Lindgren and Lindberg, in press). The monkey was fixed in a specially

designed cradle and put in the detector opening of the PET in such a way

that the radioactivity in the fetal liver and at least one placenta could be

simultaneously registered. The experiments were carried out after

approval from the Committee for Protection of Laboratory Animals at the

University. The animals were not injured by the procedure, the pregnancy

continued normally, and the offspring were healthy.

89
[

11C]Morphine (41 μ Ci and 327 μCi, respectively) and [14C]Morphine

(100 μCi; 1.79 μmol; Amersham, England) were simultaneously administered intravenously to two animals. Another monkey was given [

11C]

heroin (319 μCi) intravenously.

Instrumentation

Imaging of the uterus of the monkey started immediately after administration of the 11C-labeled opiate. A PC 384-38 positron emission

tomograph equipped with two rings of detectors (AB Scanditronix,

Uppsala, Sweden) was used. Data collection and analyses of images were

performed as described by Eriksson et al. (1982). Three horizontal

emission images were obtained for 4x10, 4x40, and 200-second periods at

predetermined intervals. Each image had a slice thickness of 13 to 14 mm

and a spatial resolution of about 8 mm. The resolution of the PET

equipment as well as other technical details are given by Bergstrom et al.

(1983). The measured radioactivity was corrected for physical decay to

the time of administration of the radioactive dose. Uptake--defined as

the relative distribution of radioactivity in different organs--was

calculated from the corrected radioactivity/cm3 and divided by the

administered radioactivity per gram of body weight where a density of 1

g/cm3 for all tissues was assumed. A measured uptake of 1.0 indicated

that the administered radioactivity was equally distributed in the whole

feto-maternal unit.

Sampling and Analysis of Drugs and Metabolites

Maternal blood was collected 0, 5, 15, 30, 60, 90, 120, 180, 240, and 360

minutes after dose. Urine and amniotic fluid were sampled at regular

intervals throughout the whole experiment. Fetal blood was obtained

from one monkey’s umbilical vein at 101 minutes after dose administration.

The 11C activity in the samples was measured immediately in a well

counter. The blood samples (about 3 ml) were centrifuged and plasma

frozen at -20°C until analyses. [

14C]morphine and [14C]Morphine-3-

glucuronide ([14C]M3G) were measured by liquid scintillation after

collection of fractions using an HPLC method (Svensson et al. 1982). The

elimination half-lives were estimated from the concentration-time curve

plotted in the log-linear scale.

RESULTS

Positron Emission Tomography

[

11C]Morphine was rapidly distributed in the body of the rhesus monkey

and maximum uptake was obtained within a few minutes in well-perfused

organs, e.g., the placenta (figure 1). The disappearance of the 11Cderived radioactivity in the placenta paralleled the decline of the total

11C-activity in maternal blood. The uptake was slow in the muscles of

the mother, as has previously been shown by Hartvig et al. (1984). The

[

11C]morphine-derived radioactivity was also rapidly transferred to fetal

blood and an uptake of 1.5 times equal distribution was seen within 5 to 10

minutes in the fetal liver. The elimination half-life of the 11C-derived

radioactivity from the fetal liver was about 20 minutes.

90
FIGURE 1. Uptake of 11C in placenta and fetal liver after intravenous

injection of [

11C]morphine to the mother. Uptake is defines as

radioactivity per cm3 tissue in realtion to total radioactivity administered

per grant body weight. denote, the placental uptake; X the fetal liver;

and maternal paraspinal muscle.

Four minutes after administration of [

11C]heroin, a maximum radioactivity uptake of 7.5 in the placenta was observed. The uptake of

[

l1C]heroin-derived radioactivity as compared to morphine was even

more pronounced in the fetal liver, and an uptake 8 times homogeneous

dilution was measured 5 minutes after dose (figure 2). The elimination

half-life from the placenta as well as from the fetal liver was estimated

to be 15 minutes.

Analysis of Plasma, Urine, and Amniotic Fluid (Figure 3)

[

14C]Morphine rapidly left the maternal plasma and was eliminated

primarily by conversion to metaboites, e.g., [

14C]M3G. Elimination

half-lives of [

11C]morphine and [14C]M3G in one monkey was 70 and 110

minutes, respectively, whereas in the other monkey, a third very slow

elimination phase could be distinguished. The ratios [

14C]M3G/[14C]

morphine 1 hour after the injection were 8 to 10 in the two mothers. In

one experiment, the fetal plasma concentration could be measured 101

minutes after dose. At that moment, the [

14C]morphine concentration

was 29 μM, or about twice that in maternal plasma (15 μM). The ratio

[

14C]M3G/[14C]morphine was 5.5 at this time.

91

483-942 0 - 85 - 4 : QL 3
FIGURE 2. PET-images of the distribution of 11C in placenta (1) and

fetal liver (2) 0.3-27 min after intravenous administration of [

11C]heroin

to the mother. To the upper right is shown the uptake in the featl liver

(upper curve), and the placental uptake (lower curve), calculated from

regions of interest on the images.

During the 4-hour study period, less than 50% of the administered

14C-derived radioactivity was excreted in the urine, more than 80% of

this fraction as [

14C]MSG.

Measurable concentrations of [

14C]morphine and [14C]M3G from 45 minutes and on indicated the excretion from the fetus into amniotic fluid.

[

14C]MSG was present at persistently high and constant levels in amniotic

fluid for at least the duration of the experiment. In one monkey, a late

amniotic fluid sample obtained 16 to 17 hours after dose had the same

concentration as at 5 to 6 hours after dose. The ratio between [

14C]M3G

and [

14C]morphine in the amniotic fluid varied between 2 and 3 in both

monkeys.

[

11C]Morphine-derived radioactivity (11C-activity) was measurable in

maternal blood and urine for at least 3 hours (figure 4). The increasing

11C-activity in amniotic fluid became equal to the maternal blood

11C-activity at about 3 hours after dose, i.e., considerably later than was

the case for [

14C]morphiie. No kinetic estimations were made for the

11C-activity curves, since this activity represents the sum of radioactivities of [

11C]morphine and metabolites.

92
FIGURE 3. [

14C]morphine and [14C]morphine-3-glucuronide concetrations in maternal plasma, amniotic fluid, and fetal plasma after

administration of [

14C]morphine to one monkey.

DISCUSSION

PET has not been used previously for the study of pharmacokinetics in the

feto-maternal unit, but it is an accepted technique for studies of brain

metabolism and for receptor studies in the brain and heart (Ell and

Holman 1982). The advantage of the method for its use during pregnancy

is that it is a noninvasive tracer technique, thus avoiding pharmacological

effects of administered compounds. The procedure may be repeated

within a few hours and performed several times during the pregnancy in

the same animal. It is thus suitable for the study of dynamic events

during various phases of fetal life.

The use of PET as an in vivo autoradiographic technique gives a unique

possibility to study the rapid initial distribution of drugs from mother to

fetus.

However, the PET technique has certain shortcomings. The PET image is

derived from all molecules labeled with the radionuclide; i.e., both the

administered drug and its metabolites. In case the drug is metabolized

within 2 hours, blood and urine must be analyzed for the distribution of

radioactivity between parent drug and metabolites by chemical analysis,

e.g., HPLC. Another approach that facilitates the interpretation of the

93
PET images is to label the molecule at different sites. The pattern of

distribution of the radioactivity when the same individual is investigated

with these compounds will give additional information on the metabolism.

FIGURE 4. [

11C]Morphine-derived activity maternal blood, fetal

blod, an amniotic fluid after administration of [

11C]morphine to the

same money as in figure 3.

PET has a fairly low spatial resolution. The distribution of morphine and

heroin in the fetal brain is of great interest to study, but we bhose to

study the liver in our initial experiments. The liver is a homogeneous

organ, in which the limited resolution is of less importance than in studies

of the central nervous system.

The radioactivity in an organ is composed of two fractions; one is limited

to the blood and the other is composed of metabolized and unmetabolized

11C-labeled moieties in the tissues. Corrections for the fraction in the

blood pool can be made by a separate determination of the blood volume

using 11C-labeled red blood cells or 68Ga-transferrin.

94
A special difficulty with fetal PET studies is that the fetus might move

during the registration even if the mother is immobilized with ketamine.

In most cases, however, no difficulties in localizing the fetal liver

placenta were experienced.

The short half-life of 11C does not permit observation times exceeding 2

hours. The simultaneous administration of the corresponding unlabeled

compound or compounds labeled with long-lived isotopes makes it possible

to perform conventional studies with sampling beyond that period. By

sampling from the mother and the fetus, the PET image may be

correlated to the results from analyses of administered drugs and their

metabolites in body fluids. By this combination of techniques, two goals

are achieved. First, the initial, rapid phase of transport between

different organs of the body is studied by PET. This is not possible by

blood sampling alone. Second, results from the analyses of radioactivity

and drug concentrations in body fluids may be used in the construction of

compartment models. Such models may then be used in future

experiments, making invasive sampling techniques necessary only in an

initial phase of the study.

The present results indicate that heroin is readily distributed to the fetus

in accordance with its high lipophilicity. The transfer rate of morphine

was somewhat slower. Our data obtained with 14C-labeled morphine

confirm that this drug is transported to the amniotic fluid within 45

minutes. Earlier sampling did not reveal measurable levels of morphine in

the fetus. The [

14C]M3G/[14C]morphine concentration ratio in maternal

blood varied between the monkeys, indicating interindividual variation in

the capacity to glucuronidate this drug in the liver (Rane et al. 1984; Sawe

et al., in press). This ratio was significantly higher than that in the

amniotic fluid at “pseudo-steady-state,” which probably reflects the

slower passage of the hydrophilic M3G metabolite as compared to the

parent drug.

No other method than PET has so far been able to reveal the events

within the body without disturbing the integrity of the feto-maternal

unit. The ultrasound-guided puncture of the umbilical vein for fetal blood

sampling has been without noticeable effect on the animals, but the

limiting factor is the volume of blood which can be drawn.

The energy metabolism and the protein synthesis are of great importance

for the development of the fetus and, in particular, its central nervous

system. Infants of drug-addicted mothers are often small for gestational

age. The reasons for this are unknown, but impaired energy utilization

and perturbed protein synthesis are possible explanations. Studies of

glucose and amino acid transport and metabolism in opiate-treated

animals may contribute to the understanding of the causes of this growth

retardation.

Another negative effect of drug abuse on the fetus is frequent abstinence

symptoms. Our results show that there was a rapid increase in radioactivity in the fetus after intravenous injection of [

11C]morphine and

[

11C]heroin to the mother. The decline of the radioactivity was also

rapid, indicating that the fetus may suffer from withdrawal effects. PET

has been shown to offer unique possibilities to register such rapid events.

95
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Correction for scattered radiation in a ring detector positron camera by

internal transformation of the projections. J Comput Assist Tomogr

7(1):42-50, 1983.

Ell, P.J., and Holman, B.L., eds. Computed Emission Tomography

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L.; Bergström, M.; Ericson, K.; and Greitz, T. A four ring positron

camera system for emission tomography in the brain. IEE Trans Nucl

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Hartvig, P.; Bergström, K.; Lindberg, B.; Lundberg, P.O.; Lundqvist, H.;

Långström, B.; Svärd, H.; and Rane, A. Kinetics of 11C-labeled opiates

in the brain of rhesus monkeys. J Pharmacol Exp Ther 230:250-255,

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Långström, B.; Antoni, G.; Halldin, C.; Svärd. H.; and Bergson, G.

Synthesis of some C-labeled alkaloids. Chem Scripta 2:46-48, 1982.

Lindgren, P.C., and Lindberg, B.S. Fetal blood sampling through ultrasound guided puncture of the umbilical vein in rhesus monkeys. J Med

Primatol, in press.

Rane, A.; Säwe, J.; Lindberg, B.; Svensson, J.-O.; Garle, M.; Erwald, R.;

and Jorulf, H. Morphine glucuronidation in the Rhesus monkeys: A

comparative in vivo and in vitro study. J Pharmacol Exp Ther

229:571-576, 1984.

Säwe, J.; Kager, L.; Svensson, J.O.; and Rane, A. Oral morphine in

cancer patients: In vivo kinetics and in vitro hepatic glucuronidation in

the individual patients J Clin Pharmacol, in press.

Svensson, J.O.; Rane, A.; Säwe, J.; and Sjöqvist, F. Determination of

morphine, morphine-3-glucuronide and (tentatively) morphine-gglucuronide in plasma and urine using ion-pair high performance liquid

chromatography. J Chromatography 230:427-432, 1282.

Wagner, H.; Burns, D.; Dannals, R.; Wong, D.; Langstrom, B.; Duelfer, T.;

Frost, J.; Ravert H.; Links, J.M.; Rosenblom, S.B.; Lukas, S.; Kramer,

A.; and Kuhar, M. Imaging dopamine receptors in the human brain by

positron emission tomography. Science 221:1264-1268, 1983.

ACKNOWLEDGMENTS

The study was supported by the Swedish Medical Research Council

(14X-04496), The Swedish Cancer Society, and The Expressen Prenatal

Research Fund.

AUTHORS

Bo S. Lindberg, M.D., Ph.D.

Department of Obstetrics and Gynecology

University Hospital

75185 Uppsala, Sweden

96
Per Hartvig, Pharm.D.

Hospital Pharmacy

University Hospital

751 85 Uppsala, Sweden

Anders Lilja, M.D.

Department of Diagnostic Radiology

University Hospital

75185 Uppsala, Sweden

Hans Lundqvist, Ph.D.

Department of Physical Biology

Gustaf Werner Institute

University of Uppsala

P.O. 531, 751 21 Uppsala, Sweden

Bengt Lågström, Ph.D.

Department of Organic Chemistry

Institute of Chemistry

University of Uppsala

P.O. 531, 751 21 Uppsala, Sweden

Petter Malmborg, M.Sc.

Department of Physical Biology

Gustaf Werner Institute

University of Uppsala

P.O. 531, 751 21 Uppsala, Sweden

Anders Rane, M.D., Ph.D.

Department of Clinical Pharmacology

Huddiige Hospital

Karolinska Institute

14186 Huddinge, Sweden

Annika Rimland, M.Sc.

Department of Organic Chemistry

Institute of Chemistry

P.O. 531, 75121 Uppsala, Sweden

Hans Svärd, M.Sc.

Department of Organic Chemistry

P.O. 531, 751 21 Uppsala, Sweden

97
Placental Transfer of Drugs, Alcohol, and

Components of Cigarette Smoke and Their

Effects on the Human Fetus

Betty R. Kuhnert and Paul M. Kuhnert

INTRODUCTION

The placenta was once thought to act as a barrier which protected the

fetus from the passage of xenobiotic compounds. Now, it is more

accurately thought of as a sieve which allows small molecules to pass by

diffusion. For example, if the mother smokes, the fetus is exposed to the

components of cigarette smoke. Furthermore, if the mother takes drugs,

the fetus is exposed to the drugs as well as to any active metabolites.

Finally, if the mother drinks alcohol, fetal blood alcohol levels may be

similar to those of the mother.

Components of cigarette smoke, alcohol, and other drugs all affect the

fetus to varying degrees. These effects can range from subtle neonatal

neurobehavioral effects following the use of meperidine during labor to

the full teratogenic effects of alcohol known as the fetal alcohol

syndrome (FAS). These effects can be short-lived, as in the case of low

doses of meperidine given to the mother, or permanent, as in the case of

the mental retardation following chronic alcohol use during pregnancy. In

some cases, such as exposure to lead or cadmium from cigarette smoke,

there may be biological effects, but no obvious clinical effects. In other

cases such as alcohol, the lower limits of the teratogenic potential are

unknown.

This chapter reviews recent work on the placental transfer of three drugs

(meperidine, phencyclidine, and alcohol) and certain components of

cigarette smoke (lead, cadmium, and thiocyanate) and examines the

effects of these compounds on the fetus.

MEPERIDINE

Meperidine is a synthetic opiate narcotic administered intravenously or

intramuscularly for analgesia during labor.

It has been in use since 1939 and today it is the most commonly used

obstetrical analgesic (Fishburne 1982). However, despite its widespread

use for many years, surprisingly little was known about its pharmacology

during the perinatal period.

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Some puzzling questions have been raised concerning the use of meperidiie

during labor. For example, meperidine has been noted to cause respiratory

depression in the newborn infant even if given to the mother in low doses

(Morrison et al. 1973). Moreover, it has also been shown to cause more

neonatal respiratory depression if given to laboring patients 2 to 3 hours

prior to delivery rather than 1 hour or less prior to delivery (Cuilhem et al.

1953; Shnider and Moya 1964). And, finally, no relationship could be demonstrated between neonatal status and the concentration of meperidine in

cord blood (Morrison et al. 1973; Shnider and Moya 1964).

Based on this information, it was hypothesized that the effects of meperidine on neonatal status were due to the formation of a toxic metabolite.

Normeperidine was thought to be this active metabolite because animal

studies had shown it to be twice as potent as a convulsant and respiratory

depressant, but half as potent as meperidine when used as an analgesic

(Miller and Anderson 1954). However, the idea of a metabolite reaching the

fetus remained a hypothesis because sensitive analytical techniques which

could distinguish between meperidine and normeperidine in plasma were

not available until the 1970s. Furthermore, it had been reported that

neither the pregnant woman nor the neonate could metabolize meperidine

to normeperidine (Crawford and Rudofsky 1966).

The mother’s ability to demethylate meperidine to normeperidine was

recently documented (Kuhnert et al. 1979b). Maternal plasma levels of

normeperidine are apparent shortly after injection of the parent drug and

levels increase throughout labor. The ratio of normeperidine to meperidine

increases steadily with time. Furthermore, normeperidine has been

reported in cord blood (Kuhnert et al. 1979a). The levels of normeperidine

in cord blood increase with time after injection of the drug in the mother

(Kuhnert et al. 1980). With long drug-to-delivery intervals, more

normeperidine can be found in cord artery than in cord vein. This reflects

uptake of the drug in fetal tissues. Thus, recent studies support the

hypothesis of an active meperidine metabolite reaching the fetus.

However, the levels of normeperidine following single doses are probably

too low to have clinical effects. Moreover, the studies do not explain the

increase in neonatal morbidity if delivery occurs 2 to 3 hours after

administration of the drug to the mother.

Cord blood levels of drugs at delivery do not necessarily reflect the

infant’s tissue levels. Since tissue is not available for study from human

infants, serial urine collections and calculations of umbilical artery to

umbilical vein ratios are used to indirectly estimate tissue equilibrium or

body burden of drugs. Using these techniques, it has been shown that

maximum uptake of meperidine by fetal tissues occurs 2 to 3 hours after a

single intravenous injection of meperidine to the mother (Belfrage et al.

1981; Kuhnert et al. 1979a). Thus, the hypothesis of a toxic metabolite

reaching the fetus and causing the neonatal morbidity noted at 2 to 3 hours

is not supported. The dose-delivery intervals resulting in maximum uptake

of the parent compound, meperidine, correspond with those resulting in

maximum neonatal depression.

The use of multiple doses of meperidine over extended time periods during

labor may result in concentrations of normeperidine that are high enough

to be clinically significant (Kuhnert et al. 1985a; 1985c). In cases of long

99
drug-to-delivery intervals (>4 hours), considerable normeperidine is formed

by both mother and fetus (Kuhnert et al. 1985a). Furthermore, following

multiple doses, the uptake of meperidine is altered from what occurs

following single doses, and the high levels of normeperiddine may add to the

adverse effects resulting from high levels of the parent compound.

Following single doses, maximum tissue uptake of meperidine occurs at 2

to 3 hours following administration of the drug to the mother. After this,

there is a diffusion gradient from fetal tissues to fetal blood to maternal

blood and the drug is cleared from the fetus. With multiple doses, the

diffusion gradient is continuously in the direction of fetal tissue uptake.

Thus, continuous uptake of the drug is occurring in fetal tissues for hours

after the last dose is given. Therefore, multiple doses of meperidine to the

mother over long time periods result in maximum exposure of the fetus to

both meperidine and normeperidiie (Kuhnert et al. 1985a, 1985c).

There was little doubt that the high levels of meperidine administered to

parturients a generation ago could result in neonatal depression (Shnider

and Moya 1964). However, obstetricians today use much lower doses and

the clinical impression is that there are usually no adverse effects on the

neonate. A study designed to test this hypothesis showed that there may

be subtle neurobehavioral effects on the fetus due to mean doses as low as

39±19 mg of meperidiie to the mother (Kuhnert et al. 1985b). Using the

Brazelton Neonatal Behavioral Assessment Scale, it was shown that infants

whose mothers received low doses of meperidine performed less well on

tests of regulation of state and had more abnormal reflexes than infants of

nonmedicated mothers. Furthermore, these effects were related to the

drug-to-delivery interval. Performance was less optimal with longer drug

to delivery intervals at both 1 and 3 days of life. Since normeperidine

levels increase with long drug-to-delivery intervals, and levels of meperidine, but not normeperidine, are very low by 3 days of age, the results of

this study again suggest that normeperidine may contribute to the adverse

effects following meperidine administration to the mother.

The clinical protocol for the administration of meperidine during labor has

changed as more has become known about the pharmacology of the drug in

the intrapartum period. It was once suggested that meperidiie should be

given in 100 to 150 mg doses at hourly intervals up to three doses and then

as required (Walker 1973). Today, we know that pharmacologically the best

time to be born following intravenous maternal analgesia with meper- idine

would be within 1 hour of a single low dose.

PHENCYCLIDINE

Phencyclidine (angel dust) was originally developed as a short acting

anesthetic. However, its hallucinogenic effects have limited its legal use

to the practice of veterinary medicine. Nevertheless, phencyclidine’s

sedative and hallucinogenic effects make it a popular drug of abuse among

adolescents. These adolescents are often the same ones with unplanned

pregnancies.

There has been one isolated report of the possible teratogenic effects of

phencyclidine (Golden et al. 1980). However, the use of phencyclidine by

pregnant women and possible effects on the fetus have only recently been

studied prospectively (Golden et al. 1984). In this study, the frequency of

100
use by pregnant women was estimated to be between 0.8 and 6.4 percent.

Most of these women were polydrug users; therefore, the isolated effects

of phencyclidine on the fetus are difficult to determine. This study

indicated that phencyclidine use during pregnancy was widespread.

Abnormal neonatal neurological and behavioral findings have been

associated with maternal phencyclidine exposure (Golden et al. 1985).

These findings included poor attention, hypertonic ankle reflex, and

depressed neonatal reflexes--grasp and rooting. A second group of

abnormal outcomes which were more frequent in study infants, but not

statistically different, included miotic pupils, increased brachial reflexes,

and decreased joint mobility. The abnormal grasp reflex was predictive of

phencyclidine use when stepwise discriminant function analysis was used.

Abnormal anatomic findings were not associated with maternal phencyclidine use in this study. These results suggested that phencyclidine

exposure to the fetus can result in abnormal neonatal neurologic findings

and behavior. Future studies must determine the long-term effects of

exposing the fetus to phencyclidine.

The pharmacology of phencyclidine in pregnant women or neonates has not

been well studied. Phencyclidine is known to cross the placenta, to be

present in neonatal urine and blood, and in breast milk (Strauss et al. 1981;

Kaufman et al. 1983a, 1983b). In nonpregnant subjects, phencyclidine is

known to be metabolized to two hydroxylated compounds (Lin et al. 1975)

and a pentanoic metabolite (Cook et al. 1982). All three metabolites may

have pharmacological activity (Domino 1978; Cohen et al. 1982).

The results of analyzing PCP metabolites in spot urine samples collected

from asymptomatic pregnant patients suggested that the pentanoic acid

metabolite is a major metabolite of PCP in these patients (Kuhnert et al.

1984). This metabolite was present in concentrations many times higher

than either the parent compound or the hydroxylated metabolites.

However, at delivery the pentanoic acid metabolite did not seem to be the

major compound excreted by parturients and this may be due to differences

in time since the drug was used. Maternal urine samples were positive for

at least 36 hours after delivery and excretion of both drug and metabolites

fluctuated.

Phencyclidine and its metabolites have been analyzed in serial samples

collected from asymptomatic neonates. The results have shown that

excretion of drug and metaholites is erratic and that the samples are

positive for at least 36 hours (Kuhnert et al. 1984). Thus, an earlier report

that urine is negative after 72 hours in symptomatic infants may have been

hampered by insensitive methodology (Strauss et al. 1981). These findings

suggest that the drug may have been sequestered in various tissues or body

compartments and more detailed pharmacological studies are obviously

needed. Furthermore, these results suggest that present methods of

screening for phencyclidine use may be inadequate.

ALCOHOL

Historically, alcohol has often been noted to have an adverse effect on

offspring. The Bible (Judges 13:7) says: “Behold, thou shalt conceive, and

bear a son; and now drink no wine nor strong drink....” In 1973, Jones et al.

101
recognized a common pattern of malformation in the offspring of

alcoholic mothers and called this the fetal alcohol syndrome. Unknown to

them, the pattern had already been described in France in 1968 (Lemoine

et al.). Since 1973, many more cases of the syndrome have been

documented throughout the world (Clarren and Smith 1978; Streissguth et

al. 1980).

The principle features of FAS include mental retardation, growth

deficiency, and characteristic facial features. These facial features are

so characteristic that the mothers can be diagnosed as alcoholics from the

children’s features alone (Lemoine et al. 1968). These features include

small head circumference, low nasal bridge, eyes with prominent

epicanthic folds and short palpebral fissures, short nose, indistinct

philtrum (groove between nose and mouth), and a thin upper lip. The

percentage of infants having the problems associated with the FAS is

directly related to the magnitude of maternal ethanol ingestion

(Seidenberg and Majewski 1978), and also to the duration of maternal

alcoholism (Majewski et al. 1976).

The lower limit of the teratogenic potential of alcohol is unknown. There

is now considerable evidence from clinical and experimental studies that

prenatal alcohol exposure may adversely affect fetal development; this is

true even in the absence of observable physical anomalies and is apparent

by behavioral deficits. In addition to lowered IQ, children exhibit various

other behavioral problems such as excessive stubbornness, aggression,

hyperactivity, and sleep disorders. Furthermore, behavioral anomalies

have been reported in neonates of social drinkera (Landesman-Dwyer et

al. 1978). In social drinkers who smoked, moderate alcohol intake coupled

with moderate smoking use exerted an interactive and deleterious effect

upon newborn learning, which was not predictable when the two drugs

were taken separately (Martin et al. 1977). Thus, alcohol can be

characterized as a potent behavioral teratogen.

There have been relatively few investigations of the disposition of ethanol

following maternal administration in humans. Brien et al. (1983) reported

that there is a relatively low ethanol concentration in amniotic fluid after

maternal administration of ethanol and then a relatively high ethanol

concentration in amniotic fluid when ethanol has been virtually eliminated

from maternal blood. Hence, the amniotic fluid appears to provide a

“chemical cloud” environment such that the fetus remains exposed to

ethanol when none is measurable in maternal blood. It is conceivable

that, during an episode of binge-type drinking, the fetus could be exposed

to a very high ethanol concentration in amniotic fluid for a longer time

period than would be predicted by the maternal blood ethanol

concentration.

There is no longer any debate that the fetal alcohol syndrome exists

(Clarren and Smith 1978). What remains to be answered are such questions

as: What is the risk to a woman, given a specific drinking history, of

producing a seriously affected offspring? How does intermittent binge

drinking as opposed to steady consumption alter the phenotype? How do

commonly associated drugs like caffeine, nicotine, and diazepam alter or

potentiate the effects of alcohol? What are the mechanisms through

which alcohol or its breakdown products produce their effect on the

embryo and fetus? And finally, can prenatal diagnostic technics be used

to detect this disorder.

102
SMOKING

Smoking may adversely affect many aspects of human reproduction. For

example, it may increase the risk of spontaneous abortion (Himmelberger

et al. 19781, retard fetal growth (Meyer et al. 1976), and increase the

number of pregnancy complications (Andrews and McCarty 1972). In

addition, children of smokers may be physically and intellectually

disadvantaged (Goldstein 1971). Precisely why these effects occur is

unknown, but many factors are believed to be involved and interrelated.

Three such factors may be the increase in the uptake of lead, cadmium,

and cyanide which occurs subsequent to exposure to cigarette smoke.

Lead

Each cigarette generates approximately 1 to 2 micrograms of airborne

lead when burned (Cogbill and Hobbs 1957). The amount of this lead that

reaches the fetus depends on how much the mother smokes. The concentration of lead in fetal blood is directly related to the concentration of

lead in maternal blood (Kuhnert et al. 1976). While it is well established

that lead is an environmental health hazard to children, its effect on the

fetus is not well documented.

The effects of low levels of lead exposure can be seen by looking at the

effect of lead on enzymes involved in the pathway of heme synthesis.

One of the earliest manifestations of toxicity from low concentration of

lead appear to be the inhibition of d-aminolevulinic acid dehydratase

(ALAD) which converts d-aminolevulinic acid to porphobilinogen; the

other manifestation is ferrochelatase which inserts iron into protoporphyrin IX to form heme. The inhibition of ALAD can be measured

directly by enzyme assay (Granick et al. 1973); the inhibition of

ferrochelatase can be measured indirectly by increases in erythrocyte

protoporphyrin levels (Sassa et al. 1973).

In the fetus there is no relationship between erythrocyte protoporphyrin

levels and blood lead at term (Kuhnert et al. 1978). This suggests that

there has been no chronic effects of lead in the fetal bone marrow. There

is, however, a strong correlation between the erythrocyte lead levels and

the activity of ALAD in human fetal erythrocytes (Kuhnert et al. 1977).

This suggests that there is a direct acute effect of lead on the enzyme in

the peripheral circulation. Enzyme inhibition due to lead averages 11

percent of the enzyme’s activity. Infants of smoking mothers were found

to have significantly more enzyme inhibition than infants of nonsmoking

mothers.

Cadmium

In addition to lead, the average cigarette contains 1 to 2 micrograms of

cadmium (Lewis et al. 1972). Heavy smokers may have an intake of 20

micrograms/day or more due to smoking, in addition to intake from

dietary sources. In animals, cadmium has been found to cause fetal

growth retardation at low concentrations (Webster 19781, and to induce

developmental anomalies and fetal death at higher concentrations

(Chernoff 1973). In humans, cadmium has been thought to be one of the

factors which may contribute to fetal growth retardation (Longo 1980).

103
Cadmium’s adverse biological effects stem from its simularity in molecular structure to the essential element, zinc. Because of this, cadmium

can substitute for zinc as a cofactor for several different enzymes,

including alcohol dehydrogenase, acid phosphatase, and others. This

substitution causes enzyme inhibition. Further biological effects may

occur because cadmium in the body is bound to metalothionein, a

transport protein which also binds zinc.

The uptake of cadmium by pregnant women who smoke and the disposition

of cadmium in the maternal-fetoplacental unit has only recently been

described (Kuhnert et al. 1982). A comparison of cadmium levels in

maternal blood, fetal blood, and placenta between smokers and

nonsmokers showed that the highest concentration of cadmium was found

in the placenta of smokers; the least amount was found in the cord blood

of infants whose mothers were nonsmokers. The placenta of nonsmokers

contained approximately seven times more cadmium than the corresponding cord blood levels; for smokers, this difference was approximately

ninefold. Significantly higher cadmium levels were found in maternal

blood and placenta of smokers. Cord blood levels were higher, but not

significantly, in smokers; the percentage increase in cadmium due to

smoking were 16 percent in cord blood, 32 percent in placenta, and 59

percent in maternal blood. It appears from this study and others (Roels et

al. 1978; Lauwerys et al. 19781 that the placenta acts as a barrier to the

transfer of cadmium to the fetus.

The fact that cadmium does accumulate in the placenta should not be

interpreted to mean that exposure to cadmium is not hazardous to the

fetus. Accumulation of cadmium in the placenta could alter its function

and thus be embryotoxic. It has been shown in animals that the placenta

is rapidly damaged after maternal injections of cadmium (Parizek 1965).

This leads to embryonic death in utero.

Cyanide

Tobacco smoke also contains cyanide, most of which is rapidly converted

to thiocyanate after absorption in the body (Pettigrew and Fell 1972).

Although low levels of thiocyanate are normally present in the body from

dietary sources, elevations in thiocyanate are believed to be one of the

mechanisms of fetal growth retardation related to smoking (Andrews 1973;

Pettigrew et al. 1977; Mebert et al. 1979; Pirani 1978). In the body,

thiocyanate and cyanide maintain a dynamic equilibrium. In higher doses,

thiocyanate and cyanide are definitely toxic. Both can act as hypotensive

agents, reduce intracellular oxygen utilization by inhibiting cytochromes,

interfere with vitamin B12 metabolism, cause degenerative neurological

disease, and alter thyroid function (Andrews 1973; Pettigrew et al. 1977;

Mebert et al. 1979; Pirani 1978; Philbrick et al. 1979).

During pregnancy, maternal thiocyanate levels are correlated with the

number of cigarettes smoked per day by the mother. Furthermore,

maternal and fetal thiocyanate levels are very closely correlated

(Pettigrew et al. 1977; Bottoms et al. 1982). Average fetal levels are

similar to those in the mother (Bottoms et al. 1982).

104
Thiocyanate can also be used as an effective biological marker of

exposure to smoking (Cohen and Bartsch 1980). This compound is

particularly attractive as a marker because of its long half-life (14 days)

in the body. Compounds such as carboxyhemoglobin (4 hours) or nicotine

(<30 minutes) have much shorter half-lives. One limitation is that the

specificity and sensitivity may be inadequate when thiocyanate levels are

close to levels due to dietary or other sources. Thiocyanate has recently

been used as a biological marker to study the effects of passive smoking

on the fetus (Bottoms et al. 1982).

Passive smoking--the exposure of nonsmokers to air contaminated with

cigarette smoke--has been suggested as producing some of the same

adverse health consequences as active smoking. There are reports of

increased frequencies of upper respiratory infection, bronchitis, pneumonia, diminished small airway function, and angina in association with

passive smoking in the workplace or in the home (White and Froeb 1980;

Tager et al. 1979; Harlap and Davies 1974; Aronow 1978). These findings

suggest that passive smoking might also affect the fetus. While there are

many studies of active smoking during pregnancy, the effects of passive

smoking during pregnancy are not well documented.

Increased fetal thiocyanate has been found in association with passive

smoking (Bottoms et al. 1982). This finding suggests that tobacco smoking

by other members in the home or workplace of a pregnant woman who

does not smoke exposes the fetus to a measurable increase in at least one

metabolic byproduct of tobacco smoke, thiocyanate. This is consistent

with the possibility that passive smoking might adversely affect the

fetus. Unfortunately, there have been no enzymatic or clinical studies to

assess possible biological or clinical effects from the relatively small

increase in thiocyanate levels associated with passive smoking.

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107
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ACKNOWLEDGMENTS

Supported in part by National Institutes of Health, United States Public

Health Service grants 5M01-RR00210, N01-RR-8-2112, R01-HD-13359,

and R01-DAU-2903.

AUTHORS

Betty R. Kuhnert, Ph.D.

Paul M. Kuhnert, Ph.D.

Perinatal Clinical Research Center

Cleveland Metropolitan General Hospital

Case Western Reserve University

3395 Scranton Road

Cleveland, Ohio 44109

109
Appendix

Maternal-Fetal Transfer of Abused

Substances: Pharmacokinetic and

Pharmacodynamic Data

Charles C. Lee and C. Nora Chiang

The objective of this appendix is to present pharmacokinetic and pharmacodynamic data in a format that allows the investigators to readily review

research findings regarding pre- and post-natal effects due to maternal

exposure to substances of abuse. It is intended to be a comprehensive

collection of clinical reports from human studies and laboratory findings

in animals as well as perfused placenta preparations. Information on the

experimental method, dosage to the mother, route of drug administration,

gestational age, and the duration of maternal/fetal exposure is also

presented. The abused substances compiled in this appendix include:

amphetamines, cannabinoids/delta-9-tetrahydrocannabinol (THC)/cannabis, cocaine, lysergic acid diethylamide (LSD), meperidine, methadone/

acetylmethadol, morphine/heroin, nicotine/tobacco, and phencyclidiie

(PCP).

The pharmacokinetic data in this appendix, where available in the mother

and fetus, include half-life (t1/2), clearance (C1), and time to peak

concentration (tmax). In most cases, the maternal to fetal concentration

ratio of drug in the tissue or biological fluid is reported because serial

blood sampling required for pharmacokinetic study is not feasible in small

animals. Since drug secretion in human breast milk is a critical concern

for breast-feeding mothers, information on this subject is also

documented for its important clinical implications.

The pharmacodynamic data encompass the broadly defined teratological

effects. Traditionally, the term teratology refers to the study of (bongenital malformations produced by exogenous agents during gestation. In

recent years, teratologists have extended their observations beyond

morphological changes and have focused on functional disorders for

drug-induced biochemical and behavioral anomalies detected at birth or

later. Teratological effects for a specific abused substance are documented and compared between animal species.

In the past decade, several tables of pharmacodynamic data for various

drug substances have been published. In 1976, Coyle et al. published a

review on behavioral teratogenesis in which they summarized prenatal

drug effects on behavior as a function of developmental stage. Of the

110
drugs examined, only THC and amphetamine belong to traditionally

defined substances of abuse. In 1977, Joneja presented a thorough study

on the teratologic effects of THC, given orally as single and multiple

doses during gestation, on the fetuses of golden syrian hamsters.

Extensive observations were made on day 15 after drug administration,

among which were percent resorption or death, number of retarded fetus

and external/skeletal abnormalities. No kinetic data were presented. In

1980, Slone et al. tabulated in a symposium proceedings some embryonically toxic drugs and chemicals of potential risk to the human fetus and

newborn, recording the number of cases studied and the elicited teratologic effects. In the same year, Abel compiled two tables documenting

prenatal effects of cannabis extract and THC. These tables include

information about dosage, route of administration, duration of drug

exposure during gestation, and teratological effects. Again, no kinetic

data were included in the compilation. Until very recently, only data on

the effects of pregnancy on drug pharmacokinetics were compiled.

Information on the fetal cord to maternal plasma ratio of drug

concentrations and drug excretion in human breast milk were compiled as

two tables by Parker (1984) for some 60 drugs, most of which were not

substances of abuse. Since Parker’s review focused on kinetic

information, those dynamic effects incurred as a result of placental drug

transfer were not documented. An overview of this research area

indicates that very little integrated kinetic/dynamic information has been

documented. Thus, this compilation is intended to integrate pharmacokinetic and pharmacodynamic data relevant to maternal-fetal transfer of

abused substances. It is hoped that this information will add to our

understanding and appreciation of this critical subject matter.

Several considerations were given in compiling the following list: (1) Only

values obtained from original papers, dating from 1974, and with data or

experimental methods which we could evaluate are included. Two review

articles for LSD are included because extensive research was performed

before that year. (2) The drug must be historically or currently abused to

a significant extent. Alcohol and benzodiazepines are not included in this

review since there exists such extensive literature on these subjects that

separate reviews would be required. (3) Information on dosage, route of

administration, and gestational age was lacking in some case studies,

particularly when human abusers were involved. However, we feel that

kinetic and dynamic effects elicited in pregnant women are sufficiently

important to warrant documentation of such effects even without

accurate recording of drug dosage regimen. (4) Some duplicate studies

using the same animal model and drug are included for the purpose of

contrasting the inter-laboratory variation of kinetic/dynamic effects.

Apparent deviations in dosage, route of administration, gestational age,

and duration of exposure are indicated. (5) The references are keyed to

individual authors so that readers can readily locate the reference

actually used.

It would obviously be most useful if there were a consensus about the

correct value for a given pharmacokinetic parameter or pharmacodynamic

measurement in a given species. In this compilation, a single value from

the literature or a range of values was selected for each parameter/

measurement. In some cases, we have summarized and interpreted the

data from the literature to determine the values reported in this table.

111
We hope that this compilation will serve as a comprehensive reference to

those research scientists faced with selection of animal models for drug

abuse. research in pregnancy. For the general public concerned with the

clinical consequences of maternal-fetal transfer of abused substances,

this compilation may be appreciated as a warning, particularly for

potential drug abusers.

112
PHARMACOKINETIC AND PHARMACODYNAMIC DATA

Model/ Dose/Route/Time Kinetic/Dynamic Effects

Preparation of Exposure

Drug Category: AMPHETAMINE/METHAMPHETAMINE

Reference

Human

Human

Human 237 out of 42,101 4% reduction in birth wt when drug

pregnancies used d-am- was taken after 28 wk of gestation;

phetamine for control no effects on birth lengths, head

of wt gain during circumferences, and perinatal mortality

pregnancy rates

amphetamine-addicted

women; gr 1: discontinued during first trimester; gr 2: throughout

pregnancy

rate of preterm delivery and perinatal motality increased for gr 2

amphetamine-addicted

women; gr 1: discontinued during first

trimester; gr 2: throughout pregnancy; gr 3:

throughout pregnancy &

children in foster home

temporary drowsiness in the infants

during the first month after birth,

normal somatic and psychomotor development at the age of 12 months;

symptoms indicating emotional disturbance were common in gr 2

Human amphetamines or other

anorectic drugs for

wt control during

pregnancy

no effect on severe congenital anomaly

(SCA) rate for live-born children

at 5. an excess of oral clefts in

the offspring of mother who took

amphetamine during early pregnancy

Naeye 1983

Eriksson et al.

1981

Billing et al.

1980

Milkovich &,

van den Berg

1977
Rat

Rat

Rat

Rat

Rat

Rat

0, 3 mg/kg/d, bid, damphetamine, SC, d 7

thru 20 of gestation

0, 0.5 mg/kg/d, dl-amphetamines, SC, during

pregnancy

0, 2 mg/kg/d, d-amphetamine, SC, d 7 of

gestation till birth

0, 0.5, 1 , 2 mg/kg/d,

d-amphetamine, SC,

d 12 to 15 of gestation

0, 2, 5 mg/kg/d, damphetamine mixed with

drinking water, 30 d

prior to mating till

parturition

0, 5 mg/kg/d, methamphetamine; 3 mg/kg/d

nicotine; bid, SC,

throughout pregnancy &

nursing

either-way avoidance learning in the

offspring was affected; pre- and postnatal amphetamine effects on avoidance

behavior dependent on nature of the

response and genetic lines

significant decrease in brain alpha but

not beta adrenergic receptor binding

without apparent changes in receptor

affinity in the adult offspring

offspring showed marked alterations

in brain neurotransmitter metabolism,

enhanced locomotor activity until 21

days of age

no detectable maternal or fetal toxicity; significant deficit in Y-maze

performance, & increased locomotor

responsiveness to postnatal challenge of amphetamine in the juvenile

offspring

delayed eye & vagina openings, slower

in behavioral tests of righting reflexes before 6 days of age and of

bridgecrossing at 14 days of age; no

differences in open-field activity

increased locomotor activity in offspring of methamphetamine-treated

group when monitored one night/month

from 3-39 months of age

Satinder &

Sterling 1983

Ramirez et al.

1983

Bigl et al.

1982

Adams et al.

1982

Monder 1981

Martin & Martin

1981
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Rat 0, 0.5 mg/kg/d, dlamphetamine, SC,

throughout pregnancy

Rat

Rat

Rat

0, 5 mg/kg/d, methamphetamine; 3 mg/kg/d,

nicotine; SC, or no

injection, throughout

pregnancy & nursing

increased susceptibility to disease.

or possible greater susceptibility to

tumor formation and growth retardation

for much of the lifespan of offspring

of methamphetamine group

1.25 mg/kg/d, N-2-cyano- increased total lipid content in fetal

ethylamphetamine, PO, heart but no differences in lipids in

d 5 to 21 of pregnancy placenta and fetal liver

0, 0.5 mg/kg/d, dlamphetamine, SC,

throughout pregnancy

no modifications of brain endogeneous

content of dopamine and noradrenaline

but modifications of catecholamine

metabolism

Rat 0, 0.5 mg/kg/d, dlamphetamine, SC,

throughout pregnancy

sexual behavior responses to ovarian

steroids were affected for female

offspring; no differences in hypothalamic monoamine content, sex cycle

duration, and ovulatory phenomena

higher motor activity in the openfield test in adults, more errors

observed in the Lashley III maze in

the first 4 days of age

Ramirez et al.

1979

Martin et al.

1979

Kulay et al.

1978

Nasello &

Ramirez 1978a

Nasello &

Ramirez 1978b
Rat

Rat

Rat

Rat

Rat

0, 5 mg/kg/d, metham- offspring of methamphetamine gr were Martin et al.

phetamine; 3 mg/kg/d significantly underweight, exhibited 1916

nicotine; SC, or no developmental delay, greater activity

injection, throughout for 8 of the first 12 monthly assesspregnancy & nursing ments begun at 90 days of age

1 or 3 mg/kg, d-amphetamine, bid, SC, from

d 5 of gestation till

term

0, 5, or 10 mg/kg/d

d-amphetamine, SC,

during d 5-9 (early)

or 12-16 (late) of

gestation

0, 1, 3, 5 mg/kg, bid,

methamphetamine.

throughout gestation

0, 0.5 mg/kg/d, dlamphetamine, SC,

throughout pregnancy

no obvious teratogenic effects but a

significant and dose-related increase

in pup mortality; surviving offspring

of 3 mg/kg gr showed increased motor

activity and decreased brain biogenic

amine levels

Hitzemann

et al. 1976

no differences among offspring in mild Seliger 1975

expression of emotionality; increased

incidences of severe emotional responses

(wild running and convulsion) in

placebo-early group

earlier delivery, decreased wt gain Martin 1975

over gestation, litter size decreased as

a function of dose, eye opening delayed,

no gross anomalities, more conditioned

avoidance responses for 5 mg/kg group

no differences in the age of eyes or Nasello et al.

vaginal aperture, or in the rate of 1974

growth; better acquisition & retention

of conditioned avoidance response at

the age of 90 days, and lower hippocampal seizure threshold
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Mouse 5 mg/kg, 3

H-d-amphet- Tmax=1 hr in placenta & fetal tissues,

amine, IP, on d 16 of Tmax=15 min in matured tissues,

gestation conc in fetal tissues lower than

maternal tissues

Mouse 0, 5 mg/kg/d d-amphet- slightly reduced body wht at birth,

amine. IP, during the altered conc of brain catecholamines

last 6-7 d of pregnancy during development and increased

activity after they had matured

Drug Category: CANNABINOIDS/DELTA-9-TETRAHYDROCANNABINOL(THC)/CANNABIS

Human daily smoking of THC in maternal blood was 2.5-6 times

marijuana in third greater than in cord blood; maternal

trimester of pregnancy level of metabolite was 4-7 times

higher than cord level

Human

Human

use of marijuana more meconium staining and longer

during pregnancy duration of labor

nursing mothers smoking THC conc in milk was higher than that

marijuana in maternal plasma; THC was absorbed

by the baby from the milk

Human use of marijuana

during pregnancy

nervous system abnormalities in

infants; visual responses were affected

in a dose-related manner

Shah and Yates

1978

Middaugh

et al. 1974

Blackard & Tennes

1984

Greenland et al.

1982

Perez-Reyes 1982

Fried 1980
Monkey

Monkey

Sheep

Dog

Rabbit

Rabbit/

Rat

Hamster

2.4 mg/kg/d, PO,

throughout pregnancy

& lactation

2.4 mg/kg/d, IV,

during gestation

and nursing period

0.5 mg/kg, pulmonary

infusion 131-140 d of

gestation

0.5 mglkg. IV,

58-63 d of gestation

15, 30, 60 mg/kg/d, SC,

7-19 d of gestation

0.5-5 mg/kg PO as THC,

5-50 mg/kg PO as crude

extract, 6-18 d of gestation

125-500 mg/kg single

dose, 25-100 mglkg

multiple dose, 7-12 d

of gestation

infants showed an alteration in visual

attention

drug-exposed mother-infant pairs were

similar to nontreated controls;

adequate maternal care was demonstrated by all mothers

decreased cardiac output, increased

blood pressure, and increased uterine

blood flow in mothers; decreased fetal

blood pressure, increased umbilical

blood flow in fetus; both mother and

fetus developed acidosis and hypoxemia

THC level in maternal brain was 3 times

higher than that in fetal brain; major

area of THC distribution remained the

same

reduction in maternal food consumption

and wt gain, embryotoxicity and

embryocidal effects

no evidence of teratogenic activity;

fetal and pup survival was reduced

only at high dose (50 mglkg of crude

marijuana extract)

higher frequency of fetal mortality

and growth retardation, no clearcut

teratogenic response

Golub et al. 1982

Golub et al. 1981

Cotterill et al.

1984

Martin et al. 1977

Sofia et al. 1979

Wright et al. 1976

Joneja 1977
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Rat

Rat

Rat

Rat 5-50 mg/kg/d, PO, from

pregnancy to d 5 of

gestation, then either

50 or 150 mg/kg/d till

21 d of gestation

dose-related decrease in pregnancies

carried to term, decrease in wt gain

during pregnancy, decrease in birth

wt

20-200 mg/kg

marijuana extract

intubation throughout

gestation

reduced food-water consumption,

reduced maternal wt gain,

decreased pup wt at birth,

increased mortality

4.2 mg/kg/d, IP, pups having lower body wt, higher

2-6 d of gestation emotionality. poor learning capacity

l-10 mg/kg, PO,

during gestation &

lactation

small doses THC suppressed luteinizing Rosencrantz &

hormone, larger doses elevated Esber 1980

follicle-stimulating hormone and

estrogens

Rat

Rat

10-150 mg/kg/d

marijuana extract

intubation 1-21 d of

gestation

3.3 mg/d. inhalation

1-19 d of gestation

Abel 1984

Abel et al. 1981

Kawash et al. 1980

impaired rotarod performance but did not Abel 1979

affect inclined plane, spontaneous

alteration, learning/memory, or openfield performance

smaller infants at birth, delayed

physiological development and less

active than the control group

fried & Charlebois

1979
Rat

Rat

Rat

Rat/

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

1.25 or 4.0 mg/kg, IV,

during nursing period

2 mg/kg/d, PO,

throughout pregnancy

25, 50, 100 mg/kg/d, SC,

6-15 d of gestation

rat: 12.5-50 mglkg, PO

mouse: 150-600 mg/kg, PO

6-15 d of gestation

50 mg THC, single oral

dose on 18th d of gestation

50 mg/kg, single oral

dose on the day of

delivery

20 mg/kg, PO, 3 times/wk

3 wk after weaning &

prior to parturition

50 mglkgld, PO, THC,

12th d of gestation

for 4 days

50 mg/kg/d, PO for

7 days from 20th d

of gestation

THC inhibited suckling-induced prolactin

release in the postpartum rat

no teratogenicity, but had passive

avoidance response

maternal wt gain depressed, fetal wt

decreased significantly at dose greater

than 50 mg/kg; some abnormalities

no severe sign of intoxication in both

rats and mice; increased litter

resorption & fetal mortality

maternal exposure altered endocrine

function and conc of brain biogenic

amines in the male offspring

maternal exposure altered male reproductive function and brain biogenic

amines in male and female offspring

decreased nest-building behavior,

litter size, and sexual behavior

maternal exposure resulted in increase

of body wt. ano-genital distance, and

testosterone conc in male and female

fetuses

reduced postnatal viability, impaired

male reproductive behavior at maturity

and decreased seminal vesicle wt

Bromley et al. 1978

Vardaris 1976

Banerjie et al. 1975

Fleischman et al.

1980

Dalterio et al.

1984a

Dalterio et al.

1984b

Frischknecht et al.

1982

Dalterio & Bartke

1981

Hatoum et al. 1981
Model/ Dose/Route/Time

Preperation of Exposure

Mouse 50 mg/kg/d, PO, 1 d

prior to parturition

and 6 d postpartum

Mouse 5 mg/kg, PO,

during lactation

Mouse 0-25 mg/kg/d

intubation

short term: 8 d

long term: 70 d

Mouse

Mouse

Mouse

25 mg/kg/d, SC, from

13th d of pregnancy

to term

3-400 mg/kg, single fetal growth retardation, increased

dose, IV, SC, & intra- external abnormalities, skeletal

gastric abnormalities

50-300 mg/kg, IP,

8-16 d of gestation

in utero death, reduction of body size, Mantilla-Plata,

offspring with cleft palate & Harbison 1976

Kinetic/Dynamic Effects Reference

reduced testes wt and elevated plasma Dalterio 1980

level of luteinizing hormone

decrease of pup body wt. some effects Frischknecht

on tail length, projection of ears, & et al. 1980

eyes opening

short term: delayed entry into proestrus, Kostellow et al.

depressed serum progesterone during 1980

luteal phase, inhibited female

receptivity to males

long term: term pregnancies were reduced,

no significant effects on length of

estrous cycles or mating

THC suppressed mammary gland lipoprotein Raine et al.

lipase activity and depressed mammary 1978

gland growth and development

Joneja 1976
Mouse 5-150 mg/kg, PO,

6-15 d of gestation

no effects on maternal wt gain, prenatal mortality, fetal wt, and frequency of gross external, internal,

& skeletal abnormalities

Fleischman

et al. 1975

Drug Category: COCAINE

Rat/Mouse 50, 60, 75 mg/kg, IP,

8-12 d of gestation

Mouse 75-175 mg/kg, SC,

7-12 d of gestati

rat: reduction in maternal and fetal

wt. increased resorption frequency

mouse: decreased fetal wt with no

increase in malformations

Fantel & Macphail

1982

on

eye and skeletal defects, teratogenic Mahal

and ontogenic 1980

ik et al.

Mouse

Human

Placenta

10 mg/kg 3H-cocaine Significant levels of cocaine in Shah et al. 1980

IP, 15-17 d of gestation placenta and the fetus

280-560 umole, cocaine decreased acetylcholine release rate

incubate with placenta by 33-50%

Sastry et al.

1977

villi for 10-15 min

Drug Category: LYSERGIC ACID DIETHYLAMIDE (LSD)

Human

Human

Human

parents used LSD before an infant with a unilateral anophthalmia

and during pregnancy

Margolis &

and normal chromosome Martin 1980

maternal LSD use during a premature baby with severe ocular Chan et al.

the first trimester malformations 1978

Maternal LSD use before baby with multiple systemic and ocular

& during pregnancy

Apple and

malformations Bennett 1974
Model/ Dose/Route/Time

Preparation- of Exposure

Kinetic/Dynamic Effects Reference

Human psychotherapeutic manage- no cytogenetic abnormality for mothers

ment before pregnancy and infants

Human 5 psychiatric patients

treated with LSD

during pregnancy

no abnormal births in 7 pregnancies

Human 140 women used LSD before

& during pregnancy (polydrug users)

out of 148 pregnancies, 83 live newborns

and 8 with major defects, 53 therapeutic

abortions, 12 spontaneous abortions

Human/

Animals

a review article five out of 161 children with limb

defects but no strong evidence of

chromosome breakage and teratogenicity

in animals or man

Human/

Animals

a review article

Rabbit 0, 50 ug/kg, IV, single

dose, at 21-25 d

of gestation

no evidence that pure LSD is teratogenic

in humans; some indication of an increased risk of spontaneous abortion in

humans; teratogenic effects reported

but not confirmed in hamsters and rats,

suggested for lower primates and confirmed in mice.

induced a disaggregation of polysomes

in fetal kidney, liver, and brain;

pretreatment with haloperidol and

pizotyline effectively inhibited

the disaggregation

Fernandez et al

1974

Robinson et al.

1974

Jacobson &

Berlin 1972

Long 1972

Dishotsky et al

1971

Heikkila et al.

1979
Rat

Mouse

3

H-LSD, 24 uCi/kg, IV,

18 d of gestation

5 ug/kg IP, on postconcept on d 12 or 18

Drug Category: MEPERIDINE

Human 100 mg, IM, 38-42 wk

of pregnancy

Human 1.5 mg/kg. IM during

labor

Human

Human

Human

50 mg, IM between 3-8 d

from childbirth

50 mg, IV, during labor

50-75 mg, IV, 4 hr

before delivery

significant fraction of the dose

appeared in the fetus

variable changes in the wt of some brain

regions; monoamine oxidase levels were

elevated

general depression of fetal heart rate

to sound stimulation was observed 30 min

after meperidine injection

fetal conc reached peak level at l-5

hr after dose; fetal-maternal drug

ratio varied between 0.35-1.5; t1/2

averaged 3.4 hr and was not different

from that in healthy control

meperidine appeared in milk, the conc

ratio between milk and serum was greater

than unity, no harmful effect in newborn

fetal conc of meperidine peaked 2-3 hr.

that of normeperidine peaked 4 hr or

later following dosing to mother

neonatal depression following meperidine

& no correlation between suppression

and route of administration or meperidine

conc at time of delivery

Back & Singh

1977

Hoff 1976

Jensen 1984

Tomson et al.

1982

Peiker et al.

1980

Kuhnert et al.

1979

Rothberg et al.

1978
Model/ Dose/Route/Time

Preparation of Exposure

Human 1 mg/kg, IV,

during amniotomy

Human 25 mg, IV infusion,

during labor

Human 50 mg, epidural

analgesia, during labor

Sheep 8.5-12.5 mg/kg, IV,

last month of gestation

Sheep 2.5 mg/kg, IV or IM,

during labor

Sheep 3.2 mg/min, IV infusion,

111-121 d of gestation

Sheep 2.5 mg/kg, IV,

111-121 d of gestation

Kinetic/Dynamic Effects

decreased pH and increased pC02, in

mother; decreased PH and base excess in

fetus

no ill effects

hypotension complication, decelerated

fetal heart rate; neonates had lower

APGAR score compared to controls

disrupted fetal sleep cycle and

increased heart rate

uptake of meperidine in fetal brain;

peak conc of meperidine in fetal brain

was 3-4 times greater from IV than IM

meperidine clearance averaged 12 ml/min;

ratio of meperidine clearance to

inulin clearance was greater than 1,

indicating tubular secretion

meperidine t1/2 approximated 30 hr;

renal clearance varied between 2.8-16.7

ml/min; meperidine was found in amniotic

and allantoic fluids

Reference

Chang et al. 1976

Eliot et al. 1975

Wingate et al.

1974

Ruckebusch

1976

Szeto et al

et al.

. 1980a

Szeto et al . 1980b

Szeto et al. 1979
Human 0.1 uCi 14C-meperidine lipid-soluble analgesics crossed

Placenta added to placenta tissue placenta by transcellular as well as

obtained 10 min after extracellular pathways; no indication

delivery of active transport

Rabbit 15 mg/hr, for 20 min, meperidine conc ratio between umbilical

Placenta IV infusion effluent and maternal plasma was 0.64;

placental clearance was reduced by

maternal protein binding, flow dependent at low umbilical flow rate and

permeability dependent at high flow

Drug Category: METHADONE/METHADYL ACETATE

Human low-dose methadone smaller head circumference, more deprestherapy sion of interactive behavior and state

controls

Human methadone therapy narcotic abstinence syndrome, smaller

head circumference, elevated systolic

blood pressure

Human

Human

50 mg/d, PO, throughout measureable but small amount of methapregnancy and perinatal done in amniotic fluid & breast milk

period t1/2 in neonate = 32.5 hr

methadone administered cord blood level of methadone was lower

to pregnant mother than maternal serum level; conc of metha

done in neonatal urine was 10-60 times

that of cord blood

Seeds et al. 1976

Hamshaw-Thomas

et al. 1984

Chasnoff et al.

1982

Rosen & Johnson

1982

Kreek 1979

Harper et al. 1977
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Human

Human

Human

Human

Sheep

Sheep

heroin & methadone

use during pregnancy

heroin and methadone

use during pregnancy

methadone maintenance

treatment

methadone treatment

program

mother: 5 ug/kg/min

fetus: 0.5 ug/kg/min

IV infusion, 130-141 d

of gestation

methadone and morphine

IV infusion, 5 hr,

130-141 d of gestation.

mother: 5 ug/kg/min

fetus: 0.25, or

0.5 ug/kg/min

induced chromosome damage in vivo

low birth rate, fetal growth

retardation

no association of maternal dosage in

last trimester of pregnancy with length

of gestation, birth wt, or withdrawal

syndrome; identical infant mortality

rate

increased infant mortality, decreased

birth wt. increased incidence of breech

presentation

both methadone and morphine resulted

in fetal arousal and suppressed

quiet sleep

maternal to fetal free drug concentration ratios were 7.6 for morphine and

2.9 for methadone; clearance from

mother to fetus was 24.9 ml/min for

morphine and 390 ml/min for methadone;

fetal nonplacental clearance was

126 ml/min for morphine and 381 ml/min

for methadone

Abrams 1975

Kandall 1975

Newman et al.

1975

Harper et al. 1974

Umans & Szeto 1983

Szeto et al.

1982
Sheep

Hamster

Rat

Rat

Rat

Rat

Rat

Rat

5-20 mg, single dose

injection to fetal

circulation, 145 d of

gestation

35 mg/kg, SC, during

critical period of

CNS organogenesis

5 mg/kg/d, methadone,

IP, throughout gestation

and/or lactation

0.2 or 2.0 mg/kg/d, PO,

1 mo prior to and

throughout pregnancy

10 mg/kg/d, methadone

PO, 5th d of gestation to term

0, 4, 16 mg/kg/d, SC,

8th d of gestation

to term

0.5-2.0 mg/kg/d, PO,

LAAM, chronic dosing

5 mg/kg/d, methadone,

IP, 5 d before breeding

& throughout gestation

and lactation

decreased carotid blood flow, decreased

frequency and amplitude in EEG;

electrical activity and heart rates

were affected

congenital malformation of CNS,

fetal anomalies

reduced social dominance in offspring

of mothers given methadone

significant cumulation of LAAM and

active metabolites in the whole fetus

and fetal brain

decreased brain levels of monoamine

and metabolites; methadone-exposed

rats exhibited more avoidance escapes

and intertrial shuttles

low body wt. high mortality, depressed

open-field activity, deficits in

behavioral development

cumulative behavioral toxicity

abnormally greater wt gain during

the postweaning period

Mann et al. 1976

Geber & Schramm

1975

Thompson & Zagon

1983

Lichtblau et al.

1982

Rech et al. 1980

Freeman 1980

Lichtblau et al. 1980

McLaughlin & Zagon

1980
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Rat 5 mg/kg/d, methadone, SC, decreased fetal testosterone and

14-19 d of gestation androstenedione level

Rat 14 mg/kg/d, methadone long-lasting growth retardation and

PO, ad lib, throughout less emotionality in open-field

gestation trials

Rat l-5 mg/kg, methadone,

SC, bid, last trimester

of pregnancy

reduction of body and brain growth

Rat 5 mg/kg, methadone, IP,

during gestation and/or

lactation

lower body wt, shorter crown to rump

length & tail length, & smaller

diameter; detrimental effect on preweaning organ and body development

Rat

Rat

Rat

5 mg/kg/d, methadone,

IP, during gestation &

lactation

2.0-8.0 mglkgld,

methadone, IM or SC,

15-21 d of gestation

5 mg/kg/d, methadone,

initially increased

to 10, 15. & 20 mg/kg/d,

PO, 8-22 d of gestation

reduction of maternal wt, lower

cerebellar wt & smaller width

of the offspring, retarded growth

and brain development of young rats

localization of methadone in fetal

rat eye

blood levels were dose related;

reduced maternal wt, increased

mortality for the mother & the young

Singh et al. 1980

Grove et al. 1979

Ford & Rhines 1979

McLaughlin et al.

1978

Zagon & McLaughlin

1977

Pertschuck et al.

1977

Hutchings et al.

1976
Rat

Rat

Rat

Mouse

Mouse

5-20 mg/kg/d, methadone, chronic fetal methadone intoxication; McDonald et al.

SC, 30 d before breeding increased neonatal death rate, 1975

& during pregnancy but no evidence of development

difficulty to 21 days of age

25 mg/kg/d, methadone, fetal growth retardation, possible Chandler et al.

oral gavage throughout positional malformation 1975

gestation

2.5, 5.0, 7.5 mg/kg/d, increased infant mortality, increased Buchenauer et al.

methadone, IP, 1 wk percentage of stillborn pups, 1974

before breeding retardation of growth, obvious

dose-response relationship at low dose

5-28 mg/kg/d, methadone, reduced maternal wt gain and food Bui et al. 1983

SC, 6-15 d of gestation consumption, increased resorptions, and

decreased survival rate; adverse effects

on growth development

0.8, 5 mg/kg, 3H-l- T max=15 min in whole fetus, fetal Shah et al. 1976

methadone, IP, brain, & liver; fetal brain conc were

15-17 d of gestation higher than maternal brain conc after

15 min

Drug Category: MORPHINE/HEROIN

Human

Human

4-6 mg, morphine, newborn had morphine-induced respira- Carlsson et al.

epidural injection tory depression; morphine was inef- 1981

in labor fective as an obstetric analgesic

pregnant heroin elevation of chromosomal breakage, mi- Van Blerk et al.

addicts tosis depression, chromosomal abberation 1980
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Human narcotic (heroin and increased pre- & post-natal growth

methadone) dependent deficiency, vascular changes,

women & hypoxic episodes

Human pregnant drug abusers

& narcotic (heroin &

methadone) dependent

women

mentally & neurologically deficient

infants. high morbidity & mortality

among newborn infants

Human 0.1 mg/kg, morphine

or heroin, IV, in

amniotomy examination

Human heroin addicts

decreased PH & increased pC02

In mother; decreased pH & base

excess in the fetus

withdrawal symptoms in infants, small

body size & circumference. intrauterine

growth retardation

Sheep 5 mg, morphine, epidural

injection, during labor

Sheep 0.66 mg/kg, morphine

Injection, to ewe's flank

near term

Rabbit 2.5-10 mg/kg, morphine,

every 6 hr. SC, from

early pregnancy till

delivery

no adverse effect in mother or fetus

altered fetal glucose & homeostasis,

decreased permeability of the

placenta to glucose

fetus weighed significantly less

than controls; morphine did not

accelerate fetal lung development

Finnegan 1979a

Finnegan 1979b

Chang et al.

1976

Vargas et al.

1975

Craft et al.

1982

Raye et al.

1980

Roloff et al.

1975
Rat

Rat

Rat

Rat

Rat

Rat

Mouse

2.5 mg/kg, morphine, IV fetal morphine conc was 1.5 times higher

bolus, or 23.3 ug/min/kg than that of maternal plasma; fetal

for 30 min, then brain conc was 4 times higher than that

3.5 ug/min/kg for 30 min. maternal plasma

18-20 d of gestation

20 mg/kg/d morphine, IV,

d 12 till end of

gestation

10 mg/kg, morphine,

5 mg/kg, naloxone, IP,

before & during

pregnancy

10 mg/kg. morphine, SC,

during gestation

5 mg/kg/hr for 4 hr,

morphine. IV infusion,

21-22 d of gestation

5 mg/kg/hr for 4 hr,

morphine, IV infusion,

near term

0.12-12 mg, morphine

SC; 0.068-6.5 mq/min.

IV infusion; 7-10 d

of pregnancy

fetus showed morphine-induced depression & later developed tolerence to

depressant effect in utero

altered regional development of

3

H-methionine binding sites in the

brains of offspring; affected the

development of opiate receptors in brain

an enhanced prostaglandin-like activity

in the homogenate of rat placenta

caused a derangement of cerebral

protein synthesis, possibly by

interfering with the availability

of m-RNA

inhibitory effect on transcription

in fetal brain resulting from intranuclear accumulation of morphine

fetal wt reduction, skeletal & soft

tissue abnormalities

Grabrielsson

& Paalzow

1983

Kirby &

Holtzmann

1982

Tsang & Nq

1980

Scoto et al.

1979

Steele &

Johannesson

1975a

Steele &

Johannesson

1975b

Ciociola &

Gautieri

1983
Model/ Dose/Route/Time

Preparation of Exposure

Human 1 pmole of 3

H-etorphine

placenta/ incubated with placenta

Rat brain mix and 0.8 mg brain

protein protein at 37°C, 1/2 hr

Human 0.4-1.0 mole, morphine

placenta incubated with acetylcholine, 37oC, 2 hr

Monkey/ 7.25-144 umole, ethylliver morphine incubated in

homogenate fetal homogenate, 12-16

wk of gestation

Drug Category: NICOTINE

Human pregnant female smokers

Human nursing female mokers

Human female smokers in 2nd

or 3rd trimester

Kinetic/Dynamic Effects Reference

human placenta exhibited lower

affinity for opioid peptide than did

rat brain opiate receptor

Valette et al.

1980

morphine reduced 3H-acetylcholine

uptake

Welsch 1976

N-demethylation of fetal liver was

l/10 of maternal liver & adult liver

Dvorchik et al.

1974

atropic and hypovascular changes in Mochizuki

placental villi, impairment of utero- et al. 1984

placental circulation, retarded fetal

growth

milk-serum conc ratio 2.92; t1/2

of nicotine in milk slightly

exceeded that in serum

Luck & Nau

1984

fetal hypoxia, elevated levels of Divers et al.

epinephrine, norepinephrine, and DOPEG 1981

in amniotic fluid
Human

Human

Human

Monkey

Monkey

Sheep

Sheep

Rat

pregnant female smokers

habitual smoking during

last trimester

pregnant female smokers

100 ug/kg/min, 20 min.

IV infusion, near term

1 mg/kg as single

injection or infused

over 20 min, 107-150 d

of gestation

15 mg/10 min, IV

infusion, 97-117 d

of gestation

0.14-0.25 mg/kg, IV

or IA, 110-120 d

of gestation

2.46 mg/d in drinking water (120 mg/l)

throughout pregnancy

infants with low birth wt, congenital

anomalies, or who died

increase of apnea and periodic

breathing movement

smaller baby, greater incidence of

premature baby abortion & stillbirth

decreased uterine blood flow, fetal

acidosis & hypoxia

disappearance of nicotine from fetal

circulation was slower than maternal

circulation; high conc in adrenal

glands, heart, kidneys, stomach wall,

and spleen of fetus

increased maternal arterial pH & blood

pressure, increased maternal and fetal

heart rates, decreased fetal arterial

PCO2 & uterine blood flow

decreased fetal PO2, maternal blood

flow, & fetal breathing movement;

fetal hypoxemia

less maternal wt gain, higher fetal

body fat, higher maternal lipolysis

rate

Weathersbee &

Lodge 1979

Gennser et al.

1975

Garrett 1975

Suzuki et al.

1981

Suzuki et al.

1974

Ayromlooi et al.

1981

Manning et al.

1978

Williams &

Kanagasabai 1984
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Rat

Rat

Rat

Rat

Rat 1.5-3.0 mg/d, SC. 1 wk

prior to mating &

continued till

parturation

15-90 mg/30 cigarette

inhalation, bid,

throughout gestation

5 mg/kg, SC, d 9 of

gestation

0.2 mg, SC, 1-8 d of prolonged gestation period, decreased

gestation fetal number & wt

6.03 mg/kg/d, PO,

O-20 d of pregnancy;

1 mg/kg, IP, single dose

d 20 of pregnancy

no difference in fetal level of

triglyceride, phospholipid, and

cholesterol compared with control

group

Mouse 5.7-28.6 mg/d in drinking

water (100 mg/l), 2 wk

before breeding &

throughout gestation

fetus weighed 12% less than control,

decreased placental wt, decreased

placental amino acid transport

no difference in wt gain of mother

and birth wt of offspring; neurological effect on offspring, prolonged

learning time

reduced maternal wt. reduced wt &

length of fetus, resorption, and

stillbirth

no significant adverse effects in

embryonic development and fetal

viability

Peters & Ngan

1982

Reznik & Marquard

1980

Lindenschmidt &

Persaud 1980

Biswas et al.

1977

Mosier et al.

1974

Rowell & Clark

1982
Drug Category: PHENCYCLIDINE

Human

Human

Human

Human

Human

Pig

Pig

Rat

PCP daily, prior to

and during pregnancy

case study of PCP

abusers

PCP during first 6 wk

of pregnancy

irregular consumption

of PCP prior to and

during pregnancy

PCP abuse during

pregnancy

2 mg/kg, IM, single

dose before general

anesthesia for delivery

0.5 g, IM, before

general anesthesia

for delivery

l0-40 mg/kg/d, IP,

15-20 d of gestation

sudden outbursts of agitation, rapid

changes in level of consciousness,

increased lability of states, and poor

consolability

presence of PCP in breast milk and

amniotic fluid

cerebellar malformation

Neonates showed jitteriness, hypertonicity, vomiting,diarrhea,

microcephaly

abnormal neonatal behavior,

dysmorphology, spasticity

PCP in piglet plasma 10 times higher

than that in maternal sow plasma,

t1/2 =6 hr in piglet, t1/2=2 hr

in adult sow

PCP in piglet plasma 10 times higher

than maternal plasma, t1/2=lO-12 hr

in piglet, t1/2 = 2-4 hr in mother

morphological & behavioral alterations

Chasnoff et al.

1983

Kaufman et al.

1983

Michaud et al.

1982

Strauss et al.

1981

Golden et al.

1980

Cummings et al.

1979

Cooper et al.

1977

Jordan et al.

1979
Model/ Dose/Route/Time Kinetic/Dynamic Effects Reference

Preparation of Exposure

Mouse 5, 10, 20 mg/kg/d. PO, delayed disappearance of cross Nicholas & Schreiber

5 d prior to mating & extensive reflex, delayed appearance 1983

throughout gestation of reflexes, decreased growth rate

Mouse 60-120 mg/kg/d, PO, malformed fetuses at 120 mg/kg/d, Marks et al. 1980

6-15 d of gestation but not teratogenic

Human 0.5 mM PCP incubated placenta was demonstrated to be an Rayburn et al. 1984

Placenta with 0.02-0.1 mg active site of PCP biotransformation

microsomal protein for

20 min, pH=8.8, T=37°C

Keys: bid=twice daily; conc=concentration; d-day; gr=group; hr=hour; IA=intraarterial; IM-intramuscular;

IP=intraperitoneal; IV=intravenous; min=minute; mo=month; PP=per OS; qd=once daily; SC-subcutaneous;

wk=week; wt=weight
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146
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Charles C. Lee, Ph.D.

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C. Nora Chiang, Ph.D.

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National Institute on Drug Abuse

5600 Fishers Lane

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NCDAI out of stock

GPO Stock #017-024-01151-7 $5 NTIS PB #251 905/AS $14.50

8 Rx: 3x/WEEK LAAM - ALTERNATIVE TO METHADONE. Jack Blaine, M.D.,

and Pierre Renault, M.D., eds.

Not available from GPO NTIS PB #253 763/AS $14.50

9 NARCOTIC ANTAGONISTS: NALTREXONE PROGRESS REPORT. Demetrios

Julius, M.D., and Pierre Renault, M.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #255 833/AS $17.50

10 EPIDEMIOLOGY OF DRUG ABUSE: CURRENT ISSUES. Louise G. Richards,

Ph.D., and Louise B. Blevens, eds. NCDAI out of stock

GPO out of stock NTIS PB #266 691/AS $22

11 DRUGS AND DRIVING. Robert Willette, Ph.D., ed. NCDAI out of

stock

GPO Stock #017-024-00576-2 $5.50 NTIS PB #269 602/AS $16

12 PSYCHODYNAMICS OF DRUG DEPENDENCE. Jack D. Blaine. M.D., and

Demetrios A. Julius, M.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 084/AS $17.50

13 COCAINE: 1977. Robert C. Petersen, Ph.D., and Richard C.

Stillman. M.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #269 175/AS $19

14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

ed. NCDAI out of stock

GPO out of stock NTIS PB #271 279/AS $22

15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wm. Sharp,

Ph.D., and Mary Lee Brehm, Ph.D., eds.

GPO out of stock NTIS PB #275 798/AS $28

16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne

Rittenhouse. Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #276 357/AS $20.50

149
17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik, M.D., Ph.D., et

al., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 353/AS $29.50

18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

Norman A. Krasnegor, Ph.D., ed.

GPO out of stock NTIS PB #276 337/AS $16

19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Petersen,

Ph.D., ed.

GPO out of stock NTIS PB #293 807/AS $28

20 SELF-ADMINISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

Norman A. Krasnegor, Ph.D., ed.

GPO out of stock NTIS PB #288 471/AS $22

21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen,

Ph.D., and Richard C. Stillman, M.D., eds.

GPO out of stock NTIS PB #288 472/AS $25

22 QUASAR: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS OF

ANALGESICS, NARCOTIC ANTAGONISTS, AND HALLUCINOGENS. Gene Barnett,

Ph.D.; Milan Trsic. Ph.D.; and Robert Wlllette, Ph.D.; eds. NCDAI

out of stock

GPO out of stock NTIS PB #292 265/AS $35.50

23 CIGARETTE SMOKING AS A DEPENDENCE PROCESS. Norman A. Krasnegor,

Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-00895-8 $6 NTIS PB #297 721/AS $19

24 SYNTHETIC ESTIMATES FOR SMALL AREAS: STATISTICAL WORKSHOP PAPERS

AND DISCUSSION. Jos. Steinberg, ed. NCDAI out of stock

GPO out of stock NTIS PB #299 009/AS $23.50

25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE ABUSE. Norman A.

Krasnegor, Ph.D., ed.

GPO ouf of stock NTIS PB #80-112428 $22

26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

ed.

GPO out of stock NTIS PB #80-118755 $17.50

27 PROBLEMS OF DRUG DEPENDENCE, 1979: PROCEEDINGS OF THE 41ST

ANNUAL SCIENTIFIC MEETING, THE COMITTEE ON PROBLEMS OF DRUG DEPENDENCE, INC. L.S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-00981-4 $9 NTIS PB #80-175482 $37

28 NARCOTIC ANTAGONISTS: NALTREXONE PHARMACOCHEMISTRY AND

SUSTAINED-RELEASE PREPARATIONS. Robert Wlllette, Ph.D., and

Gene Barnett, Ph.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #81-238875 $23.50

150
29 DRUG ABUSE DEATHS IN NINE CITIES: A SURVEY REPORT. Louis A.

Gottschalk, M.D., et al. NCDAI out of stock

GPO Stock #017-024-00982-2 $6.50 NTIS PB #80-178882 $17.50

30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES. Dan

J. Lettieri, Ph.D.; Mollie Sayers; and Helen Wallensteln Pearson,

eds. NCDAI out of stock

GPO Stock #017-024-00997-1 $10 Not available from NTIS

31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen. Ph.D.,

ed.

GPO out of stock NTIS PB #80-215171 $20.50

32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz,

Ph.D.; Allison F. Fentiman, Jr., Ph.D.; and Ruth B. Foltz.

GPO out of stock NTIS PB #81-133746 $19

33 BENZODIAZEPINES: A REVIEW OF RESEARCH RESULTS, 1980. Stephen I.

Szara, M.D., D.Sc., and Jacqueline P. Ludford. M.S., eds.

GPO Stock #017-024-01108-8 $5 NTIS PB #82-139106 $13

34 PROBLEMS OF DRUG DEPENDENCE, 1980: PROCEEDINGS OF THE 42ND

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #81-194847 $34

35 DEMOGRAPHIC TRENDS AND DRUG ABUSE, 1980-1995. Louise G.

Richards, Ph.D., ed.

GPO out of stock NTIS PB #82-103417 $13

36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS. Lorenz K.Y. Ng, M.D.,

ed.

GPO out of stock NTIS PB #81-240913 $19

37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds.

GPO Stock #017-024-01109-6 $7 NTIS PB #82-136961 $25

38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettierl, Ph.D.,

and Jacqueline P. Ludford, M.S., eds.

GPO Stock #017-024-01107-O $4.50 NTIS PB #82-148198 $14.50

39 YOUNG MEN AND DRUGS IN MANHATTAN: A CAUSAL ANALYSIS.

Richard R. Clayton, Ph.D., and Harwin L. Voss, Ph.D.

GPO out of stock NTIS PB #82-147372 $19

40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

Hendin, M.D., Ann Polllnger, Ph.D., Richard Ulman, Ph.D., and

Arthur Carr, Ph.D. NCDAI out of stock

GPO out of stock NTIS PB #82-133117 $13

151
41 PROBLEMS OF DRUG DEPENDENCE, 1981: PROCEEDINGS OF THE 43RD

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

Not available from GPO NTIS PB #82-190760 $41.50

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS. Richard L.

Hawks, Ph.D., ed.

GPO Stock #017-024-01151-7 $5 NTIS PB #83-136044 $16

43 PROBLEMS OF DRUG DEPENDENCE, 1982: PROCEEDINGS OF THE 44TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #83-252-692/AS $40

44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE SYSTEMS.

Monique C. Braude, Ph.D., and Jacqueline P. Ludford. M.S., eds.

GPO Stock #017-024-01202-5 $4 NTIS PB #85-150563/AS $14.50

45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983. Roger M.

Brown, Ph.D.; Theodore M. Plnkert. M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds.

GPO Stock #017-024-01191-6 $2.75 NTIS PB #84-184670/AS $11.50

46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT. John

Grabowski, Ph.D.; Maxine L. Stitzer, Ph.D., and Jack E.

Henningfield, Ph.D., eds.

GPO Stock #017-024-01192-4 $4.25 NTIS PB #84-184688/AS $16

47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION STRATEGIES.

Thomas J. Glynn. Ph.D.; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S., eds.

GPO Stock #017-024-01180-1 $5.50 NTIS PB #85-159663/AS $22

48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING BEHAVIOR.

John Grabowski, Ph.D., and Catherine S. Bell, M.S., eds.

GPO Stock #017-024-01181-9 $4.50 NTIS PB 84-145184 $14.50

49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 45TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01198-3 $12 NTIS PB 85-151553/AS $22

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski. Ph.D., ed.

GPO Stock #017-020-01214-9 $4 NTIS PB 85-150381/AS $14.50

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed. NCDAI out of stock

GPO Stock #017-020-01218-1 $4.50 NTIS PB 85-150365/AS $17.50

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

GPO Stock #017-024-01204-1 $4.25 NTIS PB 85-150373/AS $16

152
54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D., ed.

GPO Stock #017-024-01213-l $8.50

55 PROBLEMS OF DRUG DEPENDENCE, 1984. PROCEEDINGS OF THE 46TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01242-4 $9.50

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION. Coryl LaRue

Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

GPO Stock #0l7-024-01250-5 $6.50

57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D., Nicholas J.

Kozel, M.S., and Louise G. Richards, Ph.D., eds.

GPO Stock #0l7-024-01246-7 $4.25

58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG

ABUSERS. Rebecca S. Ashery, D.S.W., ed.

GPO Stock #017-024-01247-5 $4.25

59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

Theodore M. Pinkert, M.D., J.D., ed.

GPO Stock #017-024-01249-1 $2.50

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora Chlang.

Ph.D., and Charles C. Lee, Ph.D., eds.

IN PRESS

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John

Grabowski, Ph.D., ed.

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL

PERSPECTIVES. Nicholas J. Kozel, M.S., and Edgar H. Adams, M.S.,

eds.

62 THE NEUROSCIENCE OF DRUG ABUSE. Roger M Brown, Ph.D., and

David P. Friedman, Ph.D., eds.

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG CHILDREN AND

ADOLESCENTS. Catherine S. Bell, M.S.; and Robert J. Battjes,

D.S.W., eds.

153

U.S. GOVERNMENT PRINTING OFFICE : 1985 O - 483-942 : QL 3
DHHS Publication No. (ADM) 85-1413

Printed 1985