1 the Maternal and Neonatal Disposition of Pethidine And

Home , Orphenadrine, Pethidinic Acid

THE MATERNAL AND NEONATAL DISPOSITION OF

PETHIDINE AND BUPIVACAINE ADMINISTERED IN

CHILDBIRTH

Submitted by

Lidia Josephine Notarianni M.Sc.

for the degree of Doctor of Philosophy

of the University of London

1979

Department of Biochemical Pharmacology St. Mary's Hospital Medical School Paddington, London W.2

"Attention is drawn to the fact that copyright of this thesis rests with

its author. This copy of the thesis has been supplied on condition that

anyone who consults it is understood to recognise that its copyright

rests with its author and that no quotation from the thesis and no

information derived from it may be published without the prior consent

of the author."

This thesis may be photocopied, microfilmed or lent to other libraries

for the purpose of consultation 2

To my parents with love and gratitude "Let it, then be distinctly understood, that

the popular unprofessional use of ether and

chloroform is both immoral and injurious; that

it is highly dangerous and may produce death."

(W. Charming; in 'A Treatise on Etherization in

Childbirth' 1848). 4

CONTENTS

Page

Abstract 10

Acknowledgements 12

List of figures 13

List of tables 18

Chapter One. Perinatal pharmacology and obstetric analgesia

1.1 Introduction 23

1.1.1 Historical background 23

1.1.2 Analgesic drugs used in obstetric practice 25

1.2 The passage of maternally administered drugs to 26

the fetus 26

1.2.1 Drugs and the fetus 27

1.2.2 Placental structure and transplacental transfer 35

1.2.3 Drug distribution in the fetus 41

1.2.4 Pharmacokinetics of placental transfer at term 46

1.3 Perinatal drug metabolism 52

1.3.1 General metabolism 52

1.3.2 Drug metabolism during pregnancy 54

1.3.3 Drug retabolism by the placenta 58

1.3.4 Drug metabolism by the fetus 60

1.3.5 Drug metabolism by the neonate 63

1.4 Obstetric analgesia 69

1.4.1 Pethidine 70

1.4.2 Xylidide local anaesthetics 75

1.5 Scope of the present work 89 5

Page

Chapter Two. Materials and methods (bupivacaine) 92

Compounds 92

Syntheses 92

Animals 97

Preparation of rats with biliary fistulae 97

Human volunteers 98

Storage of biological samples 98

Spectra 99

Radiochemical techniques 99

Isotope dilution procedures 100

Chromatography 102

Visualization of compounds 103

Treatments of urine prior to analysis 104

Concentration of urinary metabolites 105

Gas-liquid chromatography (GC) 105

Mass spectrometry (MS) 105

Gas chromatography - mass spectromety (GC-MS) 106

Estimation of bupivacaine by GC and GC-MS 106

Extraction of urinary metabolites 108

Chapter Three. Materials and methods (pethidine) 111

Compounds 112

Syntheses 112

Animals 113

Administration of compounds 114

Collection of urine and faeces 114

Collection of 14CO2 in expired air of rodents 114 6

Page

Human volunteers 115

Administration of norpethidine and pethidinic acid 116

Storage of excreta 116

Collection of 14CO2 in expired air of humans 116

Radiochemical techniques 116

Quantitation of 14C in animal carcasses 117

Thin layer chromatography 117

Gas-liquid chromatography (GC) 117

Mass spectrometry (MS) 119

Gas chromatography - mass spectrometry (GC-MS) 119

Acetylation of metabolites 119

Estimation of pethidine and norpethidine by GC 119

Estimation of total pethidinic and norpethidinic acids 120 by GC

Estimation of free pethidinic and norpethidinic acids 121

Estimation of pethidine-N-oxide 122

General procedure for the separation, identification and 122 quantitation of metabolites

Chapter Four(a) The maternal and neonatal disposition of

bupivacaine administered during childbirth 125

Introduction 126

Subjects, drug administration and sample collections; 126

Results 127

(i)Mernal and neonatal cord levels at delivery 127

(ii)I`eonatal elimination 132

Discussion 134 7

Page

Chapter Four(b) The maternal and neonatal disposition

of pethidine administered in childbirth

Introduction 144

Subjects, drug administration and sample collection 144

Results 145

(i)maternal and neonatal cord levels at delivery 145

(ii)N.eonatal elimination 150

Discussion 153

Comparison of the placental transfer of bupivacaine 161 and pethidine

Chapter Five The effects of maternal analgesia (pethidine

and bupivacaine) on the fetus and neonate 165

Introduction 166

Patients and methods 166

Selection of population studied 166

Obstetric management 166

Assessment of neonate 167

Results 168

Effect of maternal analgesia on the fetal cardio- 168 vascular system

Effect of maternal analgesia on neonatal behaviour 170

Discussion 175

Implications of the results 177

Chapter Six The metabolism of bupivacaine in rat and man 182 183 Introduction

Identification of metabolites 183

Rat urine 183 8

Page

Sequential extraction of rat urine 184

Rat bile 191

Human urine 194

Sequential extraction of human urine 197

Results 199

Rat urine 199

Rat bile 203

Human urine 208

Discussion 213

Chapter Seven The identification of urinary metabolites of

pethidine in adult volunteers 227

Introduction 228

Analysis of urinary metabolites 230

Results 241

Discussion 248

Chapter Eight The metabolism of pethidine in adult volunteers

and human neonates 253

Introduction 254

Analysis of urinary metabolites 254

Adult panel study 254

Neonatal study 255

Results 255

Adult panel study 255

Neonatal study 262

Discussion 267

Page Chapter Nine Species variation in the metabolism of

pethidine 279

Introduction 280

Analysis of urinary metabolites 280

Non-human primates 280

Sub-primate mammals 281

Results 282

Non-human primates 282

Sub-primate mammals 286

Discussion 289

*References 303

Appendix A: Formulae for the dioxan and Triton based 294 scintillants used in the study.

Appendix B(1): Maternal, umbilical and neonatal blood 295 concentrations of bupivacaine; neonatal blood elimination half-life;maternal dose and last dose-delivery interval for all individuals in the bupivacaine study.

Appendix B(2): Maternal, umbilical and neonatal blood 298 concentrations of pethidine; neonatal blood elimination half-life; maternal dose and last dose-delivery interval for all individuals in the pethidine study.

Appendix C: Estimation of total drug exposure of the 301 fetus/neonate to maternally administered pethidine or bupivacaine.

Appendix D: Estimation of rate constants for the elimination of pethidine and metabolites. 302

*The first citation of any work in this thesis will be referred to in the text by all authors names and the year of publication. Subsequent references to the same work will only include the first author and year of publication. 10

ABSTRACT

(1) The maternal and neonatal disposition of bupivacaine (n=58) and pethi-

dine(n=51)administered to the mother during childbirth has

been studied.

(2) Both drugs readily pass the placenta, pethidine more extensively

than bupivacaine, and at birth the neonatal body burden of the

drug used depends partly on the time interval between the last

maternally administered dose and delivery.

(3) The infant eliminates both these drugs very slowly which,

in the case of pethidine at least, is largely due to impaired

metabolism since the blood elimination half-life of the drug is

increased by a factor of 6.1 and pethidine metabolism impaired

by a factor of 9.1 compared to the adult.

(4) The judicious use of bupivacaine and pethidine in childbirth

does not have detrimental effects on fetal physiology, but

both drugs have been shown to affect neonatal behaviour for

at least the first six weeks of life.

(5) The metabolism of bupivacaine in rat and man has been studied.

The major metabolic pathway in the rat is aromatic hydroxylation

yielding 2 isomeric phenolic metabolites of bupivacaine. In

man, the major metabolites have not been elucidated, and although

the results suggest that they are phenolic in nature, they are not 3' and 4'-hydroxybupivacaine. 11

(6) The metabolism of pethidine in human adults has been investigated

using a mixture of [ 14C] /[2H5] -pethidine to aid mass spectrometric ident-

ification- of the metabolites. The major metabolic routes of

pethidine in man are ester hydrolysis and N-demethylation

yielding pethidinic and norpethidinic acids and norpethidine.

Two minor metabolites, 4'-hydroxypethidine and pethidine-N-oxide

have been quantitated for the first time.

(7) The metabolism of pethidine in a small group (15) of adults

has been studied which has shown large inter-individual diff-

erences in its metabolism. Ester hydrolysis and N-demethylation

are competing pathways, and N-demethylation is apparently not

under the same genetic control as the aromatic, alicyclic and

aliphatic carbon oxidation of drugs such as guanoxan, phenacetin

and debrisoquine.

(8) The metabolism of pethidine in 5 neonates was studied showing

impaired metabolic ability and evidence of differential onto-

genesis of the mixed function microsomal oxidase system was

obtained.

(9) Inter-species variation in the metabolism of pethidine was

studied in 4 species of non-human primates and 3 species of

sub-primate mammals. Due to the large inter-individual diff-

erences in metabolism observed in humans, no one species was a

consistently good metabolic model for man. 12

ACMNOWLEDGEMENTS

I would like to take this opportunity to express my gratitude to Professor

Robert Smith and Dr. John Caldwell for their invaluable advice and guidance

throughout this work and their useful suggestions and comments on the

contents of this thesis. My thanks are also due to my colleagues and

technical staff of the Pharmacology Department, Professor Ted Hawes and

Mr. Lawrence Wakile for their assistance in the pethidine assay, the

Biophysics Department for making available the mass spectrometry facility

and in particular Mr. Graham Correy for his help in obtaining the mass

spectra. My gratitude also extends to all those too numerous to mention

by name who were involved in the obstetric/neonatal study for the collection

of blood and urine samples and the testing of babies at anti-social hours

which made this study possible.

Finally, but most of all, I wish to thank my parents for their love and support throughout my long education ( I hope they are not too disappointed by the result ) and my f iancē Robin for his constant help and encouragement during this work and the writing of this thesis.

This work was supported by a grant from Duncan Flockhart and Co. Ltd. 13

LIST OF FIGURES

Page

Chapter One

Fig. 1.1 Organ development and drug side-effects. 31

Fig. 1.2 The mature placenta. 37

Fig. 1.3 Changes in the circulation at birth. 44

Fig. 1.4 Drug disposition in a model of the maternal- 47

placental-fetal unit.

Fig. 1.5 Pain pathways in labour in relation to conduction 79

block.

Fig. 1.6 Blood concentrations of bupivacaine in mothers and 81

their fetuses following the epidural injection of

bupivacaine to the mothers during labour.

Fig. 1.7 Possible biotransformation scheme for lignocaine 86

in rat, guinea-pig, dog and man.

Chapter Two

Fig. 2.1 Synthesis of1 [ 4Cj-bupivacaine HC1. 93

Fig. 2.2 Analysis of bupivacaine and metabolites in urine. 110

Chapter Three

Fig. 3.1 Analysis of urinary metabolites of pethidine. 124

Chapter Four

Fig. 4.1 Umbilical artery/umbilical vein concentration ratios

for bupivacaine against last dose-delivery interval. 129

Fig. 4.2 Semi-logarithmic plot of bupivacaine concentration 130

in maternal blood at delivery against last dose-

delivery interval.

Fig. 4.3 Umbilical artery/maternal vein concentration ratios for

bupivacaine against last dose-delivery interval. 131 14

Page

Fig. 4.4 Representative semi-logarithmic plots of 132

bupivacaine blood concentration against time

from two babies whose mothers received bupi-

vacaine in labour.

Fig. 4.5 Semi-logarithmic plot of pethidine concentration 147

in maternal blood at delivery against last dose-

delivery interval.

Fig. 4.6 Umbilical artery/maternal vein concentration ratios 148

for pethidine against last dose-delivery interval.

Fig. 4.7 Umbilical artery/umbilical vein concentration 149

ratios for pethidine against dose-delivery

interval.

Fig. 4.8 Representative semi-logarithmic plot of pethidine 151

blood concentration against time from a baby whose

mother received pethidine in labour.

Fig. 4.9 Proposed transplacental passage of pethidine and 163

bupivacaine at delivery.

Chapter Five

Fig. 5.1 Effect of pethidine and bupivacaine on infant 173

behaviour.

Fig. 5.2 Effect of drug exposure on infant behaviour. 174

Chapter Six

Fig. 6.1 Radiochromatogram of O-24h rat urine following[ 14C] -

bupivacaine HC1 (30mg/kg; 20pCi/kg) i.p., in 185

system B.

Fig. 6.2 Radiochromatogram of acid treated 0-24h rat urine 185

following C 14C-bupivacaine] HC1 (30mg/kg; 20.Ci/kg) i.p., in system B. 15

Page

Fig. 6.3a Mass spectrum of desbutyl-bupivacaine via 187

GC-MS, retention time 2.2 minutes.

Fig. 6.3b Mass spectrum of peak 1 from the pH 14 extract 187

from rat urine, GC-MS retention time 2.2

minutes.

Fig. 6.4a Mass spectrum of bupivacaine via GC-MS, retention 188

time 3.5 minutes.

Fig. 6.4b Mass spectrum of peak 2 from the pH 14 extract 188

from rat urine, GC-MS retention time 3.5 minutes.

Fig. 6.5 W spectra of 'phenolic' metabolites 1 and 2 190 isolated from rat urine.

Fig. 6.6a Mass spectrum of 'phenolic' metabolite 1 isolated 192

from rat urine proposed to be 4'-hydroxybupivacaine.

Fig. 6.6b Mass spectrum of 'phenolic' metabolite 2 isolated 192

from rat urine proposed to be 3'-hydroxybupivacaine.

Fig. 6.7a Mass spectrum of 'phenolic' metabolite 1 from rat 193

urine following derivatization with BSA.

Fig. 6.7b Mass spectrum of 'phenolic' metabolite 2 from rat 193

urine following derivatization with BSA.

Fig. 6.8 Radiochromatogram of 0-24h rat bile following 195 [14 C]-bupivacaine HC1 (30mg/kg; 2OUCi/kg), i.p.,

in system B.

Fig. 6.9 Radiochromatogram of acid treated 0-24h rat bile 195

following[ 14C]-bupivacaine HC1 (30mg/kg; 20pCi/kg)

i.p., in system B.

Fig. 6.10 Radiochromatogram of 0-24h human urine following 196 14_1- C] -bupivacaine HCl (50mg; 3.8iCi) i.m. in

system B. 16

Page

Fig. 6.11 Radiochromatogram of acid treated 0-24h human 196

urine following [ 14CJ-bupivacaine (50mg; 3.8pCi)

i.m. in system B.

Fig. 6.12 Proposed mass spectrometry fragmentation pattern 204 for bupivacaine. Fig. 6.13 Proposed mass fragmentation of 3' and 4'-hydroxy- 205

bupivacaine and their TMS derivatives.

Fig. 6.14 Elimination of 14C in adult volunteers JC and 209

EMH following the administration of [ 14C]-bupi-

vacaine HCl.

Fig. 6.15 Oxidative metabolic pathways of naphthalene. 219

Fig. 6.16 Proposed N-OH intermediate in the formation of 220

4'-hydroxybupivcaine.

Fig. 6.17 A schematic representation of the proposed 222

origins of the 3' and 4'-hydroxy metabolites of bupivacaine in the rat.

Fig. 6.18 Metabolic pathways of bupivacaine in the rat. 226

Chapter Seven

Fig. 7.1a Mass spectrum of a 1:1 mixture of pethidine and 231

[2 H5]-pethidine.

Fig. 7.1b Proposed mass spectrometry fragmentation pattern for pethidine. 232 Fig. 7.2a Mass spectrum of pethidine via GC-MS, retention

time 2.1 minutes. 236

Fig. 7.2b Mass spectrum of peak 1 of pH 14 extract from human

urine, GC-MS retention time 2.1 minutes. 236

Fig. 7.3a Mass spectrum of norpethidine via GC-MS, retention 237

time 1.8 minutes.

Fig. 7.3b Mass spectrum of peak 2 of pH 14 extract from human 237

urine; GC-MS retention time 1.8 minutes. 17

Page

Fig. 7.4a Mass spectrum of 4'-hydroxypethidine via 238

GC-MS, retention time 4.9 minutes.

Fig. 7.4b Mass spectrum of acetylated 4'-hydroxypethidine 238

via GC-MS, retention time 4,9 minutes.

Fig. 7.5 ' Mass spectrum of pethidine-N-oxide via GC-MS, 239

retention time 2.0 minutes.

Fig. 7.6 Elimination of 14C by adult volunteer JC following 243

i.m. administration of [ 14C] /[ 2H5] - pethidine HC1

(50mg; 5pCi).

Fig. 7.7 Metabolic pathways of pethidine. 252

Chapter Eight

Fig. 8.1 Frequency distribution curves for metabolic pathways of pethidine in a panel of adult volunteers. 271

Fig. 8.2 Relationship between initial ester hydrolysis and 272

N-demethylation of pethidine in a panel of adult

volunteers.

Fig. 8.3 Relationship between initial and secondary ester 273

hydrolysis of pethidine in a panel of adult volunteers.

Chapter Nine

Fig. 9.1 Radiochromatograms of acid treated 0-24h urines 283

following administration of [ 14C] -pethidine HC1 to

sub-primate mammals.

Fig. 9.2 Comparative data for pethidine metabolism in primate 290

and sub-primate species 18

LIST OF TABLES

Page

Chapter One

Table 1.1 Morphological and non-morphological effects of 32

maternally administered drugs on the human fetus

and neonate.

Table 1.2 Factors modulating drug disposition in the 45

maternal-fetal-placental unit.

Table 1.3 Observed alterations in hepatic drug metabolism. 56

during pregnancy.

Table 1.4 Effect of cigarette smoking on human placental 60

benzo(a)pyrene hydroxylase activity.

Table 1.5 Drug metabolism by human fetal liver microsomes. 62

Table 1.6 Drug metabolism by human fetal adrenal homogenates 64

Table 1.7 Plasma elimination half lives of drugs in 66

infants and adults.

Table 1.8 Drug metabolism reactions by the human neonate 67

in vivo.

Table 1.9 Lumbar epidural anaesthesia with bupivacaine: 83

Maternal and umbilical blood concentrations

at delivery.

Table 1.10 Species variation in the metabolism of lignocaine. 87

Table 1.11 The metabolism of bupivacaine in rat and rhesus 88

monkey.

Chapter Two

Table 2.1 Rf values and colour reactions of bupivacaine 101

and related compounds. 19

Page Chapter Three

Table 3.1 Rf values of pethidine and related compounds 118 Chapter Four

Table 4.1 Concentration of bupivacaine in maternal and 127

umbilical cord blood at delivery.

Table 4.2 Neonatal blood levels and elimination half-lives 133

of bupivacaine.

Table 4.3 Excretion of bupivacaine and desbutyl-bupivacaine 135

in 0-24 h neonatal urine following maternal

administration of bupivacaine during labour.

Table 4.4 An analysis of the relationship between neonatal 140

jaundice, maternal smoking and type of

resuscitation required at delivery, on neonatal

blood elimination half-life of bupivacaine.

Table 4.5 Concentration of pethidine in maternal and 145

umbilical blood at delivery.

Table 4.6 Transplacental passage of pethidine and its 150

metabolites.

Table 4.7 Neonatal blood levels and elimination half-lives 152

of pethidine.

Table 4.8 Pethidine metabolism in five human neonates 153

following maternal administration during childbirth.

Table 4.9 An analysis of the relationship between neonatal 160

jaundice, maternal smoking and type of resuscitation

required at delivery, on neonatal blood elimination

half-life of pethidine.

Chapter Five

Table 5.1 Summary of data collected. 169

Table 5.2 Infant status (Apgar score and resuscitation 172

required) at birth in the three groups. 20

Page Table 5.3 Conclusions from the study. 180 Chapter Six

Table 6.1 Sequential extraction of rat urine. 186

Table 6.2 Sequential extraction of human urine. 198

Table 6.3 The elimination of.14C by the rat after the 200

administration of 1[ 4C]bupivacaine- HCl.

Table 6.4 The metabolites of [14C]-bupivacaine HC1 in 206

rat urine.

Table 6.5 The elimination of.14C by bile-duct cannulated 207

rats after administration of [1 4C] -bupivacaine

HCl.

Table 6.6 The elimination of 14C by two male volunteers 210

after administration of [1 4C]-bupivacaine HCl.

Table 6.7 The metabolites of [1 4C]-bupivacaine in human 211

urine.

Chapter Seven

Table 7.1 RadfoTLC properties of pethidine and 14C-labelled 235

metabolites isolated from human urine.

Table 7.2 Elimination of 14C by adult volunteer JC after 242

administration of [14C]/[2H5] -pethidine HCl.

Table 7.3 Sequential extraction of human urine. 244

Table 7.4 The metabolism of pethidine in adult volunteers. 246

Chapter Eight

Table 8.1 Pethidine metabolism in a panel of human volunteers: 256

(% recovery in 24h urine).

Table 8.2 Pethidine metabolism in a panel of human volunteers: 258

(data standardised to amount of pethidine in 24h urine). 21

Page

Table 8.3 Pethidine metabolism in a panel of human volunteers: 259

(data standardised to amount of pethidine and metabolites

in 24h urine) .

Table 8.4 Pethidine metabolism in a panel of human volunteers: 260

(extent of each metabolic pathway).

Table 8.5 Recoveries and rate constants for pethidine metabolites 261

in four selected volunteers. 263 Table 8.6 Pethidine metabolism in five human neonates following

maternal administration during childbirth: (recovery

in 24h urine).

Table 8.7 Pethidine metabolism in five human neonates following 264

maternal administration during childbirth: (data

standardised to amount of pethidine in 24h urine).

Table 8.8 Comparison of pethidine metabolism in human 265

adults and neonates.

Table 8.9 Differential ontogenesis of pethidine metabolism in 266

man.

Chapter Nine

Table 9.1 Metabolism of pethidine in four non-human primates. 284

Table 9.2 The elimination of 14C by the rat, guinea pig and 287

rabbit after administration of [1 4C ]pethidine- HC1. 14 Table 9.3 The metabolites of [ C]ethidine-p in rat, rabbit and 288

guinea pig urine. 22

Chapter One. Perinatal Pharmacology and Obstetric Analgesia

Page

1.1 Introduction 23

1.1.1 Historical background 23

1.1.2 Analgesic drugs used in obstetric practice 25

1.2 The passage of maternally administered drugs to the

fetus 26

1.2.1 Drugs and the fetus 27

1.2.2 Placental structure and transplacental transfer 35

1.2.3 Drug distribution in the fetus 41

1.2.4 Pharmacokinetics of placental transfer at term 46

1.3 Perinatal drug metabolism 52

1.3.1 General metabolism 52

1.3.2 Drug metabolism during pregnancy 54

1.3.3 Drug metabolism by, the placenta 58

1.3.4 Drug metabolism by the fetus 60

1.3.5 Drug metabolism by the neonate 63

1.4 Obstetric analgesia 69

1.4.1 Pethidine 70

1.4.2 Xylidide local anaesthetics 75

1.5 Scope of the present work 89 23

1.1 Introduction

1.1.1 Historical background

Childbirth is one of the few processes of life in which physical pain is an integral factor. There is little evidence to suggest that the pain modern woman experiences in labour is any different to that en- countered by her prehistoric forebears, and down through the ages, the relief of pain during childbirth has been of great interest.

Mankind's early attempts to alleviate pain are recorded in mythological writings, which indicate primitive man's association of pain with super- stition and religion. Aboriginal man believed that pain was an evil spirit, and made many efforts to ward off, appease or frighten away the pain demons by using rings worn in the nose or ears, talismans, amulets and similar charms. Incantations and spells were also used by women in labour to enable them to put the pain demons to flight. All of these methods entailed the use of suggestion, distraction or other psychological forms of analgesia. One of the most powerful forms of suggestion, which today is known as hypnotism, was used by the more advanced ancient cultures e.g. Egyptians and Chinese, to relieve the pain of childbirth.

This association of pain with religion continues into modern religious texts, notably the Christian Bible. What once had been the domain of evil spirits became punishment inflicted by an offended God: "To the woman He said, Many are the pangs, many are the throes I will give thee to endure; with pangs thou shalt give birth to children,

(Genesis 3 verse 16). Thus, with the advent of Christianity, pain became important as a means of obtaining grace, or as a sacrament, and 24

the woman in labour was expected to accept pain voluntarily. The same ready acceptance of physical pain was encouraged by all Oriental as well as the various Christian religions.

Despite these teachings, attempts to relieve pain in childbirth continued, and numerous 'medicines' were used in addition to, or instead of, psychological analgesia. Since the growth of the apothecaries' trade in the middle ages, potions consisting of mixtures of various natural extracts from plants and herbs (such as the poppy, mandragora, hemp and henbane) were used to alleviate pain, including that of childbirth.

However, as late as the sixteenth century 'witches' were prosecuted for attempting to abolish the pain of labour, the last recorded instance of such a trial ended in execution in Edinburgh in 1591.

The age of modern anaesthesia began on October 16, 1846, when William

Morton first anaesthetized a patient with ether at the Massachusetts

General Hospital. Modern obstetric anaesthesia began only three months later on January 19th, 1847, when James Young Simpson, Professor of

Midwifery at the University of Edinburgh, anaesthetized a patient in laboi_r with the same drug. On November 4th, 1847, having experimented on hi..self, Simpson discovered the anaesthetic properties of chloroform, and later that month he described its use in obstetrics before the

Medico-Chirurgical Society of Edinburgh. However, the public reception for obstetric anaesthesia was stormy; led by the Scottish Calvinist clergy, epithets of 'blasphemer', 'heretic' and 'instrument of the devil' were flung at Simpson who was accused of violating God's will. This disapproval gradually subsided but it was not until 1853 following the successful administration of chloroform to Queen Victoria for the birth 25

of her eighth child, Prince Leopold, and her positive expression of

pleasure with its effect, that obstetric anaesthesia became fashionable.

Chloroform continued to be the most commonly used anaesthetic, and it

was not until the rapid expansion of the pharmaceutical industries both

during and after the second world war that newer, safer and more effect-

ive drugs were developed. Numerous new methods of analgesia, (e.g.

regional anaesthesia) have since been introduced into obstetric practice;

general anaesthesia is seldom used for other than surgical procedures

such as delivery by caesarian section.

-1.1.2 Analgesic drugs used in obstetric practice

Although the introduction of anaesthesia in childbirth relieved pain,

experience showed that ether and chloroform were not without side-effects.

Chloroform caused ventricular fibrillation during light anaesthesia,

central necrosis of the liver, interfered with uterine activity and

depressed fetal respiration. But, in spite of these risks, it remained

until the beginning of this century the main drug used for the relief

of pain in labour. However, the search for newer safer drugs was ini-

tiated and the ideal drug for use in childbirth would ideally:

a) present no hazard to mother and child

b) abolish or diminish pain for long periods

c) not diminish uterine contractions and thereby delay labour

d) not prevent the patient from co-operating with the doctor

e) be cheap and conveniently administered.

The multiplicity of analgesics and anaesthetics used in obstetric practice

today suggests that the ideal drug for labour has not been found. Some

drugs are mainly analgesic as they tend to relieve pain without loss of 26 consciousness, e.g. opiates, while others are hypnotics and tranquillizers with little or no analgesic effect, e.g. barbiturates, benzodiazepines.

Total analgesia, i.e. complete relief from pain without loss of consciousness, can only be produced by regional anaesthesia incorporating the use of local anaesthetics. The main groups of drugs which have been used to alleviate the pain of labour are listed below:

1) Sedative-hypnotics e.g. scopolamine, chloral hydrate and glutethimide

2) Barbiturates (sedatives/hypnotics with no analgesic effect)

3) Ataractic (tranquillizing) drugs

4) Gaseous and volatile anaesthetics:

a) 'gas and air' i.e. nitrous oxide mixed with air in a 1:1 ratio

b) trichloroethylene (sometimes used for general anaesthesia)

5) Narcotics:

a) natural alkaloids present in opium e.g. morphine and codeine

b) synthetic compounds with structural resemblance to the whole

or to a part of the morphine molecule e.g. pethidine.

6) Local anaesthetics,the more commonly used ones being lignocaine,

mepivacaine and bupivacaine.

The drugs used in obstetric practice today will be discussed later.

1.2 The passage of maternally administered drugs to the fetus

The recognition of congenital malformations is as old as mankind. For instance in the Middle Ages malformations were almost invariably attributed to witchcraft and the only contributions of environmental conditions were confined to some action or slight by the mother. From about 1900 onwards however, it was shown by embryologists in a number of laboratories, initially in avian embryos and later in mammals, that drugs, other chemicals, nutritional deficiency and mechanical injury were able to alter normal 27 fetal development. Though occasional cases of apparently drug-related

adverse effects on fetuses following maternal administration had been reported, rubella virus was the first teratogen to be incriminated

beyond a doubt in humans (Gregg, 1941). Early anaesthetists noticed a

higher mortality rate in children whose mothers received morphine or

chloral hydrate during labour, and neonatal jaundice was common following

maternal chloroform administration. However these adverse neonatal

effects were ignored and the use of anaesthetics and analgesics during

labour continued as before. In the early 1960's thalidomide, a tran-

quillizer considered an ideal drug because of its apparently complete

lack of toxicity in adults, was found to produce a high incidence of

phocomelia in the fetus when given to women between the fifth and

seventh weeks of pregnancy (Lenz, 1963). This dramatic example of a

congenital malformation resulted in a greatly increased interest in

chemical teratology not only among embryologists, but also among clinicians,

pharmacologists and toxicologists.

Classically, teratology refers to the study of congenital malformations

observed grossly at birth and induced by exogenous agents during the

organogenetic period. Today however, the scope of teratology has

widened to include all of the mcrphological, biochemical, behavioural and

other adverse effects induced during fetal life, detected at birth or

later (WHO, 1966).

1.2.1 Drugs and the fetus

Although drugs are only very rarely administered for the treatment of

fetal disorders, the mother is exposed to foreign compounds during preg-

nancy, and in such cases the possible effects on the fetus must also be

considered. Such exposures of the fetus to maternally administered 28

compounds may be divided into four categories:-

1) therapy of maternal disease directly related to pregnancy and

delivery.

2) therapy of maternal disease unrelated to but coinciding with

pregnancy.

3) maternal use or abuse of drugs for nontherapeutic purposes.

4) incidental exposure of the pregnant woman to biologically active

agents e.g. food additives; accidental and deliberate contamin-

ation of the environment (air/land/water pollution).

Early studies of placental transfer indicated that most drugs administered to pregnant women will cross the placenta and enter the fetal circulation (Villee, 1965; Mirkin, 1973), and equilibration between the maternal and fetal circulation may be extremely rapid. The con- sequencesof exposure of the fetus to pharmacologically active compounds are more extensive and potentially more harmful than are generally recognised, and the possible effect upon the fetus of maternally administered drugs has become a source of increasing concern. The frequency with which therapeutic agents are administered during pregnancy appears to be high: a recent study in the United States showed the average number of drugs ingested during pregnancy, including labour and delivery, to be 10.3 (Hill, 1973), and a similar study in

Scotland gave the number of drugs as 4.2 (Forfar and Nelson, 1973), although this did not include drugs administered during labour and delivery. The most commonly ingested drugs during pregnancy were found to be analgesics, antihistamines and diuretics, other potential hazards being nicotine, caffeine, artificial sweeteners and alcohol. 29

The production of a congenital malformation depends upon the developmental stage of the embryo, its genetic susceptibility, the specificity and dose of the agent utilized and the physiological or pathological condition of the mother. Embryonic development may be divided into three main stages, characteristic malformations occurring in each stage are discussed below: a) Blastogenesis, i.e. pre-implantation development which spans from

fertilisation to implantation. In general, exogenous agents do

not produce malformations in this period which lasts approximately

7 days. The dividing ovum may be killed by severe physical or

chemical insult but when cell death does not occur the damaged

cells can be repaired or replaced so that development is resumed

without impairment of organogenesis. Penetration of drugs into

the blastocyst is a result of their secretion into the luminal

fluids of the oviduct,fallopian tubes and uterus. b) Embryogenesis, the period of organogenesis. During the formation

of the main layers, the appearance of the 'anlage' and the diff-

erentiation into organs, the embryo is most susceptible to the

effects of exogenous agents. Various malformations can be

produced, the nature of which depends to a great extent upon the

age of the embryo and the particular teratogen employed; e.g.

in the human embryo, the critical teratogenic period for the eye

is between days 24 to 40, for the heart days 18 - 40 and for

the limbs days 24 - 36 (Fig. 1.1). Thalidomide produces mainly

limb malformations in man, while rubella affects mainly the eye

and the heart. c) Fetogenesis, the period of fetal growth and functional development.

From the 9th week, when the human embryo has developed its main 30

structures and has already the shape of a miniature newborn, it

becomes a fetus. During this fetal period no major malformations

can be produced, but the differentiation of some organs, e.g. the

external genitalia, which develop only during the fetal period

can be harmed (Fig. 1.1). Various degrees of developmental impair-

ment of the genitalia leading to pseudo-hermaphroditism can be

observed when androgenic hormones are taken by the mother. Sim-

ilarly, the histogenesis of the CNS takes place during the fetal

period and therefore can be disturbed by an agent to which it is

exposed in the later stages of pregnancy.

Morphological abnormalities resulting from the influence of exogenous substances (Table 1.1) have been studied extensively and all new drugs are now tested routinely for teratogenic effects. Of topical interest here is the association between low birth weight and cigarette smoking in pregnancy, first described by Simpson in 1957. It seems likely that the effect of smoking is on fetal growth, and the association of smoking with increased number of stillbirths can be explained by the greater proportion of low birthweight babies born to smoking mothers.

In contrast to the interest in drugs producing teratogenic effects, little attention has been directed to the pharmacological effects of these agents upon the fetus, and serendipity rather than systematic research has played the major role in furthering our knowledge of these non morphological effects. There is good reason to suspect that for the newborn the effect of a drug will persist beyond the pu=rperium.

There is little hard data in this area due to lack of awareness of the extent of the problem and difficulties of finding sufficiently sensitive Fig. 1.1 Organ development and drug side-effects

female developing types 150 50 ■

male developing 45 types 90

ion gonads t ion ion a t t 37-46 iz ta la n

Chronology il t

la extremities _ -► tru r of s 24-36 Fe Imp Ga Organogenesis eves 4110- 24-40

heart 18-4o

brain 18-38 V V V BIRTH 266 days I l `` Time Axis Day O 7th 15th 42th 60th 90th 120th 150th 1st - 6th week post partum

PERIOD LSpermiogenesis IJBlastogenesis I Organogenesis I Fetogenesis (fetal development) Neonatal adaption phase

SIDE Sterility Death abortion Serious congenital Congenital Functional Adaption difficulties EFFECTS defects L defects anomalies 32

Table 1.1 Morphological and non-morphological effects of maternally

administered drugs on the human fetus and neonate

AGENT FETAL OR NEONATAL EFFECT

Antihypertensives thiazides electrolyte imbalance reserpine nasal stuffiness; hypothermia. nitrites methaemoglobinaemia ganglionic blockers paralytic ileum

CNS Depressants Chlorpromazine sedation; retinopathy chlordiazepoxide neonatal depression narcotic analgesics neonatal depression; withdrawal symptoms local anaesthetics fetal bradycardia; fetal acidosis thalidomide skeletal anomalies barbiturates increased rate of neonatal drug metabolism

Anti-Bacterials tetracycline deposition in bone; inhibition of bone growth in premature infants; discolouration of teeth streptomycin deafness (8th nerve toxicity) nitrofurantoin haemolysis chloramphenicol cardiovascular collapse ('Gray syndrome') penicillin (high conc.) convulsions sulphonamides hyperbilirubinaemia

Hormones androgens virilisation of female progesterone virilisation of female oestrogens feminization of male

Miscellaneous thiouracil goitre salicylates neonatal haemorrhage quinine deafness coumarin anticoagulants haemorrhage chloroquine retinopathy smoking impaired fetal growth 33

techniques for evaluating the behaviour of the newborn in the first few days of life. However, with the increase in popularity of obstetric anaesthesia and analgesia, it became evident that the drugs in use had a potentially detrimental effect on the neonate. This problem was realized by Clifford and Irving, who, in 1937, demonstrated a relationship between the time interval from the administration of opiates to birth and the need for resuscitation. The peak effect was found when the drug was given between four to six hours before delivery when 30% of the infants then required active resuscitation. They also noted that the delay to first cry was increased comparing a no drug group to those delivered with a combination of pentobarbital, scopolamine, rectal ether and nitrous oxide. Since this pioneer study, others have attempted to study the pharmacology of the various analgesic agents, their efficacy and the effects on the fetus and neonate, but often, evaluation of the neonate has been limited to what can be observed in the delivery room. Most studies limit testing to the first

24h of life, and the majority of published reports usually cite the

Apgar score alone (based on a 10 point scale which assesses heart rate, respiration, muscle tone, colour and response to stimulation;

Apgar, 1953) at 1 and 5 minutes after birth when the infant is re- covering from the birth process. Very few studies have examined the long-term effects of the drugs on the newborn. However, with the increased interest and knowledge of the capabilities of the newborn over the past decade, paediatricians and psychologists have devised behavioural and neurological tests which now provide evidence of subtle, but in many cases persistent effects of maternal medication on the neonate (Table 1.1). Some researchers now consider that all drugs used in childbirth, particularly general anaesthetics, 34

tranquillizers, barbiturates and muscle relaxants, have deleterious effects on the neonate, and decrease alertness and reduce orientation and responsiveness to visual and auditory stimuli (Standley, Soule,

Copans and Duchourney,1974). Pethidine and similar analgesics are considered responsible for decreased motor activity, greater irritability and lower consolability of babies, as well as reducing responsiveness to auditory stimuli (Moreau and Birch, 1974), and they may also cause feeding difficulties in the infant (Dubignon, 1969).

Babies born of mothers who received bupivacaine show a higher incidence of jerky movements, persistant startles and tremor, and are more likely to cry (Standley et al., 1974). Scanlon (1974) found that bupivacaine caused floppy muscle tone and poor head control without any decrease in alertness or sucking behaviour, while other workers have found that sucking and food intake are more affected following its epidural administration (Kron, Stein, and Goddard, 1966). In contrast to these studies, many authors failed to find any drug effects, and Scanlon (1976) has failed to confirm his earlier findings in a repeat study. This could be due to the difficulty of interpreting behavioural studies, and to the multitude of non-pharmacological factors that contribute to the condition of the infant at birth; e.g. the use of forceps, length of labour, fetal maturity at birth etc.

Over the past decades, with the increasing number of hospital deliveries, the utilization of anaesthesia and analgesia for relief of pain and anxiety during labour has become widespread. The last British birth survey (1973) showed that 46% of all births in the United Kingdom utilized pethidine and a further 23% pethilorfan (a related synthetic narcotic analgesic). The last few years have also seen the intro- 35

duction of epidural anaesthesia into obstetrics, and the drug of choice for this procedure at present is bupivacaine. Below is a list of the analgesic procedures used for the 845 deliveries during

1976 at St. Mary's Hospital, London, and shows that some 70% of mothers opted for either pethidine or epidural anaesthesia, compared with less than 20% who had no analgesic drugs.

No. of Procedure deliveries g Lumbar epidural anaesthesia with bupivacaine 298 35.3

Pethidine 290 34.3

Other procedures (N20; pudendal block with 26 3.1 lignocaine)

General anaesthesia (for Caesarian section) 76 9.0

No analgesia 155 18.3

TOTAL 845 100

There is still very little known about the placental transfer and neonatal disposition of these drugs, and it is not even known what the metabolites of bupivacaine are, much less whether they are pharmacologically active and potentially toxic towards the fetus. Thus,

the effects of such drugs on the fetus and neonate needs to be evaluated to ensure that the benefit to the mother in terms of comfort does not endanger the child in any way, and to establish whether any form of obstetric anaesthesia/analgesia is contraindicated for the child's well-being and development.

1.2.2 Placental structure and transfer

Among all the membrane systems of the body, the placenta is unique.

It separates two distinct individuals with differing genetic composition, 36

physiological responses and sensitivities to drugs. Through the placenta the fetus obtains nutrients and eliminates metabolic waste products without depending on its own immature organs, as well as receiving xenobiotics as they appear in the mother's blood.

The mature placenta (Fig. 1.2) contains a network of maternal blood sinuses into which protrude villi carrying the fetal capillaries.

These villi are covered with a trophoblastic layer beneath which is a layer of mesenchymal tissue and, finally, the capillary endothelium.

There is also a close apposition of the fetal amnion to the chorionic membrane of the uterine wall. In the early stages of gestation, the passage of substances directly into the amniotic sac and thence to the embryo may be significant, but later the amniotic fluid is in contact only with the fetal epidermis. The exchange of foodstuffs, oxygen and CO2, as well as drugs must now occur primarily across the placenta, i.e. from the maternal arterial supply by way of the intervillous spaces into the fetal capillaries in the villi, and thus into umbilical venous blood.

The possible mechanisms by which substances cross the placenta and reach the fetus may be considered, as with other natural membranes, under four main headings:- a) Simple diffusion, by which substances cross the placenta from

regions of high to low concentration, the driving force for such

transfer being molecular thermal agitation. Examples of this

include oxygen and carbon dioxide. b) Facilitated diffusion, in which a carrier substance within the Fig. 1.2 The Mature Placenta (from Gray, 1966)

Limiting or boundary Intervillous Placental septum layer space Stratum spongiosum Maternal artery

Maternal vein

Villus

/if

Marginal /i Chorion sinus Trophoblast Umbilical Amnion Umbilical vein artery U 38

placenta acts to increase the rate of transfer beyond that which

would be expected for a given substance on physico-chemical grounds.

Again, no energy is required and transfer is the direction of the

concentration gradient. Glucose and other sugars are thought to

cross the placenta in this way. c) Active transport, across a membrane involving molecular transfer

against a concentration gradient and entails the expenditure of

metabolic energy. This mechanism is responsible for the transfer

of vitamins, amino acids and essential ions such as calcium, from

mother to fetus. d) Special processes, which include (i) pinocytosis, in which small

"volumes" of plasma are engulfed by microscopic invaginations of

the cell membrane and thereby transferred directly to the fetus,

and (ii) breaks in the placental membrane, which is thought to be

the means by which red blood cell transfer occurs between the

two circulations.

Very few vital materials cross the placenta by simple diffusion, in fact, oxygen may be the only substance essential to the fetus which does this, although even this is difficult to demonstrate due to the high oxygen consumption of the placenta (Campbell, Dawes, Fishman,

Hyman and James, 1966). On the other hand, drugs and other exogenous compounds are thought to cross in the main, by simple diffusion.

Facilitated diffusion and active transport probably apply only in instances where drugs have a structural similarity to endogenous material normally transported by these mechanisms from maternal to the fetal circulation. 39

Since the majority of drugs are thought to cross by a process of simple diffusion in accordance with their physico-chemical properties such as lipid solubility, one would expect poor fetal penetration of highly ionized drugs with low lipid solubility such as d-tubocurarine, and more rapid transfer of partially ionized drugs with high lipid solubility, such as antipyrine and thiopentone. This may be a useful and valid concept in the main, but its application may be limited, e.g. when d-tubocurarine is given in large amounts as may be required to control maternal epileptic convulsions, enough of the drug may cross the placenta for the infant to be curarized at birth (Older and Harris,

1978). Additionally, salicylic acid which is almost 100% ionized at pH 7.4 shows a rapid transplacental passage (Oh and Mirkin, 1971).

Thus, any drug of suitable molecular weight, if present in sufficient concentration on'the maternal side of the barrier, will probably eventually reach the fetus. What matters however, is the exposure of the fetus which depends on the amount that effectively reaches the fetus in a given period of time, the form in which it is presented to the fetus, the ability of the fetus to eliminate the drug, and any influence of the drug on placental function.

Simple diffusion of materials across the placenta follow the general principles of Fick's diffusion equation:-

dg = KA (C2 - C1) dt d where dQ/dt = rate of transfer

K = diffusion constant for the drug

A = surface area of membrane

d = thickness of membrane 40

C1 and C2 = drug concentration on each side of membrane

so that C2 - C1 represents the gradient

across the membrane.

However, other factors are also important in influencing the rate and extent of placental transfer, and these are discussed briefly below:

1) Lipid solubility - permeability increases with lipid solubility

(Oh and Mirkin, 1971).

2) Molecular weight - compounds with a molecular weight of less than

600 readily traverse the placenta, whereas it is relatively im-

permeable to those exceeding 1,000.

3) Degree of ionization - drug molecules tend to penetrate biological

membranes more rapidly in their unionized state (King, Adam,

Schwartz and Teremo, 1971).

4) Placental blood flow - the transfer of highly lipid-soluble drugs

across the placenta appears to be proportional to placental flow.

5) Placental metabolism of drugs - numerous oxidation reactions, e.g.

hydroxylation, demethylation, N-dealkylation, can be effected

by placental homogenates (Juchau, 1972) which may theoretically

hinder drug transfer.

6) Ageing of placenta - maturation leads to a decrease in the thick-

ness of the trophoblastic epithelium, from about 25pm early in

pregnancy to 2pm at term, but despite this observation, drugs

cross the placenta more rapidly in rodents in the first trimester

of gestation than in the last (Mirkin, 1973).

7) Protein binding of drugs - those agents that are tenaciously

bound to plasma proteins will probably be transferred less rapidly

across the placenta than less strongly bound compounds because 41

the proportion of free to total drug present at any time will

be lower. Other types of binding may affect the transfer of

drugs across biological membranes, particularly if the drug has

a relatively high affinity for compounds within a specific tissue,

e.g. diphenylhydantoin is concentrated in the heart, adrenal

cortex and corpora lutea of the ovary (Mirkin, 1971).

1.2.3 Drug distribution in the fetus

Upon traversing the placenta, pharmacologically active molecules may

be (a) taken up selectively and localized in specific fetal tissues

or body compartments, (b) excreted into the amniotic fluid and uterine

lumen, (c) metabolized by fetal liver or (d) returned to the maternal

circulation via retrograde fetal-placental transfer. Some of the

basic biochemical and physiological characteristics of the fetus which

appear to determine the disposition of drugs transferred across the

placenta are discussed below:

i) The permeability of specialized membranes, such as those present

in the blood-brain barrier, renal tubules and yolk sac, markedly

influence drug distribution in the fetus. The blood-brain

barrier is highly permeable in early development of many mammal-

ian species so that the fetal brain may be exposed to relatively

higher concentrations of a given drug during uterine existence

than would the adult brain. This is partially offset by the

fact that the fetal brain contains a high percentage of water

and has a low myelin content thus reducing its affinity for

lipophilic drugs. ii) Selective uptake of drugs by fetal- tissues attributable to

several processes, such as (a) nonspecific lipid solubility, which 42

would explain why organs with a relatively high lipid content, e.g. adrenal gland, ovary and liver, are able to concentrate a wide variety of pharmacological agents, (b) specific cellular constituents which play an important role in the tissue sequestation of drugs by providing discrete binding sites for both endogenous and exogenously administered molecules, e.g. the uptake and binding of bilirubin by hepatic parenchymal cells seems to require a special transfer protein (Levi, Gatmatain and Arias, 1970) and (c) the capacity of the yolk sac, with its endodermal lining resembling the microvilli and endoplasmic reticulum of the liver, to metabolize and secrete drugs into the uterine lumen of the rat. However, it is yet to be resolved whether the yolk sac exerts an important role in eliminating drugs from the developing human fetus.

An important factor to mention here is that the binding capacity and/ or affinity of fetal plasma proteins for certain drugs may differ from that of maternal plasma, altering the quantity of free drug available for tissue distribution, e.g. in the case of bupivacaine only 51% of the drug is bound to plasma protein in fetal blood compared to 88% in adults (Tucker, Boyes and Bridenbaugh, 1970). How- ever, since the pH of maternal arterial blood is normally 0.1 to

0.15 pH units higher than that of umbilical cord blood, the concentration of undissociated base will tend to be higher in maternal blood giving a net transfer of drug from mother to fetus. In cases of fetal acidosis,the amount of abasic drug in the unionized form in the fetus will be even greater resulting in "ion trapping", and a greater than average concentration of drug in the fetus/neonate will result. 43 iii) The haemodynamics of the fetal circulation (Fig. 1.3 ) are

also of great importance in determining the relative concentrations

of drug presented to a given anatomical region in the fetus.

After a drug crosses the placenta and enters the umbilical vein,

60 - 85% of the flow into the umbilical vein enters the liver

via the portal vein and 15 - 40% bypasses the liver and goes

via the ductus venosus directly into the inferior vena cava.

Consequently, a proportion of the drug, initially present in

the umbilical vein, may be selectively taken up by the liver

which may achieve concentrations exceeding those in any of the

other fetal organs. In addition, the concentration of drug in

the inferior vena cava is also diluted by venous drainage from

the lower limbs and abdominal viscera. Upon the drug reaching

the left atrium, further dilution occurs via the superior vena

cava which drains the cephalic regions of the fetus and enters

the left atrium through the foramen ovale. These successive

dilutions reduce the blood levels of drug reaching the left

ventricle, lungs and central nervous system so that they are

similar to those of the ascending aorta, whereas the liver, ad-

renal gland and kidney are perfused by blood containing con-

centrations equivalent to that in the descending aorta (Dawes,

1968) .

It is apparent that many factors influence placental drug transfer

and fetal drug localization (Table 1.2). Each of these pro-

cesses operate concurrently in an integrated manner so that

multiple kinetic events are occurring in both maternal and fetal

environments. A schematic summary based on current concepts 44 Fig. 1.3 CHANGES IN THE CIRCULATION AT BIRTH

P F.O. L A C L BODY LEFT HEART) RIGHT HEART) E U N N T G A D.A. S T T C <

a) FETAL F.O. = Foramen Ovale D.A. = Ductus Arteriosus

b) NEONATAL

L RIGHT HEART (BODY LFT HEART! U N G S T I

c) ADULT

(After Born, Dawes, Mott and Widdicombe, 1954). TABLE 1.2 Factors Modulating Drug Disposition in the Maternal-Fetal-Placental Unit

A/ Placental transfer of drugs 1. Physicochemical properties of drugs Lipid solubility Degree of ionization Nblecular weight Protein binding 2. Placental blood flow Maternal and fetal circulation Maternal-fetal blood pH gradient 3. Stage of development of placenta 4. Placental metabolism of placenta

B/ Drug distribution in the fetus 1. Altered permeability of specific membrane-bound compartments Blood brain barrier Total body water and lipid content 2. Selective tissue uptake of drug Nonspecific lipid solubility Specific binding to cellular constituents 3. Distribution of fetal circulation 4. Fetal metabolism of drugs 5. Fetal swallowing of amniotic fluid 6. Excretion by fetal kidney 46

of drug disposition in the maternal-feto-placental unit is

presented in Fig. 1.4.

1.2.4 Pharmacokinetics of placental transfer at term

Most substances given to the mother, if present in sufficient quantity in the maternal circulation, can and will reach the fetus, and it is therefore necessary to establish how much drug reaches the fetus, how rapidly it enters the fetal circulation, and how long it remains in the fetus. It may be important to focus either on the time course of drug concentrations in the fetus/neonate (Levy and Gibaldi, 1972) or on the total drug level-time integral (Jusko, 1972) as a measure of fetal exposure to the drug. The ratio of the total area under the drug concentration vs. time curve for the fetus to that of the mother can serve as an index of relative exposure of the fetus to a drug taken by the mother. For example, the data reported by Depp,

Kind, Kirby and Johnson (1970) show an essentially identical time course of methicillin concentration in maternal and fetal serum following its intravenous administration to the mother, and this represents an index of relative exposure equal to unity. The same study showed that dicloxacillin concentrations in the fetal serum were much lower at all times than in the maternal serum, and the index of relative exposure was less than 0.2. It could therefore be postulated that drugs which are intended to reach the fetus should have a high index of relative exposure, while drugs which could potentially harm the fetus and are intended for the mother only, should have a low index of relative exposure.

Studies of placental transfer in humans during the early stages of

Fig. 1.4 Drug Disposition in a Model of the Maternal-Placental-Fetal Unit (Mirkin, 1973)

placental Fetal ether biotransformations (?) Brain Locu Fetal `of Tissues Actio P A L Fetal A Liver Plasma ► umbilical vein FREE ► I ductus 1 DRUG 1 , k venous \ ► k 1 ► 1 V t I. V. or I.M. 1 N `. I bound t T biotransformations drug . bound ■ a drug Metabolite --- _. . 1 umbilical

artery .10 maternal Metabolite liver mniotic Fluid

parent drug EXCRETION

metabolite 48

pregnancy are limited by a limited availability of suitable subjects and by ethical considerations, with most of the data being obtained from aborted fetuses, the drug being administered to the mother prior to abortion. There is the additional complication from the pharmacokinetic point of view in that only a one-point determination of drug concentration in the fetus is ordinarily feasible in humans.

Similarly, the pharmacokinetic characterization of placental drug transfer during delivery is also usually based on a composite of data obtained from different subjects at different times, i.e. the drug concentrations in maternal and cord blood are determined at the moment of delivery in a group of patients, with the variable experimental factor being the dose-delivery interval. However, a modification of the fetal scalp sampling technique introduced by Saling

(1964) now permits several blood samples to be taken from the fetus in utero for analysis, which together with analysis of the sample drawn from the umbilical cord at delivery, yields more satisfactory data from a pharmacokinetic point of view.

Multi-compartment models

Some drugs are so rapidly distributed in the body upon reaching the blood-stream that the body acts like a single compartment with respect to these drugs, and may be characterized as such for pharmacokinetic purposes. Other drugs, being less rapidly distributed, confer on the body the pharmacokinetic characteristics of a multi-compartment model. Similarly, the mother and fetus may be viewed as a two- compartment model, but more complex models can represent the mother as a multi-compartment system, with added compartments representing a drug-eliminating placenta or fetus. These different pharmacokinetic 49

models yield different types of drug concentration vs. time patterns in mother and fetus,as well as different fetal:maternal concentration ratio vs. time curves.

(i) "Single-compartment mother" plus fetus - similar to the two-

compartment open model but the fetus is too small to make any

noticeable difference. In this system, the drug is assumed to

be absorbed and distributed immediately in the body, with the

clearances (i.e. the rate constant for drug transfer times the

apparent volume of distribution), between mother (M) and fetus

(F), being equal. This rapid equilibration to a fetal:maternal

concentration ratio of about one has been found for example, with

thiopental (Moya and Thorndike, 1962).

M F

f Usually, drug absorption and distribution require a finite period

of time, which results in a lower and delayed peak of drug

concentration in the fetus than would be theoretically expected.

The difference between the theoretical and actual value depends

on the relationship between the absorption rate constant and

the elimination rate constant.

(ii) "Two-compartment mother" plus fetus - similar to the three-

compartment open model. Distribution of a drug throughout 50

the various tissues in the body is in many instances not ex-

tremely rapid compared to the elimination process. In the

three-compartment open model, it is assumed that there is a

central compartment (M) consisting of plasma and other fluids

and tissues which are instantaneously accessible to the drug.

The second and third compartments consist, in this case, of

peripheral tissues and fluids (T) and fetus (F) which are more

slowly accessible to the drug.

In this instance, the drug will initially not only be eliminated

from the central compartment, but also penetrate simultaneously

into the peripheral tissue and fetal compartments. If this

process is rapid compared to the 'back diffusion' of the drug

returning to the central compartment, a steady state will result

when the peripheral tissue and fetal concentrations parallel: plasma con

centration. The slower the penetration from the central com-

partment into the peripheral tissue and fetal compartments, the

longer it takes for the steady state to develop.

(iii) Drug elimination by the placenta or fetus. There are a few

indications that the human placenta, (P) in vitro is capable of 51

metabolizing drugs, and the fetus may also be able to contribute

to the elimination of the drug as a result of hepatic and extra-

hepatic metabolism. Because the fetal compartment (F) is so

small, drug excretion or metabolism by the fetus has usually

no discernable effect on maternal drug concentration. However,

the fetal:maternal drug concentration ratio may be decreased

in such cases; e.g. fetal:maternal lignocaine plasma concentration

is well below unity following single and repeated injections

apparently due to its fetal elimination (Fox, Houle and ?dercier,l971).

Similarly, placental drug metabolism can reduce this ratio as

the amount of drug reaching the fetus is lower than that arriving

at the placenta.

The placental transmission of numerous drugs has been demonstrated by the methods outlined previously, but detailed mathematical analysis of the transfer characteristics of individual drugs has been performed for a limited number of compounds and then only in animals (Mirkin,

1976). The time required for a drug to appear in the fetus following maternal administration, and the time required to reach fetal/maternal equilibrium, give some indication of comparative rates of placental transfer, but these are influenced by numerous variables other than 52

placental permeability and drug characteristics. Similarly, fetal:

maternal drug concentration ratios which differ from unity, largely

depend on the apparent drug distribution volumes and 'depths' of the

compartments involved. Data obtained concerning such compartments

from non-pregnant subjects are usually of little use, as physiological

changes in pregnancy occur, e.g. maternal plasma volume and extra-

cellular fluid volumes increase, the former by a maximum of 50% be-

tween 30 and 34 weeks gestation (Krauer and Krauer, 1977), glomerular

filtration and creatinine clearance are increased, and alterations

in the rate of maternal metabolism of certain drugs occur during

pregnancy as a result of hormonal changes. These factors result in

changes in drug absorption, distribution and elimination all of which

affect plasma drug concentration.

1.3 Perinatal drug metabolism

1.3.1 General metabolism

When compounds which are considered as foreign to the energy yielding

metabolism of the organism enter the body, one of three things can

happen to them. First of all, they may be metabolized by enzymes

into other compounds. Secondly, the compound may be changed spontan-

eously into other compounds without the intervention of enzymes when

it meets the appropriate physical conditions such as pH, or when it

comes into contact with a suitable physiological molecule with which

it can react spontaneously; e.g. thalidomide undergoes hydrolysis

at physiological pH values to some twelve other compounds (Williams,

1968). Thirdly, a compound can be excreted largely unchanged, and

is not chemically modified during its stay in the body; this usually occurs with highly polar, strongly acidic or basic compounds. 53

The majority of xenobiotics undergo the first fate, i.e. they are

metabolized to some extent and are transformed into other substances.

The metabolic pathways of such foreign compounds are often referred to

as detoxication mechanisms, as they usually, but not always, result

in the loss of biological activity (Williams, 1959). The result of

such metabolism is to convert polar, lipid-soluble xenobiotics into

water-soluble metabolites that may be readily excreted.

The general pattern of metabolism of foreign compounds has been divided

into two phases (Williams, 1959). In the first phase (Phase I) the

compound may undergo reactions that can be classified as oxidations,

reductions and hydrolysis, and during this phase, new functional groups,

e.g. -CH, -NH2, -COON and -SH are introduced or uncovered into the

molecule. Very often a drug is subjected to several competing path-

ways simultaneously, the extent of formation of the various metabolites

depending on the relative activities of the various enzymes. The

majority of the oxidation reactions are carried out by enzymes located

in the endoplasmic reticulum of the hepatocytes and are known to involve

a carbon-monoxide binding pigment, cytochromeP-450 (of which there

are multiple forms all under different control displaying different

spectral properties), and a flavoprotein, NADPH-cytochrome P-450 reductase.

In the second phase (Phase II), the products of the first phase, having acquired suitable functional groups, now undergo synthetic reactions that are catalyzed by enzymes occurring mainly in the liver but also to some extent in other tissues. These synthetic reactions are commonly referred to as conjugations and involve the conjugation of the parent 54

compound, or its Phase I biotransformation products, with endogenous substances such as sugars or amino acids, leading to more water- soluble and acidic products. The overall biphasic scheme may be represented as follows: oxidation Phase I reduction Phase II synthetic or Xenobiotic enzymes > hydrolysis enzymes > conjugated products products However, some drugs undergo only Phase I or Phase II metabolism, and any factor that influences the Phase I or Phase II enzymes can effect both the rate and route of metabolism of the compound. Some of these factors are listed below:

species sex

strain stress

age temperature

chronic administration time of day

presence of other foreign compounds disease

route of excretion (urine,bile) season

route of administration gut flora

diet altitude

size of dose

(After Williams, 1974).

1.3.2 Drug metabolism during pregnancy

During pregnancy there is a large and progressive increase in steroid production which falls abruptly at delivery (Amoroso and Portu, 1970), and such a disturbance of the normal endocrine equilibrium can influence the pattern of response of an animal to certain drugs (Bousquet,

Rupe and Miya, 1965). It is now acknowledged that in the rat and rabbit at least, the latter stages of pregnancy are accompanied by 55

a decrease in maternal drug-metabolizing ability. This decrease is app-

arent in both Phase I and Phase II metabolic processes. The findings on alterations in drug metabolism during pregnancy are not restricted to animals. Crawford and Rudofsky (1966) have shown that the metabolism of promazine and pethidine is impaired in pregnant women. However Kronger(1972) has found that DDT metabolism occurs at a faster rate during pregnancy.

In order to discover the reason for such changes, initial work involved the use of animal models as these permitted in vitro and in vivo studies which were not possible in humans, e.g. liver weight, cytochrome

P-450 concentration and specific activities of enzymes. The results, given in Table 1.3, showed a general reduction in drug-metabolizing ability, the extent of reduction (if any) depending on the stage of pregnancy, species and enzyme system involved.

In an attempt to assess microsomal enzyme activity in pregnant women,

Davis,Simmons, Dordoni, Maxwell and Williams (1972) measured the urinary excretion of D-glucaric acid which is formed via the glucuronic pathway in the liver by a series of microsomal enzymes and is a commonly used index of hepatic microsomal drug metabolizing activity.

Its excretion was found to increase progressively from about the 12th week of pregnancy, and this would suggest that these enzymes are induced during pregnancy. This idea is supported by the high incidence of folic acid deficiency in pregnant women, since folate is known to be a co-factor for microsomal enzymes, and the observation of Perez, Grodisch, Casavilla and

Maratto (1971) who found an increased proliferation of the smooth endoplasmic reticulum where these enzymes were situated. In an attempt to measure functional liver size, Muller and Kappas (1964) using bromosulphthalein Table 1.3 Observed alterations in hepatic drug metabolism during pregnancy

RATS (15-16 days pregnant) Liver weight increased by 33% (Neale and Parke, 1973).

RATS (19-20 days pregnant) Liver weight increased by 40%; conjugation of xenobiotics with glucuronic acid (Cessi, 1952), glutathione (Combas and Stakelum, 1966) and sulphate (Pulkkinen, 1966) have been shown to be inhibited; cytochrome P-450 concentration is decreased by 25% and the specific activities of 4-methylumbelliferone glucuronyl transferase and biphenyl-4-hydroxylase are decreased by 25% and 30% respectively, but those of biphenyl-2-hydroxylase and p-nitrobenzoic acid nitro-reductase are not changed (Neale and Parke, 1973); Above changes in activities of the enzymes nullified by pre- treatment of the animals with phenobarbitone or 3-methylcholanthrene.

RABBIT (full term) No alteration of liver weight, cytochrome P-450 level or biphenyl-4- hydroxylase activity; Glucuronyl transferase activity decreased by 20% rn and coumarin-7-hydroxylase decreased by 60%. These changes were not prevented with either phenobarbitone or 3-methylcholanthrene pre- treatment (Neale and Parke, 1973).

HUMAN (late prgnancy) Increased proliferation of the smooth endoplasmic reticulum (Perez et al., 1971); decreased metabolism of pethidine and promazine (Crawford and Rudofsky, 1966); increased metabolism of DDT (Kronger,1972); D-glucaric acid excretion increased (Davis et al.,1973); BSP liver function test shows impairment of hepatic excretory capacity (Muller and Kappas, 1964); folic acid concentration decreased. 57

(BSP), found that its excretion was impaired in pregnant women. Since

elevated levels of conjugated BSP were found in the plasma, it appears

that the transfer of dye compounds from the hepatocytes to the canal-

icular spaces was impaired.

Many explanations have been offered fer the observed alterations in

drug metabolism during pregnancy. High levels of circulating hormones

have been suggested as being responsible for the decrease in drug

metabolizing ability in animals (Pulkkinen, 1966) due to competitive

inhibition by high levels of endogenous steroids for binding sites.

However, this is not the reason for the decrease in activity observed

in some Phase I reactions since Guarino, Gram, Schroeder, Call and

Gillette (1969) found that V values were significantly lower in max 19 day pregnant rats than in control animals for aniline hydroxylase

and ethylmorphine-N-demethylase, but the K values were not signifi- m cantly different from controls. This indicated that the decreased

activity of the microsomal drug metabolizing enzymes might be due to

a reduction of the enzyme haemoprotein, although again this does not

provide a satisfactory explanation since no decline in cytochrome

P-450 concentration was observed in the pregnant rabbit. Furthermore,

the decreased metabolic activity observed in rats was reversed by

treating the rat with phenobarbitone, but this reversal was not accom-

panied by an increase in cytochrome P-450. This suggests that other

factors, e.g. cytochrome P-450 reductase, may be rate limiting al-

though this does not explain the selectivity of the decreases in enzyme activity observed in animals_. The alterations in drug metabolism found in laboratory

animals are all the result of selective inhibition of certain enzyme

activities, but in man the situation is less clear there being evidence 58

of both increased and decreased activity of drug metabolizing enzymes,

(Table 1.3). The reasons for these changes are unknown although the high levels of circulating hormones are in some way responsible since similar changes have been observed in animals and women receiving oral contraceptives, progesterone and stilboestrol.

1.3.3 Drug metabolism by the placenta

The function of the placenta as an organ of transport between mother and fetus is well established, it being known that the placenta does not represent a barrier to a wide range of compounds capable of influencing the fetus at critical stages of development. However, it is possible that the placenta may play a role as an extrahepatic drug- metabolizing organ which acts in conjunction with the fetal enzyme system to protect embryonic development from toxic compounds.

Components of the electron transport chain typical of the drug oxidizing system, i.e. cytochrome P-450 and its reductase, have been found in human placental microsomes (Miegs and Ryan, 1968; Juchau and Zachariah,

1975). Evidence for each of the four major classes of reactions

(i.e. oxidation, reduction, hydrolysis and conjugation) has been presented (Juchau and Dyer, 1972). Studies by several groups of workers

(Juchau and Yaffe, 1969; Creaven and Parke, 1965) suggests that the oxidation of substrates associated with cytochrome P-450, thought to bind

(a) to the hydrophobic sites on the oxidase (e.g. hexobarbitone, aminopyrine) or (b) directly to the haeme moiety (e.g. aniline), proceed at low or negligible rates in placental homogenates, although there appear to be exceptions, e.g. strychnine (Pecile, Chiesara, Finzi and Conti, 1969). On the other hand abundant evidence now exists that oxidation of substrates associated with cytochrome P-448 (e.g. benzo(a)pyrene, 3-methylcholanthrene) 59

will occur in placental tissues from a variety of species including humans, although this activity is not present in every placenta. The low level of the placental oxidase system observed in both man and animals may well reflect a normal 'baseline' situation where no challenge to the well being of the fetus is apparent. It is of interest to know whether the placenta can respond to such an external stimulus, and there is some evid_nce for the inducibility of placental enzymes. Studies in this field have concentrated on the effect of cigarette smoking on the drug-metabolizing capacity of the human placenta, since cigarette smoking during pregnancy increases the probability of the baby having a low birth weight. Table 1.4 shows that the human placental enzyme, benzo(a)pyrene hydroxylase is either completely absent or only present in small amounts in tissue obtained from non-smokers, whereas it is present in much greater quantities in tissue taken from smokers at delivery. The enzyme activity appears to be related to the number of cigarettes smoked/day showing that smoking induces the enzyme (Welch,

Harrison, Gommi, Poppers, Finster and Conney, 1969). Of the many components of cigarette smoke, polycyclic hydrocarbons would appear likely candidates as inducing agents, and a variety of polycyclic hydrocarbons found in cigarette smoke have been shown to induce benzo(a) pyrene hydroxylase activity in rat placenta (Welch et al.,

1969) .

Since the harmful effects of many teratogens are exerted during the first trimester of pregnancy, it is important to know whether the human placenta has significant drug-metabolizing ability during this 60

period. The evidence indicates that this is not the case - activity

being undetectable at 8 to 10 weeks and apparent only in smokers

between 11 and 13 weeks; no activity was apparent in non-smokers

between 8 and 16 weeks (Juchau, 1971; Pelkonen, Jouppila and Karki,

1972). As a result, early in pregnancy the placenta is not a 'meta-

bolic barrier' to most xenobiotics, which can therefore pass unchanged

into the fetus.

Table 1.4 Effects of cigarette smoking on human placental benzo(a)-

,pyrene hydroxylase activity (at delivery)

No. of cigarettes Benzo(a)pyrene hydroxylase smoked/day (pg-3-hydroxybenzo(a)pyrene/g/hr)

0 <0.1

10 0.5

10 5.0

20 0.6

20 17

40 23

(from Welch et al., 1969)

1.3.4 Drug metabolism by the fetus

Drug metabolism during fetal life has been investigated in various

animal species, particularly just prior to birth. In general,

negligible metabolism has been found, and the ability to metabolize

drugs increases postnatally with rates depending on the species and

drug substrate studied (Jondorf, Maickel and Brodie, 1958; Fouts

and Adamson, 1959). Since studies of the development of drug metabolizing enzymes in the human fetus have only been performed following 61

abortions and hence only cover about the first 20 weeks of gestation,

the animal data obtained was extrapolated to man. However, more recent

studies suggest that the human fetus may differ from that of other animal species with regard to drug metabolizing capabilities.

Zaurboni (1965) studied the developmental histology of the human fetal liver and found that smooth endoplasmic reticulum (SER) appeared around the third month of gestation, and deposits of glycogen and iron appeared in the hepatocytes at the same time. Further studies showed

that the appearance of SER seemed to coincide with the appearance of drug-oxidizing enzymes in the human fetal liver (Pelkonen, 1973), and the appearance of SER occurred much earlier than in animal fetal liver (Fukuda, 1974). Yaffe, Rane, Sjdgvist, Borēus and Orrenius

(1970) examined the liver from 13 aborted human fetuses with gestational ages 14 - 25 weeks, and detected cytochrome P-450 and NADPH-cytochrome c reductase in the microsomes. Their levels varied but were not related to gestational age. The level of cytochrome P-450 was of the same order of magnitude as those reported previously for human adult microsomes obtained from surgical biopsies, but the specific activity of NADPH-cytochrome c reductase was only one-third of that of the adult liver microsomes (Alvares, Shilling, Levin, Kuntzman Ft Brand, 1970).

Examples of hepatic drug metabolism in the human fetus in vitro are given in Table 1.5. Generally, fetal hepatic levels of mono-oxygenase system components and related enzyme activities in the second trimester of gestation vary from 20 - 50% of adult hepatic levels (Pelkonen, 1973), or even higher (Ackerman, 1972). Moreover, it has been shown that the liver represents about 4 - 5% of the fetal weight but only about 2% of the adult weight. As a consequence, the activities of TABLE 1-5 Drug Metabolism by Human Fetal Liver Microsomes

Enzyme Activity Reaction Age of Fetus Substrate (% adult value) Reference

Oxidation 8-22 weeks Hexobarbitone 40% Pelkonen, Vorne and Kārki (1969) Hydroxylation 8-26 weeks Benzo(a)pyrene 5-10% Pelkonen, Vorne, Joupille and Kārki (1971) Hydroxylation 8-22 weeks Aniline 40% Rane and Ackermann (1971) Sulphur oxidation 8-26 weeks Chlorpromazine 5% Pelkonen et al (1971) N-Demethylation 14-25 weeks Aminopyrine low Pomp, Schnoor and Netter (1969) N-Demethylation 8-26 weeks N-Methylaniline 5-50% Pelkonen et al (1971) -- Nitro- reduction 8-22 weeks p-Nitrobenzoic acid low Pelkonen et al (1971) — —

Alcohol dehydrogenase 8-26 weeks Ethanol low Pikkarainen and Raiha (1969) Glucuronic acid 8-22 weeks Q-Nitrophenol not detected Pelkonen -etal Conjugation (1969) 12-16 weeks o-Aminophenol 13% Dutton (1959) 1 not known 4-Methyllumbelliferone low Hirvonen (1966) 63

those enzymes per unit body weight of the fetus may approach those of human adults.

With respect to the metabolism of some substrates, the fetal adrenal glands seem to be even more active than the fetal liver (Juchau and

Pederson, 1973) (Table 1.6). Benzo(a)pyrene hydroxylase, nitro- reductase and azo-reductase activities are all very high, the highest levels being exhibited between the 12th and 16th weeks of gestation.

Again, the fetal adrenal glands are relatively larger than the corresponding adult organs and it may be concluded that the fetal adrenal glands may be important in the total drug oxidation capacity of the human fetus.

In summary, the ability of the human fetus to metabolize drugs is low compared to the adult. A notable exception exists in the case of fetuses of smoking mothers whose livers may be shown to metabolize benzo(a)pyrene and similar substrates (Welch et al., 1969). In general, it appears that the human fetus is more capable of oxidation of substrates than other species.

1.3.5 Drug metabolism by the neonate

In 1958, Jondorf, Maickel and Brodie demonstrated the inability of the newborn guinea-pig to metabolize several drugs, and the same was observed for other experimental animals including rat, rabbit and swine. With few exceptions, newborn animals have a very low capacity to metabolize drugs, but this ability is acquired quite rapidly postnatally. Maximum activity in the rat is attained at 20 - 35 days post-partum (Basu, Dickerson and Parke, 1971) and in the guinea pig

Table 1. 6 Drug Metabolism by Human Fetal Adrenal Homogenates

REACTION AGE OF FETUS SUBSTRATE ENZYME ACTIVITY REFERENCE (% fetal liver activity)

Oxidation 8-22 weeks Hexobarbitone present Pelkonen (1976)

Hydroxylation 8-26 weeks Aniline 200 Pelkonen (1976)

Nitro-reduction 8-26 weeks p-Nitrobenzoic 180 Juchau and acid Pederson (1973) N-Demethylation 8-26 weeks Aminopyrine present Pelkonen (1976)

Hydroxylation 8-26 weeks Benzo (a) pyrene 30-130 Pelkonen and Karki (1975) Azo reduction 8-26 weeks Neoprontosil 130 Juchau and Pederson (1975) 65

at 3 days after birth (Kuenzig, Kamm, Boublik and Burns, 1975), although there is a variation in the time taken for the different pathways to reach their maximum activity. This post-natal development of these enzymes has been attributed to enzyme induction resulting from the exposure of the newborn animal to foreign chemicals in its food and environment (Fouts and Hart, 1965). Rats weaned early and removed from their mothers have been found to have a higher 4- methylcoumarin hydroxylase activity and hexobarbitone oxidation than their unweaned counterparts, although aminopyrine demethylation was not affected (Henderson, 1971). The effect of weaning may be mediated by hormonal changes, particularly of progesterone and growth hormone, although food, temperature, space and other environmental influences may trigger drug metabolizing-enzyme activity.

Human neonates have also been shown to have a low capacity for drug metabolism as indicated by increased plasma half-lives of drugs in infants compared to the adult (Table 1.7). Their oxidative metabolic capacity in particular has been shown to be deficient. Furthermore, it appears that the rate of development of the various enzyme systems is not uniform as shown by the metabolism of diazepam, i.e. N-demethylation of this drug was found in premature and newborn infants as in adults, but hydroxylation reactions present in adults were absent

(Garattini, 1971). Other examples of drug metabolism reactions in the human neonate in vivo are given in Table 1.8. The low levels of enzyme activity observed in both man and animals might be attributed to the presence of an endogenous inhibitor, to a lack of cofactors or to limited amounts of the enzyme involved. Despite early reports of endogenous inhibitors (Fouts and Adamson, 1959), studies in NADP Table 1.7 Plasma Elimination Half Lives of Drugs in infants and Adults

COMPOUND NEONATAL HALF-LIFE ADULT HALF-LIFE REFERENCE

Amylbarbitone 40h 16h Krauer, Draffan, Williams, Clare, ])ollery and Hawkins (1973)

Nortriptyline 56h 17h Sjoquist, Bergfors, Borga, Lind ani Ygge (1972)

Diphenylhydantoin 72h 12h Mirkin (1971)

Phenobarbitone 77-4o4h *48-144h Jailing, Boreus, Kallberg and Agurall (1973) *Maynert (1972) Salicylic acid 70h 2.4-19h Earle (1971)

Antipyrine 59h 10.4h Murdock, Thorgeirsson, Rossigner and Davies (1973) Table 1.8 Drug Metabolism Reactions by the Human Neonate in vivo

Reaction Age of Neonate Substrate Enzyme Activity Reference

N-Demethylation 24h Pethidine low O'Donaghue (1971) Ester hydrolysis 24h Pethidine low O'Donaghue (1971). Hydroxylation 1-5days Diphenylhydantoin low Horning, Butler, Nowlin and Hill (1975) Hydroxylation 1-5days Phenobarbitone low Horning et al (1975) N-Demethylation 24h Caffeine low Horning et al (1975) Glycine conjugation 24h g-Aminobenzoic acid low Vest and Salzberg (1968) Glucuronic acid conjugation 24h E-Aminobenzoic acid low Vest and Salzberg (1968)

Acetylation Premature babies E-Aminobenzoic acid low Vest and Salzberg (1968) Oxidation O-48h Tolbutamide not detected Nitowsky, Metz and Berzofsky (1966) Oxidation Premature/Neonate Diazepam not detected Garattini (1971) N-Demethylation Premature/Neonate Diazepam low Garattini (1971) Glucuronic acid 5days Salicylamide low Stein (1970) conjugation 68

and glucose 6-phosphate enriched systems suggest an actual enzyme

system deficiency (Soyka, 1969), and Dutton (1959) demonstrated the

deficiency of glucuronic acid conjugation in the guinea pig and mouse

to be due to low levels of the cofactor UDP-glucuronyltransferases.

That the reduced drug-metabolizing ability of newborn animals is due

to an actual enzyme deficiency is suggested by the effects of enzyme

inducers since it appears that immature animals are intrinsically more

responsive to enzyme induction; e.g. orphenadrine and tofenacine

cause. significant enzyme induction in young rats although they are

without effect in older animals (Funcke and Timmerman, 1973), and

treatment of pregnant rats one week prior to delivery with phenobarbitone

resulted in increased metabolic ability in the newborn for 3 - 4 weeks,

whereas in adults induction lasted only 5 - 7 days (Mitoma and La

Valley, 1970). However, the administration of known inducing agents

to pregnant animals has no effect on neonatal drug metabolism except

when given in the last few days of gestation. This is probably due

to a maternal inhibitory factor and likely candidates are gonadal hor-

mones which decrease the mother's drug-metabolizing capacity (Fouts

and Hart, 1965; Feuer and Liscio, 1970). This phenomenon of trans-

placental enzyme induction is also observed in the human neonate where

infants born to mothers receiving phenobarbitone treatment have low serum bilirubin concentration due to an increased capacity to eliminate

the bilirubin via glucuronidation (Crigler and Gold, 1966).

It is not clear which of the components of the electron transport chain are responsible for the increase in drug-metabolizing enzyme activity observed after birth. Several studies have shown cytochrome P-450 levels to increase with drug-metabolizing ability (Danner, Siekevitz 69

and Palade, 1965; Basu et al., 1971) although other studies did not

confirm these findings (Gram, Guarino, Schroeder and Gillette, 1969).

Similarly, NADPH-cytochrome c reductase and NADPH-oxidase have been

implicated (Kato, Vassanelli, Frontino and Chiesara, 1964; Uehleke,

Reiner and Hellman, 1971). It thus appears that the complex pattern of postnatal development described cannot be correlated with a single enzyme

but perhaps must be considered in terms of the development of the

system as a whole.

1.4 Obstetric Analgesic Procedures

In the U.K. the relief of pain during childbirth is usually achieved

by one or more of the following methods.

(i) psychoprophylaxis ('breathing exercises')

(ii) narcotic analgesics (usually pethidine)

(iii) inhalation anaesthetics (a) 50% nitrous oxide and 50% oxygen

(b)0.5% trichloroethylene in air

(iv) regional anaesthesia utilizing a local anaesthetic.

Psychoprophylaxis, although taught to a large proportion of women attending ante-natal classes, is successfully practised during labour by only a minority of women, and the majority of patients using this method often require an additional method of analgesia for the second stage of labour, i.e. the expulsion of the fetus from the uterus.

Of the three inhalation agents generally used, analgesia is often inadequate with nitrous oxide and inhalation of methoxyflurane and trichloroethylene needs to be restricted to fairly short periods to avoid their accumulation in mother and fetus, so that these agents are commonly used in combination with pethidine. Pethidine is the 70

most popular obstetric analgesic in use today being practical for both hospital and home deliveries. However, the popularity of regional obstetric anaesthesia is increasing since it can give complete relief from pain without loss of maternal consciousness, its main limitation being the requirement for a trained anaesthetist. The agents used for regional anaesthesia are the xylidide local anaesthetics (lignocaine, mepivacaine and bupivacaine), of which bupivacaine is the drug of choice at present. The chemistry, pharmacological actions and use in childbirth of both pethidine and bupivacaine are discussed below.

1.4.1 Pethidine

Pethidine (meperidine, Demerol) was one of the first synthetic narcotic analgesics, being introduced into clinical use in 1939 by Eisleb and

Schaumann as a spasmolytic agent with analgesic properties. It is now, after morphine, the most widely used narcotic analgesic having approximately one-tenth the potency of morphine. Although at first sight pethidine appears chemically quite dissimilar to morphine, it shows the same spectrum of pharmacological actions, and it is presumed that its mechanism of action is the same as that of morphine.

0 C2H5

MORPHINE PETHIDINE 71

The structure of morphine and compounds with morphine-like activity

possess three characteristic features:

(i) a methyl group attached to the tertiary nitrogen atom

(ii) several oxygen-containing groups situated at a distance of 7

to 9 angstrom units from the tertiary nitrogen, and

(iii)at least one aryl nucleus attached to an asymmetric carbon

which is joined by a short hydrocarbon chain to the tertiary

nitrogen.

The apparent necessary structural backbone of the compounds led to the proposal of a complementary analgesic receptor site by Beckett and Casy in 1954, the essential features of this receptor being a) a flat portion that allows binding with the aromatic ring of the analgesic through hydrophobic bonding, (b) an anionic site that associates with the nitrogen atom of the drug in its charged state and (c) a cavity suitably orientated to accommodate the projecting hydrocarbon chain of the piperidine ring.

Pethidine, like other narcotic analgesics, exerts its chief pharmacological actions on the CNS. Therapeutic doses produce analgesia, sedation, euphoria, respiratory depression and nausea and vomiting, the latter effect due to a release of antidiuretic hormone and stimulation of the chemical trigger zone (CTZ). Pethidine produces analgesia chiefly by acting on the cerebral cortex, and this is relatively specific as the sensitivity of the CNS to other stimuli such as touch, smell and sound is little altered, although it may decrease visual perception. The perception of the pain stimulus itself is not always decreased but there appears to be an altered reaction to the painful stimulus making it more tolerable, as well as an 72

alleviation of the fear, anxiety and anticipation associated with pain.

Pethidine has been used for obstetric analgesia with increasing frequency since it was reported to cause minimal or no respiratory depression in the neonate (Schumann, 1944), but many other later reports have contradicted this observation. It remains the standard analgesic drug for obstetrics in Great Britain even though its low efficiency (25%, Holdcroft and Morgan, 1974; 60%, Beazley, Leaver,

Morewood and Bircumshaw, 1967), as an analgesic in labour has been demonstrated.

Pethidine is normally administered during labour by intramuscular injection in doses of 100 - 150 mg, usually in combination with an antiemetic drug, such as promazine, to counteract the nausea produced by CNS stimulation. Early evidence of its placental transfer was based on the clinical observation that babies born to mothers who had received pethidine during labour were more likely to exhibit respiratory depression, a known pethidine side-effect, and require resuscitation (Taylor, Fumetti, Essig, Goodman and Walker, 1955).

The placental transfer was confirmed by the finding of pethidine in neonatal urine (Way, Gimble, McKelway, Ross, Sung and Ellsworth,

1949). Because of the important place of pethidine in the management of labour, further studies were then set up to study the rate of transfer of the drug across the placenta, its concentration, pharmacological action and elimination from the neonate.

Maternal venous concentrations of pethidine have been shown to reach a peak within minutes following intramuscular injection, and transfer 73

across the placenta is extremely rapid. Crawford and Rudofsky (1965) detected pethidine in cord blood as soon as two minutes following its

intravenous administration to the mother, the concentration of pethidine

in cord blood subsequently falling exponentially, parallel to but

always lower than, the level in maternal blood. Apgar, Burns, Brodie

and Papper (1952) using a non-specific assay method found that the

plasma concentration of pethidine in cord blood varied between 45 -

106% of the maternal venous concentration at delivery, although there

appeared to be no relationship between cord concentration and dose-

delivery interval. Bonica (1957) found that peak respiratory depression in the

newborn occurred if the baby was delivered between 1 and 4h after

maternal administration, and should peak depression be related to

peak concentration, this may suggest that pethidine concentration

reaches a peak in the fetus between 1 and 4h after maternal administration.

Pethidine has been shown to undergo metabolic transformations in adults

along several pathways, particularly N-demethylation and ester hydro-

lysis. Burns, Berger, Lief, Wollack, Papper and Brodie (1955) found

that some 5% of the administered dose was excreted unchanged, with

5% as the N-demethylated product (norpethidine), 12% as the product

of ester hydrolysis (pethidinic acid) and 12% as the N-demethylated

deesterified product (norpethidinic acid). Plotnikoff, Way and

Elliot (1956) demonstrated that both the acid compounds were excreted

both free and conjugated with glucuronic acid. None of these pro-

ducts appeared to have any analgesic action although pethidine meta-

bolites have been suggested to be responsible for fetal depression,

(Morrison, Wiser, Rosser, Gayden, Bucovaz, Whybrew and Fish, 1973).

Two minor metabolic products have recently been identified but not 74

quantitated; pethidine-N-oxide (Mitchard, Kendall and Chan, 1972), and 4'-hydroxypethidine (Lindberg, Bogentoft, Bondesson and Danielson,

1975).

The reports in the literature concerning the ability of the neonate to metabolize pethidine are contradictory. Crawford and Rudofsky

(1965) failed to demonstrate metabolism in two babies (of ten) exposed to pethidine as a result of placental transfer, since only the parent drug was excreted in 0 - 48h urine. The remaining eight infants excreted norpethidine (range 10 - 203ug) and pethidine (range 62 -

420pg) in the same period, each mother being given a 50mg dose of pethidine during labour. Two infants given a lmg intramuscular injection of pethidine excreted 25% and 40% of the dose in 48h urine as pethidine. No metabolites were detected. However, O'Donaghue

(1971) identified both norpethidine and pethidinic acid in the urine of neonates who received the drug either from the mother via placental transfer or intramuscular injection (lmg). Hogg, Wiener, Rosen and

Mapleson (1977) reported that following maternal administration of pethidine during labour, the ratio of norpethidine to pethidine excreted in neonatal urine increased with time.

Pethidine produces respiratory depression in the neonate (Koch and

Wendall, 1968), and the behavioural changes attributed to pethidine in the newborn have been discussed earlier. Such changes in behaviour may be discernable for several days after birth (Brackbill, Kane,

Maniello and Abramson, 1974) or even weeks (Richards and Bernal,

1972). Even so pethidine is the most commonly used analgesic in childbirth in this country, which makes it necessary that any long- 75

term effects on the development of the neonate be assessed.

1.4.2 Xylidide local anaesthetics

Local anaesthetics are drugs which block nerve conduction when applied

locally to nerve tissue in appropriate concentrations. They act on

all parts of the nervous system, both central and peripheral, and on

every type of nerve fibre. A local anaesthetic in contact with a

nerve trunk causes both sensory and motor paralysis in the area in-

nervated, and the block is completely reversible with no permanent

damage to nerve function and structure. The clinically useful local anaesthetics have a common fundamental structure consisting of 3 parts;

(i) a hydrophilic amino group

(ii) a lipophilic aromatic group

(iii)an intermediate chain linking (i) and (ii) together The members

of this chain are usually a carboxyl group (procaine and urethane),

an amide group (lignocaine and bupivacaine), a carbamoyl group

(urethane), or a carbonyl group (falicaine).

The first local anaesthetic to be discovered was cocaine, an alkaloid found in the leaves of the coca plant, Erythroxylon coca, and in 1884, it was the only local anaesthetic in clinical use. However, its addictive properties and powerful stimulant action on the CNS led to a search for suitable synthetic alternatives and in 1948 Ldfagren synthesized the first of the xylidide local anaesthetics, 2-diethylamine-2',6'- acetoxylidide (lignocaine). Further progress was made in 1957 when

Ekenstam, Egner and Petterson synthesized a group of aromatic amides of N-alkyl pyrrolidine and N-alkyl piperidine carboxylic acids as potential local anaesthetics, among them 1 -methyl-2-(2',61-xylyl- 76

carbamoyl)-piperidine (mepivacaine) and 1-n-butyl-2-(2',6'-xylylcarbamoyl)-piperidine (bupivacaine) both of which are used extensively today.

Local anaesthetics block transmission through all nerve fibres whether they are sensory, motor or autonomic in function. This block of nerve conduction arises at the cell membrane of the nerve fibre by the prevention of the normal generation and conduction of electrical impulses since the transient permeability of the membrane to Na+

(which is necessary for the action potential) is blocked. The mechanisms involved in these changes are still obscure, although it is thought that local anaesthetics may act by mimicking and antagonizing the action ++ of Ca at its receptors in the membrane.

Not all nerve fibres are equally vulnerable to the actions of local anaesthetics. As a general rule, small diameter fibres are more easily blocked than large ones, and this explains why all sensations are not lost simultaneously.after administration of a local anaesthetic since they are probably mediated by nerves of different diameters.

The order of sensation loss is rather variable but usually pain is abolished first, followed in turn by the sensations of cold, warmth, touch and deep pressure. The sensations reappear in the reverse order as the action of local anaesthetic declines.

Other important pharmacological actions of the xylidide local anaesthetics occur on:

(a) the cardiovascular system, although in general these are only

seen when high concentrations of drug are present in the body.

Here they act directly on the myocardium, where decreases in 77

electrical excitability, conduction and in the rate and force

of contraction occur, and also on the vasculature, usually

causing arteriolar dilation. The action of mepivacaine in

spontaneously beating isolated hearts from human fetuses at mid-

gestation, when organogenesis of the heart is considered to be

complete, has also been examined. Low concentrations of mepi-

vacaine, comparable to the' concentration in blood following

epidural administration, added to the perfusion fluid, resulted

in a reduction of spontaneous contraction rate and active sys-

tolic pressure, and an increase in atrio-ventricular and intra-

ventricular conduction time. On lowering the pH of the medium

from 7.35 to 7.00, these effects were more pronounced (Andersson and

Nilson, 1970). Such effects are due to mepivacaine acting

directly on the myocardium and the above observations are probably

the result of asynchronous excitation and contraction of the

myocardial cells (Genser, 1970).

(b) the CNS, but again these are only usually seen with high concen-

trations in the systemic circulation. All local anaesthetics

have a CNS stimulant action and overdoses may lead to tremors,

restlessness and convulsions. Medullary stimulation may cause

bradycardia, hypertension and respiratory stimulation . Central

depression may occur later and death may occur from respiratory

depression.

Local Anaesthetics and Obstetric Analgesia

The basic aim of nerve blocks in obstetrics is to place a solution of a local anaesthetic in close apposition to the relevant nerve trunks or roots, so as to create an area of insensitivity distal to the 78

site of injection so as to relieve the pain of the contractions and allow obstetrical manoeuvres such as forceps extraction to be carried out. The pain of the first stage of labour originates from both the corpus and cervix of the uterus, with the sympathetic fibres from the corpus uteri originating in the posterior cervical ganglia and the pelvic plexus and ganglia before eventually entering the spinal cord with the posterior roots of the 11th and 12th thoracic segments

(Fig. 1.5). With the beginning of the second stage of labour at full cervical dilation, the descent of the fetus causes the painful impulses to shift from the uterus to the distending vagina, perineum and vulva, and painful sensations from these areas reach the spinal cord via the 2nd, 3rd and 4th posterior sacral roots, mainly by branches of the pudendal nerves (Fig. 1.5). Blocking sensory pathways from the cervix, uterus and birth canal can be accomplished by injecting a local anaesthetic solution in the vicinity of these nerve trunks or roots, and this can be achieved by several methods (listed below) which bear the name of the site where the block is performed:

(a) subarachnoid block

(b) epidural block

(i)lumbar

(ii)caudal

(iii)thoracic

(d) pudendal block

Epidural block anaesthesia is the most commonly used of the regional blocks in obstetrics and is discussed below.

The epidural space is the area between the dura of the spinal cord Fig. 1.5 Pain pathways in labour in relation to conduction block .T 10 1

Possible afferent pathway -- Upward spread T.1 accompanying the ovarian / (patient recumbent) vessels m L.2 ~ S.AA Spinal cord Main afferent sympathetic pathway for uterine pain LUMBAR EPIDURAL BLOCK Subarachanoid space Dura-arachnoid Extra dural space Downward spread (patient sitting)

Pelvic splanchnic nerve S.~ Vagina

Filium Terminale Pudendal nerve / d' Levator ani C.1 acral Hiatus Skin ,•

PARACERVICAL BLOCK PUDENDALt BLOCK CAUDAL EPIDURAL BLOCK (from Wylie and Churchill- Davidson, 1972) 80

and the ligaments and periosteum lining of the vertebral canal, and extends from the foramen magnum to the sacral hiatus (Fig. -

1.5). This space has been described as a 'potential space' since it is normally completely filled with a loose type of adipose tissue, lymphatics and blood vessels. Solutions injected into the epidural space will spread in all directions between the loose tissue structures that occupy this area thus blocking conduction in (i) the dura covered nerve roots within the epidural space, (ii) subarachanoid nerve roots after diffusion across the dura, and (iii) paravertebral nerve trunks. Lumbar epidural anaesthesia is the more commonly employed technique for obstetrics in this country, the local anaesthetic solution being introduced below the level of L2 (Fig. 1.5).

From here the anaesthetic is capable of blocking the posterior roots of the 11th and 12th thoracic segments for 1st stage labour pains as well as extending to the sacral segments for 2nd stage labour pains.

Providing the solution does not extend above the level of T11 and T12, it is possible to block all sensory impulses from the uterus without seriously interfering with motor activity.

The drug of choice for lumbar epidural anaesthesia at present is bupivacaine since it has the longest duration of action of the local anaesthetics in clinical use as well as giving rise to a high degree of sensory block in relation to motor block. Relatively little is known about its toxic effects on the fetus, and until recently it was thought that barring maternal systemic complications, bupivacaine had no demonstrable effect on the fetus. However, since bupivacaine can cross the placenta into the fetal circulation, it could possibly have adverse effects on the fetus.

Absorption of bupivacaine from the epidural space has been shown to be extremely rapid. Following lumbar epidural administration, maximum maternal venous concentrations were reached within 5 - 30 minutes (Caldwell, Moffat, Smith, Lieberman, Beard, Snedden and

Wilson, 1977; Belfrage, Raabe, Thalme and Berlin, 1975). Placental transfer of bupivacaine, shown by its presence in fetal scalp blood, was found as early as ten minutes after maternal administration, and increased slowly as the concentration in the maternal circulation declined (Fig. 1.5) (Caldwell et al., 1977).

Fig.1.6 Blood concentrations of bupivacaine in mothers and their fetuses following the epidural injection of bupivacaine to the mothers during labour. (each point indicates the mean ± S.E., N=18)

300—

Maternal

io 100 t a tr en

c 50 n co

e 30

in Fetal a ac iv

Bup 10 epidural 30 60 90 injection TIME (min)

The fetal scalp/maternal vein concentration ratio increased initially from

0.16 at 10 minutes to 0.26 at 30 minutes (Berlin, Belfrage and Magno,

1974) or from 0.17 at 20 minutes to 0.34 at 60 minutes (Caldwell et al.,'

1977), but following this, the ratio was found to remain fairly constant 82

due to a relative drug equilibrium being achieved between mother and

fetus. At delivery, the average fetal:maternal concentration ratio

was in the range 0.3 - 0.5 (Thomas and Mather, 1969;

Reynolds and Taylor, 1970; Caldwell et al., 1977), the umbilical

venous value being slightly more than the umbilical arterial concen-

tration (Table 1.9). This suggests that there is a net transfer of

bupivacaine from the mother to the fetus with some disappearance of

the drug from the fetal circulation. Furthermore, the above investi-

gations showed that there was no relation between the time from ad-

ministration of the drug to blood sampling (i.e. delivery) and the

fetal:maternal concentration ratio. Plasma protein binding of bupi-

vacaine in fetal blood has been found to be approximately half that

in maternal blood where 78 - 92% of bupivacaine was found bound to

maternal plasma protein at drug concentrations in the range 0.05 -

5.Oug/ml (Mather, Long and Thomas, 1970). This difference between

maternal and fetal plasma protein binding might influence the transfer of bupivacaine across the placenta as described previously.

The rate of disappearance of bupivacaine from the blood of newborn babies has been reported to be markedly slower than is observed for adults. The neonatal blood elimination half-life of bupivacaine is

18h while the corresponding value in adults is 1.25h (Caldwell et al.,

1977a). Only very small amounts of bupivacaine have been recovered in adult urine after its administration, indicating that its disappearance from blood is due to metabolism rather than renal elimination.

This suggests that the neonate is less able to metabolize the drug than the adult and therefore it would be-expected that any drug effects would persist for a longer length of time compared to the adult. Table1.9 Lumbar epidural anaesthesia with bupivacaine; Maternal and umbilical blood concentrations at delivery (ug/ml blood)

Maternal Number Maternal Umbilical Umbilical Dose-delivery Reference dose (mg) in study vein vein artery interval

*50 - 150 12 0.12-0.41 0.04-0.25 0.04-0.25 40-500 mins Thomas Climie and Mather(1969)

60 - 250 9 0.13-1.26 0.04-0.34 0.04-0.34 240-555 mins Reynolds and Taylor (1971)

*60 - 210 9 0.17-0.91 0.07-0.31 0.09-0.34 120-555 mins Reynolds and Taylor (1971) m * 56.7 21 0.04-1.96 0.00-0.33 0.00-0.50 11-324 mins Hyman and Shnider (1971) u'

82.9 11 0.04-0.72 0.01-0.16 0.02-0.17 22-430 mins Hyman and Shnider (1971)

* 96.4 32 0.04-0.44 0.00-0.17 0.01-0.25 36-537 mins Hyman and Shnider (1971)

50 - 500 35 0.14-1.62 0.03-0.48 0.03-0.48 38-1815mins Reynolds, Hargrove and Wyman (1973) *40 - 440 35 0.09-1.21 0.02-0.30 0.02-0.30 101-1155mins Reynolds, Hargrove and Wyman (1973) 50-- 645 23 0.10-0.76 not deterr - 0.02-0.25 24-1350mins Reynolds and Taylor (1970)

*25 - 170 33 0.07-0.59 0.01-0.26 0.02-0.14 60-970 mins Belfrage, Berlin, Raabe and Thalme (1975) **35 ± 3.2 6 0.22-0.03 0.06-0.01 0.07-0.01 no data Caldwell, Moffat, Smith, Lieberman and Beard (1977)

* Bupivacaine with adrenaline 1:200,000 **mean-S.E. 84

Schifrin (1972) has reported that the use of paracervical block in

humans with mepivacaine or bupivacaine, produced changes in fetal

heart rate in 30% of cases, 25% showing fetal bradycardia and 5% tachy-

cardia. The bradycardia was found to occur in fetuses with higher than average levels of drug in the blood, as measured by fetal scalp samples.

Teremo (1969) reported that 38% of bupivacaine paracervical block cases developed fetal bradycardia of a moderate degree, (100 - 118bpm) and that

7% developed severe bradycardia (

blood at 2 minutes before the block was 7.35, which fell to a mean of 7.30 at 15 minutes, and was still low at 40 minutes, there being a direct correlation between severity of bradycardia and the intensity of acidosis. Other authors (Whitehouse, 1968; Gudgeon, 1968) have found that paracervical block with bupivacaine has been associated with extreme fetal bradycardia resulting in mortality. Behavioural changes, e.g. an increase in the incidence of neonatal depression and a decrease in muscle strength and tone, have also been found in babies born after epidural block (Morishima, Salha and Miecyshaw, 1966; Hales,

Daily and Meyer, 1975). However it has yet to be established whether the observed bradycardia is a direct action of bupivacaine on the fetus or indirect caused as a result of physiological changes in the mother. The adverse neonatal effects reported by several authors are contradictory to each other in terms of the changes in behaviour observed, and therefore it is necessary to establish the true effects of epidural anaesthesia on the mother/fetus/neonate in order to comment on its safety.

Metahclism of the Xylidide local anaesthetics Very little work has been done on the metabolism of bupivacaine itself, 85

but it is to be expected that its metabolic fate will be similar to

that of its congeners lignocaine and mepivacaine, and hence available

information for these drugs will be reviewed in turn.

(a) Lignocaine (2-diethylamino-2',6'-acetoxylidide). Keenaghan

and Boyes (1972) studied the metabolism of lignocaine in several

species and found the main metabolic pathways to include dealkyl-

ation, aromatic hydroxylation and amide hydrolysis (Fig. 1.7).

Much interspecies variation in the relative extents of these

routes occurred and the results are tabulated in Table 1.10.

In man, approximately 84% of the dose was recovered in 24h urine,

which consisted of 73% of the dose as 4'-hydroxy-2',61 -dimethyl-

aniline and the remaining 11% as lignocaine, its mono and dide-

ethylated derivatives and their phenols. Breck and Traeger

(1971) reported that the cyclic compound N-ethyl-2-methyl-N3-

(2',6'-dimethylphenyl)-imidazolidinone is also a metabolite of

lignocaine in man accounting for less than 3% of the dose.

Mather and Thomas (1972) found that about 25% of the dose orally

administered to humans is excreted as N-hydroxy derivatives of

the amide nitrogen of lignocaine and monoethylglycinexylidide

(MEGX) .

(b) Mepivacaine (1-N-methyl-2-(2',6'-xylylcarbamoyl)-piperidine).

Thomas and Meffin (1972) found that following the oral admini-

stration of mepivacaine to human adults, approximately 1% of

the dose was recovered as unchanged mepivacaine and 1% as the

N-demethylated metabolite. Some 35% of the dose underwent

aromatic hydroxylation with 15 - 20% being excreted as 3'-hydroxy- Fig. 1.7 Possible biotransformation scheme for lignocaine in rat, guinea pig, dog and man (Keenaghan & Boyes, 1971)

CH3 CH3 O CH3 C H IOI IOI /__NH\ -C-CH -N / 2 5 \ NH-C-CH -N NH- -CH -N/ H 2 \ 2 2 C2H K C2H5 5 C2H5 CH3 CH3 3

lignocaine monoethyl g lycinexyl idide 3'-hydroxymonoethylglycinexylidide

O II NH-C-CH2-NH2 conjugates 0 C2H5 NH-C -CH2-N CH3 C 2H5 CH3

CH3 glycinexylidide 2',6'-xylidine

3'-hydroxylignocaine

COOH

conjugates conjugates 4'-hydroxy-2',6'- 2-amino-3-methyl- dimethylaniline benzoic acid 87

Table 1.10 Species variation in the metabolism of lignocaine.

% dose recovered in 24h urine COMPOUND Rats Guinea-Pigs Dogs Man

Lignocaine 0.2 0.5 2.0 2.8

MEGX 0.7 14.3 2.3 3.7

Glycinexylidide 2.1 3.3 12.6 2.3

3'-Hydroxylignocaine 31.2 0.5 6.7 1.1

3'-Hydroxy MEGX 36.9 2.0 3.1 0.3

2',6'-Xylidine 1.5 16.2 1.6 1.0

4'-Hydroxy-2',6'-xylidine 12.4 16.4 35.2 72.6

TOTAL 85.0 53.8 63.5 83.5

(from Keenaghan and Boyes, 1972).

mepivacaine and 10 - 15% as its 4'-hydroxy isomer. Both these phenolic

metabolites were excreted mainly as their glucuronides. In rats, the

major metabolite (60%) was 3'-hydroxymepivacaine. Further studies

by Meffin, Robertson, Thomas and Winkler (1973) have demonstrated that

mepivacaine can also undergo oxidation of thepiperidine ring in the

6''position to the lactam which may subsequently undergo N-demethylation

and hydroxylation of the piperidine ring. These neutral products

were reported to account for 10% of the administered dose in humans.

Goehl, Davenport and Stanley (1973) studied the metabolism of bupi-

vacaine in the rat and rhesus monkey, and the results are given in

Table 1.11. Hydroxylation in the 3'-position was the main metabolic

pathway in the rat, and amide hydrolysis in the rhesus monkey. Reynolds 88

(1971) examined the metabolism of bupivacaine in humans and

found that 6% of the dose was excreted unchanged in 24h urine

and 5% as desbutyl-bupivacaine. No other possible metabolites

were assayed.

Table 1.11 The metabolism of bupivacaine in rat and rhesus monkey

Compound Rat Rhesus monkey

Bupivacaine 2.8 5.9

Desbutyl-bupivacaine (PPX) 0 3.8

4'-Hydroxybupivacaine free O 3.8 conjugated 0 4.2

4'-Hydroxy PPX free 0 0.9 conjugated 0 4.0

3'-Hydroxybupivacaine free 2.2 0 conjugated 21.8 0

Piperidinic acid - 51.7

TOTAL 26.8 74.3

(from Goehl et al., 1973)

In conclusion, it appears that the metabolic pathways found for the xylidid.e local anaesthetics are dealkylation, amide hydrolysis and aromatic hydroxylation followed by conjugation. Different pathways are favoured by different species, the rat preferring aromatic hydroxylation particularly in the.3'-position,and the guinea pig., dog and rhesus monkey, dealkylation, amide hydrolysis and further metabolism of the resulting products. The total metabolism of bupivacaine in man remains to be elucidated. 89

Scope of the present work

Forty years ago, Clifford and Irving (1937) stated that 'the ultimate fate of the present methods of (obstetric) analgesia may well hinge on the price the infant must pay for the mother's comfort.' Numerous studies have since attempted to define this 'price' although it is only in the last decade or so that sufficiently sensitive techniques for measuring small concentrations of drugs in biological samples

(Caldwell et al., 1977) and methods of evaluating neonatal behaviour(Brazelton,

1973; Scanlon, Brown, Weiss and Alper, 1974)have been developed, both these being required for a full study of the problem.

As mentioned in the text, there are conflicting reports in the literature concerning the possible adverse effects of pethidine and bupivacaine on the fetus and neonate. Pethidine or its metabolites have been implicated in respiratory depression in the neonate, neonatal motor activity, increased irritability and feeding difficulties, and bupivacaine or its metabolites in jerky movements, persistent and exaggerated movements and greater irritability. Furthermore Dunn

(1975) has suggested that in addition to directly depressing newborn respiration, pethidine may act indirectly to cause maternal hypoxaemia followed by fetal hypoxia, and Schifrin (1972) has demonstrated decreased placental perfusion secondary to maternal hypotension following bupivacaine administration which can cause changes in the fetal heart rate pattern and the development of fetal acidosis. Since these two drugs are among the most commonly used obstetric analgesics in this country, their effects on the fetus and neonate is of contemporary obstetric importance. However, previous studies (Aleksandrowicz and

Aleksandrowicz, 1976; Richards and Bernal, 1972; Conway and Brackbill 90

1970; Standley et al., 1974; Scanlon et al., 1974) can be criticised

on one or more of the following grounds: small sample size; the use

of several analgesics/anaesthetics during labour; the selection of

infants for study after birth; and the failure to adequately control social and obstetric variables. In addition, the failure of many of these studies to measure the amounts of drugs in the fetus and neonate does not allow dose related effects to be analysed. Hence a carefully controlled study was designed at St. Mary's Hospital in

1974 to assess the effects of maternally administered pethidine and bupivacaine on the fetus and neonate, the main objectives being:

(i) To assess the effects of pethidine or bupivacaine on fetal

heart rate and pattern.

(ii) To study the pharmacokinetics of drugs in mother, fetus and

neonate.

(iii)To assess the relationship between drugs, other obstetric

variables and neonatal behaviour.

The work covered by this thesis concerns the maternal, fetal and neonatal disposition and elimination of maternally administered bupivacaine and pethidine during labour, and the results will be discussed in relation to the findings on the psychological and neurological assessment of the infants. In addition, the metabolism of both drugs has been studied in several animal species in an attempt to find appropriate animal metabolic models for the human situation. 91

Chapter Two Materials and Methods (Bupivacaine)

Page

Compounds 92

Syntheses 92

Animals 97

Preparation of rats with biliary fistulae 97

Human volunteers 98

Storage of biological samples 98

Spectra 99

Radiochemical techniques 99

Isotope dilution procedures 100

Chromatography 102

Visualization of compounds 103

Treatments of urine prior to analysis 104

Concentration of urinary metabolites 105

Gas-liquid chromatography (GC) 105

Mass spectrometry (MS) 105

Gas chromatography - mass spectrometry (GC-MS) 106

Estimation of bupivacaine by GC and GC-MS 106

Extraction of urinary metabolites 108 92

Compounds

1-n-Butyl-2-(2',6'-xylylcarbamoyl)-piperidine hydrochloride (bupivacaine,

Marcain(R)), m.p. 258°C, was obtained from Duncan, Flockhart and Co.

Ltd., London E2. 2-(2',6'-Xylylcarbamoyl)-piperidine hydrochloride

(desbutyl-bupivacaine), m.p. 258-260°C, was the gift of AB Bofors, Bofors,

Sweden. Pipecolinic acid, m.p. 281-283°C, was purchased from Aldrich

Chemical Co., Gillingham, Dorset, U.K. DL-1-[14C]-lysine monohydrochloride (specific activity lOmCi/mmol) was obtained from the Radiochemical

Centre, Amersham, U.K. All other compounds were obtained from commercial sources and purified before use where appropriate.

Syntheses [14 C1-Bupivacaine hydrochloride

The synthesis of [1 4Cj-bupivacaine hydrochloride was achieved in 14C]-L 14C - three stages. [ ysine was first cyclised yielding [

pipecolinic acid which was subsequently coupled with 2',6'-xylidine

to give labelled desbutyl-bupivacaine, and finally N-butylated to

yield [1 4Cc-bupivacaine HC1 (Fig. 2.1).

(i) Piperidine-2-[14C]-carboxylic acid hydrochloride ([14C]- pipe-

colinic acid HC1). This was prepared from DL-1-[1 4CJ-lysine

monohydrochloride according to the method of Hamilton (1952).

Nitrosyl chloride was prepared by the addition of silver nitrite

(0.6g) to ice-cold 6M-HC1 (6m1), and the supernatant decanted into

a solution of DL-1-[ 14C~-lysine monohydrochloride (200mg; 45011Ci)

dissolved in 3M-HC1 (6m1). The solution was heated at 60°C for

20 min, water and excess HCl evaporated under reduced pressure and

the residue dissolved in water (2m1). The remaining HC1 was

Fig. 2_1 Synthesis of [14C]-Bupivacaine HCI * C 00 COOH * = 14C CH. NH2 CH.C1 H2 CH2 NOC1 OH- HC1 CH CH2 2 CH N 00H \ N / COOH. HCI H2 CH2. NH2 CH. 2N H2 PC15/cH3COC1

14 [ 14 DL-14 C]-Lysine 6-Amino 2-chloro- 14C]-Pipecolinic [ C]-Pipecolinic 1-[14C]-hexanoic acid acid acid HC1

N - COC1. HC1 H

[14C]-Bupivacaine HC1 CH i 3

_ 11N- OC N —11N-OC N HC1. C4H9 KC02 3 HC1 CH 14 CH 3 [ C]-Desbutyl-bupivacaine 3 94

precipitated as AgC1 by the addition of excess solid silver sulphate, silver ions were removed by bubbling H2S gas through the solution, and sulphate removed as the barium salt by adjusting the solution to pH5 with solid barium carbonate. The solution was then filtered and evaporated to dryness leaving a residue of 6-amino-2-chloro-l- C 14 C] -hexanoic acid which was used without further purification.

Barium hydroxide (O.5M, 6m1s) was added and the solution boiled for ten minutes to cyclise the 6-amino-2-chloro-1{14 C]-hexanoic acid [ to piperidine-2- 14C]-carboxylic acid , (C14C]-pipecolinic acid).

Unlabelled pipecolinic acid (1g) dissolved in ethanol was added to the above solution, this was filtered while hot and HCl (1l.6M; 2m1s) added to remove excess barium ions as the hydrochloride. On the addition of diethyl ether to the ethanolic filtrate, piperidine _ 2- 14C]-carboxylic acid HC1, the title compound, precipitated as white crystals with m.p. 252-256°C, after recrystallization from ethanol/ether. Specific activity 113.1uCi/g; yield 0.87g; radiochemical yield based on 1[ 4C]-lysine 24.9%.

(ii) 2-(2',6'-Xylyl-L 14C]-carbamoyl)-piperidine hydrochloride

(C14C] -desbutyl-bupivacaine). This was prepared by the method of

Ekenstam, Egner and Petterson (1957). [ 14C]-Pipecolinic acid hydrochloride (O.8g) was suspended in acetyl chloride (lOmis) and stirred for six hours at 35°C with phosphorus pentachloride (0.5g).

Further phosphorus pentachloride (0.5g) was added and stirred for a further two hours. The suspension was cooled to room temperature, the product brought upon a filter and washed with dry toluene (25m1) and dry acetone (25m1). This product, piperidine-2-[14 C]-carboxylic 95

acid chloride hydrochloride, was then refluxed for thirty minutes

with 2',6'-xylidine (2m1) in acetone (6m1) whereupon white crystals

of 2-(2',6'-xylyl-[14 CJ-carbamoyl)-piperidine hydrochloride

precipitated, m.p. 251-254°C, after recrystallization from ethanol/

ether. Specific activity 97.4uCi/g; yield 0.83g; radiochemical

yield based on the 1[ 4CJ-lysine 19.3%.

(iii) 1-n-Butyl-2-(2',6'-xylyl-[ 14C]-carbamoyl)-piperidine hydro-

chloride ([14C]-bupivacaine hydrochloride). This was prepared

according to the method of Ekenstam, Egner and Petterson (1957). [14 q-Desbutyl-bupivacaine (0.7g) was dissolved in butan-1-ol,

(lOml) potassium carbonate (0.5g) and 1-bromobutane (lml) added,

and the whole refluxed for 18h. The K2CO3 was filtered off and

the solvents evaporated under reduced pressure leaving [ 14C]-bupi-

vacaine as an oil. This was dissolved in dry ethanol and a solution

of HC1 gas in ether (2.5M) added, whereby white crystals of 14C -

bupivacaine hydrochloride separated, m.p. 252-256°C after recrystal-

lization from ethanol/ether. Specific activity 76.25uCi/g; yield

0.53g; radiochemical yield based on the [1 4C]-lysine 8.8%.

Determination of purity of synthesized [1 4C-bupivacaineTJ hydrochloride. Chromatography (see Table 2.1) in systems A and B gave

a single 14C peak of Rf 0.73 and 0.80 respectively. In addition

to this, isotope dilution analysis was performed as follows.

Accurately weighed samples of [14CJ-bupivacaine hydrochloride in

the region of lmg, and unlabelled bupivacaine hydrochloride (lg)

were dissolved in water (20m1), the solution adjusted to pH14 with

10M-NaOH, and bupivacaine free base extracted with diethyl ether 96

(3 x 3 vol). The combined ethereal extracts were dried over anhydrous Na2SO4, evaporated under reduced pressure, acetone (10 ml) added, and HC1 gas in dry ether added thereby precipitating white crystals of bupivacaine hydrochloride which were recrystallized from methanol/ether to constant specific activity, m.p.

257-259°C. This isotope dilution analysis showed the [1 4Cj- bupivacaine hydrochloride synthesized to have a radiochemical purity greater than 99%.

1-n-Pentyl-2-(2',6'-xylylcarbamoyl)-piperidine hydrochloride

(pentyl PPX), was synthesized from desbutyl-bupivacaine and 1-

bromopentane by a modification of the previously described method

for the synthesis of bupivacaine according to Caldwell, Moffat,

Smith, Lieberman, Beard, Snedden and Wilson (1977), m.p. 229°C.

2-(2',6'-Xylylcarbamoyl)-1-N-2-toluenesulphonylpiperidine.

Desbutyl-bupivacaine hydrochloride (lg) was dissolved in water and the solution adjusted tō pH14 with 1OM NaOH. The solution was

extracted with diethyl ether (3 x 3 vol) and the combined ethereal

extracts dried over Na2SO4 and evaporated to dryness under reduced pressure. The residue was taken up in 2.5M-NaOH (20m1), p-toluenesulphonyl chloride (1.5g) in acetone (6m1) added, and the mixture refluxed for 20 min. The solution was then poured on to crushed

ice (l0g), and the crystals of the }- toluenesulphonyl derivative of

desbutyl-bupivacaine, the title compound, which formed were filtered

and recrystallized from aqueous ethanol, m.p. 187-189°C.

Microanalysis: C N203S requires C 65.6%; H 6.5%; N 7.3%;0 12.5%; 21H25 S 8.3%; found C 65.2%; H 6.6%; N 7.1%; 0 12.2%; S 8.6%. 97

Animals

Female Wistar albino rats, body weight 200-250g, were obtained from

Anglia Laboratory Animals, Alconbury, Huntingdon, Camb., U.K. and maintained on a diet of 41B pellets (Labsure Animal Diets, Poole,

Dorset). During metabolic studies, they were housed separately in metabolism cages designed such that urine and faeces could be collected separately; this was generally done at 24h intervals. Urine was collected directly into small conical flasks cooled with solid CO2 in insulated polystyrene boxes. Rapid freezing of urine occurred to prevent any breakdown of labile metabolites such as ester glucuronides.

The animals were allowed free access to food and water throughout the 14 experiment.p The animals were given [ C]-bupivacaine HCl (30mg/kg;

20uCi/kg) dissolved in normal saline by intraperitoneal injection.

Preparation of rats with biliary fistulae

The animals were anaesthetised with pentobarbitone sodium i.p. (Nembutal;

Abbott Laboratories Ltd., Queensborough, Kent, U.K., 70mg/kg). The procedures for cannulation of the common bile duct with a PP-10 poly- thene cannula (Portex Ltd., Hythe, Kent, U.K.)- was as described by

Abou-El-Makarem, Millburn., Smith and Williams (1967). Following administration of the above dose of bupivacaine, the rats were kept in res- training cages (Bellman, 1948) and bile was collected at hourly intervals for 6h and finally after 24h. The rats were kept warm with suitable lamps above the cages and allowed free access to a solution of dextrose

(4.3%, w/v) and NaCl (0.18%, w/v). Urine was collected in plastic trays under the cages. 98

Humans a) Metabolic study

Two healthy male volunteers, aged 29 and 36, weight 85 and 70 kg,

participated in the study. They each received an intramuscular [ injection in the buttock of 14 j -bupivacaine hydrochloride dissolved

in sterile normal saline (5m1 or 2.5m1 of a 10mg/m1 solution; 0.764

Ci/ml) which had been previously sterilized by ultrafiltration

(Millex disposable filter units; 0.22pm; Millipore S.A., France).

Urine was collected hourly for 6h, thence from 6-9, 9-12 and 12-24h

and then daily for 7 days. Faeces were collected daily for the

same period.

(b) Obstetric study

All studies had the approval of the Ethical Committee of St. Mary's

Hospital and Medical School, and all participants gave their informal

consent. 58 Obstetrically normal mothers in uncomplicated labour

were studied. Lumbar epidural anaesthesia was established with a

single dose of bupivacaine and further doses were administered as

required. Samples of maternal venous and umbilical cord arterial

and venous blood were obtained at delivery, and capillary blood

samples obtained from the warmed heel of the neonates at 2, 24,

36 and 48h after delivery. All samples were placed in heparinized

tubes. 0-24h urine from 10 male neonates was also collected.

Further details are given in Chapter 4a.

Storage of biological samples

All samples of body fluids and excreta were stored at -20°C, without pH adjustment prior to analysis. 99

Spectra

(i) Ultraviolet spectra. UV spectra of compounds were obtained on a

Pye Unicam SP 30 UV spectrophotometer

(ii)Infra-red spectra. IR spectra were recorded with a Perkin-Elmer

157G grating I.R.spectrophotometer, using Nujol mulls or KBr discs.

Radiochemical techniques

The 14C content of solutions and excreta was determined by liquid scintillation spectrometry. Urine (0.1-l.Oml), saliva (0.5-1.Oml), bile

(0.005-0.02m1) and cage washings (l.Oml) were counted directly, in duplicate, using a dioxan-based scintillation cocktail (Appendix A) with Packard Tri-Carb liquid scintillation spectrometers, models 3385 and 3320. Vials were counted after sufficient time had elapsed for cool- ing to prevent chemiluminescence, and quench correction was performed by the channels ratio method. Rat and human faeces were homogenized in water (3vol) in a Waring Blendor (human) or with an Ultra-Turrax homogeniser (rat). Aliquots of the homogenates (20m1) were made alkaline with 10M-NaOH (2m1) and H202 solution (2m1; 100 vol %) added, with a few drops of iso-octanol to control foaming. The mixture was kept at room temperature for 3 days until decolourized, aliquots (1-2m1) warmed to 60°C for 30 minutes to remove H202, and these assayed for

14 C by liquid scintillation spectrometry.

Plasma (0.5m1) obtained by centrifuging fresh blood at 2500 r.p.m. for 10 minutes, was counted directly using a Triton X-100 toluene scintillant (Appendix A).

Radiochromatogram scanning of thin-layer chromatograms was performed with 100

a Packard radiochromatogram scanner (model 7201), identification of 14 C peaks being made by comparison of Rf values with authentic compounds

(see Table 2,.1). The 14C content of peaks was estimated by scraping

0.5cm wide sections of the adsorbent from the thin layer plate and

counting in the scintillation counter.

Isotope dilution procedures

The content of bupivacaine, desbutyl-bupivacaine and pipecolinic acid

in rat and human urine were assayed using this technique:-

(i) Bupivacaine

Bupivacaine hydrochloride (lg human; 0.5g rat) was dissolved in

urine (25ml human; 5m1 rat), the solution adjusted to pH14 with

1OM-NaOH, and bupivacaine free base extracted with diethyl ether

(3 x 3 vol). The combined ethereal extracts were dried over an-

hydrous Na2SO4, evaporated under reduced pressure, acetone (lOml)

and HC1 gas in dry ether added to the solution,

precipitating white crystals of bupivacaine hydrochloride which

were recrystallized from methanol/ether to constant specific

activity and m.p. (258°C).

(ii) Desbutyl-bupivacaine

Desbutyl-bupivacaine hydrochloride (lg human; 0.5g rat) was

dissolved in urine (25m1 human; 5ml rat), the solution adjusted to

131114 with 10M-NaOH and extracted with diethyl ether (3 x 3 vol).

The desbutyl-bupivacaine so extracted was then converted to 2-(2',

6'-xylylcarbamoyl) - -toluenesulphonyl-piperidine as described

under "Syntheses", the product being recrystallized from aqueous

Table 2.1 Rf Values and Colour Reactions of Bupivacaine -and Related Compounds

Thin layer chromatography, Rf value in system: Colour with:

COMPOUND A B C D E F Ninhydrin Diazotized p Ferric Gibb's nitroaniline chloride reagent

r Bupivacaine 0.73 0.80 0.65 0.54 0.55 r0

Desbutyl-bupivacaine 0.60 0.42 0.38 0.51 0.48

* 4'-Hydroxydesbutyl- 0.55 0.21 0.16 0.44 0.30 blue blue blue bupivacaine

Pipecolinic acid 0.01 0.07 0.14 0.18 0.42 purple

TLC plates and solvents as described in chapter 6. Compounds were visualised under UV light (254nm). .* gift from Astra Parmaceutical Co., Sweden 102

ethanol to constant specific activity and m.p. (187-189°C).

(iii) Pipecolinic acid

Pipecolinic acid (lg human; 0.5g rat) was suspended in urine

(25m1 human; 5m1 rat), and sufficient ethanol added to ensure

complete solution. The solution was reduced to dryness on the

rotary evaporator, the residue extracted with ethanol (20m1), and

after filtration, HC1 gas in dry ether was bubbled through the

filtrate precipitating crystals of pipecolinic acid hydrochloride.

These were filtered off and recrystallized from ethanol/ether to

constant specific activity and m.p. (280-282°C).

Thin-Layer Chromatography (TLC)

The chromatographic properties of bupivacaine and related compounds are shown in Table 2=.1. Aliquots (0.01-0.1ml) of treated (see below) or untreated urine, (containing 104 - 105 dpm/ml), were chromatographed on precoated aluminium-backed silica gel sheets, layer thickness 60F254 0.2mm, (E. Merck, Darmstadt, W. Germany), using systems A-F. Extracted phenolic metabolites were chromatographed in system G on Imm thick TLC plates prepared by forming a slurry of silica gel (100g; E. Merck GF254 A.G., Darmstadt, W. Germany) in water 0,230ml) and spreading on glass plates (200 x 200mm). After drying at 100°C for lh, the plates were cooled to room temperature and kept in a dessicator prior to use. Both the TLC and preparative TLC chromatograms were run for about 150mm from the origin. Solvent systems used were as follows:

System A ethanol/acetone/benzene/0.88 S.G. ammonia (5:50:50:5 by volume)

System B chloroform/methanol (2:1 by volume) 103_

System C dioxane/glacial acetic acid/water (100:2:15 by volume)

System D n-butanol/glacial acetic acid/water (72:18:30 by volume)

System E iso-butanol/glacial acetic acid/water (72:18:30 by volume)

System F pyridine/water/n-butanol (1:1:1 by volume)

System G n-propanol/0.88 S.G. ammonia (7:3 by volume)

Visualization of compounds on chromatograms

The locations of the compounds on the chromatograms were determined by

the following methods:

Ultraviolet light. All the reference compounds were seen as dark quenching

spots when chromatograms were inspected under U.V. light at 254nm (Hanovia

Chromatolite).

Diazotized-p-nitroaniline. Chromatograms were sprayed with freshly

prepared diazotized p-nitroaniline as described by WickstrSm and Salvesen

(1952). Phenols gave strong blue spots which were not permanent.

Gibb's reagent. Freshly prepared 2,6-chlorobenzoquinone-4-N-chloroimine

(0.1%) in ethanol was sprayed onto TLC plates which were then oversprayed

with saturated aqueous sodium bicarbonate solution (Smith, 1960). A

deep blue colour indicates the presence of phenols, the colour being most

intense for 'meta' or 3' substituted phenols.

Ferric chloride. Equal volumes of 1% potassium ferricyanide and 1%

ferric chloride (aqueous) solutions were mixed just prior to spraying.

Dark blue spots developed in the presence of phenols due to a complex

between the Fe3+ ion and the phenol, which were made permanent by over-

spraying the plate with 2M-HC1.

Ninhydrin spray. Amines were seen as a purple spot on heating the

chromatogram to 100° for several minutes after spraying with a solution of ninhydrin (0.25%) and 2,4,6-collidine (2%) in isopropanol.

104

Treatments of urine prior to analysis

(i) Treatment with a-glucuronidase Portions (2m1) of urine, adjusted

to pH5 with glacial acetic acid were incubated with 5000 units of

S-glucuronidase (Ketodase; General Diagnostics, Morris Plains, N.J.,

U.S.A.) at 37°C for 24h. Two controls were set up, one with boiled

enzyme and the other containing phenolphthalein glucuronide. The

liberation of phenolphthalein from its glucuronide, indicated by

the pink colouration on the addition of base, ensured that the Keto-

dase was working. The incubation mixture was applied directly to

chromatograms.

(ii)Treatment with sulphatase Portions (2m1) of urine, adjusted to

pH5 as above and buffered with 0.2M pH5 acetate buffer (lml), were

incubated at 37°C for 24h with 0.5m1 of a sulphatase preparation

(H-2, Sigma Chemical Co.). Saccharo-1,4-lactone (Sigma Chemical

Co.)(1 x 1014M) was added to inhibit the S-glucuronidase present

in the enzyme preparation.

(iii)Acid hydrolysis Portions (5m1) of urine were refluxed with an

equal volume of 10M-HC1 for 2h. The hydrolysate was neutralized

with 5M-NaOH and NaHCO3 prior to chromatography.

(iv) Treatment with titanous trichloride (TiCl3) Urine (2ml) was ad-

justed to pHl4 with 5M-NaOH and extracted with ether. The aqueous

phase was then allowed to react with TiC13 (0.2m1) and 10M-HC1

(0.2m1) for lh at room temperature. 105

Concentration of urinary metabolites by extraction on XAD-2 resin for thin-layer and preparative chromatography

Amberlite XAD-2 resin (BDH Chemicals Ltd., Poole, U.K.) was washed and packed into a column 30 x 1 cm) as described by Caldwell, Koster, Smith and Williams (1975), irrigated with urine (10-50m1) obtained from animals and humans dosed with C 14CJ-bupivacaine, and the effluent collected. The column was washed with water (50m1) and the metabolites eluted with methanol (50-lOOmi). The methanol was reduced to a small volume (1-5m1) in vacuo and portions (0.05m1) used for chromatography.

The methanol eluates contained about 98% of the 14C applied to the column.

Gas-Liquid Chromatography (GC)

A Packard-Becker 417 gas chromatograph equipped with a flame ionization detector was used. The column was.of glass 1.8 x imm i.d. and packed with

OV-17 (3%) on Chromosorb W(HP), 80-100 mesh. Operating conditions were: o oven temperature 2400; detector 260 ; injection port 2400; nitrogen flow rate 60m1/min; hydrogen, 30m1/min; and air, 300m1/min. The column was conditioned prior to use by allowing N2 flow at 30m1/min at an oven temperature of 260° for 24h, and silanized in situ with BSA (N,0-bis trimethylsilyl acetamide; Pierce Chemical Co.), 4 x 251.11. The retention times (min) of bupivacaine and related compounds under these conditions were: bupivacaine, 3.1; desbutyl-bupivacaine, 1.9; pentyl PPX, 4.0.

Mass Spectrometry (MS)

Mass spectrometry was performed using a Varian-MAT CH5 mass spectrometer by direct insertion technique. Operating conditions: electron energy,

70eV; ionizing electron current, 30011A; ion accelerating voltage, 3kV; electron multiplier voltage, 2.6kV. Spectra were acquired and processed 106

in a 620/i computer (Varian Data Machines). Perfluoroalkane 250 was used for calibration of the data system.

Gas Chromatography-Mass Spectrometry (GC-MS)

The Varian-MAT CH5 mass spectrometer. was used, coupled with a Varian

Aerograph 1700 GLC through a Watson-Biemann two-stage separator. The column was of glass, 2m x 3mm i.d. packed with SE-30 (3%) on chromosorb

W(HP) 80-100 mesh. The temperatures were: GC injection port, 270°; GC oven 220°; MS Line of sight inlet 220°c; separator, 260°; ion source

212°. The helium carrier gas flow rate was 30m1/min. The electron energy was 70eV; ionising electron current, 300uA; ion accelerating volate 3kV, and the electron multiplier voltage 2.6kV. The retention times (min) of reference compounds under these conditions were: bupivacaine, 3.5 ; desbutyl-bupivacaine, 2.2; pentyl PPX, 4.5.

Derivatization of phenolic metabolites for GC and GC-MS

Residues after evaporation of dichloromethane extracts of urine at pH8.5 as such, (or following isolated of individual metabolites by preparative

TLC), were derivatized with N,0-bis-(trimethylsilyl)-acetamide (BSA,

Pierce Chemical Co.). The residues were taken up in pyr_dine (25p1),

BSA (25u1) added, and the mixture allowed to stand at room temperature for 15 min. The solvents were evaporated off under a stream of dry nitrogen, and the residue taken up in CS2 (lOpl) and aliquots used for

GC and GC-MS.

Estimation of bupivacaine by GC

Bupivacaine in urine and blood was assayed by GC according to the method of Caldwell et al.(1977). Whole blood or urine (lml) was placed in a 107

7m1 glass-stoppered tube and pentyl PPX (lug; l0}►1 of a lOCug/ml solution) added. The solution was made alkaline with 5M-NaOH (0.5m1)

and extracted for 15 minutes with freshly distilled diethyl ether

(3m1). The ethereal extract was placed in a second 7m1 glass-stoppered

tube and extracted with M-HC1 (lml). After centrifuging to separate the layers, the ether layer was discarded and the aqueous phase made alkaline with 5M-NaOH (0.5m1). This was extracted with ether (3m1) and the ether evaporated in a 'silli-vial' (Pierce Chemical Co.) under a stream of dry nitrogen. The dried residue was taken up in carbon disulphide (10ul) and aliquots (1-2ul) were taken for GC. Bupivacaine concentration in maternal blood was calculated from the peak height ratio bupivacaine/pentyl PPX, using a previously established standard curve, which was linear over the range 100-2000ng bupivacaine.

Assay for bupivacaine in neonatal blood

The small size of the neonatal samples (20-100u1) did not permit the accurate measurements of their volume, and the blood volume was obtained by the gravimetric method of Caldwell, Moffat, Smith, Lieberman, Beard,

Sneddon and Wilson (1977).

To the diluted alkaline blood was added pentyl PPX (long; l0ul of a bOug/ ml solution), and the extraction was carried out as described above.

After evaporation of the final ethereal extract, the residue was taken up in CS2 (20u1) and aliquots (3-5u1) analysed by GC (as above) or

GC-MS using single ion monitoring (GC-MS SIM) as described by Caldwell et al. (1977). Bupivacaine concentration using the GC assay was obtained from the peak height ratio bupivacaine/pentyl PPX, reference being made to a previously established standard curve which was linear over the 108

range 100-2000ng. Reproducibility of assay from blood at 10Ong ml-1 was ± 5% (S.D., n = 6). The amount of bupivacaine present in neonatal blood assayed by GC-MS SIM was obtained from the peak height ratio m/e 140/ m/e 154, reference being made to a previously established standard curve which was linear over the range 1-100ng bupivacaine. At long the reproducibility of assay from blood was ± 6% (S.D., n = 5) while at ing this rose to ± 13% (S.D., n = 5).

General procedure for the separation, identification and quantitation of metabolites

An outline of the procedure used for the separation and identification of bupivacaine and its metabolites in urine is given in Figure 2.2.

Aliquots of urine as such, or after incubation with 8-glucuronidase, sulphatase or acid treatment as described, were adjusted to pH14, and basic compounds extracted with diethyl ether (3 x 3 vol). The ethereal extracts were combined, dried over Na2SO4 and evaporated under reduced pressure, the residue taken up in a small volume of methanol (5m1) and aliquots taken for radio-TLC, GC and GC-MS examination. A portion of the aqueous phase was then treated with TiC13/HC1 as described and reextracted at pH14 and taken for GC and TLC analysis. The remainder of the aqueous phase was adjusted to pH8.5, saturated with sodium chloride to increase the ionic strength,and extracted with dichloromethane

(3 x 3 vol). The organic phase was concentrated as above and the residue taken up in methanol (5m1). Individual metabolites were isolated by preparative TLC and examined by GC-MS as such, and following derivatization with BSA, (see Chapter 6). The pH8.5 aqueous phase was then adjusted to pH5 with 10M-HC1, extracted with dichloromethane (3 vol) and the extract evaporated to dryness. The residue was taken up in 109

methanol (2m1) and examined by radioTLC. The aqueous phase was subsequently examined by radioTLC, treated with TiC13/HC1 and reexamined by TLC. Fig.2_2 Analysis of Bupivacaine and Metabolites in Urine

URINE XAD-2 column, evaporate methanol, take up residue in water, adjust to pH14, extract with ether.

4. 4, ETHER PHASE AQUEOUS PHASE incubated with (3-glucuronidase adjusted to pH8.5, saturated with NaCl and extracted examined by TLC,GC,and GC-MS extracted with dichloromethane. 0 bupivacaine desbutylbupivacaine 1 DICHLOROMETHANE PHASE examined by TLC,GC,and GC-MS AQUEOUS PHASE adjusted to pH5, extracted with dichloro- 3'and4'-hydroxybupivacaine (RAT) methane

unknown (MAN) i y DICHLOROMETHANE AQUEOUS PHASE PHASE examined by TLC not characterized pipecolinic acid unknown Compounds underlined also confirmed by reverse isotope dilution 111

Chapter Three Materials and Methods (Pethidine)

Page

Compounds 112

Syntheses 112

Animals 113

Administration of compounds 114

Collection of urine and faeces 114

Collection of 14CO2 in expired air of rodents 114

Human volunteers 115

Administration of norpethidine and pethidinic acid 116

Storage of excreta 116

Collection of14CO2 in expired air of humans 116

Radiochemical techniques 116

Quantitation of 14C in animal carcass 117

Thin layer chromatography 117

Gas liquid chromatography (GC) 117

Mass spectrometry (MS) 119

Gas chromatography - mass spectrometry (CG-MS) 119

Acetylation of metabolites 119

Estimation of pethidine and norpethidine by GC 119

Estimation of total pethidinic and norpethidinic acids by GC •120

Estimation of free pethidinic and norpethidinic acids 121

Estimation of pethidine-N-oxide 122

General procedure for the separation, identification and 122

quantitation of metabolites 112

Compounds

(N-C14 CJ-Methyl)-pethidinehydrochloride, specific activity 4.44mCi/mmol,

was purchased from Mallinckrodt Inc.,St. Louis Mo., U.S.A., pethidine

hydrochloride (m.p. 184-185°C) from Roche Products, Welwyn Garden City,

U.K., lignocaine hydrochloride (m.p. 76-79°) from Astra Products, Watford,

U.K., deuterium oxide (2H2O) (98 atom %D) and sulphuric acid-[2H2] in

2H20 (98% at 99 atom %D) from British Oxygen Co., Deer Park Road, London

SW19, U.K. Norpethidine hydrochloride (m.p. 110-112°) was a gift from

Sterling-Winthrop Research Institute, Rensslaer, N.Y., U.S.A. and 4'-

hydroxypethidine (m.p. 138-140°) and pethidine-N-oxide (m.p. 48-50°)

were gifts from Dr. Claes Lindberg, Department of Organic Pharmaceutical

Chemistry, Biomedical Centre, Uppsala, Sweden.

Syntheses

[ 2H51-Pethidine hydrochloride: Pethidine, labelled with five deuterium

atoms in the benzene ring, was synthesized from pethidine and 90% sulphuric

acid-[2H2] in 2H2O as described by Lindberg, Bogentoft and Danielsson

(1974). Pethidine hydrochloride (2g) was dissolved in water, made alkaline with 5M-NaOH and the free base extracted into ether (3 x 3 vol).

The ethereal extracts were combined, dried over Na2SO4and evaporated under reduced pressure leaving an oily residue of pethidine. This was stirred in an air-tight flask for seven days with 90% sulphuric acid-2H2](16.25g) in 2H2O. The solution was cooled to 0°C and diluted first with 2H2O

(5m1) and then with H2O (50m1), made alkaline with 5M-NaOH and extracted with diethyl ether (2 x 2 vol). The combined ethereal extract was dried over Na2SO4, evaporated under reduced pressure and HC1 in ether added precipitating white crystals of [2HO-pethidine hydrochloride, m.p. 184-

185°C after recrystallization from ethanol/ether. Chemical purity of 113

the product was demonstrated by TLC and m.p., and isotope abundance was determined by mass spectrometry and estimated to be >95 atom % 2H.

Pethidinac acid: Pethidine base was refluxed in equal quantities of

96% ethanol and 2M-NaOH for 2h, the mixture diluted fivefold with water and acidified (pH3) whereby white crystals of pethidinic acid were precipitated, m.p. 302-304° after recrystallization from benzene (literature m.p. 304-306°, Lindberg, Bogentoft and Danielsson, 1974).

Norpethidinic acid: This was synthesized from norpethidine base, as described for pethidinic acid, m.p. 320-324° (literature m.p. 320-322°,

Lindberg, Bogentoft and Danielsson; 1974).

Animals

Female Wistar albino rats (200-25Og), female Dunkin-Hartley albino guinea-pigs (250-300g), four species of Old World monkeys and female

Dutch rabbits (2.5kg) were used in the metabolic studies. The rats

(Anglia Laboratory Animals, Alconbury, Huntingdon, U.K.), - guinea-pigs

(Redfern Animal Breeders Ltd., Jason's Farms, Brenchley, U.K.) and rabbits (Ranch Rabbits, Crawley Down, Sussex, U.K.) were fed on 41B pellets, RGP pellets and R14 pellets respectively (Labsure Animal Diets,

Poole, Dorset, U.K.).

The monkeys were all housed in the Primate Colony of the Department of

Biochemistry, University of Ibadan, Nigeria. Vervets (Cercopithecus aethiops, 2.5kg), patas monkeys (Erythrocebus patas patas, 3-4kg), a mona monkey (Ceropithecus mona, 4kg) and a cherry-crowned mangabey

(Cercocebus tarquinus, 5.5kg) were studied. All the monkeys with the 114

exception of one of the vervets were female.

Administration of compounds 14C] -Pethidine hydrochloride was dissolved in sterile normal saline and

administered by intraperitoneal injection to rats (30mg/kg; 15pCi/kg),

guinea pigs (30mg/kg; 15.Ci/kg) and rabbits (20mg/kg; 6.42pCi).

Pethidine hydrochloride dissolved in sterile normal saline was admini-

stered at a dose of 5mg/kg to the monkeys by intramuscular injection in

the thigh muscle.

Collection of urine and faeces

The rats and guinea-pigs were kept singly in all glass metabolism cages

(Metabowls; Jencons, Hemel Hempstead, Herts, U.K.) which enabled the

urine and faeces to be collected separately. The rabbit was housed in

a wire-mesh cage. Monkeys were housed singly in cages and urine coll-

ected into a tray containing 5m1 of 2% HgC12 as preservative. All the

animals were allowed free access to food and water, urine and faeces

being collected daily for up to 3 days.

Collection of radioactive carbon dioxide in the expired air of rodents

A pump was used to draw the air from the Metabowls through a CaC12 trap

to remove moisture, and two wash bottles containing 250m1 and 150m1 of

freshly distilled 2-methoxyethanol-ethanolamine (2:1, v/v) to trap CO2

in the expired air. The air passing through the absorbent was at a

rate which just prevented condensation inside the Metabowls. The

carbon dioxide absorbent was replaced every 24h and the CaC12 as

necessary for 3 days. Aliquots (lml) of. the CO2 absorbent were counted

directly using a dioxan-based scintillant (Appendix A). 115

Human volunteers a) Metabolic study

Two healthy male adult volunteers participated in the labelled

study. Each received by intramuscular injection into the buttock

lml of a solution containing pethidine HC1 (24.7mg), [2H5]-pethidine

HCl (25mg), and [14CI-pethidine HCl (0.3mg; 511Ci) in sterile

normal saline, previously sterilized by ultrafiltration as described

for bupivacaine. A nylon cannula was placed in a vein in the ante-

cubital fossa, and blood samples (lOml) were withdrawn at regular

intervals for Sh. Urine was collected hourly for 12h, every two 14 hours from 12-24h, and at longer intervals for the next 24h. CO2

in the expired air was collected at intervals for 24h (see below).

Fifteen further adult volunteers (2 females, 13 males), also part-

icipated in the study, each receiving an i.m. injection of pethidine

HC1 (50mg). Their 0-24h urine was collected for analysis.

b) Obstetric study

5L Ostetrically normal mothers in uncomplicated labour were studied.

Pethidine was administered by intramuscular injection as required.

Samples of maternal venous and umbilical arterial and venous blood

were taken at delivery and neonatal blood samples obtained at 2, 6 ,

24 and 36h after delivery as described for bupivacaine. The first

24h urine from 5 male neonates whose mothers received pethidine was

also collected. Further details are given in Chapter Four(b).

Collection of 14CO2 in expired air of volunteers

14CO2 in the expired air was estimated at 15 minute intervals for the 116

first 4h, hourly from 4-12h and every two hours from 12-24h. The volunteers expired into a mouthpiece, connected via a CaC12 drying tube, to a scintillation vial containing hyamine hydroxide (1M in methanol) coloured with thymolphthalein. Titration of the indicator, with M-HC1, showed that the colour changed from blue to colourless when 0.37mmoles

CO2 had been trapped.

Administration of norpethidine and pethidinic acid to human volunteers

One male subject (age 30 years, wt. 80kg) received by i.m. injection,

25mg of norpethidine hydrochloride in lml normal saline, the solution having been sterilized by ultrafiltration. One female volunteer (age

25,, wt. 62kg) took 20mg pethidinic acid by mouth in a hard gelatine capsule. Both subjects collected their urine for 24h.

Storage of biological samples

All biological material was stored at -20°, without prior pH adjustment except during analytical procedures. Blood samples were divided into two, one half being stored as such in heparinized tubes, the other centrifuged at 2500 rpm for 10 minutes to separate plasma from red blood cells, and the plasma stored separately.

Urine from the monkeys was preserved with 2% HgC12, filtered and frozen prior to transport by air to London for analysis.

Radiochemical techniques

The 14C content of biological samples was estimated as described for bupivacaine. 117

Determination of activity in animal carcasses

Rat and guinea pig carcasses were dissolved in 300-400m1 of 20% w/v NaOH in 30% aqueous ethanol. Aliquots were neutralised with l2M-HC1 and adjusted to pH7.0 with NaHCO3. Samples (lml) were counted by liquid scintillation counting after bleaching as described for faeces.

Thin-Layer Chromatography (TLC)

TLC was performed on silica gel pre-coated aluminium sheets, layer 6OF254 thickness O.2mm (E. Merck, Darmstadt, W. Germany), which were developed with the following systems:

System B chloroform/methanol (2:1 by volume)

System D n-butanol/glacial acetic acid/water (45:5:40 by volume)

System G n-propanol/0.88 S.G. ammonia (7:3 by volume)

The chromatographic properties of pethidine and its derivatives are listed in Table 3.1.

Gas Chromatography (GC)

A Packard-Becker 417 gas chromatograph equipped with a flame ionization detector was used. The column and packing was identical to that used for bupivacaine (see Chapter 2) and the operating conditions were: oven temperature 200°; detector, 2400; injection port, 240°; nitrogen flow rate, 40m1/min; hydrogen, 3Om1/min; and air 30Om1/min. The retention times (min) of reference compounds under these conditions were as follows: pethidine, 1.8; norpethidine, 3.0; pethidine-N-oxide, 3.2;

4'-hydroxypethidine, 5.5; 4'-acetoxypethidine, 5.4; pethidinic acid methyl ester, 1.75; norpethidinic acid methyl ester, 2.9; lignocaine

(internal standard), 5.7. Table3 .1 Rf Values of Pethidine and Related Compounds

Thin layer chromatography, Rf value in system:

COMPOUND B D G

Pethidine 0.47 0.30 0.69

Norpethidine 0.53 0.48 0.61

Pethidinic acid 0.01 0.15 0.31

Norpethidinic acid 0.02 0.25 0.08

Pethidine-N-oxide 0.25 0.27 0.50

4'-Hydroxypethidine 0.39 0.25 0.69

TLC plates and solvents as described in the text. Compounds were visualised under UV light (254nm). 119

Mass spectrometry (MS)

MS was performed using a Varian-Mat CH5 mass spectrometer using identical

conditions to those used for bupivacaine (see Chapter 2).

Gas chromatography - Mass spectrometry (GC-MS)

GC-MS conditions were again similar to those used for bupivacaine with

the following alterations in operating conditions; the column was packed

with 3% SE 30 on AW-DMCS treated chromosorb W, 80-100 mesh; oven temp-

erature, 190°; injection port 2500; MS line of sight, 200°; separator, o 0 220 ,ion source, 205 , with a helium carrier gas flow of 24m1/min.

The retention times (min) of standard compounds in this system were:

pethidine, 1.6; norpethidine, 1.8; pethidine-N-oxide, 2.0; 4'-hydroxy-

pethidine, 4.9; 4'-acetoxypethidine, 4.9; lignocaine (internal standard),

5.4

Acetylation of 4'-hydroxypethidine

Residues were taken up in acetic anhydride (20u1) and allowed to react

at room temperature for 15 min. The solvents were evaporated off using

a stream of dry nitrogen, the residues taken up in CS2 (lOpi) and aliquots

(2-5p1) used for GC and GC-MS.

Estimation of pethidine and norpethidine

These compounds in blood, plasma and urine samples were assayed by GC .

0.5-1.0 samples of the body fluid were taken, lignocaine (lug; 0.1ml of

a lOUg/ml solution) added as internal standard and extracted with ether

.(3m1). The ether was transferred to a clean tube, extracted with M-HC1

(lml) and the ether phase discarded. The aqueous phase was made

alkaline, basic compounds extracted into ether (3m1), and the ether 120

evaporated in a 'silli-vial' (Pierce Chemical Co.) under a stream of dry nitrogen. The dried extracts were dissolved in lOul freshly distilled

CS2 for GC. Pethidine and norpethidine concentrations were obtained from the peak height ratio pethidine/lignocaine and norpethidine/lignocaine, using previously established standard curves, which were linear over the range 100-2000ng for both. compounds. Reproducibility of the assay from blood at 10Ong/m1 was ± 4% (S.D., n = 6).

Assay for pethidine in neonatal blood

The volumes of the neonatal blood samples were obtained by the gravimetric method of Caldwell et al. (1977). To the diluted alkaline blood was added lignocaine (long; lOUl of a lOug/mi solution) and the extraction was carried out as described above. After evaporation of the final ethereal extract, the residue was taken up in CS2 (20u1) and aliquots (3-5u1) assayed by GC (as above) or GC-MS SIM as described by

Caldwell, Wakile and Notarianni (1978). Pethidine concentration was obtained from the peak height ratio, pethidine/lignocaine (GC) or m/e

218/m/e 234 (GC-MS SIM). In each case reference was made to previously established standard curves which were linear over the ranges 100-1000ng

(GC) or 1-10Ong (GC-MS SIM). Reproducibility of the assay from blood at 10Ong/m1 was ±4% (S.D., n = 6) for the GC method, and at long and 2ng using GC-MS SIM, ± 5% (S.D., n = 6) and ± 11% (S.D., n = 7) respectively.

Estimation of total pethidinic acid and norpethidinic acid lml aliquots of urine were made alkaline and extracted with 3 x 5 vol diethyl ether to remove basic compounds. The aqueous phase was then refluxed for lh with 10M-HC1 (1m1) to hydrolyse conjugates of these acids.

Preliminary experiments showed that this treatment did not hydrolyse 121

pethidine and norpethidine. Lignocaine as internal standard (lug;

O.lml of a 10pg/m1 solution) was added to the hydrolysate which was passed down an 8 x 2cm column of Amberlite XAD-2 resin, prepared as described previously, and the metabolites eluted from the column with methanol (25m1). This was reduced to dryness on a rotary evaporator, the residue taken up in dry ethanol (lOml), 98% H2SO4 (lml) added and the whole refluxed for 2h. The ethyl esters of pethidinic and norpethidinic acid so produced (i.e. pethidine and norpethidine) were extracted and assayed by GC as for pethidine and norpethidine. Cali- bration curves were established for both acids carried through this procedure, and these were linear over the concentration range 500-5000ng.

Estimation of free pethidinic and norpethidinic acids

Since the acid esterification used above would hydrolyse conjugates of these acids present in urine, a milder esterification technique was devised to assay the free acids only. Urine was adjusted to pH14 with

10M-NaOH and extracted with 3 x 5 vol ether to remove pethidine and norpethidine. Lignocaine as internal standard (0.5ug; O.05m1 of a lOUg/ ml solution) was added to the aqueous residue, this passed down an

Amberlite XAD-2 column as above, the column eluted with 25ml methanol and the methanol removed by rotary evaporation. The residue was then dissolved in dry methanol (lOml), 0.2m1 BF3-methanol complex (Aldrich

Chemical Co., Gillingham, Dorset, U.K.) added and the whole refluxed for 3h. The methyl esters of pethidinic and norpethidinic acids were extracted and assayed by GC as for pethidine and norpethidine. Cali- bration curves for both acids carried through this procedure were linear over the concentration range 20o-5000ng. 122

Estimation of pethidine-N-oxide

lm1 aliquots of urine were adjusted to pHl4 and pethidine and nor-

pethidine removed by ether extraction (3 x 5 vol). The aqueous phase

was treated with 0.2m1 10M-HC1 and 0.2m1 titanous chloride solution (15%

w/v in 10M-HC1; BDH Chemicals, Poole, U.K.) for lh at room temperature,

lignocaine (0.5ug; O.05m1 of a lOpg/ml solution) added as internal

standard, and the pethidine formed by the reduction of the N-oxide assayed

by GC as before. A calibration curve for pethidine-N-oxide carried

through this procedure was linear over the concentration range 200-2000ng.

General procedure for the separation, identification and quantitation of

metabolites

An outline of the procedure used for the separation, identification and quantitation of pethidine and its metabolites in urine is given in Fig.

3. 1. Aliquots of urine as such, or after incubation with S-glucuronidase

(Ketodase; General Diagnostics, Morris Plains, N.J., U.S.A.) at 37° overnight, were passed down a 40 x 15cm column of Amberlite XAD-2 resin, and pethidine and metabolites eluted with 200m1 methanol which was removed by rotary evaporation. The residue was then dissolved in loml water, adjusted to pHl4 and pethidine and norpethidine assayed by GC as described. This extract was also examined by GC-MS and radioTLC. A portion of the aqueous phase was then assayed for pethidine-N-oxide as described, and the remainder adjusted to pH9 with an equal volume of

0.2M phosphate buffer pH9, extracted with 3 x 3 vol ether, the extract dried over anhydrous Na2SO4 and the ether removed on a rotary evaporator.

The residue was dissolved in 0.3m1 methanol and divided into 3. One portion was examined by GC and GC-MS, one by radio TLC, and one dried and acetylated for GC and GC-MS. The pH9 aqueous phase was then adjusted 123

to pH5 with 10M-HCl, extracted with 3 vol chloroform, the extract evaporated to dryness and the residue dissolved in methanol. This was examined by GC and GC-MS as such, and after acetylation, and also by radioTLC as described for the pH9 extract. The pH5 aqueous phase was then assayed for free and total pethidinic and norpethidinic acids by

GC as described, and also subjected to radioTLC. The product of the esterification procedure for total acids was examined by GC-MS. Fig.3.1 Analysis of Urinary Metabolites of Pethidine

URINE

pH14/ether

ORGANIC PHASE AQUEOUS PHASE Pethidine Norpethidine pH5/ether

Pethidine-N-oxide 4'-Hydroxypethidine (assayed as pethidine after TiC13/HC1 reduction)

Esterification 1) BF3/CH3OH Free pethidinic acid Free norpethidinic acid 2) H2SO4/C2H5OH Total pethidinic acid Total norpethidinic acid

Assay of metabolites by GC, GC-MS and radioTLC as appropriate 125

Chapter Four (a). The maternal and neonatal disposition of

bupivacaine administered during childbirth

Page

Introduction 126

Subjects, drug administration and sample collection 126

Results 127

(i)Maternal and neonatal cord levels at delivery 127

(ii)Neonatal elimination 132

134 Discussion

Chapter Four (b). The maternal and neonatal disposition of pethidine

administered in childbirth

Introduction 144

Subjects, drug administration and sample collection 144

Results 145

(i)Maternal and neonatal cord levels at delivery 145

(ii)Neonatal elimination 150

Discussion 153

Comparison of the placental transfer of bupivacaine and pethidine

161 126

Introduction

In recent years there has developed a considerable interest in fetal and neonatal body burdens of drugs used during childbirth which may pass across the placenta following maternal administration. Although the use of bupivacaine has been associated with changes in the fetal heart rate pattern and alterations in the behaviour of the newborn

(see Chapter One), little is known about its maternal and fetal disposition. Thus as part of the interdisciplinary study previously described on the safety of obstetric analgesia, the maternal and fetal disposition of bupivacaine was investigated as well as its subsequent elimination from the neonate.

Subjects, drug administration and sample collection

Fifty-eight obstetrically normal mothers in uncomplicated labour were studied, lumbar epidural anaesthesia being established with a dose of

35 ± 3.2mg (mean ± S.E.) of bupivacaine, and further epidural injections of bupivacaine were given as requested. The total dose given during labour was 120 ± 17.4mg with only eight women receiving a single dose.

At delivery, samples (3 - 4m1) of maternal venous blood, taken from a vein in the dorsum of the hand, as well as samples (3 - 4m1) of umbilical arterial and umbilical venous blood, were obtained.

Capillary blood samples were obtained by pricking the warmed heel of the neonate at 2, 24, 36 and 48h after birth, and the first 24h urine from 10 of the male neonates was collected. All blood samples were placed in heparinized containers and all specimens stored at -20°C prior to analysis.

Bupivacaine in maternal and cord blood was assayed by GC and in neonatal 127

blood by GC or GC-MS SIM as described in Chapter Two. Bupivacaine

in urine samples was also assayed by GC as described in the text.

Results

(i) Maternal and cord levels at delivery

The concentrations of bupivacaine in maternal venous, umbilical venous

and umbilical arterial blood at delivery, after its administration to

the mother by epidural injection during labour, in each of the indivi-

duals in the study is listed in Appendix B(1). The mean concentrations

of bupivacaine with S.E. and ranges in maternal venous umbilical

venous and umbilical arterial blood are given in Table 4.1.

Table 4.1. Concentration of bupivacaine in maternal and umbilical

cord blood at delivery

Blood concentration (ng/ml) MEAN S.E. LOW HIGH

Maternal vein (MV) n*=54 231.2 14.0 63 485

Umbilical vein (UV) n*=52 80.5 6.6 10 274

Umbilical artery (UA) n*=49 79.3 6.8 21 160

UV/UA n*=47 1.0 0.1 0.5 1.7

UA/MV n*=47 0.35 0.02 0.04 0.67

(*n * number of women in study due to difficulties in obtaining

complete sets of samples)

The concentration of bupivacaine in maternal blood was always greater

than that in cord blood with the mean fetal (i.e. UA): maternal (i.e. MV) 128

concentration ratio being 0.34. The drug concentration in the UV was

usually higher than the corresponding UA value, but with increasing

time interval between the last bupivacaine injection and delivery the

UA/UV drug concentration ratio rose and was sometimes greater than

unity (Fig. 4.1). There was a large variation in the concentration of bupivacaine within each of the three sample groups, i.e. MV, UV and

UA, as can be seen from Table 4.1. These variations could be accounted for in part by the differences in the dose-delivery interval, i.e. the time interval between the last administration of bupivacaine and delivery, as well as the maternal dose. A plot of bupivacaine concentration in maternal blood at delivery against dose-delivery interval was a scattergram (Fig. 4.2), but with the exception of eight patients, all the women in the group received more than one dose of bupivacaine which could account for these findings. If instead of considering the whole group, only data from these eight mothers were used, a monoexponential fall in bupivacaine concentration in maternal blood with increasing dose-delivery interval was seen. Although it is not possible from the results of the whole graph to calculate the blood elimination half-life of bupivacaine, an estimate of the maternal elimination half-life can be made using this group of eight mothers.

From Fig. 4.2, the blood elimination 'half-life' of bupivacaine was

2.4h. When the UA/MV concentration ratio was plotted against dose- delivery interval, the highest values were concentrated around 90 minutes. A histogram was plotted using the mean result for each 25 minute period and appeared to 'peak' between 75 and 100 minutes (Fig.

4.3). This implies that the highest concentration of bupivacaine in the fetus relative to the mother will occur between 75 - 100 minutes following drug administration. A plot of UA/UV concentration

) Fig. 4.1 Umbilical artery/Umbilical vein concentration ratio against dose-delivery interval

ine (bupivacaine) a ac iv 0 The histogram indicates the mean UA/UV 0 (bup

concentration ratio in each 25 minute period. io t a r n 0 io t a tr en 0 nc 0

co 0 in

e 0 l v a 0 0 ic il b 8 0

/Um 0 0 0 0 0 0

tery 000 00 0 0 l ar a

ic 0 il 0 0 b Um

TIME (min) 100 200 300 400 500 Fig.4.2 Semi-logarithmic plot of bupivacaine concentration in maternal blood at delivery against last dose-delivery interval

1000

• mothers receiving one dose of bupivacaine

0 mothers receiving more than one dose of bupivacaine

500 -- 0 0 8 0 0 0 O 0 0 TRATION 0 ab O 00 — — 231.2 + 14.0 200 — • mean + S.E. 0 0 00 INE CONCEN 0 0 • O O 100 — BUPIVACA

I I I 100 200 300 400 500 DOSE-DELIVERY INTERVAL (MN) Fig.4.3 Umbilical artery/maternal vein concentration ratios for bupivacaine against last dose-delivery interval

( The histogram indicates the mean UA/MV concentration ratio in each 25 minute period )

0.8-

0 • 4 0. ~7! LOG •

FQa' 0.6 • z • 6 • 0 0.5 • • z W • 0.4 ••

0.33 + 0.02 0.3 • • • • • mean + S.E. • • • rx • • • 0.2 • 8 • • 0.1 - • P •

100 200 300 400 500

DOSE-DELIVERY INTERVAL (min) 132

ratio against dose-delivery (Fig. 4.1) again gave no clear-cut pattern,

but the highest ratios tended-to occur between 75 and 100 minutes

following maternal administration.

NEONATAL ELIMINATION

Fig. 4.4 shows typical blood level-time curves for bupivacaine in neo-

natal blood for up to 48h after birth. In 16 of the 51 curves examined,

Fig. 4.4 Representative semi-logarithmic plots of bupivacaine blood concentration against time from two babies whose mothers received bupivacaine in labour

t Umbilical artery 24 48 concentration AGE (hours) 133 there was an initial rapid fall in drug concentration between 0 (i.e.

UA value) and 2h, followed by a slower exponential decline from which the elimination half-lives were calculated. The curves from the other 35 babies showed only the slower elimination phase from which the half-life was calculated. Half-life (t1/2) was calculated using the equation

t' = loge 2

kel where kel is the slope of the semilogarithmic plot of blood concentration versus time curve, i.e. the slope of the terminal phase in Fig. 4.4.

Individual neonatal blood drug levels and elimination half-lives are given in Appendix B(1) and the mean values are listed in Table 4.2.

Table 4.2 Neonatal blood levels and elimination half-lives of

bupivacaine

Blood concentration (ng/ml) MEAN S.E. LOW HIGH

Umbilical artery at delivery (n=49) 79.3 6.8 21.0 160.4

2h neonatal blood (n=50) 44.6 4.1 8.9 198.0

24h neonatal blood (n=48) 16.9 1.8 0.9 58.0

36h neonatal blood (n=31) 8.7 1.7 0 48.0

48h neonatal blood (n=15) 3.4 1.0 0 29.0

Blood elimination half-life (h) 14.0 1.1 3.2 50.2 (n=51)

The first 24h urine from ten male neonates whose mothers received bupivacaine during labour was analysed for bupivacaine and desbutyl- 134- bupivacaine by GC as described in Chapter Two. The results, given in Table 4.3, show that there was considerable variation in the amount of bupivacaine excreted by these babies (170 - 4400 ng), which may be related in part to the amount of drug administered to the mother, the amount of drug in each infant at birth, the ability of the infant to metabolize/excrete the unchanged drug etc. No desbutyl-bupivacaine could be detected in any of the urines examined.

Discussion

The use of sensitive and specific GC and GC-MS techniques has permitted the assessment of the pharmacokinetics of bupivacaine in mothers and their babies after its epidural administration to the mother during childbirth. Bupivacaine traverses the epidural space into the maternal circulation very rapidly following its administration as shown by its presence in MV samples at delivery even when the dose-delivery interval is short. Peak maternal blood concentrations of bupivacaine following its .epidural injection to mothers have been found to occur between 5 and 15 minutes after administration (Caldwell et al., 1977). An estimation of the maternal blood elimination half-life of bupivacaine based on the MV concentration at delivery of the eight mothers who received only one dose during delivery, the variable factor being the dose-delivery interval, was 2.4h. This is of the same order as the figure of 1.25h obtained by Caldwell et al.(1977a) and Scott, Jebson and Boyes (1973) in non-pregnant adult volunteers using more conventional methods for half-life determinations, although Belfrage,

Berlin, Raabe and Thalme (1975) estimated the half-life of the drug in 6 mothers at 9.0 ± 0.6h (mean ± S.D.). Bupivacaine crosses the placenta and enters the fetal circulation very rapidly following

Table 4.3 Excretion of bupivacaine and desbutyl-bupivacaine (PPX) in 0-24h neonatal urine following maternal administration of bupivacaine during labour

Baby No. Bupivacaine concentration Urine Urine Bupivacaine in PPX in urine Blood elimination Umbilical Umbilical vol. pH urine (ng) (ng) half-life (h) vein(ng/ml) artery(ng/ml) (ml)

2 65 NS 66.0 5.9 4400 ND 3.2 3 57 NS 5.8 6.7 170 ND •7.0 w u, 11 30 55 30.5 7.1 1322 ND 17.7 18 175 160 8.4 6.4 245 ND 30.1 19 115 105 12.5 5.9 4385 ND 11.6 29 90 75 17.0 6.6 2139 ND 9.1 36 50 50 6.2 5.8 299 ND 23.2 40 55 55 5.0 6.5 363 ND 11.6 52 274 323 8.6 6.5 3533 ND 9.8 53 92 55 13.0 6.1 791 ND 12.6

NS sample not supplied for analysis; ND not detected normal volume of urine in infants 1-2days old 15-60m1/24h (Kolmer and Boerner, 1945) 136

maternal administration as shown again by its presence in umbilical cord blood when the dose-delivery interval was short. The UA/UV concentration ratio at birth varied from 0.42 to 4.0. When the UA/UV ratio is less than 1, there is a net passage of drug from mother to fetus with some removal of the drug by the fetal tissues. When the

UA/UV concentration ratio is greater than 1 however, there is a net transfer of drug from the fetus to the mother. Since in most cases analgesics are administered more than once during labour, if the dose interval is less than or approximately equal to the blood elimination half-life of the drug there will be an accumulation of the drug in the mother which may also occur in the fetus. While the umbilical circulation is intact, any drug which has entered the fetus may be returned to the mother via the placenta, but after birth the infant must rely on its own resources for metabolizing and excreting any remaining drug. The ability of the neonate to metabolize drugs is known to be less than in adults (Table 1.8), and in the newborn human infant, kidney function, as indicated by the glomerular filtration rate and renal plasma flow, is approximately 30-40% of that of the adult (Mirkin, 1976), indicating that the newborn may have difficulty in eliminating drugs in the urine. Hence it becomes desirable for delivery to occur at a time when the amount of drug in the fetus is as low as possible. There may therefore be a 'time window' after the last administration of the drug during which, if delivery were to occur, the infant would be comparatively more 'at risk' from the pharmacological effects of the drug than if delivery were to occur either close to or long after maternal drug administration when the body burden is low in comparison. In this study, the UA/MV or

UV/MV concentration ratios tended to be highest between 75 and 100 137

minutes following drug administration (Fig. 4.3). Caldwell et al.

(1977) have shown by serial scalp blood sampling of the human fetus

in utero that the concentration of bupivacaine in the fetus increases

steadily for 75-90 minutes following maternal epidural administration

of bupivacaine, while the maternal venous concentration declined over

the same time period (Fig. 1.6). This suggests that there is a net

passage of drug from the mother to the fetus for about 75-100 minutes

after administration with the maximum fetal concentration probably

being achieved around this period. It therefore appears that, for

bupivacaine, it is desirable that the infant is delivered either well

before or after this period to ensure minimal drug levels in the neo-

nate. This concept of the 'time-window' is more obvious with

pethidine than bupivacaine and will be discussed later. However, it

must be remembered that any fetal drug exposure in utero could lead

to detrimental effects in the neonate and hence it is not only residual

body burdens of a drug in the newborn which could be hazardous.

The UA/UV concentration ratio was found to increase with increasing dose-delivery interval. Since the UV carries substances into the fetus and the UA is the mechanism by which they are excreted from the fetus, the UA/UV ratio is indicative of what is happening in the umhilical- fetal circulation, and may indirectly indicate what is happening in the fetal tissues where the drug will be having its effect, although this may not necessarily be the case. Assuming that the drug concentration in the umbilical circulation does indeed reflect the concentration in the fetal tissues, the above observation could possibly be explained by tissue uptake of the drug by the fetus. Initially, the drug passes from mother to fetus with some of the drug being sequestered 138

by the tissues, and hence the UA/UV ratio is less than 1. Once the

concentration of bupivacaine in fetal blood drops below the level where

there is relatively more in the tissues than the circulation, there will

be a net outflow of drug from the tissues back into the fetal circulation,

and hence the UA/UV concentration will be greater or equal to 1.

There have been no reports in the literature on the disposition of bupi-

vacaine in the fetus or neonate, but several workers have examined the

tissue distribution of some closely related local anaesthetics in

animals using whole-body autoradiography. The results showed that on

examining the fetus lh following maternal intravenous administration of [ 14C J -mepivacaine (Kristerson, Hoffmann and Hansson, 1965); 4CJ-[1

lignocaine (Katz, Gershwin and Houd, 1968) and CJ[14 rilocaine-p (Katz,

1969), radioactivity was not localized in any particular tissue, and

the amount penetrating to the fetus was extremely low. However, in

the guinea-pig, the concentration of 1[ 4C]-lignocaine was found to be

higher in fetal liver than in maternal liver as early as 1 minute

following maternal administration, although no other fetal tissue had

a concentration higher than the corresponding maternal tissue (Finster,

Morrishima, Boyes and Covino, 1972).

The rate of disappearance of bupivacaine from neonatal blood was

markedly slower than is observed in adults. The mean neonatal blood

elimination half-life in the 51 babies studied was 14h as opposed to the adult value of l.25h. Furthermore, a biphasic elimination pattern was seen in 16 of the 51 babies studied with a rapid fall in drug level from O-2h post delivery, followed by a slower elimination phase (Fig.

4.4). The initial fast phase could be due to the changes that occur 139

in the fetal circulation at birth (see Fig. 1.3). At birth, the

pulmonary circulation commences and the lungs are fully perfused for

the first time thus commencing the transition to the adult circulatory

system. Local anaesthetics exhibit an extensive first pass uptake iln lung

(Scott, Jebson and Boyes,1973).and this may therefore contribute to

the initial rapid fall in drug concentration. The variation in

neonatal half-life could not be correlated to neonatal jaundice,

maternal smoking or resuscitation required at birth, (Table 4.4)and may well reflect the relative maturity of the infants at birth.

The metabolism of bupivacaine in man has not been fully elucidated

(see Chapter Six), and consequently could not be studied fully in the

neonate. However, the amounts of bupivacaine and desbutyl-bupivacaine

excreted in the first 24h urine of ten male infants were measured

(Table 4.3). In the adult, although N-desbutylation is not quantitatively a major metabolic pathway, desbutyl-bupivacaine is excreted in urine in approximately a 1:1 ratio with the unchanged drug.

Desbutyl-bupivacaine could not be detected in any of the urines of the

babies although considerable amounts of bupivacaine, ranging from 170 to 4400 ng, were present. This indicates that the ability of the human

neonate to N-dealkylate bupivacaine is impaired relative to the adult.

Since only a small proportion of a dose of bupivacaine is excreted

unchanged in adult urine (see Chapter Six), its disappearance from blood is almost exclusively the result of its metabolism. The blood

elimination half-life of bupivacaine is prolonged by nearly tenfold

(on average) in the neonate compared with the adult, and this suggests that other pathways of bupivacaine elimination (metabolism and excretion) are impaired in the neonate. Table 4.4 An analysis of the relationship between neonatal jaundice, maternal smoking and type of resuscitation

required at delivery, on neonatal blood elimination half-life (t2) of bupivacaine

** (1) Neonatal 01 bupivacaine/neonatal jaundice on day 1 and/or 3 after birth

standard degrees of ***Critical F No mean 01 (h) *F ratio deviation freedom ratio at P=0.05 no jaundice 23 12.58 5.33 2.529 (1,40) 4.08 N.S. jaundice 19 16.6 10.63

(ii) Neonatal 01 bupivacaine/maternal smoking

standard degrees of ***Critical F No. mean 02 (h) *F ratio deviation freedom ratio at P=0.05 maternal smoking 15 12.43 5.31 0.742 (1,47) 4.00 . N.S. no smoking 34 14.52 8.65

(continued overleaf) Table 4.4. (continued)

(iii) Neonatal t' bupivacaine/type of resuscitation required at delivery

standard degrees of ***Critical F No. mean t1/2 (h) *F ratio deviation freedom ratio at P=0.05 routine suction 44 14.26 8.06 0.232 (2,48) 3.23 -> N.S. oxygen 6 12.85 5.39

IPPR 1 9.80

* Results were calculated using an analysis of variance. The F ratio is the ratio of the between groups

variance divided by the within groups variance.

** Jaundiced babies included those classified as jaundiced by the psychologists on the basis of yellow

pigmentation of the skin alone (sub-clinical jaundice) and those infants with confirmed elevated

bilirubin levels (clinical jaundice), i.e. total plasma concentration of bilirubin >10 mg/100 ml.

* * * F > critical F ratio indicates a significant difference between distribution at 5% level. N.S.

indicates no significant difference in neonatal blood elimination half-life between the groups. 142

It is interesting to speculate on the size of the body burden of bupivacaine in the infant at birth. There is however no way of estimating the total dose to the fetus on a one-point determination of fetal or cord blood concentration, but, by making a number of assumptions a rough estimate can be obtained. Assuming the UA concentration is representative of the fetal circulation at birth, the minimum amount of bupivacaine in the neonate will be that contained in the circulation; i.e.

minimum amount of bupivacaine UA x blood volume in the neonate at birth

(volume of blood in the neonate is about 80m1/kg and the

average baby weighs about 3kg).

Hence, using the UA concentration values for the 10 babies in Table 4.3, the minimum body burden of bupivacaine ranges from 15pg for baby No. 36 to 93pg for baby No. 52. However, any amount of tissue sequestation would increase these values. A useful assessment of the degree of tissue localization of a drug can be obtained from its apparent volume of distribution, Vd. The Vd for bupivacaine in adults has been estimated at 1.821/kg (Caldwell, Moffat and Lieberman; unpublished data) where

Vd was calculated as : dose administered/theoretical concentration in whole blood at the moment of injection. Assuming that the Vd for the neonate is the same as in adults, then;

Body load of bupivacaine at birth = Vd x weight x UA

Hence the estimated amount of bupivacaine in the infants at birth using the adult Vd value of 1.82 1/kg ranges from 0.33mg for baby No. 36 to

2.12mg for baby No. 52. However, the largest amount of bupivacaine excreted in the first 24h urine by these infants was only 4.4ug (Baby

No. 2) and this was far lower than the smallest estimated body burden 143

on zero tissue sequestation. There therefore appears to be a large discrepancy between the calculated body burden and the actual amount of bupivacaine excreted unless (a) the neonates were good metabolizers of bupivacaine (b) their renal function was greatly impaired or (c) bupivacaine was eliminated by means other than renal excretion. Im- paired renal function could account for some of the discrepancy between bupivacaine body burden and excretion, but since metabolism is apparently necessary for elimination (see Chapter Six) and the long blood elimination half-lives in these infants suggest impaired metabolic activity, the poor urinary recoveries compared to the estimated body burdens cannot be attributed solely to impaired renal function. Also, some of the drug could have been excreted (a) into the amniotic fluid or (b) in the bile and subsequently eliminated in the faeces, which were not examined. However, unless bupivacaine was 'stored ) within the neonate

(which is unlikely on the basis of tissue distribution experiments with similar compounds in animals), it is very likely that the above method greatly overestimates the body burden of bupivacaine in these babies.

A difference in the Vd in neonates compared to adults is to be expected since body composition in the newborn, especially in premature babies, differs greatly from that in adults; e.g. there is a relative lack of fatty tissue in the newborn (infants of diabetic mothers are a notable exception) which is more pronounced in premature babies. There are in addition, differences in the distribution of the body water with a higher extracellular fraction in the newborn compared to adults. It is therefore probable that the Vd for bupivacaine is different to that in adults which may account for some of the variation between calculated body burden and the amounts actually recovered. 144

Chapter Four (b) The maternal and neonatal disposition of pethidine

in childbirth

Introduction

Pethidine is widely used for the relief of pain in childbirth being

the most popular obstetric analgesic in the United Kingdom at the present time. However, pethidine can affect the newborn infant; its use has been associated with respiratory difficulties and signs of CNS depression in the neonate (see Chapter One). Paradoxically, the incidence of such effects apparently increase with increasing time interval between maternal drug administration and delivery (Morrison, et al., 1973). To study this problem further, three aspects of pethidine disposition in childbirth were investigated, namely (a) the transplacental passage of the drug as a function of the dose-delivery interval (b) accumulation of pethidine by the fetus and (c) elimination of the accumulated pethidine burden by the neonate.

Subjects, drug administration and sample collection

Fifty-one obstetrically normal mothers were studied in uncomplicated labour. They received pethidine (100 or 150mg) combined with metaclo-. promide (10mg) to prevent nausea, by intramuscular injection and similar injections were repeated as requested. Eleven women received more than one dose. The total amount of pethidine administered during labour was 163 ± 12mg (mean ± S.E.). Samples of maternal venous and umbilical arterial and venous blood were obtained at delivery, and serial neonatal blood samples were obtained at (ideally) 2, 6, 24 and

36h after birth as described in Chapter Three. The first 24h urine from five male neonates Nos 15,19,20,25,44 was also collected. Storage of samples was as for bupivacaine samples. 145

Pethidine in maternal and cord blood and in urine samples was assayed by GC, and in neonatal blood by GC or GC-MS SIM as described in

Chapter Three.

Results

Maternal and cord levels at delivery

The concentration of pethidine in maternal venous (MV), umbilical venous (UV) and umbilical arterial (UA) blood at delivery, following its maternal administration in each of the 51 mothers studied is given in Appendix B(2). The mean concentrations of pethidine with S.E. and ranges in MV, UV and UA blood are given in Table 4.5.

Table 4.5. Concentration of pethidine in maternal and umbilical

blood at delivery

Blood concentration (ng/ml) MEAN S.E. LOW HIGH

Maternal vein (MV) n*=38 256.3 23.4 81 640

Umbilical vein (UV) n*=39 213.2 18.5 20 435

Umbilical artery(UA) n*=35 222.0 22.4 28 540

UV/UA (n*=35) 1.2 0.1 0.7 2.1

UA/MV (n*=34) 0.86 0.1 0.16 3.21

*n # number of women in study due to difficulties in obtaining complete

sets of samples.

The mean fetal (UA):maternal (MV) concentration ratio was 0.86 showing extensive placental transfer of pethidine. There was a large variation between the subjects in the amount of pethidine in the maternal and cord bloods at delivery, in some cases the greater drug concentration being found in cord blood rather than in maternal blood. These variations 146

in drug levels and ratios are accounted for by the differences in the time between the last maternal administration of pethidine and delivery.

The drug concentration in the UV was usually higher than the corresponding

UA value, but with increasing time interval between the last pethidine injection and delivery, the UA/UV drug concentration ratio rose until it was greater than 1 (see below).

A plot of pethidine concentration in maternal venous blood against dose- delivery interval showed a monoexponential decline in the 2 groups of single-dose mothers - one group receiving 100mg of pethidine, the other group 150mg pethidine i.m. (Fig. 4.5). Again, as in the case of bupivacaine, it is not possible to calculate the blood elimination half-life of pethidine from this data, but an estimate of the maternal blood elimination half-life can be made. From Fig. 4.5, the blood elimination

'half-life' of pethidine is 2h, both dose groups giving the same answer.

The UA/MV concentration ratio rose with increasing dose-delivery interval from about 0.5 at 40 minutes to in excess of 2.5 at 500 minutes (Fig.

4.6). A similar plot was obtained for the UV/MV concentration ratio when plotted against dose-delivery interval. Fig. 4.6 shows that when the dose-delivery interval is longer than 170 minutes, the concentration of pethidine in umbilical cord blood is greater than that in maternal blood, the implications of which will be discussed later. In only one case was the UA/MV ratio less than 1 when the dose-delivery was longer than 170 minutes. Fig. 4.7 shows that the plot of UA/UV concentration ratio against dose-delivery interval follows a quadratic-type curve with a peak at approximately 290 minutes following maternal administration.

The UA/UV concentration ratio was greater or equal to 1 when the dose-delivery interval was longer than 95 minutes.

147

Fig. 4.5 Semi-logarithmic plot of pethidine concentration in maternal blood at delivery.against last dose - delivery interval

600— • • maternal dose 100mg

400_ • • 'ti' = 2h • 300— •

200_ •

••

100 • I - I r I I 100 200 300 400 500 Dose-delivery interval (min) ō N

maternal dose 150mg 400 — • 'ti' = 2h • 300 — • • • 200 — •

•

100 — •

• 50 I 100 200 300 400 500 Dose-delivery interval Fig. 4.6 Umbilical artery/maternal vein concentration ratios for Pethidine against last dose-delivery interval

• 3 -

=0.70) 2 • • • • • • • • • Fetal concentration higher •• • Maternal concentration higher • • • • • • •• • • • •

100 200 300 400 500 LAST DOSE-DELIVERY INTERVAL (min) Fig. 4.7 Umbilical artery/Umbilical vein concentration ratio against dose-delivery interval for pethidine

0 cd a 0 0 2.0

a U a 0 0 U 1.8

•v 2 0 0 0 0 0 y= a2x + a lx + ao ca U "r4 1.2

1.1 a = 0.583 a' 0.8 0 as a I = 0.007

cd a2 = 0.00003 U ri 0.4 5

100 200 300 400 500 TIME (min) 150

Samples of maternal and cord blood from two patients were analysed for

pethidine metabolites using plasma obtained from the samples by centri-

fugation. Plasma (20m1) was concentrated using Amberlite XAD-2 resin

and assayed for all known metabolites of pethidine using the sequential

extraction and derivatization procedures described in Chapter Three for

urine samples followed by GC analysis. The results given in Table 4.6

show that norpethidinic acid was the only metabolite present in the

umbilical circulation. Norpethidine was not detected in either maternal

or umbilical blood suggesting that its rate of formation was less than

its clearance rate. Pethidinic acid, although present in the maternal

samples, was not detected in umbilical blood.

Table 4. 6 Transplacental passage of pethidine and its metabolites.

Concentration of: (ng/ml) plasma) PETHIDINE NORPETHIDINE PETHIDINIC NORPETHIDINIC ACID* ACID*

PATIENT 1 UV 455 ND ND 23

UA 450 ND ND 21

PATIENT 2 UV 436 ND ND 66

UA 422 ND ND 57

MATERNAL BLOOD 2h after 61 ND 13 72 administration

ND = not detected. *total acids estimated (i.e. free and conjugated)

Neonatal elimination

The concentration of pethidine in the capillary blood of the neonates 151

studied fell monoexponentially with time over the 48h period, and Fig.

4.8 shows a representative plot from which the half-life was calculated from the slope of the line as described for bupivacaine.

Fig. 4.8 Representative semi-logarithmic plot of pethidine blood concentration against time from a baby whose mother received pethidine during labour

E 100 c

n io t a 50 — tr en nc co e in id h t 20 — Pe

t 1 I Umbilical artery 24 48 concentration AGE (hours) 152

The mean neonatal elimination half-life of pethidine calculated from

the curves for each infant was 22.4h, and there was considerable inter-

subject variation as shown in Appendix B(2). The mean neonatal blood

levels and elimination half-life of pethidine are listed in Table 4.7.

Table 4.7 Neonatal blood levels and elimination hjlf-life of pethidine

Blood concentration (ng/ml) MEAN S.E. LOW HIGH

Umbilical artery at delivery n=35 222.0 22.4 28.4 540.5

2h neonatal blood n=36 164.0 18.2 53.0 450.0

6h neonatal blood n=12 89.4 12.1 32.0 161.0

24h neonatal blood n=35 81.3 10.8 23.3 323.0

36h neonatal blood n=12 78.7 11.6 22.0 134.0

Elimination half-life(h) n=40 22.4 4.7 3.2 38.1

The first 24h urine of five babies whose mothers received pethidine during labour was analysed for pethidine and all known pethidine metabolites as described in Chapter Three. The results are given in

Table 4.8; the neonatal metabolism of pethidine will be discussed fully in Chapter Eight.

Discussion

The concentration of pethidine in maternal venous blood fell monoexponentially with increasing dose-delivery interval as shown in Fig.

4.5. This plot gives an estimate of the blood elimination 'half-life' for the mothers of 2h. This is of the same order as the value of 3.2h obtained by Mather, Tucker, Pf lug, Lindop and Wilkerson (1975) in non- pregnant adult volunteers using more conventional methods for half-life Table 4.8 Pethidine metabolism in five human neonates following maternal administration during childbirth

The first 24h urine from 5 male neonates was collected and analysed as described in the text. Figures are expressed as pg in that form in 24h urine **

Baby No. 15 19 20 25 44

Urine pH 5.8 6.8 5.8 6.2 5.9 9 Urine volume (ml) 12.5 20.5 26.0 21.0 11.4 Blood elimination half-life (h) 18.7 23.3 22.7 15.0 26.5 ,

Pethidine 15.7 177.4 46.5 123.5 109.1 ul w Norpethidine 0.4 43.7 7.5 57.1 6.4 Pethidine-N-oxide 0.03 0.64 0.21 0.40 0.24 Pethidinic acid free 3.6 10.6 12.4 8.5 4.0 conjugated 1.0 5.9 16.6 11.0 0.2 total 4.6 16.5 29.0 19.5 4.2 Norpethidinic acid free 2.3 6.3 19.2 8.8 4.4 conjugated 5.3 11.9 17.9 24.7 3.7 total 7.6 18.2 37.1 33.5 8.1

Total 28.3 256.5 120.3 234.0 128.0

* 4'-Hydroxypethidine was not detected in any of the urines ** see Chapter Eight for details UA and UV concentrations (pethidine concentration in umbilical blood) are given in Appendix B(2) 154

determinations.

The transplacental passage of pethidine as measured by its concentration in UA and UV blood at delivery, suggests that its transfer to the fetus is both rapid and extensive. The mean UA/MV concentration ratio was

0.86 and in some cases the concentration of pethidine in cord blood was much higher than that in maternal blood. The UV/MV and UA/MV concentration ratios rose with increasing dose-delivery interval, and when the dose-delivery interval was longer than 170 minutes, the concentration of pethidine in fetal blood was (with only one exception) higher than that in maternal blood. This is in accord with the principles governing drug distribution into tissue depots. The distribution of drugs in man frequently follows a so-called two compartment model in which the drug equilibrates between a central compartment, M, commonly identified with the plasma and highly perfused tissues; and a peripheral compartment, T, which may represent other less-well perfused tissues of the body (Wagner, 1973). The extent of entry of the drug into the tissues of the body is governed by its physico-chemical characteristics. The concentrations of a drug in the circulation and the tissues based on the two-compartmental model suggested above may be typically represented as shown below.

M plasma and well- a perfused tissue

111 T M T less-well perfused tissue 155

TIME

The slopes of the lines shown represent the elimination rate constants

(kel) of the drug from each of the compartments, more usually expressed in terms of biological half-life, t1/2, (t' = 0.693/kel).

Thus, although the tissue level of a drug is related to that in the circulation, the peak level in the tissues is generally, but not always, lower than that in the circulation, and the presence of the drug in the tissues is sustained for longer. There will be a period in which the tissue level is still rising when the blood level is declining. In pregnancy, the presence of the fetus and its associated circulation may constitute a third compartment, F. The penetration of drugs into the fetus is governed both by their physico-chemical properties and the haemodynamics of the two circulations, and it is generally the case that the fetal circulation is less accessible to the drug than are the other well perfused tissues of the body (Dawes, 1973). Hence, the passage 156

of pethidine to the fetal circulation will occur at a considerably

lower rate than it gains access to the maternal circulation, and there-

fore the peak amount in the fetus will be reached later in time than

the peak in the mother. Hence the fetus can act as a 'sink' due to

sequestation by fetal tissues. The concentration of pethidine in the

maternal circulation decreases as a result of metabolism and excretion,

and thus the fetal blood concentration also falls with time. When the

concentration of pethidine in fetal blood drops below the level where

there is relatively more in the fetal tissues than the circulation,

there will be a net outflow of drug from the fetal tissues back into

the fetal circulation and thence back to the mother via the UA. The

lowest concentration of drug in the fetus may therefore not coincide

with the times at which the level in the maternal circulation and tissues

is lowest. Fig. 4.6 shows that the concentration of pethidine in cord

blood relative to that in maternal blood increases with time, and the

concentration of pethidine in maternal blood decreases with an elimination

half-life of 2h (Fig. 4.5). Thus, the amount of pethidine in the fetus

will at first increase (at the same time that the amount in the mother

is decreasing) and reach a maximum before declining, and it would

obviously be desirable for delivery to occur at a time when the amount

°of drug in the neonate is as low as possible. There may therefore be

a 'time-window' after the last administration of the drug during which

if delivery were to occur, the infant would be comparatively more 'at

risk' than if delivery were to occur either close to or long after the

final dose of the analgesic. Such a concept is supported by several

groups of workers: Bonica (1S57) and Schnider (1970) both found that

respiratory depression in the newborn was greatest if the infant was

born between one and four hours after pethidine administration to the 157

mother, and conversely that it was minimal if the baby was born before

one hour or more than four to six hours after maternal administration.

Similarly, Morrison et al. (1973) showed that babies born less than

one hour after the last maternal dose of pethidine showed much less

evidence of drug-induced depression than those born one or two hours

after pethidine. Thus, the peak-neonatal depression as suggested by

these studies occurs between 1 and 4 hours after maternal administration

indicating that delivery would be more desirable either before or after

this period.

There have been no reports in the literature on the disposition of pethi-

dine in the fetus or neonate, but Burns et al. (1965) have examined the

tissue distribution of pethidine in the dog. Forty minutes following

intraperitoneal administration, the concentration of pethidine in the

tissues was at most six times higher than in the plasma, the only tissue

where the concentration was less than that found in the plasma being

fat. Thus, general tissue uptake of pethidine by the dog appears to be

large, although how far this data may be extrapolated to the human fetus

remains to be seen.

An estimate of the neonatal body burden of pethidine can be made in a similar manner to the procedure used for bupivacaine. Assuming that the UA concentration is representative of the fetal circulation at birth, then;

minimum amount of pethidine UA x blood volume in the neonate at birth

Hence, using the UA values for the five babies in Table 4.8, the minimum body burden of pethidine varies from 19.7ug for baby No. 20 to 158

120Pg for baby No. 25. Table 4.8 shows that the 24h urinary recovery

of pethidine and all known metabolites was between 28ug for baby No.

15 and 256ug for baby No. 19 showing that some tissue sequestation had

taken place on the basis of the above values. The Vd of pethidine in

adults, where Vd = Dose/theoretical concentration in whole blood at

moment of injection, has been estimated at 4.33 ± 0.85 1/kg (mean ±

S.D., n = 16) (Bennett,unpublished data). Thus, assuming that the

neonate has a similar Vd to that of adults the esimated body burden of

the neonates allowing for tissue sequestation, varies from 1.07mg for

baby No. 20 to 6.50mg for baby No. 25. However, there is now once

again a large discrepancy between the estimated body burden using the

adult Vd value and the actual total recovery of pethidine and all known

metabolites in 0-24h neonatal urine. It is possible that this discrepancy

could be accounted for should (i) pethidine or its metabolites be elimin-

ated by uninvestigated routes, e.g. faeces, (ii) be 'stored' in the

tissues or (iii) retained by the neonate due to impaired renal function.

Since metabolism is apparently necessary for renal elimination in the

adult and neonatal metabolism of pethidine is impaired (see below) the

difference between the observed urinary recoveries and the estimated body

burdens of the neonates cannot be attributed solely to impaired renal

function. Thus, barring tissue sequestation and elimination by other

routes, the most likely reason for this discrepancy is that the estimated

body burden of pethidine in the neonate was inaccurate. For the same

reasons discussed for bupivacaine, the Vd is expected to differ in adults

and neonates due to differences in body composition, and hence, the use

of the adult Vd in calculating neonatal body burdens was in all probability not valid.

The sensitivity and specificity of the GC-MS SIM method for the assay of 159

pethidine has enabled its measurements in small samples of neonatal

capillary blood. The elimination half-lives for the babies ranged

from 3.2 to 38.1 hours with a mean of 22.4 hours. Thus in general,

the rate of pethidine elimination from the neonatal circulation was

far lower than that found in adults (3.2h, Mather et al., 1975), and

since only a small amount of pethidine is excreted unchanged in the

urine of adults (see Chapters Seven and Eight), this indicates that

its disappearance from blood is principally due to metabolism. It

therefore appears that the metabolism of pethidine is defective in the

newborn, and the differences in the elimination rate may well indicate

the relative maturity of the drug metabolizing systems of the babies.

Again, there was no correlation between neonatal blood elimination

half-life of pethidine and maternal smoking, neonatal jaundice and

type of resuscitation required at delivery (Table 4.9).This suggestion that the

neonate is relatively unable to metabolize pethidine is further supported by the analysis of the 0-24h neonatal urines collected from five babies for pethidine and all metabolites (Table 4.8). This aspect of impaired metabolic ability of the neonate to metabolize pethidine will be discussed fully in Chapter Eight. Table 4.9. An analysis of the relationship between neonatal jaundice, maternal smoking and type of resuscitation required at delivery, on neonatal blood elimination half-life (t2) of pethidine

(1) Neonatal 02 pethidine/neonatal jaundice on day 1 and/or 3 after birth

standard No. mean 01 (h) F ratio degrees of critical F deviation freedom ratio at P=0.05

no jaundice 22 17.57 10.04 0.003 (1,33) 4.17 4 N.S. jaundice 13 17.79 11.13

(ii) Neonatal el pethidine/maternal smoking

standard critical F No mean 02 (h) F ratio degrees of deviation ratio at P=0.05 freedom

maternal smoking 11 14.76 5.56 1.429 (1,38) no smoking 28 19.10 11.48 4.07 -> N.S.

(iii) Neonatal 02 pethidine/type of resuscitation required at delivery

standard degrees of critical F No mean 02 (h) F ratio deviation freedom ratio at P=0.05 routine suction 31 19.07 9.87 oxygen 6 11.03 12.69 1.663 (2,36) 3.27 N.S. IPPR 2 14.50 0.85

Abbreviations and details are as in Table 4.4 161

Comparison of the Placental Transfer of Bupivacaine and Pethidine

Although bupivacaine and pethidine are basic compounds (pKa bupivacaine

8.1; pKa pethidine 8.63) with similar molecular weights (bupivacaine

288; pethidine 247), this work has shown that there are large differ-

ences in their transplacental passage in women at term, notably that

(a) the concentration of bupivacaine in maternal blood was always

greater than that in cord blood, whereas this was not always the

case for pethidine.

(b) the UA/MV concentration ratio when plotted against dose-delivery

interval gave a steady rise for pethidine, whereas for bupivacaine

the highest ratioswere concentrated around 75-100 minutes after

maternal administration.

These differences suggest that the fetus is able to accumulate pethidine

to a far greater extent than bupivacaine. This might be expected on

the basis of the degree of plasma protein binding of the two drugs in

adults: bupivacaine 88% bound (Tucker et al., 1970), pethidine 38-42%

bound (Mather et al., 1970). It may be argued from this data that

since only unbound drug crosses the placenta, there will be relatively

more pethidine than bupivacaine available for fetal distribution.

However, the pH of maternal venous blood (pH 7.31-7.43) is generally

0.1 to 0.15 pH units higher than that of umbilical blood and thus the

amount of unionized drug on either side. of the placenta will differ.

Typically, for pH values of 7.4 for maternal blood and pH 7.15 for

umbilical blood, the degree of ionization for pethidine will be 95% and

97% respectively, and for bupivacaine 83% and 90% respectively. Since

only unionized drug crosses the placenta there will be a net transfer of both drugs from mother to fetus, and this phenomena of fetal'ion- trapping' may be especially important in cases of fetal acidosis. 162

Thus, more pethidine than bupivacaine is available for fetal distribution on the basis of plasma-protein binding, but less is available on the basis of degree of ionization. However, since it is the total amount of unbound, unionized drug that is important, comparatively more pethidine than bupivacaine will be involved in transplacental passage.

As- mentioned previously, the distribution of drugs in the human body frequently follows a so-called two compartment model in which the drug equilibrates between the circulation, compartment M, and the tissues of the body, compartment T, (Wagner, 1973). In the pregnant women, the presence of the fetus and its associated circulation may constitute a third compartment, but the small volume of this compared with the volume of the maternal body compartment generally means that from the viewpoint of maternal pharmacokinetics, the baby appears to be continuous with the other tissues of the mother's body (Levy and Heyton, 1973).

On the basis of the results obtained, it appears that this concept is valid in the case of bupivacaine, when the fetus can be regarded as a well perfused 'tissue' that sequesters little of the drug and cannot be mathematically distinguished as a separate compartment (Fig. 4.9).

However, in the case of pethidine, more of the drug is sequestered by the fetus which in this instance can act as a 'sink' for the drug and may be regarded as a separate compartment (Fig. 4. 9). The implications of this are that babies whose mothers received pethidine during delivery are likely to have a larger body burden of drug than those whose mothers were given bupivacaine, and are therefore conceivably more 'at risk'.

Furthermore, the'time window' concept discussed earlier, where there appears to be an interval at some time following maternal drug administration where should delivery occur the infant would have a higher body Fig. 4.9 Proposed transplacental passage of pethidine and bupivacaine during labour following maternal administration

T M F PETHIDINE F BUPIVACAINE

4 T

t t en en tm tm ar M>F ar mp comp co in in n io ion t t a a tr tr en Ī • \N en nc F>M conc co E Drug Drug

Time Time 164

burden than if he were born either side of this period, may well be

more important for pethidine than bupivacaine on the basis of the results obtained.

This study has shown that both bupivacaine and pethidine pass the placenta from the maternal to the fetal circulation very rapidly after maternal administration although the transplacental passage of pethidine is more extensive than that of bupivacaine. The fetus sequesters relatively more pethidine than bupivacaine and therefore the infant whose mother received pethidine during labour is likely to have a relatively larger body burden of drug residue than the infant whose mother received bupivacaine. Both these drugs have a long blood elimination half-life in the neonate, probably as a result of impairment of their metabolism, which means that the pharmacological activity of these drugs is much longer than in adults. The transition from intra- uterine to extrauterine life involves a number of major physiological changes for the fetus. While many of these changes are of a functional nature, e.g. expansion of the lungs, regulation of body temperature etc.; the cardiopulmonary changes also involve gross anatomic alterations

(Fig. 1.3), and some of these functions could possibly be affected by these drugs. It remains to be established whether either pethidine or bupivacaine have any adverse effects on the fetus and neonate and the infant's future development, particularly since the CNS is not fully developed at term. This aspect of the effect of residual drug in the neonate is discussed in the following chapter. 165

Chapter Five The effects of maternal analgesia (pethidine and

bupivacaine) on the fetus and neonate

Page

Introduction 166

Patients and Methods 166

Selection of population studied 166

Obstetric management 166

Assessment of neonate 167

Results 168

Effect of maternal analgesia on the fetal cardiovascular system

Effect of maternal analgesia on neonatal behaviour 170

Discussion 175

Implications of the results 177 166

Introduction

The aims of the inter-disciplinary study on the safety of obstetric

analgesia have been listed in Chapter One. This work constitutes an

attempt to study the perinatal pharmacology of maternally administered

bupivacaine and pethidine during labour, while controlling the many

variables that can contribute to the state of the baby at birth. The

varied scope of this study necessitated the involvement of several

groups of workers in different disciplines; i.e. obstetrics, paediatrics,

pharmacology,'psychology and statistics. This chapter gives a brief

outline of the entire study, its results and conclusions, which have

been correlated with the extent of placental transfer, and half-life of

the two drugs in each individual baby.

Patients and Methods

Selection of population studied

Married Caucasian women with no known medical, familial or obstetric

disorders which could adversely affect the outcome of pregnancy were

invited, during the first half of pregnancy, to participate in this

study. They were allowed to select the method of analgesia as random

allocation to a drug group was considered unethical and impractical.

Of 145 patients, 36 elected not to have any drugs during labour, 51

requested pethidine and 58 elected to have epidural injections of

bupivacaine. The patients received routine antenatal care and were

prescribed only iron and folic acid as necessary.

Obstetric management

Labour was induced in 72 of the 145 patients, all of these subsequently

receiving either pethidine or bupivacaine, and at the onset of labour 167

a standard procedure was followed. Continuous fetal heart-rate monitoring was undertaken in all patients, and uterine contractions in

the oxytocin-induced and augmented labours. Pethidine was administered

to the 51 women by intramuscular injection (100 or 150mg) combined with

10mg metaclopromide to prevent nausea,similar injections were repeated

as required. The mean dose of pethidine administered during labour

to the mothers was 163 ± 12mg (mean ± S.E.). In the epidural bupi-.

vacaine group, a 0.375% solution of bupivacaine (10-14m1) without

adrenaline was administered by the lumbar epidural route, and again, injections repeated as necessary. The mean total dose of bupivacaine

administered during labour was 120 ± 17.4mg (mean ± S.E.). At delivery, blood samples (5ml) were taken from a maternal vein in the dorsum of the hand, and the umbilical vein and artery. Neonatal heel pricks were also taken at intervals up to 48h after birth as described previously. All samples were subsequently analysed for pethidine or

bupivacaine; the results are presented in Appendices B(1) and B(2).

Assessment of the neonate

The Apgar score (Apgar, 1953) was recorded at one and five minutes after

delivery, and also the immediate post-delivery care of the infant, i.e.

type of resuscitation required - none; oxygen by face mask; or inter-

mittent positive pressure ventilation (IPPR); which was determined by

the clinical condition of the baby at birth at the discretion of the

attending medical or nursing staff. The behaviour of the infants was

evaluated using the Brazelton Neonatal Assessment (Brazelton, 1973) at

selected intervals from delivery to six weeks of age by four psychologists.

All observations and assessments during the study were made without knowledge of the type of maternal medication, and schedules were arranged 168

to avoid consecutive assessment by the same psychologist. Inter- observer reliabilities between the four psychologists remained above

0.87 throughout the study.

In addition to the behavioural assessment, an independent neurological examination of each infant was carried out by a paediatrician using the Prechtl Neurological Examination (Prechtl and Beintema, 1964) on day 5. Records of sleep and waking patterns, frequency and consumption of each feed, developmental progress and temperament of the neonate was kept by the mother throughout the first six weeks.

A summary of the data collected is given in Table 5.1.

Results

Effect of maternal analgesia on the fetal cardiovascular system

Changes in the fetal heart rate pattern following maternal administration of either pethidine or bupivacaine were analysed in 2 ways:

(i) by looking for the presence of late decelerations (i.e. deceleration

occurring in the latter stages of a contraction and hence likely to

be associated with the contraction) on the fetal heart rate trace.

(ii)the derivation of a deceleration index, this index being expressed

as:

deceleration index = no. of decelerations in a 30min period no. of contractions in the 30min period x 100

Late decelerations in the fetal heart rate pattern occurred in

eight patients following the use of bupivacaine. In 5 of these

8 patients maternal hypotension was present (BP < 6O mm Hg) and

in the other 3 patients uterine hyperstimulation by oxytocin was

evident. In this study, uterine hyperstimulation (contractions 169

Table 5.1. Summary of data collected

Antenatal Obstetric history of mother; details of present pregnancy, i.e. mood, illnesses, symptoms, etc.

Labour Continuous fetal heart rate monitoring; uterine contractions; blood pressure at 5 minute intervals.

Delivery Blood samples (maternal venous, umbilical vein and artery); *Apgar score at 1 and 5 min; selective items of Brazelton examination; resuscitation necessary.

DAY 1 **Brazelton examination; maternal questionnaire of labour, sleep and feed charts on infant. Blood elimination half-life of drug by infant.

DAY 3 Brazelton examination

DAY 5 Prechtl neurological examination

DAY 7 Brazelton examination.

DAY 21 Brazelton examination

DAY 42 Brazelton examination

* Apgar test was defined in Chapter One.

** Brazelton Neonatal Assessment contains 23 items which score on a

9 point scale which characterizes 4 areas of newborn behaviour, i. e.

(i) attention and orientation to the environment (ii) muscle tone and motor organisation (iii) control and organisation of states of sleep and wakefulness (iv) physiological response to stressful stimuli. 170

occurred at the rate of 1 or more every 3 minutes) and maternal

hypotension were always accompanied by late decelerations.

There was no evidence of fetal acidosis (i.e. umbilical blood pH > 7.15) in

either of the two drug groups. Umbilical blood was generally 0.05-0.25 (UA)

and 0.01-0.15 (UV) pH units below that of maternal venous blood.

Effect of maternal analgesia on neonatal status (at birth) and neonatal

behaviour

Table 5.2 shows that apart from routine mucus extraction twelve of the fifty-

one infants (24%) in the pethidine group and six of the fifty-eight (10%) in

the bupivacaine group were given oxygen by face mask. Three infants (6%)

in the pethidine group and three (5%) in the bupivacaine group required IPPR.

No babies in the control (no-drug) group required resuscitation.

Table 5.2 also compares the 1 minute Apgar scores for the three groups.

Bearing in mind that the higher the score the better the condition of the baby,

ten (20%) of the babies in the pethidine group had a low (i.e. (7)\ 1 minute

Apgar score compared with two (3.5%) in the bupivacaine group and none in

the no-drug group. There were no significant differences in the 5 minute

Apgar scores between the groups.

At delivery, infants in the epidural group were less responsive to the human

voice but no other differences were observed between the groups by the Brazelton

Assessment tests. Subsequent assessments also failed to reveal any significant differences in neonatal behaviour between the three groups on day 1, 7, 21 and 42 of life with the single exception of the inability of the pethidine babies to ignore the repeated ringing of a bell on day 3 (i.e. habituation response was impaired).

There were no significant differences in the sleep and feed patterns or Prechtl neurological examination on day 5. Hence, apart from the 1-minute Apgar score for the pethidine group, between group 171

analysis shows that maternal analgesia has little effect on subsequent

neonatal behaviour.

However, when the two drug groups were examined separately in relation

to certain parameters of neonatal body burden, differences in neonatal

behaviour attributable to maternal analgesia were evident. Attempts

were made to correlate (i) neonatal blood elimination half-life (t1/2)

(ii) maternal and umbilical blood concentrations and (iii) total drug

exposure of the infant (both during and after birth), with the various behavioural measurements in the Brazelton neonatal assessment. Fig.

5.1 correlates t1/2, MV, UA and UV concentration at delivery with adversely (negatively) affected behaviour. The longer the t1/2 or the higher the MV, UA or UV concentrations, the worse the performance of

the baby in certain items of the Brazelton test. Fig. 5.2 correlates total drug exposure (calculated as the area under the fetal/neonatal concentration - time curve as described in Appendix D) with behaviour; incidences of both adversely (negatively) affected and improved behaviour (with respect to no-drug babies) were observed in cases of high drug exposure. The results show that the degree of neonatal exposure to the drugs used may be correlated with changes in neonatal behaviour for at least six weeks after birth, it being interesting to note that in the pethidine group more items of the Brazelton test are adversely affected after day 3 than before this in babies exposed to a relatively large amount of drug. The importance of these changes and whether the infant's future behaviour and development is also affected remains to be seen. Table 5.2 Infant status (Apgar score and resuscitation required) at birth in the three drug groups

Apgar score at Number of Number of infants requiring resuscitation at birth 1 minute infants suction oxygen IPPR

No drug group

7 0 0 0 0 8 2 2 0 0 9 27 27 0 0 10 6 6 0 0

Pethidine group

7 10 2 . 5 3 8 15 9 6 0 9 21 20 1 0 10 5 5 0 0

Bupivacaine group

7 2 0 0 2 8 19 15 4 0 9 31 29 2 0 10 6 6 0 0 173

Fig. 5.1 EFFECT OF PETHIDINE AND BUPIVACAINE ON INFANT BEHAVIOUR

ITEMS WHICH ARE NEGATIVELY AFFECTED BY DRUGS

DAY 1 3 7 21 42 ATTENTION/SOCIAL RESPONSIVENESS

ORIENTATION •❑ * A` ❑ 0 ❑

ORIENTATION (RATTLE) ❑ * ORIENTATION FACE A 0

ORIENTATION VOICE A❑ 0

ORIENTATION FACE & VOICE 4. 0

ALERTNESS ♦ ❑ * ❑

CONSOLABILITY • ❑ ■

MUSCLE TONE AND MOTOR ORGANISATION

TONE •A ❑ ❑

ACTIVITY LEVEL ' ❑ * HAND-TO-MOUTH *A ■• ■

DEFENSIVE MOVEMENTS ♦ 0 ❑• ❑ PULL-TO-SIT

COTROLLING STATE OF CONSCIOUSNESS

RESPONSE TO LIGHT •

RESPONSE TO RATTLE •

RESPONSE TO BELL Al

BUILD UP TO CRYING

PEAK OF EXCITEMENT

IRRITABILITY

SELF-QUIETING ABILITY

LABILITY OF STATE

PHYSIOLOGICAL RESPONSE TO STRESS

STARTLES

TREMORS • ♦❑

•ABOVE ARE ALL THE ITEMS INVOLVED IN THE BRAZELTON ASSESSMENT OF THE INFANT WITH THE ITEMS NEGATIVELY AFFECTED BY DRUGS INDICATED; i.e. A WITHIN GROUP ANALYSIS.

KEY ADVERSE EFFECTS CORRELATED WITH

• LONG HALF LIFE PETHIDINE A LONG HALF LIFE BUPIVACAINE

• SHORT HALF LIFE PETHIDINE O SHORT HALF LIFE BUPIVACAINE

• HIGH CORD LEVEL PETHIDINE ❑ HIGH CORD LEVEL BUPIVACAINE

*HIGH MATERNAL DOSE PETHIDINE *'HIGH MATERNAL DOSE BUPIVACAINE

174

FIG.5.2 EFFECT OF URIC EXPOSURE ON NEONATAL BEHAVIOUR

DRUG EXPOSLRE = TOTAL DRUG CONCENTRATION-TINE INTEGRAL (FETAL & NEONATAL DRUG EXPOSURE)

DAY t 3 7 21 42 ATTENTION/SOCZAL RESPONSIVENESS

ORIENTATION 0

ORIENTATION SOUND (RATTLE)

ORIENTATION FACE ■O

ORIENTATION VOICE

ORIENTATION FACE & VOICE

ALERTNESS

CONSOLABILITY

MUSCLE TONE & MOTOR ORGANISATION

TONE * ACTIVITY LEVEL

HAND-TO-MOUTH 0 0

DEFENSIVE MOVEMENTS ■

PULL-TO-SIT *0

CONTROLLING STATE OF CONSCIOUSNESS

RESPONSE TO LIGHT

RESPONSE TO RATTLE

RESPONSE TO BELL

BUILD UP TO CRYING

PEAK OF EXCITEMENT 0 O ■O

IRRITABILITY ■

SELF-QUIETING ABILITY ■O 0 0

LABILITY OF STATE ■ ■ 0

PHYSIOLOGICAL RESPONSE TO STRESS

STARTLES

TREMORS

`ABOVE ARE ALL THE ITEMS OF TIE BRAZELTON NEONATAL ASSESMENT WITH THE ITEMS AFFECTED BY PETHIDINE OR BUPIVAC.AINE INDICATED: i.e. A WITHIN GROUP ANALYSIS COMPARING DIFFERENCES IN BABIES WITH A HIGH DRUG EXPOSURE TO THOSE WITH A RELATIVELY SMALL EXPOSURE. DRUG EXPOSURE DEPENDS ON MATERNAL DOSE. LENGTH OF LABOUR. NEONATAL ELIMINATION.

KEY ITEM NEGATIVELY AFFECTED BY HIGH DRUG EXPOSURE 0 PETHIDINE; ■ BUpI VAC AINE

ITEM POSITIVELY AFFECTED BY HIGH DRUG EXPOSURE *PETHIDINE: *BUPIVACAINE 175

Discussion

Fetal bradycardia (less than 120 beats/minute) has been the most fre-

quently reported and the most serious complication of regional obstetric

anaesthesia. While the documented incidence of fetal bradycardia

varies widely, it has been reported to occur in 2-70% of carefully observed cases (Thiery and Vorman, 1972). The bradyca-.dia could be the result of a direct action of the local anaesthetics USES (i.e. lignocaine,

mepivacaine, bupivacaine) or their metabolites, on the fetal myocardium

(Asling, Shnider, Margolis, Wilkinson and Way, 1970; Heymann, 1972).

Others have suggested that the cardiovascular changes are secondary to stimulation of the CNS and convulsions induced by these agents (Teramo,

Benowitz, Heymann, Kahampaa, Sumes and Rudolph, 1975), or are a result of decreased placental perfusion secondary to maternal hypotension

(Schifrin, 1972). Pethidine, as prescribed in this study did not

affect the fetal heart-rate pattern, although late decelerations in

the fetal heart-rate pattern was seen in 8 of the 58 patients who had epidural bupivacaine. These late decelerations were associated with maternal hypotension in 5 patients and uterine hyperstimulation in 3 patients. Hyperstimulation and maternal hypotension did not occur in any other patients. These findings are consistent with the reports of Schifrin (1972) and Wingate (1974) and suggest that it is these additional complications which adversely affect the fetal heart rate pattern and not the epidural anaesthetic.

Although significantly more of the babies born to mothers who received pethidine had an Apgar score of 7 or less at one minute after delivery, and required oxygen by face mask or IPPR, there were no observable differences between the 3 groups at 5 minutes. The absence of adverse 176

'drug' effects on neonatal behaviour between the three groups during the first six weeks of life, as judged by the Brazelton test, is consistent with the findings of Scanlon et al. (1974) and Tronick et al. (1976).

However, by examining the within-group behaviour of babies whose mothers received pethidine or bupivacaine, it becomes clear that the amount of drug in the baby at delivery; i.e.UA or UV concentrations; and the infant's ability to eliminate the drug, bears a strong relation to its subsequent behaviour. In the pethidine group for example, no adverse behavioural effects were discernable at delivery, but infants with a longer than average half-life showed significantly depressed alertness and ability to respond to external stimuli in the first week of life.

At three weeks, infants who had high cord levels of pethidine, or a relatively high drug exposure were more irritable, difficult to console and less likely to smile, although the result of the Prechtl examination on day 5 was not affected by pethidine. In the epidural group, the cord level at delivery was more predictive of behaviour than half-life, although again there were no effects at delivery. In the first week of life, high cord levels of bupivacaine predicted consistently poor alertness and responses to external stimuli. These babies tended to be very irritable, were more likely to cry on day 3 and show more tremulousness on day 7. At 3 and 6 weeks, high cord levels, high maternal dose, long blood elimination half-life and high drug exposure were still associated with poor orientation ability, greater physiological reaction to stress and less smiling. As with pethidine the neurological score was not affected. 177

The alterations in neonatal behaviour attributed to pethidine and bupivacaine follow no systematic pattern and are difficult to interpret.

Such alterations could be attributed to either a direct action of the drug on the neonate or be a secondary effect; e.g. the reduction of attentive behaviour observed in the pethidine babies could be attributed to (i) the persistance of the drug in the infant or (ii) to a drug- induced delay in the functional development of the CNS. Since there seems to be a delay in the adverse behaviour appearing, it is more likely that these drug induced effects are secondary in nature. Almost all the infants had a prolonged blood elimination half-life of the drug compared to the adult, and therefore the pharmacological action of the drug would be increased in the neonate. However, pharmacological data on the action/effects of a drug in adults cannot be extrapolated to the newborn due to factors such as an immature CNS, possible differences in tissue receptor sites etc., and therefore the pharmacology of the newborn is not equivalent to that of the adult. It may not therefore be assumed that the action of a drug in an adult will be the same as that in the neonate, and therefore the administration of any drug during pregnancy and delivery may have unexpected adverse effects on the infant.

The conclusions from the study are listed in Table 5.3.

IMPLICATIONS

The major implication of these findings is that if the drug dosage and timing, and the management of labour are carefully controlled, then the effects of the drug on the fetus/neonate are minimal despite the fact that both drugs cross the placenta to a considerable extent and are slowly eliminated by the neonate. Indeed it appeared that many mothers 178

who received either of the methods of analgesia had babies who

performed as well as, or better than, babies whose mothers received

no drug. The effect of such pain relief may well reduce maternal

hypertension and stress to a manageable level so that the baby is less

likely to experience fetal heart rate changes and subsequent serious

respiratory distress. However, large quantities of drug, as indicated

by one of the following: high maternal dose, high umbilical blood

concentration, long neonatal blood elimination half-life or a relatively

high drug exposure, were seen to affect the infant's behaviour. When

the drugs were administered in relatively large amounts or when the

neonatal blood elimination half-life was particularly long, both drugs

exerted a depressive effect on behaviour throughout the first week of

life. Large quantities of bupivacaine appeared to be more deleterious

than pethidine at 3 and 6 weeks. It is interesting to note that

neither drug significantly affected the delivery room behaviour of the infant and suggests that most infants are aroused enough by the 'trauma' of birth to exhibit sufficient spontaneous and elicited behaviour.

The results obtained from this study suggest no reason for witholding maternal analgesia during childbirth providing the amount of drug used is. kept within certain limits. Obviously it would be desirable if the drug used was unable to cross the placenta but this 'ideal' drug has not been found. One possible method of limiting placental transfer would be to use an epidural injection of a drug hydrolysed by maternal plasma esterases since this would at least limit the amount of drug available for transfer. Such a compound, 2-chloroprocaine, is undergoing clinical trials in the U.S.A. This compound has a maternal plasma elimination half-life of 6-7 seconds and has not been detected in 179

cord blood.. However, the metabolites of such a drug may still cross the placenta and have possible deleterious effects on the neonate.

This study has shown that adverse effects on the fetus, such as alterations in heart rate pattern, are related to changes in maternal physiology and unrelated to the analgesic used. Pethidine and bupivacaine can affect neonatal behaviour for up to six weeks after birth, but whether the future development of the infant is also affected remains to be seen. This aspect is currently being studied. 180

Table 5.3. Conclusions from the study

(1) Fetal bradycardia (less than 120 beats/minute) following maternal

epidural administration of bupivacaine (in the doses used) is a

consequence of maternal hypotension or uterine hyperstimulation,

and is not the result of a direct action of bupivacaine on the

fetal myocardium or fetal CNS.

(2) The judicious use of pethidine or epidural bupivacaine in normal

pregnant women, compared to a no-drug group, is not associated

with systematic adverse effects on their offspring; i.e. routine

low doses have minimal effects on neonatal behaviour.

(3) A within-group analysis (bupivacaine or pethidine) reveals that

certain aspects of neonatal behaviour are adversely affected by a

high maternal dose; high MV, UA, or UV concentrations; long

neonatal blood elimination half-life; high fetal/neonate drug

exposure. However, there are no apparent effects of drugs at

delivery suggesting that the birth process itself is a powerful

enough stimulus to swamp any other effects.

(4) Some obvious or important effects;

Orientation - Pethidine; depression of orientation on day 1

in babies with a comparatively long half-life

Bupivacaine; continued depression until 6 weeks in

babies with a high drug concentration in cord blood.

Motor activity- Pethidine; no apparent effect

Bupivacaine; depressed in the first week of life,

but predicted by high cord level and long half-

life. 181

Irritability - Pethidine; high cord levels predict a baby

who cries more, cannot console himself, and

is more difficult to console at 3 weeks.

Bupivacaine; high cord level is predictive of

similar behaviour to above at 3 days.

Maternal handling at delivery - Pethidine; after large doses of drugs mothers

are less likely to interact with their baby.

Bupivacaine; no consistent effects 182

Chapter Six. The metabolism of bupivacaine in rat and man

Page

Introduction 183

Identification of metabolites 183

Rat urine 183

SequFntial extraction of rat urine 184

Rat bile 191

Human urine 194 197 Sequential extraction of human urine

199 Results 199 Rat urine 203 Rat bile 208 Human urine

213 Discussion 183

Introduction

Bupivacaine is at present the drug of choice for lumbar epidural

anaesthesia in childbirth. It is almost completely metabolized in

human volunteers, mothers in childbirth and by their babies (Caldwell

et al., 1977), but little is known of the metabolic pathways involved.

Accordingly, the fate of bupivacaine was studied in man in an attempt

to identify the products of metabolism. The metabolism of bupivacaine

was also followed in the rat with the view of the animal being a pot-

ential metabolic model for man.

In order to perform metabolic studies on a drug in animals, it is

necessary to have a highly specific and sensitive means Of detecting

the drug and its metabolites in body fluids. The best way to achieve

this is by isotopically labelling one or more atoms in the drug mole-

cule, which with current techniques is most conveniently done by using 14 radioactive isotopes e.g. either radioactive carbon .( C) or hydrogen

(3H; tritium). The position of the label within the molecule is cri-

tical as this decides the extent of degradation undergone which may be

detected. In this study, bupivacaine was labelled with 14C at the amide

linkage (see Fig. 2.1) enabling minor modifications of the intact mole-

cule (e.g. N-dealkylation and ring hydroxylations) as well as residues

containing the pipecolinic acid moiety to be detected.

Identification of metabolites

Rat Urine

Rats were dosed with [14Cj-bupivacaine HC1 (30mg/kg; 201;Ci/kg) and

excreta collected for three days as described in Chapter 2. Their

0-24h urine after concentration with XAD-2 resin, was subjected to thin- 184

layer chromatography in system B and the chromatogram scanned for

14 C. Seven peaks were found whose centres had Rf values of 0.06,

0.19, 0.41, 0.48, 0.61, 0.75 and 0.80 (Fig. 6.1). Treatment of the

urine with HC1 or S-glucuronidase caused a decrease in the peak of

Rf 0.06, with concomitant increases in those of Rf 0.48 and 0.61 (Fig.

6.2), indicating that these two compounds were excreted in part as

their glucuronides. Both the latter peaks gave positive reactions

with diazotized ,-nitroaniline and Gibb's reagent. The peaks with

Rf values of 0.06, 0.41 and 0.80 correspond in Rf values to pipecolinic

acid, desbutyl-bupivacaine, and bupivacaine respectively, and these

three compounds were quantitated by isotope dilution. The peaks of

Rf 0.19 and 0.75 did not correspond to the Rf of any standard compound.

Sequential extraction of rat urine

Rat urine was subjected to the sequential extraction procedure shown

in Fig. 2.2. The percentage 14C extracted at the various pH's is

shown in Table 6.1 and each extract was examined by TLC, GC and GC-

MS as described in Chapter 2. The residue from the pH 14 extract

was chromatographed in systems A, B and C. In each case radio-

chromatogram scanning revealed two 14C peaks with Rf values of 0.72

and 0.59 in system A; 0.80 and 0.42 in system B; and 0.63 and 0.37

•in system C; which correspond in each system to the Rf values of

bupivacaine and desbutyl-bupivacaine respectively. Neither the

percentage 14C extracted nor the nature of the metabolites was altered

by prior treatment of the urine with HCl or S-glucuronidase. GC

examination of this residue showed the presence of two peaks with

retention times of 1.9 min (peak A) and 3.1 min (peak B) which corre-

spond to the retention times of desbutyl-bupivacaine and bupivacaine

respectively. Mass spectra were obtained of these two peaks and are

given in Fig. 6.3b and Fig. 6.4b. A portion of the aqueous phase 185

Fig.6.1 Radiochromatogram of 0-24h Rat Urine following [14C]-Bupivacaine HC1 (30mg/kg; 20uCi/kg) i.p., in system B

Origin Front

Fig.6.2 Radiochromatogram of acid treated 0-24h Rat Urine following [14 C]-Bupivacaine HC1 (30mg/kg; 20uCi/kg) i.p., in system B

0.06 0.75 0.41 0.80

0.19

1 1 Origin Front Table 6.1 Sequential Analysis of Rat Urine

CONDITIONS OF EXTRACTION AND SOLVENT UNTREATED AFTER R-GLUCURONIDASE pH 14 (ether) 4.6 (4.1-5.3) 4.7 (4.6-5.3) pH 8.5 (dichloromethane, 18.3 (10.7-24.3) 74.0 (65.7-79.5) NaC1 saturation) pH 5.0 (dichloromethane) 1.5 (0.7-2.1) 2.4 (1.8-79.5) remaining in aqueous phase 75.3 (66.3-79.5) 19.1 (9.9-25.4)

Figures are expressed as % of 14C in 24h urine extracted in each case; the mean of 3 animals is given with ranges in parentheses.

Fig.6.3a Mass spectrum of desbutyl-bupivacaine via GC-MS, retention time 2.2minutes 8 0 84

232 (M+ ) 120 1 148 ity O II 1 I I tens 100 200 300 M In ive t Fig.6.3b la Mass spectrum of peak 1 from the pH14 extract from rat urine, GC-MS retention time 2.2minutes

Re 0- 84

120 148 232 (M+ ) 0 I I II I I I 100 200 300 m/e Fig.6.4a Mass spectrum of bupivacaine via GC-MS, retention time 3.5minutes g o - • 140

148

44 84 240 288 (M+ ) 0 11 1 1 H

100 200 300 CO 9

Mass spectrum of peak 2 from the pH14 extract from rat urine, GC-MS retention time 3.5minutes 140

44 240 148 84 288 (M+ )

0 1 I li i i I 100 200 300 m/e 189

was subsequently treated with TiC13/HC1 as described in Chapter 2 and

reextracted with ether at pH 14. Examination of this extract by GC

and radioTLC showed it to contain no drug related compounds.

The residue from the pH 8.5 extract showed two 14C peaks on radioTLC

with Rf values 0.48 and 0.61 in system B and Rf 0.21 and 0.52 in

system E. Both peaks in each system gave a positive reaction with

Gibb's reagent and diazotized E-nitroaniline. The percentage 14C

extracted at this pH increased following treatment of the urine with

HC1 or R-glucuronidase showing that the metabolites were excreted, in part, as their glucuronic acid conjugates.

Table 6.1 shows that the residue from the pH 8.5 extract of urine treated with 0-glucuronidase or HC1 contained the major excretion products of bupivacaine. This residue was chromatographed on Imm thick

TLC plates in system G and the chromatogram scanned for 14C by analysing the 14C content of 1 x lcm areas of silica gel along the length of the plate perpendicular to the solvent front. Two radioactive peaks of

Rf 0.29 (metabolite 1) and 0.88 (metabolite 2) were identified, and colour tests with Gibb's reagent and diazotized - nitroaniline were positive in each case. The areas of silica gel corresponding to each 14 C peak were scraped off the plate, placed on a glass sinter and metabolites eluted with methanol for further examination. The UV spectra of the eluates of both peaks were similar to that of bupivacaine, but there was a shift in the A from 210 to 235nm for metabolite 1 max and from 240nm to 250nm for metabolite 2 on changing the pH of the solution from acid (pH 1) to alkali (pH 14) (Fig. 6.5). GC of these compounds using the OV-17 column and conditions described in Chapter 2, Fig.6.5 W spectra of 'phenolic metabolites 1 and 2 isolated from rat urine

Metabolite 1 Metabolite 2

3 - - 3

2 - -2

0 • 0 • Will pH 14 pH 1 + } pH 14 - 1 1 200 220 240 260 200 220 240 260 280 X (nm) X (nm) 191

showed that metabolite 1 had a retention time of 15.4 min and metabolite 2 of 15.9 min. Following treatment with BSA, the trimethylsilyl (TMS) derivative of metabolite 1 had a retention time of 9.2 min, and that of metabolite 2 of 10.1 min. Elucidation of the structures of the two metabolites was obtained by GC-MS since only very small quantities were available which would not permit analysis by other methods. Both metabolites were examined by GC-MS as such, and as their TMS derivatives. Their mass spectra given in Fig. 6.6 and

Fig. 6.7, indicated that the insertion of oxygen in the aromatic ring had occurred (see below).

The residue from the pH 5.0 extract was subjected to radioTLC in system

A and a small 14C peak of Rf 0.19 was found. The percentage 14C extracted at this pH was very small (<2%) and there was not sufficient material to permit GC-MS analysis.

The aqueous residue remaining from the sequential extraction was partially purified with Amberlite XAD-2 resin and chromatographed in system B. On scanning the plates for 14C,a large peak of Rf 0.75 and a small one of Rf 0.14 were observed. The smaller peak gave a positive result with the ninhydrin spray and corresponded in Rf tō pipecolinic acid. Again there was insufficient material (<3.5%) to permit GC-MS analysis. Neither the radioTLC trace nor the amount of pipecolinic acid present, as quantitated by isotope dilution, were altered on prior treatment of the aqueous phase with TiC13/HC1.

Rat bile

Biliary cannulated rats were dosed with [14CJ-bupivacaine HC1 as before Fig.6.6a Mass spectrum of 'phenolic' metabolite 1 isolated from rat urine proposed to be 4'-hydroxybupivacaine

GC-MS retention time 15.4min 44 140

164 261 136 304 (M~ ) 1 100 200 300

Fig.6.6b Mass spectrum of 'phenolic' metabolite 2 isolated from rat urine proposed to be 3'-hydroxybupivacaine

0 44

ity 140

tens GC-MS retention time 15.9min in

iv 164 t 261 la 304 (M+) Re 0 I t ~ 100 200 300

Fig. 6.7a Mass spectrum of 'phenolic' metabolite 1 from rat urine following derivatization with BSA

g 0 73 140 75 212 GC-MS retention time 9.2min 44 448

84 280 336 308 1348 433 I I ' I I 100 200 300 400

6.7b Mass spectrum of 'phenolic'metabolite 2 from rat urine following derivitization with BSA

73 140 44 GC-MS retention time 10.1min

212 448 75 433n 280 308 336 I I 1348 0 I I 1 100 200 300 400 194

and 0-24h urine and bile collected as described in the text. The

0-24h rat bile was subjected to TLC in system B and scanned for 14C.

Three 14C peaks were seen with Rf values of 0.05 (major), 0.48 and

0.61 (both minor) Fig. 6.8. The peak with Rf 0.05 disappeared

following treatment of the bile with HC1 or S-glucuronidase prior to

chromatography, and the other two peaks were correspondingly in-

creased (Fig. 6.9). Similar chromatograms of bile developed in

systems E and F showed the same pattern of three peaks of Rf 0.02,

0.21 and 0.52 in system E and 0.04, 0.48 and 0.66 in system F. Again,

prior treatment of the bile with HCl or $-glucuronidase caused the

peak of Rf 0.02 in system E and 0.04 in system F to disappear with

concomitant increases in the other two peaks. Colour tests with

Gibb's reagent and diazotized E-nitroaniline were positive for both

remaining peaks in all three systems. The Rf values obtained are

identical with those obtained for the major metabolites in urine

(system B) and in the residue from the pH 8.5 extract (systems B and

E), indicating that the major metabolites excreted in urine are also

excreted in bile, again extensively conjugated with glucuronic acid.

Human urine

The two adult male volunteers were dosed with [14C]-bupivacaine HC1

50mg; 0.38pCi (JC) or 25mg; 0.19pCi (EMH) as described in the text,

and excreta collected for 7 days. The 0-24h (JC) or 0-72h (EMH)

urine, after concentration with XAD-2 resin, was examined in system

B and the chromatograms scanned for 14C. Seven peaks, whose centres

had Rf 0.01, 0.07, 0.15, 0.31, 0.42, 0.68 and 0.80, were found (Fig.

6.10). Treatment of the urine with HC1 or S-glucuronidase caused a decrease in the peak of Rf 0.01 and corresponding increases in the peaks

195

Fig. 6.8 Radiochromatogram of 0-24h rat bile following C14Cj - bupivacaine HCl (30mg/kg; 20 Ci) i.m. in system B

0.48 0.61 0.05

T 1 Origin Solvent Front

Fig. 6.9 ytdiochromatogram of acid treated 0-24h rat bile following CJ-bupivacaine HC1 (30mg/kg; 20 Ci) i.m. in system B

1 Origin Solvent Front 196 Fig.6.10 Radiochromatogram of 0-24h Human Urine following [14C]-Bupivacaine HC1 (50mg;3.81aCi) i.m., in system B

Origin Front

Fig.6.11 Radiochromatogram of acid treated 0-24h Human Urine after r14C]-Bupivacaine HCI (50mg;3. 8uCi) i.m., in system B

0.80

Origin Front 197 of Rf 0.31 and 0.68 (Fig. 6.11). Colour tests with Gibb's reagent and diazotized E-nitroaniline were positive for the peaks of Rf 0.31 and 0.68. The peaks of Rf 0.07, 0.42, and 0.80 corresponded in Rf value to pipecolinic acid, desbutyl-bupivacaine and bupivacaine respectively. All three compounds were quantitated by isotope dilution. The peaks of Rf 0.01 and 0.15 did not correspond to the Rf of any standard compound.

Sequential extraction of human urine

Urine from the two volunteers given[ 14C]-bupivacaine HC1 was subjected to the sequential extraction procedure used for the rat, and the percentage 14C extracted at each pH is given in Table 6.2. The concentrated pH 14 extract was subjected to radioTLC in system B which revealed two 14C peaks with Rf values 0.42 and 0.80 which correspond to the Rf values of desbutyl-bupivacaine and bupivacaine respectively.

Neither the percentage 14C extracted nor the nature of metabolites was altered by prior treatment of the urine with HC1 or S-glucuronidase.

GC examination of this residue showed the presence of two peaks with retention times of 1.9min and 3.1min which correspond to the retention times of desbuty-bupivacaine and bupivacaine respectively. Treatment of a portion of the aqueous phase with TiC13/HC1 as described in

Chapter 2 followed by re-extraction with ether at pH 14 and subsequent analysis by GC and radioTLC showed the extract to contain no drug related compounds.

The concentrated pH 8.5 extract from S-glucuronidase treated urine was subjected to TLC in system B, and the chromatogram scanned for 14C.

Two radioactive peaks were present whose centres had Rf 0.31 (minor) Table 6.2 Sequential Analysis of Human Urine

CONDITIONS OF EXTRACTION AND SOLVENT UNTREATED AFTER pi-GLUCURONIDASE

JC EMH JC EMH

pH 14 (ether) 2.1 8.9 4.0 8.8 pH8.5 (dichloromethane, 40.7 9.3 53.1 29.0 NaC1 saturation) pH 5.0 (dichloromethane) 1.4 3.6 2.3 9.7 remaining in aqueous residue 52.1 72.4 38.2 50.6 total 96.3 94.p 97.6 98.1

Figures are expressed as 4 of 14C in 24h (JC) or 72h (EMH) urine extracted in each case. 199

and 0.68 (major). Both these peaks gave positive reactions with Gibb's reagent and diazotized 2-nitroaniline. GC of this extract using the

OV-17 column and conditions described in Chapter 2, showed that the

minor metabolite had a retention time of 9.9min and the major metabolite

of 12.4min. Following derivatization with BSA, the TMS derivative of the minor metabolite had a retention time of 6.9min, and that of the major metabolite 9.Oriin. Elucidation of the structures of the two metabolites was attempted by GC-MS as such, and as their TMS derivatives.

TLC of the concentrated pH 5 extract gave a single 14C peak of Rf 0.22 in system A and 0.15 in system B. The percentage 14C extracted at

this pH was very small (<6%) and was not altered on treating the urine

with R-glucuronidase or HC1. This metabolite was not characterised further.

Analysis of the remaining aqueous phase was performed by radioTLC in system B. Two peaks of Rf 0.01 (major) and 0.07 (minor) were observed,

and that of Rf 0.07 gave a positive result with the ninhydrin spray and corresponds in Rf value to pipecolinic acid. Neither the radioTLC trace

nor the amount of pipecolinic acid present, as quantitated by isotope

dilution, was altered on prior treatment of the aqueous phase with

TiC1 /HC1. 3

RESULTS

Rat

Table 6.3 shows the elimination of 14C by rats following the administration 14 of [ c]-bupivacaine hydrochloride (50mg/kg) by i.p. injection. Approx- imately 74% of the dose was excreted in 4 days (33% in the urine and Table 6.3 The elimination of 14C by the rat after the administration of[14C]-bupivacaine hydrochloride

14 [ C]-Bupivacaine hydrochloride in normal saline was administered to rats at a dose of 30 mg/kg; 2011Ci/kg; 14 by i.p. injection. Values given are % of C dose in excreta, averages of 3 animals with ranges in parentheses.

In urine on day 1 26.8 (25.6-28.6)

2 5.2 ( 4.2-5.8)

3 1.1 ( 0.5-1.6) b) 0 0 4 0.4 ( 0.3-0.6)

Total 33.5 (30.6-36.7)

In faeces on day 1 28.7 (24.2-33.8)

2 9.6 ( 6.1-13.3)

3 1.7 ( 1.4-2.2)

4 0.4 ( 0.3-0.6)

Total 40.4 (32.0-49.9)

Total excretion 73.9 (62.6-86.6) 201

41% in the faeces), with 56% (27% urine, 29% faeces) appearing in the first 24h.

The sequential extraction procedure showed that some 5% of the 14C in

24h urine could be extracted at pH 14 (Table 6.1). TLC and GC analysis showed the presence of bupivacaine and desbutyl-bupivacaine, and reverse isotope dilution for these compounds showed that bupivacaine accounted for 3.4% and desbutyl-bupivacaine for 1.2% of the total 14C in 24h urine. Thus, all strongly basic compounds excreted i.e. those extracted at pH 14, were accounted for as bupivacaine or desbutyl-bupivacaine. GC-MS analysis of this extract showed that the mass spectra of both compounds were identical to those in Fig. 6.3a and 6.4a for authentic desbutyl-bupivacaine and bupivacaine respectively. The mass spectra of bupivacaine and desbutyl-bupivacaine from the pH 14 extract are given in Fig. 6.3b and 6.4b.

The greatest proportion of 14C was extracted at pH 8.5 following HC1 or S-glucuronidase treatment of the urine. TLC showed the presence of two compounds both of which were excreted principally as glucuronic acid conjugates. Colour tests, conditions of extraction and the conjugation of the metabolites with glucuronic acid, all strongly suggested that the compounds were phenolic in nature. Furthermore, the UV bathochromic alkaline shift observed with both metabolites is consistent with the metabolites being phenolic compounds. In the presence of alkali, a proton is removed from the phenol resulting in a highly resonance stabilized anion which strongly absorbs at a higher wave- length than the unionized form, resulting in a shift of the X towards max the red end of the spectrum. 202

Elucidation of the structures of these metabolites was obtained by

GC-MS and their mass spectra are shown in Fig. 6.6. The mass spectrum

of bupivacaine (Fig. 6.4a) shows that the fragmentation pattern is

dominated by cleavage at the a-piperidinocarbonyl atom giving effectively

two groups of ions - one being attributed to fragments containing the

aromatic residue, e.g. m/e 13, and 148 and the other incorporating the

piperidine ring, e.g. m/e 84 a-id 140 (Fig. 6.12). The mass spectra of

the two metabolites isolated from the pH 8.5 extract were identical to

each other in terms of their fragmentation pattern but there were

differences in the relative intensities of the major ions. The mole-

cular ion was at m/e 304, which was an increase of 16amu over the

molecular ion of bupivacaine, strongly suggesting the insertion of a

single oxygen atom into the molecule. The presence of all the peaks

associated with the piperidine moiety in the mass spectrum of bupi-

vacaine in the mass spectrum of the metabolites showed that this piper-

idine ring was unaltered. However, all ions containing the aromatic

ring were replaced by analogues 16amu higher, showing that the oxygen

was contained within this structure. The absence of peaks 18amu less

than residues containing the aromatic ring (including M+-18) indicated

that the new oxygen function was not an alcohol and hence the metabolites

did not arise from oxidation of an aliphatic centre. These results

are therefore indicative of the oxygen function being contained in the

aromatic ring. Further evidence was obtained by examining the mass spectra of the TMS derivatives of the two metabolites (Fig. 6.7).

The molecular ion was now at m/e 448 showing the insertion of two TMS groups into the molecule. Again ions characteristic of the unaltered piperidine ring were evident and ions previously attributed to the aromatic portion of the molecule were all replaced by new ions 72 or 203

144 amu higher (Fig. 6.13). Thus, by inference, one TMS residue was associated with the oxygen function on the aromatic ring, and the other with the amide nitrogen. These spectra are consistent with the presence of two isomeric phenolic metabolites of bupivacaine, i.e. 3' and 4'-hydroxybupivacaine. However, on this evidence, it was not possible to assign the position of the hydroxyl group in the two phenolic metabolites (1 and 2) and this was therefore done by analogy with similar compounds (see discussion for full details). On this basis,

3'-hydroxybupivacaine was taken to be metabolite 2 and 4'-hydroxy-bupi-

vacaine metabolite 1.

The pH 5 residue was not characterized by GC-MS since insufficient

material was present. However, conditions of extraction suggested

that it was neutral in character, and accounted for 0.6% of the dose

in 24h urine.

The remaining aqueous phase contained an acidic water-soluble compound(s),

(3.5%) which was not characterised, and pipecolinic acid, quantitated

by isotope dilution, accounting for 1.6% of the dose. No evidence of

N-oxidation products was found on the basis of negative results obtained

with the TiC13/HC1 treatment of the relevant extracts described previously.

Quantitative details of the metabolites of [ 14C' -bupivacaine in the 0-

24h urine of rats are given in Table 6.4.

Rat bile

The excretion of 14C by the bile duct cannulated rat over a period of

24h is given in Table 6.5. In this period some 16% of the dose is

excreted in the urine with 75% in the bile. Analysis of the bile by Fig. 6.12 Proposed mass spectrometry fragmentation pattern for bupivacaine

CH 0 m/e 288 II —C — NH - N .+ C4H9 CH3

CO-NH- -C3H 7 CH3 m/e 140

CH m/e 164 C4H9 0 II C—NH_. CH3 C4H8 II CH2 CH3

m/e 245 m/e 84 H2N

H Fig. 6.13 Proposed mass fragmentation of 3'and 4'-hydroxybupivacaine and their TMS derivatives (details of hydroxy metabolites are shown in red) (details of TMS derivatives are shown in black) CII OTMS 0 m/e 448 II OH 304 N - N -♦- I TMS C1}II9 CH3 CH3 0 cn OTMS O II OH C—N- -+- TMS m/e 140 m/e 308 CH3 180 OTMS OH

OTMS m/e 280 OH m/e 84

H m/e 208 CH3 136 TABLE 6.4 The metabolites of [14C]-bupivacaine in rat urine

Dose 30mg/kg; 2.51tCi/animal. The first 24h urine was analysed as described in the text, and the results are expressed as averages of 3 animals with ranges in parentheses.

% of 14C dose in that form Metabolite Sought in 24h urine

Bupivacaine 0.9 (0.8 - 1.1) Desbutyl-bupivacaine 0.3 (0.27 - 0.34) Pipecolinic acid 1.6 (1.0 - 2.3) 3'-Hydroxybupivacaine free 3.0 (2.9 - 3.1) conjugated 9.1 (6.2 - 13.4) total 12.1 (9.1 - 16.5) 4'-Hydroxybupivacaine free 1.9 (0.7 - 3.1) conjugated 5.8 (4.6 - 7.1) total 7.7 (5.3 - 10.2) Neutral unknown (s) 0.6 (0.3 - 0.7) Other unknown 3.5 (2.6 - 4.9) Total of above metabolites 26.7 (24.1 - 29.3) Total 14C in urine examined 26.8 (25.6 - 28.6) Table 6.5 The elimination of 14C by bile-duct cannulated rats after administration of [i4C]-bupivacaine hydrochloride

[14 C]-Bupivacaine hydrochloride in normal saline was administered to bile-duct cannulated rats at a dose of 30mg/kg; 14 20}LCi/kg; by i.p. injection. Values g iven are °% of C dose in excreta, averages of 3 animals with ranges in parentheses.

In bile at 0 - lh 14.1 (0.5 - 32.8) 1 - 2h 8.8 (5.0 - 14.6) 2 - 3h 8.3 (4.3 - 11.1) 3 - 4h 5.1 (3.4 - 7.9) 4 - 5h 4.3 (2.1 - 5.9) ō 5 - 6h 3.8 (1.6 - 5.7) 6 - 24h 31.0 (6.5 - 54.7)

Total 75.4 (71.2 - 78.4)

In urine in 24h 16.3 (12.0 - 18.4)

Total excretion 91.7 (83.2 - 96.7) 208 radioTLC showed the presence of two metabolites, in approximately equal quantities, which were excreted both free and conjugated with glucuronic

acid in the proportion 1:4. The Rf values of these metabolites were identical to the Rf values of the two metabolites in the pH 8.5 urine extract, which were identified by GC-MS as 3' and 4'-hydroxybupivacaine.

However, the total excretion of 14C (urine and bile) by the bile-duct cannulated rats was higher than the 14C excretion by intact rats which is indicative of enterohepatic circulation. Furthermore the relative proportion of the two phenolic metabolites in urine was different; 3:2 in the 0-24h urine of the intact rats, compared to 1:1 in 0-24h bile.

Human urine

Table 6.6 shows the excretion of 14C in the 2 adult male volunteers following the administration of [14( -bupivacaine HCl. Although the

total 14C recovered in 7 days was similar in both subjects ("78%), the rate of 14C excretion was very different as shown in Fig. 6.14, JC excreting the drug some 3 -times faster than EMH. The elimination of both 14C and bupivacaine from plasma was followed in both subjects as described in the text, and whereas the plasma half-life (t1/2) for bupivacaine was similar in both subjects (1-2h),the t1/2 of the radioactivity was about twice as long in EMH.(Table 6.6).Thefate of [14Cl-bupivacaine HCl in the two volunteers is shown in Table 6.7.

Table 6.2 gives the results of the sequential extraction of the 24h urine. Basic compounds extracted at pH 14 accounted for some 5% of 14 urinary C, and isotope dilution confirmed that bupivacaine accounted for 2%, and desbutyl-bupivacaine 3% of urinary 14C. RadioTLC and GC showed these to be the only 14C-labelled compounds in the pH 14 extract. Fig. 6.14 Elimination of 14C in adult volunteers following the administration of 14C-bupivacaine IIC1

Dose administered 50mg; 3.8 Ci (JC) or 1.9 Ci (EMIT)

80 —

N 0

20 % dose in 7day faeces

.IC 0

1 1 I 1 10 20 30 40 50 60 70 80 90 100 Ito 120 Administration of TIME (MIN ) Ī C1-Uupivacaine Hdl 210

TABLE 6.6 The elimination of 14C by two male volunteers after administration of [14C]-bupivacaine hydrochloride

Administered dose 50mg; 3.8uCi (JC)or 25mg; 1.911Ci (EMH) in normal saline by i.m. injection. Figures given are % of 14C dose in urine.

Subject JC EMH

Body weight: 87kg 73kg

In urine at 0-lh 2.2 0.4 1-2h 3.6 0.8 2-3h 5.9 1.1 3-4h 4.3 1.0 4-5h 3.3 0.8 5-6h 4.3 0.7 6-12h 13.7 2.9 12-24h 18.3 5.9

In urine on day 1 55.6 13.6 2 9.2 21.4 3 3.8 14.9 4 1.5 8.5 5 0.4 4.3 6 0.4 1.8 7 0.4 1.2 Total 71.3 65.7

In faeces on day l 1.3 0.2 2 1.0 3.4 3 5.4 1.3 4 1.3 0.6 5 0.8 6 3.0 7 0.7 Total 13.5 5.5

Total excretion 84.8 71.2

Bupivacaine blood elimination half-life: 115min (JC), 54min (EMH) 14 C Plasma elimination half-life: 524min (JC), 1002min (EMH) TABLE 6.7 The metabolites of [14C]-bupivacaine in human urine

Dose 50mg; 3.811Ci (JC) or 25mg; 1.911Ci (EMH) i.m. The first 24h (JC)or 72h (EMH) urine was analysed as described in the text, and the results are expressed as % of 14C administered in that form in 24h (JC) or 72h (EMH) urine.

Metabolite Sought JC EMH

Bupivacaine 1.1 0.5

Desbutyl-bupivacaine 1.0 1.9 N Pipecolinic acid 1.2

"Phenolic" metabolites Untreated urine 22.6 4.6 (extracted at pH 8.5) after (i-glucuronidase 29.5 14.5

"Neutral" metabolites (extracted at pH 5) 1.3 4.8

"Acidic" metabolites (remaining after pH 5 extraction) 22.0 25.3

Total recovery 56.1 49.6 212

Both volunteers excreted large amounts of 14C metabolites which were

extracted from urine at pH 8.5. TLC showed the presence of two meta-

bolites in this residue, both being excreted in part conjugated with

glucuronic acid, and both giving positive reactions with Gibb's reagent

and diazotized 2-nitroaniline. TLC and GC characteristics of these

metabolites were not the same as those from the equivalent extract in

the rat. GC-MS analysis was attempted on the isolated metabolites

but was unsuccessful due to the presence of large amounts of impurities

in the samples. In an attempt to identify the metabolites from the

impurities, ENE was given a mixture of bupivacaine HC1 and [149- 2 [H 93-bupivacaine HC1, where the 9 hydrogen atoms of the n-butyl group were replaced with deuterium atoms. It was hoped that the mass spectra

of these metabolites would then be easily recognisable from any impurities

by the presence of doublets 9amu apart in all fragments containing the

n-butyl group. This technique using a mixture of [1 4C]-pethidine and

[2 H5]-pethidine proved invaluable in studying the metabolic fate of

pethidine in man (see Chapter 7), but impurities again interfered with

the production of good mass spectra in the case of bupivacaine. Thus,

the identities of the major metabolites of bupivacaine in human urine

remain unknown, although their conjugation with glucuronic acid, con-

ditions of extraction,, and colour tests all suggest that they are

likely to be phenolic in nature. The two major metabolites of bupi-

vacaine in rat urine, 3' and 4'-hydroxybupivacaine had Rf values in

system B of 0.48 and 0.61 and GC retention times of 15.4 and 15.9min

respectively. The major metabolites in human urine had Rf values in

system B of 0.31 and 0.68 and GC retention times of 9.9 and 12.4min.

Hence, the major metabolites in human urine are not 3' and 4'-hydroxy-

bupivacaine, and therefore, the rat is not a good metabolic model for 213 bupivacaine in man.

Two minor E14C]-metabolites in urine were isolated but not characterised. One compound was extracted at pH 5, and therefore likely to be neutral in character, and the other remained in the aqueous phase after the sequential extraction and therefore probably an acidic water-soluble product. Pipecolinic acid, also remaining in the aqueous phase,accounted for some 2% of the dose in 24h urine as shown by isotope dilution.

No evidence of N-oxidation products was found on the basis of negative results obtained with the TiC13/HC1 treatment of the relevant extracts.

DISCUSSION

The findings show that in the rat bupivacaine is metabolized along several metabolic pathways, namely aromatic hydroxylation and subsequent conjugation with glucuronic acid, N-desbutylation, amide hydrolysis and two minor unidentified pathways giving rise to the 9 metabolites listed in Table 6.4. The total recovery in,4 days was some 74% of the dose,

40% in the faeces and 34% in the urine, with most of the radioactivity

(29% faeces, 27% urine) being excreted in the first 24h. The major excretion products were the two isomeric phenolic metabolites of .. bupivacaine, but the GC-MS properties of these compounds did not permit unequivocal assignment of the positions of hydroxylation. Insufficient material was available for NMR analysis. Goehl, Davenport and Stanley

(1973) found that the rat excreted only the 3'-hydroxy isomer of bupivacaine and, other work reported in the literature on the fate of structurally related local anaesthetics, indicates that in the rat, 3'- hydroxylation of such drugs is quantitatively more important than attack in the 4'-position (Keenaghan and Boyes, 1972; Thomas and Meffin, 1972). 214

Assignment of the structures of these phenolic metabolites is therefore by analogy with these other compounds, and thus metabolite 2 is

taken to be 3'-hydroxybupivacaine, (accounting for 12% of the dose in

24h rat urine) and similarly metabolite 1 is taken to be 4'-hydroxybupivacaine (accounting for 8% of the dose in 24h rat urine).

Additional evidence that metabolite 1 is the 4'-hydroxy isomer and metabolite 2 the 3'-hydroxy isomer is supplied by the difference in their UV bathochromic alkaline shifts. Larger bathochromic shifts would be anticipated for ortho and para aryl amides as opposed to meta hydroxy amides since they are most highly resonance stabilized,(Kemp,1973). In this instance metabolite 2 gave the large UV alkaline shift indicating that it is the 3'-hydroxy isomer. These two phenolic metabolites were excreted both free and conjugated with glucuronic acid, the proportion of free to conjugated being of the order 1:4 in both cases.

The excretion of 14C in the urine and bile of bile-duct cannulated rats, following the i.p. injection of' 14C }bupivacaine HC1 is given in

Table 6.5. 75% of the dose appeared in the bile in 24h with a further

26% of the activity being excreted in the urine. However, in the intact animal, only 56% of the dose (29% faeces, 27% urine) was excreted in the same period which is indicative of extensive enterohepatic circulation of the drug and/or metabolites. Examination of the bile by radioTLC showed that the sole 14C constituents of bile were 3'- and

4'-hydroxybupivacaine which were present in approximately equal quantities.

Both metabolites were excreted mainly as their glucuronic acid conjugates, which was the same ratio as in urine. These results are to be expected from the results of Millburn, Smith and Williams (1967). They showed that for significant biliary excretion (defined as more than 5-10% of 215

the dose) in rats, compounds should be polar and possess a molecular

weight of not less than 325 ± 50. In the case of bupivacaine, the

only(known)metabolites that meet these requirements are the iso-

meric 3'- and 4'-hydroxybupivacaines (M.W. 304) and their glucuronides

(M.W. 480).

The results show that although the 3'- and 4'-hydroxy metabolites are

present in bile in approximately equal proportions, in urine there is

about 50% more 3'-hydroxybupivacaine than 4'-hydroxybupivacaine. There

are a number of possible explanations which would be in accord with this

data:-

(i) 4'-hydroxybupivacaine, whether free or conjugated, is more favoured

in enterohepatic circulation so that its transport to the kidney

is delayed.

(ii)4'-hydroxybupivacaine is preferentially excreted in faeces.

(iii)renal clearance of the metabolites is by a common saturatable,

active transport process and competition occurs, with 3'-hydroxy-

bupivacaine being preferentially excreted.

(iv) the rate of formation of the 3'-hydroxybupivacaine is initially

higher than that of the 4'-hydroxy isomer, although in 24h

approximately equal amounts have been synthesized.

Differences in the relative extents of biliary and urinary excretion between closely related molecules have been observed previously, and examples include isomeric phenolic compounds. The main metabolites of LSD in the rat are the glucuronides of 13'- and 14'-hydroxy LSD which 216

account for 70-75% of the total radioactivity in the 5h bile or 24h

urine samples (Siddik, 1975). In the bile the 13'- and 14'-hydroxy

LSD glucuronides were in the ratio 0.6 but in urine this ratio was

reversed to 1.5. However, this difference was accounted for by the

14'-hydroxy metabolite predominating over the 13'-hydroxy isomer in

the faeces, the ratio here of 13'-:14'-hydroxy LSD glucuronide being

0.45. It is therefore possible that this may also be the case for

the phenolic metabolites of bupivacaine in the rat, where 4'-hydroxy-

bupivacaine may be preferentially excreted in the faeces and 3'-hydroxy-

bupivacaine in the urine. However, difficulties in extracting the

faecal metabolites, which were probably 3'- and/or 4'-hydroxybupivacaine

since these were the only metabolites found in bile, did not permit

their quantitation in faeces.

Meffin and Thomas (1973) have shown that in man, the relative rates at

which the urinary metabolites of mepivacaine, 3'- and 4'-hydroxymepivacaine, varied as the interval between administration and urine collection increased. Initially, the 3'-hydroxy metabolite was excreted in higher concentration, but by the end of the 24h period, relatively more of the 4'-hydroxy metabolite had been produced. Entero- hepatic circulation was not involved in this case as no metabolites could be detected in bile collected from one patient, and this is to be expected as the molecular weight of hydroxymepivacaine glucuronide

(438) is below the threshold of 500 postulated as necessary for significant biliary excretion in man (Smith, 1973). Kinetic data showed that the renal: clearance of the metabolites was not saturated and it was therefore concluded that the rate of appearance of the two phenolic metabolites in the urine reflected the relative rates at which they 217

were synthesized, with the proportion of 4'-hydroxymepivacaine relative

to 3'-hydroxymepivacaine increasing with time.

It is well known that the formation of isomeric phenolic metabolites

of a range of compounds does not give equal quantities of each isomer

e.g. aniline is hydroxylated by the rabbit in the ortho-, meta-, and

para-positions in the proportions 1:100:500 (Parke, 1968); the

formation of 6'- and 7'-hydroxybutamoxane in 48h rat urine in the

proportion 2:1 (Murphy, Bernstein and McMahon, 1974); and the formation

of 3'- and 4'-hydroxylignocaine in 24h rat urine in the proportion 3:1

(Mather and Thomas, 1972). Such variations are thought to be largely

due to the different metabolic pathways involved in the formation of

isomeric phenols, the rate of appearance of the two phenolic metabolites

in the urine reflecting the relative rates at which they are synthesized.

The proposed mechanisms of enzyme-mediated aromatic hydroxylation will

be discussed below.

There is considerable evidence in the literature that aromatic hydroxy-

lation can involve the formation of an arene oxide intermediate which

can undergo a number of fates including non-enzymic rearrangement to form a phenol, hydration by epoxide hydrase to a dihydrodiol (i.e. 1,2- dihydro-1,2-dihydroxy derivative), or interaction with cellular nucleo- philes. Dihydrodiols can subsequently dehydrate to form a phenol or be oxidized by a dehydrogenase to generate a diphenolic metabolite; e.g. napthalene-1,2-oxide has been identified in vitro as the obligatory intermediate in the oxidative metabolic pathways of naphthalene (Daly,

Jenina and Witkop, 1972). This intermediate isomerizes nonenzymically to i~ naphthol or is converted to a dihydrodiol having the trans config- 218

uration as a result of hydrolytic cleavage of the epoxide ring catalysed by epoxide hydrase. This can subsequently be dehydrated enzymically to

yield 1- naphthol or be oxidized by a dehydrogenase to a catechol,l,2- dihydroxynaphthalene (Fig. 6.15).

The route involving opening of the epoxide intermediate would be the most obvious mechanism by which the aromatic hydroxylation of bupivacaine, could occur. The chemical reactivity of the 2', 6'-xylidine ring of bupivacaine is dominated by the two methyl groups ortho to the nitrogen. These methyl groups inhibit the resonance between the lone pair of electrons on the amide nitrogen and the electrons in the aromatic ring, by forcing the nitrogen out of the plane of the ring. In such circumstances, where the influence of the nitrogen lone pair is reduced, the 2',6'-xylidine ring behaves towards electrophiles in a similar way to m-xylene. Kaubisch, Daly and Jerina (1972) have demonstrated that the main product of microsomal hydroxylation of m-xylene is 2',4'-dimethylphenol, with a small amount of 2' 6'-dimethylphenol also being formed.

However, the acid-catalysed rearrangement of 1', 3'-dimethyl-4',5'- dihydrobenzene-4', 5'-oxide, the m-xylene epoxide corresponding to the proposed bupivacaine epoxide, only gives rise to the 2',4'-dimethylphenol and not the 2',6'-dimethylphenol. This indicates that where the reactivity of the 2',6'-xylidine ring is dominated by the effect of the ortho methyl groups, as is proposed for bupivacaine, rearrangement of the proposed bupivacaine epoxide would give rise to the 3'-hydroxy metabolite exclusively (Fig. 6.17). All of the above considerations strongly suggest that the 3'-hydroxy metabolite arises via the proposed epoxide intermediate but that this intermediate is not the immediate precursor of the 4'-hydroxy metabolite. Fig. 6.15 Oxidative metabolic pathways of naphthalene

OH 9

2-Naphthol

Naphthalene Naphthalene-1,2- oxide

./ OH i

1,2-Dihydrodiol naphthalene 1,2-Dihydroxynaphthalene 220

The finding of two N-oxidised metabolites of lignocaine in rat urine

(Mather and Thomas, 1972) has led to the suggestion that the formation of 4'-hydroxybupivacaine occurs by rearrangement of the proposed 2- butylpipecolo-N-hydroxy-2',6'-xylidide intermediate (Fig. 6.16).

Fig. 6.16 Proposed N-hydroxy intermediate in the formation of 4'-hydroxybupivacaine

C H3 0 CN

C4 H 9 C H3

Enzymic rearrangement of N-hydroxy amides to phenolic amides has been demonstrated by Miller and Miller (1960) and by Booth and Boyland

(1964). Such a rearrangement would proceed via a positively charged amidonium ion formed as the result of the loss of an hydroxyl ion, as proposed by Gutmann and Erickson (1969), or through the same intermediate formed by the loss of water. Such a rearrangement could also occur via a concerted mechanism, with the simultaneous attack of OH in the 4'- position and the removal of an hydroxyl ion or a water molecule from the nitrogen. However, no evidence of urinary N-hydroxy metabolites were found in either rat or man on the basis of the titanous chloride/

HC1 results, although it is possible that any N-hydroxy amides formed 221

could be completely converted to 4'-hydroxybupivacaine prior to excretion. The N-oxide metabolite was also not deteeted in rat urine.

It is also possible that some of the 3' and 4'-hydroxy metabolites result from the dehydration of a dihydrodiol intermediate, as postulated for naphthalene, or direct phenol formation by an insertion mechanism similar to the formation of alcohols. This latter mechanism has been proposed for benzene and simple aromatic derivatives, and in a few cases evidence has been obtained that this direct phenol formation does actually occur. For example, the benzodioxane derivative butamoxane yields a catechol metabolite, and studies using oxygen isotopes have shown the generation of this metabolite to occur by consecutive hydroxylation rather than the epoxide-dihydrodiol pathway (Murphy, Bernstein and McMahon, 1974).

BUTAMOXANE 6- or 7- HYDROXY- 6,7-DIHYDROXYBUTAMOXANE BUTAMOXANE In summary, therefore, biological and chemical evidence is consistent with the proposal that 3'-hydroxybupivacaine may arise from an arene oxide intermediate and that both isomers could result from the dehydration of a dihydrodiol intermediate or as a result of direct hydroxyl insertion (Fig. 6.17). Fig.6_I7 A schematic representation of the proposed origins of the 3' and 4'-hydroxy metabolites of bupivacaine in the rat

ĪI N ~ NH-C C4H9 /' '/ NH-CU/ N' C4H9 . N

bupivacaine N CH3

arene oxide intermediate

N N N

dihydrodiol intermediate O 1/ N — NH- C HO —NH- C ~ C4H9 C4H9

HO CH 3

3'-hydroxybupivacaine 4'-hydroxybupivacaine 223

A marked difference occurs between the metabolism of bupivacaine in the rat and man. The route of excretion varied between the two species, since the rat was found to excrete 29% of the dose in the faeces compared to some 9% in man. The much smaller amount of the dose excreted in the faeces by the human volunteers suggests that biliary excretion has less influence on overall drug disposition in man than in the rat.

Furthermore, the major metabolites of bupivacaine in the two species were different, and although the major metabolites in human urine were not identified, they were shown not to be 3'- and 4'-hydroxybupivacaine.

However, other workers have shown that aromatic hydroxylation of the anilide local anaesthetics is their main metabolic fate in man. Thomas and Meffin (1972) identified 3' and 4'-hydroxymepivacaine as the major human urinary metabolites of mepivacaine, and similarly for lignocaine where 4'-hydroxy-2',6'-dimethylaniline accounted for some 73% of the dose (Keenagham and Boyes, 1972). However, the major metabolite of bupivacaine in the rhesus monkey was the amide hydrolysis product, pipecolinic acid (Goehl, Davenport and Stanley, 1973).

In both rat and man, amide hydrolysis and N-desbutylation were minor metabolic pathways. This fits in well with Hollunger's theory that

N-dealkylation is a prerequisite for amide hydrolysis. In his studies with lignocaine (Hollunger, 1960) he showed that the amide-hydrolysing enzyme showed marked substrate specificity having a high activity towards the secondary amine (monoethylglycinexylidide), but very low activity towards the primary amine (glycinexylidide) and the tertiary amine,lignocaine'(diethylglycinexylidide). In the present study, the amount of desbutyl-bupivacaine and pipecolinic acid excreted by both species was low and no N-butyl pipecolinic acid was detected. Thus it appears that like lignocaine, N-dealkylation of bupivacaine is necessary for 224

amide hydrolysis. However, the extent of N-dealkylation and consequently amide hydrolysis of bupivacaine is much lower (3% of the dose in 24h rat urine) than in the case of lignocaine (52% of the dose in 24h rat urine (Keenaghan and Boyes, 1972)), and this is likely to be due to the size of the N-n-butyl substituent and to steric factors associated with the aromatic ring. Another indication of the resistance

N-alkyl groups to dealkylation is given by Thomas and Meffin (1972) who reported that N-demethylation is a minor metabolic route for mepivacaine accounting for 1% of the dose in urine. The resistance of the amide bond of bupivacaine to chemical hydrolysis is evident since both acid & alkali hydrolysis of bupivacaine under pressure and at 100°C in our laboratory, failed to yield, 2',6'-xylidine. These results therefore contrast with those cf Goehl, Davenport and Stanley (1973) who reported that some 52% of a dose of bupivacaine in the rhesus monkey was excreted in the urine as pipecolinic acid. Both species studied excreted a small amount of neutral material which was not identified.

Thomas and Winkler (1973) have identified three minor neutral metabolites of mepivacaine in humans as a result of oxidation of the piperidine ring at the 6 position to the lactam. This oxidation may occur either prior to or subsequent to N-demethylation and hydroxylation of the piperidine ring. Since the metabolism of bupivacaine._in the rat has been shown to be similar to that of the other amide-type local anaesthetics, it is possible that in the rat at least, the unidentified minor neutral metabolite(s) may be the result of such aliphatic carbon oxidation.

The excretion of 14C after the administration of [149-bupivacaine

HC1 to human volunteers was similar in both cases in terms of metabolism 225

and total recovery. However, the rate of excretion was very different, radioactivity being voided some 3 times slower by EMH. Other metabolic studies in our laboratory have indicated that EMH is a genetically determined 'slow' metabolizer as shown by pharmacokinetic analyses of debrisoquine and phenacetin, two compounds whose metabolism is determined by a large gene effect rather than by environmental factors (Sloan,

Mahgoub, Lancaster, Idle and Smith, 1978).

In conclusion, the major metabolic pathway of bupivacaine in the rat is aromatic hydroxylation to yield two isomeric phenolic metabolites, with hydroxylation in the 3' position predominating. The major metabolites of bupivacaine in man, although appearing to have the extraction characteristics of phenols, were not 3' and 4'-hydroxybupivacaine and remain unidentified. The rat in this instance is therefore not a good metabolic model for man. The metabolic pathways of bupivacaine in the rat are shown in Fig. 6.18.

Fig.6.18 Metabolic pathways of bupivacaine in the rat

N ~* minor CONH C4H9

CH3 major bupivacaine

N CONH H

C113

desbutyl-bupivacaine N CONH C4H9

~~ COOH and 4'-hydroxybupivacaine Īi pipecolinic acid conjugates 227

Chapter Seven. The identification of urinary metabolites of

pethidine in adult volunteers

Page

Introduction 228

Analysis of urinary metabolites 230

Results 241

Discussion 248 228

Introduction

Pethidine is a synthetic narcotic analgesic introduced into clinical use in 1939 which finds wide application for the relief of pain post- operatively and in childbirth. The fate of this drug in animals and man was first examined in the 1950's (Burns et al., 19 55; Plotinikoff et al., 1956) using N- [14C]-methyl labelling and colorimetric analysis. The results indicated two pathways of metabolism, namely ester hydrolysis to give the corresponding acid and N-demethylation. More recently, two minor metabolites, 4'-hydroxypethidine (Lindberg et a1,1975) and pethidine-N-oxide (Mitchard et al., 1972) have been reported in man but not quantitated (see Chapter 1). This chapter describes a re-evaluation of the metabolism of pethidine in man, using combined radioactive (14C) and stable (2H) labelling of the molecule, and GC-

MS characterisation of the metabolites.

14C, 35S, 131 Unstable (radioactive) isotopes, e.g. I,have been used in pharmacology for the last 30 years primarily as tracer labels for drug distribution and metabolism studies. With radioactive labels, liquid scintillation counting provides a convenient and inexpensive means of detecting the emitted radiation and hence the drug or metabolite. However, the use of radioactive isotopes does not by itself allow differentiation between the various molecular forms in which the isotope may be present, and they do not aid identification of compounds by mass spectral analysis. Stable isotopes on the other 13C, 15 hand, e.g. 2H, N, lack a simple isotope-specific method of quantitation comparable to liquid scintillation counting, but are potentially very useful qualitatively when used in combination with the unlabelled compound. Such a mixture would, when subjected to 230

mass spectrometry, produce doublets in the mass spectrum as a consequence of the two different molecular weights of the corresponding fragments. These doublets thus serve as an easy means of identi- fying stable-labelled compounds from other structures present in the mixture. To this end, pethidine was synthesized with five deuterium atoms in the aromatic ring, [2H51-pethidine, which increased its molecular weight by 5amu. The mass spectrum of a mixture of [ 2H57 - pethidine (MW 252) and unlabelled pethidine (MW 247) in an 'L 1:1 ratio shows conspicuous doublets 5amu apart in all fragments containing the intact aromatic ring (Fig. 7.1a). This would also be true of pethidine metabolites. By adding a small amount of[ 14c3_ pethidine to this mixture, the advantages of both radioactive and stable isotope labels are obtained for both quantitative and qualitative drug metabolism studies. The proposed mass fragmentation pattern of pethidine is shown in Fig. 7.1b.

Analysis of Urinary Metabolites 14 2 Two adult male volunteers were each given a mixture of [ CJ /[ H5J - pethidine HC1 (50mg; 5pCi) i .m. as described in Chapter 3, and their urine collected for up to 4 days. One volunteer (JC) collected the 14CO2 in expired air as described on page 115 and the 14C content of this estimated by scintillation counting. The O-24h urines were subjected to the sequential extraction procedure at various pH's performed as described in the text, and each extract was examined by TLC, GC and GC-MS.

The residue from the PH 14 extract was examined by TLC in systems

B, D and G and the plates scanned for 14C. In all three systems a

Fig. 7.1a Mass spectrum of a 1:1 mixture of pethidine (MW 247) and 2H5-pethidine (MW 252)

_ 100 71 ions attributed to 2I15- pethidine

ions attributed to pethidine

rl 252 (M ) 247 96 (M ) ity 177 ns 174 te 218 I 223 In

to3 1s 1 179

ive 108 1/ 172 II t II 1 136 II II la II 11 91 Re I I II 4 II 232 I I 14o 163 'I n 1 1 15 237 t58 ; p I u 120 I~ II ' I I u u 1 1 1 E' ` II 1 I I II I II d 'II 1 I 50 100 150 200 250 300 m/e Fig. 7.1b Proposed mass spectrometry fragmentation pattern for pethidine

C2H5 y\ COOC2H5

0--C=0 N m/e 140 CH LH3 m/e 169 m/e 15a~ CH3 m/e 232

m/e 174 m/e 96

CH3 m/e 218

CH CH /// 2~ CC=CH2 CH2 VII N= CH2 CH CH2 C...-7--CH 2 CH 3 m/e 91 m/e 131 m/e 115 233

single 14C peak was observed with Rf 0.45 in system B; 0.29 in system D and 0.68 in system G; which corresponds in each case to pethidine. GC analysis of this residue using conditions specified in Chapter 3, showed the presence of two compounds with retention times of 1.8 and 3.0 minutes which correspond to the retention times of pethidine and norpethidine respectively. The mass spectra of these peaks are given in Fig. 7.2 and Fig. 7.3 along with the mass spectra of standard pethidine and norpethidine for comparison. These two compounds were quantitated by GC as described in Chapter 3.

The pH 9 extract was subjected to TLC in systems B, D and G and the plates scanned for 14C. A single peak was observed in each case whose Rf value corresponded to standard 4'-hydroxypethidine (Table

7.1). GC analysis of this extract showed it to contain a single drug- related compound with retention time identical with that of 4'-hydroxypethidine (5.5min), which was shortened following acetylation to 5.4 min. The extract, accounting for some 3% of the urinary 14C, contained insufficient material to obtain a full scan (i.e. 0-600amu) mass spectrum, and therefore single ion monitoring (SIM) for ions diagnostic of 4'-hydroxypethidine and its 0-acetyl derivative was performed on the appropriately treated extracts. The full mass spectra of authentic 4'-hydroxypethidine and its acetylated derivative are given in Figs. 7.4a and 4b, and the ions chosen for SIM were:-

4'-hydroxypethidine, m/e 263 (M+), 267 (M++4): 268 (M++5)

4'-aceto m/e 276 (carboxylate ions (M ++29);

280 (M++33); 281 (M++34) 234

The pH 5 extract was shown by radioTLC to contain one 14C peak which had an Rf identical with that of authentic pethidine-N-oxide in systems B, D and G (Table 7.1). GC analysis showed the residue to contain a single compound with retention time 3.2 minutes which was identical with that of pethidine-N-oxide. Some 1% of urinary 14C was contained in this residue which was insufficient for full scan MS, and again SIM was employed on the extract.

The full scan mass spectrum of authentic pethidine-N-oxide is given in Fig. 7.5 and the ions chosen for SIM were:

m/e 263 (M+); 267 (M++4); 268 (M++5)

Additional demonstration that this extract contained pethidine-N- oxide was provided by treating this extract with TiC13/HC1 as described in Chapter 3, after which analysis by GC showed the presence of a single compound with retention time identical to that of pethidine (1.8 min). Furthermore, radioTLC showed the 14 presence of one C peak in systems B, D and G which correspond in each case to pethidine (Table 7.2). Pethidine-N-oxide was quantitated by GC following its reduction to pethidine with

TiC13/HC1 as described in Chapter 3.

A portion of the aqueous phase remaining from the above sequential extraction was treated with S-glucuronidase and examined by radioTLC in Table7.I RadioTLC properties of pethidine and 14C-labelled metabolites isolated from human urine

Rf value of 14C peak in system: B D G pH14 extract 0.45 0.29 0.68 (pethidine)

pH9 extract 0.38 0.27 0.70 (4'-hydroxypethidine)

pH5 extract 0.25 0.29 0.48 (pethidine-N-oxide)

Aqueous residue (pethidinic acid) as such 0.03 0.15 0.43 after esterification* 0.46 0.29 0.67

Details of TLC systems in chapter 3 . * Esterification with ethanolic H2SO4 as for total pethidinic and norpethidinic acids.

Fig.7.2a Mass spectrum of pethidine via GC-MS, retention time 1.61min ō 91 1-1 131 115 140 172 218 247 (M+ ) 129 232 77 190 158

169 202

i I N W 100 150 200 250 300 ity tens In

Fig. 7.2b Mass spectrum of peak 1 of pH14 extract from human urine, GC-MS retention time 1.6 min ive 0 t 247(M+ ) la 172

Re 140 252 (Mt )

96 177 115 232 77 82 131 218 222 237 158 190 202 ` 207 0 I~ it I t I t I fl 1 100 150 200 250 m/e 300 Fig.7 3a Mass spectrum of norpethidine via GC-MS, retention time 1.8 minutes

0 7 I"

233 (M+ ) 91 103 115 82 160 126 155 204 218 I I 11131 0 I I L

100 200 300 ity s Fig.7 3b Mass spectrum of peak 2 of pH14 extract from human urine, GC-MS retention time 1.8 minutes ten

In 57

e 0 H 44 iv t la 233 (Mt ) Re 238 (M+ ) 103 115 165 91 16o 204 82 120 223 (2o9J

0 IIB I 21 100 200 300 Fig. 7.4a Mass spectrum of 4'-hydroxypethidine via GC-MS, retention time 4.9 minutes

0 71 H

263 ( M+)

96 140

169 192 245

0 i 1 ity 100 200 300 tens In

ive Fig.7.4b Mass spectrum of acetylated 4'-hydroxypethidine via GC-MS, retention time 4.9 minutes t la g 0 Re H 71

(M+) 14o 305 96 188 276 0 H 1 1 1 100 200 300 Fig. 7.5 Mass spectrum of pethidine N-oxide via GC-MS, retention time 2.0 minutes

71 0 60

247

172 ity s ten In

e 143 iv t la 190 Re 218

0 50 100 150 200 250 m/e 240

systems B, D and G when in each case, a single 14C peak which had an

Rf identical with that of authentic pethidinic acid was found (Table

7.1). The remainder of the aqueous phase was then treated in the following ways:

(i) acid hydrolysis follo':ed by ethanolic H2SO4 treatment converting

pethidinic and norpethidinic acids to their ethyl esters. GC

analysis as described showed two peaks with retention time 1.8

and 3.0min, corresponding to pethidine and norpethidine respect-

ively, and radioTLC in systems B, D and G one peak with Rf

corresponding to pethidine (Table 7.1). Norpethidinic and

pethidinic acids were quantitated as described in Chapter 3.

(ii)BF 3-methanol esterification of free pethidinic and norpethidinic

acids to their methyl esters. GC analysis of the appropriate

extract of the reaction mixture showed two peaks of retention

time 1.75min (pethidinic acid methyl ester) and 2.9min (norpeth-

idinic acid methyl ester), which were quantitated as described

in Chapter 3.

(iii)incubation at pH 5 with R-glucuronidase followed by BF3-methanol

esterification as in (ii) which showed the presence of the methyl

esters of norpethidinic and pethidinic acids. The excretion of

the ester glucuronides of these acids was thus calculated from

the difference between assays (iii) and (ii).

These procedures permitted the identification and quantitation of all known urinary metabolites of pethidine.

The metabolism of norpethidine and pethidinic acid was investigated 241

by administering these substances to 2 volunteers as described in

Chapter 3. Their O-24h urines were collected and analysed for pethidine metabolites as above.

RESULTS

The elimination of 14C in the urine and expired air of the volunteer

JC after the administration of [14C]/[ 2H5J-pethidine HC1 determined as described in Chapter 3 is given in Table 7.2 and Fig. 7.6. 31% of the activity was excreted in the urine in 24h and 33% in the expired air as 14CO2 in the same period. The 14CO2 was the result of N- demethylation of pethidine. The volunteer JD'S excreted 45% of the

administered activity in 24h urine, but the amount excreted in the

expired air was not estimated.

Table 7.3 shows the results of the sequential extraction of the 24h urine in both volunteers, and the various extracts were shown by GC

and/or by radioTLC to contain pethidine and norpethidine (pH 14);

4'-hydroxypethidine (pH 9); pethidine-N-oxide (pH 5) and pethidinic

and norpethidinic acids (aqueous residue). GC-MS data for the pH 14

extract (Fig. 7.2b and 7.3b) gave spectra similar to those for authentic

pethidine and norpethidine (Fig. 7.2a and 7.3a), with the addition of

5amu doublets for several of the major peaks with approximately the

same relative intensities. These extra:peaks were the result of the

increase in molecular weight of fragments containing the deuterated rings, the metabolism of 2H5 pethidine being identical to that of

pethidine. Hence, fragments containing the aromatic residue would

show a peak at m/e +5 compared to similar fragments from unlabelled

pethidine and norpethidine. GC-MS of the aqueous phase remaining Table 7.2 Elimination of 14C by adult volunteer JC after administration of [14c],[2H5]_ pethidine HC1

Pethidine HC1 (50mg; 5uCi) in normal saline was administered by intramuscular injection. Figures given are % dose (cumulative) excreted.

in urine in expired air total 0 - lh 1.5 1.3 2.8 1 - 2h 4.6 3.4 8.0 2 - 3h 7.1 5.3 12.4 3 - 4h 8.7 6.9 16.6 4 - 5h 11.11 8.1 29.2 5 - 6h 13.4 9.0 22.4 6 - 8h 15.4 10.5 25.9 8 - 10h 18.0 11.5 29.5 10 - 12h 19.7 13.0 32.7 12 - 14h 21.6 14.0 35.6 14 - 16h 23.9 15.0 38.9 16 - 18h 26.2 16.0 42.2 18 - 20h 27.7 17.2 44.9 20 - 22h 29.5 17.9 47.4 22 - 24h 31.1 18.2 49.3 24 - 36h 38.7 36 - 48h 42.5 243

Fig.7.6 Elimination of 14C by alt volunteer JC following i.m. administration of 5 C C] /[ H ~ - pethidine HC1 (50mg; SuCi)

40 -1 urine

) e

iv 30 t la d (cumu te e

excr 20 e s do

I I I I 1 1 8 16 24 32 40 48

TIME (hours)

Table 7.3 Sequential extraction of human urine

%14C Figures are expressed as in 24h urine extracted in each case

Conditions of extraction Untreated urine Urine treated with s-glucuronidase and solvent

volunteer JC JD'S JC JD'S

pH 14 (ether) 25.8 26.7 28.1 28.2 pH 9 (ether) 2.5 1.5 4.8 1.8 pH 5 (ether) 3.0 2.7 3.1 , 2.5 remaining in aqueous 68.7 70.1 64.0 67.5 phase

Total 100 100 100 100 245

from the sequential extraction procedure following ethanolic H2SO4 treatment, showed the presence of two peaks corresponding to pethidine and norpethidine which

gave identical spectra to those obtained from the pH 14 extract. This confirms

that pethidinic and norpethidinic acids had been converted to pethidine and

norpethidine.

GC-MS SIM on the pH 9 residue (GC-MS retention time 5.5minutes) displaced large

peaks at m/e 263 (the molecular ion of 4'-hydroxypethidine) and m/e 267. The

peak at m/e 263, an increase of 16amu over pethidine, suggests the incorporation of a single oxygen atom into the pethidine molecule, while the peak at m/e 267 indicates that the oxygen attack had occurred at the aromatic ring with the

displacement of a deuterium atom. Similarly, the molecular ion of the acetyl-

ated pH 9 residue (CSC-MS retention time 5.4minutes) occurred at m/e 276 and that of its deuterated analogue at m/e 280, again showing a loss of a deuterium atom as a result of oxygen incorporation.

GC-MS SIM on the pH 5 residue (GC-MS retention time 3.2minutes) displayed large

peaks at m/e 263 (the molecular ion for pethidine-N-oxide) and m/e 268. Thus, once again this residue contained a compound with an increase of 16amu over pethidine strongly suggesting the incorporation of oxygen, while the peak at m/e

268 showed the oxygen attack to be independent of the aromatic residue since the ion corresponding to 2H5 -pethidine still maintained a 5amu increase over the corresponding pethidine ion.

These results are consistent with the pH 9 residue being 4'-hydroxypethidine and the pH 5 residue being pethidine-N-oxide.

The metabolites of 14C -pethidine HC1 in 24h urine are listed in

Table 7.4. Six of these metabolites namely norpethidine, pethidine-N- oxide, pethidinic acid and its glucuronide,. norpethidinic acid and its glucuronide, as well as unchanged pethidine urine assayed by GC.

Four metabolites, 4'-hydroxypethidine, pethidine-N-oxide, pethidinic Table 7.4 The metabolism of pethidine in adult volunteers

Each subject received 50mg[14C]/ ~25H ~ - pethidine HC1 by i.m. injection and excreta was collected and analysed as described in the text

% dose excreted as volunteer JC JD'S

Pethidine 5.7 7.8 Norpethidine 12.1 10.1 Pethidine-N-oxide 1.6 0.6 4'-Hydroxypethidine 1.3 2.3 Pethidinic acid free 6.1 3.9 glucuronide 8.3 13,2 total 14.4 17.1 Norpethidinic acid free 6.8 8.4 glucuronide 16.3 22.7 total 23.1 31.1

Total 58.2 69.0

% 14C dose in 24h urine 22.2 26.2 14 % C dose in expired air 33.3 ND (24h)

247

acid and its glucuronide as well as pethidine, were also quantitated by 14C, and the results are shown below for comparison purposes:

% dose in 24h urine by EXTRACT COMPOUND 14 C GC pH 14 pethidine 8 7 pH 9 4'-hydroxypethidine 1.8 not determined pH 5 pethidine-N-oxide 0.3 1 aqueous residue pethidinic acid 21 16

Thus there was a good correlation between the GC analysis and 14C quantitation of the metabolites. Furthermore, the 14CO2 in expired air accounted for some 33% of the administered dose, and N-demethylated metabolites, i.e. norpethidine and norpethidinic acid, by GC methods some 35%; again giving good agreement between the two methods of analysis.

The metabolites of 14C-pethidine in 24h adult urine as quantitated by

GC (where possible) are listed in Table 7.4. The major urinary metabolite was norpethidinic acid accounting for some 27% of the administered dose of which about 60% was conjugated with glucuronic acid.

Pethidinic acid, also the result of an hydrolysis pathway accounted for ti16% of the dose, and norpethidine, largely the result of N- demethylation of pethidine for 11%. Both 4'-hydroxypethidine and pethidine-N-oxide were minor excretion products.

Metabolism of Norpethidine and Pethidinic acid

The metabolic fates in human volunteers of norpethidine and pethidinic 248

acid are shown below. Norpethidine was extensively hydrolysed to norpethidinic acid (45% of dose) and some unchanged material was also excreted (27% of dose). Urine was treated with TiC13 after extraction of basic compounds, as described for pethidine, but no increase in the amount of norpethidine occurred. This indicates that metabolic N-oxidation of norpethidine had not taken place. Pethidinic acid was largely excreted unchanged and as its glucuronic acid conjugate

(total 79% of dose) but a small amount of N-demethylation to norpethidinic acid was observed (total norpethidinic acid + glucuronide 9%).

L7N 20mg % dose in 24h urine as: JC 25mg pethidinic acid norpethidine l.m. per os

Norpethidine 28.3

Norpethidinic acid 45.4 9.0

Pethidinic acid 79.0

Total 73.7 88.0

DISCUSSION

The combination of radioactive and stable isotope labelling employed enabled the identification of seven metabolites of pethidine in addition to the unchanged drug in the urine. Quantitative data on the metabolism of pethidine in the two volunteers is listed in Table 7.4. A total of ti 64% of the dose was accounted for as the unchanged drug and seven metabolites. The major route of metabolism was ester hydrolysis giving rise to pethidinic and norpethidinic acids which together account for some 43% of the dose. These acids are excreted free and as their ester glucuronides, the ratio of free to conjugated acids being about 70%. N-Demethylation was also an important metabolic 249

pathway, accounting for some 61% of the dose metabolites excreted,

this pathway leading to norpethidine and norpethidinic acid. N-

Oxidation and 4'-hydroxylation were minor pathways.

This study enabled both a 14C determination and GC assay for several

of the metabolites as well as unchanged drug in the O-24h urine. The

two methods were found to correlate well showing the validity of the

GC method which was subsequently used to quantitate the urinary meta-

bolites in a small group of adults and neonates which will be discussed

fully in Chapter 8. There was also a very good agreement between

the extent of N-demethylation, as determined by GC analysis of nor-

pethidine and norpethidinic acid, and the amount of 14CO2 eliminated

in the expired air. La Du, Gaudette, Trousof and Brodie (1954) have

shown that the methyl group is oxidatively removed as formaldehyde.

This study has shown that in man, the removed N-methyl group is almost

completely excreted as CO2.

There is some speculation in the literature concerning the origin of norpethidinic acid - whether it is the result of ester hydrolysis of norpethidine and/or N-demēthylation of pethidinic acid. The results showed that ester hydrolysis of norpethidine gives rise to about 90% of norpethidinic acid excreted in the urine indicating that there are two distinct hydrolysis pathways. La Du et al. (1954) have shown that N-demethylation of pethidine and other alkylamines is catalysed by an enzyme system present in liver microsomes and that reduced NADPH and oxygen are required. Gaudette, La Du and Brodie (1955) have found that the microsomal enzyme system does not catalyse the N-demethylation of pethidinic acid, a finding which is in accord with the 250

observations in this study that, following the administration of pethidinic acid to man, it is N-demethylated to norpethidinic acid to only a very small extent.

Pethidine-N-oxide was quantitatively a comparatively minor excretion product but the N-oxide found may not represent the end product of metabolism. Tertiary amine drugs are converted into dealkylated and

N-oxide metabolites by liver microsomal enzymes. The two reactions are catalyzed by NADPH-dependent microsomal electron transfer chains, the first involving NADPH- cytochrome C reductase and cytochrome

P-450, the latter a different flavo-protein and no cytochrome P-450.

N-oxides can be further metabolized by dealkylation and/or by reduction in extra-microsomal comparments; thus:-

R R A R' - N R' - N + HCHO CH2OH R / R' - N:

CH3 C R

R' - N ->0 CH3

Isotope trapping experiments using imipramine and imipramine-N-oxide as substrates showed the simultaneous occurrence of all four reactions

(i.e. A, B, C, D, in the above schen) in NADPH-fortified liver homogenates. When the individual reaction rates were studied it was found that the rate constant for reaction D was greater than for reaction B 251

and C (Bickel, 1971). Further experiments with liver slices, perfused livers and whole animals with the same substrates also suggest that the four reactions (A, B, C, D) are functioned in vivo, and therefore N-oxide formation may well be a major pathway, at least in the case of imipramine. Since the pethidine-N-oxide excreted represents the amount that has escaped hepatic and extrahepatic N-oxide reduction/dealkylation, the amount of N-oxide actually formed as an intermediary metabolite may be more considerable than the amount excreted. Thus N-oxidation of pethidine may conceivably be a quantitatively more important route than is suggested by the small amount

of pethidine-N-oxide excreted in the urine.

This study is in good agreement with the results of Brodie et al.

(1955) who used colourimetric analysis. They reported that approximately 5% of a dose of pethidine is excreted unchanged and about 12% as pethidinic acid. However their values of 5% of the dose being

excreted as norpethidine and 12% as norpethidinic acid are less than

half the values found in this work. Both pethidine-N-oxide and 4'-

hydroxypethidine excreted in 24h urine have been quantitated for the

first time. The metabolic fate of pethidine in man is given in

Fig. 7.7. Fig.7.7 Metabolic pathways of pethidine

C6H5 COOC2H5

ESTER GLUCURONIDE N, CH3 \` C2H5 000C2H 5

pethidine pethidinic acid / N U1

norpethidine

C6H5 COOC2H5 C6H COOH 5

2< ESTER GLUCURONIDE CH3 0 \ 3 CH

4'-hydroxypethidine pethidine N-oxide norpethidinic acid

Major route Minor route 253

Chapter Eight The metabolism of pethidine in adult volunteers and

human neonates

Page

Introduction 254

Analysis of urinary metabolites 254 254 Adult panel study

Neonatal study 255

Resu Its 255

Adult panel study 255

N eonatal study 262

Discussion 267 254

Introduction

Since pethidine was introduced into clinical use in 1939 it has gained wide acceptance as an obstetric analgesic agent even though serious deleterious effects on the respiratory capacity of infants has been attributed to this compound (Roberts, Kane, Percival, Snow and Please,

1957). As a result, considerable research has been carried out on the toxic effects of pethidine and its metabolites on both mother and fetus, and some studies have reported a relationship between pethidine and/or metabolites concentration (particu]arly norpethidine) to neonatal CNS depression (Miller and Anderson, 1954; Morrison et al.,

1973). The work described in this chapter was performed, (a) to assess inter-individual variation in pethidine disposition in adult volunteers and (b) to examine neonatal metabolism of residual body burdens of pethidine, acquired as a result of transplacental transfer following maternal administration during childbirth, to see whether impaired metabolic ability accounts for the prolonged blood elimination half-life of the drug in infants reported in Chapter Four.

Subjects and Methods

Adult panel study

Fifteen volunteers (13 males, 2 females) were given pethidine HC1 (50 mg) by an intramuscular injection in the buttock, and their 0-24h urine collected. Details of age, weight, urinary pH and volume are given in Table 8.1. The urines were taken through the sequential extraction procedure outlined above and pethidine and all known metabolites assayed by GC as described in Chapter Three.

Four of the above volunteers consented to a repeat of the above experiment 255

when serial urine collections were made for

36h, and each urine sample assayed for pethidine and metabolites.

Two of these volunteers were classified as poor metabolizers (PM)

and the other two as extensive metabolizers (EM) in their ability to

4'-hydroxylate debrisoquine (see later).

Neonatal urine

The first 24h urine from five male neonates (subject numbers, 15, 19,

20, 25, 44) whose mothers received pethidine during labour was analysed

for all known metabolites of pethidine by GC. The procedure followed

was the same as for adult urine (described in Chapter Three) with the

difference that both the amount of internal standard and the concentra-

tions used for the standard curves were decreased by a factor of 10 in

order to bring them more in line with the concentrations being analysed.

Results

Adult panel study

The qualitative and quantitative aspects of the urinary excretion of

pethidine and its metabolites in the fifteen volunteers are shown in

Table 8.1. The overall recovery of the administered dose in the 0-24h

urine as shown by the total of the unchanged drug and its metabolites

varied considerably, ranging from 11.9% to 68.7%. In all subjects

six urinary metabolites were identified and quantitated in addition to

the parent drug, namely pethidinic acid and norpethidinic acid and their

glucuronic acid conjugates, norpethidine and pethidine-N-oxide. 4'-

Hydroxypethidine was identified in three volunteers but not quantitated since only small amounts were present. Table 8.1 Pethidine metabolism in a panel of human volunteers (figures are % recovery in that form in 0-24h urine following a 50mg dose of pethidine HCl i.m.)

** Subject JC LJN JD'S TS TM TE EH AL JĒC J0'G RLS BD JCR SL PS Weight (kg) 87 67 80 60 68 74 73 74 60 64 89 76 83 70 70 Age 31 25 23 27 25 31 35 22 25 28 45 32 22 33 34 Oxidation phenotype EM EM PM EM EM PM EM EM PM PM PM - PM EM - Urine pH 6.69 6.02 6.03 6.04 6.63 6.22 6.26 5.82 5.48 6.26 5.81 5.78 5.43 6.16 5.95 Urine volume (ml) 2680 930 1510 1341 1020 1510 2200 1320 985 1325 1168 1375 1820 1160 2164 Pethidine 5.7 3.4 7.8 3.6 4.2 10.1 7.1 5.2 5.2 3.5 2.0 6.9 3.6 4.0 10.1 Norpethidine 12.1 10.5 10.2 4.7 3.3 2.9 4.3 4.3 0.8 3.7 5.8 7.1 1.0 5.1 0.8 N 01 Norpethidinic acid free 6.8 4.8 8.8 2.1 1.2 2.6 3.8 3.2 2.8 4.6 0.0 9.9 4.6 2.9 0.3 conjugated 16.3 10.5 22.7 9.0 3.4 2.6 1.6 7.1 11.4 3.0 0;9 3.4 4.2 3.9 2.9 total 23.1 15.3 31.5 11.1 4.6 5.2 5.4 10.3 14.2 7.6 0.9 13.3 8.8 6.8 3.2 Pethidinic acid free 6.1 4.8 3.9 3.5 4.4 8.4 9.3 0.4 5.8 3.1 1.5 9.9 2.1 5.8 3.8 conjugated 8.3 23.1 13.2 21.7 19.1 33.9 11.8 8.8 10,6 11.0 1,6 8.4 4.7 10.2 1.5 total 14.4 27.9 17.1 25.2 23.5 42.3 20,1 9.2 16.4 14.1 3.1 18.3 6.8 16.0 5.3 Pethidine-N-oxide 1.6 0.1 0.6 0.3 0.2 0.7 0.4 0.2 0.2 0.4 0.1 0.6 1.7 0.4 0.2 *4/ -Hydroxypethidine 0.8 ND 1.5 ND ND ND ND ND ND ND ND ND ND ND ND

Total recovery 57.8 57.2 68.7 44.9 35.8 61.2 38.3 30.2 36.8 29.3 11.9 46.2 21.9 32.3 19.6

ND not determined; * assayed by 14C; ** female 257

The relative amounts of unchanged pethidine and its metabolites excreted

in 0-24h urine varied widely within the group, and apart from the two

subjects with the lowest recoveries, the major metabolite in each case

was either pethidinic acid or norpethidinic acid which were both ex-

creted to a large extent conjugated with glucuronic acid. There was

a large variation in the amount of norpethidine (range 0.8-10.5% of

the dose) and pethidine (2-10%) excreted, but the amount of pethidine-

N-oxide in the urine was consistently low (1,0.5%) in all subjects.

In order to compensate for the different recoveries and hence permit

direct comparison between the individuals on the extent of metabolism

the data was treated in several ways.

(i) standardised to the amount of pethidine excreted, i.e. the amount

(%) of each metabolite was divided by the amount (%) of pethidine

in the urine (Table 8.2).

(ii)expressed relative to the dose recovered; i.e. the amount (%) of

each metabolite was divided by the total recovery (Table 8.3).

(iii)expressed as the dose recovered undergoing each of the individual

reactions; e.g. extent of:-

N-demethylation = % norpethidine + % norpethidinic acid % dose recovered

initial ester hydrolysis = % pethidinic acid/% dose recovered

secondary ester hydrolysis = % norpethidinic acid/% total

N-demethylation (Table 8.4).

The tables clearly show that the major metabolic pathways of pethidine

in man are ester hydrolysis and N-demethylation with the major metabolites

being pethidinic or norpethidinic acids. • Frequency distribution curves for the-different pathways (Fig. 8.1) drawn from the data obtained Table 8.2 Pethidine metabolism in a panel of human volunteers

Data standardised to the amount of pethidine in 24h urine, i.e. pethidine = 1, metabolite x = % metabolite x/ % pethidine

Subject JC LJN JD'S TS TM TE EMH AL JEC J0'G RLS BD JCR SL PS

Pethidine 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Norpethidine 2.12 3.09 1.31 1.31 0,79 0.29 0.61 0.83 0.15 1.06 2.9 1.03 0.28 1.28 0.08

U1 Pethidinic acid m free 1.07 1.41 0.50 0.97 1.05 0.82 1.31 0.08 1.12 0.89 0.75 1.43 0.58 1.45 0.38 conjugated 1.46 6.79 1.69 6.03 4.55 3.31 1.66 1.69 2.04 3.14 0.80 1.22 1.31 2.55 0.15 total 2.53 8.20 2.19 7.00 5.60 4.13 2.97 1.77 3.16 4.03 1.55 2.65 1.89 4.00 0.53

Norpethidinic acid free 1.19 1.41 1.13 0.58 0.29 0.26 0.54 0.62 0.54 1.31 - 1.43 1.28 0.73 0.03 conjugated 2.86 3.09 2.91 2.50 0.81 0.26 0.23 1.37 2.19 0.86 0.45 0.49 1.17 0.98 0.29 total 4.05 4.50 4.04 3.08 1.10 0.52 0.77 1.99 2.73 2.17 0.45 1.92 2.45 1.71 0.32

Pethidine-N-oxide 0.28 0.03 0.08 0.08 0.05 0.07 0.06 0.04 0.04 0.11 0.05 0.09 0.46 0.10 0.02

Total 9.98 16.82 8.62 12.47 8.54 6.01 5.41 5.63 6.37 8.37 5.95 6.69 6.08 8.09 1.95 Table 8.3 Pethidine metabolism in a panel of human volunteers

Data standardised to total recovery of pethidine and metabolites in 24h urine ; i.e. pethidine + metabolites in urine = 1

Subject JC LJN JD'S TS TM TE EMH AL JEC J0'G RLS BD JCR SL PS

Pethidine 0.10 0.06 0.12 0.08 0.12 0.17 0.19 0.18 0.14 0.12 0.17 0.15 0.16 0.12 0.52

Norpethidine 0.21 0.18 0.15 0.10 0.09 0.05 0.11 0.15 0.02 0.13 0.49 0.16 0.05 0.16 0.04

Pethidinic acid free 0.11 0.08 0.06 0.08 0.12 0.14 0.24 0.01 0.16 0.11 0.13 0.21 0.10 0.18 0.19 conjugated 0.15 0.41 0.20 0.48 0.54 0.55 0.31 0.30 0.29 0.38 0.13 0.18 0.21 0.32 0.08 total 0.26 0.49 0.26 0.56 0.66 0.69 0.55 0.31 0.45 0.49 0.26 0.39 0.31 0.50 0.27

Norpethidinic acid free 0.12 0.08 0.13 0.05 0.03 0.04 0.10 0.11 0.08 0.16 - 0.21 0.21 0.09 0.21 conjugated 0.29 0.19 0.40 0,20 0.10 0.04 0.04 0.25 0.31 0.10 0.08 0.08 0.19 0.12 0.15 total 0.41 0.27 0.53 0.25 0.13 0.08 0.14 0.36 0.39 0.26 0.08 0.29 0.40 0.21 0.36

Pethidine-N-oxide 0.03 0.001 0.009 0.008 0.005 0.011 0.009 0.006 0.007 0.014 0.01 0.013 0.08 0.013 0.01 Table 8.4 Pethidine metabolism in a panel of adult volunteers

Data standardised to total recovery of pethidine and metabolites in 24h urine; i.e. pethidine + metabolites in urine = 1

Subject JC LJN JD'S TS TM EMH AL JEC J0'G RLS BD JCR SL PS TE

Extent of demethylation 0.62 0.45 0.62 0.35 0.22 0.25 0.50 0.41 0.39 0.56 0.44 0.45 0.37 0.20 0.13 (norpethidine + norpethidinic acid) N ol 0 Extent of initial ester hydrolysis 0.26 0.49 0.25 0.56 0.66 0.55 0.31 0.45 0.48 0.26 0.40 0.31 0.50 0.27 0.69 (pethidinic acid)

Extent of secondary ester hydrolysis 0.41 0.27 0.53 0.25 0.13 0.14 0.36 0.39 0.26 0.08 0.29 0.40 0.21 0.16 0.08 (norpethidine -4- norpethidinic acid)

Extent norpethidine undergoing ester 0.66 0.59 0.76 0.70 0.58 0.56 0.70 0.95 0.67 0.13 0.65 0.90 0.57 0.80 0.64 hydrolysis (% norpethidine > norpethidinic acid)

Extent of acids undergoing glucuronic acid conjugation Pethidinic acid 0.58 0.83 0.77 0.86 0.81 0.56 0.96 0.65 0.78 0.52 0.46 0.69 0.64 0.28 0.80 Norpethidinic acid 0.71 0.69 0.72 0.81 0.74 0.30 0.69 0.80 0.40 1.00 0.26 0.48 0.57 0.91 0.50 Table 8.5 Recoveries and rate constants for pethidine metabolites in four selected volunteers

Each subject was given an intramuscular injection of pethidine HC1 (50mg) and 24h urine urine collected for analysis as described in Chapter Three.

RIS EMH JC U N

Oxidation phenotype Poor(PM) Poor(PM) Extensive(EM) Extensive(EM) Urine volume (ml) 2230 1940 2200 1110 Urine pH 6.01 6.20 6.11 5.51 % dose excreted as Pethidine 5.3 3.0 5.0 2.9 Norpethidine 3.4 7.7 5.2 3.7 rn Pethidinic acid (total) 22.7 17.6 11.9 6.6 ~ Norpethidinic acid (total) 4.5 21.3 16.4 4.4 Total recovery (%) in 24h urine 35.9 49.6 38.5 17.6 Calculated infinite recovery (%) 45.8 62.6 47.9 21.2 Overall elimination rate constant (Kh-1) 0.052 0.047 0.055 0.054 Elimination rate constant for pethidine (h-1) 0.0077 0.0028 0.0072 0.0088 Rate of formation of: (h-1) Pethidinic acid 0.0329 0.0167 0.0170 0.0204 Norpethidinic 0.0065 0.0202 0.0235 0.0135 Norpethidine 0.0050 0.0073 0.0074 0.0113 Rate of N-demethylation (h-1) 0.0114 0.0275 0.0308 0.0248 Overall elimination half-life for pethidine (h) 13.4 14.8 12.6 12.8 262

from Table 8.4 tend towards a normal distribution within the 'population',

although it must be emphasised that these curves were drawn from only

15 adults, and hence may not be representative of a large population.

Serial urine samples were collected from four selected volunteers for

36h following an i.m. injection of pethidine EC]. (50mg). Two of the

volunteers (RLS and EE) belong to a genetically determined minority group

that are known to be relatively slow oxidizers of drugs such as debri-

soquine, phenacetin and guanoxan, while the other two (JC, LJN) are

phenotypically rapid metabolizers of these compounds. The serial

urine collections were analysed for all known metabolites of pethidine

from which the rate of formation of each of the metabolites was calculated.

The results (Table 8.5) show that there is no significant difference

between the overall rate constant of pethidine (and metabolites by all

routes) or the rates of formation of any of the metabolites. The method

used for the calculation of the rate constants is given in Appendix D.

Neonatal Urine

The first 24h urine from five neonates whose mothers received pethidine

during labour was analysed for all known metabolites of pethidine, and

the results are listed in Table 8.6. In all five babies, the major

urinary excretion product was pethidine with smaller amounts of nor-

pethidine and both free and conjugated pethidinic and norpethidinic

acids being present. The amount of pethidine-N-oxide in the urine

was very small and 4'-hydroxypethidine was not detected.

The variation in the amount of pethidine and metabolites in the urines of the five babies could be partly attributed to differences in the Table 8.6. Pethidine metabolism in five human neonates following maternal administration during childbirth

The first 24h urine from 5 male neonates was collected and analysed as described in the text. Figures are

expressed as pg in that form in 24h urine.

Baby No. 15 19 20 25 44 MEAN S.E.

Urine pH 5.8 6.8 5.8 6.2 5.9 Urine volume (ml) 12.5 20.5 26.0 21.0 11.4 Blood elimination half-life (h) 18.7 23.3 22.7 15.0 26.5 21.2 2.0 Pethidine 15.7 177.4 46.5 123.5 109.1 94.4 28.7 Norpethidine 0.4 43.7 7.5 57.1 6.4 23.0 0.1 Pethidine-N-oxide 0.03 0.64 0.21 0.40 0.24 0.3 0.1 Pethidinic acid free 3.6 10.6 12.4 8.5 4.0 7.8 1.8 conjugated 1.0 5.9 16.6 11.0 0.2 6.9 3.1 total 4.6 16.5 29.0 19.5 4.2 14.8 6.6 Norpethidinic acid free 2.3 6.3 19.2 8.8 4.4 8.2 3.7 conjugated 5.3 11.9 17.9 24.7 3.7 12.7 3.9 total 7.6 18.2 37.1 33.5 8.1 20.90 9.35 Total 28.3 256.5 120.3 234.0 128.0 153.4 41.5

*41 -hydroxypethidine was not detected in any of the urines. Table 8.7 Pethidine metabolism in five human neonates following maternal administration during childbirth

Figures are standardised to the amount of pethidine in 0-24h urine, i.e. pethidine=1

Neonate 15 19 20 25 44 mean S.E.

Pethidine 1 1 1 1 1 Norpethidine 0.03 0.25 0.16 0.46 0.06 0.19 0.07 Pethidinic acid free 0.23 0.06 0.27 0.07 0.04' 0.13 0.04 conjugated 0.06 0.03 0.36 0.09 0.002 0.11 0.06 0.29 0.09 total 0.63 0.16 0.042 0.24 0.10 N Norpethidinic acid free 0.15 0.04 0.41 0.07 0.04 0.14 0.06 conjugated 0.34 0.07 0.38 0.20 0.03 0.20 0.06 total 0.49 0.11 0.79 0.27 0.07 0.34 0.06 Pethidine-N-oxide 0.002 0.004 0.005 0.003 0.002 0.003 0.001 Total N-demethylation 0.28 0.24 0.37 0.39 0.11 0.28 0.05 Initial ester hydrolysis 0.16 0.06 0.24 0.08 0.03 0.11 0.03 % N-Demethylated product 94.2 30.6 83.2 37.0 53.9 59.8 12.53 undergoing ester hydrolysis % ester glucuronide of: pethidinic acid 20.7 33.3 57.1 56.3 4.8 34.4 9.0 norpethidinic acid 69.4 63.6 48.1 74.1 42.9 51.6 10.8 Table 8.8 Comparison of pethidine metabolism in human adults and neonates

Adult volunteers were given pethidine HC1 (50mg) by intramuscular injection and 0-24h urine analysed for pethidine metabolites by GC as described in the text. Neonates received pethidine as a result of placental transfer following maternal administration during delivery. All data is standardised to pethidine = 1; mean values N=15 (adults) or N=5 (neonates) are given.

Adult Neonate Adult/Neonate

Pethidine 1 1 1

Norpethidine 1.14 0.19 6.00 N Pethidine -N-oxide 0.10 0.003 33.33 tri

4'-Hydroxypethidine 0.17 0.001 170

Pethidinic acid free 0.92 0.13 7.10 conjugated 2.56 0.11 23.30 total 3.48 0.24 14.50

Norpethidinic acid free 0.76 0.14 5.43 conjugated 1.36 0.20 6.80 total 2.12 0.34 6.24

Total (pethidine + metabolites) 8.01 1.77 4.53

Total metabolites 7.01 0.77 9.10 Table 8.9 Differential ontogenesis of pethidine metabolism in man

Mean values are given, N=15 (adult) or N=5 (neonate)

Adult (A) Neonate (N) A/N

*Total N-demethylation 3.25 0.53 6.15

*Initial ester hydrolysis 3.48 0.24 14.50

*N-Oxidation 0.10 0.003 33.33

*Aromatic hydroxylation 0.17 0.001 170

% N-Demethylated product 66.32 64.15 1.03 undergoing ester hydrolysis

% ester glucuronide of: pethidinic acid 68.10 45.83 1.48 norpethidinic acid 64.85 58.82 1.09

* data standardised to pethidine=l 267

dose each baby received from the mother before birth. Since there was no way of knowing the exact amount of pethidine remaining in the baby at birth (although rough estimates in Chapter Four varied from 19.711g

- 6.5 mg) the results were standardised and expressed relative to the amount of pethidine excreted: i.e. pethidine - 1; norpethidine

0.19 etc. (Table 8.7). This enabled the metabolism of the five infants to be compared to each other as well as to the adult data.

Table 8.8 compares the metabolism of pethidine in adults and neonates.

The amount of each metabolite excreted by the babies was far lower

(relative to pethidine) than that found in adult urine, and the extent of N-demethylation and hydrolysis pathways, calculated as above, was also lower in the neonate. The total amount of all metabolites in neonatal urine accounted for 44% of the excreted material, i.e. the amount of pethidine excreted exceeded the amount of all the urinary metabolites, whereas in the adult, the metabolites accounted for 86% of the drug related material in the urine, i.e. the total of the metabolites exceeded the amount of pethidine excreted. By dividing the standardised adult data (A) by the standardised neonatal (N) data a ratio (A/N) indicative of the degree of impairment by the neonate to make each metabolite was obtained (Table 8.9). This ratio varied from 5.4 for free norpethidinic acid to >170 for 4'-hydroxypethidine.

The overall metabolism of pethidine in the neonate was impaired by a factor of 9.1.

A similar table was obtained for the impairment of the individual metabolic pathways (Table 8.9).

Discussion

The rate and extent to which a drug is metabolized and the chemical 268

nature of the products can be a major determinant of the therapeutic and toxic responses to many drugs. The metabolism of a drug may behighly var-

iable between individuals and this is frequently the source of major intersubject variation in response to drugs (Williams, 1967). Large interindividual variations in drug response can arise from multiple genetic, environmental and physiological factors affecting drug ab- sorbtion, distribution, biotransformation, excretion, interaction with receptor sites or combinations of these. Pethidine is an interesting compound to look at in respect of interindividual variations occurring as a result of qualitative and quantitative differences in metabolism since it is metabolized by five different metabolic pathways giving rise to eight metabolites (see Chapter Seven), all but one of which have been assayed by GC in this study.

The results for the adult panel study (Table 8.1) show that pethidine is metabolized in man along at least five different metabolic pathways, namely N-demethylation, hydrolysis of the ester link, N-oxidation, aromatic hydroxylation and glucuronic acid conjugation of pethidinic and norpethidinic acids. There was a large variation in both the dose recovered (range 12-69% of the dose in 24h urine) and quantitative differences in the metabolites formed. The two most important pathways from a quantitative point of view were N-demethylation and ester hydrolysis which give rise to norpethidine, norpethidinic and pethidinic acids.

The previous chapter showed that norpethidinic acid was largely the result of a secondary ester hydrolysis, that of norpethidine, and since these two metabolites along with pethidinic acid account for more than 269

90% of all the drug-related material excreted in 24h urine, it is

reasonable to suggest that the initial metabolites formed in the

body are largely norpethidinic and pethidinic acids:

i.e. PETHIDINE

initial ester N-demethylation hydrolysis

PETHIDINIC NORPETHIDINE ACID

secondary ester hydrolysis

NORPETHIDINIC ACID

When the extent of initial ester hydrolysis is plotted against N-

demethylation for each individual studied, a linear relationship is

observed (Fig. 8.2). Thus it appears that for the major two pathways,

N-demethylation and initial ester hydrolysis, compete for pethidine with one or other of the pathways dominating in most individuals.

A plot of extent of initial ester hydrolysis against extent of secondary ester hydrolysis also yields a linear relationship (Fig. 8.3), indicating that competition again exists, and this time the two substrates compete for the same enzyme. The above discussion implies that knowledge of the extent of any of the three major pathways, i.e. N-demethylation, initial and secondary ester hydrolysis, allows an estimation of the other two to be made in an individual.

There is considerable interest in whether the metabolism of a drug is under genetic or environmental control. Some of the interindividual 270

differences in the metabolism of drugs are due to environmental factors (Conney, 1967), but the metabolism of others have been clearly demonstrated to be genetically determined: e.g. N-acetylation of iso- niazid is under genetic control in man (Evans, Manley and McKusick,

1960). Often, the results from a population study (e.g. elimination half-life; % dose administered undergoing a particular metabolic pathway) are expressed in the form of a frequency distribution histogram or curve. Where such a frequency distribution has more than one mode, then each mode may represent a genetic phenotype and the measured parameter is under the control of a single gene, i.e. polymorphic control. On the other hand when a single mode only is observed, the drug parameter is normally under the control of several genes and this is known as polygenic control. In an attempt to discover whether the individual metabolic pathways of pethidine are under polygenic control, the extent of each metabolic pathway was plotted as a frequency distribution curve using the results obtained from the panel study. The curves obtained (Fig. 8.1) all tended towards normal unimodal distri- butions, but the small numbers used in the study (N = 15) do not allow a valid conclusion to be made on the genetic control of pethidine metabolism.

Significant differences in pethidine metabolism were observed in four selected individuals (RLS, EMH, LJN, JC) when each was readministered with the same dose of pethidine as used in the panel study. Table

8.1 shows the qualitative and quantitative aspects of pethidine metabolism in these volunteers following the initial intramuscular injection of pethidine EC1 (50mg), and Table 8.5 shows the results obtained with an identical dose administered some 6-8 months later

In all four subjects, both the dose and the relative proportions of the

Fig. 8.1 Frequency distribution curves for metabolic pathways of pethidine in a panel of adult volunteers

4 _ 4 4 •

3 3

2 • 2 -

1 1

1 1 I 1 I 1 I 1111111 1 I 1 I I I I I I I 1 0.1 0.3 0.5 0.7 0.1 0.3 0.5 0.7 0.1 0.3 0.5 0.7 0.9

i) initial ester hydrolysis ii) N-demethylation iii) conjugation of pethidinic acid

4 - 4 -

3 3 - •

2 2 _

1 •

I 1 I I 1 1 I I 1 1 0,2 0.4 0.6 0.8 1.0

iv) secondary ester hydrolysis v) ester hydrolysis of norpethidine vi) conjugation of norpethidinic acid Fig.8.2 Relationship between initial ester hydrolysis and N-demethylation of pethidine in a panel of adult volunteers

0.7' • •

0.8-

•

is s •

ly 0.5 • • dro hy

ter s 0.4- l e ia t i in

f • 0.3- o t • ten • Ex

0.2 I 1 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Extent of N-demethylation Fig. 8.3 Relationship between initial and secondary ester hydrolysis of pethidine in a panel of adult volunteers

0.7 • •

0.6 -

• is s 0.5 - ly • • dro • hy ter 0.4 - l es ia it in f 0.3 - • o t ten • • • Ex

0,2 I I I 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Extent of secondary ester hydrolysis 274

urinary metabolites recovered varied considerably between the two

administrations. Numerous environmental factors, e.g. diet, stress,

temperature, disease and time of administration (time of day, season)

are known to affect the basal, genetically controlled level of drug

metabolism. For example, Vessell, Shively and Passananti (1971) have

shown that temporal variations in antipyrine half-life, ranging from

an increase of 173% to a decrease of 42% occur in man in a 12h period,

and diurnal variations in the activity of hepatic microsomal hexobarbital

oxidase in the rat have been found (Chedid and Nair, 1972). Alterations

in the metabolism of drugs due to changes in diet (Kappas, Anderson,

Conney and Alvares, 1976) and following exposure to environmental chemicals

(Kolmodin, Azarnoff and SjSgvist,1969) have also been reported in man.

Hence, although the number of volunteers in the panel study was too

small an assessment of the contribution of specific, single environmental

factors to the total interindividual variation in pethidine metabolism

to be made, a reevaluation of pethidine metabolism in four individuals,

following a large time interval when their 'environment' might be expected

to alter, has shown that environmental conditions may substantially

affect the metabolism of pethidine.

The most common Phase I reaction in the metabolism of drugs is oxidation at a carbon centre which may be an aliphatic, alicyclic or aromatic carbon. Work in our laboratory has reported the occurrence in Caucasians of a polymorphism in respect of carbon oxidation of the antihypertensive drug debrisoquine, where about 5% of the population have a reduced ability to effect the alicyclic hydroxylation of this drug. Apparently this

4'-hydroxylation of debrisoquine is under the control of alleles at a single autosomal locus and defective alicyclic hydroxylation is a 275

recessive characteristic (Mahgoub, Idle, Dring, Lancaster and Smith,

1977). Further work has shown that the genetically determined

hydroxylation polymorphism described for debrisoquine also controls

the oxidative metabolism of guanoxan and phenacetin (Sloan, Mahgoub,

Lancaster, Idle and Smith, 1978).. Thus, the same genetic polymorphism appears to influence metabolic processes at such dissimilar carbon atoms as the aromatic 6- and 7-carbon atom of guanoxan, the alicyclic 4-carbon atom of debrisoquine and the aliphatic a-carbon atom of the ethyl residue of phenacetin. Hence the defect apparently influences some component common to the processes concerned in the oxidation of several chemically dissimilar carbon centres. It was thought therefore conceivable that the N-demethylation pathway of pethidine may also be affected since this involves an initial oxidative attack on the carbon atom of the methyl group. To investigate this possibility, the rate of formation of pethidine metabolites, particularly norpethidine and norpethidinic acid ,were studied in

4 volunteers, 2 of whom were phenotyped as extensive metabolizers

(EM) and 2 as poor metabolizers (PM) in respect of debrisoquine-4- hydroxylation, 6- and 7-hydroxylation of guanoxan and the a-carbon oxidation of phenacetin. The results given in Table 8.5 showed that there were no significant differences in the rate of any metabolic pathway of pethidine, including N-demethylation, in the two phenotypically poor metabolizers as compared to the two phenotypically extensive metabolizers. This suggests that the large gene effect controlling the defective hydroxylation observed for debrisoquine, phenacetin and guanoxan is probably not operating in the N-demethylation pathway of pethidine. 276

Since pethidine is excreted principally in the form of metabolites

by adults, it is tempting to speculate that the prolonged blood

elimination half-life seen in babies compared with adults (Chapter

Four), is due to impaired metabolic ability in the newborn. To

investigate this possibility, pethidine together with all of its

known metabolites were assayed in the O-24h urines of 5 babies whose

mothers received pethidine during labour, and the results compared with

those from normal adults. It is thought that the metabolites found

in neonatal urine (Table 8.6) are true neonatal metabolites and not

maternal metabolites that had crossed the placenta before birth.

Analysis of maternal venous and umbilical venous and arterial blood

for all known metabolites showed that norpethidinic acid was the

only metabolite present in the umbilical circulation, and therefore

the other metabolites at least are almost certainly of neonatal

origin. The difference in the amount excreted (i.e. pethidine and

metabolites) in the babies was attributable to variation in body

load at birth and elimination by the kidney of the unchanged drug

and/or metabolites.

The metabolites of pethidine in neonatal urine were expressed rela-

tive to the amount of pethidine excreted in order to compare metabolism

between the infants. In all five babies pethidine was the major ex-

cretion product (Table 8.6). Table 8.8 compares the mean standardised

data for adults and neonates. 14% of the excreted material (pethidine

and all metabolites) in O-24h adult urine was unchanged pethidine

whereas in the neonate 56% of the excreted products was the unchanged drug. The relative amount of each metabolite excreted was greatly reduced in the neonate compared to the adult showing that the infant 277

is deficient in drug metabolizing ability. The degree of metabolic

impairment by the neonate was estimated by dividing the standardised

adult data by the standardised neonatal data (Tables 8.8 and 8.9).

This ratio was extremely variable for the various metabolites/

metabolic pathways; e.g. the mean metabolic impairment ratio for

N-demethylation was 6.2; for N-oxidation 33.3; for aromatic

hydroxylation >170 etc. The overall metabolism of pethidine in the

neonate was impaired by a factor of 9.1. This data would support

the idea that all five metabolic pathways of pethidine develop at

different rates in neonates, and that the three oxidative reactions

which this drug may undergo area carried out by different forms of

the mixed function oxidase system. Further evidence for this

conclusion is provided by a study of pethidine metabolism in the

panel of adult volunteers which also shows the independence of the

individual reactions. Differential ontogenesis of drug metabolizing

enzymes have been previously reported for animals. Atlas, Boobis,

Felton, Thorgeirrsson and Nebert (1977) have reported evidence for

at least 3 forms of cytochrome P-450 in rabbit liver each of which

is under different genetic control and develops at different rates.

However, so far no reports on differential ontogenesis of drug

metabolizing enzymes in humans have been reported in the literature,

and this study provides good, but indirect, evidence that this does

in fact occur.

The overall metabolic impairment of pethidine in the neonate was

estimated at 4.5 and the mean prolongation of blood elimination half- life in the neonate was 6.1. These two values, metabolic impairment and increase in half-life are very similar and although this is 278

probably fortuitous, the fact that they are of the same order of magnitude allows us to conclude that in the case of pethidine, one of the main reasons for the prolongation of half-life in the neonate is impaired metabolism. 279

Chapter Nine Species variation in the metabolism of pethidine

Page

Introduction 280

Analysis of Urinary Metabolites 280

Non-human primates 280

Sub-primate mammals 281

Results 282

Non-human primates 282

Sub-primate mammals. 286

Discussion 289 280

Introduction

It is now widely recognised that species may differ in the way they

metabolize drugs both in respect of rates and routes of metabolism

(Parke and Smith, 1977). Such inter-species differences in metabolism

are a source of major problems in attempting to extrapolate animal

pharmacological and toxicological findings to the human situation.

The most commonly used animals for the pharmacological and toxicological

testing of new drugs are rats, guinea pigs, rabbits, cats and dogs, for

both economic reasons and because of the wealth of data already available on them. However, in recent years interest has focussed on the possibility that non-human primate species may provide more adequate metabolic models for the human situation than non-primate species since they are nearest to man in the evolutionary 'tree'. In a recent review it was demonstrated that in the majority of the cases considered this was so, although not invariably the case (Smith and Caldwell, 1976).

This chapter presents a study of the species variation in the metabolism of pethidine in order to compare the suitability of both sub-primate mammals and non-human primates as metabolic models for man in this instance.

Analysis of Urinary Metabolites

Non-human primates,

Four species of Old World Monkeys (vervet, mona monkey, patas monkey and cherry-crowned mangabey) housed in the Primate Unit of the Depart- ment of Biochemistry, University of Ibadan, Nigeria, were dosed with pethidine HCl (5 mg/kg) dissolved in sterile isotonic saline (lml) by intramuscular injection in the thigh muscle. Urine was collected for

24h into a tray containing 2% HgC12 solution as preservative, filtered 281

and frozen prior to transport by air to London for analysis. Mercuric

ions were removed from the bulked 24h urines by precipitation with

1OM-NaOH, this centrifuged and the supernatant subjected to a pre-

liminary purification by an Amberlite XAD-2 resin column. The

resulting purified urine was then taken through the sequential extraction

procedure outlined previously for adult urine, and all metabolites

assayed by GC as described in Chapter Three.

Sub-primate mammals 14 C C3-Pethidine HC1 dissolved in sterile isotonic saline was administered to rats (30mg/kg; 15pCi/kg); guinea pigs (30mg/kg; 15jCi/kg) and rabbit (20mg/kg; 6.4pCi) by i.p. injection and excreta collected for three days as described in Chapter Three. The 24h urine from each animal was concentrated using an Amberlite XAD-2 resin column which gave a recovery of >95% in each case, and subjected to radioTLC in system G when qualitative similarities were observed. In all three species four radioactive peaks were found whose centres had Rf 0.07,

0.42, 0.50 and 0.68. Prior treatment of the urines with HCl or S- glucuronidase as described in Chapter Three caused the peaks at Rf

0.07 to disappear with an equivalent increase in the peaks at Rf 0.42.

The peaks at Rf 0.42 and 0.50 correspond to pethidine acid and pethidine-

N-oxide respectively. Both pethidine and 4'-hydroxypethidine have very similar Rf values in this system which correspond to the peak at

Rf 0.68 on the chromatograms. Chromatography of the acid treated urines in system B again gave the same qualitative results for the three species but the 14C content of the peaks varied between the species.

Four 14C peaks of Rf 0.02, 0.22, 0.35 and 0.47 were found which correspond in Rf to pethidinic acid, pethidine=N-oxide, 4'-hydroxypethidine and 282

pethidine respectively (Fig. 9.1).

The 24h urine of each species was subjected to the sequential extraction

procedure outlined in Chapter Three and each fraction examined by TLC,

GC and GC-MS. Each metabolite was subsequently assayed by GC as

described in Chapter Three.

The rats and guinea pig were sacrificed by cervical dislocation 72h

following administration of pethidine,and the carcasses digested in

20% NaOH in 30% ethanol. Aliquots were subsequently bleached and

counted for 14C as described in Chapter Three.

Results

Non-human primates

The qualitative and quantitative aspects of the urinary excretion of

pethidine and its metabolites in four non-human primate species are

shown in Table 9.1. The overall recovery of the administered drug

in the 0-24h urine, as shown by the total of unchanged drug and its

metabolites, was reasonably good ("60% of the dose) for the vervets;

moderate (30-40%) for the mona monkey and mangabey, and poor (15%) in the case of the patas monkey. The poor urinary recoveries exper- ienced with the patas and mona monkeys and mangabey are probably attributable to the difficulties in obtaining a complete urinary collection under the experimental conditions used.

In all four species, six urinary metabolites were identified and quantitated in addition to the parent drug, namely, pethidinic acid and norpethidinic acid and their glucuronic acid conjugates, norpethidine 283

Fig. 9.1 Radiochromatograms of acid treated urines following administration of 14C-pethidine HC1 to sub-primate mammals developed in system B

GUINEA PIG (30mg/kg; 15 Ci/kg) 0.35

0.47

1 origin solvent front

RAT (30mg/kg; 15 Ci/kg)

origin solvent front

RABBIT (20mg/kg; 6.4 Ci)

0.35

0.22 0.02 0.47

origin solvent front

TABLE 9.1 Metabolism of pethidine in four non-human primates

0-24h urines of monkeys dosed with pethidine hydrochloride (5mg/kg i.m.) were analysed for pethidine and metabolites by G.C. methods as described in the text. Results are expressed as % dose recovered in that form.

Vervet Patas monkey Mona monkey Mangabey

Pethidine 21.9, 25.6 3.7, 1.8 7.8 2.4 Norpethidine 1.1, 11.8 3.2, 3.0 12.8 2.6 4'-Hydroxypethidine <1, <1 n.d., n.d. n. d. n. d. Pethidine N-oxide 0.3, 0.8 0.4, 0.6 0.3 0.1 Pethidinic acid free 1.1, 1.1 0.2, 0.3 0.5 1.5 glucuronide 6.1, 14.7 1.7, 1.4 3.3 6.5 Norpethidinic acid free 3.3, 1.5 0.4, 6.3 3.8 3.0 glucuronide 19.4, 16.3 4.1, 5.5 14.3 14.1 Total of metabolites 54.2, 72.8 13.7, 18.9 42.8 30.2

n.d. not detected 285

and pethidine-N-oxide. The relative amounts of unchanged pethidine

and metabolites excreted varied between the four monkey species.

Unchanged pethidine accounted for a much larger proportion (40%) of

the total drug excretion in the vervets than in the mangabey where it

accounted for only 18%.

The major urinary metabolite in all four species was norpethidinic

acid which was excreted partly free but largely in a conjugated form.

The conjugate is a glucuronic acid conjugate as indicated by the fact

that it is hydrolysed on treatment with a-glucuronidase to afford

norpethidinic acid, and it is probably the ester glucuronic acid

conjugate of this acid. Treatment of the urine with 10M-HC1 did not

release any more of the acid from its conjugates than did S-glucuronidase.

With the exception of norpethidinic acid excretion, major species

differences occurred in respect of the urinary excretion of the other

metabolites of pethidine. In the mona monkey and patas monkey, the

second major metabolite was norpethidine, whereas for the vervet and

mangabey this was pethidinic acid. The latter was excreted, like

norpethidinic acid, both free but largely in a conjugated form which

was thought to be an ester of glucuronic acid because it yielded

pethidinic acid after treatment with R-glucuronidase. Again, concen-

trated HC1 treatment did not cause the appearance of any more of the

acid than did S-glucuronidase. Pethidine-N-oxide was also detected

in the urine of all four species but the amount was small (<1% of dose). 4'-hydroxypethidine, a product of the aromatic hydroxylation of pethidine, was detected in the urine of the vervet (<1% of dose). 286

Sub-primate mammals

The elimination of 14C in the urine, faeces and expired air of the rats

and guinea pigs after the administration of [14 CI-pethidine HC1 is

given in Table 9.2. Some 66% of the dose was recovered in 3 days;

56% in urine, 3% in faeces and 7% in expired air (in the form of 14CO2).

The majority of the activity (60%) was excreted in the first 24h.

The rabbit excreted 43.6% of the dose in the same 3 day period; 43%

in urine, 0.6% in faeces, and once again most of this activity (37%)

was recovered in the first 24h. The excretion of 14CO2 was not

estimated for the rabbit.

The rodents were killed after 72h and their carcasses digested and analysed for 14C, as described in Chapter Three, when 22% of the dose 14 was recovered. Thus the total C recovered from the rats and guinea pigs was about 90% of the administered dose. The amount of radioactivity remaining in the rabbit carcasses after 3 days was not estimated.

The qualitative and quantitative aspects of the urinary metabolites of pethidine in rat, guinea pig and rabbit are given in Table 9.3. In each case the major urinary metabolite was pethidinic acid which was excreted partly free but mainly as its glucuronic acid conjugate, but apart from this, major species differences occurred in respect of the other urinary metabolites of pethidine. In the rat and guinea pig the second major urinary metabolite was norpethidine whereas in the rabbit it was norpethidinic acid, the rabbit being the more extensive convertor of norpethidine to norpethidinic acid. Pethidine-N-oxide was excreted in very small amounts in all three species (<1. of the dose), and urinary 4'-hydroxypethidine accounted for about 6% of the 14C TABLE 9 .2 The elimination of by the rat, guinea pig and rabbit after administration of [14C]-pethidine hydrochloride

[14c] -Pethidinehydrochloride in normal saline was administered to rats (30mg/kg; 1511Ci/kg); guinea pigs (30mg/kg; 15uCi/kg) and rabbit (20mg/kg; 6.4211,Ci) by i.p. injection. Values given are % of 14C dose in excreta; ;ndividual figures for rabbit and rat, average of 3 guinea pigs with ranges in parentheses.

Rat Guinea Pig Rabbit

In urine on day 1 53.1, 44.7 55.6 (51.3 - 64.1) 37.1 2 2.9, 6.0 1.3(1.2 - 1.3) 4.4 3 1.8, 0.9 0.4(0.3 - 0.5) 1.3 Total 57.8, 51.6 56.7(51.3 - 65.7) 42.8

In faeces on day 1 2.9, 3.5 1.8(15. - 2.9) 0.3 2 0.7, 1.0 0.6(0.5 - 0.8) 0.3 3 0.1, 0.5 0.2(0.1 - 0.3) 0.1 Total 2.7, 5.0 2.4(1.5 - 3.7) 0.6

In expired air on day 1 7.4, 6.3 6.6(4.1 - 8.5) 0.3, 0.7(0.5 - 0.8) 2 0.4 not 3 0.1, 0.2 0.2(0.1 - 0.3) determined Total 7.8, 6.9 7.1(4.7 - 9.6)

Total excretion 69.3, 63,5 66.1(5919 - 74.3) 44.1

14C recovered from carcass 22.3, 27.2 22.4(15.9 - 27.3) not determined TABLE 9.3 The metabolites of [14C]-pethidine in rat, rabbit and guinea pig urine

11 [ ~C]-Pethidine hydrochloride in normal saline was administered to rats (30mg/kg; 1530i/kg); guinea pigs (30mg/kg; 15p.Ci/kg) and rabbit (20mg/kg; 6.42p.Ci) by i.p. injection. The first 24h urine was analysed as described in the text, and the results are expresses as % of dose administered.

Metabolite Sought Rat Guinea Pig Rabbit

*Pethidine 1.9, 2.9 7.6(5.2 - 10.0) 14.1 Norpethidine 9.4, 15.5 14.1(8.9 - 22.8) 3.7 *Pethidine N-oxide 0.3, 0.4 0.5(0.1 - 1.0) 0.1 *Pethidinic acid free 2.9, 4.6 4.1(2.3 - 5.4) 2.4 conjugated 30.9, 33.6 35.2(27.8 - 39.6) 15.8 total 33.8, 38.2 39.3(30.1 - 43.7) 18.2 Norpethidinic acid free 0.6, 0.3 1.2(0.3 - 2.9) 3.4 conjugated 5.3, 3.3 9.8(7.9 - 12.4) 13.6 total 5.9, 3.6 11.1(8.4 - 15.3) 17.0 *4'-Hydroxypethidine 8.3, 7.2 4.7(4.0 - 5.1) 7.2

Total of above metabolites 59.6, 67.8 72.5 (66.8 - 80.4) 60.3 Total 14C in urine examined 44.7, 53.1 55.6(51.3 - 64.1) 37.1 Total i4C metabolites 44.3, 48.7 47.4(35.7 - 54.7) 39.6

*E14C]-metabolites 289

dose. The amount of unchanged pethidine excreted in 24h urine

varied from 2% for the rat to 14% for the rabbit.

Discussion

The findings show that pethidine can be metabolised along at least

five different metabolic pathways namely, N-demethylation hydrolysis

of the ester link, N-oxidation, aromatic hydroxylation and conjugation

of pethidinic and norpethidinic acid with glucuronic acid. In all

species a large amount of the dose remained unaccounted for in 24h

urine, and the 14C animal studies showed that the remaining activity

was not excreted after this period. Subsequent digestion of the rodent

carcasses showed the 14C label to be contained within the tissues,

although the location and molecular form of the label was not ascertained.

The 14C content of the 24h urine in rodents correlated well with the 14 total of the 14C metabolites assayed by GC, but the CO2 recovered

from the rodents did not approach the amount of N-demethylated meta-

bolites (i.e. norpethidine and norpethidinic acid) excreted. This

suggests that at least some of the 14C from the N-methyl group was incorporated into general anabolic metabolism, which is substantiated

by the presence of the label in the guinea-pig and rat carcasses.

Fig. 9.2 presents comparative data for the metabolism of pethidine in four non-human primates and three sub-primate mammals together with the mean result for the adults previously studied. It should be borne in mind that the results for the mona monkey, mangabey and rabbit are for single animals only, those for the vervet, patas monkey and rat are for two of each species, and those for the guinea pig for three animals. The human data represents the mean values for fifteen volunteers. Assuming that the results for the animals studied are

Fig. 9.2 Comparative data for pethidine metabolism

GUINEA PIG RABBIT 60 - MAN RAT

l as ia 40 ter d ma te 20 - cre x ❑ ❑ % e 0 ❑ ❑ ❑

0— ❑ 0 ❑ ❑

60 -

VER VET MONA MONKEY PATHS MONKEY MANGABEY 40

20 — ❑ 0 ❑ ❑ ❑ ❑ ❑ ❑ 0 —

❑ ❑ Pethidine Norpethidine Pethidinic acid ❑ ❑ Norpethidinic acid 291

representative of the species, then it can be seen that considerable species differences in metabolism exist in respect of the relative importance of the various metabolic pathways involved in pethidine disposition. The main differences which emerged from this study are:-

(i) Pethidine is least metabolized in the vervet, and undergoes

extensive metabolism in rodents anti in the cherry-crowned mangabey.

(ii)The extent of total N-demethylation is low in all the sub-primate

mammals and in man and the vervet. The patas and mona monkeys

and mangabey were fairly extensive N-demethylators, although

the form of the excretion product, i.e. norpethidine or norpeth-

idinic acid, varied between the species.

(iii)The extent of aromatic hydroxylation was greatest in the sub-primate

mammals.

(iv) The sub-primate mammals appeared to be poor N-demethylators

compared to the primate species.

The results from this study show that significant species variation in the metabolism of pethidine exist. Species variation in drug metabolism can occur both in respect of the extent at which metabolism occurs (quantitative differences) and in the metabolic pathways employed (qualitative differences). These differences in the pattern of metabolism commonly arise because of one or more of the following reactions:

(a) competing reactions where a compound may be metabolized along

two or more competing pathways but the importance of the in-

dividual pathways may vary with species. In this instance the

same metabolites are formed but the relative amounts vary with

species. 292

(b) the occurrence of metabolic defects where some species may have

relative defects in their capacity to carry out certain metabolic

reactions, e.g. the inability of the cat to conjugate a wide

range of compounds with glucuronic acid and the pig to conjugate

with sulphate.

in primate species, phenylacetic acid is largely conjugated with

glutamine whereas in sub-primates glycine is utilized; and 3,5-

diiodo-4-hydroxybenzoic acid is 0-methylated to the corresponding

4-methoxy derivative in man, the rhesus, cynomolgus, squirrel

and capuchin monkey, but not in the rat and rabbit.

Species variation in the metabolism of pethidine is due to quantitative differences in competing reactions since' pethidine underwent the same metabolic reactions in all species but the relative proportions of the metabolites formed varied. The two major competing reactions appeared to be ester hydrolysis and N-demethylation for the formation of pethidinic acid or norpethidine respectively, assuming that, like humans, norpethidinic acid is the result of a secondary pathway due to further metabolism of norpethidine. N-oxidation was apparently a minor metabolic pathway in all species examined and as mentioned in Chapter Seven, N-oxides can be further metabolized by dealkylation and/or reduction. However, since the amount of N-oxide excreted was consistently low in all animals studied, it appears that N-oxide formation of pethidine is probably a minor pathway.

The interest in species variation in metabolism has largely stemmed from the stringent safety evaluation of new drugs that is now necessary, and a comparative study of metabolism could be of value for suggesting 293

a species likely to be useful for comparing the activity of a new

drug with that in man. The testing of new chemicals of potential

economic value is initially carried out in animals lower down the

evolutionary scale than man, with sub-primate mammals being used

extensively. In recent years interest has focussed on the possibility

that non-human primate species may provide more adequate models for

the human situation, and while this has been shown to be so in the

majority of cases, it is not invariably the case (Smith and Caldwell,

1977). Furthermore, such studies have also indicated that although only a dozen non-human primate species of the 200 or so available have

been examined there exists significant inter-primate species differ-

ences in metabolism. Often, studies using non-human primates fail to state the species and refer only to 'monkey' and this study has shown that not all the 'monkeys' used were suitable metabolic models for man in the case of pethidine. Fig. 9.2 shows that there is no really adequate model for pethidine disposition in man, with the most suitable (for the population mean) being the guinea-pig, and the mangabey and rabbit being less acceptable. The vervet, mona and patas monkey were found to be unsuitable. However, this discussion has implied that there exists no metabolic variation in man, and that one animal model can represent a whole 'population'. However, the previous chapter has shown that there exists a considerable interindividual variation in the metabolism of pethidine in man. Thus it poses the question of which animal model is best representative of the population or even of an individual, and therefore only general conclusions can normally be made in extrapolating animal data to man. 294

APPENDIX A

Dioxan scintillant

600g napthalene

40g 2,5-diphenyloxazole (PPO)

2g 1,4-bis E2-(5-phenyloxazoylj-benzene (POPOP)

1000m1 methanol

200m1 ethanediol made up to 10 litres with dioxan

Triton X-100 toluene scintillant

3.33 1 triton X-100

6.66 1 toluene

55g PPO

lg POPOP Appendix B(1) Maternal, umbilical and neonatal blood concentrations of bupivacaine; neonatal blood elimination half-life; maternal dose and last dose-delivery interval for all individuals in the bupivacaine study

No. Maternal Dose- Maternal Umbilical Umbilical neonatal blood conc. half- dose delivery vein conc. vein conc. artery conc. 2h 24h 36h 48h life (mg) interval (ng/ml) (ng/ml) (ng/ml) (hours) (min) (ng/ml)

1 68 107 200 50 64 57 37 20 7 6.5 2 188 41 120 65 NS 59 9 8 4 3.2 3 83 172 125 57 NS 52 5 2 ND 7.0 4 45 80 142 42 108 61 35 18 4 12.5 5 75 105 305 95 85 53 NS NS NS 6 83 238 180 55 30 27 NS 4 NS 12.0 7 188 150 325 40 30 28 16 NS 2 11.8 8 75 165 245 10 40 20 7 ND ND 14.3 9 45 70 120 30 27 13 1 ND NS 5.9 10 45 155 150 40 60 NS 3 ND ND 11 45 149 125 30 55 28 13 8 5 17.7 12 53 73 200 30 65 23 12 7 ND 20.1 13 75 277 150 15 40 17 9 1 ND 9.4 14 83 172 164 NS NS 26 15 5 1 9.7 15 75 90 239 32 NS 28 5 NS ND 9.1 16 110 295 65 102 32 23 7 ND 16.7 17 143 63 NS 36 14 ,4 ND ND 10.4 18 80 485 175 160 NS 12 7 7 30.1 19 94 165 255 115 105 39 29 8 3 11.6 20 268 25 NS 205 140 108 21 2 NS 6.4 No. Maternal Dose- Maternal vein Umbilical Umbilical neonatal blood conc. half- dose delivery vein conc. vein conc, artery conc. 2h 24h 36h 48h life (mg) interval (ng/ml) (min) (ng/ml) (ng/ml) (ng/ml) (hours)

21 98 125 260 175 130 69 25 3 ND 7.7 22 105 463 355 97 105 36 32 30 7 17.7 23 60 90 210 77 105 52 13 NS ND 11.2 24 139 20 365 65 27 9 3 NS NS 15.1 25 110 158 89 50 37 14 5 ND 11.2 26 65 108 67 75 48 9 1 ND 6.7 27 56 123 115 65 30 15 2 ND ND 6.7 28 113 195 260 77 77 NS 3 ND ND 29 94 50 440 90 75 28 13 2 ND 9.1 30 150 130 153 NS 95 25 12 5 ND 15.1 31 38 168 155 75 NS 36 NS 4 NS 10,8 32 150 158 130 63 NS 53 19 10 ND 13.7 33 45 58 220 80 55 26 3 NS NS 7.5 34 158 135 240 60 45 23 14 10 6 25.1 35 900 93 215 NS NS 47 27 15 7 16.7 36 45 120 115 58 50 44 24 17 11 23.2 37 65 238 77 65 47 38 24 8 20.1 38 65 182 55 62 21 9 4 ND 15.1 39 20 346 102 103 53 39 23 12 21.5 40 75 65 205 55 53 NS 14 7 ND 11.6

41 30 80 160 48 84 NS NS NS NS No. Maternal Dose- Maternal Umbilical Umbilical neonatal blood cone. half-life dose delivery vein conc. vein conc. artery conc. 2h 24h 36h 48h (h) ( mg) interval (ng/ml) (ng/ml) (ng/ml) (ng/m1) (min)

42 113 320 80 62 40 16 NS NS 16.0 43 135 109 72 72 66 32 NS NS 18.0 44 83 11 NS 83 61 60 NS 48 29 50.2 45 101 113 270 94 79 59 20 NS NS 13.7 46 150 120 375 116 91 85 25 NS NS 12.0 47 38 197 275 125 93 72 58, NS NS 23.2 48 90 155 52 77 NS NS NS NS 49 195 342 116 96 30 NS NS NS 12.6 50 188 430 131 150 48 17 9 NS 16.0 51 179 195 103 71 44 20 12 NS 21.4 52 263 485 274 323 198 34 NS NS 9.8 53 120 340 92 53 38 11 1 ND 12.6 54 180 325 102 104 85 5 NS NS 7.0 55 90 205 50 55 37 NS NS NS 6.0 56 75 140 72 61 56 5 NS NS 5.2 57 NS NS NS NS NS NS NS 58 NS NS NS NS NS NS NS

NS sample not supplied; ND bupivacaine not detected in sample; Appendix B(2) Maternal, umbilical and neonatal blood concentrations of pethidine; neonatal blood elimination half-life; maternal dose and last dose-delivery interval for all individuals in the pethidine study

No. Maternal Dose- Maternal Umbilical Umbilical Neonatal blood concentration half-life(h) dose delivery vein conc. vein conc. artery conc. 2h 6h 24h 36h (mg) interval (ng/ml) (ng/ml) (ng/ml) (ng/ml) (min) ( or other specified time )

1 100 112 100 125 125 152 32(7.5h) NS NS 3.4 2 100 60 515 265 82 53 37(7.5) NS NS 7.0 3 100 202 87 135 137 90 NS 62 NS 28.8 4 300 318 215 195 405 135 92(6.5) NS NS 3.2 5 100 45 405 260 205 NS NS 62 NS 13.9 6 450 145 640 390 405 231 77 54 NS 9.7 7 150 130 200 95 98 298 67 30 NS 10.1 8 150 60 368 328 128 145 131 NS NS 17.6 9 100 67 110 60 90 NS NS NS NS 10 200 127 115 70 95 89 62(8.5) NS NS 13.9 11 150 157 65 28 38 NS NS 24 NS 36.1 12 250 125 235 200 115 110(2.5)NS 31(25.5) NS 13.1 13 400 146 312 307 415 194 NS 42 NS 8.5 14 100 105 255 155 170 NS NS NS NS 15 100 122 349 20 85 22 NS 20 NS 18.7 16 150 305 101 91 150 158 NS 85 NS 26.9 17 150 155 270 225 260 NS NS 39 22 9.8 18 100 25 530 310 240 NS NS 110 60 14.0 No. Maternal Dose- Maternal Umbilical Umbilical Neonatal blood cone. half-life (h) dose delivery vein cone. vein cone. artery cone. 2h 6h 24h 36h (mg) interval (ng/ml) (ng/ml) (ng/ml) (ng/ml) (min)

19 150 82 325 317 292 250 NS 170 130 30.0 20 100 98 167 103 82 76 NS 54 NS 22.7 21 100 80 260 367 314 320 NS 120 61 15.0 22 150 292 203 183 264 244 NS 140 80 27.0 23 150 211 290 273 300 276 NS 65 45 12.0 24 100 98 270 223 344 NS NS NS NS 25 325 165 430 378 500 450 NS 150 90 15.0 26 100 320 575 360 NS 385 NS 116 92 20.0 27 300 310 92 315 NS 300 NS 200 134 34.0 28 150 75 271 129 NS 221 NS 23 NS 6.8 29 150 67 NS 435 540 350 NS 75 45 10.0 30 100 330 255 270 220 172 NS 73 NS 24.9 31 300 480 153 266 319 NS NS NS NS 32 150 166 236 225 214 159 NS 53 NS 12.8 33 150 200 81 87 100 77 NS 66 35 29.5 34 100 93 151 107 146 129 NS 23 NS 8.9 35 100 152 243 113 113 57 NS 28 NS 19.7 36 100 50 199 135 136 117 NS 23 NS 9.6 37 150 306 131 256 421 NS NS 69 NS 9.5 38 150 472 153 92 NS 153 NS 57 NS 15.1 39 100 27 382 420 223 337 NS 323(12h) NS 43.0 40 150 47 NS NS NS 242 NS NS NS No. Maternal Dose- Maternal Umbilical Umbilical Neonatal blood concentration Half- dose delivery vein cone. vein cone., artery cone. 2h 6h 24h 36h life (h) (mg) interval (ng/ml) (ng/ml) (ng/ml) (ng/ml) (min)

41 150 220 NS NS NS 167 113 79 NS 23.8 42 100 275 NS NS NS 107 NS 29(25h) NS 12.1 43 250 102 NS NS NS 161 161 82 NS 21.0 44 100 105 NS NS NS 124 87 67 NS 26.5 45 100 NS NS NS 338 NS 200 150(48h) 38.0

46 - 51 no data available/samples not supplied

w 0 0

NS sample not supplied; ND pethidine not detected in sample

301

APPENDIX C Estimation of total drug exposure of the fetus/neonate

to maternally administered pethidine or bupivacaine

To estimate the total drug exposure to maternally administered drugs one needs to calculate;

(i) drug exposure of the fetus in utero

(ii) drug exposure of the neonate

Neonatal drug exposure may be calculated from the area under the curve, blood concentration against time, for each individual baby.

Fetal drug exposure however, cannot be calculated on an individual basis since the only indication of drug concentration in any one fetus is the concentration in umbilical blood at birth. Therefore an estimate of fetal blood concentration against time was obtained by plotting UA concentration against dose-delivery interval for each baby_ in the group to which a regression line (of slope M) was fitted. The UA concentration and dose-delivery interval are known for each infant and the fetal drug exposure is thus calculated from the area under the fitted regression line of slope M against dose-delivery interval, adjusting the position of the line to the UA concentration for each baby (see below)

[UA] "fitted" regression line slope M

} Maternal Birth Time (h) Administration 302

APPENDIX D Estimation of rate constants for the elimination of pethidine and

metabolites

Rate constant for formation of K x infinite recovery of metabolite x metabolite x total infinite recovery (k ) x where k is the overall elimination rate constant of pethidine being the slope of the plot: log rate of excretion against time, i.e. e

loge rate of slope = K excretion

Tf TIME (h) (last collection period)

Infinite recovery of metabolite x = amount actually recovered + estimated recovery from T -> For a rate plot, the amount excreted is given by the area under the curve, and the amount excreted from T } = is given by rate at time T k 303 REFERENCES

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