Mechanisms of Interaction Between Anticholinesterases

MECHANISMS OF INTERACTION BETWEEN ANTICHOLINESTERASES

AND OPIOIDS UPON RODENT NOCICEPTION

Paul GREEN, BSc (Hons)., MSc

A thesis submitted in accordance with the requirements of the University of Surrey for the degree of Doctor of Philosophy

Department of Biochemistry November, 1985 University of Surrey Guildford, Surrey ProQuest Number: 10798494

INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest

ProQuest 10798494

Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author.

ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ACKNOWLEDGEMENTS

I am very grateful to my supervisor, Dr Ian Kitchen, who has given invaluable

advice and encouragement throughout my doctoral studies.

I would like to thank my mum and dad, brothers and Julie for all their help

and support.

I would also like to thank Mr Paul Bishop (Biochemistry Workshop) for the

skilled construction of locomotor activity monitor and the hot-plate, Dr John

Shorter (Beecham Pharmaceuticals) for the locomotor activity monitor

software, and Dr Martin Crowder (Mathematics Department) for the

statistical models used in the analysis of the antinociceptive data.

This work has been carried out with the support of the Procurement

Executive, Ministry of Defence.

I am grateful to Miss Tracey Bakall for her excellent typing of this thesis. SUMMARY

The literature describing the role of the cholinergic system and opioids on

antinociception is reviewed. The effects of anticholinesterase agents and drugs

used in the treatment of anticholinesterase poisoning were studied for their

effects on opioid antinociception and locomotion. In both mice and rats, the

irreversible anticholinesterase, DFP, produced apparent antinociception, but only at doses which caused motor incapacitation. Pre-treatment with DFP potentiated the antinociceptive potency of the opioid drug alfentanil but had little effect on morphine or fentanyl using the hot-plate test in mice. The reversible anticholinesterases, neostigmine and pyridostigmine, did not affect alfentanil antinociception in this species. In rats, DFP potentiated alfentanil and fentanyl antinociception using the paw pressure test, but had no effect on morphine. In mice, drugs used to treat anticholinesterase poisoning (atropine, pralidoxime and diazepam) did not alter the effect of DFP on alfentanil antinociception. DFP produced a decrease in locomotor activity in mice which was reversed following atropine and pralidoxime administration. DFP decreased the hyperlocomotory activity of alfentanil in mice, probably by increasing alfentanil catatonia, but had little effect on morphine or fentanyl. When diazepam was administered as part of the treatment for anticholinesterase poisoning, locomotor scores of opioid treated mice were markedly reduced. In mice radiolabelled distribution studies showed that DFP treatment enhanced the entry of alfentanil into all brain regions, whilst morphine and fentanyl levels were unaltered. Studies on plasma protein binding of alfentanil showed this was, in part, due to a displacement of alfentanil bound to plasma proteins by DFP, enabling free drug to enter the brain. These studies suggest that fentanyl would least likely produce interaction in individuals poisoned by irreversible anticholinesterase agents. In addition, the new opioid drug, alfentanil, may be liable to interactions when used clinicall^with other drugs by displacement from plasma protein binding sites to alter its antinociceptive activity. CONTENTS

ACKNOWLEDGEMENTS . i

SUMMARY ii

LIS T OF TABLES AND FIGURES x

PUBLICATIONS xiii

CHAPTER 1 - INTRODUCTION 1

1.1. General Introduction. 2

1.2. Choice of Drugs Used. 2

1.3. Uses of Anticholinesterases. 4

1.3.1. Reversible Anticholinesterases: 4

C linical Uses.

1.3.2. Reversible Anticholinesterases: 3

Non-clinical Uses.

1.3.3. Irreversible Anticholinesterases: 5

C linical Uses.

1.3.4. Irreversible Anticholinesterases: 6

Non-clinical Uses.

1.3.3. Anticholinesterases as Chemical 6

Warfare Agents.

1.4. Aim of the Study. 8

1.5. Terminology. 8

1.6. A Review of the Literature on the Role 11

of the Cholinergic System and Opioids

on Antinociception.

1.6.1. Introduction. 11

1.6.2. Nociceptive Models. 11

1.6.3. Anticholinsterases and Antinociception. 14

(a) Neostigmine 14

(b) Physostigmine 15 1.6.4. Irreversible Anticholinesterases 16

1.6.5. Other Anticholinesterase Agents. 17

1.6.6. Cholinom im etics and A ntinociception. 19

(a) Muscarinic Agonists 19

(b) N icotinic Agonists 21

1.6.7. Cholinergic Antagonists and A ntinociception 26

1.6.8. Miscellaneous Compounds. 29

1.6.9. Acetylcholine Histopharmacology and 30

Opioids

(a) Role of Acetylcholine in Nociceptive 30

Pathways.

(b) A cetylcholine and Acupuncture- 32

Induced Antinociception

in Non-Nociceptive Systems.

1.6.10. Conclusions. 35

CHAPTER 2 - ANTINOCICEPTIVE ACTIVITY OF MORPHINE, 61

FENTANYL AND ALFENTANIL IN MICE AND

RATS: EFFECTS OF DFP AND DRUGS USED

IN THE TREATMENT OF DFP POISONING

INTRODUCTION 62

2.1. Antinociceptive Models. 62

2.2. In itia l Experiments. 64

MATERIALS AND METHODS 65

2.3. Animals and Experimental Conditions. 65

2.4. Drugs. 66

2.5. Safety Procedures for DFP. 66

2.6. Dosing Protocols in Mice. 67 2.7. Dosing Protocols in Rats. 69

2.8. Antinociceptive Testing in Mice. 71

2.9. Antinociceptive Testing in Rats. 71

2.10. S tatistical Procedures. 73

2.10.1. Hot-Plate Data. 73

2.10.2. Paw Pressure Data. 74

RESULTS 73

2.11. Pilot Studies. 73

2.12. Effect of DFP on Nociception in Mice. 75

2.13. Effect of DFP and Drugs Reversing the 81

Effects of DFP on Opioid Antinociception

in Mice.

2.14. E ffe c t of Atropine and Pralidoxim e and 83

Diazepam on Nociception.

2.15. Effect of Neostigmine and Pyridostigmine 83

on Opioid Antinociception.

2.16. Antagonism of A lfe nta nil, Alone and W ith 88

DFP, on Naloxone.

2.17. Effect of DFP Upon Nociceptive Responses 88

in the Rat.

2.18. Effect of DFP Pre-Treatment on 89

Opioid Antinociception in Rats.

DISCUSSION 94

2.19. Drug Injection Times. 94

2.20. Effect of DFP on Nociceptive Responses 94

in Mice and Rats.

2.21. Effect of DFP on Morphine Antinociception. 96

2.22. Effect of DFP in Fentanyl and Alfentanil 97

Antinociception. - 2.23. Proposed Mechanisms for DFP Potentiation 98

of Alfentanil.

2.23.1. Absorption 98

2.23.2. Distribution 98

2.23.3. Receptor Action 99

2.23.4. Metabolism 99

2.23.3. Excretion 100

2.24. Effect of Atropine and Pralidoxime 100

on DFP Potentiation of Alfentanil Antinociception.

2.25. Effect of Atropine and Pralidoxime 101

on the Antinociceptive Activity of Morphine.

2.26. Effect of Diazepam on the Antinociceptive 102

Activity of Opioids in Mice Receiving DFP,

Atropine and Pralidoxim e.

2.27. Variation in Antinociceptive Potency of 102

Alfentanil in Mice.

CHAPTER 3 - LOCOMOTOR ACTIVITY OF MICE: EFFECT OF DFP 104

AND DRUGS USED IN THE TREATMENT OF DFP

POISONING

INTRODUCTION 105

3.1. Assessment of Drug Effect on Spontaneous Locomotor 105

A c tiv ity .

MATERIALS AND METHODS 106

3.2. Animal and Experimental Conditions. 106

3.3. Drugs. 106

3.4. Dosing Protocols. 106

3.5. Locomotor Activity Testing. 106

3.6. Statistical Procedures. 107 3.7. Effect of DFP on Locomotor Activity. 108

3.8. Effect of Atropine and Pralidoxime on Locomotor 108

A c tiv ity .

3.9. Effect of Diazepam on Locomotor Activity. 108

3.10. Effect of Various Treatments on Alfentanil- 108

Induced Hyperlocomotion.

3.11. Effect of Various Treatments on Fentanyl- 112

Induced Hyperlocomotion.

3.12. Effect of Various Treatments on 112

Morphine-Induced Hyperlocomotion.

DISCUSSION 115

3.13. Effect of DFP on Locomotor Activity. 115

3.14. E ffe c t of Atropine and Pralidoxim e Alone and 116

With DFP on Locomotor Activity.

3.15. E ffe c t of Diazepam Alone and W ith DFP on 116

Locomotor Activity.

3.16. Effects of DFP on Opioid-Induced Hyperlocomotion. 116

3.17. The E ffe c t of Atropine and Pralidoxim e on the 118

Locomotor Activity of DFP and Opioid-Treated Mice.

3.18. Effect of Diazepam on Opioid-Induced Locomotion. 118

3.19. Interpretation of Locomotor Data. 119

CHAPTER 4 - DISTRIBUTION OF 3H MORPHINE, FENTANYL 120

AND ALFENTANIL IN MOUSE BRAIN: EFFECT

OF DFP ADMINISTRATION

INTRODUCTION 121

4.1. Distribution Studies. 121

MATERIALS AND METHODS 122

4.2. Animals and Experimental Conditions. 122 4.3. Drugs. 122

4.4. Dosing Protocols. 123

4.5. Distribution Studies Protocol. 123

4.6. Scintillation Counting. 123

RESULTS 125

4.7. Effect of DFP Pre-Treatment on Alfentanil Brain 125

and Plasma Levels.

4.8. Effect of DFP Pre-Treatment on Morphine and 125

Fentanyl Brain and Plasma Levels.

DISCUSSION 131

4.9. Opioid Lipophilicity and Access to the CNS. 131

4.10. Effect of DFP on Opioid Brain Levels. 131

4.11. Mechanisms of Increased Levels of Alfentanil 132

Produced by DFP.

CHAPTER 5 - IN VIVO AND IN VITRO PLASMA PROTEIN BINDING 135

OF 3H ALFENTANIL AND 3 H FENTANYL: EFFECT

OF DFP ADMINISTRATION

INTRODUCTION 136

5.1. Plasma Protein Bind of Drugs. 136

5.2. Plasma Protein Binding of Alfentanil, Fentanyl and 136

DFP.

5.3. Assessment of Drug Binding to Plasma Proteins. 137

MATERIALS AND METHODS 140

5.4. Animal and Experimental Conditions. 140

5.5. Drugs. 140

5.6. In Vivo Protein Binding Protocol. 140

5.7. In Vitro Protein Binding Protocol. 140

5.8. Ultracentrifugation Protocol. 141 5.9. Calculation of Free and Bound Drug 141

Concentration.

5.10. Determination of Drug Adsorption in 141

Uitracentrifugation Apparatus.

5.11. Recovery Experiments. 144

5.12. Percentage of Plasma Protein Binding of 144

Fentanyl and Alfentanil.

5.13. Effect of DFP on Binding Fentanyl and 144

Alfentanil to Plasma Proteins.

DISCUSSION 147

5.14. DFP Binding to Plasma Proteins. 147

5.15. Plasma Protein Binding of A lfe n ta n il and 147

Fentanyl.

5.16. Effect of DFP on Plasma Protein Binding of 149

Alfentanil.

5.17. Consequences of Alfentanil Displacement from 151

Plasma Protein Binding Sites.

5.18. Protein Binding and Relationship to Variation 152

in Alfentanil Potency.

CHAPTER 6 - GENERAL DISCUSSION 153

REFERENCES 156

APPENDIX A 185

A P P E N D IX B 186

APPENDIX C 189

APPENDIX D (i) 190

APPENDIX D (ii) 191 LIST OF TABLES AND FIGURES

Fig. 1.1. Three Dimensional Structures and Formulae 3

of Morphine, Fentanyl and Alfentanil.

F ig.1.2. British troops will go to war on valium. 9

Table 1.1. Methods of Nociceptive Assessment. 12

Table 1.2. Anticholinesterases and Antinociception. 37

Table 1.3. Cholinom im etics and A ntinociception. 46

Table 1.4. Cholinergic Antagonists and Antinociception. 57

Fig. 2.1. Dosing Protocols for Antinociceptive Testing 68

in Mice.

Fig. 2.2. Dosing and Testing Protocols in Rats 70

Fig. 2.3. Use of the Ugo Basile Analgesy Meter. 72

Table 2.1. Effect of Repeated Exposure to Antinociceptive 76

Testing Procedures on Hot-Plate Times of

Naive Mice.

Table 2.2. Effect of Multiple Saline Injections on Hot- 77

Plate Response Times in Mice.

Table 2.3. Effect of DFP on Hot-Plate Reaction Times 78

in the Mouse.

Table 2.4. Effect of Different Vehicles on the Antinociceptive 79

Activity of DFP.

Table 2.5. Effect of Atropine and Naloxone on 2mg/kg 80

DFP Induced Antinociception in Mice.

Table 2.6. Effect of DFP (Img/kg) and of Drugs Reversing 82

DFP Poisoning on Opioid-Induced Antinociception

in Mice. Table 2.7. Effect of Intramuscularly-Administered

Diazepam and Injection Vehicle on

Hot-Plate Reaction Times in Mice.

Table 2.8. Effect of Various Doses of Neostigmine

on the Antinociceptive Activity of Alfentanil.

Table 2.9. Effect of Pyridostigmine (lmg/kg) on

Antinociceptive Potencies of Morphine, Fentanyl

and A lfe n ta n il.

Table 2.10 Naloxone Antagonism of Antinociception Induced

by Alfentanil With and Without DFP.

Fig. 2.3. Time Course for the Effect of DFP on the

Nociceptive Responses in the Rat Paw Pressure Test.

Fig 2.4. Effect of DFP on Morphine Antinociception in the

Rat Paw Pressure Test.

Fig 2.5. Effect of DFP on Fentanyl Antinociception in the

Rat Paw Pressure Test.

Fig 2.6. Effect of DFP on Alfentanil Antinociception in the

Rat Paw Pressure Test.

Fig. 3.1. Graph Showing the Effect of Various Drug

Treatments on Locomotor Activity in Mice.

Fig. 3.2. Table Showing Effect of Various Drug Treatments

on the Locomotor Activity in Mice

Fig. 3.3. Effect of DFP on Alfentanil-Induced Hyperlocomotion

Over 15 and 75 Minutes.

Fig. 3.4. Effect of DFP on Fentanyl-Induced Hyperlocomotion

Over 15 and 75 Minutes.

Fig. 3.5. Effect of DFP on Morphine-Induced Hyperlocomotion

Over 15 and 75 Minutes. Fig. 4.1. Effect of DFP on Brain Levels of (dpm g~^) 126

A lfe n ta n il

Fig. 4.2. Effect of DFP on Brain Levels of (dpm g~^) 127

Fentanyl.

Fig. 4.3. Effect of DFP on Brain Levels of (dpm g~^) 128

Morphine.

Table 4.1. Effect of DFP (lmg/kg”'*') on Plasma Levels of 129

Morphine, Fentanyl and Alfentanil.

Table 4.2. Effect of DFP (Img/kg-^) on Brain Plasma Ratios for 130

Morphine, Fentanyl and Alfentanil.

Fig. 4.4. Scheme for Explanation of Observed Increased 133

Brain Alfentanil Levels of DFP (lmg/kg)

Administration.

Fig. 5.1. Protocol for Plasma Protein Binding Experiments 139

In Vivo and In V itro .

Fig. 5.2. The Amicon Micropartition MPS-1 System. 143

Table 5.1. E ffe c t of DFP Treatm ent on In Vivo and In V itro 145

Plasma Levels of Alfentanil.

Table 5.2. E ffe c t of DFP Treatm ent on Levels of In Vivo and 146

In Vitro Free Plasma Levels of Alfentanil and

Fentanyl.

Fig. 5.3. DFP Phosphorylation of Protein by an Alkyl 148

Phosphorylation Reaction.

APPENDIX A 185

APPENDIX B 186

APPENDIX C 189

APPENDIX D(i) 190

APPENDIX D(ii) 191 PUBLICATIONS

Publications resulting from work in this thesis are listed below:

KITCHEN, I. and GREEN, P.G. (1983) Differential effects of diisopropylfluorophosphate poisoning and its treatment on opioid antinociception in the mouse. Life Sciences 33: 669-679.

GREEN, P.G. and KITCHEN, I. (1984) Diisopropylfluorophosphate enhances entry of alfentanil into mouse brain. British Journal of Pharmacology. 82: 297P.

GREEN, P.G. and KITCHEN, I. (1985) Different effects of diisopropylfluorophosphate on the entry of opioids into mouse brain. British Journal of Pharmacology. 84: 657-661.

GREEN, P.G. and KITCHEN, I. (1985) Displacement of ^H alfentanil from plasma protein binding sites by diisopropylfluorophosphate in the mouse British Pharmacological Society Communication, September 1985. (in press).

GREEN, P.G. and KITCHEN, I. (1986) Antinociception, opioids and the cholinergic system. Progress in Neurobiology (in press) C H A P TER 1

INTRODUCTION 1.1 General Introduction

Opioids have been used by humans for many thousands of years. Opium was

first used around 4000 BC when it was mixed with wine by the Sumerians. Its

popularity as a drug remained, and is reflected by Sydenham who, in 1680, wrote 'among the remedies which it has pleased Almighty God to give to man to relieve his suffering, none is so universal and efficacious as opium1. The main active constituent of opium, morphine, is still used therapeutically as the standard analgesic against which new agents are compared.

Despite Sydenham's praise, the therapeutic use of opioids have a number of drawbacks, the most important of which are respiratory depression and a dependence liability. Even though there has been extensive research in the development of novel opioids, these problems have not been completely overcome. Another approach has involved the investigation of non-opioid drugs which either possess inherent analgesic activity or are able to potentiate the desirable (analgesic) but not the undesirable action of opioids.

Cholinergic drugs, such as anticholinesterase agents, have received a lot of attention in this area and their interaction with opioids and nociceptive systems is considered in Section 1.6.

1.2 Choice of Drugs Used

Three opioids were used in the studies. Morphine as the standard analgesic agent, and two synthetic opioids, fentanyl and alfentanil. These latter two are of a new generation of opioids which are very potent and are extremely rapidly acting. These factors make them suitable for use as intravenous (i.v.) analgesics for use in surgery. The structure of these opioids is given in

Figure 1.1. , ,h n ,o i co-oh » i oh ■ o ic h . c o o n ;.. c f h. : *• FENTANYL

l?4h3?n?0;' ALFENTANIL CHr°-CH,

N - C - CH - CH,

Fig. 1.1. Three-dimensional Structures and Formulae of Morphine, Fentanyl and Alfentanil Three anticholinesterases were used though the compound di

isopropylfluorophosphate (DFP) was investigated most thoroughly.

Anticholinesterases are considered further in the next Section.

1.3. Uses of Anticholinesterases

Anticholinesterases inhibit the cholinesterase enzyme which results in a

build-up of excessive acetylcholine. The therapeutic and toxic effects of

these compounds are due to the accumulation of acetylcholine at muscarinic

and nicotinic cholinergic receptors (Karczmar, 1967).

Anticholinesterases are normally divided into two groups, reversible and irreversible, which correspond to carbamyl esters and organo-phosphorous compounds respectively. However both classes of drugs react with the cholinesterase enzyme in the same way, and so this classification reflects only quantitative differences. There are, in fact, very few truly reversible compounds and even neostigmine is not considered to be in this class (Usdin,

1970). Nevertheless, for the sake of clarity this classification will be used in the following discussion.

1.3.1. Reversible Anticholinesterases: Clinical Uses

Therapeutically, reversible anticholinesterases are indicated in certain neuromuscular abnormalities such as myasthenia gravis, where neuromuscular transmission is to be enhanced (see British National Formulary, 1983).

Neostigmine, pyridostigmine, ambenonium, physostigmine and distigmine are the anticholinesterases prescribed for myasthenia gravis, pyridostigmine reported to have the fewest side-effects. These drugs are also used to tre a t urinary retention, and because they

increase intestinal motility, they may be used as stimulant laxatives

particularly post-operatively in case of paralytic ileus.

Probably the most common use for this class of drug is in surgery. Non-

depolarising muscle relaxants which are used as a premedication in surgery,

compete for the acetylcholine receptor at the neuromuscular junction. Their

action may be reversed by increasing the synaptic concentration of

acetylcholine with anticholinesterases. The usual drugs used to reverse post

operative muscle relaxation are neostigmine and pyridostigmine.

Anticholinesterases are also used in the treatment of glaucoma and indeed

the first therapeutic use for this class of drug was when physostigmine was

used for glaucoma (Laquer, 1877). Physostigmine is still used for this purpose.

Reversible anticholinesterases are also used in the treatment of anticholinesterase poisoning (see Section 1.3.3.).

1.3.2. Reversible Anticholinesterases: N on-clinical Uses

The major non-clinical use for reversible anticholinesterases is as

insecticides. Since their development in the 1950's, compounds such as

carbamyl and propoxur have been used extensively for th e ir highly selective

insecticidal action.

1.3.3. Irreversible Anticholinesterases: C linical Uses

The first organophosphorous anticholinesterase agent was synthesised in the

1850's (De Clermont, 1854). The first therapeutic use, however, was only made some hundred years later when DFP was used to treat glaucoma (Quillam, 1947). DFP became the drug of choice in the treatment of

glaucoma as it had the advantage over the carbamate drugs in that it was

very long acting and could therefore be applied infrequently. However, it

was established some years later that continued treatment with long acting

anticholinesterase agents carried a high risk of a specific type of cataract

(Axelsson and Holmberg, 1966). Currently in the UK the only

organophosphorous compound used therapeutically, for the treatment of

glaucoma, is ecjhiopiate.

1.3.4. Irreversible Anticholinesterases: N on-clinical Uses

As for reversible anticholinesterases, the irreversible organophosphorous

agents are extensively used as agricultural insecticides. The first

organophosphorous compound synthesised, tetraethyl pyrophosphate (TEPP)

(De Clerm ont, 1854), has been used as an insecticide though in recent years,

more selective agents have been developed. One of the most widely used insecticides is parathion, and this compound also probably accounts for more accidental poisonings and fatalities than any other organophosphorous compound (Koelle, 1985). Even more selective compounds were developed to reduce the toxicological risk to humans. For example, malathion has a UD^g mouse:LD^g housefly ratio of 68 compared to only 6 for parathion (Usdin,

1970).

1.3.5. Anticholinesterases as Chemical Warfare Agents

The potential for anticholinesterases as chemical warfare agents was realized initially in Germany by Lange in 1935 (Karczmar, 1970). This was followed by the synthesis and investigation of new compounds (tabun and sarin) developed specifically for their toxicity. Though these compunds were synthesised on a large scale, they were never actually deployed due to the fear of retalliation by Allied forces using their own organophosphates. Work on chemical warfare agents continued in post-war years, though research proceeded at a low level, and Britain stopped its development of new compounds in the 1950's. However, the USA, USSR and France still manufacture these compounds so the development of effective antidotes is an important area of reseach.

Protection against organophosphorous poisoning may be afforded by the administration of several agents. The prophylactic effect of carbamates has been known for some time (Koster, 1946). Research in subsequent years has shown that many carbamates protect against poisoning effectively, and that activity is dependent on many factors such as absorption, distribution and metabolism (Gordon et al, 1978). The mechanism for the protective action of carbamates is dependent primarily on the formation of a semi-stable carbamylated acetylcholinesterase; the enzyme in this state cannot be phosphorylated by an organophosphate. The carbamylated enzyme can spontaneously break down to release the active enzyme (Wilson et al, 1960) and as the organophosphate is broken down relatively rapidly, sufficient released enzyme is present to maintain life (Berry and Davis, 1970).

Further protection from organophosphate poisoning may be achieved by pharmacologically antagonising the effects of excess acetylcholine, by administering an anticholinergic such as atropine. In addition, the inhibited cholinesterase enzymes may be reactivated by the administration of oxime such as pralidoxime mesylate. This is achieved by accelerating the spontaneous reactivation of the inhibited cholinesterase by promoting hydrolysis of the inhibitor-enzyme complex (Wills, 1970). This additional treatment with an oxime enhances the protective potency of the carbamate and anticholinergic regimen (Gordon et al, 1978). The most recent addition to the battery of drugs used to treat organophosphate poisoning is diazepam. It has been shown to greatly enhance the protective activity of atropine against physostigmine lethality in mice

(Klemm, 1983). It is believed to have this effect by virtue of its anti convulsant activity. It does have the disadvantage, however, of impairing m otor function and perform ance (Gall, 1981; Fig. 1.2.).

1.4. Aim of the Study

The initial objective of the research described in this thesis was to study potential interactions between irreversible anticholinesterase agents and opioid analgesics, in an attempt to highlight drug interaction problems which might occur in individuals poisoned with anticholinesterase agents, treated for such poisoning and given opioid drugs for the relief of pain. This situation is clearly most pertinent in a military context, if in the field, personnel exposed to organophosphorous compounds and receiving antidotes also sustain injuries requiring surgery. In a broader sense, the aim of this thesis was to study the nature and attempt to analyse the mechanisms of interaction between opioids and anticholinesterase agents.

1.5. T erminoloqy

In this thesis, in describing the effects of opioids on pain responses in animals, the term 'antinociception1 is used, as distinct from the word

'analgesia' used in the human context.

Analgesia is defined as 'absence of pain' and pain as 'suffering distress of body or mind' (Oxford English Dictionary). As suffering and distress are subjective British troops will go to war on valium

New Scientist 25 November 1982

, REVISED Af?H\ h y m n a l

“Hymn number 69, ‘He who would Valiumed be ’gainst all disaster'.’’ phenomena, animals therefore cannot be described as perceiving pain per se.

However, they are able to sense and respond to noxious stim uli, so the term s

'nociception' and 'antinociception' are used.

The term 'opioid' has been used throughout this thesis to describe analgesic drugs. This is distinct from the term 'opiate', the use of which should be confined to those products of the opium poppy and related synthetic alkaloids. 'Opioid' describes all drugs which are opiate-like in their action

(see Kitchen, 1984). 1.6. A Review of the Literature on the Role of the Cholinergic

System and Opioids on Antinociception

1.6.1 Introduction

The potentiating effect of anticholinesterase agents on the activity of opioid

analgesics has been known for several years (Flodmark and Wramner, 1945;

Slaughter, 1950) and there is now a large body of literature describing the

interaction between cholinergic agents and opioids. This review summarizes

the present literature and suggests mechanisms underlying interactions.

Most of the available literature concerns work using animal models of pain

perception though there are reports for human models and for clinical pain,

and it is these studies which indicate a potential therapeutic role for

cholinergic agents in enhancing the desirable analgesic effects of opioids,

whilst reducing the undesirable components such as respiratory depression.

Furthermore, these studies have gone someway in elucidating the physiology

of pain perception and have indicated possible future developments in the

pharmacology of pain control. .

1.6.2. Nociceptive Models

The assessment of the potency of analgesic drugs is usually initially carried

out in experimental animals. A variety of tests have been developed, using

several animal species to determine the antinociceptive activity of drugs and

a brief description of the tests described in this review is given in Table 1.1.

Even though all the models described are responsive to opioids, it is

important to consider that differences in the nociceptive stimuli (heat, pressure, electrical) may well involve distinct pathways utilizing different neurotransmitters and that processing of nociceptive information may vary TABLE 1.1. METHODS OF NOCICEPTIVE ASSESSMENT

Test Species Method of End-point or Reference nociceptive stimulation quantification

Hot plate Rat Animals placed on Lifting, shaking Woolfe and Mouse heated metal plate or licking paw, MacDonald, (48 to 55°C) or jumping 1944.

Tail flick Rat Tail exposed to Flicking of tail D'Am our and Mouse localized heat source away from heat Smith, Cat e.g. focussed light 1941.

Tail immersion Rat Terminal part of Flicking or Janssen Mouse tail immersed in water lifting tail out et al., (50-55°C) of water 1963.

Hot wire Rat Tail heated by Flicking of tail Davies hot wire away from heat et al., 1946.

Tail clip Rat Application of Attempt to bite H affner, Mouse pressure by a clip or remove clamp. 1929. Rabbit on base of ta il Quantalresponse Cat

W rithing Mouse Intraperitoneal Number of Hendershot injection of irritant abdominal and substance (e.g. acetic constrictions Forsaith, acid, phenoxybenzo- observed in 1939. quinone) set perioid

Formalin test Rat Subcutaneous injection Scoring of O'Keefe, Cat of 5% form alin into intensity and 1964. forepaw pad duration of lifting, licking, shaking of paw

Paw pressure Rat Pressure applied to Withdrawal of Randall and normal or inflamed hind foot, struggling S elitto, paw. Pressure increasing or vocalization 1957. w ith tim e

Ear clamp Rabbit Varying pressure applied Attempt to Weinstock, Dog by clip on ear withdraw or 1980. vocalization

Toe pinch Guinea Varying pressure applied Attempt to C ollier, pig by clip on toe withdraw or 1962. Cat vocalization Prim ate Test Species Method of End-point or Reference nociceptive stimulation quantification

Electroshock Rat Application of electric Vocalization, Evans, Mouse current, usually to vocalization 1961. feet after discharge or withdrawal of paws

Shock Rat Electric current to Time spent at Weiss and titra tio n Mouse feet with stepwise each shock level Laties, Primate increase in current before 1970. w ith tim e responding by pressing lever to reduce current

Tail Rat Electric current via As for tail Carol and electrica l Mouse electrodes in clamp or Lim , stimulation base of ta il electroshock 1960.

Tooth pulp Rabbit Electric stimulation Threshold to Hoffmeister electrical to upper incisors jaw opening or 1968. stimulation licking response

Radiant heat Many Part of skin (may Varies with Hardy Species be blackened) exposed species: escape et al., 1940. (eg. dog, to intense focussed response (Human human) light or laser (rodent), skin testing) twitch (dog), verbal report (human)

N ote: References refer to the first report of a particular method, or to a widely used adaptation of a method. There are of course, numerous variations and refinements of many of the tests given in this table. between species. Modulation of nociception by some drugs may, therefore,

be test and species dependent. In addition some opioid receptor selective

agonists exhibit varying degrees of antinociception depending on the test

used. For example, k-opioid agonists are more effective against pressure

than thermal stimuli (Tyers, 1980).

1.6.3. Anticholinesterases and Antinociception

a) Neostigmine

The first studies of the antinociceptive activity of anticholinesterase in man

were carried out by Flodmark and Wramner (1945). They showed

neostigmine, a quaternary reversible anticholinesterase, had antinociceptive

activity and also potentiated morphine antinociception. A similar

potentiation was observed for other opioids (Slaughter, 1950). In contrast to

man, animal studies have failed to show antinociceptive activity of

neostigmine (Saxena and Gupta, 1957). However, there are reports of

potentiation by neostigmine of morphine antinociception (Knoll and Komlos,

1952; Saxena and Gupta, 1957). These observations have not been confirmed

by other workers except when neostigmine is administered i.c.v. (Pedigo et

al., 1975). By this route the antinociception is antagonised by naloxone and

atropine but not mecamylamine indicating that both opioid and muscarinic

systems mediate the effect (Pedigo et al., 1975).

As the quaternary compound neostigmine does not readily cross the blood-

brain barrier it exerts its action peripherally following systemic injection. It

is possible that the potentiation of opioids may be due to impaired

metabolism or altered distribution. The observed antinociceptive effect in humans (Flodmark and Wramner, 1945) may be due to the test used (radiant heat). As the authors suggest, neostigmine may increase cutaneous blood flow and so dissipate the radiated heat more rapidly. Alternatively, there may be an effect on peripheral pain sensitive neurons. b) Physostiqmine

Table II shows that physostigmine is reported to possess antinociceptive

activity in most of the tests described. Most of the reports showing no

antinociceptive activity involved central administration of physostigmine.

Since physostigmine readily crosses the blood-brain barrier, the lack of effect

of central administration suggests that physostigmine may be metabolized in

the periphery to the active compound. Indeed, FUrst et al. (1982) studied the

antinociceptive activity of eseroline, the primary metabolite of

physostigmine, and showed it to be a naloxone reversible analgesic when

administered either peripherally or centrally. The structure of eseroline is

similar to morphine and the antinociceptive effect appears independent of its

anticholinesterase activity.

Studies w ith atropine and hyoscine (Harris et al., 1969; Wong and Bentley,

1978; Dayton and Garret, 1973) and naloxone (Harris et al., 1969; Romano

and King, 1981) indicate that physostigmine exerts its action through either

the cholinergic or opioid system or both. The ability of naloxone to

antagonise physostigmine was not found in some studies (Pleuvrey and Tobias,

1971; Lipman and Spencer, 1980; Conzantis et al., 1983) and has even been

shown to potentiate physostigmine (Romano and King, 1981). Other evidence

suggesting that physostigmine’s effect may be independent of the opioid

system are that tolerance may be induced following multiple administrations

of physostigmine, whilst morphine antinociception is not affected by this regimen (Dayton and Garret, 1973; Little and Rees, 1974). In studies in the

rhesus monkey, physostigmine appears to increase morphine antinociception

(Pert, 1975). However, this effect is observed only at doses that also

produced general behavioural depression. The antinociceptive effects of physostigmine may be inhibited by

pretreatment with compounds depleting central 5-HT (Bhattacharya and

Nayak, 1978). 5-HT has been implicated in antinociception (Messing and

Lytle, 1977), and may be part of a pathway also involving cholinergic neurons.

It is worth considering that the neurotoxins used to deplete 5-HT do not have

absolute specificity and may well disrupt other systems.

Widman et al. (1978) described the effect of centrally administered calcium ry chloride on physostigmine antinociception. They observed that Ca + ions n reduced antinociception, and conclude that Ca + appeared to exert its effects intraneuronally and postsynaptically. Divalent ions are also involved

in acetylcholine induced antinociception (Section 1.6.6.(c)) and are also

discussed in Section 1.6.8.

1.6.4 Irreversible Anticholinesterases

Diisopropylfluorophosphate (DFP) has been investigated by several workers.

In studies with mice, DFP was not found to be antinociceptive by one group up to 2 mg/kg (Cox and Tha, 1972). Morphine and fentanyl antinociception appear unaffected by DFP, but alfentamil antinociception is potentiated

(Kitchen and Green, 1983). Bhargava and Way (1972), however, did conclude

that DFP does potentiate morphine antinociception, though the effect was not statistically significant.

The effect of DFP in rats has been studied thoroughly by one group (Koehn and Karczmar, 1977; Koehn and Karczmar, 1978; Koehn et al., 1980). They have determined that DFP has antinociceptive activity in hot plate and tail flick tests from 0.5 mg/kg. Antagonist studies indicate that this effect is mediated through both cholinergic and opioid systems. DFP's antinociceptive action was stereoseiectively inhibited by (-)benzomophan antagonists. The

(+)isomers were effective only to a much lesser degree. The role of this

stereoselectivity is considered fu rth e r in section 1.6.6. It is worthy of note,

however, that in animals made tolerant to morphine, DFP's effect was

unaltered (Koehn et al., 1980). Saxena (1958) failed to show an

antinociceptive effect of DFP in rats, or any morphine potentiating activity.

This may be due either to the utilization of pressure as the nociceptive

stim ulus, or the lower dose (200 ug/kg) employed.

Clement and Copeman (1984) have studied the very potent anticholinesterase

agents, soman and sarin. Both these agents produce antinociception that

persists for up to 96 hours after administration. Further studies with soman

showed only an additive effect with morphine, and that naloxone was only a

partial antagonist up to 10 mg/kg, whereas atropine completely antagonised

soman's effect at 2.2 mg/kg. This information suggests that at least some of

soman's antinociceptive activity is exerted through non-opioid systems.

1.6.5. Other Anticholinesterase Agents

There are a few reports concerning antinociceptive activities of some less

familiar anticholinesterase compounds, some of which are naturally occurring

plant products. Galanthamine is a natural alkaloid, isolated from

Amaryllidaceae, which possesses anticholinesterase activity. It also has a

chemical structure similar to codeine. Its antinociceptive activity has been studied in the rat hot plate and the mouse writhing tests (Cozantis et al.,

1983). In both these models galanthamine was antinociceptive, and was shown to be able to potentiate morphine antinociception in the rat. Its mode of action appears to be species dependent: its antinociceptive activity is antagonized by naloxone (0.1 mg/kg) in the rat, but is refractory to naloxone up to 0.5 mg/kg in the mouse. It is also possible that the nociceptive stimulus may be a determining factor as to whether the action of galanthamine is opioid mediated.

Tetrahydroaminoacridine (THA) was found to be inactive up to 3 mg/kg in the mouse hot plate antinociceptive test (Wooten and Klemm, 1980) though it appears to prolong the antinociceptive effects of morphine. Using a higher dose of 7.5 mg/kg and a d iffe re n t test model, Wong and Bentley (1978) observed antinociceptive activity for THA which was partially antagonized by atropine at 2 mg/kg. THA also potentiated the action of morphine; this action was also antagonized by atropine, and in addition the antagonistic activity of naloxone was enhanced with THA pretreatment.

Antinociceptive effects of dibromopyruvic' acid have been reported (Martin et al., 1958). This compound has weak anticholinesterase activity (1/400 that of neostigmine) and may also possess muscarinic agonist activity. However, its antinociceptive activity was not blocked by atropine at 2 mg/kg. Ibogaine is an indole alkaloid from Tubernanthe iboqa and is able to inhibit cholinesterase (Vincent and Sero, 1942). Though ibogaine itself is not antinociceptive up to 40 mg/kg, lower doses are able to potentiate morphine antinociception and lethality (Schneider and McArthur, 1956).

Recently, the organophosphorus insecticide triorthotolyl phosphate has been reported to produce a six-fold potentiation of heroin's potency in the mouse writhing test (Cohen, 1984). 1.6.6. Cholinom im etics and A ntinociception a) Muscarinic Agonists

The data for oxotremorine shown in Table 1.3. clearly shows that it is able to induce antinociception in four species using many forms of nociceptive stimulation. Only the rat paw pressure test failed to show clear antinociceptive activity as the effects were small and variable (Bill et al.,

1983). Oxotremorine has been shown to be very potent on a molar basis in all the studies shown, with an ED5Q as low as 11 ug/kg being reported by Metys et al., 1969. It appears to exert its action through both cholinergic and opioid systems as atropine, hyoscine and other anticholinergics are effective antagonists (Bill et al., 1983; Harris et al., 1969; Leslie, 1969; Ireson, 1970;

Paalzow and Paalzow, 1973; Wong and Bentley, 1979; Lewis et al., 1983) as are naloxone, naltrexone and benzomorphans (Harris et al., 1969; Bartolini et al., 1979; Ben-Sreti and Sewell, 1982; Lewis et al., 1983). It is possible that catacholamines may also be involved as Pleuvrey and Tobias (1971) reported potentiation by treatments which lower noradrenaline. Slater (1981) however, reports that neuronal lesioning with 6-hydroxydopamine, which destroys catecholaminergic neurons, acts through the noradrenergic system to antagonize oxotremorine’s effect. Other reports also indicate a role for noradrenaline in the mediation of oxotremorines antinociceptive actions

(Sethy et al., 1971; Barar and Madan, 1976). Oxotremorine is believed to be the active metabolite of the compound tremorine (Cho et al., 1961), and tremorine itself has been shown to be antinociceptive in mice (van Eick and

Bock, 1971; Lenke, 1938).

A recent study using stereospecific opioid antagonists has yielded important imformation on the mode of action of oxotremorine (Ben-Sreti and Sewell,

1982). They report a reciprocal stereospecific sensitivity for oxotremorine and opioid analgesics toward opioid antagonists. The (-)benzomorphan isomers, Mrl452 and Mr2266, antagonized opioid induced antinociception but had no effect on oxotremorine-induced antinociception. Conversely the

(+)benzomorphans, Mrl453 and Mr2267 antagonized oxotremorine but not opioid antinociception. Furthermore (+)benzomorphans antagonized oxotremorine antinociception, but not tremorogenic or hypothermic actions

(Ben-Sreti et al, 1982), suggesting muscarinic receptor subpopulations mediating these physiological effects, Koehn et al (1980) have shown the effects of (+) and (-) opioid antagonists on DFP antinociception in the rat to be opposite to that observed by Ben-Sreti and Sewell (1982) in mice. This species difference may be due to the involvement, only in the mouse, of noradrenaline in oxotremorine's antinociceptive action. Alternatively, DFP may. act through the endogenous opioid system (Koehn et al 1980) whilst oxotremorine may not as it is refractory to (-)benzomorphan antagonism

(Bensreti and Sewell, 1982).

Other muscarinic agonists, arecoline, RS 86, and pilocarpine have also been reported to be antinociceptive in a number of species and tests, though potentiation of opioids has only been reported fo r pilocarpine (Knoll and

Komlos, 1952; Wong and Bentley, 1979) and this effect is not universal

(Kaakkola and Ahtee, 1977; Malec and Langwinski, 1982). An important exception in the antinociceptive activity of these muscarinic agonists is in studies in non-human primates. Using the rhesus monkey, Pert (1975) failed to show any antinociception for arecoline or pilocarpine. Similarly the nociceptive response of the squirrel monkey was unaffected by pilocarpine

(Houser and Houser, 1973). This does not appear to be due to the nociceptive test employed (electric shock titration), since pilocarpine has been shown to be active in electrofoot shock in rats (Houser and van Hart, 1973; Kaakkola and Ahtee, 1977) and arecoline was e ffe ctive in ta il ele ctrica l stim ulation

(Metys et al., 1969). In addition, arecoline has antinociceptive activity, as

observed following electrical stimulation of rabbit tooth pulp (Metys et al.,

1969). The differing results, therefore, lie in species differences. Pert (1975)

suggests that the responses of rodents may involve polysynaptic spinal

reflexes which may be sensitive to cholinergic agents. Alternatively the

neurochemical coding of pain may be different in primates. These results

indicate the possible pitfalls when extrapolating data obtained in rodents to

primates, including humans.

b) N ico tin ic Agonists

In the preceding sections most of the papers describing antinociceptive actions of cholinomimetics and the effects of antagonists, have indicated that cholinomimetics exert their action through CNS muscarinic receptors.

Indeed, the use of nicotinic antagonists has been shown to be ineffective in

antagonizing cholinergic-induced antinociception (Pedigo et al., 1975; Dewey et al., 1975). However, there have been some reports describing potent antinociceptive actions of nicotine and dimethylphenylpiperazine. Nicotine has been shown to be antinociceptive in mice, rats and dogs using a variety of nociceptive stimuli. It is very rapidly acting, exerting an effect 30 seconds after s.c. administration (Tripathi et al., 1982). It appears to be acting only through nicotinic receptors as atopine and hyoscine were ineffective in antagonizing nicotines antinociceptive effect, (Phan et al., 1973; Tripathi et al., 1982), whilst mecamylamine was effective (Phan et al., 1973; Sahley and

Berntson, 1979; Tripathi et al., 1982). However, Sahley and Berntson (1979) observed that hyoscine did antagonise nicotine and proposed that nicotines antinociceptive activity was mediated via release of acetylcholine (see

Section 1.6.6.(c)). Species differences in nicotines action have been observed. Very close correlations between nicotine brain levels and antinociception is seen in the mouse, but there is a poor correlation in the rat (Tripathi et al., 1982).

Further studies indicated that this finding is probably mainly due to the rapid development of tachyphylaxis only in the rat. This same group also observed that naloxone had very little effect in the rat, producing a maximum of 23% inhibition of nicotine induced analgesia at a high dose of 10 mg/kg. In mice, however, naloxone was effective from 0.1 mg/kg, though complete anatagonism could not be obtained. Sahley and Berntson (1979) also showed that naloxone was ineffective in the rat.

The specific ganglionic stimulant, dimethylphenylpiperazine has been shown to be antinociceptive in mice (Phan et al., 1973) and this appears to be a nicotinic effect as mecamylamine is an effective antagonist.

These data, therefore, indicate that stimulation of the nicotinic receptor in the CNS produces potent antinociception and on a molar basis equivalent to morphine (Tripathi et al., 1982). Again species differences are apparent as in the mouse nicotine’s action has an opioid component and does not exhibit tolerance. In the rat, antinociception is mediated through the nicotinic receptor and rapid tolerance occurs.

c) Choline Esters

The intraventricular administration of acetylcholine has been shown to induce antinociception in mice. This effect can be blocked with atropine, though not with the quaternary derivative atropine methylnitrate or the nicotinic antagonist mecamylamine, indicating that acetylcholine exerts its antinociceptive effect through central muscarinic receptors (Pedigo et al.,

1975). Acetylcholine has also been reported to inhibit morphine antinociception (Mudgill et al., 1974). However, the effect is small and occurs 30 minutes after i.c.v. injection. As peak antinociception of acetylcholine occurs around 10 minutes post injection, its antagonist effect on morphine antinociception probably acts through a mechanism distinct from its antinocicpetive effect. There is also evidence that a component of acetylcholine's antinociceptive action is via opioid receptors. Its antinociceptive actions are antagonised by naloxone (Pedigo et al., 1975;

Pedigo and Dewey, 1981) and the rank order of potency of five narcotic antagonists is the same for both acetylcholine and morphine (Pedigo et al.,

1975). Furthermore, the divalent ions Mg^+, Mn^+ and Ca^+ but not Sr^+ or

Ba^+ also inhibit both acetylcholine (Widman et al., 1978) and morphine

(Harris et al., 1977) antinociception.

It has also been reported that i.c.v. adm inistration of 40 pg acetylcholine differentially altered the level of four peptide and non-peptide brain fractions which had opioid-like activity (Chan et al., 1982). It is possible that these opioid-like materials mediate the antinociceptive effect of acetylcholine.

There are, however, indications that acetylcholine exerts its antinociceptive effect through mechanisms distinct from that for morphine. For example,

Pedigo et ai. (1975) found that the 1-isomers of pentazocine and cyclazocine blocked morphine whilst the d-isomers were without effect; the converse is true for acetylcholine. Pedigo and Dewey (1981) have observed this reverse stereoselectivity with the (+) and (-) isomers of naloxone (cf. oxotremorine). In addition, studies with naloxone have yielded different apparent pA 2 values

for naloxone versus morphine and acetylcholine (6.86 and 6.09 respectively)

and different slopes on the Schild plot (-0.944 for morphine, -1.46 for

acetylcholine) (Pedigo and Dewey, 1981). These marked differences indicate

that naloxone inhibits acetylcholine-induced antinociception non-

competatively. They also showed that there is an assymetrical cross

tolerance between morphine and acetylcholine. The rapid tolerance to the

antinociceptive effect of acetylcholine only slightly decreased morphine

antinociception, however, tolerance to morphine produced a 12-fold increase

in the ED^g of acetylcholine.

With respect to interaction and involvement of amine systems, though

reserpine and tetrabenazine pretreatment appeared to inhibit acetylcholine

antinociception, these compounds lack specificity, and more specific

depletors of dopamine, noradrenaline or 5-HT were without effect (Pedigo

and Dewey, 1981).

In summary, the evidence indicates that acetylcholine exerts its

antinociceptive through muscarinic receptors (Dewey et al., 1975) and that

the mediation is independent of opioid pathways.

Choline administration has been shown to increase brain turnover and levels of acetylcholine (Cohen and Wurtman, 1975). Knoll and Komlos (1952) found that choline appeared to potentiate morphine antinociception. However, the dose of 200 mg/kg used is probably toxic. A t this dose of choline morphine

LD^g is reduced from 705 mg/kg to 66 mg/kg (Ho et al., 1979). A non-specific behavioural depression may therefore be responsible for an increased hot-plate latency. The opposite effect of choline was observed by

Botticelli et al. (1972) who found a dose-dependent attenuation of morphine antinociception using 30 and 60 mg/kg. At 60 mg/kg, choline alone did not affect nociception. The authors Concluded that morphine's action may be due to its ability to decrease acetylcholine release (Jhamandas ^ t al., 1971) and so choline, which has been shown to increase acetylcholine levels (Cohen and

Wurtman, 1975) may therefore attenuate the antinociceptive effects of morphine. This hypothesis however, is not easy to reconcile with the literature concerning i.c.v. administration of acetylcholine (vide supra) and the literature concerning the effects of opioid analgesics on brain acetycholine levels is contradictory (see Dewey et al., 1976). It is possible that choline may be acting on the post-synaptic cholinergic receptor; thishas been proposed by Fredrickson and Pinsky (1975).

Carbachol does not cross the blood-brain barrier but several studies using i.c.v. and intracerebral (i.e.) administration have demonstrated antinociceptive activity of carbachol. Furthermore, i.e. application studies have gone someway in identifying the particular groups of cells within the brain that are responsive to carbachol. Metys et al, co-workers (1969) showed that i.c.v. carbachol (at 1.25 gg) was antinociceptive in both rat and rabbit. Local i.e. application indicated that the septal areas and midbrain reticular formation - central grey region were both sensitive to 0.3 gg carbachol, whereas cudate putamen and dosal hippocampal administration only showed effects between 5 and 10 gg, doses at which transventricular diffusion may well occur.

Brodie and Proudfit (1984) examined the role of the cholinergic system on the nucleus raphe magnus (NRM). This structure in the caudal medulla has been shown to affect nociceptive processing (see Fields and Basbaum, 1978), and also to possess cholinergic terminal markers. For example, cholinergic receptors (Kobayashi et al., 1978) acetylcholinesterase (Palkovitz and

Jacobowitz, 1974) and choline acetyltransferase (Kobayashi et al., 1975).

Brodie and Proudfit (1984) showed that carbachol (i.e.) produced antinociceptive responses, which could be reversed by atropine or prevented by atropine pretreatment. The authors suggest that carbachol acts on the

NRM to activate descending raphe-spinal neurons, which impinge on spinal cord dosal horn neurons. It is these neurons which are sensitive to noxious stim ulation (see Brodie and P roudfit, 1984 fo r references).

In the cat, Katayama et al. (1984a,b) found a group of cells in the parabrachial region (PBR) very sensitive to the antinociceptive effects of carbachol. This appears to be solely a muscarinic response as it is inhibited by atropine, but not mecamylamine or naloxone. Morphine at a dose of 25 gg had no effect when injected into the same site. Although a role for these groups of cells which appear to mediate nociceptive information via muscarinic receptors is not clear, it is possible that they are the substrate for environmentally induced antinociception. This form of antinociception is discussed in Section 5.

1.6.7. Cholinergic Antagonists and Antinociception

The previous sections have described the literature concerning cholinomimetic agents and give a strong indication that antinociception is at least in part mediated by acetylcholine in the CNS. The use of antagonists in nociceptive studies is shown in Table 4 and provides further support for the. role of acetylcholine in nociception and opioid function.

Hemicholinium decreases acetylcholine levels in the cerebral cortex

(Rodriguez de Lores Arnaiz et al., 1970), by interfering with choline transport (Marchbanks, 1968) and pretreatment with this compound slightly antagonises the antinociceptive action of morphine (Bhargava et al., 1974).

Hemicholinium itself has no antinociceptive activity. This would suggest that part of morphine's action involves cholinergic neurones. Hemicholinium has also been shown to inhibit acupuncture antinociception following i.c.v. administration (Ren et al., 1980). The role of the cholinergic system in acupuncture antinociception is discussed in Section 1.6.7.(b).

The muscarinic antagonist, hyoscine, administered by the peritoneal route is not antinociceptive in mice (Bill et al., 1983) or rats (Houser and van Hart,

1973; Bill et al., 1983; Lewis et al., 1983) and indeed causes significant hyperalgesia in rat tail flick tests at 1 mg/kg (Watkins et al., 1984).

However, s.c. administration has been shown to produce potent antinociception using the mouse writhing test, which may reflect route of administration or perhaps a peripheral interaction with the writhing agent phenylbenzoquinone.

Data from primate studies (Houser and Houser, 1973; Pert, 1975) indicate that hyoscine is antinociceptive at low doses. Though co nflicting w ith the rodent data these results are consistant with the findings described in

Section 1.6.6.(a), on the study of muscarinic agonists in primates, indicating again that pain pathways may be species dependent. When hyosine was administered into discreet brain regions that had been shown to be responsive to morphine, no antinociception was observed. Ventricularly administered hyoscine was effective however, thus strongly suggesting a cholinergic pathway that modulates nociception at least in part distinct from opioid sensitive systems (Pert and Maxey, 1975). Opioid induced antinociception has been shown to be inhibited in two species by hyoscine (Bili et al., 1983) though not in the writhing test, but this could be related to hyoscine's own antinociceptive activity in this test. Malec and

Langwinski (1982) found that hyosine enhanced codeine's effectiveness whilst leaving morphine, fentanyl and pentazocine unaffected.

The use of cholinergic antagonists has indicated possible transmitter substrates for endogenous environmentally induced antinociception.

Endogenous antinociception may be induced by stressful stimuli, such as cold water swimming (Bodnar et al., 1978), electric shocks (Jackson, 1979), and fighting (Rodgers and Hendrie, 1983). In many cases the antinociception is naloxone reversible implying a role for endogenous opioid systems. However, some forms of environmentally induced antinociception are refractory to narcotic antagonists, for example brief continuous, as opposed to prolonged intermittant, foot shock (Lewis et al., 1980), and hind paw, as opposed to front paw, foot shock (Watkins and Mayer, 1982) appear to utilize non-opioid mechanisms. The cholinergic system has been implicated by some workers in environmentally induced antinociception, as hind paw foot shock induced antinociception and antinociception induced by classical conditioning are blocked by hyosine (1 mg/kg), but not the quaternary derivative, methylhyoscine (Watkins et al., 1984). Prolonged interm ittant shock was dose dependently antagonised by hyoscine from 0.1 mg/kg (Lewis et al., 1983), similarly long term opioid antinociception induced by inescapable foot shock followed by mild reinstating shocks 24 hours later was also antagonized by hyoscine at 2.5 mg/kg. The indications are that some forms of environmentally induced antinociception are mediated via central muscarinic system, whilst others act through endogenous opioid systems which employ intermediary muscarinic cholinergic pathways. 1.6.8 Miscellaneous Compounds

The role of the cholinergic system in nociception has been implicated by

pharmacological treatments not directly involving the use of cholinergic

drugs. There are several neurotransmitters which have been reported to be

involved in nociception, one of which is the inhibitory amino acid

Y -am inobutyric acid (GABA). The role of GABA as an analgesic has been

suggested from studies with an analogue, baclofen (Saelens et al., 1980). The

lack of antagonism by naloxone indicates that baclofen acts through an

opioid-independent system.

Kendall et al. (1982) investigated the role of GABA in nociception using

directly acting agonist (kojic amine and THIP), a GABA-degradation inhibitor

(y-vinylGABA) and an inhibitor of high affinity transport (nipecotic acid

ethyl ester). As with previous studies, naloxone (5 mg/kg) failed to alter the

antinociceptive activity of these compounds in mice using hot plate and tail

immersion tests. In contrast atropine (5 mg/kg) inhibited the antinociceptive

activity of all treatments whilst mecamylamine (5 mg/kg) was ineffective.

Those drugs which modify GABAergic transmission probably do not exert

their effect by acting directly on muscarinic receptors as only nipecotic acid

showed significant affinity for the receptor in binding studies. The results

indicate then, that GABA is exerting its antinociceptive effect via

intermediary cholinergic neurons.

2+ 2 The divalent cations barium (Ba +) and strontium (Sr +) are believed to be

able to substitute for calcium ions to induce release of neurotransmitter.s 2+ (Rubin, 1970) and Ba has been shown to release acetylcholine (Silinsky,

1977). Using the mouse tail-flick model Welch et al. (1983) demonstrated 2+ 2+ that in traven tricular administered Ba and Sr induced antinociception. This effect was reversed by atropine but naloxone antagonism was not dose 2 2+ related. These results suggest that Ba +'s and Sr +,s inherent antinociceptive activity is mediated via acetylcholine release.

The involvement of the cholinergic system in antinociception may be of clinical significance with the recent development of the analgesic drug m eptazinol. This drug appears to possess both opioid and cholinergic antinociceptive components as Bill et al. (1983) demonstrated in several rodent antinociceptive tests (see Table 1.4.) that meptazinol’s effects were inhibited by naloxone, atropine and hysocine. It has been reported recently that m eptazinol possesses cholinergic a c tiv ity by virtue of its marked anticholinesterase activity (Galli, 1983). The possibility of utilising the cholinergic component clinically is of great interest as the drug seeking behaviour and addictive liability of cholinergic or opioid-cholinergic analgesics may be less than that observed with opioids.

1.6.9 Acetylcholine Histopharmacology and Opioids a) Role of Acetylcholine in Nociceptive Pathways

The previous sections have described how the cholinergic system is involved in nociceptive processing. The question arises as to whether there is an anatomical correlate of these pharmacological observations.

Morphine has been shown to inhibit acetylcholine release from the guinea-pig ileum (Paton, 1957; Schaumann, 1957) and to increase levels in the brain (e.g.

Herken et al., 1957). These studies demonstrating morphine action in increasing brain acetylcholine levels was observed only at doses above that required for antinociception. However, these were measurements in whole brain, and more recent studies have measured regional changes in acetylcholine, with antinociceptive doses. Green et al. (1976) showed that in mice, 10 mg/kg morphine increased acetylcholine levels significantly in the striatum and at higher doses (300 mg/kg) in the hippocampus. In rats increases in striatal acetylcholine was observed after 30 mg/kg, and after

90 mg/kg increased levels were measured in the hippocampus; these effects were shown to be naloxone reversible. There is m vitro evidence that this effect is due to morphine's action on high-affinity Na+-dependent choline transport (HANDCU), an indicator of cholinergic neuronal activity m vivo

(Kuhar and M urrin, 1978). It has been shown that at antinociceptive doses morphine stimulates HANDCU in mouse striatal (Vallano et al., 1982) and rat hippocampal (Vallano and McIntosh, 1980) synaptosomes. In addition, enkephalin at 40 nM was shown to decrease neocortical HANDCU (Wenk,

1984). It is also possible that opioids increase acetylcholine levels by inhibiting its release, i.e. decreasing cholinergic neuronal activity. Indeed, release of cortical acetylcholine may be evoked by electrical stimulation of nerves in the forepaw of anaesthetized rats (Jhamandas and Sutak, 1983) and this is enhanced following systemic administration of the narcotic antagonists naloxone or naltrexone. This suggests that there is an inhibitory role for endogenous opioids on acetylcholine release. Opioid receptor regulation appears to be limited to certain pathways (Wood et al., 1984) viz, septal- hippocampal and substantia innominata-cortical pathways where agonists at p, 6 and e receptors exert inhibitory regulatory influences, whilst k-agonists appear to be ineffective. In addition, there is a co-localization of enkephalin and acetylcholine in the lateral system of olivocochlear cells (Altschuler et al., 1983) and several preganglionic parasympathetic neurons (Glazer and

Basbaum, 1980). This co-existence, and possibly co-release, may also occur in other neuronal

systems, and the literature describing the relationship between opioid and

acetylcholine turnover and release suggest a close relationship between these

two systems. It is quite plausible, that acetylcholine plays a key role in the

actions of endogenous and exogenous analgesia.

b) Acetylcholine and Acupuncture-Induced A ntinociception

In China, acupuncture is used routinely as a method to induce surgical

analgesia. The mechanism of this effect is still poorly understood. It is only

in recent years that this technique has, at least in the West, received any

credence and undergone scientific enquiry. The possible mechanisms involved

\d acupuncture-induced analgesia have been reviewed recently (Han and

Terenius, 1982). There are several reports describing the role of the

cholinergic system in acupuncture antinociception. Acupuncture causes an

increase in acetylcholine in the CSF and the increase correlates with increase

a nalgesia (He et al., 1979). In rats, acetylcholine levels were found to

increase during electroacupuncture-induced antinociception in the locus

coeruleus and dorsal raphe (Ge, 1979) hypothalamus and nucleus caudatus

(Wang et al., 1979). In addition, increased acetylcholinesterase activity was

observed in the thalamus following electroacupuncture and locus coeruleus

though activity was reduced in the substantia gelatinosa (Ai et al., 1979).

Pharmacological manipulation of the cholinergic system has added to the

evidence for a role of acetylcholine in acupuncture-antinociception.

Inhibition of acetylcholine synthesis with hemicholinium (i.c.v.) was shown to

inhibit electroacupuncture in rats (Han, 1979; Guan, 1979), and this could

partially be reversed with the acetylcholine precursor choline chloride (Han,

1979). Blockade of muscarinic receptors with atropine in rats and rabbits (Han, 1979) and with hyoscine in rabbits (He et al., 1979) was found to block analgesia induced by acupuncture, whilst the anticholinesterase physostigmine has been shown to potentiate acupuncture analgesia in rats

(Han, 1979; Guanetal., 1979; Ai, 1979).

There is also evidence for the involvement of other transmitter systems (e.g. endogenous opioid and 5-HT) suggesting a multiple neurtransmitter system m ediating the effe cts of acupuncture (See, Han and Terenius, 1982, fo r review).

c) Acetylcholine-Qpioid Interaction in Non-Nociceptive Systems

There are a number of reports in the literature describing interactions between central cholinergic and opioid systems not involved in nociception, but having potential clinical applications. For example, anaesthesia has been shown to be potentiated or prolonged following anticholinesterases (Pellanda,

1933; Green and Davis, 1956). Another potential beneficial effect of the opioid-cholinergic interaction has been investigated by Weinstock and her colleagues. The respiratory depressant effect of morphine, or other opioids that are routinely given as part of pre-operative medication is usually not important, however, in patients with lung diseases or breathing problems such an effect is potentially fatal. However, physostigmine pretreatment has been shown to antagonize respiratory depression in experimental animals

(Weinstock et al., 1980) and humans (Snir-Mor et al., 1983). This property of physostigmine has been exploited successfully in the treatment of severe respiratory depression resulting from overdose in heroin addicts (Rupreht et al., 1984). The authors point out that physostigmine treatment is preferable to the usual procedure of administration of naloxone, as acute withdrawal is not precipitated. Treatment of opioid addiction is an important area of research in opioid

pharmacology, and there is evidence that the cholinergic system modulates

the processes of tolerance and dependence to opioids. The literature has

been extensively reviewed (Wahlstrbm, 1978); short-term morphine

treatment ( 3 days) appear to increase acetylcholine turnover initially

(Bhargava and Way, 1975; Cheney et al., 1975), whilst several studies indicate

that longer treatment (2-4 weeks) appears to result in increase levels or

decreased acetylcholine utilization (e.g. Domino and Wilson, 1973). A possible clinical application to the use of a cholinergic drug in the treatment of dependence and withdrawal has been proposed by Albin et al. (1975). They reported that nalorphine-precipitated withdrawal signs following constant morphine plus an anticholinesterase (tetrahydroaminoacridine, THA) dosing in rhesus monkeys were markedly less than that observed after morphine alone

treatment. Indeed, an earlier study indicates that THA administration reduces physical dependence in humans (Stone et al., 1961).

There is evidence for a cholinergic involvement in affective disorders (e.g. depression) in humans, as following administration of physostigmine in normal subjects resulting symptoms include many of those.associated with depressive symptomatology (Risch et al., 1980). In addition, in patients with a history of affective disorder, but with clinical remission, infusion of low doses of physostigmine produces transient depressive symptomatology (Janowsky et al., 1974). More recent studies (Risch et al., 1981) have shown that administration of physostigmine or ajrecoline to both normal and affective- disorder subjects produces altered mood and affective changes, resulting in increased depression. These observed effects were highly correlated with increased plasma 3-endorphin immunoreactivity (but not cortisol or prolactin) and the authors suggest the existence of a cholinergically mediated

3-endorphin pathway modulating affective and cognitive states in humans. A classical response to opioid administration in mice is to increase their

locomotor activity. This stereotypic running is believed to be dependent on

dopamine (Kushinsky and Hornykiewicz, 1974; Eidelberg and Erspamer,

1975), but there is also evidence that a component of morphine's action

involves the cholinergic system. Administration of the muscarinic receptor agonist oxotremonine (Seidel et al., 1979), or the anticholinesterase agent,

DFP (Su and Loh, 1975), attenuate morphines effect on locomotion, whilst the

muscarinic antagonist, atropine enhances morphines action (Seidel et al.,

1979). Cholinergic drugs appear then to influence opioid-induced running.

The mechanism is unclear, but may well involve other neurochemicals, such as catecholamines.

1.6.10. Conclusions

Several agents which interfere with the cholinergic system have been shown to be antinociceptive. These include reversible and irreversible anticholinesterases and agonists at muscarinic and nicotinic receptors. To affirm that cholinergic drugs potentiate the analgesic action of opioids is perhaps an overgeneralisation. However many experimental studies have been able to show that enhancement of cholinergic activity can lead to enhancement of opioid antinociception. In addition there is evidence to suggest that endogenous analgesia, induced by stress may in part be modulated by cholinergic inputs.

The interactions between the cholinergic and opioid systems probably primarily occurs within the central nervous system as studies with quaternary compounds have been mostly negative whilst interactions following central administration are generally observed. The molecular mechanisms involved are not understood but most of the evidence points to a pharmacodynamic rather than a pharmacokinetic interaction.

Too few studies have used receptor selective antagonists and where cholinergic antinociception has been observed the involvement of both cholinergic and opioid receptors is often the case. Our understanding of interactions at the receptor level require more selective pharmacological tools espeically with respect to opioid pharmacodynamics. It is also probably an oversimplification to think of an opioid/cholineric interaction involving only one mechanism and there may be interrelationships with other neuronal systems. In particular the evidence points to a close relationship with monamines. Whether manipulation of cholinergic and opioid system produces more successful pharmacological agents are analgesics remains to be seen. >> 0 m o 4-3 cxi NO c — 1 1 m i" . r - 0 ON 0 1 ON T3 m On O n > ^ 0 CO h ■*-’ LCJ 0 C- CO • a o O 0 C 4-3 c_ CO 0 > 0 cn O 0 cn C co ■CT> & co0 O u in 0 4—5 CO c «+- c: t_ r^- Z EX Q. D cj c o z 0 1 1 0 0 0 ON c o 0 0 - 2 D Cd Q- h- □ CD Q_ l—i Z in o 1 cn 2

c o 0 “ 0 c 0CTi . £ o 4-J Q . X ‘5- O O o c t-H < 4-> 0 4-3

0 CO c 0 0 0 0 c Z 4-3 . £ 4-3 C CL CL 0 Z a z 0 Z CL C-c c_ 4 J CL H— CL o o c Lh (-> C 4-> o H— t-i O E E 0 O 0 O 4-3° 5 <*- 3 a 4-3 E E c c o O E O 0 CL CO c T3 0 ,) d o o o _ 0 cn 0 4-3 4-3 c n _ x 4-3 <*- a a o 0 U a o 0 0 « cn cn 4-3 O c E c 0 s i ? CL 0 O 4-3 o a c o Z i~Hg EI? Q. r—1 o 0 -

cn cn cn cn cn -X cn 0 cn cn cn CL cn 0 4-3 CL CL CL cn CL in CL CO CJ m O O O O O O O o o o o o cn CX| CM o o o O • 4-3 4-3 4-3 m > CL o cl a C L 6 D c j c l a X CO D « X CO LlI .d D CO

0 > I 4-J ■n a . c 0

< 1 CO o o o c o 0 Z Z > z

o CL o CL -X 0 IZ JC CO 0 4-3 E CO 0 4-3 4-3 cn o a — I CO Ld 0 c 0 _ 0 f—

c0 E cn 4-> cn oW CJC-i 0 Q Z CM UN o r- UN ON CM i—! CO ON ON UN 0 "8 4-> c CO ON r- 0 0 0 I"' C- 0 c ~ C O N O 4-4 r. H •8 §5 ^ « s Cm CO 0 rH C XI > 0 _0 cn c cn to'«CO '.x r- t- O s ? 4-J m 'nO o CD JZZ C-4 c o o ON J$2 ON X I 0 ON cn U CD z * U !j I i—I CO ^ H rH

0 -“ .£ o o□ L o X i j 7a < Z

w Cl 1 0 C^j 0 0 0 co .CD. 4-J 4 - j 0 CO 0 X X g *0 x 4-J H .5 CO co CM X CM o 4 - j C CO CO _ •co S - E C JZ c Cm O !E 0 0 c X rH c o -a 0 4-4 0 0 cn CL § ° CO 0 4-J Cm LC UN 0 C CO 0 o I CM 03 03 c c CL 0 0 C CL t CD o o “ 4-4 o 0 Q 0 • - CO t-i •— 0 •0 O £ 0 ■- Q. c c l .E c E L lI Cm ■E j-) •O c 03 Q ~ J Z 0 0 ° s* 0 v O T3 j? 0 0 CL 4 - j CO j? 0 c *“■ 0 a o .5 2- o 0 £ c O •=P. c H C H 0 c l c •- •- O co o *0 0 O 4-J 4 J a cn E cn E a "a xi 0 JZ — c c *- cn o . cn -1 Q. E p 0 o 0 ^ a. E cn 0 Z3 cm c 0 a E cn cn t_ c E 4-J Cm XI o o UN E UN 0 n o c e ! E °CM o C ^ 0 co c co co O CO H KN C KN TO E rHg P C cn E X3 Q- c Ll I CM t

0 0£ —i "a cn 0 o _x t- x: cn cn cn cn o CO cn cn -X c 0 n _x _ x CL —* Cm cn cn ^ .c o cn 01 cn o cn o' ■*-> CO n ZL CM 0 1 01 m CM a c co O CD CM o CM o CO o CO rn ’wq co _ I—I NO 4-J CM cl a 4-j a Q a C L o 4-J O _b P 3 CO CO L lI co 3 .J < CO#

CO CO CO CO CO 0 O 0 0 0 0 o > z >- > > > z

0 0 JZ -Xa z 4 -j I d < c cn jo 0 3 7o- 4-J 3 0 O 0 0 X IY. 1 H

0 C t iN E cn

co o >.V) JC CL o CD 00 CO CO o a On ON CO ON 4-J r - 0

cn 3 □ r - c o 0 o c cn 4-J 0 ’ o . 4-J o o c LA C i < 0 O ( J LlI

0 0 c c ■D v O 0 a '' 1c ! c CO CNJ 3 3 c-> 0 fA C 4-J o 0 _ O o H O ° < £ E E a l a Ld Ld 0 Q O Ti 0 o TD c 0 .E a < r - 0 0 •- 73 Id ^ 4-J 4-j x : £ . 2 a o 0 co 0 0 CL SP A3 LlI a o 0 C ON • n c-< ^ c O c u— • - O 4-J M- 1c 1-1 C 3 ^ 0 Q - O 0 0 0 -3 . Q _ U 4-J c-. < f 4-J (-1 t- o rro

cn cn cn cn ZJ z l cn DC * > z j 0 c n co £ cn cn cn cn 0 4-J CX| Q_ 3 3 cn 3 cn 3 CO 3 3 O O CD 2 ° \ 8 O CD CD CD CD o cn Q £ i—1 c CD CD l*A rH fA LA LA LA 1 A 3 r H CD • o . 3 . C CJ o . O CD O CD O 4-J > 4-J > 4-J L A 4-J fA L A > O 44 H * . . . Q 6 Q - a O C «t— D- CL o CL £ Q CJ 3 4-J CJ CJ Q o L d . d D . d L A 0 0 . d D . d D . d L d to* D < CO CO Ld to

0 > I 4J i & <■§ o CO CO CO CO c o 0 O o 0 O 0 0 Z > - z Z > z > >

ZJ0 o JZ CO CO cn 0 4 - j — I to 3 O c (-< a 0 4-J o o s . * - 0 cn * 0 f - id

cn 3 t - Q f A 4-J CM cn 0 cn 00 c_ ON c ON c_ c CO fA rA 5 5 CJ 00 00 O n •a *0 ON •8 C » «a o c o co .£ C c c 0 .acn c 0 .Xicn CO o cn - j 0 o £ cn ^ m £ c 4 - j rA -TO 0 cn E CO CO E: c 00 CO £ 03 s :z? o 0 o ON rboN JZ ON ° ON 0 £ U _ r H CL Q □ Z CL! rH 2 _ I Q Z

0 0 0 c o £ c X o ‘El o 0 |> . 4->s Z I <

cn 0 4-J c 0 o j z - 0£2 <4— 0 CL 0 0 <*~ O£s —H c U 0 C 0 t * a 1 | 0 cz ° C o ° ^ Q- o 4-J — T o c 0 > , o 9 s «+- C N cn CO 0 zi 0 4-J zl cn C LA 0 LA z5 . *+— S D.

I O c o D. cn cn ZJ cn cn -X O -NC _ x 0 0 cn cn cn cn cn cn ZL z r Z l cn ZL ZL S “ a c n o 2 j? O cn cn CZ) rH ZL f A 0 CO o ZL LA ZL rH o (H n c c CO CM o . o o O CM IA ■L-» > LA . rH LA . Q) m fA 4-J Q . -9 - o O cj 4 - j CJ O CJ o Q - J5 - a . < .J D Ll I cn < £ cn U CO Q_ < .J D .J

cn cn cn cn cn 0 O 0 0 0 O 0 O > Z > > > z >- z

0 -X JO4-J a 0 . 4-J 0 0 1 H ON n - ON -X 1 i—1 0 1 CO I m 0 __X 3 CO 1 *3 0 > . 011 CD c ON m 0 I 0 Cl co rH a 4-J o * 3 c cn CD co 0 0 1 0 .VC 0 c O o 0 4-j SZ o ON 4-J ~ c 4-J C cn 0 CD C rH a c rH E j z a B f- c c 0 cn j c >-, 0 ~ zl CO t_ 0 N c-0 : x ? 0 jr c “ nj 4-j 0 4-J 0 3 4 - Ex: s ^ 0 4-j E ? D. O CD n 03 > Fhro £ D N 0 0 P '£ ° On o 0 0 o .£ o 0 CL ON Ol X _ ! _J O i—i CO £ ^ I m ( £ Q CD i—1

cn CO 4-j CO C -n * O a ^ cn C_ —1 0 4-J CO 2 c cn i j < E c □rN °0

S £

.JL c P C 4J o o c • - P 4-J CL c .c ■o 0 o m— O —1 ■ g -1 q - .ir; a Ld Q. o O c c 0 4-J o a .

cn cn cn cn 0 cn cn cn cn cn cn ZL J* 0 4-J D. cn ZL co D cn cn O O cn cn cn cn cn cn CL n CL a CL cn E CL E O 01 CL in o □ o O a UN O o o o o CM CM o o vo in . m m < r in m vo in 4-J 4 - j q . Q o 4-j a 4—J Q. 4-j CJ CL CJ < .X I d co

0 > I *4J 4-j 0 - c 0

o cn CO CO cn CO CO CO c 0 0 0 0 O 0 0 0 0 o > > > - > z > - > > > z

0 L-. 0 c o cn CO o o cn 0 4-j M— 0 — CO ‘ cn o c- CJ 0 C-c u, w CL 0 0 4—J SJ. a o g - 1- =3 E 4-J _0 o tn 0 0 o s 1 Ld w CL Ll . 4-J

cn x

Q LA 1 1 r 4-> ON 1 0 * 0 1 1 rH (D -X LA a a a > a A' o "if t- c o c o ON ffll o ht 0 0 a 4-J 4 J rH 4-J t - CO co E CO E | 0 c O c C o 4-J « rH c CM H- 0 f-H M £ CO CO 0 0 * 3 ^ * 0 CO S 2 b oo 0 ON Os JT ON 0 .to ON ON ON 0 1 U i-H Q - CO rH > i—1 U_ i—I

c 0 o c cn o 0 4-J X c o < 1 3 Z 0 c 'j z 0 Q. c 0 c_ o 0 cn E “I E •i. c c j * CO 0 C 4-» o JO c * * - u j o cn 0 o 4-J ° < ’•§. .5 i O -rj 0 2 E “ Q - o E 0 .2 o < r o c cn cn cn oj f c . 2 a 1 5 ! c_ 0 E L d CL o Dl ° .2 O C JC g>E ^ O Q . c u "ct 0 ° * ■ _ 0 U. 0 0 2 M- o « w o ^ O 4- I— ° o o c LA C < f i—i a) c CL -O rH O

c n cn c n 0 c n c n 0 4-J .X cn CO CJ ZL E o o c n cn c n n n CM n c n c n CM Q 01 LA (A o o a E E o a □ O • O i-H i—i LA i—I L A > LA 4-j cd - > 4-J > 4-> > 4-> a Q a Q

< .d < .J < .j < < CO U J U s . c

0 > —i Q. •£ 0

<•§ co co CO CO o >- OT 0 0 0 0 c s ~ > > > >

4 -j 0 0 c 0 CL CL x 0 c 0.2 JO 0 CO 03 -H CO c > 0 4-> 0 4-> CL CD E 4-J _ 0 Z .2 3 V E J 0 — CO t ! D .2 < (H c O 0 CD O -q 4-3 CO - X 4-1 ^ 4-J 20 «CO 0 CD _h CD a Ld 0 0 4> 0 •OC 0 TO g . 1- a ° T ° h -2 CO t - S co < * 0 o £ O & - L 4-J D « 30 0 co 0 < O 01 h- H Ld Q Ld 01 co X3 X Ld CO CL 0 a cr: i

Q cn u 03 0 C-H CO Q UJ r- CO CO r - ON ON ON LfNCO CO CM 01 ON JZ co h- > on •ag 0 1 •8 CD rH 0 cn c c o c CM c~* ^ JZ E S' s i 0 CD 0 u 0 r"- 8 ” X CD ON £ J- O O 0 o 0 oz U CD ^ z z z z to c 0 SI CL f-> o c o 0 £ E cn (Z o co J- 0 .= o .5 § N C-H O Q . X Q. X C 0 X c O O o o 0 c o < ij 0 h Id ;=<•O oE 0ro < Z < Z 1 co " 7

0 .5 IE 0 trCL C 4-3 c O o E t . I ■ ° < £ . C ■ O -rj 0 c o 0.-0 o c °- c 4-1 t .2 O *" 0 “ o L lI CL O 2 S 8 o 0 O c - —*00 cn 0 c E c g c CM co.E 0

cn .x cn -X cn cn cn cn cn 0 .x .X .X-X n co0 4-1D cn O O E cn cn cn cn 0 E E E E 0 o of ■ CM CM m in i-H in O O 4-1 o CD CD 4-3 CL O 4-) O 4-1 0 4-1 0 4-3 0 CL 0 ZD co CO < £ CO < CO CO ZD CO

i §- < 1 o CO CO CO CO c o 0 0 0 0 o Z >* > > > z

CL CO 0 -X .X E 0 4-3 4-1 o o —I CO L J 0 _0 0 o 0 D j cn □ S 0 < r ^ o 0 0 ^ I I- Of I h- H

s * cn §■0- o C-. .2 8 Q 5 Q. < r co CO CO cn n o O CO I-' rH 4-J ON 0 ON r , c n c 4-J c 0 CD CD c c c 4-J 0 E 0 E c c ^ 0 CD 0 0 0 CO 3

0 0 0 c o c c X o "S. _o ’o . X o o o C- 73 ■ ( j 0 Z <4- z

0 c x 0 Q- 0 t-< c .£ O x X ■O £ C L ON Q . O ON x ° x t7 cn cn 4-J Q ° S O X E 5 LU cm cn e I? 0 TJ X cn 2 0 X C L 2 E > .5 g ? « t- XN x 0 F "Zj CL 0 f-< C-« pH b 0 KtD -O o a , o t 0 Q o < 2 . 5 to Q G 4 - j

cn cn x cn x x cn cn cn cn x cn cn x cn cn x E x cn E E cn cn x cn m cn x CO CO E cn 3 . E r-« E cn CL XN XN CXJ XN O UN E O cxi O O o o O lA I—1 LTN . UN UN , 4-J UN ^ * 4-J 0 -M 6 Q o O o Q > O c j Q. 4-J 4—j a < C CO <£ to Ld to G to G .d G co D . < < to

to to CO CO to CO CO 0 0 0 0 0 o 0 0 2 2 2 2 2 Z 2 2

CL 0 0 0 0 4-J 4 -j cn E . 4-J cn G G 0 0 G c L d 0 G 0 c CL cn iS cn c n - - * □ - Q- ^ a X a D D _ J 4-J 4-j 4—J 4-J b * j O o O o < o o z 0 7 3 o £ "tZ *0 2 1 2 X OZ I 2 ^ 2 H 2 1

a c o E u c a x > E S 4-J c c 2 « <2 0 c 0 2 ■o E ^ " 2 "a &-• o c3 *0 >> O cn cn GJ Ee 3 0 X 0 2 u0 4-J • *■4 0 Q h- VO m cn

cn u (-> c T30 jr3 4-J '3 C- D

c o c n co 4-J c <

0 c 3 CL C 4-> O C-H O E co ° < £ 3 ° TO 03 CO o TO 0) . M U cn co 0 0 c c- 4-j o c 0 C £ .2 3 0 O LlI Q. O cn > SI * 0 O ' c 3 c 0 4-J 4-J c b C-. 3 JO 0 4 - j 0 o 4 -j vo C C-I o 4-J 3 E 0 - 2 CO CL 3 3 o TO 3 o 3 4-J cn C-. t o 0 C-H 0 c . 4-J 0 > c en 3 o > > 0 cn o C-h JO 0 co JO cn =3 0 TO 0 0 4 -j c_ 0 4-J co 3 c n C_ E E 0 c O O E 0 o 0 o M- Q c n C i 0 Q. i n < t 4-J C-H 0 TO CO o o c 4-J C l 4-J 4-J c CO O 0 JC Q - c j CL 4-J 0 D CO D CO 3 C 0 0 O > > O Q. JO co 3 0 0 0 TO c > 0 C-H 0 C 0 C CO I 3 o 0 4-J JO CO i & 0 O 0 3 Ci i n 0 TO j : 0 0 cn CO 4-J D. o c o O 0 c 0 o o Ci 0 c c_ 3 4-J Ci Z Z a u 0 TO 0 CL O c- CL C 0 0 cn CO > 0 0 4-J > co Ld .b O 3 0 co TO O 0 cn [r; 3 CL ’dj H E o 0 0 co &»- 0 > CJ cn cn Q *3 c 03 5 o 4-J 3 C£ Li S H 0 C-. 4—J o O T0 3 0 C O 3 JO 0 c 3 0 c It o cn co 0 i n Ld 4—J 0 O c c Ld < < cn 0 CJ 03 c_ o o JO Is 0 i V CNJ • 13 1 t"- XJ ° Q 00 1 0 I"' 0 —H I 0 1 131 xj 1 CO ON ON 0 1 ON •3 a jC x j iH •—J | 0 1 x j x j i 0 0 I—I 0 1 r f— 1 H 1 0 1 c S-r XJ 0 1 0 CO f_ CO H 1 to to dT > 0 •8 0 0 >po\ 5 ON r A c rA O n E CO j- j x £! 1 1 zz co 0 Lh NO ■o r - 0 '5s ^ 0 0 0 Is i o ON 0 0 ■ H ON £ ON ON On •H a: U rH CO r H rn rH CD cn CD r H D - r H X i—1 X rH cn c 0 c0 JC cn HQ. CL CO E o 0 0CO iaON 'c CL 0 E o O c o 0 0 £ > § g CO c Z > _o 0 O O CM+ LA9 < 9 o 13 i j 13 0 D no .d Z + co 1 < Z CJ Ld

C J-> o ° < ’■£ O -Q 0 "0 0 .i: o 0 t .2 o m CD Ld CL o .2 C O C C JC I £- o ° CL E

cn -N£ cn cn _y 0 cn cn cn cn cn cn 0 X J zi cn cn Q1 cn cn cn -X co 0 .x ZL ZL zi O O o cn cn cn cn cn o cn cn ZL ZL cn ZL ZL CM zi D DC CM ZL ZL zi fA CM O O LA A O A □ O O O O • A m CM i—1 rH CM i-\ tH A . A . A > t“H Q o x j cj XJ CJ x ) Q. XJ CJ XJ cj XJ O XJ CJ Q O Q o O o XJ D. Ld n < to < to < < to < CO < CO < CO Ld co Ld co Ld .J < . d

0 > i j-> i & < “ o o CO CO CO CO CO CO CO CO CO CO CO CO c 0 0 0 0 0 0 0 0 0 0 0 0 X > - > X X > X > > > - > >

to .x 0 XJ o —< to cn Ld 0 O 0 H ^ 'a. t—Hto cn z O 3 0 o X I h- a < u 0 zM .E M fe £ E < 0 cn U b D cn o C-H D X Q 2 O ON vo oox n > > rs ON ON x n 0 c oo o oo 0 4-1 ON TO o n 03 o c ON o O n ON 0 ON r - r-> 0 v o r " r- o \ CD ON J ON ON 4-> I — ON — t - CD 0 1 c i—i 0 I 0 I C rH CC 4-J 5

co o ‘ Eh C-. 0 0 £ 0 0 0 0 0 0 0 o 0 0 c c o c o £ . £ x E o £ £ JC £ CD a ‘ o ' a X ‘ a . ‘ a a ‘ 5 . 'EL X CO CO CO o o o o jD 0 0 C-H 0 4 J C-. C-H 5 S *0 c 0 > > * 0 1 z 1 < Z I Z 1

c n lQ*TnC; Q c co Ll I jE £ .2 c o s i C -C o .■y a 4Ja P- c-* H o ° ^ <5 IE Q. E o

c n c n . x c n c n cn cn cn c n c n c n c n c n CsC cn CL c n c n c n J* o t: c n CL c n cn c n c n O c n c n CL c n CL c n CL c n CL CO c n CL c n CL CL CL CL CL C l CL □ O o o o o a CO o O O CD O O LfN o LfN CM rH XN 1—1 LCN . r—\ |H LfN rH LfN . LfN L/N XN - . • . 4-1 Q. 4-1 Q. 4-1 o 4-1 a 4-1 CJ 4-1 n 4 -i a O CJ 4-> o 4-1 Q. 4-1 a 4-1 CJ Q o ' _ _ 4-1 CJ • < s < CO I d to < to < < to < CO Ld to < .d < .d < CO < CO < CO < CO

to to to to CO CO CO co CO CO CO CO CO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 > > > > - > > > > > > - > > >

J* U CL o JC E c n to o c £ o C-H 7 j 4-1 mD 0 h - cc E cc E 0 0 c * 0 »■' t < * 0 I— £ h - Ld 1 - ON ON O n ON VO NO VO VO VO ON ON ON r*N ON ON LTN co r - ■3" ON ON OO 00 CO I CO I i—I UN • 8 •15 £ r - o n ON rH 03 I 03 I £ Ci r 0 0 I 0 1 03 00 0 rH „ o o CO CO CO CO N N 0 CJi ~ 0 O co 0 i C-r J * 4-1 4-3 "co 13 O « 4-3 O O C a 0 0 TO 0 U D 0 0 O 0 CC 2 2 a. c l u a: s 2 CD CD _J

c o 0 cn 0 .5 4-3 '5l c IHo < 4-3 <

c C 4J o ° < ’■£ o - a 0 0.-0 fc .2 o Ld c l o O c

CO C cn o CO cn 0 cn "ai cn cn 0 0 j* C-4 • cn cn > E 0 J* cn cn cn co c n 3 cn 3 4 J UN UJ —] -X E 3 3 p H O CM O O LfN CJI 0 0 UN cn CO I c rH 0 (2 3 . UN E rH 3 UN CO 1 CM 0 | 3 CM 0 Z UN O > O O O 3 o LfN r^N 4-3 UN rH > X Ci CO UN . O o 4-3 4-3 O 4-1 cj £ 3 4-3 > 4-3 O a cLQ CJ < co < . J < G < .J < .J

0 > I 4J i « < “ co CO CO CO CO CO CO o 0 0 0 O CO 0 0 0 0 o 2 2 2 Z 2 2 2 c £ >

I—•CD 4-3o J o o 0 o £ M— . f e 4-3 I 3 4-3 < g Ld 0 , , UJ J 2 o ►H CO Ld .2 o £ CD 3 +-> CD -C . Z °- 3 r - o o CD n D •— —1 E 5 ^ 5 c n JD o 0 < o .£ -J1 o O £ D L I— co Ld w 0 . 01 I - Ld O h- 2 I

0 I—c oL i cn E 3 o C-. L i O H OV Ov OV ov 1— 1 Ov VO vo VO VO CO VO Ov Ov Ov Ov Ov Ov r H r H i-H r H iH r H r- — VO *V VO CO X i * I X i ^ . XI X 1 a jrrH I —H 1 co A- LA 01 0 1 01 0 1 0 1 0 1 ov 0 1 0 1 LTN ON ON r H 4-J I 4-J 1 4-J I 4-J 1 •H 1 4J 1 4 - j I 4-1 I OV r H 0 1 0 1 0 1 0| 0 1 0 1 0 1 0 1 o« - 0 1 f-H C CO CO CO CO c CO 0 , 0 >> c ' CO x >. >> :h JH X >> £0 X rH X 0 X c x rj 4J 4-j 4-J H 0 4-J CJ 4-J A 4-J A x CO 4-j a 0 0 0 0 o 0 o 0 ov 0 ov 0 0 ON t o I-H UO r H CJ _J t o I-H XXX X X X * 5

C-l O CO 4-> 0 E Cl ‘ c c O 0 0 o Q) X I J , c • 8 O 0 X c {- 0 0 O 0 p CL .= I 0 > p O c C P >> .5 ■ 0 < Q-X O o C 4-) C L X CJ Q_ X 0 c Js 0 x — • 2 f-l< & X .2 (J 0 X 0 ° l O CO -H X IX CL TO t- 0 n CO f4-J | l S « J £ C l 0 0 i j (j 4-J I P 0 C£ X X CL 5 X < CCL X < I H c£ x x ®8

c o H CL 0 - ^ 0 0 .2 a 5= .2 a U CL O O C

cn cn cn cn cn cn x x cn cn x x cn x x x X x cn cn cn cn cn cn cn cn cn cn cn E E E E j* E x E E E cn cn LA LA A- A Ov X x cn a CTl Ov A A* VO cn cn E CM CD CM CD E CM CM rH fA CM E E a a o CD a a O a o a CM CM LA LA LA • CM _LA . LA J A . LA ,. J A . LA Q D - Q Q. Q E 4-j a Q o Q CL Q o Q a Q o Q o 4-j a X° L U X Ll ! X LJ X

to to to to CO CO CO CO CO CO CO CO CO 0 0 0 0 0 0 0 0 0 0 0 0 0 X X X X X X X X X X > X X

X c a X o C l o . o o X 0 X E 0 q- o X co a cn 4-> CL _0 co LJ CO 0 JO O OCi .2 0 *3 4-J x + J X *o o 4-J o o x E 0 o ’co 0 •- 0 O o 0 0 "C < o 0 H f— CO LJ X X h- h ~ £ 1 H LJ m

o a 0 L. < O n ON ON ON Ov VO VO VO VO VO O v Ov ON o v Ov CO r—i i—1 r—H i—1 O Ov •v *v £ Ov —5 1 —-4 1 —H | —4 1 rH O v O CD in 0 1 0 1 0 1 0 1 a r - 4-1 I 4-1 1 L J 1 L J I >-> c Ov 0 1 0 1 0 1 0 1 o * £ CD t-> CO CO CO CO 0 > 4 > 4 > 4 *—] cn c 4 - LJ 4-1 4-1 LJ 0 o C 0 0 0 0 0 0 0 0 c ° CD CL X X X 2 X

C o 0 CJI c 0 ’5. 4-1 o c t-. < 4-1 <

0 c co 0 0 X 4-J c .£ 0 Q l 0 jc > C-. c o LJ < t o o ov 4 -j c cn 4-1 CL E 0 a 0 c CJ 0 a o w o 4 - B o> 4 - .5 'a o £ £ ^ cn Ld CL o a> £ s eE c <+- j - L CJlDC **- CO o m ■*— c c 0 g -

cn cn cn cn cn DJ j * DC DJ cn cn cn cn — 0 cn cn cn cn 0 4-J E E E CL j : DJ co 3 CN| m ON O O cn CD cn cn ZL m m I"- CM E O DC m c-1 CD nj E CD in CD o CD CD a CD in in . in in CM Qo Oo Oo Oq < .j: D.S Ld co LlI co Ld co LlI co

0 > CO CO CO CO CO CO co I L J 0 o 0 0 0 0 0 0 > > >- > > a 8 - > Z > < 3 O c Ld Z o CL j* 0 o 0 . 3 . 1 co 4-1 o E cn o 4-1 cn ►—ir ~ □_ l to j Ld 0 0 c JC Id 0 n D ^ co £ CD JC . CO DJ co X CL I- CO Q O Ld O D ^ I- O 4-1 D 4-> CO < _0 o O 4-1 < o S i s O £ 0 0 o o cn h - Ld cn tn 2 I H h- cn S X

a c ‘EL c3 cn so o a CO o C— CO O cn CM CD TA ON IA fA r - ON r-- O n VO th ^ i—l r - ' ON T3 OO » 8 ON ON I—I "D rH A 0 i—i C “ • 8 4-T _ 0 r - co .5 * So ON * tT 4-J r> c 0 o 0 ^ I u ® j z o o 5 o c - 0 0 •J“ 0 “ 0 cn 4-J c_ 4-1 cn ^ > > 0 0 JZ d C-I D O CL o ® ell 0 o 0 < I S 0 0 ZJ N 2 _J O I > 1 CL f 1 Dl. q

0 0 c £ ’ o ‘5 . cn o Q. o c-> >> 4-1 X <

c £O g c 0 r 0 ° - e -8 c T3 C o 0 o a j z cn . . CL 0 fe co 0 a cn*+- c 5 E ro " 0 4->0 < C «f= < £

cn cn cn J* -N£ cn cn cn cn cn cn cn cn ZL DL cn E cn cn E cn o O cn L.p E CD A ZL CM LA E A CM CD CM O O O O CD O A 4-> LA i—l A . CM CL CJ o 0 4-1 a Q D. D cn LlI cn < cn Ld < .j : 5 3 < 3

cn cn cn cn cn 0 0 0 0 o 0 > > > > Z > > LJ > Ld z * O c Z 0 o 2 - 5 C-r D cn 0 J* co Ld S cn CJ a: z3 0 0 A 8 D c- "a. iS a cn ^ 4-i D. H a 4J Ld o 0 4-1 0 o T o t- i < O 0 _ L r ; ■*-» 30 JZ 8 < 0 £ I H LJ ° O' cn Xj CO CO (X CL

fc- a c0 E co 1 CO UN rA i—I rA rA r A rA a ~ A- a ~ A- J' ON —4 1 ON ON ON ON ON CM r H r-H r—I i—1 i-H O n A» 0 1 "0 1 A* ON CO #N ON r—| ON • . 4-> 1 j- 1 1 • * 1 * | 0 1 1—1 0 1 i—! To 1 0 1 0 1 0 1 0 C • 8 c 4-J 1 r~ 4~> 1 4-J 1 4-» 1 *-> 1 C o >> 1c CO 0 I 4-J 0 1 0 1 0 1 0 1 > > O 0 4 -j CO 4-J 0 0 c c c r~ 0 c D. 4-J . 2 c ^ 1—1 n 0 0 0 0 0 x : u jc C_ 0 CO x : tH JC JC sz -C 0 0 0 0 -H ON "C, 0 _ H Q. 0 . CL CL to CD to CD _ 1 h 1—

E 0 2 c c o E E E E 0 JC 0 0 0 0 0 C ■a I 0 4-J0 I § E I F E FE ■= o E F o•- EF CD §S CD ” g » •- 0 CD co Cu cn 0 1 1 O >, O .2 X JZ O O o O o O j= o 0 r: 0 CO 0 o 0 0 >> 0 >, 0) o S 0£ —7 « 2 ZIIII > 2 T ^

C C 4-J o ° < £ O -q 0 0 .z, a P .2 o L±J Q. o O c:

cn cn cn CJi .x 0 1 _x cn cn cn cn cn 0 cn jx. . x cn cn -X o E n cn CL E cn cn cn Cl cn <3 o CM o O LA E EE H '< o LA LA CL o LA . LA LA LA i—1 LA ^ • j j a Q o 4-J Q_ 4-J D_ -LJ Q_ 4-J D o < .J Ld co* < .J < .X < ■X < Ll ! co

CO CO CO CO CO CO co CO CO CO 0 0 0 0 0 0 0 0 0 0 > > > - > * > > > >• > >

0 J* 0 4-1 o cn 4-j o LlI 0 c _0 !c "a. 4-J to D °- f- 4-J H 0 ’iu < o 0 I—4cn 25 I ° H OZ I 1- z o □ <

I-HCJ z 0 P .£ R o H-4CJ o z z m ON o n - r- r - r - | CO I O n ON ON co I CM r-H , 0 I * 8 LP> c c “I cn CT U i—I c CD ~ to 0 C £ ON .. 4-1 N 4-J —H m m ^ C 0 Q) 0 I “ ■2 fc £ «h C j z ° r “ 0 co | ! 0 *CO tI - CD CO , 0 CD 0 CD CO CD ON ■ii 0 JZ oc cn cn J h *! CL Q. _ I X

0 0 c c CO E E 'c 0 0 o cn 0 E E 4-> 0 0 c a O < 0 0 2

c 0 C 4-1 o S" TD s « c ° < ’■& 0 ^ t D -n O 0,0 0 .zl a T f s I . £ 5= .2 o 0 0 "Z Ld a o O c c < - g _ .2 Q 0 O LJ ^ s- C CL O 0

c cn 0 0 0 cn 0 4-1 cn ZL co D "co j * 0 cn cn O O O cn cn cn cn o ZL ZL Q QC ZL ZL cn m 0 ■* ZL CD O 0 cn O O o in . o . in > 4 -j i— i > I— I > Q. in in 4-> O 4-1 O 4-J > CL > ± 2 O < . j < < .J < .J D < . j : <

CO CO CO CO C0 CO I 4-> 0 0 o 0 0 ■- Q- o 0 0 Z > > >- > Z > > C 03 < • 5 o > c LJ

0 z £-, o z SZ 0 co 0 0 SI 0 u CO 4-> JZ in c J* c 0 o 0 LJ 0 n C_ D 0 £ CL in 4-i CL I CD^ s L d o 0 h - ~ £ cn . £ * ■ 3 T O L 0 O - L r~ 4-J < "co 9 ° Q. Q cn 2 QC cn Z» QC f— in 2 1 i c£ u c H 0 cn JZ LJ a u LJ O > * . £ JC N 4 -) (Q _ j cn 0 £ n **» z c 0 Ssd 0 c- .2 D. I u Q Q *a (J < i n r " r - i n i n t ''- ON ON t " i" - ON On On

CO O'. CD O c c * £ 0 1 0 o 0 o 0 o e- Cn 0 cn 0 E S' ^ Cn »4- *"5 ■O TO “ XJ ‘■5 0 0 0 G 3 0 0 CL CL Q CL

0 c CO + CM ‘ n c c n co CO o 0 “ c c cn 0 0 . 2 o 4 -) Q . X o o + "f" c CM CM COo 2 < 4J 0 0 ^ 0 0 0 < Z U 2 a lh - Z

C 4-J o 4-J 4-) JC o c n 0 .-j <+— • 4— 00-0 0 o ® C (D 5= .2 O L lI a . o .o - 0 © ) 02 O c JC 2 03 □ l c *-* Cl u o o 0 TJ m - a E TO 0 O § 3 * ° • 0.2 .2 o jc CD c n c n O a. 0 < c n 0 4-> cn 3 3 cn 3 • 3 CO 3 i—1 CO vo> - 1 O O in O Dl CO °2 i—i r—1 cn m > in m m > m > Q a Q o □ Q Q o Q o U U U U U .-5 U .JI

0 > CO CO CO co CO ‘Xj 0 0 0 0 0 a 0 > >- > > > o ‘ a o c

_X O cn r) "j c j c Q .' 4-J CO 0 H

cn 3 c-, Q la ON ON NO On ON NO ON On

cnj CO CO NO LA 4-J j O n ON 1 0 0 1 P" CD ON rH 1—1 ON o c 0 I •a 4-T ° * 8 0 CO cn 0 C-H 0 XJ .2x3- 0 x CJ XJ CJ co *4— 5 o "e 4-J O 0 O 0 0 0 0 0 c o o C-H C-H C- C-H 0 O al Q CD CD Ql CO Q. 2 X

c o 0 X cn c [£ 0 4-J Q.Z c o . <

4J 0 0 C cn 0 X zc c Q. C 4J (-4 O O C 4-J E & 4J < Q_ E 0 -rj 0 CO NO° tP 0 CJ 0 cn h-H — Q.zl •— a i 4-J XD 0 Ld 0- o 0 *4J cn £ « O c c 0 E a i 4-J 0 IA 0 O CD □_ CNJ <$ % 0

cn an _y 0S£C 0 cn 0 4-J cn n CO CJ cn o o d cn CD cn CD 5 2 5 2 CL LA O C£ Cl NO LA CnJ i n * LA %■ CM IA CNJ . O _• / rA > # 4-J CD ^ 0 iH > CD 4-J O lj 4-J O CJ Q- D. t i ^ - n 4J o < - d CO D . d < . d < . d < . d C . d

0 > CO CO CO co co I 4J 0 0 0 0 0 0 O 3 8- > Z >- >* > > Z < •5 O C

4J o CO DC 0 0 _y o 0 4-> 4J o *+- Ld .2 Ld CO 0 o ’a cn J2 ^ . . 0 4 -j J * CL d i: D J CJ o cn Q ’S O wo <& ^ O 0 0 jU o X f- X X a : x O' X h- h- LJ «

o JC 0 0 o E c 0 cn 0 to 4-) .Q a XJ 0 o C-. 0 □ O JC 0 Q CD co 0 (J U On H 0 JD > -JD 0 >> a g a $ S 0 0 -. 4— CO m 03 CO 0 0 « g a- 0 ON 0 ON 0 a: 2 Y r—i * 1—1 I

0 C u. 0 o 0 ■7—1 cn c c c c o 0 s s i -D 4-J 'a . "EL "EL ‘S. 0 c o i S . 2 o o o Lh t-. CO < -c c 4 - j wC t ° l >)- => < <

c0 c ’co 4-J o 0 c c-< 4J < CL 4 - j 0 1 X3 CO CJ U CLI a "a o LJ "EL o x: ">L c 4-J x: O 3 4-J 0 0 E 3 i 3 e -a n H- Cl -o Q. u 0 _0 > .£ 3 O a c-> X) CO JD co 0 c 0 ■a 3 0 4-J Cl 0 3 cn C cn cn cn cn E 0 c. cn O O CL cn cn cn 0 CJ 3 o a: CL 3 3 3 3 3 3 (-> 0 0 LA 4-J C-i 0 a CD CD CD CD CD CD CO O 0 0 JC JC 4-J CJ 4-J CJ4-J CJ 4 -j CJ 4-J CJ 4-J CJ 4-J o 4-J CL 0 < . J < < < < < < -X CO 3 0 c 0 C-H O CO CL 3 co JD _0 0 0 0 •a > 0 c_ 0 3 CO CO CO CO CO CO CO c c 3 i 0 0 0 0 0 0 0 0 C o JD > > > > > > 4-J i & 0 O 0 3 Lh LA XJ <.s 0 0 X o CL a cn CO o O 0 .£ 0 C£ t~> C-. c 4-J 3 4-J Z c 3 a •a 0 o 0 C- Ch CL cn a 0 0 H f l £ C 0- co > 3 o 0 LC O 4-J n t Q- 4-1 01 0 co -a 4-J 0 CD JC W 3 c CL 3 ® h- X ) q E 4-J o 0 0 XI CL r ~ h - - J C f- to u O tn 5 o ° 0 0 > 0 <£ ' 0 1= .E O 0 0 < O “ c 4-J 4J "a 3 ££ h- 0 U H X CL Ll_ I— f— 0 C l cr 4-J a o c 0I 0 c 0 a 0 4J 3 JD C-< 0 c o c 4 - j 0 Q- CO c n gonist drugs given in parentheses indicate only partail antagonism obtaine a 0 LA u 0 4-J Q < c LJ < CL < cn 3c_ Q m CNJ oo a m m m 03 ON 03 C O 0 0 CO ON Q\ ON ON ON r H 1— 1 i H 0 1 CD U 0 * • . cn 0 Ch c > “co 1 " 1 *0 •8 .2 0 0 I CD CD X J 1 J -3 ' cn X J 1 0 a 5 JZ CO CD c-, ON 01 01 0 cn C ; ,__ , t .5 «4- CD — 1 § 0 JZ 0 ■ 1 —* 5 § *0 0 fX CD cd £ £ £ 2 . j _!

o 0 2r 0 0 0 .£ CL >> o O XJ CO o 1 c CO 2 N 0 0 0 12 XJ C-. XJ 0 CL XJ c o 0 c CO o 0 ’ o . 0 .2 .2 0 O 'n *0 CL S c # TJ C-. CO ‘5 & .2 cn 1.2 to O O 0 O Zj -U >> CtJ CO CO I CL J Z 0 CL O _ 0 J -3 .2 ■O 0 cn 0 co ^ C XJ . 2 c .2 i? O C o .2 JZ E 0 E 0 £ cn LPi N vO N cn o 0 0 CL . 2 c ™ 0^ 0 f-i 0 0 d * c XJ XJ §•§ u h a O Lh <5 « J -3 O J -3 « 0 c 0 O -n 0 "O 0 cl in CL cn co 7 3 c : 0 c C f - 0 a 0.-0 CL J -3 CJI 0 0 ZL JZ c **- D 9? cc 0 D c cn co 0 5= .2 O CO ON 'co E E CD 0 0^ -X •a I d CL O 0 0 O *0 cn c c CO i n J -3 CO CO VO O c J -3 J -3 iH cn O "2 0 O ■° § E c ^ 1 5 ZL CO cn “ 0 9 ^ 0 Q . Id 15 O co cn c l CO 0 ^ c- m _ CO H X 0 0 xj -J J -3 jz 0 15 in O E & O C-. c -o 2 O 33 ° O c c Q 0 XJ 1—( Ld JZ □ 0 *— 1 E ►— 1 t— 1 O m E rH CO < .2

cn j* cn cn cn .x cn ^ 0 cn j* cn 0 X J ZL cn cn CO ZJ cn E 5 ° O E E E Q Dd o o

CL C L C L C L C L Q o D . D .d D .J Ld co D.S-

co = & o o 0 o < • 2 Z Z >- Z o o c

to H co to 0 0 2 ^ o ►— XJ cn XJ "o w tO Z Ld 0 c £ 0 “ O' o to 7 ? CL ' O h- C3 3 .S' CO F— < 0 <> □ Z 1 2 X c o O n fA f A 00 F'- •8 4-J ON f A 0 O'. VO LALA F-" r-> c 00 c ON LA ON ON ON 0 1 cJT 0 *T- 0 > 1 « 8 0 0 12 0 1 CO -1- CO 0 0 CO CO 4-3 X .2 8 4-3 o 3 C 0 Cl X Ci 0 0 0 0 0 0 o o 0 JO 0 O 0 0 £ £ a 2 CQ I > X CL 1 1 u u

E c - 0 " 0 4-3 T 3 1 C u 0 c n o C 0 f < 0 C C c 4-3 0 o o ■0 CL . £ 0 U_ 3 CO 0 o IE . § 1 ■H » 4-3 O c Q- CO J Z 3 C-i C 4-3 O * 0 C 4-3 3 t o c c 3 ‘n O 0 0 o o O 0 ° < 3 3 o 0 lc C 4-3 0 E 3 E 3 c 0 - O -n 0 •- ’S.■ " 0 CL 4-3 CO 0 .Id O 0 ’co 0 0 c n o c 0 c n c 0 c 5= .2 o 0 0 d c 0 E c n Ld CL O ‘S. 1? o / —s Cj T 3 co Cl c n 4-3 3 O C E ^ ■0 o 0 f-4 c o . 0 0 0 c o c E 0 0 4 -j CL o 4-3 ’o. LA T3 JZ zO o id 03 c C c < Ol 0 CNJ I CL CL □ T 3

c n z j c n CJI c n c n c n 0 ZJ 0 0 4-3 c n ZJ c n z i CO '0 E c n ZJ LA O O c n c n c n CO o E 0 c n 0 CNJ 0 CO O D i c n I—I 0 J fA z j CNF o o o o o CO o ♦ o CO O c n o 4-3 4-3 LA LA # 1—1 > # LA . E 4-3 CL 4-3 > 4-3 0 0 cj CL CJ • t r ^ < < < D < .d D CO

0 > cn CO cn 4 J o o 0 0 o 0 o CL o Z Z Z > - > z > Z c 0 0 > < Ld. 3 > - o c Ld z 0 c Z o 0 c-> o 4J 0 4-3 0 3 o z —I 0 cn c-. s z cn o Id 4-3 q— cn .- 0 01 C i O z 3 f 1 ^ D ^ a CL h Z J cn h—I dC ■ ® £ 0 l . l Q - f“ o u Ld o D uo £ _0 o o 0 X £ 0 JZ D 0 Q. Ed m 0 1 cn cn cn I 0 1

c n 03 C- □ CSI 00 o KN ON KN 00 o r —1 00 CO CO ON oo NO •- ON n O n CJ\ O r- 1—1 rH On XJDC ON • a r— CO • a 0 cn C co' U c 0 c c 0 O 0 0 3 O 0 3 D^ 0 o CO -C CO JD .£ =5 0 cn 0 ! XJ D D* CO E c -NC r— D f :d? 0 D CL 0 c- 3 L— (X O O . Q- Z 0 Z *0 Ch C-. 0 0 _J CO 5 2 _J 5 CD CL * 1

0 C-. d 0 0 4-1 c C o CJI c D ^ JC 3 D CL CL CL cn u Cl D 0 E O o O c o E E 0 3 r H o o CL 1 o CJ C- LfN O 3 0 CNJ E x> 0 0 -Q JC .5 4-3 c c .£ c c 0 0 3 X0 3 ‘o 03 CL 0 0 Q. O (D c-> CO c. N C 4-3 o *0 . 5 O 0 ; C l 3 3 4-1 -C c E E .5 0 CL Q. 0 C CL "O c 5 1 X J C-i t-, co 0 Cl D f —4 O o TO c o 0 O 0 4-3 ~ 3 > 3 E E 4-J cn 0 O 5 1 cn CL CO o > > CO 0 E c ^j CO 4-> CL QJ ’co 4-1 =1 £ XJ 0 C o C ■»-> ° Qo * c 0 LfN 0 JEP ■h irt2 > 0 _ JD 4-) C 3 o £ 4-j Q -C o d 0' c < lxl *5 JD 0 Q 0

cn cn cn DC DC .l: CJI cn cn D f CJI E E E CJI CM LfN f-"N E O O CD 4-1 4-3 r H n 5 cj* 4-J CL CL 4-> CL D . D . <

O o o o Z Z Z Z

0 dc: j* D^ I CJ o 0 O o o H Q. + J CO H O 4—3 4-1 0 o 0 < o 0 I— a i x LJ °

0 c ‘EL t*o ia VO O * 3 NO ON 0 1 c CO ON °0 ON 0 . 0 13 E w 0 cn to .2 % ~o CJ XJ cj c 2 o CJi Cx c 0 0 0 CJ -C (-4 g b . 0 CC 2 3 2 O CD Q

TJ 0 CO TJ Cx _ 0 JC ^ cn LA~ c0 CO jx . J3 S a cn q0 cn c >> I s XJ 0 U m n ^ O t- o 2 c CL0 "2C O o 0 ^ O <1- ® 0 in O 0 fH (0 _ D -C 0 c C co O 0 CO c q - O O © •- cn .2 x O 'co 0 CL XJ 0 C c-> XJ 0 J i C J-> o t-i 0 C 0 XJ CO o =3 J ? ■R 0 - co ca 'I- TJ —< t- CL Ci E 0 O ° 0 2 ° ° < £ T J £ < r c ■E e £ O -Q 0 CO C C 0 0 .2 13 0 .E o 0 cl EE CO c - .2 03 cn o Cx 3 8 CO TJ "o 5= .2 o IS — 05 m R 5 g 0 o .2 LJ CL o C m CO O E • 2 0 co X 'cn CO ^ - a a 0 O C x ^ o CJI T J R - ^ XJ 0 c 0 cn 0 ^ 0 cn o xj c 0 “ CJ Cx . 2 o 0 £ ^ X E 0 0 Cx 0 CJ c -a E A jc o cn . XJ O -n J-» co 0 CO XJ JC Cx — ■I i “ o C 0 c 2 ■ 0 O O • - C VX0 0 0 LJ Q 3 3 a TJ 0 .2 Cx *EXJ x Cx TJ 0 0 C. Cx > 0 o >, CL Cx X 0 x II "X . 0 cn a 0 T3 0 X J n 0 Cx 0 cc CO CJ x 0 C CD o o o XJ o 0 a CL □ a: c c Cx 0 m Cx 0 CQ T) to o XJ Cx a XJ c co o CL 0 x 0 XJ D X CO CJ XJ C 0 o >N CL X 0 0CO TJ 0 Cx 0 C 0 C o XJo X «E 2 0 a 0 c ’eU Cx LfN 0 0 TJ < •5 CL O cn CO o o 0 c 0 c c-> Cx "a XJ CJ a . c TJ 0 O Cx 0 IXJ a CL 0 0 CO JZ CO Q) 0 0 > CO > 0 JJ a D. Z XJ o "n OT o t | N CO TJ XJ O < CD E cj CJ CL o £ f l . 0 0 CL i— <—i •H O Cx 0 I n ' CO D 0 0 > .2 < *0 *0 D 0 CJ § 1 1 c XJ XJ ‘o o I - I VC I cn VC h- o 0 Cx h r XJ 0 a O U I 0 C o 0 XJ X Cx 0 c i CJ XJ 0 o co c ii 0 0 c C co 0 LA LJ ’S. XI 0 ntagonist drugs given in parentheses indicate only partail antagonism obtained. o LJ < is 0 cn N XJ > l»N c O C u 0 0 N 0 CD 2 £ CHAPTER 2

Antinociceptive Activity of Morphine, Fentanyl and Alfentanil

in Mice and Rats:

Effect of DFP and Drugs Used in the Treatment of DFP Poisoning INTRODUCTION

2.1. Antinociceptive Models

There are many tests available to determine the antinociceptive activity of

drugs. The majority of these tests utilize the response to experimental pain

in animals (Linebery, 1981). Animal testing can only serve as a model of

human clinical pain as the pain response in man is a subjective parameter

with large variability between individuals. Extrapolation from animal data to

humans must be viewed with caution as false negatives and positives may be

encountered.

Wood (1984) presented a list of five criteria for consideration when

interpreting data from animal screening tests:

1 Test Validity: Is the test predictive of the clinical utility of known

analgesics?

In considering the most appropriate antinociceptive test in this

study, these points were taken into consideration. It was decided to

employ the hot-plate test (Woolfe and MacDonald, 1944) for mice

and the paw pressure test (Randall and Selitto, 1957) for rats.

2 Test Reliability: Is the test response reproducible throughout an

experimental day and from day to day?

3 Test Simplicity: Does the procedure use a standard defined noxious

stimulus, measure a well-defined animal response, and is the test

easily utilized? The mouse hot-plate is an extensively used procedure and unlike the tail flick test does not rely on a reflexive response. The paw lick response in the hot-plate test relies on integrated behaviour involving processes occuring at a high level within the central nervous system. The rat paw pressure test was chosen to determine antinociceptive drug activities in an additional species using a different noxious stimulus, and in this test the responses are not reflexive in nature.

Antinociceptive responses are subject to wide variations, so care was taken to account for and control environmental variables.

Test S ensitivity: Can d iffe re n t analgesic doses be discrim inated and can the procedure detect weak and agonist/antagonist analgesics?

The paw pressure test, but not the mouse hot-plate test is sensitive to agonist/antagonist analgesics. In this study, this would not be so important as only strong analgesics were investigated.

Data Analysis: Varies between laboratories. Is the method of analysis appropriate? Methods of analysis should be described.

The analysis of the data was given full consideration, and a model was developed to accurately fit the form of the observed data (see

Section 2.10.). 2.2. Initial Experiments

Ample evidence has been presented in Section 1.6. for the role of cholinergic agents in opioid antinociception. The initial studies, which are presented in this section, were designed to determine the interactions between the anticholinesterase agents and opioid antinociception in the models described above. MATERIALS AND METHODS

2.3. Animals and Experimental Conditions

Male albino mice (CD-I strain; 25-30g) were purchased from Charles River.

Male albino Wistar rats (Surrey University strain; 140-210g) were bred at the

University's Animal Unit. Upon delivery, animals were housed in the Animal

Unit under controlled temperature, humidity and lighting conditions (12h light/dark cycle; lights on 0700h). Mice remained in the Unit for at least 5 days before use. Twenty-four hours before experimentation, the animals were equilibrated in a quiet laboratory where all procedures were carried out.

Animals had free access to food and water up to experimentation. All experiments were carried out between 0900h and 1300h.

The quiet laboratory (3.3m x 3.5m x 2.75m high) was windowless and had the same lighting conditions as the Animal Unit. The room was ventilated but had no independent heating or humidity controls. Ambient temperature was monitored during experimentation, and stayed within the range 20-25°C throughout the year. So as to minimise disturbance there was restricted access to the room before and during experimental procedures. 2.4. Drugs

The follow ing drugs were used:

DRUG NAME SOURCE

Di-isopropylfluorophosphate Sigma

Atropine sulphate Sigma

Morphine sulphate Macfarlan Smith

Fentanyl citrate Janssen Pharmaceutical*

Alfentanil hydrochloride Janssen Pharmaceutical*

Pralidoxime methyl sulphonate CDE, Porton Down*

Diazepam Roche*

Neostigmine bromide Sigma

Pyridostigmine bromide Roche*

Naloxone hydrochloride Endo*

* These compounds were generously donated by the manufacturers.

Drugs were dissolved in 0.9% saline (Travenol) immediately prior to use, with the following exceptions: in some experiments DFP was dissolved in peanut oil (Sigma), and as diazepam is not soluble in saline it was first dissolved in polyethylene glycol 200 (PEG) to which an equal volume of saline was added to give the correct drug concentration. Great care was taken in handling

DFP due to its highly toxic nature. The supplied Safety Data Sheet was used as a guideline to handling procedures outlined below:

2.5. Safety Procedures for DFP

DFP was stored, refrigerated at about 4°C, and kept within the lidded metal container in which it was supplied. The vial containing DFP was handled only in a fume cupboard, with the user wearing gloves and an overall with tight cuffs. Appropriate quantities of DFP were obtained by penetrating the seal with a

fixed needle Hamilton syringe and withdrawing liquid. The DFP was

immediately transfered to a volumetric flask to which saline was added. The

flask was shaken thoroughly for at least 2 minutes to ensure that the DFP had

dissolved. The fume cupboard also contained a bucket containing 2% sodium

hydroxide. Any accidental spillages or contaminated equipment could be neutralised in copious quantities of the sodium hydroxide solution.

DFP degrades with time due to hydrolysis (Leopold and Krishna, 1963) and therefore stock was replaced every six months. In addition, alfentanil potentiating a c tiv ity was confirm ed (see Section 2.13.).

2.6. Dosing Protocols in Mice

Mice were scruffed and drugs were injected in 0.1ml volumes subcutaneously

(s.c.) in the back of the neck, with the exception of diazepam and neostigmine. Diazepam was injected in a 0.03ml volume intramuscularly

(i.m.) into the hind leg, because of a local interaction with DFP (D. Green,

CDE, personal communication), and neostigmine was injected intraperitoneally (i.p.) in 0.1ml volumes.

Drug dosing protocols are illustrated in Figure 2.1. When used, atropine

(17mg/kg) and pralidoxime (15mg/kg) were mixed and injected together 1 minute after DFP, according to Gordon et al (1978). Diazepam (6.25pmol/kg) was injected immediately prior to the atropine/pralidoxime dose (D. Green, personal communication). zUJ _J _ l < Z CO < < 'DC f t CD o 0. z Z UJ ft CL LL LL f t < CO _1 UJ O < Q < ft <3> <3£> <3> 1L_ i 1 H i— LO O CO o LO CO • 0 c O -P E G II G ra LU •H 0 0 0 s H 7 3 f t o 0 G 0 •H •H i—I O G 0 ft 0 G 0 0 •H 0 ft 0 hC 73 hC O G 0 G5 •H P 0 -P UJ ft 0 P 0 ft G •> O 0 0 0 LO -J E-i •H E 0 < < > G g p i i CO cr z CD 0 •H f t G L_ CL < > E 0 G 0 o •H G N H £ :NJ ft -P G G •H CL z CO a •H ft UJ UJ 0 0 G O Q < 0 u. O CO P P ft •H G <3> <3c> o <3> O 0 G II I 1--- H- —I o o O G G 73 CO G •H 0 •H c LO CO •H i—I ~ s o ft G 0 -H •H LO E G 0 S ft ftiO < o LU X P P 0 G 0 O O -H ft 0 X3 hJC'H £ P rX G *H P J3 .G o hC ft ftP P o P -H G UJ o pH 73 O £ -p ^ G £ ^ o G 73 0 UJ p ft 0 0 P < —I ft ft 0 f> P O CO < z CD H O 0 P Z Q G - £ X he •H 0 0 -O G 0 Q. CL ft G a O -r-3 fl G N X CO •H 73 O 0 Q. X O 111 0 •H p p U . h — O O P Q < Q ft Q <3> CO <3> <3£> o —I 1----- 1--- —f t I 1 o •H II O rH CO O i- CO G c CO rH O 0 >s G 0 G G • G P E 1 0 p G G *H G 0 P 0 ft UJ be 0 P G f t •H O G G 0 ft ft ft ft ft rH f t G G For the neostigmine experiments, either saline or neostigmine were administered (i.p.) at time 0, alfentanil 25 minutes later, and antinociceptive testing carried out at 30 minutes.

Pyridostigmine (lmg/kg) was administered 1 hour before antinociception. The times for the injection of morphine, fentanyl and alfentanil were 30, 50 and

55 minutes after pyridostigmine, respectively.

In those experiments when naloxone was used, it was administered 30 minutes before testing.

2.7. Dosing Protocols in Rats

Rats were scruffed for s.c. injection (0.1ml volumes) in the back of the neck.

DFP was administered at time 0 and morphine, fentanyl or alfentanil administered 30, 50 and 55 minutes later respectively. Each animal was tested four times; once immediately before the first injection and again at three times to include peak antinociceptive effect. The timings for

\ injections and for post-drug testings are given in Fig 2.2. a p CD O z o 0 •H •n < O £ H- o •H £ i_ m •H CO ° 2 P d LUh-H £ Q— I LLC0C0 C0 d 0 —I LULU LU £ \k< < t - h - h- d •H Q CO 0 .£ £■ » £ I------— 1 1 l— f O CO lO N - O 10 C 1 0 1 0 (0 CO E LU

>> -P >! •H 0 > S i •H P > - P Z o n d *h u-LLI < O °2 K" h" H d a ZC 0 CO CO o o Q___I LULU LU UJ l l < O O LL h- H h- i- 1 *H QCO O o £ 1 < 1 d o m o O S co £ m m cd N- d c r£ P a £ 0 wE O £ LU 0 CO e. 0 0 rH T3 •H o LU £ z a 0 a l-LU X •H CL -P ° Z h- I- I- X -P 0 ___I CO CO c o £ o LU LU LU 0 «H U - < £ «H QCO I- H I— 0 -H

0 minutes after DFP, and animals were tested 20, 30 and 60 minutes after receiving the opioid. Fentanyl •H +-> 0 «H bD 0 o •H O £ X!

The hot-plate test (Woolfe and MacDonald, 1944) was employed for

antinociceptive testing. The hot plate was made in the Biochemistry

workshop, University of Surrey, and consisted of an aluminium plate (30cm

xl5cm) surrounded by a transparent perspex wall (22cm high). This was

placed in a thermostatically controlled water bath with the water maintained

at 33°C. The same apparatus and tem perature setting was used throughout

all experiments.

When tested mice were placed on the hot-plate and a hand-held digital

stopwatch started as soon as the mouse touched the plate, the nociceptive

end-point was taken as paw-licking (in most cases this was the fore-paw) or jumping on the few occasions when the licking response was absent. If the

mice failed to respond after 60 seconds they were removed from the plate

and scored as >60 seconds. As the walls of the hot-plate were perspex and unheated, occasionally some mice spent long periods on their hind paws with

fore-paws resting on the walls; these mice were excluded.

2.9. Antinociceptive Testing in Rats

The non-inflamed paw pressure test was employed for nociceptive determinations (Randall and Selitto, 1957) using an Ugo Basile Analgesy meter

(A.R.Horwell, London). This apparatus (Fig. 2.3. ) consists of a plastic pusher

(P) positioned about 2mm above a Teflon plinth (B). Animals were held firmly, but not tightly around the upper part of their body, so they could be held in the correct position without the animal struggling. The animals paw, was placed under the pusher and subjected to a force increasing linearly with time as a pivoted weight (W) moved horizontally. A foot-pedal controlled the M i mm 'M: s m v

'* ' w4

Basile AnalgesS Met

■ fig weight's movement which v/asmarked off on a graduated scale (SC). The score on the graduated scale was converted to the force applied in grams by m ultiplying by 30.

The nociceptive end-point was taken as a withdrawal or attempted withdrawal of the animal's paw, or when the animal struggled. With practice it was possible^ distinguish between struggling in response to the noxious stimulus and non-specific struggling. Both hind paws, in turn, were placed under the pusher and a mean value taken as the score for that animal. To avoid damage to the animal's paws, a maximum cut-off weight of 750g was employed. Those animals which did not respond before the 750g cut-off was reached were scored as 750g.

2.10. S ta tistica l Procedures

2.10.1. H ot-P late Data

Four doses for each opioid were used to obtain a dose-response curve. Each point was the mean of 12 or 18 observations made on 2 or 3 separate days.

The dose-response relationship was represented by an AD^-g value, which was defined as the dose required to increase the hot-plate latency to 30 seconds

(ie., 30% of the maximal response time). Unpaired Student's t-test (see

Appendix A) was used to.compare AD<-g values.

All dose response curves included censored values, that is mice failing to respond before the 60 seconds cut-off, which were scored as >60 seconds.

Though employing a cut-off time is essential to avoid inducing tissue damage in the animal's paws, it does distort the dose-response relationship. However, this distortion may be compensated for by using appropriate mathematical models. Such a model was applied to the data to obtain AD^g values and is described in Appendix B. The Fortran listings for the test procedures (run on the Prime computer operating system) are given in Appendix B. 2.10.2. Paw Pressure Data

A mathematical model was applied to data giving the time course of drug effect to obtain values for peak responses for each dose. Comparison between groups was by unpaired *t’ test. Results were normally mean values of at least six animals. To obtain a value for peak response, a quadratic equation was fitted to the data: y = a - b (x-c) , where: a = peak response score, b = curvature of line and c = time of peak response RESULTS

2.11. Pilot Studies

Repeated exposure to antinociceptive testing procedures of naive mice (ie., non-injected) was assessed on the hot plate (Table 2.1.). Re-testing the same mouse resulted in significantly increased latency. All subsequent antinociceptive data presented represent latencies from mice tested once only.

Table 2.2. shows the effect of repeated saline injection on nociceptive responses of mice. There were significant changes (eg., single injection 11.7 -

0.73, triple injection 9.2 - 0.85, t-test p<0.05), but these were quantitatively small.

2.12. Effect of DFP on Nociception in Mice

Table 2.3. shows that DFP at lmg/kg did not exhibit antinociceptive activities at the times studied. However, at a 2mg/kg dose a 2- to 3-fold increase in hot-plate response time was observed over this period. At this higher dose, mice exhibited symptoms of anticholinesterase poisoning, such as tremors and fasiculations. Mice showed hind-limb abduction and impaired and reduced ambulation.

When peanut oil was used as the vehicle fo r DFP (Table 2.4.) no alteratio n in hot-plate response time was observed compared with saline.

The effect of the opioid antagonist, naloxone, and the muscarinic cholinergic antagonist, atropine, on the antinociceptive activity of DFP at 2mg/kg is shown in Table 2.5. Table 2.1. Effect of Repeated Exposure to Antinociceptive Testing

Procedures on Hot-Plate Times of Naive Mice

H ot-P late Latency (Secs)

One exposure 9.3 - 2.0

Second exposure (+ 30 mins) 17.0 - 3.3

Third exposure (+ 4 hours) 16.8 i 2.0

Each value represents the mean - SEM of 12 observations. Table 2.2. Effect of Multiple Saline Injections on Hot-Plate Response Time in Mice

H ot-P late Latency (Secs)

Number of Time from last injection to test

Injections

30 mins 10 mins 3 mins

1 11.7 - 0.7 (56) 10.3 - 1.1 (12) 9.6 - 0.7 (18)

2 9.3 - 0.8 (12) 14.6 - 1.4 (6) 11.1 - 1.6 (18)

3 9.2 - 0.8 (17) 10.4 - 1.2 (11) 9.3 - 1.0 (12)

Each value represents mean - SEM. Number of observations are given in parentheses.

Mice receiving two injections were first injected 60 minutes before testing, and those receiving three injections had their first two injections 60 and 59 minutes before testing. Table 2.3. Effect of DFP on Hot-Plate Reaction Times in the Mouse

H ot-P late Latency (Secs)

Time after Injection

0.3 hours 1 hour 2 hours 4 hours

Saline 11.9 - 1.6 13.2 - 1.1 12.7 - 1.5 11.7 - 1.0

DFP (lmg/kg) 9.7 - 0.8 9.4 - 0.7 11.8 - 1.2 12.0 - 0.9

DFP (2mg/kg) 28.4 - 5.6* 19.0 - 2.0* 18.7 - 2.8 33.0 - 8.7*

Each value represents mean - SEM of at least 6 observations. Unpaired t- test vs. saline *p<0.05. Table 2.4. Effect of Different Vehicles on the Antinociceptive Activity of DFP

H ot-P late Latency (Secs)

Vehicle used lm g/kg 2mg/kg

Peanut Oil 9.8 - 1.1 9.2 t 2.3 18.6 +4.5

Saline 9.4 - 0.7 19.0 - 2.0

Each value represents the mean - SEM of 6 observations. Table 2.5. Effect of Atropine and Naloxone on 2mg/kg

DFP Induced Antinociception in Mice

Treatm ent H ot-P late Latency (secs)

Saline 33.9 - 5.9 (13)

Naloxone 0.1 mg/kg 26.0' - 4.9 (12)

1.0 mg/kg 29.9 - 7.5 (6)

10 mg/kg 33.6 - 5.0 (12)

Saline 28.1 - 2.6 (6)

Atropine 1 mg/kg 16.4 - 2.1 (6)*

10 mg/kg 16.2 - 3.8 (6)*

20 mg/kg 12.1 - 0.9 (6 )**

Each value represents mean - SEM. Number of observations are in parentheses. Unpaired t-test vs saline *p<0.005 **p<0.001. Doses of naloxone between 0.1 and lOmg/kg had no effect on the hot-plate reaction times of mice receiving DFP (2mg/kg). However, when mice received atropine (1 to 20mg/kg) hot-plate reaction time was significantly reduced. A 20mg/kg, response times were not significantly different from mice receiving saline injections.

2.13. Effect of DFP and Drugs Reversing the Effects of DFP on Opioid

Antinociception in Mice

The antinociceptive activities of morphine, fentanyl and alfentanil are shown in Table 2.6. Activities are represented in terms of computed ADj-g values as a measure of antinociceptive potency.

In mice pre-treated with lmg/kg DFP the AD^g values of morphine and fentanyl were unaltered. However, a two-fold increase in the antinociceptive activity of alfentanil (as shown by a reduced AD^g), was observed following

DFP pre-treatment.

The atropine-pralidoxime mix was administered as a treatment for DFP poisoning. The effect of DFP on the antinociceptive activities of fentanyl and alfentanil was unaltered by the atropine-pralidoxime mix treatment, however, an increased antinociceptive activity of morphine (denoted by significant decreases in AD^g) was observed by this treatment.

Diazepam was used as an additional component in the treatm ent of DFP poisoning. No significant changes in AD^g values for the opioids were observed following additional administration of diazepam (Table 2.6.). Table 2.6. Effect of DFP (Img/kg) and of Drugs Reversing DFP

Poisoning on Opioid-Induced Antinociception in Mice

A D cn Values

Treatment Morphine Fentanyl Alfentanil

(mg/kg) (gg/kg) (gg/kg) .

Saline 20.7 - 1.9 66.4 - 6.7 329.7 - 23.3

DFP 24.7 - 2.5 52.5 - 5.6 164.3 - 11.0**

DFP + Atropine + 12.0-1.0* 54.3 - 5.9 175.0 - 1 5 .0 **

Pralidoxime

DFP + Atropine + 10.7 ± 1.7* 53.3 ± 8.3 207.3 ± 3 3 .0 **

Pralidoxime + Diazepam

AD^g values are the mean - SEM (of at least 9 observations). AD^g values are doses required to increase hot-plate reaction time to 30 seconds and are calculated from the full dose response curves for each agonist. Normal deviate for comparison of means vs. saline **p<0.01; ***p<0.001. 2.14. Effect of Atropine and Pralidoxine and Diazepam on Nociception

As a control, atropine-pralidoxime mix, and diazepam were administered alone, to determine their effect on mouse hot-plate latency.

When the atropine-pralidoxime mix was administered at 1 minute, with saline given at 0 and 30 minutes, testing at 60 minutes gave a response time of 10.5

- 0.7 seconds (n=12), which is not significantly different from saline control values.

The effect of diazepam on nociceptive responses is shown in Table 2.7.

Diazepam did not affect hot-plate response times, even at doses which produced clear motor incapacitation.

Using a PEG/saline injection vehicle o£ injecting saline i.m. did not give hot plate reaction times significantly different from saline s.c. (Table 2.7.)

2.15 Effect of Neostigmine and Pyridostigmine on Opioid Antinociception

The effect of the quaternary anticholinesterase agents neostigmine and pyridostigmine opioid antinociception is shown in Tables 2.8 and 2.9.

Neostigmine did not produce any significant potentiation of alfentanil over the dose range studied. Pyridostigmine pre-treatment did not significantly alter AD^g values for the opioids studies, though there was a small enhancement of alfentanil antinociception (p=0.105). Table 2.7. Effect of Intramuscularly-Administered Diazepam

and Injection Vehicle on Hot-Plate Reaction Times in Mice

Treatm ent Hot-Plate Response

Time (Seconds)

Saline 8.7 - 1.3 (12)

PEG/Saline 8.2 - 0.5 (6)

Diazepam 6.25 pmol/kg 8.7 - 1.6 (6)

Diazepam 12.5 pmol/kg 10.2 - 2.1 (6)

Values show means - SEM of hot-plate response times. Number observations is shown in parentheses. PEG = polyethylene glycol. Table 2.8. Effect of Various Doses of Neostigmine on the

Antinociceptive Activity of Alfentanil

Alfentanil AD,-g (gg/kg)

Saline 143 - 18

Neostigmine 50gg/kg 146 1 13

Saline 178 - 13

Neostigmine lOOgg/kg 170 - 11

Saline 155 - 14

Neostigmine 200gg/kg 128 - 14

Values are mean + SEM of 6 observations on hot-plate reactions times.

Control (saline) AD^g values were obtained on the same day for each dose of neostigmine. Table 2.9. Effect of Pyridostimine (Img/kg) On Antinociceptive

Potencies of Morphine,Fentany 1 and Alfentanil

AD^g Values

Treatm ent Morphine Fentanyl A lfe n ta n il

(mg/kg) (pg/kg) (pg/kg)

Saline 16.8 37.6- 1.3- 4.3 220 - 17.9

Pyridostigmine lm g/kg 16.1 - 1.4 36.7 - 2.8 172 - 18.9

Values are mean - SEM of 6 observations on hot-plate reaction times.

Control (saline) AD ^q values were obtained on the same day for each opioid. Table 2.10. Naloxone Antagonism of Antinociception Induced by

Alfentanil With and Without DFP

Treatm ent Naloxone a d 50+s e m Dose Ratio Slope of Apparent pA2 values

(pg/kg) pg/kg pA2 plot (95% confidence interva

A lfe n ta n il 0 0.22 - 0.02

10 0.26 - 0.03 1.18

100 0.57 - 0.07 2.59

1000 3.20 - 0.30 14.55 -1.00 8.30 (8.26 - 8.35)

DFP + 0 0.13 - 0.07 -

A lfe n ta n il 10 0.25 - 0.06 1.92

100 0.56 - 0.06 4.31

1000 3.30 - 0.09 25.39 -0.99 8.95 (6.30 - 11.60)

AD^g values are the mean doses - SEM required to increase hot-plate reaction time to 30 seconds. 2.16 Antagonism of Alfentanil, Alone and With DFP, on Naloxone

The effect of naloxone antagonism on the antinociceptive activity of

alfentanil and the effect of naloxone on DFP plus alfentanil antinociception

is shown in Table 2.10.

Naloxone antagonism was measured as 'apparent pA£' according to Hayashi

and Takemori (1971). The pA 2 value represent the affinity for the antagonist

naloxone for its receptor. The values obtained for 'apparent PA 21 were very

similar indicating that the compounds were acting through the same

receptors. The slope of the pA 2 plot were very similar to the theoretical

value of -1 for competitive antagonism.

2.17. Effect of DFP upon Nociceptive Responses in the Rat

Figure 2.3. shows the time course for the effect of DFP administration on the nociceptive responses of the rat in the paw pressure test.

Saline base-line responses remained constant over the time studied and with repeated testing. At lmg/kg, DFP did not affect the nociceptive response.

However, there were significant (p<0.001) increases in response thresholds fo r all the time points studied following DFP at 2mg/kg (Fig 2.3.) At the

2mg/kg dose DFP produced clear signs of anticholinesterase poisoning, such as marked tremors. However, the leg withdrawal and struggling responses were not impaired. 2.18 Effect of DFP Pre-Treatment on Opioid Antinociception in Rats

The e ffe c t of the sub-antinociceptive dose of DFP (Img/kg) on the antinociceptive activities of morphine, fentanyl and alfentanil are shown in

(Figs. 2.4., 2.5. and 2.6.). DFP pre-treatment did not affect the antinociceptive activity of morphine, but both fentanyl and alfentanil were significantly potentiated at low doses of these opioids, (p < 0.01, unpaired t- test). RESSURE(g) 00

0 0 -

00 -

0 0 -

00 -

y y Pre ao 45 60 90 TIME(mins)

Fig. 2.3. Time Course for the Effect of DFP on the Nociceptive Responses in the Rat Paw Pressure Tests

Closed circles: saline treated animals. Open triangles: DFP (lmg/kg) treated animals. Closed triangles: DFP (2mg/kg) treated animals. Points represent means and SEM of 10 observations. PRESSURE(g) 8OO1

700-

100 -

2.5 20 DOSE(mg/kg)

Fig. 2.4. Effect of DFP on Morphine Antinociception in the Rat Paw Pressure Test

The abscissa shows dose for morphine (s.c.) in mg/kg Closed circles: control treated. Open circles: DFP (lmg/kg) treated. Each histogram represents mean and SEM of 10 observations.

Means represent response at peak.antinociceptive times PRESSURE(g)

800 - i

700

600

500-

400-

300-

2 0 0 -

100 -

5h T5o 260 400 DOSE(jjg/kg)

Fig. 2.5. Effect of DFP on Fentanyl Antinociception in the Rat Paw Pressure Test

The abscissa shows dose for fentanyl (s.c.) in yg/kg. Closed circles: control treated. Open circles: DFP (lmg/kg) treated. Each histogram represents mean and SEM of 10 observations.

I Means represent response at peak antinociceptive times PRESSURE(g)

700-

600-

500

400-

300

200

100

20 40 80 DOSE(|jg/kg)

Fig 2.6. Effect of DFP on Alfentanil Antinocoception in the Rat Paw Pressure Test

The abscissa shows dose for alfentanil (s.c.) in yg/kg. Closed circles: control treated. Open circles: DFP (lmg/kg) treated. Each histogram represents mean and SEM of 10 observations.

Means represent response at peak antinociceptive times DISCUSSION

2.19. Drug Injection Times

The choice of injection times and initial doses of drugs were determined from pilot experiments or were based on available literature.

Neostigmine administration (30 minutes before antinociceptive testing) and in itia l dose were based on studies of Lawrence and Livingstone (1979).

Pyridostigmine has a longer duration of action than neostigmine (British National

Formulary, 1983) and so for these studies pyridostigmine was injected one hour before testing. A high dose of lmg/kg O-D^g = 1.3mg/kg mice; Mestinon data sheet) was used so th a t any e ffe c t would more likely be revealed.

Naloxone was administered 30 minutes before testing, a time generally given in the literature (eg, Koehn and Karczmar, 1978).

2.20. Effect of DFP on Nociceptive Responses in Mice and Rats

Though at lmg/kg DFP did not appear to show any appreciable antinociceptive activity in the mouse hot-plate test, at 2mg/kg the increase in hot-plate reaction time was marked. This apparent antinociceptive activity of DFP was long- lasting. However, at this higher dose mice showed pronounced signs of anticholinesterase poisoning, including tremors and fasiculations, decreased ambulation and apparent sedative ataxia. The nociceptive response in the hot plate test involves the animal lifting and licking one or more of its paws. As this response requires a complex set of co-ordinated non-reflexive movements, it is possible that the apparent antinociceptive effect observed for DFP at 2mg/kg was due to impaired muscular function. This impairment could be of central origin, eg, disruption of control of movement or peripheral origin, eg, muscular weakness, or indeed, both.

It has been reported that in rats at 2mg/kg s.c. DFP caused hind-limb abduction

(Fernando et al, 1984) and at 2.2mg/kg i.m. the animals appear behaviourally depressed and lethargic (Samson, 1984). Even at doses below lm g/kg, DFP is reported to produce physical incapacitation (Harris and Stitcher, 1984). It is not unreasonable to presume from observations in this study and the available literature, that DFP administration could impair the nociceptive response without necessarily possessing antinociceptive activity. The effect of DFP on the locomotor activity of mice and the implications in nociceptive testing is given full consideration in Section 3.13.

The data presented in this thesis are consistent with the findings of Cox and Tha

(1972) who did not observe any antinociceptive for DFP up to 2mg/kg in the mouse hot-plate test.

In contrast, studies in the rat indicate that DFP is antinociceptive in this species. Doses as low as 0.lmg/kg s.c. have been shown to induce antinociception in the rat hot-plate test (Koehn et al, 1980) and 0.5mg/kg s.c. in the rat tail flick test (Koehn and Karczmar, 1978). The results presented in this thesis show that

DFP at 2mg/kg produced marked antinociception in the paw pressure test.

Though this antinociceptive dose is much higher than that reported by Koehn et al (1980), this may be a reflection on the use of pressure as the nociceptive stimulus. The nociceptive response to pressure is believed to be predominantly mediated through k -opioid receptors, whilst heat noxia are believed to act through p-opioid receptors (Tyers, 1980; Upton et al, 1983). The observation that naloxone up to lOmg/kg did not alter the reaction time

following administration of DFP (Table 2.3.) suggests that the apparent antinociceptive activity of DFP. is not mediated through an opioid system.

Atropine, however, did antagonize the increased latencies, and at 20mg/kg totally reversed DFP's effect. The apparent antinociceptive activity is therefore only mediated via muscarinic receptors and this contrasts with effects in the rat where both opioid and muscarinic receptors mediates its action (Koehn and

Karczm ar, 1978).

Clear species differences emerge, with mice being insensitive to an antinociceptive action of DFP whilst in rats, DFP may product potent antinociception.

2.21. Effect of DFP on Morphine Antinociception

DFP had no effect on morphine antinociception (Table 2.6.) and this contrasts w ith the findings of Bhargava and Way (1972). They reported potentiating activity of DFP upon morphine antinociception, though it should be stressed that these effects, while consistent, failed to reach statistical significance. The possibility of vehicle-related effects must be entertained since Bhargava and

Way (1972) used peanut oil for DFP adm inistration and 0.9% saline was used in the experiments described in this thesis. However, though peanut oil has been shown to induce sedation (Pinsky et al, 1982) no antinociceptive effects for DFP in this vehicle could be shown (Table 2.4.) and this possibility can be realistically excluded. At a dose of 0.2mg/kg, Saxena (1938) found that DFP did not potentiate morphine in the rat tail clip test. Though there are many reports of cholinergic drugs potentiating opioid antinociception (see Table 1.2.) the available data appears to militate against a potentiating effect of DFP in morphine-induced antinociception.

2.22. Effect of DFP on Fentanyl and Alfentanil Antinociception

Though the activity of the synthetic opioid analgesic fentanyl was not found to be potentiating by DFP in the mouse hot-plate test, the structurally-related compound alfentanil was potentiated two-fold (Table 2.6.).

In addition, alfentanil antinociception in the rat paw pressure test appeared to be potentiated by DFP pre-treatment. Though this potentiating effect was only observed at low doses of alfentanil, it seems likely that employment of a cut-off weight masks potentiation at higher doses. This problem was also encountered in the hot-plate data (see Section 2.11.1).

Fentanyl also appeared to be potentiated in rats by DFP in the same way as alfentanil. So alfentanil is potentiated by DFP in both mouse and rat antinociceptive test, though fentanyl was only potentiated in the rat. This could be a reflection of different nociceptive mechanisms in the rat compared to those in mice. 2.23. Proposed Mechanisms for DFP Potentiation of Alfentanil

Though morphine, fentanyl and alfentanil are all primarily u-opioid agonists,

DFP has been shown in this study to have a differential potentiating effect on these opioids. There are several ways by which DFP could potentiate the activity of opioids; these are alterations in absorption, distribution, receptor action, metabolism or excretion.

2.23.1. Absorption

There are a number of factors which affect absorption of drugs from s.c. injection sites. A major factor is cutaneous blood flow and DFP treatment is believed to decrease circulatory rate (Ramachandran, 1967). In addition, DFP dose dependently induces hypothermia (Kenley, et al, 1982) which would decrease peripheral blood flow. These factors would tend to reduce drug absorption from a s.c. site and furthermore would not discriminate between the opioids studied.

It is also probable that drug absorption is not a limiting factor for fentanyl and alfentanil. Both these drugs are lipid-soluble with octanokwater partition coefficients of 860 and 130 for fentanyl and alfentanil respectively (Meuldemans et al, 1982). Morphine has low lipid solubility with an octanokwater partition coefficient of 1.42 (Kaufman et al, 1975). A differential interaction between

DFP and alfentanil at the site of injection is therefore unlikely.

2.23.2 Distribution

Many drugs are bound to plasma proteins and may compete for the same binding sites. Such competition could result in displacement and altered pharmacokinetic behaviour of one drug. This type of drug interaction would be particularly important for drugs which have high fractional binding (see

MacKichan, 1984 for review). Both fentanyl and alfentanil have high fractional binding to blood proteins

(Meuldemans, 1982) as does DFP (Martin, 1985). It is possible that DFP could

displace alfentanil from binding sites, allowing more alfentanil to enter the

brain. This possibility was studied and is described in Chapter 5.

2.23.3 Receptor Action

DFP may act at the receptor level to increase the potency of alfentanil. For

example, DFP may be producing opioid receptor super-sensitivity to alfentanil.

Pilot binding studies using alfentanil were performed, but were inconsistent.

This is most probably due to the very rapid dissociation from the receptor site

that has been reported for this ligand (Leysen et al, 1983).

In fact, an interaction between DFP at the receptor level would not explain the

differential action of DFP on alfentanil, since all three opioids studied exert

pharmacological effects through p-opioid receptors (Upton et al, 1983; Leysen et al, 1983).

2.23.4. Metabolism

In addition to being a potent inhibitor of cholinesterase, DFP may well inhibit other enzymes including those involved in drug metabolism (see O’Neill, 1981).

For example, a significant inhibition of mitochondrial aldehyde dehydrogenase occurs for at least 85 days following administration of 0.5mg/kg DFP (Messiha, et al, 1983).

DFP potently inhibits the enzymes hydrolysing substance P (Kato et al, 1980) and metabolising enkephalins (Snead et al, 1980). Alfentanil is rapidly metabolised following i.v. administration, with 50-60% metabolised after 4 minutes (Michiels et al, 1981) by N-dealkylation and O-demethylation (Meuldemans, 1980). However the termination of alfentanil's action is predominantly dependent on a redistribution mechanism (Stanski and Hug, 1982). It is conceivable that DFP could inhibit the enzymes responsible for the metabolism of alfentanil, thereby increasing its antinociceptive activity. However, it must be borne in mind that antinociceptive testing was carried out five minutes after alfentanil administration and over such a short time period, DFP would have to be a very potent inhibitor to produce a two-fold potentiation of alfentanil.

2.23.5.Excretion

If, as mentioned above, redistribution is the main means of termination of action, an interaction between DFP and alfentanil in excretion processes would probably not affect alfentanil action. Moreover in rats only 0.2% of the parent drug is excreted in the urine (Meuldemans, 1980b), and so it is unlikely th a t impaired renal excretion would have any effect on the antinociceptive activity of alfentanil.

2.24. Effect of Atropine and Pralidoxime on DFP Potentiation of Alfentanil

Antinociception

The potentiating effect of DFP on alfentanil antinociception may be independent of an action on the cholinergic system as the administration of the muscarinic receptor antagonist atropine and pralidoxime to regenerate cholinesterase enzymes did not alter the A D ^q value for DFP plus alfentanil. However, pralidoxime is unable to cross the blood-brain barrier, so that brain cholinesterase would remain largely inhibited by DFP even after pralidoxime administration. Though atropine would block muscarinic receptors, it is possible that an interaction between DFP and alfentanil could involve unprotected central nicotinic receptors. Other possibilities regarding the nature of DFP and alfentanil interaction are given in Section 4.11. 2.25. Effect of Atropine and Pralidoxime on the Antinociceptive Activity of

Morphine

DFP alone did not affect the AD^g for morphine, but when atropine and

pralidoxime was also administered morphine's antinociceptive activity was

enhanced. As mentioned in Section 2.24., the atropine-pralidoxime mix does not

protect central nicotinic receptors and so it may be that by blocking the

muscarinic receptors with atropine, more of the non-metabolised acetylcholine would be able to interact with nicotinic receptors. In other words, the presence of a muscarinic antagonist in a cholinesterase inhibited system could lead to increased nicotinic response. In consideration of this, it is worth noting that there have been several studies in mice, ra t and dog showing potent antinociceptive a c tiv ity of nicotinic agonists (see Table 1.3.).

This interaction, however, would have to be specific for morphine and the nicotinic response since neither fentanyl or alfentanil had altered AD^g follow ing atropine and pralidoxim e.

An alternative explanation for these findings is that morphine may be interacting with atropine. Indeed, it has been reported that in the rat hot-plate test, atropine at 2mg/kg significantly potentiated the antinociceptive activity of morphine (Malec and Langwinski, 1982). This report also notes that atropine did not alter the antinociceptive activity of fentanyl or of pentazocine. The results of this study showing differential potentiation of morphine antinociception by atropine are supportive of the results presented in this thesis.

An additional mechanism for the potentiation of morphine by atropine which should be considered is that respiratory alkalosis enhances the percentage of unionised morphine in the circulation and thereby increasing the amount entering the brain (Schulman et al., 1984). It is possible that the additional administration of atropine and pralidoxime produces a degree of respiratory alkalosis, which through pharmacokinetic mechanisms potentiates the antinociceptive activity of morphine plus DFP.

2.26. Effect of Diazepam on the Antinociceptive Activity of Opioids in Mice

Receiving DFP, Atropine and Pralidoxime

Diazepam administered alone did not produce any increase in hot-plate latency in the mouse. Diazepam has, in fact, been reported to possess antinociceptive activity in humans (Haas et al, 1979) though at doses much higher than those described in this thesis.

Diazepam has been shown to both reduce (Mantegazza, 1982) and to potentiate

(Matla and Langwinski, 1982) the antinociceptive activity of opioids including morphine and fentanyl. In this study, co-administration of diazepam did not alter the AD^g values of the three opioids studied. It should be realised, however, that the animals were receiving four different drugs in addition to the opioid, so that any effects of diazepam on antinociceptive activities could be obscured by non-specific drug interactions.

2.27. Variation in Antinociceptive Potency of Alfentanil in Mice

The values given for AD^g scores for alfentanil in Tables 2.6. and 2.8. are different. The origin of this disparity is not obvious as the animal strain, test conditions and experimentor were the same for both sets of experiments. Two differences do exist however, and these are, firstly, that different alfentanil batches were used and, secondly, these experiments were carried out at different times of the year. Considering the second proposition, it has been reported that stimulation-induced antinociception is subject to circadian variation (Buckett,

1981) and this has been confirm ed w ith morphine using the same species and apparatus described in this thesis (P.G.Green, unpublished observations). In addition, there are reports of circannual variation in opioid response as Buckett

(1981) reports that stimulation-induced antinociception as measured by latencies in mice vary throughout the year. In vitro response of vasa deferentia to 2 5 morphine and D-Ala , Met enkephalinamide have also been shown to exhibit circannual variation, with a five-fold variation in potency (De Ceballos and De

Felipe, 1984). The variation of alfentanil potency is also considered in Section

5.18. This information described above highlights the importance of carrying out appropriate controls in parallel with the experiment. CHAPTER 3

Locomotor Activity of Mice

E ffect of DFP and Drugs Used in the Treatment of DFP Poisoning INTRODUCTION

3.1 Assessment of Drug Effect on Spontaneous Locomotor Activity

As a behavioural measure, locomotor activity is probably one of the most simple. Its simplicity enables many experiments to be carried out simultaneously and data to be collected automatically. On the other hand, its simplicity means that only general ambulation is measured and information on stereotypic resonses (eg, sniffing and grooming), co-ordination and muscular rigidity cannot be recorded. It is well known that potentially useful information goes unrecorded (Krsiak et al, 1970). The information gained from these locomotor studies should therefore be viewed as only a basic measure of alterations in behaviour and too detailed an interpretation of the results should be avoided. Nevertheless, whilst bearing in mind the limitations of these techniques, drug-inducing alteration of locomotor function does give an indication of certain drug-receptor interactions within the central nervous system. Indeed, it has been suggested that in order to assess the behavioural effects of a drug, data must be obtained on its influence on spontaneous motor activity (Robbins, 1977).

Data presented in this thesis shows that DFP at 2mg/kg produced apparent antinociception (Table 2.3.). Though the hot-plate reaction time was significantly increased compared to saline controls, mice exhibited reduced ambulation and muscular weakness (as evidenced by hind limb abduction). It is possible that impaired muscular function and co-ordination could delay the nociceptive end-point (the licking response). Experiments were therefore devised to assess the effects of DFP on spontaneous motor activity, and activity following opioid administration. The effects of drugs used in treating anticholinesterase poisoning were also assessed fo r e ffe cts on locomotor activity. MATERIALS AND METHODS

3.2. Animals and Experimental Conditions

Male albino mice (CD-I strain; 25-30g) were used as described in Section

2.2.1. Procedures for equilibration were also as those previously described.

3.3. Drugs

The drugs used were obtained from sources listed in Section 2.6.

3.4. Dosing Protocols

Mice were injected subcutaneously using the same timings to parallel the

antinociceptive procedures described in Section 2.3.3. Doses of opioids used

were 20mg/kg morphine, 80ug/kg fentanyl and 400pg/kg alfentanil.

3.3. Locomotor Activity Testing

Locomotor activity in mice was measured using a system identical to that described by Francis et al (1983) in which a photobeam monitoring system was linked to a microcomputer. The apparatus consisted of pairs of industrial grade infra-red source and detector pairs. Three infra-red light beams cross each test box 0.3cm above the box base, and are detected by appropriate sensors. When the beam path is blocked or unblocked, a score is registered.

Data is collected and displayed on a microcomputer (BBC B) via an interface.

The software employs an interrupt routine to assess the state of the photocells (on or off) every 10ms, and a change in state is recorded as a score. Locomotor hardware was built in the Biochemistry Workshops, University of

Surrey. Original software written for this apparatus was adapted to display

the mean and standard deviation of counts (see Appendix C for listing).

Individual mice were placed in each testing box (18.3 x 11cm), allowing five

mice to be tested simultaneously. Locomotor activity was measured

immediately after the last injection, with the exception of some experiments

where DFP or saline were injected 1 hour before testing. Injection timings

were the same as those described in Section 2.2.3.(a). Testing was monitored

for 15 5-minute epochs, so that changes in activity over time could be

assessed.

3.6. Statistical Procedures

Locomotor scores were analysed following data reduction. Mean locomotor

scores for each 5-minute period were summed over the first 15 and total 75

minute periods. Means for the 15 and 75 minute periods were obtained from

10 or 15 animals recorded on one, two or three separate days.

Effect of drug treatment was analysed using multiple analysis of variance

followed by a posteriori examination using Duncan's Multiple Range Test

(Duncan, 1955). These statistical analyses were carried out using the SPSS software on the University's Prime computer (Statistical Packages for the

Social Sciences, Pennsylvania State University, Version M, Release 9.1.). RESULTS

3.7. Effect of DFP on Locomotor Activity

DFP at lmg/kg produced a significant decrease in locomotor activity only

over 75 minutes compared to saline (Figure 3.1. and Figure 3.2.). However,

when DFP was injected 1 hour before testing, DFP significantly decreased

exploratory activity over the first 15 minutes. This depression of locomotor

activity was further decreased following DFP 2mg/kg .

3.8. Effect of Atropine and Pralidoxime on Locomotor Activity

The atropine-pralidoxime mix produced hyperlocomotion which was not decreased in animals pre-treated with DFP. The effect of DFP alone was reversed by the atropine-pralidoxime mix administration.

3.9. Effect of Diazepam on Locomotor Activity

Diazepam alone greatly depressed locomotor activity and further decreased the hyperlocomotory effect of DFP.

3.10. Effect of Various Treatments on Alfentanil Induced Hyperlocomotion

The hyperlocomotion following alfentanil administration was significantly decreased by DFP (Fig. 3.3.).

Over the first 15 minutes, atropine-pralidoxime mix administration did not significantly alter the DFP effect, but over 75 minutes there was a reversal, with increased locomotor activity. The additional injection of diazepam produced a marked decrease in activity. SCORE 800 i

700-

600-

500-

400-

300-

2 0 0 -

1 0 0 -

5 10 15 20 25 30 35 40 45 50 55 6 0 65 70 75 TIME(mins)

Fig 3.1. Graph Showing the Effect of Various Drug Treatments on Locomotor Activity in Mice

Close circles: saline controls. Closed triangles: DFP (lmg/kg) treated. Open triangles: DFP (2mg/kg) treated. Open squares: morphine (20mg/kg) treated. Each graph represents the mean of at least 10 observations. P CO 02: 05:ts O P * a; ♦ 0: fl P CD —^ p- I i < < < in * O £ (D -P Cl P cij 0- 5/ k - 1 P CD a : < O < ♦ a : 0 Jh ° £ r S: P bo ££< C bJO m < 5 o < o 2 * * •H p a O£ T3 0\k < -> X3 ♦ c l CQ W H 1 2: Q- o ' P a : h 0 O z * u < I 1 I 0 < 1—I -H u . ♦ 5 ♦ ,Q Jh 1 cti d Q; a Eh > * -p - c l L. u. o. 7 l*- O O _J L. - y * - > u O < C u. ♦ 2 * X I—I _) ocj 6 < Z < O in CO < in<>« ♦ a CL -j/ -J a : < O < ♦ CL OS: ° £ 12: « cL 3 < o 5 < 5 O < O < * ♦ a^ -J _j £ < 0 < < ♦ CL QC ° £ 1 2 : . a Q- oc CL h- u a cl & -Jti. H u < O < <0 < u . ♦ 2 ♦ c c r X o d Cl a t- p. CL ti_ L_a 7 li- _iii. u O 0 O < C u . ♦ 2 ♦ 1 V” -J ac: _) < uZ 1/1 < O in l i Ti u . ♦ 2 ♦ <— «p DFP DFP = Di-isopropylfluorophosphate, ATR/PRAL = Atropine and Pralidoxime, DIAZ = Pralidoxime, DIAZ = Diazepam

< < < < CL CL c l c a . a a a a: K c? a _ j a a a w* L_ U_ L . a a tn O O O _ j a a a u . u . c. << u . u . u . O O u ut: u 5 c c o 1 X O (j o a Q. Z c O z z z Z Z C L CL II (I M lj u UJ U U O O Abbreviations: ALF Abbreviations: ALF = Alfentanil, FENT = Fentanyl, MORPH = Morphine, SAL = Saline < < < < < u . u . u . 2 2 | ->•-> Arrows in this table indicate significant changes in locomotor activity mice in 10000-J

frJ Ri FF jfiL+flLFENT DFP+ftLFENT DFP+RTR/PR OFP+DIflZ. SRLINE+RTR +RLFENTRNIL +R/P+RLF /F’RhL+hLF

Fig 3.3. Effect of DFP on Alfentanil-induced hyperlocomotion over 15 and 75 minutes

Black histograms: locomotor score over 15 minutes Grey histograms: locomotor score over 75 minutes SAL + ALFENT: Saline and alfentanil treated. DFP + ALFENT: DFP and alfentanil treated. DFP + ATR/PR DFP, atropine and pralidoxime + ALFENTANIL: and alfentanil treated.

DFP + DIAZ + A/P + ALF: DFP, diazepam, atropine and pralidoxime and alfentanil treated.

Values represent mean - S.E.M. of at least 10 observations.

Saline control scores: 15 mins 1442 - 108

(n=5) 75 mins 5630 ^479 3.11. Effect of Various Treatments on Fentanyl Induced Hyperlocomotion

The hyperlocomotory activity of fentanyl was not significantly altered by administration of DFP or DFP + atropine/pralidoxime over 15 or 75 minute periods (Fig. 3.4.). The additional injection of diazepam, however, significantly depressed locomotor activity scores.

3.12. Effect of Various Treatments on Morphine Induced Hyperlocomotion

Morphine-induced hyperlocomotion was significantly decreased following DFP pretreatment, but only over the 75 minute period (Fig.3.5.). The atropine- pralidoxime mix had no effect on morphine hypolocomotion. Diazepam produced marked decreases in locomotor scores, and though this was significant over the first 15 minutes statistical significant differences were not observed over the 75 minutes, despite there being nearly a 34-fold difference; very high pooled standard deviations existed in these groups. LOCOMOTOR SCORE 10000-

B$^^/+++////B

5000-1

FENTRNIL SflL+FENT DFP+FEHT DFP+ftTR/PR QFP+DIflZ, SflLINE+flTR +FENT fi/P+FENT /P+FENT

Fig. 3.4. Effect of DFP on fentany1-induced hyperlocomotion over 15 and 75 minutes

Black histograms locomotor score over 15 minutes Grey histograms: locomotor score over 75 minutes SAL + FENT: Saline and fentanyl treated. DFP + FENT: DFP and fentanyl treated. DFP + ATR/PR + FENTANYL: DFP, atropine and pralidoxime and fentanyl treated. DFP + DIAZ + A/P + FENT: DFP, diazepam, atropine and pralidoxime and fentanil treated.

Values represent mean t S.E.M. of at least 10 observations.

Saline control scores: 15 mins 1384 - 141

75 mins 5957 - ~531 LOCOMOTOR SCORE 1000th ^147901691^8944691576436^1079456^14795

5000-

' ••••

PH SfiL+MORPH DFP+hORPH OFP+HTR/PR DFP+DI.RZ, SRLINE+flTR tilORPH I HE +ii/P+nuRPH /P+MmPPm

Fig 3.5. Effect of DFP on morphine-induced hyperlocomotion over 15 and 75 minutes

Black histograms locomotor score over 15 minutes Grey histograms: locomotor score over 75 minutes SAL + MORPH: Saline and morphine treated. DFP + MORPH: DFP and morphine treated. DFP + ATR/PR + MORPHINE: DFP, atropine and pralidoxime and morphine treated.

DFP + DIAZ + A/P + MORPH: DFP, diazepam, atropine and pralidoxime and morphine treated.

Values represent mean - S.E.M. of at least 10 observations.

Saline control scores: 15 mins 1541 - 122

(n=15) 75 mins 6095 - 404 DISCUSSION

3.13. Effect of DFP on Locomotor Activity

There are several reports in the literature describing the effects of

anticholinesterase agents in impairing or reducing ambulatory behaviour in

rodents. This e ffe c t has been shown fo r DFP (Koehn and Karczm ar, 1977,

1978; Koehn et al, 1980; Raslear and Kaufman, 1983; Samson, 1984; M artin,

1985), physostigmine (Chaturvedi, 1984; Gonzales, 1984; Harris et al, 1984;

Wolthuis and Vanworsch, 1984), pyridostigmine (Harris et al, 1984; Wolthuis

and Vanworsch, 1984), sarin (Landauer and Romano, 1984b), soman (Wolthuis

and Vanworsch, 1984) and tabun (Landauer and Romano, 1984a; W olthuis and

Vanworsch, 1984). The well documented effect of cholinergic agents on

locomotor activity has been described as alert non-motile behaviour (ANMB)

(Karczmar, 1977) and is believed to involve a cholinergic-dopaminergic dipole

in nigrostriatal and extrapyramidal pathways.

In this study, significant and marked decreases in locomotor activity were

observed for DFP at lmg/kg in mice. When administered 1 hour before testing

to parallel the antinociceptive testing procedures, DFP reduced exploratory

motor activity. In interpreting the consequences of this data, it is not

unreasonable to suggest that DFP induced 'antinociception' was observed as a result of impaired motor co-ordination and muscular function rather than an

inhibitory action on nociceptive systems. This suggestion gains credence when the antagonist data is taken into consideration (Table 2.5). The opioid antagonist naloxone did not alter the DFP antinociception but atropine which reversed the hypolocomotor effects of DFP (see below), also reversed antinociceptive activity. 3 . 1 4 . Effect of Atropine and Pralidoxime Alone and With DFP on Locomotor Activity

DFP induced reduction of locomotor activity appeared to involve the cholinergic system as DFP's effects were completely reversed following atropine-pralidoxime mix administration. When administered alone, atropine and pralidoxime produced hyperlocomotion. Hyperlocomotion has been previously reported for atropine (Chatervedi, 1984; Seidel et al, 1979) and for another muscarinic antagonist, hyoscine (Sansone, 1983; Kuribara and

Tadokar, 1983).

3.15. Effect of Diazepam Alone and With DFP on Locomotor Activity

Inhibition of locomotor activity following diazepam administration was marked and this is in contrast with some reports in the literature. For example, it has activity been shown that in mice, diazepam up to Img/kg did not affect locomotion (Sansone, 1982). In rats at 2.5mg/kg diazepam enhanced ambulatory activity whilst at 20mg/kg it was inhibited (Matsubara and

Matshushita, 1982). In this study the dose of diazepam used was 1.78mg/kg, which suggests that a marked motor depression was observed, mice may be more sensitive than rats to the locomotor depressant action of diazepam.

When administered after DFP the locomotor activity of diazepam was decreased still further, so that the hypolocomotory effects of these drugs were addictive.

3.16. Effects of DFP on Opioid-Induced Hyperlocomotion

The action of opioids in mice is to produce a stereotypic running response (eg,

C arroll and Sharp, 1972) and in this study morphine, fentanyl and a lfen tan il all produced this running response. This hyperlocomotion was reduced for all opioids by DFP pretreatment. This is consistent with the literature as DFP at 2mg/kg has been reported to decrease morphine-induced running in mice

(Su and Loh, 1975). and oxotrem orine has also been shown to decrease morphine hyperlocomotion (Seidel, 1979). It has been suggested that the augmentation of the opioid-induced running response by cholinergic agents may be due to an increase in dopamine utilization produced by muscarinic agonists (Seidel, 1979).

The data indicates that the effect of DFP on opioid induced running is most pronounced with alfentanil. However, if DFP was enhancing the effect of alfentanil in the same way as that observed for the antinociceptive activity, one might have expected increased locomotor scores. A possible explanation of this apparent anomaly highlights a drawback encountered in this form of measurement of locomotor activity. Observation of mice receiving opioids, especially alfentanil, revealed that during peak opioid activity mice had running fits ie, periods of intense running activity interspersed with periods of immobility (cf. dopamine catatonia). These periods of catatonia appeared with greater frequency and duration with increasing dose, at 800pg/kg, half or sometimes all of the 60 seconds antinociceptive testing period mice were catatonic. If DFP potentiated alfentanil, the measured locomotor activity score could have been reduced following DFP if the mice spent a greater proportion of their time in a catatonic immobile state. Activity is not a unitary measure and photocell measured locomotor activity cannot detect such stereotyped activities such as circling, excessive grooming, licking or biting or differentiate between walking and running activities. More sophisticated activity monitors or scored human observation would be required to identify these specific changes in activity. So if DFP potentiated alfentanil's activity and produced increased catatonia, this would be reflected in a decreased locomotor activity score. 3.17. The Effect of Atropine and Pralidoxime on the Locomotor Activity of

DFP and Opioid-Treated Mice

In DFP and opioid-treated mice the effect of the atropine-pralidoxime mix treatment on locomotor activity was only significant for alfentanil over 75 minutes. It is quite likely that no significant effect was observed for the other opioids due to the interaction of the four drugs administered and their effects on locomotor activity. For example, the atropine-pralidoxime mix in addition to possessing hyperlocom otory a c tiv ity (see Section 3.18.), would tend to antagonize the hypolocomotory effects of DFP. This antagonism of

DFP could therefore decrease the incidence of alfentanil catatonia (see

Section 3.20.). Furthermore, atropine is reported to potentiate morphine- induced circling (Seidel, 1979).

In summary, the combination of opioid, DFP, and the atropine-pralidoxime mix produce a complex effect on locomotor behaviour. The observed enhancement of alfentanil locomotion with atropine-pralidoxime mix is likely to have been a result of both a reduced degree of catatonia and an additive cholinergic hyperlocomotion.

3.18. Effect of Diazepam on Opioid-Induced Locomotion

The large decrease in locomotor score following diazepam was common to all three opioids studied. This effect has been reported previously for morphine- and fentanyl-induced hyperlocomotion (Matla and Langwinski, 1982).

Benzodiazepines are known to have a sedative action and it could well be this action on benzodiazepine GABA receptors which produced the decrease in locomotor score. Alternatively, diazepam could be acting through another receptor system; the role of the dopaminergic system has already been mentioned in its involvement in locom otor a c tiv ity (Section 3.17.) and it is possible that the observed effect of diazepam on locomotion was due to its action inhibiting dopamine function (Reubi et al, 1977). It may also be possible that opioid catalepsy (and hence immobility) was enhanced following diazepam, as catalepsy has been shown to involve inhibition of the central dopaminergic pathway (Kushinsky and Hornykiewicz, 1972).

3.19. Interpretation of Locomotor Data

Locomotor measurement is a simplistic measure of alteration of behaviour.

It is subject to many errors and it is well known that large quantitative differences between individual animal activity scores are observed (Izazole-

Conde et al, 1983).

Observation of animals during locomotor testing procedures suggested that at least part of the large variation of scores between animals were probably due to behaviours such as grooming. Mice were occassionally observed to be grooming directly in the path of the infra-red light beam so that their rapid head movements increased the locomotor score.

As mentioned above, production of catalepsy is produced with high doses of opioids and would tend to increase variation between animals. This form of error has been reported in the lite ra tu re where alcohol at certain doses produces unchanged or increased locomotor activity and simultaneously large increases in immobility (Smoothy and Berry, 1984). Observation revealed that this was due to alcohol's effect of decreasing non-locomotor activity such as rearing which is not recorded by simple locomotor activity monitors. These limitations notwithstanding, the locomotor activity monitor used in this study gave a general indication of the effects of drugs on locomotor activity of mice. CHAPTER 4

Distribution of H Morphine, Fentanyl and Alfentanil

In Mouse Brain: Effect of DFP Administration INTRODUCTION

4.1 Distribution Studies

The differential potentiation of alfentanil by DFP led to the conclusion that the mechanism probably involved an effect on pharmacokinetics (Section

2.28) rather than altered pharmacodynamic parameters. A simple measure of distribution utilizes radiolabel tracing methodologies, wherein plasma and brain levels of a radiolabelled drug are determined. Drug concentration in control and treated animals can then be compared.

A drawback to the method used in this thesis is that only total radioactivity was determined. The presence of radioactive metabolites, which may or may not possess pharmacological activity are not distinguished. However, for fentanyl and alfentanil the short time period between administration of the drug and killing would mean that it is likely that the majority of the radioactivity in the brain is the parent drug. For morphine the glucuronide metabolites do not contribute significantly to uptake into the brain of labelled morphine (Hartvig et al, 1984). However, morphine-3-glucuronide does possess some antinociceptive a c tiv ity (P.G.Green, unpublished observations). MATERIALS AND METHODS

4.2. Animals and Experimental Conditions

Male albino mice (CD-I strain; 25-30g) were used as described in Section

2.2.1. Procedures for equilibration were also as those previously described.

Radiolabelled morphine and fentanyl were supplied in ethanol, and

radiolabelled alfentanil was suplied in water:acetonitrile 30/70, ethanol 100.

The solvent was removed under a gentle stream of dry nitrogen at room

temperature.

4.3. Drugs

The drugs used were obtained from sources described in Section 2.4. In addition, the following radiolabelled compounds were used:-

DRUGS

^ H Morphine sulphate Amersham International

Specific Activity 20-24 Ci/mmol

3 H Fentanyl citrate IRE, Fleurus (Belgium)

Specific Activity 15-18 Ci/mmol

3 H Alfentanil hydrochloride Janssen Pharmaceuticals

Specific Activity 23.1 Ci/mmol

3 H Hexadecane tritium standard Amersham International

Specific Activity 1.69 qCi/ml

* 3 H Alfentanil was generously donated by Janssen Pharmaceutical 4.4. Dosing Protocols

Drug administration procedures were carried out by identical to that described in Section 2.6. for DFP and opioid administration.

Mice received either 0.9% w/v saline or DFP (Img/kg) s.c. This was followed by s.c. injections of radiolabelled morphine, fentanyl or alfentanil administered 30, 30 or 33 minutes later respectively. Two doses of each opioid were studied and each injection contained 7pCi of ^H-labelled opioid.

4.5. Distribution Studies Protocol

At times of peak opioid antinociceptive activity (see Figure 2.1.) (also 1 hour after DFP or saline administration) animals were killed by decapitation and trunk blood collected into heparinised tubes. The brains were rapidly removed and cooled over ice on a moistened filter paper covered petri dish.

Brains were then dissected into eight regions, following a protocol similar to that described by Glowinski and Iversen (1966). Each region was weighed and transferred to glass scintillation vials to which 1ml of Soluene-100 (Packard) was added. Plasma was obtained by centrifugation at 2500g for 10 minutes and a 20pl of plasma was solubilized with 0.5ml Soluene-100. Brains were left overnight to solubilize. With the larger brain regions, cerebellum and cortices, solubilization was promoted by warming the vials to 50°C for 10 to

30 minutes, and allowing to cool.

4.6. Scintillation Counting

Following solubilization, scintillation fluid consisting of 1.5 litres of sulphur- free toluene, 750ml of synperonic NX PO POP 225mg and PPO 12g (Wood et 3 al, 1975) was added to samples. H activity was determined in an LKB 1210 Ultrobeta scintillation counter. Chemiluminescence was avoided by acidifying the scintillant with 10ml glacial acetic acid per 2.5 litres of scintillant.

Presence of solubilised brain tissue has a quenching effect on detection of scintillations. As varying amounts of brain tissue were present in each vial, it was important to take into account and compensate for variable quenching.

A quench correction curve was therefore set up. Brain tissue weighing 10, 20,

40, 60, 80, 100 and 120pg were solubilized in 1ml of Soluene-100. To each vial,

10 pi of hexadecane standard was added, and the vials counted on the 1210

Ultrobeta. Decay tables for allowed total activity per vial to be calculated. With c.p.m. and R values from these vials, the coefficients of the data were calculated by applying a Basic regression programme (see Appendix

D (i). This program yielded values for AO, Al, A2 and A3 which were programmed into the scintillation counter, enabling radioactivity to be given in d.p.m. The quench curve was also plotted using the d.p.m. package on a

RackBeta 1215 (see Appendix D (ii)).

A similar procedure was carried out for plasma as occasionally samples were haemolysed. Plasma samples of varying degree of haemolysis were counted with 10pi hexadecane standard. The coefficients were determined as for the brain samples. RESULTS

4.7. Effect of DFP Pretreatment on Alfentanil Brain and Plasma Levels

Figure 4.1. represents the effect of DFP on levels in various brain regions of alfentanil, expressed in d.p.m./g wet weight of tissue.

Pretreatment with DFP at Img/kg caused a significant increase in the levels in all brain regions of alfentanil at 400pg/kg (Figure 4.1.). This increase was most marked in the hypothalamus with a 3.2-fold increase in levels compared to saline controls. There was a marked reduction in plasma alfentanil ra d io a ctivity (Table 4.1.). A t the 200pg/kg dose of alfentanil, DFP appeared to little affect brain levels. If brainrplasma ratios are considered (Table

4.2.), DFP pre-treatment produced a significant effect in all brain regions and at both doses.

4.8. Effect of DFP Pretreatment on Morphine and Fentanyl Brain and Plasma

Levels

DFP appeared to have little effect on brain levels of fentanyl or morphine

(Figure 4.2. and 4.3.). There was a slight decrease in morphine levels following DFP but this only reached significance in the hypothalamus at the higher dose. Similarly, DFP did not significantly alter plasma radioactivity of either morphine or fentanyl, (Table 4.1.) and so brain:plasma ratios fo r these two opioids were very similar in control- and DFP-treated animals

(Table 4.2.). DPM g'"1' { x10-3) 400

380 -

360

340

320 200

180

160

120

100

80 ■

60 •

40 -

20 -

CB PM HYP STR MB HIP HCX FCX CB PM HYP STR MB HIP HCX FC>

Fig. 4.1. Effect of DFP on brain levels (dpm g -*-) of alfentanil (A = 200yg/kg“l, B = 400yg/kg_^)

Open histograms: control treated. Dotted histograms: DFP (ling/kg--1-) treated. Each histogram represents the mean * s.e. mean of at least six observations. CB = cerebellum, PM = pons and medulla, HYP = hypothalamus, STR = striatum, MB = mid brain, HIP = hippocampus, HCX = hind cortex, FCX = frontal cortex. DPM g '1 (x10“3) B 2 4 0 i 220 1

200

160

80 i

60 i

40 -j 20 i

CB PM HYP STR MB HIP HCX FCX CB PM HYP STR MB HIP HCX FCX

Fig. 4.2. Effect of DFP on brain levels (dpm g_l) of fentanyl ( A = 40yg/kg“l, B = 80yg/kg--*-)

Open histograms: control treated. Dotted histograms: DFP (lmg/kg-l) treated. Each histogram represents the mean 1 s.e. mean of at least six observations. CB = cerebellum, PM = pons and medulla, HYP = hypothalamus, STR = striatum, MB = mid brain, HIP = hippocampus, HCX = hind cortex, FCX = frontal cortex. 3 D M g " 1

1 x 10~3) A B -5 i

40 -

I [I 1 30 1 X I 25 jL I II 1 X JL 20 1 I 4- JL X JL X LL X 15 -

10 -

i l CB PM HYP STR MB HIP HCX FCX CB PM HYP STR MB HIP HCX FCX

Fig. 4.3. Effect of DFP on brain levels (dpm g-^-) of morphine (A = lOmg/kg-1, B = 20mg/kg-1)

Open histograms: control treated. Dotted histograms: DFP (lmg/kg-1) treated. Each histogram represents the mean t s.e. mean of at least six observations. CB = cerebellum, PM = pons and medulla, HYP = hypothalamus, STR = striatum, MB = mid brain, HIP = hippocampus, HCX = hind cortex, FCX = frontal cortex. Plasma Radioactivity dpm ml" (x 10” )

Opioid Control DFP-Treated

M orphine, 197 - 7 174 - 6* (10mg/kg~ )

Morphine 118 - 10 97 - 8 (20mg/kg" )

Fentanyl , 73-7 90 -13 (40pg/kg" )

Fentanyl , 67 - 4 57-3 (80pg/kg” )

Alfentanil . 166 - 10 110 ± 12* (200pg/kg )

Alfentanil 147 +34 91-6 (400pg/kg" )

Each value is the mean of six observations, t test, DFP vs. control * p < 0.05

Table 4.1. Effect of DFP (lmg/kg"^) on Plasma Levels of H Morphine, Fentanyl and A lfe n ta n il i—i CL * * * * * 1 * * * , Ll. CM cn 1 .1 1 .6 1 .0 1 .9 Q 1 .3 <—1 3 .3 rH c5 ■* 4_i cn + C S3- 0 A vO VO v o vO vO *4- O vO CO O CD CD CD a CD CD CD < §

CL * * * ** ** * A CO vO — c n Ll- vo r H CO A c Q CD 1— 1 CD CD CD CD CD CD O 4-1 CJ1 + C CD. H-CD o VO A A CD CD CD < S CD O CD CD CD

CL c o Is 0 \ ^ t A N I— On i L. — . c n cmojaaaacmcm > > ^ Q c _ + CD ^ 4-1 H - A A CM O Ov r- c ••••. •.• co CD O O CM CM A A CM CM CM CM •—I L l . ° o H •H < A S3 Ql O CL d < cm cm vo a CD (H HHH r -P la- OCQ S3 _ c n Cl V co > > . * Q. CD < /~ \ ‘H 0 S ' * _ l 4 -1 H - —I ^ H CL c co vo A vO I o < o) a hfl'H Ll. O ’ ^ ' d z C o '- * * CO c < o bfl o ctf { £ S *H CD o5 H P H > CL A r-» O f'- Ov w d >5 r —1 CO A CL P3 S3 1 Ll. CM 1—1 A CM CM A «H rH Pm d 0 c p .... * • •• Ll. Q O 0 CD CD CD h c i P c O 0 O Q o e c X CD + CO CD Q_ £ °H d Pp f-i A a A A A Ov r ^ CO 5 O CM CM A CM CM CM r —1 rH 0 rH . • . •• • •• O 1 »v X CM OO a OO a 0 -P 0 CD O d S3 cn 0 *H *H C ‘H d X3 o 4-1 ‘H fH f t H CL A CD CO CM A A •H O CO pq pq h 1 Li_ •—1 —( rH 1—( rH .. . •• • • • O (U CJ> Q C_ c O a 0 a O OO CD 0 cn x cn + X A 0 iH A O £■t-H ^ Ov A CD CD >H •—1 H rH

2 L

c 0 0 E 0 -C D X cn 0 X) D 4-1 C 0 d X o t- E Q. 0 o 0 E 0 4-1 ’&> D E (-. O D 0 •O "co o c 0 0 la CC "0 0 X o U > X 4-1 XI o c 0 cn O I CL - a x ’co c_ C CL ■a CL c o c_ 0 o 0 CD L> CL X CO 1 Ld DISCUSSION

4.9 Opioid Lipophilicity and Access to the CNS

Opioids exert their antinociceptive effect in the central nervous system. To gain access to their site of action, opioids must cross the blood-brain barrier.

Generally speaking, the greater the lipophilicity of an opioid (or any other drug) the greater the ease with which it is able to cross this barrier and enter the brain (Jacobson, et al, 1977).

A measure of lipophilicity is the octanohwater partition coefficient at pH7.4 and the literature values for the opioids studied are 1.42 for morphine

(Kaufman et al, 1975), 130 and 860 fo r alfen tan il and fentanyl respectively

(Meuldermans et al, 1982). These values parallel the order of brain:plasma ratios obtained in this study. Levels of radioactivity in the brain compared with the plasma are highest for fentanyl, followed by alfentanil and then morphine. The times for measurement of morphine, fentanyl and alfentanil levels were chosen to correspond to peak antinociception (see Section 2.6.).

These times agree with literature values for these opioids (Gardocki and

Yelnoski, 1963; Johannesson and Becker, 1973). There is also a good correlation between levels of these opioids in the brain and their antinociceptive effects (Johannesson and Becker, 1973;Hug and Murphy, 1981).

4.10. Effect of DFP on Opioid Brain Levels

DFP pretreatment produced a disparate effect on the brain levels and distribution of radiolabelled morphine, fentanyl and alfentanil. The most marked effect of DFP is on the distribution of alfentanil where plasma levels are lower and at 400pg/kg alfentanil there is a concomitant rise in alfentanil in the brain. This enhanced entry could account for the observed increase in the antinociceptive activity of alfentanil following DFP (see Section 2.24.).

The absence of an apparent increase in brain levels of alfentanil at 200pg/kg by. DFP may be partly due to the decrease in plasma levels, since the blood present in the brain was not accounted for and contributes to the total radioactivity. Alternatively, DFP may be altering pharmacokinetic parameters so that peak brain alfentanil levels could occur earlier. Peak opioid receptor occupancy or antinociceptive effect could persist for a short time after peak drug level (see Figure 4.4.)

4.11. Mechanism of Increased Levels of Alfentanil Produced by DFP

There is no literature at present describing a potentiation of alfentanil by other drugs. However, there are some reports in the literature describing the action of some drugs in increasing the potency of morphine by a mechanism of increasing the plasma and brain levels of this opioid. For example, a synergism has been demonstrated between diazepam and morphine (Sanchez et al, 1982). This appears to be dependent on diazepam competitively inhibiting morphine glucuronization and demethylation. This results in higher plasma and hence higher brain levels of morphine. Brain uptake of morphine was shown to be increased following administration of the antihistamine tripelennamine (Vadlamani, 1984) possibly by increasing permeability of the blood-brain barrier. By contrast, the potentiation of morphine by physostigmine did not appear to be dependent on alteration in morphine plasma or brain levels (Bhargava and Way, 1972).

It is possible that DFP could increase alfentanil's entry into the brain also by affecting the blood-brain barrier. Indeed, DFP has been shown to increase BRAIN DFP+400pg/kg ALFENTANIL ALFENTANIL CONC. \ N N 400pg/kg'' S \ \\ ALFENT/ \ ANILX f \ / ~ ^tNpFP+20Qjjg/kg / \ / * \/ )>

Fig. 4.4. Scheme for Explanation of Observed Increased Brain Alfentanil Levels for DFP (lmg/kg) Administration

This figure illustrates that by assessing brain levels at one time point (5 minutes), misleading information may be obtained (see text). the permeability of capillaries in the anterior segment of the eye (von

Sallman and Dillon, 1947). Furthermore, in considering the mechanism of potentiation of morphine antinociception by the two anticholinesterases, physostigmine and neostigmine, Rypka and Navratil (1982) showed that the blood-brain barrier permeability to acridine orange was markedly increased following administration of physostigmine and neostigmine. If however, DFP exerted this same effect on blood-brain barrier permeability, this would be non-selective and it might be expected that all three opioids would all have enhanced entry into the brain. Moreover, as alfentanil and fentanyl are highly lipid soluble, the blood-brain barrier would not normally lim it the entry of these drugs into the brain. On the other hand, the low lipid solubility of morphine accounts for the low penetration of morphine into the brain

(Hartvig et al, 1984). This means that an increase in the permeability of the blood-brain barrier would tend to increase the entry of a low permeability agent such as morphine, rather than an agent which is highly lipid permeable.

The observation that morphine levels are unchanged by DFP tends to m ilitate against an action on blood-brain barrier permeability by DFP.

The evidence suggests that DFP enhancement of alfentanil entry into the brain is probably more likely to be dependent on pre-blood-brain barrier mechanisms. One such candidate for this mechanism is alteration of alfentanil binding to plasma proteins. CHAPTER 5

3 In Vivo and In Vitro Plasma Protein Binding of H Alfentanil 3 and H Fentanyl: Effect of DFP Administration INTRODUCTION

I A3 5.1 Plasma Protein Bind[of Drugs

The ability of plasma proteins to bind and transport chemicals and drugs has been known for many years (Rosenthal, 1926). The potential binding capacity is enormous with an estimated total surface area of plasma proteins per person being between 130,000 and 735,000m (Bennhoid, 1967). In practice, only a fraction of this area is available as compounds tend to bind to specific sites (Bickel, 1978).

The binding of drugs to plasma proteins has been comprehensively reviewed over the years (Goldstein, 1949; Meyer and Guttman, 1968; Jusko and Gretch,

1976). It has become apparent that the protein binding of a drug can influence its distribution which directly and indirectly influences the action of the drug by determining its availability at its sites of action.

5.2 Plasma Protein Binding of AlfentaniljFentanyl and DFP

Studies in rat, dog and man have shown that fentanyl and alfentanil have high fractional binding to blood proteins (Meuldemans et al, 1982). As only free unbound drugs may cross the blood-brain barrier increased entry of drug could occur if, for example, the proportion of opioid bound to plasma proteins is altered following DFP administration. It is possible that DFP may alter binding of opioids to plasma proteins, as its administration may produce either acidosis or alkalosis by either stimulating respiration at high doses or depressing it at low doses (Karczmar, 1967) since fentanyl binding is markedly affected by alterations in blood pH (Meuldermans et al, 1982). In addition, alteration in pH would alter the per cent ionization (ionized drugs cannot pass through the blood-brain barrier) and this could be important for fentanyl where only 9% is unionized at normal pH. Though alfentanil would be little affected as 89% is unionized, change in per cent ionization has been shown to markedly potentiate the brain uptake of morphine as shown in alkalotic rats

(Schulman et al, 1984). However it appears that altered blood pH is not an important factor as of the three opioids studied alfentanil pharmacokinetics are the most pH-independent.

DFP has also shown to bind, irreversibly, to plasma proteins (Truhaut, 1966) and so it is worth entertaining the notion that DFP competes for or inhibits alfentanil binding, thereby increasing the proportion of free alfentanil which is able to enter the brain. This form of interaction is well known, and has also been shown for irreversible anticholinesterase agents; it has been suggested that the potentiation of sodium pentobarbital anaesthesia by soman is dependent on competition for plasma protein binding (Clement, 1984). This mechanism of interaction is considered in this section.

5.3 Assessment of Drug Binding to Plasma Proteins

There are two main methods for determining free drug levels: equilibrium dialysis and ultracentrifugation. Equilibrium dialysis is the most used of these methods but suffers from a number of methodological drawbacks.

These include Donnan effects, changes in ion concentration which may compete with drugs for binding or alter protein conformation (Bowers et al,

1984). Ultrafiltration has the advantage of being a more rapid procedure though one drawback is that there may be non-specific adsorption of drug to the ultrafiltration membrane (Zhirkov and Piotrovski, 1984), however this adsorption may be determined and compensated for. It has been concluded that ultracentrifuga'cion is a more sensitive method for determining small changes in protein binding (Coyle and Denson, 1984) and better reflects the real values than those obtained by equilibrium dialysis (Tuila, 1984). IN VITRO IN VIVO

Mice injected with DFP or saline

Mice injected with -opioid

Trunk blood collected ^

Plasma obtained. by centrifugation

Plasma aliquot incubated with -opioid Ultracentrifugation to obtain unbound drug fraction

^ Liquid scintillation , counting

Fig. 5.1. Protocol for Plasma Protein Binding Experiments Iri Vivo and Iri Vitro. (See text for details) MATERIALS AND METHODS

5 .4 Animal and Experimental Conditions

Male albino mice (CD-I strain; 25-30g) were used as described in Section

2.2.1. Procedures for equilibration were also as those previously described.

5.5 Drugs

The drugs used were obtained from sources described in Section 2.6. and 4.6.

5.6. In Vivo Protein Binding Protocol

Timings of drug administrations were the same as the previous experiments

(Section 4.4) where DFP or 0.9% w/v saline was given s.c. at time 0, fentanyl

(80pg/kg) at 50 minutes, and alfentanil (400pg/kg) was administered at 55 minutes. Each injection contained 7pCi of opioid. Trunk blood was collected following decapitation at 60 minutes, and plasma obtained by centrifugation, (Fig 5.1.).

5.7 In Vitro Protein Binding Protocol

Mice received s.c. DFP or 0.9% w/v saline and were sacrificed by decapitation 60 minutes later. A 250pl aliquot of plasma obtained following centrifugation was incubated for 30 minutes at 37°C with 25pl ^ H alfentanil or ^ H fentanyl. The incubation medium contained additional cold opioid so that drug concentrations paralleled those following a s.c. antinociceptive dose (Fig 5.1.). 5.8. Ultracentrifugation Protocol

Free drug concentrations were determined by ultracentrifugation of plasma samples in an Amicon micropartition MPS-1 kit (Figure 5.2.). A 200pl aliquot was pipetted into the sample reservoir of the assembled unit. The unit was centrifuged in a bench top centrifuge at 1500g for 10 minutes. A 20pi aliquot of the ultrafiltrate (i.e. unbound drug) and 20pi of the plasma (i.e. total drug) were added to polythene tubes in duplicate. 3-5mls of Unisolve-1 scintillation fluid (Koch Light) was added and tubes were counted in a RackBeta 1215 or

1216.

5.9. Calculation of Free and Bound Drug Concentration

Calculation of the free and bound drug concentrations were made from data of ultrafiltrate and plasma radioactivities using the equation:

Total drug concentration = bound concentration + Free concentration Plasma Concentration)

So, % Bound = Total - Free 100 or % Bound = 100 - % Free

5.10. Determination of Drug Adsorption in Ultracentrifugation Apparatus

Recovery experiments were performed to determine whether any loss of alfentanil or fentanyl occurred during the ultracentrifugation process through adsorption on the membrane or plastic reservoir. A stock of mouse plasma 3 3 contaimrKj either H alfentanil or H fentanyl was aliquoted and ultracentrifuged as described in Section 5.6. Radioactivity was determined for the stock and for ultrafiltrates in duplicates. Recovery could then be determined by comparing radioactivities in the original stock with the ultrafiltrate. RESERVOIR ' I CAP ______i i ]! i !

SAMPLE RESERVOIR

CLIP

ORING YM MEMBRANE

MEMBRANE SUPPORT BASE FILTRATE CAP FILTRATE CUP______

Fig 5.2. The Amicon Micropartition MPS-1 System RESULTS

5.11. Recovery Experiments

Recovery experiments for alfentanil and fentanyl showed that the alfentanil ultrafiltrate contained 103 - 11% (n=4) of the original stock and the fentanyl ultrafiltrate contained 80.7 - 1.3% (n=6).

5.12. Percentage of Plasma Protein Binding of Fentanyl and Alfentanil

Control binding of alfentanil were fairly similar following in vivo and in vitro binding protocols (Table 5.1). The figures for fentanyl binding in control mice were quite different, dependent on whether _in vivo or in vitro protocols were used.

5.13. E ffect of DFP on Binding Fentanyl and Alfentanil to Plasma Proteins

Table 5.1. shows the effect of DFP (lmg/kg) on the levels of plasma total and plasma free alfentanil and fentanyl following incubation in vitro. The values presented have been adjusted fo r losses during ultracentrifugation in the in vitro experiments. There was no alteration in free or bound fentanyl in the presence of DFP (Table 5.2.), however, there was a greater than 40% increase in free alfentanil in DFP treated plasma.

The effect of DFP on m vivo administration of alfentanil (Table 5.2.) is more marked with nearly a 60% increase in free alfentanil. For in vivo binding of fentanyl, DFP has very little effect on free levels (Table 5.2.) Table 5.1. Effect of DFP Treatment on In Vivo and In Vitro

Plasma Levels of Alfentanil

and fentanyl

Treatment Alfentanil Fentanil

ng/ml ng/ml in vivo In vivo In vitro In vitro

Total 485 - 25.2 519 - 5 16.87^0.36 13.25 - 0.76

Saline

Free 118 - 6.1 140 ± 10 2.01 - 0.14 7.09 - 0.54

DFP Total 286 - 40.2 517 - 9 16.30 - 0.59 12.25 - 1.51

Img/kg Free 110 - 13.0 195 - 12 1.83 - 0.06 5.63 - 0.68

Values are means ^ SEM of 6 observations. Table 5.2. Effect of DFP Treatment on Levels of In Vivo and in Vitro

Free Plasma Levels of Alfentanil and Fentanyl

% Free levels

Treatment Alfentanil Fentanyl

In Vitro In Vivo In Vitro In Vivo

Saline 24.3 26.9 53.5 11.9

DFP lmg/kg 38.5 37.8 46.0 11.2 DISCUSSION

5.14 DFP Binding to Plasma Proteins

DFP is known to bind plasma proteins in several species including man

(Jandoff and McNamara, 1950; Cohen and Warringa, 1954; M urachi, 1963;

Truhaut, 1966; Ramachandran, 1967; Hansen et al, 1968; Schuh, 1970; Martin,

1985). DFP is capable of combining covalently with most proteins (Martin,

1985) in a general type of alkyl phosphorylation reaction (Murachi, 1963; see

Figure 5.3.). The percentage of DFP bound to plasma proteins is high following injection; a dose of lmg/kg DFP i.v. in mice results in over 95% of plasma DFP being bound.

There has been little work on attempting to identify to which protein DFP is bound. Schuh (1970) however, has indicated that albumin is the main protein as the same values for percentage DFP binding can be obtained by substituting a 3.2% albumin solution, equivalent to normal plasma concentrations.

5.15. Plasma Protein Binding of Alfentanil and Fentanyl

Both alfentanil and fentanyl have been shown to have high fractional protein binding (Meuldemans et al, 1982). The results in this thesis showing around

75% binding for alfentanil are consistent with those obtained in the lite ra tu re : for ra t, dog and man and the fraction bound is 84, 73 and 92% respectively (Meuldermans, et al. 1982). Values obtained in this thesis for fentanyl binding vary depending on the method used. Following in vitro methodology, fentanyl binding was found to be similar to that obtained in rat, D "71 TJ 0 TJ x O X o O X o co -N| -Nj •Nl C 0 ° c o ° ...... ' . II + 3 "U r-t* O 2 o ■D 5‘ O r+ 2 T| x X o o % ■Nj CO° I I Z3 % + 7T a o CD 0> + TI c o c r s ® O O ■D ■D TJ

Fig. 5.3. DFP Phosphorylation of Protein by an Alkyl Phosphoyrlation Reaction dog and man (Meuldermans, et al, 1982). In vivo binding yields a lower percentage level which is similar to that obtained by Murphy et al (1979) in the dog. The discrepancy observed in the study presented here between jn vivo and jn vitro binding could depend on several factors; fentanyl binding is temperature and pH dependent (Hollt and Teschemacher, 1975) so that change from pH 7.4 to pH 7.55 increases fentanyl binding from 67% to 76%. The

37°C incubation step in the in vitro binding protocol could produce the observed increase in fentanyl binding compared to the in vivo protocol.

Though structurally similar, these drugs do not apparently bind to the. same sites. Fentanyl has been shown to bind red blood cells, glycoproteins and lipoproteins, whilst alfentanil is predominantly bound to the acute phase reactant protein a^-acid glycoprotein (a^-AGP) (Meuldemans et al, 1982). In fact, Meuldemans et al (1982) have calculated that if a^-AGP were the only binding protein, it could account for all alfentanil binding in human plasma but only about half of fentanyl binding. Volunteer plasma samples showed a linear relationship between a^-AGP concentration and free drug fraction only for alfentanil (Meuldermans et al, 1982).

5.16. Effect of DFP on Plasma Protein Binding of Alfentanil

The presence of DFP decreased the fractional binding of alfentanil to plasma proteins. As alfentanil is highly lipophilic, the blood-brain barrier does not lim it the entry of free alfentanil into the brain. It is reasonable to suppose, therefore, that by increasing free alfentanil increased brain levels and increased antinociceptive effect would follow. DFP did not alter fentanyl plasma protein binding and so an explanation for the observation of differential potentiation by DFP of alfentanil antinociception could be made on the basis of DFP specifically inhibiting the binding of alfentanil to plasma proteins. Though Schuh (1970) suggested that DFP binds to albumin (see Section 5.12.),

this does not rule out the possibility that DFP also binds a^-AGP because as already mentioned, DFP reacts with most proteins and will phosphorylate several classes of proteins when incubated with plasma in vitro (Murachi,

1963).

Moreover, it has been recently shown that albumin potentiates the binding of propanolol to a^-AGP (Chauvelot-Moachon et al, 1985) so that if this held true for alfentanil, binding to a^-AGP could be affected by DFP phosphorylation of albumin.

a-^-AGP is a high affinity low capacity binding protein (Piafsky, 1981;

Mackichan and Zola, 1982), so that if DFP were to compete for and saturate binding sites on the a-^-AGP molecule this would lead to raised levels of free alfentanil. Fentanyl, on the other hand, if displaced from these sites, would not necessarily result in increased free levels as it could be sequestered in other high capacity binding depots such as red blood cells.

Several drugs have been shown to bind a-^-AGP and there is often a m utual displacement of drugs as there is only a single binding site (MUller et al, 1983).

Furthermore, the binding site might represent a remote hydrophobic area within the protein part of the glycoprotein molecule (Brunnmer and MUller,

1985). So not only is the binding site probably a protein to which DFP could covalently bind, but it is also a hydrophobic region where the DFP molecule would tend to go due to its lipophilic nature (Karczmar, 1967). There is a good correlation between displacing potency against ^ H-imipramine from a^-AGP and lipophilic parameters for a variety of compounds (MUller et al,

1983). 5.17. Consequences of Alfentanil Displacement from Plasma Protein

Binding Sites

As the effect of a drug appears to be more closely related to the free

unbound levels rather than the total concentration (Lima et al, 1981) the

question arises as to what the consequences of the displacement of bound

alfentanil by DFP.

The degree of protein binding of alfentanil limits its entry into the brain.

This is reflected in the brainrplasma ratios which average about 0.6 at

400ug/kg (see Table 4.2.). Literature values measuring unchanged alfentanil

rather than total radioactivity show braintplasma ratios averaging around 0.13

in the rat for 10 minutes following i.v. administration (Michaels et al, 1981).

If binding to plasma proteins limits alfentanil entry into the brain,

displacement by DFP would tend to cause a proportional increase in entry of

alfentanil. In this situation, displacement of alfentanil would not alter the

average free drug concentration, though there would be a transient rise in

free drug concentration immediately following displacement (Mackichan,

1984). This is borne out in the data presented in Table 5.1. following in vivo

binding as free drug concentrations were little changed. The jn vitro binding

data shows that DFP increases free alfentanil concentrations. In vivo this

free alfentanil is able to redistribute into other tissues including the brain, so

that the actual steady state concentration is not altered jn vivo following

DFP whilst the average total drug concentration decreases. 5.18. Protein Binding and Relationship to Variation in Alfentanil Potency

As described in Section 2.27. initial studies with alfentanil showed AD^g values different from that obtained in experiments some months later. One explanation for this anomaly was that there is a variation in circannual sensitivity to alfentanil, however, though this may be true, the data from the binding experiments which show the relevance of alfentanil binding to its potency, introduces the possibility that variation in binding could markedly affect potency. If, for example, the batch of mice in the initial experiments were carried out had a low level viral infection, this could greatly increase a^-AGP levels and hence binding capacity. This could effectively increase the true AD^-g value. CHAPTER 6

GENERAL DISCUSSION 6 GENERAL DISCUSSION

In the foregoing chapters an account has been given of the potentiation of alfentanil, but not morphine or fentanyl antinociception by DFP pre-treatment in mice. This effect was not reversed by drugs used to treat DFP poisoning. In addition, DFP has differential effects on opioid-induced running behaviour, with the most marked effect observed in alfentanil-treated mice. Further experiments identified that DFP enhanced the entry of alfentanil into the brain, whilst the distribution of the other opioids was unaffected. This appeared to be due to a displacement of alfentanil from plasma protein binding sites, thereby allowing more drug to enter the central nervous system.

The differential potentiation of alfentanil may well be dependent on the observation that this opioid primarily binds to a^-AGP whilst fentanyl binds more than one protein (Meuldermans et al, 1982). Morphine's low fractional binding means that displacement from these sites would not greatly increase free drugs.

There are important implications to the findings presented here, demonstrating functional increase in antinociceptive potency of alfentanil following displacement from its plasma protein binding sites. In these studies, the data shows that in mice, 75% of alfentanil is bound to plasma protein, whilst in humans the figure is 92% bound (Meuldemans et al, 1982). The higher the fractional binding the greater the percentage increase in free drug following displacement, thus in humans, potentiation of alfentanil action could be even greater than that observed in mice. Alfentanil binding may vary in patients if they are not receiving any drug

therapy, as the normal levels of a^-AGP varies in normal healthy volunteers

from 55 to 140mg/100ml with a mean of 66mg/100ml (Piafsky et al, 1978).

Increased levels are probably due to subclinical or minor infections. a^-AGP

levels are also increased as a result of the physiological stress associated with

inflammation, cancer (Piafsky, 1980) and surgery (Fremsted et al, 1978).

This information suggests that alfentanil potency may be sensitive to

pharmacological and physiological variables, which may be important clinically.

It should be borne in mind, however, that it is an assumption to extrapolate the

demonstrated interaction in an experimental animal to an interaction in humans.

An assessment of the potential interactions of alfentanil with other drugs should

therefore be carried out with other drugs and using human plasma.

The property of the potentiating activity of DFP does not appear to be

dependent on its anticholinesterase activity per se, as the carbamate

anticholinesterases neostigmine and pyridostigmine, did not alter alfentanil

antinociception. A plausible interpretation of this is that DFP may produce a

stable phosphorylated plasma protein (which competes for alfentanil binding) and

this may hold true for other organophosphorous compounds including

organophosphates. Given that in DFP treated mice, morphine antinociception is

altered following atropine and pralidoxime administration, fentanyl appears to be

the opioid of choice and the least likely to cause interaction problems with

organophosphates or drugs used in the treatment of anticholinesterase poisoning.

This study has shown the existance and probable mechanisms of DFP potentiation of alfentanil antinociception. This is of importance not only as an example of

the relevance of plasma protein binding on drug effect, but also in terms of the

possible implications for the clinical use of alfentanil following administration of other drugs. There is clearly scope for further research in this area. REFERENCES

Ai, M.K., Ru, L.Q. and Luo, Q.Y. (1979). Influence of electroacupuncture on the cholinesterase activity in thalamus of rat. Proc. of Natn. Symp. Acupuncture, Moxibution and Acupuncture Anaesthesia, p.445, Beijing.

Ai, M.K. and Wu, Z.J. (1979). The influence of electroacupuncture on the ultrastructure of synapses in medial region of thalamus. Proc. Natn. Symp. Acupuncture, Moxibustion and Acupuncture Anaesthesia, p.451, Beijing.

Albin, M.S., Orr, M.D., Bunegin, L. and Henderson, P.A. (1975). Tetrahydroaminoacridine antagonism to narcotic addiction. Expl. Neurol., A6, 644-648.

Altschuler, R.A., Parakkal, M.H. and Fex, J. (1983). Localization of enkephalin-like immunoreactivity in acetylcholinesterase-positive cells in the guinea-pig lateral superior olivary complex that project to the cochlea. Neuroscience, 9, 621-630.

Axelsson, U. amd Homberg, A. (1966). The frequency of cataract after miotic therapy. Acta.Ophthalmol., 44, 421-423.

Barar, F.S.K. and Madan, B.R. (1976). Tremorine-oxotremorine-induced tremor, hypothermia and analgesia, and physostigmine toxicity in mice after pretreatment with 8-adrenoceptor antagonists. J. Pharm. Pharmac., 28, 286-289.

Bartolini, A., Bartolini, R., Aiello-Malmberg, P., Biscini, A. and Reni, G. (1979). New data concerning the interaction between cholinergic, enkephalinergic and serotonergic systems during analgesia. In: Adv. Pharmac. Res. Prac. Volume V, Opiate receptors and neurochemical correlates of pain, Ed. FUrst, S., pp.171-182.

Ben-Sreti, M.M. and Sewell, R.D.E. (1982). Stereospecific inhibition of oxotremorine-induced antinociception by (+)-isomers of opioid

antagonists: comparison with opioid receptor agonist. J. Pharm.

Pharmac., 34, 501-505. Ben-Sreti, M.M., Sewell, R.D.E. and Upton, N. (1982). Some observations on the effects of enantiomers of 2 benzomorphan narcotic antagonists and atropine on analgesia, tremor and hypothermia produced by oxotrdmorine. Arch. Int. Pharmacodyn. Ther., 256, 219- 2.2.9-

Bennhold, H. (1967). Transport function of the serum proteins: Historical review and report of recent investigations on the transport of dyestuff and of iron. In: Transport Function of Plasma Proteins. Ed. P.Desgrez and P.M. De Traverse, Elsevier Publishing Company, 1-12.

Berry, W.K. and Davies, D.R. (1970). The use of carbamates and atropine in the protection of animals against poisoning by 1,2,2- trimethylpropylmethylphosphonofluoridate. Biochem.Pharmacol., 19, 927-934.

Bhargava, H.N., Chan, S.L. and Way, E.L. (1974). Influence of hemicholinium (HC-3) on morphine analgesia, tolerance, physical dependence and on brain acetylcholine. Eur. 3. Pharmac., 29, 253- 261.

Bhargava, H.N. and Way, E.L. (1972). Acetylcholinesterase inhibition and morphine effects in tolerant and dependent mice. 3. Pharmac. exp. Ther., 183, 31-40.

Bhargava, H.N. and Way, E.L. (1975). Brain acetylcholine and choline following acute and chronic morphine treatment and during withdrawal. 3. Pharmac. exp. Ther., 194, 65-73.

Bhattacharya, S.K. and Nayak, B.B. (1978). Suppression of nociceptive responses in the rat by physostigmine. Role of serotinin. Neurosci. L e tt., 9, 245-248.

Bickel, M.H. (1978). Pharmacological consequences of plasma protein and tissue binding of drugs. In: Transport by Proteins. Ed. G.Blauer and H.Sund, Walter de Gruyter, 325-339. Bill, D.J., Hartley, J.E., Stephens, R.J. and Thompson, A.M. (1983). The antinociceptive activity of meptazinol depends on both opiate and cholinergic mechanisms. Br. J. Pharmac., 79, 191-199.

Bodnar, R.J., Kelly, D.D., Spiaggia, A., Ehrenberg, C. and Glusman, M. (1978). Dose-dependent reductions by naloxone of analgesia induced by cold-water stress. Pharmac. Biochem. Behav., 8, 667-672.

Botticelli, L.J., Lytle, L.D. and Wurtman, R.J. (1972). Choline-induced attenuation of morphine analgesia in the rat. Commun. in Psychopharmac., 1, 519-523.

Bowers, W.F., Fulton, S. and Thompson, J. (1984). U ltra filtra tio n vs equilibrium dialysis for determination of free fraction. Clin.Pharm acokin., 9, 49-60.

British National Formulary (1985). British Medical Association, London.

Brodie, M.S. and Proudfit, H.K. (1984). Hypoalgesia induced by the local injection of carbachol into the nucleus raphe magnus. Brain Res., 291, 337-342.

Brunner, F. and MUller, W.E. (1985). Prazosin binding to human a^-acid glycoprotein (orosomucoid), human serum albumin, and human serum. Further Characterization of the ’single drug binding site1 of orosomucoid. J.Pharm.Pharmacol., 37, 305-309.

Buckett, W.R. (1981). Stimulation produced analgesia is dependent on the influences of both circadian and circannual rhythms. Br.J.Pharmacol. 74, 281P.

Carroll, B.J. and Sharp, P.T. (1972). Monoamine metabolism of the morphine-induced activity of mice. Br.J.Pharmacol., 46, 124-139.

Carroll, M.N. and Lim, R.K.S. (1960). Observations on the neuropharmacology of morphine and morphine-like analgesia. Arch. Int. Pharmacodyn. Ther., 125, 383-403. Ceballos,de, M.L. and de Felipe, C. (1984). Circannual variation in opioid receptor sensitivity in mouse vas deferens. Eur.J.Pharmacol., 106, 227-228.

Chan, S.H.H. and Yip, M.K. (1979). Central neurotransmitter systems in the morphine suppresion of jaw-opening reflex in rabbits: The cholinergic system. Expl. Neurol., 63, 201-210.

Chaturvedi, A.K. (1984). Effects of meamylamine, nicotine, atropine and physostigmine on the phenydidine-induced behavioural toxicity. Pharmacol.Biochem.Behav., 20, 554-566.

Chan, T.T., Izazola-Conde, C. and Dewey, W.L. (1982). Evidence for the existance of peptide and non-peptide morphine-like materials in mouse brain: effect of an analgesic intracerebroventricular dose of acetylcholine on their levels. J. Pharmac. exp. Ther., 222, 612-616.

Chauvelot-Moachon, L., Marquis, O. and Giroud, J.P. (1985). Modification of propanolol binding to a^-acid glycoprotein by serum albumin. Biochem.Pharmacol., 34, 1591-1594.

Chen, G. (1958). The antitremorine effect of some drugs as determined by Haffner's method of testing analgesia in mice. J. Pharmac. exp. Ther., 124, 73-76.

Cheney, D.L., Trabucchi, M., Hanin, I. and Costa, E. (1975). Morphine dependence and in vivo turnover of ACh in whole mouse brain. 3. Pharmac. exp. Ther., 195, 288-295.

Cho, A.K., Haslett, W.L. and Jenden, D.J. (1961). Biochem. biophys. Res. Commun., 5, 276-279.

Christensen, E.M. and Gross, E.G. (1948). Analgesic effects in human subjects of morphine, meperidine and methadon. 3. Amer. Med. Assoc., 137, 594-599.

Clement, J.G. (1984). Sodium pentobarbital alteration of the toxicity and distribution of soman (pinacolyl methylphosphonofluoridate) in mice. Biochem.Pharmacol., 33, 683-685. Clem ent, J.G. and Copeman, H.T. (1984). Soman and sarin induce a long- lasting naloxone-reversible analgesia in mice. Life Sci., 34, 1415- 1422.

Clerm ont,de, P. (1854). Note sur la preparation de quelques ethers (Seance du lundi, 14 aout, 1854). C.R.Acad.Sci., 39, 388-340.

Cohen, E.L. and Wurtman, R.J. (1975). Brain acetylcholine : increase after systemic choline administration. Life Sci., 16, 1095-1102.

Cohen, S.D. (1984). Mechanisms of toxicological interactions following organophosphate insecticides. Fund.Appl.Toxicol., 4, 315-324.

Cohen, S.D. (1984). Mechanisms of toxicological interactions involving organophosphate insecticides. Fund. appl. Toxicol., 4, 315-324.

Cohen, J.A. and Warringa, M.G.P.J. (1954). The fate of P ^ labelled diisopropylfluorophosphate in the human body and its use as a labelling agent in the study of the turnover of blood plasma and red cells. J.Clin.Invest., 33, 459-467.

Collier, H.O.J. (1962). Multiple toe-pinch test for potential analgesic drugs. In: The assessment of pain in man and animals. Ed. C.A. Keefe and R. Smith, pp.262-270. Latimer Press, Plymouth.

Cox, B. and Tha, S.J. (1972). The antinociceptive activities of oxotremorine, physostigmine and dyflos. J. Pharm. Pharmac., 24, 547-551.

Coyle, D.E. and Denson, D.D. (1984). Protein binding of lidocaine in canine serum and plasma: Effects of an acidic pH and the technique of study. Biopharm.Drug Disp., 5, 399-404.

Cozantis, D.A., Friedman, T. and FUrst, S. (1983). Study of the analgesic effects of galanthamine, a cholinesterase inhibitor. Arch. Int. Pharmacodyn. Ther., 266, 229-238. Crocker, A.D. and Russell, R.W. (1984). The up-and-down method for the determination of nociceptive thresholds in rats. Pharmac. Biochem. Behav., 21, 133-136.

D'Amour, F.E. and Smith, D.L. (1941). A method for determining loss of pain sensation. J. Pharmac. exp. Ther., 72, 74-79.

Davies, O.L., Raventos, J. and Walpole, A.L. (1946). A method for evaluation of analgesic activity using rats. Br. J. Pharmac. Chemother., 1, 233-264.

Dayton, H.E. and Garrett, R.L. (1973). Production of analgesia by cholinergic drugs. Proc. Soc. expl. Biol. Med., 142, 1011-1013.

Denisenko, P.O. (1964). Pharmacological blocking of central cholinoreactive systems and the possibilities of its therapeutic application. In: Pharmacology of Cholinergic and Adrenergic Transmission, pp.147-152. Ed. G.B. Koelle et al., Pergamon Press, London.

Dewey, W.L., Cocolas, G., Daves, E. and Harris, L.S. (1975). Stereospecificity of intraventricularly administered acetylmethylcholine antinociception. Life Sci., 17, 9-10.

Dewey, W.L., Pedigo, N., Karbowski, M., Forbes, T. and Harris, L.S. (1976). Antinociceptive activity of acetylcholine and its involvement in the actions of morphine and other analgesics. In: Tissue Responses to Addictive Drugs. Ed. Ford, D.H. and Clouet, D.H. Spectrum Publishing.

Dirksen, R. and Nijhuis, G.M.M. (1983). The relevance of cholinergic transmission at the spinal level to opiate effectiveness. Eur. J. Pharmac., 91, 215-221.

Domino, E.F. and Wilson, A.E. (1973). Enhanced utilization of brain ACh during morphine withdrawal in the rat. Nature, 243, 285-286.

Duncan, D.B. (1955). Multiple range and multiple F tests. Biometrics, U, 1-42. ''an Eick, A. 3. and Bock, J. (1971). Comparison of analgetic, cholinomimetic anticholinergic and sympathomimetic drugs by means of the hot-plate test. Arch. Int. Pharmacodyn., 189, 384-387.

Eidelberg, E. and Erspamer, R. (1975). Dopaminergic mechanisms of opiate action in brain. J. Pharmac. exp. Ther., 192, 50-57.

Evans, W.O. (1961). A new technique for the investigation of some analgesic drugs on a reflexive behaviour in the rat. Psychopharmacology. (Berlin), 2, 318-321.

Fernando, J.C.R., Hoskins, B., Ho, I.K. (1984). A striatal serotonergic involvement in the behavioural effects of anticholinesterase organophosphates. Eur.J.Pharmacol., 98, 129-132.

Fields, H.L. and Basbaum, A.I. (1978). Brainstem control of spinal pain- transmission neurons. Ann. Rev. Physiol., 40, 217-248.

Flodmark, S. and Wramner, T. (1945). The analgetic action of morphine, eserine and prostigmine studied by a modified Hardy-Wolff-Goodall method. Acta Physiol. Scan., 91, 88-96.

Francis, G.R., Needham, P.L. and Shorter, J.H. (1983). An inexpensive microcomputer-controlled activity monitor for mice. Br.J.Pharmacol., 79, 424P.

Frederickson, R.C.A. and Pinskuj, C. (1975). E ffects of cholinergic and anticholinergic drugs and a partial cholinergic agonist on the development and expression of physical dependence on morphine in the rat. J.Pharmac.Exp.Ther., 193, 44-55.

Fremstad, D., Bergerud, K., H affner, J.F.W. and Lunde, P.K.N. (1976). Increased plasma binding of quinidine after surgery: A preliminary report. Eur.J.Clin.Pharm acol., 10, 441-444.

Fllrst, S., Friedmann, T., Bartolini, A., Bartolini, R., Aiello-Malberg, P., Galli, A., Somogyi, G.T. and Knoll, J. (1982). Direct evidence that eseroline possesses m orphine-like effects. Eur. J. Pharmac., 83, 233-241. Gall, D. (1981). The use of therapeutic mixtures in the treatment of cholinesterase inhibition. Fundam.App.Toxicol., L 214-216.

Galli, A. (1985). Inhibition of cholinesterase by the opioid analgesic meptazinol. Biochem. Pharmac., 34, 1579-1581.

Gardocki, J.F. and Yelnosky, J. (1964). A study of some of the pharmacologic actions of fentanyl citrate. Toxicol.Appl.Pharmacol., 6, 48-62.

George, R., Haslette, W.L. and Jenden, D.J. (1962). The central action of a metabolite of tremorine. Life Sci., 1, 361-363, 1962.

Ge Z., Huang W.M. (1979). A comparison between the influence of acupuncture and morphine upon locus coeruleus and raphe nucleus. Proc. Natn. Symp. Acupuncture, Moxibustion and Acupuncture Anaesthesia, Beijing, pp.431.

Glazer, E.J. and Basbaum, A.I. (1980). Leucine enkephalin: Localization and axoplasmic transport by basal parasympathomimetic preganglionic neurons. Science, 208, 1479-1480.

Glowinski, J. and Iversen, L.L. (1966). Regional studies of catecholamines in the rat brain. I. Disposition of ^H-norepinephrine, ^H-dopamine 3 and H-dopa in various regions of the brain. J.Neurochem., 13, 665- 669.

Goldstein, A. (1949). The interaction of drugs and plasma proteins. Pharmacol.Rev., L 102-165.

Gonzales, L.P. (1984). Quantitative analysis of phsostigmine-induced changes in behaviour. Pharmacol.Biochem.Behav., 21, 551-554.

Gordon, J.J., Leadbeater, L. and Maidment, M.P. (1978). The protection of animals against organophosphate poisoning by pre-treatment with a carbamate. Toxicol.Appl.Pharmacol., 43, 207-216. Green, J.P., Glick, S.D., Crane, A.M. and Szilagyi, P.I.A. (1976). Acute effects of morphine on regional brain levels of acetylcholine in mice and rats. Eur. J. Pharmac., 39, 91-99.

Green, V.A. and Davis, J.E. (1956). Modification of CNS drug action by anticholinesterases. Fed. Proc., 15, 431.

Guan, X.M. and Yu, Z. (1979). The role of cholinergic nerves in electroacupuncture analagesia: influence of eserine, acetylcholine and hemicholinium-3 on electroacupuncture analgesia. Proc. Natn. Symp. Acupuncture, Moxibution and Acupuncture Anaesthesia, p.443, Beijing, 1979.

Haffner, F. (1929). Experimentelle PrUfung Schmerzstillender Mittel. Deut. Med. Wochenschr., 55, 731-733.

Han, J.S. (1979). The role of some central neurotransmitters in acupuncture analgesia. Proc. Natn. Symp. Acupuncture, Moxibustion and Acupuncture Anaesthesia, Beijing, pp.27.

Han, J.S. and Terenius, L. (1982). Neurochemical basis of acupuncture anaesthesia. Ann. Rev. Pharmac. Toxicol., 22, 193-220.

Hardy, J.D., Wolff, H.G. and Goodell, H. (1940). A new method for measuring pain threshold: observation on spatial summation of pain. J. Clin. Invest., 19, 649-657.

Harris, L.S., Dewey, W.L., Howes, J.F., Kennedy, J.S. and Pars, H. (1969). Narcotic-antagonist analgesics: interactions with cholinergic systems. J. Pharmac. exp. Ther., 169, 17-22.

Harris, R.A., Yamamoto, H., Loh, H. and Way, E.L. (1977). Discrete changes in brain calcium with morphine analgesia, tolerance- dependence, and abstenance. L ife Sci., 20, 501-506.

Haas, S., Emrich, H.M. and Beckmann, H. (1982). Analgesic and euphoric effects of high dose diazepam in schizophrenia. Neuropsychobiol., 8, 123-128. Hansen, D., Schaum, E. and Wasserman, O. (1968). Serum level and excretion of diisopropylfluorophosphate (DEP) in cats. Biochem.Pharmacol., 17, 1159-1162.

Harris, L. and Stitcher, D. (1984). Protection against diisopropylfluorophosphate intoxication by pyridostigmine and physostigmine in combination with atropine and mercamylamine. Nauyn-Schmeidelberg’s Arch.Pharmacol., 327, 64-69.

Harris, L.W., Lennox, W.J. and Talbot, B.G. (1984). Toxicity of anticholinesterases: * Interactions of pyridostigmine and physostigmine with soman. Drug Chem.Toxicol., 7, 507-526.

Hartvig, P., Bergstrbm, K., Lindberg, B., Lundberg, P.O., Lunqvist, H., Langstrbm, B., SvHrd, H. and Rane, A. (1984). K inetic of "C -labelled opiates in the brain of rhesus monkeys. J.Pharmac.Exper.Ther., 230, 250-255.

Hayes, R.L., Katayana, Y., Watkin, L.R. and Becker, D.P. (1984). Bilateral lesions of the dorsolateral funiculus of the cat spinal cord: effects on basal nociceptive reflexes and nociceptive suppression produced by cholinergic activation of the pontine parabrachial region. Brain Res., 311, 267-280.

He, L.F., Xu, 5.F. and Jiang, C.C. (1979). The role of the caudate nucleus in acupuncture analgesia. Proc. Natn. Symp. Acupuncture, Moxibution and Acupuncture Anesthesia, p.32, Beijing.

Hendershot, L.C. and Forsaith, J. (1959). Antagonism of the frequency of phenylquinone-induced writhing in the mouse by weak analgesics and non-analgesics. J. Pharmac. exp. Ther., 125, 237-240.

Herken, H., Maibauer, D. and MUller, S. (1957). Acetylcholingehalt des Gehirns und Analgesia nach Einwirkung von Morphine und einigen 3- Oxymorphinanen. Arch. expl. Path. Pharmac., 230, 313-324.

Hayashi, G. and Takemori, A.E. (1971) The types of analgesic-receptor j interaction involved in certain analgesic assays. Eur.J.Pharm., 16, I 63-66. Herz, A. and Teschemacher, H.J. (1971). Activities and sites of antinociceptive action of morphine-like analgesics and kinetics and distribution following intravenous, intracerebral and intraventricular a p p lica tio n . A dv.D rug Res., 6, 79-118.

Ho, I.K., Loh, H.H. and Way, E.L. (1979). Toxic interaction between choline and morphine. Toxicol. Appl. Pharmac., 51, 203-208.

Hoffmeister, F. (1968). Tierexperimentelle Untersuchungen Uber den Schmerz und seine pharmakologische Beeinflusung. Cantor Edition, Wbrttenberg.

Httllt, V. and Teschemacher, H. (1975). Hydrophobic interactions responsible for unspecific binding of morphine-like drugs. Naunyn- Schm iedeberg’s A rch.P harm acol., 288, 163-177.

Holtzman, S.G. and Jewett, R.E. (1971). Interactions of morphine and nalophine with physostigmine on operant behavior in the rat. Psychopharmacology (Berlin), 22, 384-395.

Houser, V.P. (1976). Modulation of the aversive qualities of shock through a central inhibitory cholinergic system in the rat. Pharmac. Biochem. Behav., 4, 561-568.

Houser, V.P. and Houser, F.L. (1973). The alteration of aversive threshold with cholinergic and adrenergic agents. Pharmac. Biochem. and Behav., .1, 433-444.

Houser, V.P. and van Hart, D.A. (1973). The effects of scopolamine and pilocarpine upon the aversive threshold of the rat. Pharmac. Biochem. Behav., 1, 427-431.

Hug, C.C., Jr. and Murphy, R. (1981). Tissue redistribution of fentanyl and termination of its effects in rats. Anaesthesiology, 55, 369-375.

Ireson, J.D. (1970). A comparison of the antinociceptive actions of cholinomimetic and morphine-like drugs. Br. J. Pharmac. 40, 92- 101. Izazole-Conde, C., Garcia, E., Mate-Moreno, E. and Rodriguez, R. (1983). Quantitative and temporal characteristics of photocell measured locomotor activity in a sample of mice. Proc.West.Pharmac.Soc., 26, 269-272.

Jackson, R.L., Maier, S.F. and Coon, D.J. (1979). Long term analgesic effects of inescapable shock and learned helplessness. Science, 206, 91-93.

Jacobson, A.E., Klee, W.A. and Du, W.J. (1977). A quantitative relationship betwwen antinociceptive activity, opiate receptor affinity and lipophilicity. Eur.J.Med.Chem., 12, 49-52.

Janowski, D.S., El-Yousef, M.K. and Davis, J.M. (1974). Acetylcholine and depression. Psychosomatic Med., 36, 248-257.

Janssen, P.A.J., Niemegeers, C.J.E. and Dony, J.G.H. (1963). The inhibitory effect of fentanyl and other morphine-like analgesics on the warm water induced tail withdrawal reflex in rats. Arzneimittelforsch., 13, 502-507.

Jhamandas, K., Phillis, J.W. and Pinski, C. (1971).Effects of narcotic analgesics and antagonists on the in vivo release of acetylcholine from the cerebral cortex of the cat. Br. J. Pharmac., 43, 53-66.

Jhamandas, K. and Sutak, M. (1976). Morphine-naloxone interaction in the central cholinergic system: the influence of subcortical lesioning and electrical stimulation. Br. J. Pharmac., 58, 101-107.

Johansson, T. and Becker, B.A. (1973). Morphine analgesia in rats at various ages. Acta.Pharmacol.Toxicol., 33, 429-441.

Jondorf, B.J., and McNamara, P.D. (1950). Distribution of radiophosphorus in rabbit tissues after injection of phosphorus-labelled diisopropylfluorophosphate. J.Pharmac.Exp.Ther., 98, 77-84.

Jusko, W.J. and Gretch, M. (1976). Plasma and tissue protein binding of drugs in pharmacokinetics. Drug Metab.Rev., 5, 43-140. Kaakkola, S. and Ahtee, L. (1977). Effect of muscarinic cholinergic drugs on morphine-induced catalepsy, antinociception and changes in brain dopamine metabolism. Psychopharmacology, 52, 7-15.

Karczmar, A.G. (1967). Pharmacologic, toxicologic and therapeutic properties of anticholinesterase agents. Physiol.Pharmac., 3, 163- 322.

Karczmar, A.G., (1970). History of the research with anticholinesterase agents. In: International Encyclopedia of Pharmacology and Therapeutics, Section 13, Vol. 1., Anticholinesterase Agents. Ed. A.G.Karczmar, Pergamon Press, 1-46.

Karczmar, A.G. (1977). Acute and long lasting central actions of organophosphorus agents. Fund.Appl.Toxicol., 4, S1-S17.

Katayama, Y., DeWitt, D.S., Becker, D.P. and Hayes, R.L. (1984a). Behavioural evidence for a cholinoceptive pontine inhibitory area: descending control of spinal motor output and sensory input. Brain Res., 296, 241-262.

Katayama, Y., Watkins, L.R., Becker, D.P. and Hayes, R.L. (1984b). Non opiate analgesia induced by carbachol microinjected into the pontine parabrachial region of the cat. Brain Res., 296, 263-283.

Katayama, Y., Watkins, L.R., Becker, D.P. and Hayes, R.L. (1984c). Evidence for involvement of cholinoceptive cells of the parabrachial region in environmentally induced nociceptive suppression in the rat. Brain Res., 299, 348-353.

Kato, T., Nemoto, R., Mori, H., Unno, K., Goto, A. and Homma, M. (1980). Changes in prolyl endopeptidase during maturation of rat brain and hydrolysis of substance P by the purified enzyme. J.Neurochem., 35, 527-535.

Kaufman, I.J., Somo, N.M. and Koski, W.S. (1975). Microelectrometric

titration measurements of the pKa's anc* partition and drug distribution coefficients of narcotics and narcotic antagonists and their pH and temperature dependence. J.Med.Chem., 18, 647-655. Kendall, D.A., Browner, M. and Enna, S.J. (1982). Comparison of the antinociceptive effect of GABA agonists: Evidence for a cholinergic involvement. J. Pharmac. exp. Ther., 220, 482-487.

Kenley, R.A., Howd, R.A. and Uyeno, E.T. (1982). Effects of PAM proPAM, and DFP on behaviour, thermoregulation, and brain AChE in rats. P harm acol.Biochem .Behav., 17, 1001-1008.

Kitchen, I. (1984). The rise and fall of endogenous opioid nomenclature. Prog.Neurobiol., 22, 345-358.

Kitchen, I. and Crowder, M. (1984). Assessment of the hot-plate antinociceptive test in mice: A new method for the statistical treatment of graded data. J.Pharmacol.Meth., 13, 1-7.

Klemm, W.R.. (1983). Efficacy and toxicity of drug combinations in treatment of physostigmine toxicosis. Toxicology, 27, 41-53.

Knoll, J. and Komlos, E. (1952). Einfluss des Peptons auf die Wirkung der Analgetika und Parasympathomimetica. Acta Physiol. Acad. Scient. Hung., 3, 127-136.

Kobayashi, R.M., Palkovits, M., Hruska, R.E., Rothschild, R. and Yamamura, H.I. (1978). Regional distribution of muscarinic cholinergic receptors in rat brain. Brain Res., 154, 12-23.

Kobayashi, R.M., Browstein, M., Saavedra, J.M. and Palkovits, M. (1975). Choline acetyltransferase content in discrete regions of the rat brain stem. J. Neurochem., 24, 637-640.

Koehn, G.L. and Karczmar, A.G. (1977). The behavioural effects of diisopropyl phosphofluoridate interactions with morphine and naloxone in the rat. Pharmacologist, 9, 141.

Koehn, G.L. and Karczmar, A.G. (1978). Effect of diisopropyl phosphofluoridate on analgesia and motor behaviour in the rat. Prog. Neuropsychopharmac., 2, 169-177.

Koehn, G.L., Henderson, G. and Karczmar, A.G. (1980). Diisopropyl phosphofluoridate-induced antinociception: possible role of endogenous opioids. Eur. J. Pharmac., 61, 167-173. Koehn, G.L., Lazaron, L.M. and Moon, B.H. (1981). Role of pituitary opioid peptides in physostigmine- or arecoline-induced antinociception in rats. Fed. Proc., 40, 283.

Koster, R. (1946). Synergisms and antagonisms between physostigmine and di-isopropyl fluorophosphate in cats. J.Pharmacol.Exp.Ther., 88, 39- 46.

Kuhar, M.J. and Murrin, L.C. (1978). Sodium-dependent, high affinity choline uptake. J. Neurochem., 30, 115-121.

Krsiak, M., Steinberg, H. and Stolerman, I.P. (1970). Uses and limitations of photocell activity cages for assessing effects of drugs. Psychopharmacology, 17, 258-274.

Kuribara, H. and Tadokar, S. (1983). Circadian variation in susceptibility to the ambulation-increasing effect of scopolamine in mice. Jap.J.Pharmacol., 33, 927-931.

Kushinsky, K. and Hornykiewicz, O. (1972). Morphine catelepsy in the rat. Relation to striatel dopamine metabolism. Eur.J.Pharmacol., 19, 119-122.

Kushinsky, K. and Hornykiewicz, O. (1974). Effects of morphine on striatal dopamine metabolism: possible mechanism of its opposite effect on locomotor activity in rats and mice. Eur. J. Pharmac., 26, 41-50.

Landauer, M.R. and Romano, J.A. (1984a). Acute behavioural toxicity of the organophosphate sarin in rats. Neurobehav.Toxicol.Teratol., 6, 239-243.

Landauer, M.R. and Romano, J.A. (1984b). The acute behavioural toxicity of tabun in rats. Soc.Neurosci.Abstr., 10, 1070.

Laquer, L. (1877). Eber atropin und physostigmin and ihre wirkung auf den intracularen druck. (Ein beitrag zur therapie des glaucomas). A rch.O phthalm ol., 23, 149-176. Laskawiec, G. and Herman, Z.S. (1982). The effect of certain neurotransmitters on the analgesic action of enkephalinamide. Acta Physiol. Pol., 33, 559-566.

Lawrence, D. and Livingstone, A. (1979). The effect of physostigmine and neostigmine on ketamine anaesthesia and analgesia. Br. J. Pharmac., 67, 426P.

Lenke, D. (1958). Narkosepotenzierendende und analgetische Wirkung von l,4-Dipyrrolidino-2-butrin. Naunyn-Schmiedebergs Arch, fur Pharmakol. und exp. Path., 234, 35-45.

Leopold, I.H . and K rishna, N. (1963) Local use o f anticholinesterase agents in ocular therapy. In: Cholinesterases and anticholinesterase agents. Ed. G.B.Koelle, Handbook of Experimental Pharmacology, Vol. 15, S pringer-V erlag, 1051-1080.

Leslie, G.B. (1969). The effect of anti-Parkinson drugs on oxotremorine- induced analgesia in mice. J. Pharm. Pharmac., 21, 248-250.

Lewis, J.W., Cannon, J.T. and Liebeskind, J.C. (1980). Opioid and non opioid mechanism of stress analgesia. Science, 208, 623-625.

Lewis, 3.W., Cannon, J.T. and Leibeskind, J.C. (1983). Involvement of central muscarinic cholinergic mechanisms in opioid stress analgesia. Brain Res., 270, 289-293.

Leysen, J.E., Gommern, W. and Niemegeers, C.J.E. (1983). ^H Sufentanil, a superior ligand for p-opiate receptors: Binding properties and regional distribution in rat brain and spinal cord. Eur.J.Pharmac., 87, 209-225.

Lima, J.J., Boudoulas, H. and Blanford, M.F. (1981). Concentration dependence of disopyramide binding to serum protein and its influence on kinetics and dynamics. J.Pharmac.Exper.Ther., 219, 741-747. Linebery, C.G. (1.981). Laboratory animals in pain research. In: Methods in Animal Experimentation, Vol. VI. Ed. W.I.Gay, Academic Press, 238-311.

Lipman, J.J. and Spencer, P.S.J. (1980). A comparison of muscarinic cholinergic involvement in the antinociceptive effects of morphine and clonidine in the mouse. Eur. J. Pharmac., 64, 249-258.

Little, H.J. and Rees, J.M.H. (1974). Tolerance development to the antinociceptive actions of morphine, amphetamine, physostigmine and 2-aminoindane in the mouse. Experientia, 30, 930-932.

Llewelyn, M.B., Azami, J., Grant, C.M. and Roberts, M.H.T. (1981). Analgesia following microinjection of nicotine into the periaqueductal gray matter (PAG). Neurosci. Lett., Supp.(7), S277.

Maclennan, A.J., Drugan, R.C. and Maier, S.F. (1983). Long-term stress- induced analgesia blocked by scopolamine. Psychopharmacology, 80, 267-268.

MacKichan, J.J. and Zola, E.M. (1982). Carbamazepine binding to plasma, albumin and a^-acid glycoprotein. Clin.Pharmac.Ther., 31, 246-247.

MacKichan, J.J. (1984). Pharmacokinetic consequences of drug displacement from blood and tissue proteins. Clin.Pharmacokin., 9, (Suppl.l), 32-41.

Malec, D. and Langwinski, R. (1982). Cholinergic influence on opioid activity in rats. Pol. J. Pharmac. Pharm., 34, 85-92.

Mantegazza, P., Parenti, M., Tammiso, R., Vita, P., Zambotti, F. and Zonta, N. (1982). Modification of the antinociceptive effect of morphine by centrally administered diazepam and midazolam. Br.J.Pharmac., 75, 569-572.

Marchbanks, R.M. (1968). The uptake of ("^C)choline into synaptosomes in vitro. Biochem. J., 110, 533-541. Martin, L.J., Doebler, J.A., Shih, T.M. and Anthony, A. (1985). Protective effects of diazepam on soman-induced brain lesion formation. Brain Res., 325, 287-289.

3 Martin. B.R. (1985). Biodisposition of H diisopropylfluorophosphate in mice. Toxicol.Appl.Pharmac., 77, 275-284. — *

Martin, W.R., Abdulian, D.H., Unna, K.R. and Busch, H. (1958). A study of the peripheral and central actions of dibromopyruvic acid. J. Pharmac. exp. Ther., 124, 64-72.

Matla, J. and Langwinski, R. (1982). Effect of benzodiazepines on the central action of narcotic analgesics. Pol.J.Pharmacol.Pharm., 34, 135-144.

Matsubara, K. and Matshushita, A. (1982). The binding of drugs by plasma proteins. J.Pharm.Sci., 57, 895-918.

Messiha, F.S., Hartman, R.J. and Geller, I. (1983). Biochemical toxicity of diisopropylfluorophosphate in the rat. Proc.West.Pharmacol.5oc., 26, 211-213.

Messing, R.B. and Lytle, L.D. (1977). Serotonin-containing neurons: their possible role in pain and analgesia. Pain, 4, 1-21.

Metys, J., Wagner, N., Metysova, J. and Herz, A. (1969). Studies on the central antinociceptive action of cholinomimetic agents. Int. J. Neuropharmac., 18, 413-425.

Meuldemans, W., Hurkmans, R., Hendrickx, J. and Heykants, J. (1980a). The excretion and metabolism of tritium-labelled alfentanil after intravenous administration in rats. Preclinical Research Report R39209/11, Janssen Pharmaceutica.

Meuldermans, W. Hurkmans, R., Hendrickx, J., Woestenborghs, R., Thijssen, J., Lenaerts, F. and Heykants, J. (1980b). Plasma levels, excretion and metabolism of tritium-labelled alfentanil after intravenous administration in dogs. Preclinical Research Report R 39209/12, Janssen Pharmaceutica. Meuldemans, W.E.G., Hurkmans, R.M.A., and Heykants, J.J.P. (19gl). Plasma protein binding and distribution of fentanyl, sufentanil, alfentanil and lofentanil in blood. Arch.Int.Pharmacodyn., 257, 4-19.

Meyer, M.C. and Guttman, D.E. (1968). The binding of drugs by plasma proteins. J.Pharm.Sci., 57, 895-918.

Michiels, M., Hendriks, R., Michielsen, !_., Heykants, J. and Lenaerts, F. (1981). Plasma levels and tissue distribution of alfentanil (R39209) in the male Wistar rat after a single intravenous dose of O.I6mg/kg. Janssen Pharmaceutica, Preclinical Research Report R39209/13.

Migdall, W. and Frumin, M.J. (1963). Amnesic and analgesic effects in man and centrally acting anticholinergics. Fed. Proc., 22, 188.

Mudgill, L., Friedhoff, A.J. and Tobey, J. (1974). Effect of intraventicular administration of epinephrine, norepinephrine, dopamine, acetylcholine and physostigmine on morphine analgesia in mice. Arch. Int. Pharmacodyn., 210, 85-91.

Mliller, W.E., Stillbauer, A.E. and El-Gamal, S. (1983). Psychotropic drug competition for ^H imipramine binding further indicates the presence of only one high-affinity drug binding site on human acid glycoprotein. J.Pharm.Pharmacol., 35, 684-686.

Murachi, T. (1963). A general reaction of diisopropylphosphofluoridate with proteins without direct effect on enzymic activities. Biochim.Biophys.Acta., 71, 239-241.

Murphy, S.D. (1980). Pesticides. In: Casarett and Daill’s TOxicology. The Basic Science of Poisons. Ed. C.D.Klaassen and M.O.Amdur, Macmillan, 357.

Niemegeers, C.J.E. and Janssen, P.A.J. (1981). Alfentanil (R39209) - A particularly short-acting intravenous narcotic analgesic in rats. Drug Develop.Res., J, 83-88. Oguchi, K., Arakawa, K., Nelson, S.R. and Sanson, F. (1982). The influence of droperidol, diazepam and physostigmine on ketamine- induced behavior and brain regional glucose utilization in rat. Anesthiology, 57, 353-358.

O'Keefe, J. (1964). Spinal cord mechanisms subserving pain perception. Master's Thesis, McGill University.

O'Neill, J.J. (1981). Non-cholinesterase effects of anticholinesterase. Fund. Ap p l.T o xico l., 154-160.

Paalzow, G. and Paalzow, P. (19 ). Antinociceptive action of oxotremortrine and regional turnover of rat brain noradrenaline, dopamine and 5-HT. Eur. J. Pharmac., 31, 261-272.

Piafsky, K.M., Borga, O., Odar-Cederlbf, I., Johansson, C. and Sjbqvist, F. (1978). Increased plasma protein binding of propanolol and chlorpromazine mediated by disease-induced elevations of plasma a^-acid glycoprotein. New Engl.J.Med., 299, 1435-1439.

Palkovitz, H. and Jacobowitz, D.M. (1974). Topographical atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. II. Hirjbrain (mesencephalon, rhombencephalon). J. Comp. Neurol., 157, 29-42.

Paton, W.D.M. (1957). The action of morphine related substances on contraction and acetylcholine output of coaxially stimulated guinea- pig ileum. Br. J. Pharmac. Chemother., 12, 119-129.

Pedigo, N.W. and Dewey, W.L. (1981). Comparison of the antinociceptive activity of intravenously administered acetylcholine to narcotic antinociception. Neurosci. Lett., 26, 85-90.

Pedigo, N.W., Dewey, W.L. and Harris, L.S. (1975). Determination and characterization of the antinociceptive activity of intraventricularly administered acetylcholine in mice. J. Pharmac. exp. Therap., 193, 845-852. Pellanda, C. (1933). La Geneserine - morphine, adjuvant de l’anesthesie generate. Lyon Med., 151: 653-657.

Pert, A. (1975). The cholinergic system and nociception in the primate: interactions with morphine. Psychopharmacologica (Berlin), 44, 131-137.

Pert, A. and Maxey, G. (1975). Assymetrical cross-tolerance between morphine and scopolamine induced antinociception in the primate:Differential sites of action. Psychopharmacologica (Berlin), 44, 139-145.

Phan, D.V., Doda, M., Bite, A., Gybrgy, L. (1973). Antinociceptive activity of nicotine. Acta Physiol. Acad. Scient. Hung., 44, 85-93.

Pinsky, C., Dua, A.K. and LaBella, F.S. (1982). Phenylmethylsulfenyl fluoride (PMSF) given systemically produces naloxone-reversible analgesia and potentiates effects of B-endorphin given centrally. L ife Sciences, 31, 1193-1196.

Pleuvrey, B.J. and Tobias, M.A. (1971). Comparison of the antinociceptive activities of physostigmine, oxotremorine and morphine in the mouse. Br. J. Pharmac., 43, 706-714.

Quillam, J.P. (1947). Di-isopropylfluorophosphate (DFP): Its pharmacology and its therapeutic uses in glaucoma and myasthenia gravis. Postgrad.Med.J., 23, 280-282.

Ramachandran, B.V. (1967). The influence of DFP, atropine and pyridinium oaldoximes on the rate of clearance of diisopropyl 32 phosphate (DI P) from the mouse circulatory system. Biochem.Pharmacol., 16, 2061-2068.

Randall, L.O. and Selitto, J.J. (1957). A method for measurement of analgesic activity on inflamed tissue. Arch. Int. Pharmacodyn. Ther., I ll, 409-419. Raskar, T.G. and Kaufman, L.W. (1983). Diisopropyl Phosphofluoridate (DFP) disrupts circadian activity patterns. N eurobehav.T oxicol.T eratol., 3, 407-411.

Reubi, J.C., Iversen, L.L. and Jessel, T.M. (1977). Dopamine selectivity increases ^H-Gaba release from slices of rat substantia nigra in vitro. Nature, 268, 632-654.

Ren, M.F., Tu, Z.P., Han, J.S. (1980). The effects of hemicholine, choline, eserine and atropine on acupuncture analgesia in the rat. In: Adv. Acupuncture and Acupuncture Anaesthesia, pp.329-440, Beijing, China.

Risch, S.C., Cohen, R.M., Janowsky, D.S., Kalin, N.H. and Murphy, D.L. (1980). Mood and behavioral effects of physostigmine on humans are accompanied by elevations in plasma 8-endorphin and cortisol. Science, 209, 1545-1546.

Risch, S.C., Janowsky, D.S., Kalin, N.H., Cohen, R.M. and Murphy, D.L. (1981). Cholinergic-endogenous opioid mechanism in mood and cognition in man. Psychopharmacology Bull., 17, 132-137.

Robbins, T.W. (1977). A critique of the methods available for the measurement of spontaneous motor activity. In: Handbook of Psychopharmacology, Vol. 7. Ed. L.L.Iversen, S.D.Iversen and S.H.Snyder, Plenum Press, 37-82.

Rodgers, R.J. and Hendrie, C.A. (1983). Social conflict activates status- dependent endogenous analgesic or hyperalgesic mechanisms in male mice: effects of naloxone on nociception and behaviour. Physiol. Behav., 30, 775-780.

Rodriguez de Lores Arnaiz, G., Zieher, L.M. and Robertis, E.De. (1970). Neurochemical and structural studies on the mechanisms of actions of hemicholinium-3 in central cholinergic synapses. J. Neurochem., 17, 221-229. Romano, J. and King, 3 . (1981). Naloxone differentially affects the analgetic actions of physotigmine on three behaviour measures of nociception. Soc. Neurosci. Abst., 7, 879.

Romano, J. and Shih, T.-M. (1983). Cholinergic mechanisms of analgesia produced by physostigmine, morphine and cold-water swimming. Neuropharmacology, 22, 827-833.

Rosenthal, S.M. (1926). The liberation of adsorbed substances from the proteins II. The effect of addition of sodium oleate to whole blood upon non-protein nitrogen in blood filtrates. J.Biol.Chem., 70, 129- 133.

Rubin, R.P. (1970). The role of calcium in the release of neurotransmitter substances and hormones. Pharmac. Rev., 22, 389-428.

Rupreht, J., Dworacek, B., Oosthoek, H., Dzoljic, M.R. and Valkenberg, M. (1984). Physostigmine versus naloxone in heroin-overdose. Clin. Toxicol., 21, 387-397.

Rypka, M. and Navratil, J. (1982). A contribution to the mechanism of effect of physostigmine and neostigmine (syntostigmine) in the CN5. Activ.Nerv.Sup. (Praha)., 24, 277-278.

Saelens, J.K., Bernard, P.S. and Wilson, D.E. (1980). Baclofen as an analgesic. Brain Res. Bull., 5, 533-579.

Sahley, T.L. and Berntson, G.G. (1979). Antinociceptive effects of central and systemic administrration of nicotine in the rat. Psychopharmacology, 65, 279-283.

von Sallmann, L. and Dillon, B. (1947). The effect of di isopropylfluorophosphate on the capillaries of the anterior segment of the eye in rabbits. Am.J.Physiol., 30, 1244-1262.

Samson, F.E., Pazdernik, T.L., Cross, R.S., Giesler, M.P., Mewes, K., Nelso, S.R. and McDonough, J.H. (1984). Soman induced changes in brain regional glucose use. Fund.Appl.Toxicol., 4, S173-S183. Sanchez, E., Del Villar, E., Letelier, M.E., Vega, P. and Cirio, R. (1982). Mecanismos de la sinergia entre diazepam y morfina. Rev.Med.Chile, 110, 7-14.

Sansone, M. (1982). Scopolamine-induced locomotor stimulation in mice: Effect of diazepam and a benzodiazepine antagonist. Psychopharmacol. (Berl.)., 77, 292-293.

Saxena, P.N. (1958). Mechanism of cholinergic potentiation of morphine analgesia. Ind. J. Med. Res., 46, 653-658.

Saxena, P.N. and Gupta, G.P. (1957). Potentiating effect of prostigmine on morphine-induced analgesia. Ind. J. Med. Res., 45, 319-3256.

Schaumann, W. (1957). Inhibition by morphine on the release of acetylcholine from the intestine of guinea-pig. Br. J. Pharmac. Chemother., 12, 115-118.

Schneider, J.A. and McArthur, M. (1956). Potentiation action of Ibogaine (Bogadin TM) on morphine analgesia. Experientia, 12, 323-324.

Schuh, F. (1970. On the binding of diisopropyl-phosphoro-fluoridate (DFP) to plasma proteins. Arch.Toxikol., 26, 262-272.

Schulman, D.S., Kaufman, J.J., Eisenstein, M.M. and Rapoport, S.I. (1984). Blood pH and brain uptake of C-morphine.Anesthesiology, 61, 540-543.

Seidel, E.R., Beaton, J.M. and Teague, R.S. (1979). The effects of cholinergic agents on morphine-induced circling behavior in the intact mouse. Eur. J. Pharmac., 56, 75-80.

Sethy, V.H., Naik, S.R. and Sheth, U.K. (1971). Effects of drugs influencing amine synthesis on the analgesic action of tremorine. Psychopharmacologia, 19, 73-80.

Silinsky, E.M. (1977). Can barium support the release acetylcholine by nerve impulses? Br. J. Pharmac., 59, 215-217. Sitram, N., Buchsbaum, M.S. and Gillin, J.C. (1971). Physostigmine analgesia and somatosensory evoked responses in man. Eur. J. Pharmac., 42, 285-291.

Slater, P. (1974). Effect of 6-hydroxydopamine on some actions of tremorine and oxotremorine. Eur. J. Pharmac., 25, 130-137.

Slater, P. (1981). The effect of 6-hydroxydopamine on the antinociceptive action of oxotremorine. Psychopharmacology, 74, 365-368.

Slaughter, D. (1950). Neostigmine and opiate analgesia. Arch. Int. Pharmacodyn. Ther., 83, 143-148.

Slaughter, D. and Munsell, D.W. (1940). Some new aspects of morphine action effects on pain. J. Pharmac. exp. Ther., 68, 104-112.

Smoothy, R. and Berry, M.S. (1984). Alcohol increases both locomotion and immobility in mice: An ethological analysis of spontaneous motor activity. Psychopharmacol., 83, 272-276.

Snead, O, III and Bearden, L.J. (1980). Anticonvulsants specific for petit mal antagonize epileptogenic effects of leucine enkephalin. Science, 210, 1031-1033.

Snir-Mor, I., Weinstock, M., Davidson, J.T. and Bahar, M. (1983). Physostigmine antagonizes morphine-induced respiratory depression in human subjects. Anesthesiology, 59, 6-9.

Spaulding, T.C., Ma Minh, G., Dunn, R.W. and Fielding, S. (1984). Lack of antinociceptive activity of clonidine in two pain models: Comparison with other analgesics and CNS active drugs. Drug Dev. Res., 4, 217- 222.

Stanski, D.R. and Hug, C.C. Jr. (1982). Alfentanil - a kinetically predictable narcotic analgesic. Anesthesiology, 57, 435-438.

Stone, V., Moon, W. and Shaw, F.H. (1961). Treatment of intractable pain with morphine and tetrahydroaminoacridine. Br. Med. J., 1, 471- 473. Su, C.-Y. and Loh, H.H. (1975). Cholinergic influence on the morphine- induced running response in the mice. J. Formosan Med. Ass., 74, 455-460.

Takemori, A.E., Tulunay, F.C. and Yano, I. (1975). Differential effects on morphine analgesia and naloxone antagonism by biogenic amine modifiers. Life Sci., 17, 21-28.

Tiula, E. (1984). A pressure ultrafiltration method for serum free drug determination. Comparison with equilibrium dialysis using normal and uraemic sera. Meth.and Find.Exptl.Clin.Pharmacol., 6, 51-55.

Tripathi, H.L., Martin, B.R. and Aceto, M.D. (1982). Nicotine induced antinociception in rats and mice:correlation with nicotine brain levels. J. Pharmac. exp. Ther., 221, 91-96.

Truhaut, R. (1966). The transportation of toxic compounds by plasma proteins. In: Transport Function of Plasma Proteins. Ed. P.Desgrez and P.M.de Traverse, E lsevier, 147-172.

Tyers, M.B. (1980). A classification of opiate receptors that mediate antinociception in animals. Br. J. Pharmac., 69, 503-512.

Upton, N., Sewell, R.D.E. and Spencer, P.S.J. (1983). Analgesic action of M- and k-opiate agonists in rats. Arch.Int.Pharcodyn.Ther., 262, 199- 207.

Usdin, E. (1970). Reaction of cholinesterase with substrates, inhibitors and reactivators. In: International Encyclopedia of Pharmacology and Therapeutics, Section 13, Vol. 1. Anticholinesterase Agents. Ed. A.G.Karczmar, Pergammon Press, 47-354.

Vallano, M.L. and McIntosh, T.K. (1980). Morphine stimulates cholinergic neuronal activity in the rat hippocampus. Neuropharmacology, 19, 851-853.

Vallano, M.L., Spoerlein, M.T. and Van der Wende, C. (1982). Morphine enhances high-affinity choline uptake in mouse striatum. Pharmac., Biochem. Behav., 17, 419-423. Vincent, D. and Sero, M. (1942). Action inhibitrice de Tabernanthe Iboga sur la Cholinesterase du serum, c.r. Soc. Biol., 136, 612- .

Vadlamani, N.L., Pontani, R.B. and Misra, A.L. (1984). Increased brain uptake of morphine in the presence of the antihistamine tripelennamine. J.Pharm.Pharmacol., 36, 61-63.

Wahlstrbm, G. (1978). Addictive drugs and some neurotransmitters (Acetylcholine and gamma-aminobutyric acid). In: The Bases of Addiction. Ed. J. Fishman, pp.353-374, Berlin, Dahlem Konferenzen.

Wang, C.Y., Yu, B. and Liu, X.C. (1979). The influence of acupuncture on the acetylcholine level in various regions of rat brain. Proc. Natn. Symp. Acupuncture, Moxibustion and Acupuncture Anaesthesia, p.444, Beijing.

Watkins, L.R., Katayama, Y., Kinscheck, I.B., Mayer, D.J. and Hayes, R.L. (1984). Muscarinic cholinergic mediation of opiate and non opiate environmentally induced analgesias. Brain Res., 300, 231- 242.

Watkins, L.R. and Mayer, D.J. (1982). Organization of endogenous opiate and non-opiate control systems. Science, 216, 1185-1192.

Weinberg, V.E., Lewis, J.W., Cannon, J.T. and Liebeskind, J.C. (1981). Evidence for the involement of central cholinergic mechanisms in opioid stress analgesia. Soc. Neurosci. Abst., 7, 879.

Weinstock, M., Davidson, J.T., Rosin, A.J. and Scheiden, A. (1982). Effect of physostigmine on morphine-induced postoperative pain and somnolence. British J. Anaesth., 54, 429-434.

Weinstock, M., Erez, E. and Roll, D. (1981). Antagonism of the cardiovascular and respiratory depressant effect of morphine in the conscious rabbit by physostigmine. J. Pharmac. exp. Ther., 218, 504-508. Weinstock, M., Roll, D., Erez, E. and Bahar, M. (1980). Physostigmine antagonizes morphine-induced respiratory depression but not analgesia in dogs and rabbits. Br. 3. Anaesth., 52, 1171-1175.

Weiss, B. and Laties, V.G. (1970). The psychophysics of pain and analgesia in animals. In: Animal Psychophysics. The design and conduct of sensory experiment, pp.185-216. Ed. W.C. Stebbins, Appleton- Century-Crofts, New York.

Welch, S.P., Vocci, Jr. F.J. and Dewey, W.L. (1983). Antinociceptive and lethal effects of intraventricularly administered barium and strontium: Antagonism by atropine sulphate or naloxone hydrochloride. Life Sci., 33, 359-364.

Wenk, G.L. (1984). Pharmacological manipulations of the substantia inominata-cortical cholinergic pathway. Neurosci. Lett., 51, 99- 103.

Widman, M., Rosin, D. and Dewey, W.L. (1978). Effects of divalent cations, lanthanum, cation chelators and an ionophore on acetylcholine antinociception. J. Pharmac. exp. Therap., 205, 311- 318.

Willis, J.H. (1970). Toxicity of anticholinesterases and its treatment. In: International Encyclopedia of Pharmacology and Therapeutics, Section 13, Vol. 1. Anticholinesterase Agents. Ed. A.G.Karczmar, Pergammon Press, 357-469.

Wilson, I.B., Hatch, M.A. and Ginsburg, S. (1960). Carbamylation of acetylcholinesterase. J.Biol.Chem., 235, 2312-2315.

Wong, C.-L. and Bentley, G.A. (1978). The role of the cholinergic system in the development of increased naloxone potency in mice. Eur. J. Pharmac., 50, 221-230.

Wong, C.-L. and Bentley, G.A. (1979). Further studies on the role of cholinergic mechanisms in the development of increased naloxone potency in mice. Eur. J. Pharmac., 56, 115-121. Wood, P.L. (1984) Animal models in analgesic testing. In: Analgesic: Neurochemical, Behavioural and Clinical Perspectives. Ed. M.Kuhar and G .Pasternak, Raven Press, 175-194.

Wood, P., English, J., Chakraborty, J. and Hinton, R.H. (1975). The use of non-ionic industrial detergents in liquid scintillation counting. Lab.Pract., 24, 739-740.

Wood, P.L., Stotland, L.M. and Rackham, A. (1984). Opiate receptor regulation of acetylcholine metabolism: Role of mu, delta, kappa and sigma narcotic receptors. In: Dynamics of Neurotransmitter Function. Ed. I. Hanin. Raven Press, New York.

Woolfe, G. and MacDonald, A.D. (1944). The evaluation of the analgesic action of pethidine hydrocholoride (Demerol). J. Pharmac. exp. Therap., 80, 300-307.

Wooten, W.C. and Klemm, W.R. (1980). Cholinergic involvement in morphine-induced analgesia and dependence: evaluation of tetrahydroaminoacridine. Research Commun. Subst. Abuse, 1, 39- 47.

Zhirkov, Y.A. and Piotrovskii. (1984). On the usefulness of ultrafiltration in drug-protein binding studies. J.Pharm.Pharmacol., 36, 844-845. aj-PENDIX a

Equation for Unpaired Student’s t-test

t = x

(SEM^2 + (SEM2 )2 APPENDIX B

Computer Program for Antinociceptive Data in Mouse Hot-Plate Test

A set of data has three components, the mean response time, the variation of response time about the mean, and the error distribution. For this data, it was found that a Weibull distribution could successfully be applied to this last component. The data may also be linearlized, such that log (mean response time) is linear in dose. Straight-line regression is calculated by a method similar to the usual least-squares method, using a general procedure called maximum likelihood estimation. The sum of squares is replaced by the likelihood function, which represents the probability of obtaining data under the model. This model enables predictions to be made on the percentage of response times that exceed 60 seconds. This model also allows for the inclusion of zero-dose (ie, saline) data. A fuller description of this model is given by Kitchen and Crowder (1985). ANAlGfM SCREEN i;ATA FEB S3): CENSORED RESPONSE HUES UNDER DRUGS

ANA < O L S U . • >: REFN DATA . 1 0 ) . WX C c»X ; .WY< 6 0 0 > . R ( 1 5 ) . B L U ( 2 . 1 5 / DOUBLE PRF.C I S IO N WI ( I 5 / . W 2( 1 5 ) , W 3(1 5 . 1 5 ) . P I V ,T G L .L L K D COMMON -'R ID A T / Y < 6 0 0 > . N . YMS . VCNSR. X ( 6 0 0 . 10 > . NDRG EXTERNAL CRT012 DATA IX/600. . IW/TOC/I . D - 1 0 / READ C.AfA ETC. READ; 1 .* > I RF>. IwR, I CHECK 10 CALL IkRTOl( IRD. ICHECK) ZF(N.lE.O) STOP 2 0 UR IT E M i PC-1) R E A D H . * ) NO IE STOP no so j«i,is Rl.U< 1 , J ) = 0 . 3 0 D L U ( 2 .* J ) = 1 0 0 . B L U M . N P > * . 01 - CALL FMIN!CRT012.NP.W1,LLKD»W2.W3.W3,IW.BLU) r PARAMETER COVARIANCE MATRIX A - W K 1 ) DO 5 0 J s l.N D R G 50 B G A M =W 1(NP ) WRITE(1.90©) READ( I .* > ICOV IF 67 WRITE!IWR,802) EDI*SD1.ED2.SD2 C R E S ID U A L S 70 WRITE!1.909) READM.*) IRES IF!IRES.EQ .1) CALL CRT022!A.B.GAM.WY,IWR) GOTO 2 0 801 FORMAT(2014) 802 FORMAT(1P8G10.3) 90? FORMAT!/IOHPARAMETERS/!1P6G12.5)) 903 FORMAT(5HPIV0T.1 2 .IPG10.3) 904 FORMAT(/45HFIT: ORDER OF REGN (l-LINEAR.2-QUADRATIC.ETC)) 906 FORMAT(/4HED50) 907 FORMAT!/10HSTD ERRORS/!1P80I0.3)> 903 FORMAT!/27HPARAMETER COVARIANCE MATRIX) 909 FORMAT(/9HRESIDUALS) END SUBROUTINE IKRT01( IRD.IWR) C ANALGESIC SCREEN CENSORED RESPONSE TIME DATA (FEB 83) C X HAS COLS (DRUG NO.. DOSE.RESPONSE TIME) IN TE G E R N O ( 2 0 ) REAL DS(20) COMMON/RTDAT/Y(600)> N.YMS.YCNSR, X(6 0 0 .1 0 ).NDRG YUS— 9 9 . Y C N S R -6 0 . D0SDIV-100. N * 0 READ!IRD,803) NDRG IF(NDRG.LE.0) RETURN DO 3 0 J D R G - l. NDRG READ(IRD.804) NDS READ *UDRG 20 X(N,?)-nS!.J)/DOSDIV READ!IRD.#) !X (I*3).I-I1*N > 30 Ci-in TINIIF VCNSR-ALOG( YCNSR> DO 4 0 1 = 1 ,N YI = X < 1 .3 ) I F ! V I . G T . O. ) Y ( I ) = 01 .00 ( Y I 5 40 CONTINUE IF!IW R .LE.0) RETURN WRITE!IW R,901> DO 6 0 1 - 1 . N 60 WRITF!IWR.80?> r X ( I ,..»> . J = 1 ..? ) , Y ! I > RETURN 802 FORMAT(1P8G10. 3■ 803 FORMAT!5X.13) 804 FORMAT(J5X. 13.) 90) FORMAT!/10H0HFCK DATA) END SUBROUTINE CRT012

SUBROUT INE CRT002!A.6 ,GAM.LLKD,GL»HL, IH,IGR,IW R) MINUS LOG-LIKD AND DERIVS TOR LOG-WEIBULL CENSORED RESPONSE TIMES f. MU/SIG MODEL SUPPLIED BY S/RTNE CRT003 REAL MU, B! 15)» GP ! 2 ). GC! 2 )»HP! 2» 2 )» HC! 2» 2 ). GM! J 3 )• OS! 15)» 1 HM<15.15),H S(15.15) DOUBLE PRECISION LLKD,GL(IH ),HL( IH ,IH ) C0MMON/RTDAT/YI6O0),N.YMS.YCNSR,XI600,10).NDRG L L K D * 0 . N P -N D R G + 2 IF(NP,G T.15) STOP IF { IG R . I.E . 0 > GOTO 2 0 DO 1 5 J * 1 , NP G I „ ! J > * 0 . IF(IG R .LE .l) GOTO 15 DO 10 K=1,NP 10 HL(J.K)=0. 15 CONTINUE 2 0 DO 4 0 1 * 1 ,N YI*Y!I) IF!Y I,FJ3. YMS) GOTO 40 ID *IFIX!X!T»l)+.5> D S * X ( 1 , 2 ) CALL CRT003(A,B.NDRG.GAM, ID.DS.MU.SIG.GM.GS.HM.HS,15.IGR) IFIYI.LT.YCNSR) CALL WBLL01*GM!K)-GP<1)*HH*GS!K)-GP!2)#HS!J.K> 30 HL(K.J)*HL(J.K) 35 GL! J) *GL.! J) -G P!t)«GM !U)-GP!2)*GS RETURN 801 FORMAT!20I4) 802 FORMAT!1P8G10.3) 803 FORMAT!IPG12.5) 901 FORMAT!/25HMINUS LOG-LIKD AND DERIVS) 906 FORMAT!2I3.1P6G12.5/!6X,1P6G12.5)) END SUBROUTINE CRT003!A,B»NB•GAM, ID,DS,MU.SIG,GM,OS.HM.HS,IH.IGR) C CENSORED RESPONSE TIMES C REGN MODEL TYPE MU-A+B!ID)*DS !FOR DRUG ID,DOSE DS), SIG*OAM»MU C RETURN MU,SIG AND DERIVS WRT RETURN HS i 1 , N P ) * 1 . HS1NP,1)*I. HS! ID + 1»NP)«=DS HS1NP,ID+1)*DS RETURN END SUDROUTINE CRT022!A,B,GAM.OR»IWR) C 10G-WEIBULL CENSORED RESPONSE TIMES! EXPONENTIAL RESIDUALS C O R !1 ),,...OR!N) CORRESP TO NON-CENSORED Y'S INTEGER NW!600) REAL MU,B(15)»OR!600),GM!1>.OS!1 ),HM!1 ,1 ),HS!1»1) COMMON/RTDAT/Y!6 0 0 ).N,YMS,YCNSR. X!6 0 0 ,1 0 ),NDRG DATA EG/O.577215664?/.P6/1.2825498/ IF ! IWR.GT.0) WRITE!IWR,901) N,NDRG.YMS.YCNSR DO 20 1*1.N ID *IFIX (X !t.t)+.5> D S - X ! 1 , 2 ) CALL CRT003!A.B.NDRG,GAM.ID,DS.MU,SIG,CM.OS.HM,HS,1.0) YD*P6«!Y!I)-MU)/SIG OR!I>*EXP!YO-EG) IF!IW R.GT.0) WRITE!IWR.803) ID,DS.M U.Y!I).YD,OR!I) 7.0 C O N TIN UE RETURN 803 FORMAT! I3.9F 8.4) 901 FORMAT!/? !HEXPONENTIAL RESIDUALS.214,F 6 .1.F 7.4) END SUBROUTINE WBt.LOl (Y,MU. SIG ,PP . CD, GP,GC, HP. HC, IGR) C LOG-WEIDULL DISTN: PD-LOG DENSITY FN, CD*l OG!1-DISTN FN). AND r D E R IV S WRT M U ,S IG RFAL MU,GP!2 ),G C!2).HP!2 ,2 ).HC(2.2) DATA EG/O.5772156649/.P6/1.2825498/ YD=P6*!Y-MU)/SIG EY*EXP(YP-EG) PD*ALOG!P6/SIG )+YD-EG-EY CD*-EY IF ! IGR. LE. 0) RETltRN GP!1)*-P 6*!1.-EY)/SIG 0P !2>*-!!.+Y0*!1.-EY>)/SIG GC!1>*P6«EY/SIG GC!2>*YD«EY/SIG IF!IG R .LE .t) RETURN S02*SIG*SIG HP!1 ,I>*-P6*P6*EY/S02 H P !I,2 )*P6+!1 .-EY-YP#EY)/ $ G 2 HP!2»1)«HP!1,2) HP!2,2)*!1.+!YD+YD)*!1 .—EY)-YD*YD*EY)/SG2 HC(1 .1 )»-P6*P6*EY/SG2 HC!1.2)=-P 6*(1 .+YD)+EY/SG2 M C!2,1)*HC!J,2) HC!2» 2 )*-!2 .+YD>»FY*YD/SG2 RETURN END E p P 0 O p -P 0 O Q s 0 0 0 p> 0 1—1 TJ 0 d CQ P >£ P -p •H □ 5 S S *H - £ £ £ a5 p> m P -H p h si s i l l 7 - i 7 be p> 0 i '.Z2 0 0 m S S :^s« • i * P c p 5 C Sc 2<<£ ft 0 <55- 5Sls:;‘=us p CQ MM I! i s r p 0 • > - > «*si& > •N -0710-:5ts:& • » •• 5*. -O* t- £5 - ill! lllfl^lls 0 +J T5 +J 0 0 s lb s Isslst Isslllsslls P s P il.J l| Slil P: O d E O P O O «H O ftl p •H cp o

I; H n t 111) •; | "‘

l l j ! s li!Js|^!P!f*!f*! ” - ==Esi*fl!!fl! s ?s . t-, siiss;-!?:?::!:.?:?;::!: !? -1% si 5 nn:ni«uii s iiii^iiiiiiitiiip ,is | fsj. HssliiHiiiiiiiiififill. 235 11*1 is fs^l^l|jsel!!,32:sss3ss33ssfll§sslr .islil JssIMssiMSSssSsssssSssse Jds-ssssSsS Jsfl » *

u 0 -p pi ft s £ O I I O 5 : , = - O ^

Z:: S 5■ '• ^ IN ^ u Q 10• • £ j ?1 ?sf - . J * -H CO I I 'f : n . i U U ;. ;- v >y a ?: s L.r i £ =’t £ - i 5 = S CQ O . •. ?i s ; ;a5:5' ?. ;5 5, i; leg 2 -> pq o 'r_1H *• -.I «* si I n r « si?!?l ; i ?5 i u .|U ! : : ® S ?! V- 51 !? I *21. S!*m m |» .5 1! 7 I c'lMs1 ' ? 1* n © I1 \u,\\ :i 1 Ui IlIIMsii it is It ;\ ] IPIpi .,2 i ,..:sa; : i!?li $ g ri.i«||!i I? -ssHL ihiUsnl'?**!? is _ o«'- ‘in?1 _ _ . *fm C * «- *♦♦♦♦*.*♦i-mmsi*** S K «6 < “ ,5‘ £??I= -= " sv?; si;? ■*' :;£? Josssssssss-S icq 8 lsLusIH-s 1HH isl??IIIIISS! 8*555^ a ‘ i;??£S££ ilritstSSSJS£ = 5li*£-s^“- ,I' ,e ail|a2‘i|!|2Si«i??2«ssr !»»*■,2 5;5iti:5:MM-i-i;uo;-. iiiini^iiii .,u mu e a ^ n --0T--.llllfllllll* >_ ^ CO

P CtJ p *n d S ft a P: O PbC aJ x o >» QI—I pft "H-p HX CQ-H > ft -h p> ft -P o <3 ■ Eh APPENDIX D(i) Program to obtain values for AO, Al, A2 and A3, as parameters for quench curve for 1210 Ultrobeta

10 REN NAME STANDARD PARAMETERS 20 REN PROGRAHHER A/SCOTT 30 REH DATE 2 4 .0 7 .8 0 40 REH THIS PR0GRAHHE CALCULATES THE COEFFICIENTS OF THE GIVEN DATA 50 REM BEST-FITTING THIRD DEGREE P0LYN0HIAL 60 REH E=A0+A1*R+A2*R*2+A3*R*3 BY THE HETH0D OF LEAST SQUARES 70 DEFINE FILE #1= 'SCOTT",ASC,120 71 REM AT FIRST N, THE TOTAL NUHBER OF POINTS. 72 REH THEN A, THE ACTIVITY. 73 REH THEN THE VALUES FOR R AND CPH IN TURNS. 75 REH INSERT DATA IN FILE til. AT FIRST N, THE TOTAL NUMBER OF POINTS 80 REH AND A, THE ACTIVITY, THEN THE VALUES 90 REH FOR R AND CPH IN TURNS. 95 DIM R<28) 100 READ til ,N,A 110 FOR H=1 TO 10 120 S(H)=0 130 NEXT H 140 FOR 1=1 TO N 150 READ ti1,R(I ) , C 160 FOR J=1 TO 6 170 S(J)=S(J)+R(I)*J 180 NEXT J 190 FOR K=7 TO 10’ 200 S(K)=S(K) + 10"0*C*R(I) A (K-7)/A 210 NEXT K 215 PRINT 'R = /,R (I)/E = ' ,INT<10A4*C/A+.5)/100 220 NEXT I 230 D=S(2)-S(1)*2/N 240 E=S(3)-S(1)*S(2)/N 250 F=S(4)-S(1)*S(3)/N 260 G=S(8)-S(1)*S(7)/N 270 A=EA2-(S(4)-S(2)"2/N)*D 280 B=(S(5)-S<2)*S(3)/N)*D-E*F 290 C=(S*S(7)/N)*D-F*G)+B*C) 300 A3=Q/(A*((S(6)-S(3)A2/N)*D-F*2)+B"2) 310 A2=(B*A3-C)/A 320 A1=(G-A2*t-A3*F)/D 330 A0=(S(7)-A1*S(1)-A2*S(2)-A3*S<3))/N 335 PRINT 340 PRINT 'AO = ' , INT<10*A0+.5)/10 350 PRINT 'Al =',INT(10*A1+.5)710 360 PRINT 'A2 = ',INT(10*A2+.5)/10 370 PRINT 'A3 = ' , INT(10*A3+.5)/10 371 PRINT 3.72 FOR i= 1 TO N i74 Y=A3*R(I)A3+A2*R(I) ‘ 2+A1*R(I )+A0 376 PRINT 'Y =',INT(10"2*Y+.5)/100 378 NEXT I 400 END APPENDIX D (ii)

Quench curve obtained from d.p.m. package on RackBeta 1215 * ’’

Ou6+JL+\ C^l EFF1Z RATIO 8947.0 25.06 1.312 8019.0 22.46 1.187 7492.0 20.99 1.146 4523.0 18.27 1.050 5985.0 16.76 1.009 4275.0 11.97 .848 2531.0 7.09 .672 1774.0 4.97 .525 1743.0 4.88 .591 1070.0 3.00 .460

PLOT QUENCH CURVES ->R

IUSY CALCULATING

ISOTOPE 1. UINDOU 1

EFFZ 25.0*4 *| I **» I »* I * 22.50+ I * I 0* I «* 2 0 . 00+ * I « + I «* I *0 17.50+ « I I I • I *« 15. 00+ « I ♦« I * I ** 12.50+ ** I I * I *» 10.00+ *• I • I *• I * 7.50+ ♦* I *0 I ♦ I +* 3.00+ •#«*«•* I •• I * 10 I I------♦------♦------♦------♦------♦...... ♦...... ♦------♦...... 0 .500 .A00 .700 .000 .900 1.000 ' 1.100 1.200 1.300