mechanisms of interaction between anticholinesterases and opioids...
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MECHANISMS OF INTERACTION BETWEEN ANTICHOLINESTERASES
AND OPIOIDS UPON RODENT NOCICEPTION
by
Paul GREEN, BSc (Hons)., MSc
A thesis subm itted in accordance w ith the requirements o f the University o f Surrey fo r the degree of
Doctor o f Philosophy
Department of B iochemistry University o f Surrey Guildford, Surrey
November, 1985
ProQuest Number: 10798494
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ACKNOWLEDGEMENTS
I am very g ra te fu l to my supervisor, Dr Ian K itchen, who has given invaluable
advice and encouragement throughout my doctoral studies.
I would like to thank my mum and dad, brothers and Julie fo r a ll th e ir help
and support.
I would also like to thank Mr Paul Bishop (Biochem istry Workshop) fo r the
skilled construction o f locom otor a c tiv ity m onitor and the hot-p la te , D r John
Shorter (Beecham Pharmaceuticals) fo r the locomotor a c tiv ity m onitor
software, and Dr M artin Crowder (Mathematics Department) fo r the
s ta tis tica l models used in the analysis o f the antinociceptive data.
This work has been carried out w ith the support o f the Procurem ent
Executive, M in is try o f Defence.
I am gra te fu l to Miss Tracey Bakall fo r her excellent typing o f th is thesis.
SUMMARY
The lite ra tu re describing the role o f the cholinergic system and opioids on
antinociception is reviewed. The e ffec ts o f anticholinesterase agents and drugs
used in the treatm ent o f anticholinesterase poisoning were studied fo r the ir
e ffects on opioid antinociception and locomotion. In both mice and rats, the
irreversib le anticholinesterase, DFP, produced apparent antinociception, but only
at doses which caused m otor incapacita tion. P re-trea tm ent w ith DFP
potentiated the antinociceptive potency of the opioid drug a lfen tan il but had
li t t le e ffe c t on morphine or fentanyl using the hot-p la te test in m ice. The
reversible anticholinesterases, neostigmine and pyridostigm ine, did not a ffe c t
a lfen tan il antinociception in this species. In rats, DFP potentiated a lfen tan il and
fentanyl antinociception using the paw pressure test, but had no e ffe c t on
morphine. In m ice, drugs used to trea t anticholinesterase poisoning (atropine,
pralidoxime and diazepam) did not a lte r the e ffe c t o f DFP on a lfen tan il
antinociception. DFP produced a decrease in locom otor a c tiv ity in m ice which
was reversed fo llow ing atropine and pralidoxime adm in istration. DFP decreased
the hyperlocomotory a c tiv ity o f a lfen tan il in m ice, probably by increasing
a lfen tan il catatonia, but had l i t t le e ffe c t on morphine or fentanyl. When
diazepam was administered as part o f the trea tm ent fo r anticholinesterase
poisoning, locomotor scores o f opioid treated mice were markedly reduced. In
mice radiolabelled d istribu tion studies showed that DFP trea tm ent enhanced the
entry of a lfen tan il into a ll brain regions, w hilst morphine and fen tanyl levels
were unaltered. Studies on plasma protein binding o f a lfen tan il showed this was,
in part, due to a displacement o f a lfen tan il bound to plasma proteins by DFP,
enabling free drug to enter the brain. These studies suggest tha t fentanyl would
least like ly produce in teraction in individuals poisoned by irrevers ib le
anticholinesterase agents. In addition, the new opioid drug, a lfen tan il, may be
liable to interactions when used clin icall^w ith other drugs by displacement from
plasma protein binding sites to a lte r its antinociceptive a c tiv ity .
CONTENTS
ACKNOWLEDGEMENTS . i
SUMMARY ii
LIST OF TABLES AND FIGURES x
PUBLICATIONS x iii
CHAPTER 1 - INTRODUCTION 1
1.1. General In troduction. 2
1.2. Choice of Drugs Used. 2
1.3. Uses o f Anticholinesterases. 4
1.3.1. Reversible Anticholinesterases: 4
C lin ica l Uses.
1.3.2. Reversible Anticholinesterases: 3
N on-clin ica l Uses.
1.3.3. Irreversib le Anticholinesterases: 5
C lin ica l Uses.
1.3.4. Irreversible Anticholinesterases: 6
N on-clin ica l Uses.
1.3.3. Anticholinesterases as Chemical 6
W arfare Agents.
1.4. Aim of the Study. 8
1.5. Term inology. 8
1.6. A Review o f the L ite ra tu re 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 Antinociception. 19
(a) Muscarinic Agonists 19
(b) N ico tin ic Agonists 21
(c) Choline Esters 22
1.6.7. Cholinergic Antagonists and Antinociception 26
1.6.8. Miscellaneous Compounds. 29
1.6.9. Acetylcholine Histopharmacology and 30
Opioids
(a) Role o f Acetylcholine in Nociceptive 30
Pathways.
(b) Acetylcholine and Acupuncture- 32
Induced Antinociception
(c) Acetylcholine-O pioid Interaction 33
in Non-Nociceptive Systems.
1.6.10. Conclusions. 35
CHAPTER 2 - ANTINOCICEPTIVE ACTIVITY OF MORPHINE, 61
FENTANYL AND ALFENTANIL IN M ICE AND
RATS: EFFECTS OF DFP AND DRUGS USED
IN THE TREATMENT OF DFP POISONING
INTRODUCTION 62
2.1. An tinociceptive Models. 62
2.2. In it ia 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 ta tis tica l Procedures. 73
2.10.1. H ot-P la te Data. 73
2.10.2. Paw Pressure Data. 74
RESULTS 73
2.11. P ilo t Studies. 73
2.12. E ffe c t of DFP on Nociception in M ice. 75
2.13. E ffe c t o f DFP and Drugs Reversing the 81
E ffects o f DFP on Opioid Antinociception
in Mice.
2.14. E ffe c t o f Atropine and Pralidoxime and 83
Diazepam on Nociception.
2.15. E ffe c t o f Neostigmine and Pyridostigm ine 83
on Opioid Antinociception.
2.16. Antagonism of A lfen tan il, Alone and W ith 88
DFP, on Naloxone.
2.17. E ffe c t of DFP Upon Nociceptive Responses 88
in the Rat.
2.18. E ffe c t of DFP Pre-Treatm ent on 89
Opioid Antinociception in Rats.
DISCUSSION 94
2.19. Drug Injection Times. 94
2.20. E ffe c t of DFP on Nociceptive Responses 94
in Mice and Rats.
2.21. E ffe c t of DFP on Morphine Antinociception. 96
2.22. E ffe c t o f DFP in Fentanyl and A lfe n ta n il 97
Antinociception.
- 2.23. Proposed Mechanisms fo r DFP Potentia tion 98
of A lfen tan il.
2.23.1. Absorption 98
2.23.2. D istribution 98
2.23.3. Receptor Action 99
2.23.4. Metabolism 99
2.23.3. Excretion 100
2.24. E ffe c t o f Atropine and Pralidoxime 100
on DFP Potentiation o f A lfe n ta n il Antinociception.
2.25. E ffe c t o f Atropine and Pralidoxime 101
on the Antinociceptive A c tiv ity o f Morphine.
2.26. E ffe c t o f Diazepam on the Antinociceptive 102
A c tiv ity o f Opioids in Mice Receiving DFP,
Atropine and Pralidoxime.
2.27. Variation in Antinociceptive Potency o f 102
A lfe n ta n il 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 o f Drug E ffe c t 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 A c tiv ity Testing. 106
3.6. S ta tis tica l Procedures. 107
3.7. E ffe c t of DFP on Locom otor A c tiv ity . 108
3.8. E ffe c t of Atropine and Pralidoxime on Locomotor 108
A c tiv ity .
3.9. E ffe c t o f Diazepam on Locomotor A c tiv ity . 108
3.10. E ffe c t o f Various Treatments on A lfe n ta n il- 108
Induced Hyperlocomotion.
3.11. E ffe c t of Various Treatments on Fentanyl- 112
Induced Hyperlocomotion.
3.12. E ffe c t o f Various Treatments on 112
Morphine-Induced Hyperlocomotion.
DISCUSSION 115
3.13. E ffe c t o f DFP on Locomotor A c tiv ity . 115
3.14. E ffe c t o f Atropine and Pralidoxime Alone and 116
W ith DFP on Locomotor A c tiv ity .
3.15. E ffe c t o f Diazepam Alone and W ith DFP on 116
Locomotor A c tiv ity .
3.16. E ffects o f DFP on Opioid-Induced Hyperlocom otion. 116
3.17. The E ffe c t o f Atropine and Pralidoxime on the 118
Locomotor A c t iv ity o f DFP and Opioid-Treated M ice.
3.18. E ffe c t o f Diazepam on Opioid-Induced Locomotion. 118
3.19. In te rpre ta tion o f Locomotor Data. 119
CHAPTER 4 - DISTRIBUTION OF 3H MORPHINE, FENTANYL 120
AND ALFENTANIL IN MOUSE BRAIN: EFFECT
OF DFP ADMINISTRATION
INTRODUCTION 121
4.1. D istribution Studies. 121
MATERIALS AND METHODS 122
4.2. Animals and Experimental Conditions. 122
4.3. Drugs. 122
4.4. Dosing Protocols. 123
4.5. D istribution Studies Protocol. 123
4.6. S c in tilla tion Counting. 123
RESULTS 125
4.7. E ffe c t o f DFP Pre-Treatm ent on A lfen tan il Brain 125
and Plasma Levels.
4.8. E ffe c t o f DFP Pre-Treatm ent on Morphine and 125
Fentanyl Brain and Plasma Levels.
DISCUSSION 131
4.9. Opioid L ipoph ilic ity and Access to the CNS. 131
4.10. E ffe c t o f DFP on Opioid Brain Levels. 131
4.11. Mechanisms of Increased Levels o f A lfen tan il 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 o f Drugs. 136
5.2. Plasma Protein Binding o f A lfen tan il, Fentanyl and 136
DFP.
5.3. Assessment o f Drug Binding to Plasma Proteins. 137
MATERIALS AND METHODS 140
5.4. Anim al and Experimental Conditions. 140
5.5. Drugs. 140
5.6. In Vivo Protein Binding Protocol. 140
5.7. In V itro Protein Binding Protocol. 140
5.8. U ltracen trifuga tion Protocol. 141
5.9. Calculation o f Free and Bound Drug 141
Concentration.
5.10. Determ ination o f Drug Adsorption in 141
U itracen trifuga tion Apparatus.
5.11. Recovery Experiments. 144
5.12. Percentage o f Plasma Protein Binding of 144
Fentanyl and A lfen tan il.
5.13. E ffe c t o f DFP on Binding Fentanyl and 144
A lfe n ta n il to Plasma Proteins.
DISCUSSION 147
5.14. DFP Binding to Plasma Proteins. 147
5.15. Plasma Protein Binding o f A lfen tan il and 147
Fentanyl.
5.16. E ffe c t o f DFP on Plasma Protein Binding of 149
A lfen tan il.
5.17. Consequences o f A lfen tan il Displacement from 151
Plasma Protein Binding Sites.
5.18. Protein Binding and Relationship to Variation 152
in A lfen tan il Potency.
CHAPTER 6 - GENERAL DISCUSSION 153
REFERENCES 156
APPENDIX A 185
APPENDIX B 186
APPENDIX C 189
APPENDIX D (i) 190
APPENDIX D (ii) 191
LIST OF TABLES AND FIGURES
Fig. 1.1.
F ig .1.2.
Table 1.1.
Table 1.2.
Table 1.3.
Table 1.4.
F ig. 2.1.
F ig. 2.2.
Fig. 2.3.
Table 2.1.
Table 2.2.
Table 2.3.
Table 2.4.
Table 2.5.
Table 2.6.
Three Dimensional Structures and Formulae 3
of Morphine, Fentanyl and A lfen tan il.
B ritish troops w ill go to war on valium . 9
Methods o f Nociceptive Assessment. 12
Anticholinesterases and Antinociception. 37
Cholinom im etics and An tinociception. 46
Cholinergic Antagonists and Antinociception. 57
Dosing Protocols fo r A n tinociceptive Testing 68
in M ice.
Dosing and Testing Protocols in Rats 70
Use of the Ugo Basile Analgesy M eter. 72
E ffe c t of Repeated Exposure to Antinociceptive 76
Testing Procedures on H ot-P la te Times of
Naive Mice.
E ffe c t of M u ltip le Saline Injections on H ot- 77
Plate Response Times in M ice.
E ffe c t o f DFP on H ot-P la te Reaction Times 78
in the Mouse.
E ffe c t of D iffe re n t Vehicles on the Antinociceptive 79
A c t iv ity o f DFP.
E ffe c t of A tropine and Naloxone on 2mg/kg 80
DFP Induced Antinociception in M ice.
E ffe c t o f DFP (Img/kg) and o f Drugs Reversing 82
DFP Poisoning on Opioid-Induced Antinociception
in M ice.
Table 2.7.
Table 2.8.
Table 2.9.
Table 2.10
Fig. 2.3.
Fig 2.4.
Fig 2.5.
Fig 2.6.
F ig. 3.1.
Fig. 3.2.
F ig. 3.3.
Fig. 3.4.
F ig. 3.5.
E ffe c t o f Intram uscularly-Adm inistered
Diazepam and In jection Vehicle on
H ot-P la te Reaction Times in Mice.
E ffe c t of Various Doses of Neostigmine
on the Antinociceptive A c tiv ity o f A lfen tan il.
E ffe c t of Pyridostigm ine (lm g/kg) on
A n tinociceptive Potencies o f Morphine, Fentanyl
and A lfe n ta n il.
Naloxone Antagonism of Antinociception Induced
by A lfe n ta n il W ith and W ithout DFP.
Time Course fo r the E ffe c t o f DFP on the
Nociceptive Responses in the Rat Paw Pressure Test.
E ffe c t o f DFP on Morphine Antinociception in the
Rat Paw Pressure Test.
E ffe c t o f DFP on Fentanyl Antinociception in the
Rat Paw Pressure Test.
E ffe c t o f DFP on A lfen tan il Antinociception in the
Rat Paw Pressure Test.
Graph Showing the E ffe c t o f Various Drug
Treatments on Locomotor A c t iv ity in Mice.
Table Showing E ffe c t o f Various Drug Treatments
on the Locom otor A c t iv ity in Mice
E ffe c t o f DFP on A lfentanil-Induced Hyperlocom otion
Over 15 and 75 Minutes.
E ffe c t o f DFP on Fentanyl-Induced Hyperlocom otion
Over 15 and 75 Minutes.
E ffe c t o f DFP on Morphine-Induced Hyperlocom otion
Over 15 and 75 Minutes.
Fig. 4.1.
F ig. 4.2.
Fig. 4.3.
Table 4.1.
Table 4.2.
Fig. 4.4.
Fig. 5.1.
Fig. 5.2.
Table 5.1.
Table 5.2.
Fig. 5.3.
E ffe c t of DFP on Brain Levels o f (dpm g~^)
A lfen tan il
E ffe c t o f DFP on Brain Levels o f (dpm g~^)
Fentanyl.
E ffe c t of DFP on Brain Levels o f (dpm g~^)
Morphine.
E ffe c t o f DFP (lm g/kg” '*') on Plasma Levels o f
Morphine, Fentanyl and A lfe n ta n il.
E ffe c t o f DFP (Im g/kg- ^) on Brain Plasma Ratios fo r
Morphine, Fentanyl and A lfen tan il.
Scheme fo r Explanation o f Observed Increased
Brain A lfe n ta n il Levels o f DFP (lmg/kg)
Adm in istra tion.
Protocol fo r Plasma Protein Binding Experiments
In Vivo and In V itro .
The Amicon M icropartition MPS-1 System.
E ffe c t o f DFP Treatm ent on In Vivo and In V itro
Plasma Levels o f A lfen tan il.
E ffe c t o f DFP Treatm ent on Levels of In Vivo and
In V itro Free Plasma Levels o f A lfe n ta n il and
Fentanyl.
DFP Phosphorylation o f Protein by an A lky l
Phosphorylation Reaction.
126
127
128
129
130
133
139
143
145
146
148
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)
D iffe re n tia l e ffec ts o f diisopropylfluorophosphate poisoning and its
trea tm ent on opioid antinociception in the mouse.
L ife Sciences 33: 669-679.
GREEN, P.G. and KITCHEN, I. (1984)
Diisopropylfluorophosphate enhances entry o f a lfen tan il in to mouse brain.
B ritish Journal o f Pharmacology. 82: 297P.
GREEN, P.G. and KITCHEN, I. (1985)
D iffe re n t e ffec ts o f diisopropylfluorophosphate on the entry o f opioids in to
mouse brain.
B ritish Journal o f Pharmacology. 84: 657-661.
GREEN, P.G. and KITCHEN, I. (1985)
Displacement of ^H a lfen tan il from plasma protein binding sites by
diisopropylfluorophosphate in the mouse B ritish Pharmacological Society
Communication, September 1985. (in press).
GREEN, P.G. and KITCHEN, I. (1986)
Antinociception, opioids and the cholinerg ic system.
Progress in Neurobiology (in press)
CHAPTER 1
INTRODUCTION
1.1 General In troduction
Opioids have been used by humans fo r many thousands o f years. Opium was
f irs t used around 4000 BC when i t was mixed w ith wine by the Sumerians. Its
popularity as a drug remained, and is re flec ted by Sydenham who, in 1680,
w rote 'among the remedies which i t has pleased A lm ighty God to give to man
to relieve his suffering, none is so universal and efficacious as opium1. The
main active constituent o f opium, morphine, is s t i l l used therapeu tica lly as
the standard analgesic against which new agents are compared.
Despite Sydenham's praise, the therapeutic use o f opioids have a number o f
drawbacks, the most im portant o f which are respiratory depression and a
dependence lia b ility . Even though there has been extensive research in the
development of novel opioids, these problems have not been com plete ly
overcome. Another approach has involved the investigation o f non-opioid
drugs which either possess inherent analgesic a c tiv ity or are able to
potentia te the desirable (analgesic) but not the undesirable action o f opioids.
Cholinergic drugs, such as anticholinesterase agents, have received a lo t o f
a ttention in this area and the ir in te raction w ith opioids and nociceptive
systems is considered in Section 1.6.
1.2 Choice o f Drugs Used
Three opioids were used in the studies. Morphine as the standard analgesic
agent, and two synthetic opioids, fentanyl and a lfen tan il. These la tte r two
are of a new generation of opioids which are very potent and are extrem ely
rapid ly acting. These factors make them suitable fo r use as intravenous (i.v .)
analgesics for use in surgery. The structure o f these opioids is given in
Figure 1.1.
FENTANYL, , h n , o i co-oh » i oh ■ o i c h . c o o n ;. . c f h . : *•
ALFENTANIL l?4h3?n?0;'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 d i
isopropylfluorophosphate (DFP) was investigated most thoroughly.
Anticholinesterases are considered fu rthe r in the next Section.
1.3. Uses of Anticholinesterases
Anticholinesterases inh ib it the cholinesterase enzyme which results in a
build-up of excessive acetylcholine. The therapeutic and tox ic e ffec ts o f
these compounds are due to the accumulation of acetylcholine a t muscarinic
and n ico tin ic cholinergic receptors (Karczmar, 1967).
Anticholinesterases are norm ally divided into two groups, reversible and
irreversib le , which correspond to carbamyl esters and organo-phosphorous
compounds respectively. However both classes o f drugs react w ith the
cholinesterase enzyme in the same way, and so this c lassification re fle c ts
only quantita tive differences. There are, in fac t, very few tru ly reversible
compounds and even neostigmine is not considered to be in this class (Usdin,
1970). Nevertheless, fo r the sake of c la r ity this classification w ill be used in
the fo llow ing discussion.
1.3.1. Reversible Anticholinesterases: C lin ica l Uses
Therapeutically, reversible anticholinesterases are indicated in certa in
neuromuscular abnormalities such as myasthenia gravis, where neuromuscular
transmission is to be enhanced (see B ritish National Form ulary, 1983).
Neostigmine, pyridostigm ine, ambenonium, physostigmine and distigm ine are
the anticholinesterases prescribed fo r myasthenia gravis, pyridostigm ine
reported to have the fewest side-effects.
These drugs are also used to trea t urinary re tention, and because they
increase in testina l m o tility , they may be used as stim ulant laxatives
pa rticu la rly post-operative ly in case o f para ly tic ileus.
Probably the most common use fo r this class o f drug is in surgery. Non-
depolarising muscle relaxants which are used as a premedication in surgery,
compete fo r the acetylcholine receptor at the neuromuscular junction. The ir
action may be reversed by increasing the synaptic concentration o f
acetylcholine w ith anticholinesterases. The usual drugs used to reverse post
operative muscle re laxation are neostigmine and pyridostigm ine.
Anticholinesterases are also used in the trea tm ent o f glaucoma and indeed
the f irs t therapeutic use fo r this class o f drug was when physostigmine was
used fo r glaucoma (Laquer, 1877). Physostigmine is s t il l used fo r this purpose.
Reversible anticholinesterases are also used in the trea tm en t o f
anticholinesterase poisoning (see Section 1.3.3.).
1.3.2. Reversible Anticholinesterases: N on-clin ica l Uses
The major non-clin ica l use fo r reversible anticholinesterases is as
insecticides. Since the ir development in the 1950's, compounds such as
carbamyl and propoxur have been used extensively fo r the ir highly selective
insectic idal action.
1.3.3. Irreversib le Anticholinesterases: C lin ica l Uses
The f irs t organophosphorous anticholinesterase agent was synthesised in the
1850's (De Clerm ont, 1854). The f ir s t therapeutic use, however, was only
made some hundred years la te r when DFP was used to tre a t glaucoma
(Quillam , 1947). DFP became the drug o f choice in the trea tm ent o f
glaucoma as i t had the advantage over the carbamate drugs in tha t i t was
very long acting and could therefore be applied in frequently. However, i t
was established some years la te r that continued trea tm ent w ith long acting
anticholinesterase agents carried a high risk o f a specific type o f ca ta ract
(Axelsson and Holmberg, 1966). C urrently in the UK the only
organophosphorous compound used therapeutica lly, fo r the trea tm ent o f
glaucoma, is ecjhiopiate.
1.3.4. Irreversible Anticholinesterases: N on-clin ica l Uses
As fo r reversible anticholinesterases, the irreversib le organophosphorous
agents are extensively used as agricu ltu ra l insecticides. The f irs t
organophosphorous compound synthesised, te trae thy l pyrophosphate (TEPP)
(De C lerm ont, 1854), has been used as an insecticide though in recent years,
more selective agents have been developed. One of the most w idely used
insecticides is parathion, and this compound also probably accounts fo r more
accidental poisonings and fa ta lit ie s than any other organophosphorous
compound (Koelle, 1985). Even more selective compounds were developed to
reduce the toxico logica l risk to humans. For example, malathion has a UD^g
mouse:LD^g housefly ra tio o f 68 compared to only 6 fo r parathion (Usdin,
1970).
1.3.5. Anticholinesterases as Chemical Warfare Agents
The potentia l fo r anticholinesterases as chemical warfare agents was realized
in it ia lly in Germany by Lange in 1935 (Karczmar, 1970). This was fo llowed by
the synthesis and investigation o f new compounds (tabun and sarin) developed
specifica lly fo r the ir to x ic ity . Though these compunds were synthesised on a
large scale, they were never actually deployed due to the fear of re ta llia tio n
by A llied forces using the ir own organophosphates.
Work on chem ical warfare agents continued in post-war years, though
research proceeded at a low level, and B rita in stopped its development o f
new compounds in the 1950's. However, the USA, USSR and France s t il l
manufacture these compounds so the development o f e ffe c tive antidotes is an
im portant area of reseach.
P rotection against organophosphorous poisoning may be afforded by the
adm in istration o f several agents. The prophylactic e ffe c t o f carbamates has
been known fo r some tim e (Koster, 1946). Research in subsequent years has
shown tha t many carbamates pro tect against poisoning e ffe c tive ly , and tha t
a c tiv ity is dependent on many factors such as absorption, d is tribu tion and
metabolism (Gordon et al, 1978). The mechanism fo r the pro tective action o f
carbamates is dependent p rim arily on the form ation o f a semi-stable
carbamylated acetylcholinesterase; the enzyme in th is state cannot be
phosphorylated by an organophosphate. The carbamylated enzyme can
spontaneously break down to release the active enzyme (Wilson e t al, 1960)
and as the organophosphate is broken down re la tive ly rapid ly, su ffic ien t
released enzyme is present to maintain life (Berry and Davis, 1970).
Further protection from organophosphate poisoning may be achieved by
pharmacologically antagonising the e ffects o f excess acetylcholine, by
adm inistering an anticholinergic such as atropine. In addition, the inh ib ited
cholinesterase enzymes may be reactivated by the adm in istra tion o f oxime
such as pralidoxime mesylate. This is achieved by accelerating the
spontaneous reactiva tion o f the inhib ited cholinesterase by prom oting
hydrolysis o f the inhib itor-enzym e complex (Wills, 1970). This additional
trea tm ent w ith an oxime enhances the pro tective potency of the carbamate
and anticholinergic regimen (Gordon et al, 1978).
The most recent addition to the ba tte ry o f drugs used to tre a t
organophosphate poisoning is diazepam. I t has been shown to greatly enhance
the pro tective a c tiv ity of atropine against physostigmine le th a lity in mice
(Klemm, 1983). I t is believed to have this e ffe c t by v irtue o f its a n ti
convulsant a c tiv ity . I t does have the disadvantage, however, o f im pairing
m otor function and performance (Gall, 1981; Fig. 1.2.).
1.4. Aim of the Study
The in it ia l objective o f the research described in this thesis was to study
potentia l in teractions between irreversib le anticholinesterase agents and
opioid analgesics, in an a ttem pt to h ighlight drug in teraction problems which
m ight occur in individuals poisoned w ith anticholinesterase agents, trea ted
fo r such poisoning and given opioid drugs fo r the re lie f o f pain. This s ituation
is c learly most pertinent in a m ilita ry context, i f in the fie ld , personnel
exposed to organophosphorous compounds and receiving antidotes also sustain
in juries requiring surgery. In a broader sense, the aim of this thesis was to
study the nature and a ttem pt to analyse the mechanisms of in te rac tion
between opioids and anticholinesterase agents.
1.5. T erminoloqy
In this thesis, in describing the e ffects o f opioids on pain responses in
animals, the term 'antinociception1 is used, as d is tinc t from the word
'analgesia' used in the human context.
Analgesia is defined as 'absence of pain' and pain as 'suffering distress o f body
or mind' (Oxford English D ictionary). As suffering and distress are subjective
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phenomena, animals therefore cannot be described as perceiving pain per se.
However, they are able to sense and respond to noxious stim u li, so the terms
'nociception' and 'antinociception ' are used.
The term 'opioid' has been used throughout this thesis to describe analgesic
drugs. This is d is tinc t from the term 'opiate', the use o f which should be
confined to those products o f the opium poppy and re lated synthetic
alkaloids. 'Opioid' describes a ll drugs which are opiate-like in th e ir action
(see K itchen, 1984).
1.6. A Review of the Literature on the Role of the Cholinergic
System and Opioids on Antinociception
1.6.1 Introduction
The potentia ting e ffe c t o f anticholinesterase agents on the a c tiv ity o f opioid
analgesics has been known fo r several years (Flodmark and Wramner, 1945;
Slaughter, 1950) and there is now a large body o f lite ra tu re describing the
in te raction between cholinergic agents and opioids. This review summarizes
the present lite ra tu re and suggests mechanisms underlying in teractions.
Most o f the available lite ra tu re concerns work using animal models o f pain
perception though there are reports fo r human models and fo r c lin ica l pain,
and i t is these studies which indicate a potentia l therapeutic ro le fo r
cholinergic agents in enhancing the desirable analgesic e ffects o f opioids,
w h ilst reducing the undesirable components such as respiratory depression.
Furthermore, these studies have gone someway in elucidating the physiology
o f pain perception and have indicated possible fu tu re developments in the
pharmacology o f pain contro l. .
1.6.2. Nociceptive Models
The assessment o f the potency o f analgesic drugs is usually in it ia lly carried
out in experimental animals. A varie ty o f tests have been developed, using
several animal species to determine the antinociceptive a c tiv ity o f drugs and
a b rie f description of the tests described in this review is given in Table 1.1.
Even though a ll the models described are responsive to opioids, i t is
im portant to consider that differences in the nociceptive s tim u li (heat,
pressure, e lec trica l) may well involve d is tinc t pathways u tiliz ing d iffe re n t
neurotransm itters and that processing o f nociceptive in form ation may vary
TABLE 1.1. METHODS OF NOCICEPTIVE ASSESSMENT
Test Species Method of nociceptive stim ulation
End-point or quantifica tion
Reference
Hot plate RatMouse
Animals placed on heated m etal plate (48 to 55°C)
L ift in g , shaking or lick ing paw, or jumping
Woolfe andMacDonald,1944.
Tail f lic k RatMouseCat
Tail exposed to localized heat source e.g. focussed ligh t
F licking o f ta il away from heat
D'Amour and Smith,1941.
Tail immersion RatMouse
Term inal part ofta il immersed in water(50-55°C)
F lick ing or lif t in g ta il out o f water
Janssen et al., 1963.
Hot wire Rat Tail heated by hot w ire
F lick ing o f ta il away from heat
Davies et al., 1946.
Tail c lip RatMouseRabbitCat
Application of pressure by a c lip on base o f ta il
A ttem p t to b ite or remove clamp. Quantalresponse
H affner,1929.
W rithing Mouse Intraperitoneal in jection o f ir r ita n t substance (e.g. acetic acid, phenoxybenzo- quinone)
Number o f abdominal constrictions observed in set perioid
HendershotandForsaith,1939.
Form alin test RatCat
Subcutaneous in jection o f 5% form alin into forepaw pad
Scoring of in tensity and duration o f lif t in g , lick ing , shaking o f paw
O'Keefe,1964.
Paw pressure Rat Pressure applied to normal or inflamed hind paw. Pressure increasing w ith tim e
W ithdrawal o f foo t, struggling or vocalization
Randall andS e litto ,1957.
Ear clamp RabbitDog
Varying pressure applied by clip on ear
A ttem p t to w ithdraw or vocalization
Weinstock,1980.
Toe pinch Guineapig
CatPrim ate
Varying pressure applied by clip on toe
A ttem p t to w ithdraw or vocalization
C o llie r,1962.
Test Species Method of End-point or Referencenociceptive stim ulation quantification
Electroshock
Shocktitra t io n
Taile lec trica lstim ulation
Tooth pulp e lec trica l stim ulation
Radiant heat
Note:
Rat Application o f e lec tricMouse current, usually to
fee t
Rat E lec tric current toMouse feet w ith stepwisePrim ate increase in current
w ith tim e
Rat E lec tric current viaMouse electrodes in
base o f ta il
Rabbit E lec tric stim ulationto upper incisors
Many Part o f skin (maySpecies be blackened) exposed (eg. dog, to intense focussedhuman) ligh t or laser
Vocalization, Evans,vocalization 1961.a fte r dischargeor w ithdrawalo f paws
Time spent at Weiss andeach shock level Laties,before 1970.responding bypressing leverto reduce current
As fo r ta il Carol andclamp or L im ,electroshock 1960.
Threshold to H o ffm e is te rjaw opening or 1968.lick ing response
Varies w ith Hardyspecies: escape et al., 1940.response (Human(rodent), skin testing)tw itc h (dog),verbal report(human)
References re fe r to the f irs t report o f a particu la r method, or to a w idely used adaptation o f a method. There are o f course, numerous variations and refinem ents o f 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 exhib it varying degrees o f antinociception depending on the test
used. For example, k-op io id agonists are more e ffec tive against pressure
than therm al s tim u li (Tyers, 1980).
1.6.3. Anticholinesterases and Antinociception
a) Neostigmine
The f irs t studies o f the antinociceptive a c tiv ity o f anticholinesterase in man
were carried out by Flodmark and Wramner (1945). They showed
neostigmine, a quaternary reversible anticholinesterase, had antinociceptive
a c tiv ity and also potentiated morphine antinociception. A s im ila r
potentia tion was observed fo r other opioids (Slaughter, 1950). In contrast to
man, animal studies have fa iled to show antinociceptive a c tiv ity o f
neostigmine (Saxena and Gupta, 1957). However, there are reports of
potentia tion by neostigmine o f morphine antinociception (Knoll and Komlos,
1952; Saxena and Gupta, 1957). These observations have not been confirm ed
by other workers except when neostigmine is administered i.c .v . (Pedigo e t
al., 1975). By this route the antinociception is antagonised by naloxone and
atropine but not mecamylamine indicating tha t both opioid and muscarinic
systems mediate the e ffe c t (Pedigo et al., 1975).
As the quaternary compound neostigmine does not readily cross the blood-
brain barrier i t exerts its action peripherally fo llow ing systemic in jection . I t
is possible that the potentia tion o f opioids may be due to im paired
metabolism or altered d istribution. The observed antinociceptive e ffe c t 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 rapid ly. A lte rna tive ly , there
may be an e ffe c t on peripheral pain sensitive neurons.
b) Physostiqmine
Table II shows tha t physostigmine is reported to possess antinociceptive
a c tiv ity in most o f the tests described. Most o f the reports showing no
antinociceptive a c tiv ity involved centra l adm in istration o f physostigmine.
Since physostigmine readily crosses the blood-brain barrier, the lack o f e ffe c t
o f centra l adm in istra tion suggests tha t physostigmine may be m etabolized in
the periphery to the active compound. Indeed, FUrst et al. (1982) studied the
antinociceptive a c tiv ity o f eseroline, the prim ary m etabolite o f
physostigmine, and showed i t to be a naloxone reversible analgesic when
administered either peripherally or cen tra lly . The structure o f eseroline is
s im ilar to morphine and the antinociceptive e ffe c t appears independent o f its
anticholinesterase a c tiv ity .
Studies w ith atropine and hyoscine (Harris et al., 1969; Wong and Bentley,
1978; Dayton and G arret, 1973) and naloxone (Harris e t al., 1969; Romano
and King, 1981) indicate tha t physostigmine exerts its action through e ither
the cholinergic or opioid system or both. The a b ility o f naloxone to
antagonise physostigmine was not found in some studies (Pleuvrey and Tobias,
1971; Lipman and Spencer, 1980; Conzantis e t al., 1983) and has even been
shown to potentia te physostigmine (Romano and King, 1981). O ther evidence
suggesting tha t physostigmine’s e ffe c t may be independent of the opioid
system are that tolerance may be induced fo llow ing m ultip le adm inistrations
o f physostigmine, w hilst morphine antinociception is not a ffected by this
regimen (Dayton and G arret, 1973; L it t le and Rees, 1974). In studies in the
rhesus monkey, physostigmine appears to increase morphine antinociception
(Pert, 1975). However, this e ffe c t is observed only at doses tha t also
produced general behavioural depression.
The antinociceptive e ffects of physostigmine may be inhib ited by
pre trea tm ent w ith compounds depleting centra l 5-HT (Bhattacharya and
Nayak, 1978). 5-HT has been im plicated in antinociception (Messing and
Ly tle , 1977), and may be part o f a pathway also involving cholinergic neurons.
I t is worth considering tha t the neurotoxins used to deplete 5-HT do not have
absolute spec ific ity and may w e ll disrupt other systems.
Widman et al. (1978) described the e ffe c t o f cen tra lly administered calciumry
chloride on physostigmine antinociception. They observed tha t Ca + ionsn
reduced antinociception, and conclude that Ca + appeared to exert its
e ffec ts intraneuronally and postsynaptically. D ivalent 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 w ith m ice, 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 a lfen tam il antinociception is potentia ted
(K itchen and Green, 1983). Bhargava and Way (1972), however, did conclude
that DFP does potentia te morphine antinociception, though the e ffe c t was
not s ta tis tica lly s ign ificant.
The e ffe c t of DFP in rats has been studied thoroughly by one group (Koehn
and Karczm ar, 1977; Koehn and Karczm ar, 1978; Koehn et al., 1980). They
have determined that DFP has antinociceptive a c tiv ity in hot plate and ta il
f lic k tests from 0.5 mg/kg. Antagonist studies indicate that this e ffe c t is
mediated through both cholinergic and opioid systems. DFP's antinociceptive
action was stereoseiectively inhib ited by (-)benzomophan antagonists. The
(+)isomers were e ffe c tive only to a much lesser degree. The role o f this
stereose lectiv ity is considered fu rthe r in section 1.6.6. It is worthy o f note,
however, that in animals made to leran t to morphine, DFP's e ffe c t was
unaltered (Koehn et al., 1980). Saxena (1958) fa iled to show an
antinociceptive e ffe c t o f DFP in rats, or any morphine potentia ting a c tiv ity .
This may be due e ither to the u tiliza tio n o f pressure as the nociceptive
stimulus, 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 fo r up to 96 hours a fte r adm in istration. Further studies w ith soman
showed only an additive e ffe c t w ith morphine, and tha t naloxone was only a
pa rtia l antagonist up to 10 mg/kg, whereas atropine com plete ly antagonised
soman's e ffe c t at 2.2 mg/kg. This in form ation suggests tha t a t least some of
soman's antinociceptive a c tiv ity is exerted through non-opioid systems.
1.6.5. Other Anticholinesterase Agents
There are a few reports concerning antinociceptive ac tiv itie s o f some less
fam ilia r anticholinesterase compounds, some of which are natura lly occurring
plant products. Galanthamine is a natural alkaloid, isolated from
Am aryllidaceae, which possesses anticholinesterase a c tiv ity . I t also has a
chemical structure s im ilar to codeine. Its antinociceptive a c tiv ity has been
studied in the ra t hot plate and the mouse w rith ing tests (Cozantis et al.,
1983). In both these models galanthamine was antinociceptive, and was
shown to be able to potentia te morphine antinociception in the ra t. Its mode
o f action appears to be species dependent: its antinociceptive a c t iv ity is
antagonized by naloxone (0.1 mg/kg) in the ra t, but is re frac to ry to naloxone
up to 0.5 mg/kg in the mouse. I t is also possible that the nociceptive stimulus
may be a determ ining fac to r as to whether the action of galanthamine is
opioid mediated.
Tetrahydroam inoacridine (THA) was found to be inactive up to 3 mg/kg in the
mouse hot plate antinociceptive test (Wooten and Klemm, 1980) though i t
appears to prolong the antinociceptive e ffects o f morphine. Using a higher
dose o f 7.5 mg/kg and a d iffe re n t test model, Wong and Bentley (1978)
observed antinociceptive a c tiv ity fo r THA which was pa rtia lly antagonized by
atropine at 2 mg/kg. THA also potentiated the action o f morphine; th is
action was also antagonized by atropine, and in addition the antagonistic
a c tiv ity o f naloxone was enhanced w ith THA pretreatm ent.
A n tinociceptive e ffects of dibromopyruvic' acid have been reported (M artin et
al., 1958). This compound has weak anticholinesterase a c tiv ity (1/400 tha t o f
neostigmine) and may also possess muscarinic agonist a c tiv ity . However, its
antinociceptive a c tiv ity was not blocked by atropine at 2 mg/kg. Ibogaine is
an indole alkaloid from Tubernanthe iboqa and is able to in h ib it
cholinesterase (Vincent and Sero, 1942). Though ibogaine its e lf is not
antinociceptive up to 40 mg/kg, lower doses are able to potentia te morphine
antinociception and le th a lity (Schneider and M cArthur, 1956).
Recently, the organophosphorus insecticide tr io rth o to ly l phosphate has been
reported to produce a s ix-fo ld potentia tion o f heroin's potency in the mouse
w rith ing test (Cohen, 1984).
1.6.6. Cholinom im etics and Antinociception
a) Muscarinic Agonists
The data fo r oxotremorine shown in Table 1.3. clearly shows that i t is able to
induce antinociception in four species using many forms o f nociceptive
stim ula tion. Only the ra t paw pressure test fa iled to show clear
antinociceptive a c tiv ity as the e ffec ts were small and variable (B ill e t al.,
1983). Oxotremorine has been shown to be very potent on a molar basis in a ll
the studies shown, w ith an ED5Q as low as 11 ug/kg being reported by Metys
et al., 1969. I t appears to exert its action through both cholinergic and opioid
systems as atropine, hyoscine and other anticholinergics are e ffe c tive
antagonists (B ill et al., 1983; Harris e t 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; B a rto lin i e t
al., 1979; Ben-Sreti and Sewell, 1982; Lewis et al., 1983). I t is possible tha t
catacholamines may also be involved as Pleuvrey and Tobias (1971) reported
potentia tion by treatm ents which lower noradrenaline. Slater (1981)
however, reports that neuronal lesioning w ith 6-hydroxydopamine, which
destroys catecholam inergic neurons, acts through the noradrenergic system
to antagonize oxotrem orine’s e ffe c t. Other reports also indicate a role fo r
noradrenaline in the mediation o f oxotremorines antinociceptive actions
(Sethy et al., 1971; Barar and Madan, 1976). Oxotremorine is believed to be
the active m etabolite o f the compound trem orine (Cho et al., 1961), and
trem orine itse lf has been shown to be antinociceptive in mice (van Eick and
Bock, 1971; Lenke, 1938).
A recent study using stereospecific opioid antagonists has yielded im portan t
im form ation on the mode of action of oxotremorine (Ben-Sreti and Sewell,
1982). They report a reciprocal stereospecific sensitiv ity fo r oxotrem orine
and opioid analgesics toward opioid antagonists. The (-)benzomorphan
isomers, M rl452 and Mr2266, antagonized opioid induced antinociception but
had no e ffe c t on oxotremorine-induced antinociception. Conversely the
(+)benzomorphans, M rl453 and Mr2267 antagonized oxotremorine but not
opioid antinociception. Furtherm ore (+)benzomorphans antagonized
oxotremorine antinociception, but not tremorogenic or hypotherm ic actions
(Ben-Sreti et al, 1982), suggesting muscarinic receptor subpopulations
mediating these physiological e ffects, Koehn et al (1980) have shown the
e ffec ts of (+) and (-) opioid antagonists on DFP antinociception in the ra t to
be opposite to tha t observed by Ben-Sreti and Sewell (1982) in m ice. This
species d ifference may be due to the involvement, only in the mouse, o f
noradrenaline in oxotremorine's antinociceptive action. A lte rn a tive ly , DFP
may. act through the endogenous opioid system (Koehn et al 1980) w h ils t
oxotremorine may not as i t is re frac to ry 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 o f species and tests, though
potentia tion o f opioids has only been reported fo r pilocarpine (Knoll and
Komlos, 1952; Wong and Bentley, 1979) and this e ffe c t is not universal
(Kaakkola and Ahtee, 1977; Malec and Langwinski, 1982). An im portant
exception in the antinociceptive a c tiv ity of these muscarinic agonists is in
studies in non-human primates. Using the rhesus monkey, Pert (1975) fa iled
to show any antinociception fo r arecoline or pilocarpine. S im ila rly 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 (e lec tric shock titra tio n ), since pilocarpine has been shown to
be active in e lectro foo t shock in rats (Houser and van H art, 1973; Kaakkola
and Ahtee, 1977) and arecoline was e ffe c tive in ta il e lec trica l s tim ula tion
(Metys et al., 1969). In addition, arecoline has antinociceptive a c tiv ity , as
observed fo llow ing e lec trica l s tim ula tion of rabb it tooth pulp (Metys et al.,
1969). The d iffe ring results, therefore, lie in species differences. Pert (1975)
suggests tha t the responses o f rodents may involve polysynaptic spinal
reflexes which may be sensitive to cholinergic agents. A lte rn a tive ly the
neurochemical coding o f pain may be d iffe ren t in primates. These results
indicate the possible p itfa lls when extrapolating data obtained in rodents to
primates, including humans.
b) N ico tin ic Agonists
In the preceding sections most o f the papers describing antinociceptive
actions of cholinom im etics and the e ffects o f antagonists, have indicated
tha t cholinom im etics exert the ir action through CNS muscarinic receptors.
Indeed, the use o f n ico tin ic antagonists has been shown to be in e ffec tive in
antagonizing cholinergic-induced antinociception (Pedigo et al., 1975; Dewey
et al., 1975). However, there have been some reports describing potent
antinociceptive actions o f nicotine and dimethylphenylpiperazine. N icotine
has been shown to be antinociceptive in m ice, rats and dogs using a va rie ty o f
nociceptive stim u li. I t is very rapid ly acting, exerting an e ffe c t 30 seconds
a fte r s.c. adm in istra tion (T ripa th i et al., 1982). I t appears to be acting only
through n ico tin ic receptors as atopine and hyoscine were in e ffec tive in
antagonizing nicotines antinociceptive e ffe c t, (Phan et al., 1973; T ripa th i et
al., 1982), w h ils t mecamylamine was e ffe c tive (Phan et al., 1973; Sahley and
Berntson, 1979; T ripa th i e t al., 1982). However, Sahley and Berntson (1979)
observed that hyoscine did antagonise nicotine and proposed tha t nicotines
antinociceptive a c tiv ity was mediated via release o f acetylcholine (see
Section 1.6.6.(c)).
Species differences in nicotines action have been observed. Very close
corre lations between nicotine brain levels and antinociception is seen in the
mouse, but there is a poor corre lation in the ra t (T ripath i et al., 1982).
Further studies indicated tha t th is finding is probably mainly due to the rapid
development o f tachyphylaxis only in the ra t. This same group also observed
tha t naloxone had very l i t t le e ffe c t in the ra t, producing a maximum o f 23%
inh ib ition of nicotine induced analgesia at a high dose o f 10 mg/kg. In mice,
however, naloxone was e ffe c tive from 0.1 mg/kg, though complete
anatagonism could not be obtained. Sahley and Berntson (1979) also showed
tha t naloxone was ine ffec tive in the ra t.
The specific ganglionic stim ulant, dimethylphenylpiperazine has been shown
to be antinociceptive in mice (Phan et al., 1973) and this appears to be a
n ico tin ic e ffe c t as mecamylamine is an e ffe c tive antagonist.
These data, therefore, indicate that stim ulation o f the n ico tin ic receptor in
the CNS produces potent antinociception and on a molar basis equivalent to
morphine (T ripath i et al., 1982). Again species differences are apparent as in
the mouse nicotine’s action has an opioid component and does not exh ib it
tolerance. In the ra t, antinociception is mediated through the n ico tin ic
receptor and rapid tolerance occurs.
c) Choline Esters
The in traven tricu la r adm in istration o f acetylcholine has been shown to induce
antinociception in mice. This e ffe c t can be blocked w ith atropine, though not
w ith the quaternary derivative atropine m ethy ln itra te or the n ico tin ic
antagonist mecamylamine, indicating tha t acetylcholine exerts its
antinociceptive e ffe c t through centra l muscarinic receptors (Pedigo e t al.,
1975). Acetylcholine has also been reported to inh ib it morphine
antinociception (M udgill et al., 1974). However, the e ffe c t is small and
occurs 30 minutes a fte r i.c.v. in jection. As peak antinociception o f
acetylcholine occurs around 10 minutes post in jection, its antagonist e ffe c t
on morphine antinociception probably acts through a mechanism d is tinc t from
its antinocicpetive e ffe c t. There is also evidence tha t a component o f
acetylcholine's antinociceptive action is via opioid receptors. Its
antinociceptive actions are antagonised by naloxone (Pedigo e t al., 1975;
Pedigo and Dewey, 1981) and the rank order o f potency o f five narco tic
antagonists is the same fo r both acetylcholine and morphine (Pedigo e t al.,
1975). Furtherm ore, the divalent ions Mg^+, Mn^+ and Ca^+ but not Sr^+ or
Ba^+ also inh ib it both acetylcholine (Widman et al., 1978) and morphine
(Harris et al., 1977) antinociception.
I t has also been reported tha t i.c.v. adm in istration o f 40 pg acetylcholine
d iffe re n tia lly altered the level o f four peptide and non-peptide brain
fractions which had opio id-like a c tiv ity (Chan et al., 1982). I t is possible tha t
these opio id-like m ateria ls mediate the antinociceptive e ffe c t o f
acetylcholine.
There are, however, indications tha t acetylcholine exerts its antinociceptive
e ffe c t through mechanisms d is tinct from that fo r morphine. For example,
Pedigo et a i. (1975) found that the 1-isomers o f pentazocine and cyclazocine
blocked morphine w h ils t the d-isomers were w ithout e ffe c t; the converse is
true fo r acetylcholine. Pedigo and Dewey (1981) have observed this reverse
stereose lectiv ity w ith the (+) and (-) isomers o f naloxone (cf. oxotremorine).
In addition, studies w ith naloxone have yielded d iffe ren t apparent pA2 values
fo r naloxone versus morphine and acetylcholine (6.86 and 6.09 respectively)
and d iffe re n t slopes on the Schild p lot (-0.944 fo r morphine, -1.46 fo r
acetylcholine) (Pedigo and Dewey, 1981). These marked differences indicate
tha t naloxone inh ib its acetylcholine-induced antinociception non-
com petatively. They also showed tha t there is an assym etrical cross
tolerance between morphine and acetylcholine. The rapid tolerance to the
antinociceptive e ffe c t o f acetylcholine only s ligh tly decreased morphine
antinociception, however, tolerance to morphine produced a 12-fo ld increase
in the ED^g of acetylcholine.
W ith respect to in te raction and involvement o f amine systems, though
reserpine and tetrabenazine pretreatm ent appeared to inh ib it acetylcholine
antinociception, these compounds lack spec ific ity , and more specific
depletors o f dopamine, noradrenaline or 5-HT were w ithout e ffe c t (Pedigo
and Dewey, 1981).
In summary, the evidence indicates tha t acetylcholine exerts its
antinociceptive through muscarinic receptors (Dewey et al., 1975) and tha t
the mediation is independent o f opioid pathways.
Choline adm inistration has been shown to increase brain turnover and levels
of acetylcholine (Cohen and Wurtman, 1975). Knoll and Komlos (1952) found
tha t choline appeared to potentia te morphine antinociception. However, the
dose o f 200 mg/kg used is probably tox ic. A t this dose o f 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 fo r an
increased hot-p la te latency. The opposite e ffe c t o f choline was observed by
B o ttic e lli et al. (1972) who found a dose-dependent attenuation o f morphine
antinociception using 30 and 60 mg/kg. A t 60 mg/kg, choline alone did not
a ffe c t nociception. The authors Concluded tha t morphine's action may be due
to its a b ility 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 e ffec ts of
morphine. This hypothesis however, is not easy to reconcile w ith the
lite ra tu re concerning i.c.v. adm in istration o f acetylcholine (vide supra) and
the lite ra tu re concerning the e ffects o f opioid analgesics on brain
acetycholine levels is contrad ictory (see Dewey et al., 1976). I t is possible
tha t choline may be acting on the post-synaptic cholinergic receptor; th is has
been proposed by Fredrickson and Pinsky (1975).
Carbachol does not cross the blood-brain barrier but several studies using
i.c.v. and in tracerebra l (i.e.) adm in istration have demonstrated
antinociceptive a c tiv ity o f carbachol. Furtherm ore, i.e. application studies
have gone someway in identify ing the particu la r groups o f cells w ith in the
brain tha t are responsive to carbachol. Metys et al, co-workers (1969)
showed tha t i.c.v. carbachol (at 1.25 gg) was antinociceptive in both ra t and
rabb it. Local i.e. application indicated tha t the septal areas and m idbrain
re ticu la r form ation - centra l grey region were both sensitive to 0.3 gg
carbachol, whereas cudate putamen and dosal hippocampal adm in istra tion
only showed effects between 5 and 10 gg, doses at which transventricu la r
d iffusion may w e ll occur.
Brodie and P roud fit (1984) examined the role o f the cholinergic system on the
nucleus raphe magnus (NRM). This structure in the caudal medulla has been
shown to a ffe c t nociceptive processing (see Fields and Basbaum, 1978), and
also to possess cholinergic term inal markers. For example, cholinerg ic
receptors (Kobayashi et al., 1978) acetylcholinesterase (Palkovitz and
Jacobowitz, 1974) and choline acetyltransferase (Kobayashi et al., 1975).
Brodie and P roud fit (1984) showed that carbachol (i.e.) produced
antinociceptive responses, which could be reversed by atropine or prevented
by atropine pre trea tm ent. The authors suggest that carbachol acts on the
NRM to activa te descending raphe-spinal neurons, which impinge on spinal
cord dosal horn neurons. It is these neurons which are sensitive to noxious
s tim ulation (see Brodie and P roud fit, 1984 fo r references).
In the cat, Katayama et al. (1984a,b) found a group o f cells in the
parabrachial region (PBR) very sensitive to the antinociceptive e ffec ts o f
carbachol. This appears to be solely a muscarinic response as i t is inhib ited
by atropine, but not mecamylamine or naloxone. Morphine at a dose o f 25 gg
had no e ffe c t when in jected into the same site . Although a role fo r these
groups of cells which appear to mediate nociceptive in form ation via
muscarinic receptors is not clear, i t is possible that they are the substrate fo r
environm entally induced antinociception. This form of antinociception is
discussed in Section 5.
1.6.7. Cholinergic Antagonists and Antinociception
The previous sections have described the lite ra tu re concerning
cholinom im etic agents and give a strong indication that antinociception is at
least in part mediated by acetylcholine in the CNS. The use o f antagonists in
nociceptive studies is shown in Table 4 and provides fu rthe r support fo r 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 in te rfe ring w ith choline transport
(Marchbanks, 1968) and pretreatm ent w ith this compound s ligh tly antagonises
the antinociceptive action o f morphine (Bhargava et al., 1974).
Hem icholinium itse lf has no antinociceptive a c tiv ity . This would suggest tha t
part o f morphine's action involves cholinergic neurones. Hem icholinium has
also been shown to inh ib it acupuncture antinociception fo llow ing i.c .v .
adm in istra tion (Ren et al., 1980). The role o f 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 (B ill e t al., 1983) or rats (Houser and van H art,
1973; B ill et a l., 1983; Lewis et a l., 1983) and indeed causes s ign ifican t
hyperalgesia in ra t ta il f lic k tests at 1 mg/kg (Watkins et al., 1984).
However, s.c. adm in istration has been shown to produce potent
antinociception using the mouse w rith ing test, which may re fle c t route o f
adm in istra tion or perhaps a peripheral in teraction w ith the w rith ing agent
phenylbenzoquinone.
Data from prim ate studies (Houser and Houser, 1973; Pert, 1975) indicate
tha t hyoscine is antinociceptive at low doses. Though con flic ting w ith the
rodent data these results are consistant w ith the findings described in
Section 1.6.6.(a), on the study o f muscarinic agonists in primates, indicating
again tha t 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. Ventricu la rly administered
hyoscine was e ffe c tive however, thus strongly suggesting a cholinerg ic
pathway tha t modulates nociception at least in part d is tinct from opioid
sensitive systems (Pert and Maxey, 1975).
Opioid induced antinociception has been shown to be inhib ited in two species
by hyoscine (B ili et al., 1983) though not in the w rith ing test, but this could
be re lated to hyoscine's own antinociceptive a c tiv ity in this test. Malec and
Langwinski (1982) found tha t hyosine enhanced codeine's effectiveness whilst
leaving morphine, fentanyl and pentazocine unaffected.
The use o f cholinergic antagonists has indicated possible transm itte r
substrates fo r endogenous environm entally induced antinociception.
Endogenous antinociception may be induced by stressful s tim u li, such as cold
water swimming (Bodnar et al., 1978), e lec tric shocks (Jackson, 1979), and
figh ting (Rodgers and Hendrie, 1983). In many cases the antinociception is
naloxone reversible im plying a role fo r endogenous opioid systems. However,
some forms of environm entally induced antinociception are re frac to ry to
narcotic antagonists, fo r example b rie f continuous, as opposed to prolonged
in te rm itta n t, foo t shock (Lewis et al., 1980), and hind paw, as opposed to
fro n t paw, foo t shock (Watkins and Mayer, 1982) appear to u tilize non-opioid
mechanisms. The cholinergic system has been im plicated by some workers in
environm entally induced antinociception, as hind paw foo t shock induced
antinociception and antinociception induced by classical conditioning are
blocked by hyosine (1 mg/kg), but not the quaternary deriva tive ,
methylhyoscine (Watkins et al., 1984). Prolonged in te rm itta n t shock was dose
dependently antagonised by hyoscine from 0.1 mg/kg (Lewis e t al., 1983),
s im ila rly long term opioid antinociception induced by inescapable foo t shock
followed by m ild re instating shocks 24 hours la te r was also antagonized by
hyoscine at 2.5 mg/kg. The indications are tha t some form s of
environm entally induced antinociception are mediated via centra l muscarinic
system, whilst others act through endogenous opioid systems which employ
in term ediary muscarinic cholinergic pathways.
1.6.8 Miscellaneous Compounds
The role of the cholinergic system in nociception has been im plicated by
pharmacological treatm ents not d irec tly involving the use o f cholinergic
drugs. There are several neurotransm itters which have been reported to be
involved in nociception, one o f which is the inh ib ito ry amino acid
Y -am inobutyric acid (GABA). The role o f GABA as an analgesic has been
suggested from studies w ith an analogue, baclofen (Saelens et al., 1980). The
lack o f antagonism by naloxone indicates that baclofen acts through an
opioid-independent system.
Kendall et al. (1982) investigated the role o f GABA in nociception using
d irec tly acting agonist (kojic amine and THIP), a GABA-degradation inh ib ito r
(y-v iny lG A B A ) and an inh ib ito r o f high a ff in ity transport (n ipecotic acid
e thyl ester). As w ith previous studies, naloxone (5 mg/kg) fa iled to a lte r the
antinociceptive a c tiv ity o f these compounds in mice using hot plate and ta il
immersion tests. In contrast atropine (5 mg/kg) inhib ited the antinociceptive
a c tiv ity of a ll treatm ents w hilst mecamylamine (5 mg/kg) was in e ffec tive .
Those drugs which m odify GABAergic transmission probably do not exert
the ir e ffe c t by acting d irec tly on muscarinic receptors as only n ipecotic acid
showed sign ificant a ff in ity fo r the receptor in binding studies. The results
indicate then, that GABA is exerting its antinociceptive e ffe c t via
in term ediary cholinergic neurons.
2+ 2 The divalent cations barium (Ba +) and strontium (Sr +) are believed to be
able to substitute fo r calcium ions to induce release o f neurotransmitter.s
2+(Rubin, 1970) and Ba has been shown to release acetylcholine (Silinsky,
1977). Using the mouse ta il- f l ic k model Welch et al. (1983) demonstrated
2+ 2+that in traven tricu la r administered Ba and Sr induced antinociception.
This e ffe c t was reversed by atropine but naloxone antagonism was not dose
2 2+related. These results suggest that Ba +'s and Sr +,s inherent antinociceptive
a c tiv ity is mediated via acetylcholine release.
The involvement o f the cholinergic system in antinociception may be o f
c lin ica l significance w ith the recent development o f the analgesic drug
meptazinol. This drug appears to possess both opioid and cholinerg ic
antinociceptive components as B ill e t al. (1983) demonstrated in several
rodent antinociceptive tests (see Table 1.4.) that m eptazinol’s e ffec ts were
inhib ited by naloxone, atropine and hysocine. It has been reported recently
that meptazinol possesses cholinergic a c tiv ity by v irtue o f its marked
anticholinesterase a c tiv ity (G alli, 1983). The possibility o f u tilis ing the
cholinergic component c lin ica lly is o f great in terest as the drug seeking
behaviour and addictive lia b ility o f cholinergic or opio id-cholinergic
analgesics may be less than that observed w ith opioids.
1.6.9 Acetylcholine Histopharmacology and Opioids
a) Role o f 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
anatom ical corre late of these pharmacological observations.
Morphine has been shown to inh ib it 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 tha t
required fo r antinociception. However, these were measurements in whole
brain, and more recent studies have measured regional changes in
acetylcholine, w ith antinociceptive doses. Green et al. (1976) showed tha t in
mice, 10 mg/kg morphine increased acetylcholine levels s ign ifican tly in the
stria tum and at higher doses (300 mg/kg) in the hippocampus. In rats
increases in s tria ta l acetylcholine was observed a fte r 30 mg/kg, and a fte r
90 mg/kg increased levels were measured in the hippocampus; these e ffec ts
were shown to be naloxone reversible. There is m v itro evidence tha t this
e ffe c t is due to morphine's action on h ig h -a ffin ity Na+-dependent choline
transport (HANDCU), an ind ica tor o f cholinergic neuronal a c tiv ity m vivo
(Kuhar and M urrin, 1978). I t has been shown tha t at antinociceptive doses
morphine stim ulates HANDCU in mouse s tria ta l (Vallano et al., 1982) and ra t
hippocampal (Vallano and McIntosh, 1980) synaptosomes. In addition,
enkephalin at 40 nM was shown to decrease neocortical HANDCU (Wenk,
1984). I t is also possible tha t opioids increase acetylcholine levels by
inh ib iting its release, i.e. decreasing cholinergic neuronal a c tiv ity . Indeed,
release o f co rtica l acetylcholine may be evoked by e lec trica l s tim u la tion o f
nerves in the forepaw o f anaesthetized rats (Jhamandas and Sutak, 1983) and
this is enhanced fo llow ing systemic adm in istration o f the narcotic antagonists
naloxone or naltrexone. This suggests tha t there is an inh ib ito ry ro le fo r
endogenous opioids on acetylcholine release. Opioid receptor regulation
appears to be lim ite d to certa in pathways (Wood et al., 1984) viz, septal-
hippocampal and substantia innom inata-cortica l pathways where agonists a t
p, 6 and e receptors exert inh ib ito ry regulatory influences, w h ils t k-agonists
appear to be ine ffec tive . In addition, there is a co-loca liza tion o f enkephalin
and acetylcholine in the la te ra l system of olivocochlear cells (A ltschu ler 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 lite ra tu re describing the relationship between opioid and
acetylcholine turnover and release suggest a close relationship between these
two systems. I t is quite plausible, tha t acetylcholine plays a key ro le in the
actions o f endogenous and exogenous analgesia.
b) Acetylcholine and Acupuncture-Induced Antinociception
In China, acupuncture is used routine ly as a method to induce surgical
analgesia. The mechanism of this e ffe c t is s t il l poorly understood. I t is only
in recent years tha t th is technique has, at least in the West, received any
credence and undergone sc ie n tific enquiry. The possible mechanisms involved
\d acupuncture-induced analgesia have been reviewed recently (Han and
Terenius, 1982). There are several reports describing the role o f the
cholinergic system in acupuncture antinociception. Acupuncture causes an
increase in acetylcholine in the CSF and the increase correlates w ith increase
analgesia (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 a c tiv ity was
observed in the thalamus fo llow ing electroacupuncture and locus coeruleus
though a c tiv ity was reduced in the substantia gelatinosa (A i et al., 1979).
Pharmacological manipulation of the cholinergic system has added to the
evidence fo r a role o f acetylcholine in acupuncture-antinociception.
Inh ib ition o f acetylcholine synthesis w ith hemicholinium (i.c.v.) was shown to
inh ib it electroacupuncture in rats (Han, 1979; Guan, 1979), and this could
pa rtia lly be reversed w ith the acetylcholine precursor choline chloride (Han,
1979). Blockade of muscarinic receptors w ith atropine in rats and rabbits
(Han, 1979) and w ith hyoscine in rabbits (He et al., 1979) was found to block
analgesia induced by acupuncture, w h ils t the anticholinesterase
physostigmine has been shown to potentia te acupuncture analgesia in rats
(Han, 1979; G u a n e ta l., 1979; A i, 1979).
There is also evidence fo r the involvement o f other transm itte r systems (e.g.
endogenous opioid and 5-HT) suggesting a m ultip le neurtransm itter system
mediating the e ffec ts o f acupuncture (See, Han and Terenius, 1982, fo r
review).
c) Acetylcholine-Q pio id In te raction in Non-Nociceptive Systems
There are a number o f reports in the lite ra tu re describing in teractions
between centra l cholinergic and opioid systems not involved in nociception,
but having potentia l c lin ica l applications. For example, anaesthesia has been
shown to be potentiated or prolonged fo llow ing anticholinesterases (Pellanda,
1933; Green and Davis, 1956). Another potentia l benefic ia l e ffe c t o f the
opioid-cholinergic in teraction has been investigated by Weinstock and her
colleagues. The respiratory depressant e ffe c t o f morphine, or other opioids
tha t are routine ly given as part of pre-operative medication is usually not
im portant, however, in patients w ith lung diseases or breathing problems such
an e ffe c t is po ten tia lly fa ta l. However, physostigmine pretreatm ent has been
shown to antagonize respiratory depression in experimental animals
(Weinstock et al., 1980) and humans (Snir-Mor et a l., 1983). This property o f
physostigmine has been exploited successfully in the trea tm ent o f severe
resp ira tory depression resulting from overdose in heroin addicts (Rupreht et
al., 1984). The authors point out tha t physostigmine trea tm ent is preferable
to the usual procedure of adm in istration o f naloxone, as acute w ithdraw al is
not precip ita ted.
Treatm ent of opioid addiction is an im portant area of research in opioid
pharmacology, and there is evidence that the cholinergic system modulates
the processes of tolerance and dependence to opioids. The lite ra tu re has
been extensively reviewed (Wahlstrbm, 1978); short-term morphine
trea tm ent ( 3 days) appear to increase acetylcholine turnover in it ia lly
(Bhargava and Way, 1975; Cheney et al., 1975), w h ilst several studies indicate
that longer treatm ent (2-4 weeks) appears to result in increase levels or
decreased acetylcholine u tiliza tio n (e.g. Domino and Wilson, 1973). A
possible c lin ica l application to the use o f a cholinergic drug in the trea tm ent
o f dependence and w ithdrawal has been proposed by Albin et al. (1975). They
reported that nalorphine-precipitated w ithdrawal signs fo llow ing constant
morphine plus an anticholinesterase (tetrahydroam inoacridine, THA) dosing in
rhesus monkeys were markedly less than that observed a fte r morphine alone
treatm ent. Indeed, an earlie r study indicates that THA adm in istra tion
reduces physical dependence in humans (Stone et al., 1961).
There is evidence fo r a cholinergic involvement in a ffec tive disorders (e.g.
depression) in humans, as fo llow ing adm inistration o f physostigmine in normal
subjects resulting symptoms include many o f those.associated w ith depressive
symptomatology (Risch et al., 1980). In addition, in patients w ith a h istory o f
a ffe c tive disorder, but w ith c lin ica l remission, infusion o f low doses o f
physostigmine produces transient depressive symptomatology (Janowsky et
al., 1974). More recent studies (Risch e t al., 1981) have shown tha t
adm inistration of physostigmine or ajrecoline to both normal and a ffe c tiv e -
disorder subjects produces altered mood and a ffe c tive changes, resulting in
increased depression. These observed e ffects were highly corre lated w ith
increased plasma 3-endorphin im m unoreactiv ity (but not co rtiso l or
pro lactin) and the authors suggest the existence o f a cholinerg ically mediated
3-endorphin pathway modulating a ffec tive and cognitive states in humans.
A classical response to opioid adm inistration in mice is to increase the ir
locomotor a c tiv ity . This stereotypic running is believed to be dependent on
dopamine (Kushinsky and Hornykiew icz, 1974; Eidelberg and Erspamer,
1975), but there is also evidence that a component o f morphine's action
involves the cholinergic system. Adm inistra tion of the muscarinic receptor
agonist oxotremonine (Seidel et a l., 1979), or the anticholinesterase agent,
DFP (Su and Loh, 1975), attenuate morphines e ffe c t on locomotion, w h ils t the
muscarinic antagonist, atropine enhances morphines action (Seidel e t al.,
1979). Cholinergic drugs appear then to influence opioid-induced running.
The mechanism is unclear, but may w e ll involve other neurochemicals, such
as catecholamines.
1.6.10. Conclusions
Several agents which in te rfe re w ith the cholinergic system have been shown
to be antinociceptive. These include reversible and irreversib le
anticholinesterases and agonists at muscarinic and n ico tin ic receptors. To
a ffirm that cholinergic drugs potentia te the analgesic action o f opioids is
perhaps an overgeneralisation. However many experimental studies have
been able to show that enhancement o f cholinergic a c tiv ity can lead to
enhancement o f opioid antinociception. In addition there is evidence to
suggest that endogenous analgesia, induced by stress may in part be
modulated by cholinergic inputs.
The in teractions between the cholinergic and opioid systems probably
prim arily occurs w ith in the centra l nervous system as studies w ith quaternary
compounds have been mostly negative whilst in teractions fo llow ing centra l
adm inistration are generally observed. The molecular mechanisms involved
are not understood but most o f the evidence points to a pharmacodynamic
rather than a pharmacokinetic in teraction.
Too few studies have used receptor selective antagonists and where
cholinergic antinociception has been observed the involvement o f both
cholinergic and opioid receptors is often the case. Our understanding o f
in teractions at the receptor level require more selective pharmacological
tools espeically w ith respect to opioid pharmacodynamics. I t is also probably
an overs im plifica tion to th ink o f an opio id/cholineric in te raction involving
only one mechanism and there may be in terrelationships w ith other neuronal
systems. In particu la r the evidence points to a close relationship w ith
monamines. Whether manipulation o f cholinergic and opioid system produces
more successful pharmacological agents are analgesics remains to be seen.
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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. An tinoc icep tive Models
There are many tests available to determ ine the antinociceptive a c tiv ity of
drugs. The m a jo rity o f these tests u tilize the response to experim ental pain
in animals (Linebery, 1981). Anim al testing can only serve as a model of
human c lin ica l pain as the pain response in man is a subjective param eter
w ith large va riab ility between individuals. Extrapolation from animal data to
humans must be viewed w ith caution as false negatives and positives may be
encountered.
Wood (1984) presented a lis t o f five c r ite r ia fo r consideration when
in te rpre ting data from animal screening tests:
1 Test V a lid ity : Is the test predictive o f the c lin ica l u t i l i ty o f known
analgesics?
In considering the most appropriate antinociceptive test in this
study, these points were taken in to consideration. I t was decided to
employ the hot-p la te test (Woolfe and MacDonald, 1944) fo r mice
and the paw pressure test (Randall and Se litto , 1957) fo r rats.
2 Test R e lia b ility : Is the test response reproducible throughout an
experimental day and from day to day?
3 Test S im p lic ity : Does the procedure use a standard defined noxious
stimulus, measure a well-defined animal response, and is the test
easily utilized?
The mouse hot-p la te is an extensively used procedure and unlike the
ta il f lic k test does not re ly on a re flex ive response. The paw lick
response in the hot-p la te test relies on integrated behaviour
involving processes occuring at a high level w ith in the centra l
nervous system. The ra t paw pressure test was chosen to determ ine
antinociceptive drug a c tiv itie s in an additional species using a
d iffe re n t noxious stimulus, and in this test the responses are not
re flex ive in nature.
An tinociceptive responses are subject to wide variations, so care
was taken to account fo r and contro l environmental variables.
Test S ensitiv ity : Can d iffe ren 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-p la te test is sensitive
to agonist/antagonist analgesics. In th is study, this would not be so
im portant as only strong analgesics were investigated.
Data Analysis: Varies between laboratories. Is the method of
analysis appropriate? Methods o f analysis should be described.
The analysis o f the data was given fu ll consideration, and a model
was developed to accurately f i t the form of the observed data (see
Section 2.10.).
2.2. In it ia l Experiments
Ample evidence has been presented in Section 1.6. fo r the role of cholinergic
agents in opioid antinociception. The in it ia l studies, which are presented in
this section, were designed to determ ine the in teractions between the
anticholinesterase agents and opioid antinociception in the models described
above.
MATERIALS AND METHODS
2.3. Anim als and Experim ental Conditions
Male albino mice (CD-I strain; 25-30g) were purchased from Charles R iver.
Male albino Wistar rats (Surrey U nivers ity strain; 140-210g) were bred at the
University 's Anim al U n it. Upon delivery, animals were housed in the Anim al
U n it under contro lled temperature, hum id ity and lighting conditions (12h
ligh t/da rk cycle; lights on 0700h). Mice remained in the U n it fo r at least 5
days before use. Tw enty-four hours before experimentation, the animals
were equilibrated in a quiet laboratory where a ll procedures were carried out.
Animals had free access to food and water up to experim entation. A ll
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 Anim al U n it. The room was ventila ted but
had no independent heating or hum id ity controls. Am bient tem perature was
monitored during experimentation, and stayed w ith in the range 20-25°C
throughout the year. So as to minimise disturbance there was res tric ted
access to the room before and during experimental procedures.
2.4. Drugs
The fo llow ing drugs were used:
DRUG NAME SOURCE
Di-isopropylfluorophosphate Sigma
A tropine sulphate Sigma
Morphine sulphate M acfarlan Smith
Fentanyl c itra te Janssen Pharm aceutical*
A lfe n ta n il hydrochloride Janssen Pharm aceutical*
Pralidoxim e m ethyl sulphonate CDE, Porton Down*
Diazepam Roche*
Neostigmine bromide Sigma
Pyridostigm ine bromide Roche*
Naloxone hydrochloride Endo*
* These compounds were generously donated by the manufacturers.
Drugs were dissolved in 0.9% saline (Travenol) im m ediate ly prior to use, w ith
the fo llow ing exceptions: in some experiments DFP was dissolved in peanut
o il (Sigma), and as diazepam is not soluble in saline i t was f irs t 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 tox ic nature. The supplied Safety Data Sheet was used
as a guideline to handling procedures outlined below:
2.5. Safety Procedures fo r DFP
DFP was stored, re frige ra ted at about 4°C, and kept w ith in the lidded m eta l
container in which i t was supplied. The v ia l containing DFP was handled only
in a fume cupboard, w ith the user wearing gloves and an overall w ith tig h t
cuffs.
Appropriate quantities o f DFP were obtained by penetrating the seal w ith a
fixed needle Ham ilton syringe and w ithdrawing liquid. The DFP was
im m ediate ly transfered to a vo lum etric flask to which saline was added. The
flask was shaken thoroughly fo r at least 2 minutes to ensure tha t 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 o f the sodium hydroxide solution.
DFP degrades w ith tim e due to hydrolysis (Leopold and Krishna, 1963) and
therefore stock was replaced every six months. In addition, a lfen tan il
potentia ting a c tiv ity was confirm ed (see Section 2.13.).
2.6. Dosing Protocols in M ice
Mice were scruffed and drugs were in jected in 0.1ml volumes subcutaneously
(s.c.) in the back o f the neck, w ith the exception o f diazepam and
neostigmine. Diazepam was in jected in a 0.03ml volume in tram uscularly
(i.m .) into the hind leg, because o f a local in teraction w ith DFP (D. Green,
CDE, personal communication), and neostigmine was in jected
in traperitonea lly (i.p.) in 0.1ml volumes.
Drug dosing protocols are illus tra ted in Figure 2.1. When used, atropine
(17mg/kg) and pralidoxime (15mg/kg) were mixed and in jected together 1
minute a fte r DFP, according to Gordon et al (1978). Diazepam (6.25pm ol/kg)
was in jected im m ediate ly prior to the atropine/pralidoxim e dose (D. Green,
personal communication).
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For the neostigmine experiments, e ither saline or neostigmine were
administered (i.p.) at tim e 0, a lfen tan il 25 minutes la te r, and antinociceptive
testing carried out at 30 minutes.
Pyridostigm ine (lm g/kg) was administered 1 hour before antinociception. The
times fo r the in jection o f morphine, fentanyl and a lfen tan il were 30, 50 and
55 minutes a fte r pyridostigm ine, respectively.
In those experiments when naloxone was used, i t was administered 30 minutes
before testing.
2.7. Dosing Protocols in Rats
Rats were scruffed fo r s.c. in jection (0.1ml volumes) in the back o f the neck.
DFP was administered at tim e 0 and morphine, fentanyl or a lfen tan il
administered 30, 50 and 55 minutes la te r respectively. Each animal was
tested four times; once im m ediate ly before the f irs t in jection and again at
three times to include peak antinociceptive e ffe c t. The tim ings fo r\
in jections and fo r post-drug testings are given in Fig 2.2.
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2.8. Antinociceptive Testing in Mice
The hot-p la te test (Woolfe and MacDonald, 1944) was employed fo r
antinociceptive testing. The hot plate was made in the Biochem istry
workshop, University o f Surrey, and consisted o f an aluminium plate (30cm
xl5cm) surrounded by a transparent perspex w a ll (22cm high). This was
placed in a therm osta tica lly contro lled w ater bath w ith the water maintained
at 33°C. The same apparatus and temperature setting was used throughout
a ll experiments.
When tested mice were placed on the hot-p la te and a hand-held d ig ita l
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 lick ing response was absent. I f the
mice fa iled to respond a fte r 60 seconds they were removed from the plate
and scored as >60 seconds. As the walls o f the hot-p la te were perspex and
unheated, occasionally some mice spent long periods on the ir hind paws w ith
fore-paws resting on the walls; these mice were excluded.
2.9. Antinociceptive Testing in Rats
The non-inflamed paw pressure test was employed fo r nociceptive
determ inations (Randall and Se litto , 1957) using an Ugo Basile Analgesy m eter
(A .R .Horwell, London). This apparatus (Fig. 2.3. ) consists o f a p lastic pusher
(P) positioned about 2mm above a Teflon p lin th (B). Animals were held
f irm ly , but not tig h tly around the upper part o f the ir body, so they could be
held in the correct position w ithout the animal struggling. The animals paw,
was placed under the pusher and subjected to a force increasing linea rly w ith
tim e as a pivoted weight (W) moved horizonta lly. A foot-pedal contro lled the
M i mm 'M: s m v
'* ' w4
B a s i le A n a lg e s S M et
■ fig
weight's movement which v/as marked o ff on a graduated scale (SC). The
score on the graduated scale was converted to the force applied in grams by
m ultip ly ing by 30.
The nociceptive end-point was taken as a w ithdrawal or attem pted
w ithdrawal o f the animal's paw, or when the animal struggled. With practice
i t 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 fo r tha t animal. To
avoid damage to the animal's paws, a maximum c u t-o ff weight o f 750g was
employed. Those animals which did not respond before the 750g c u t-o ff was
reached were scored as 750g.
2.10. S ta tis tica l Procedures
2.10.1. H ot-P late Data
Four doses fo r 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-p la te latency to 30 seconds
(ie., 30% of the maximal response tim e). Unpaired Student's t- te s t (see
Appendix A) was used to.compare AD<-g values.
A ll dose response curves included censored values, tha t is mice fa iling to
respond before the 60 seconds c u t-o ff, which were scored as >60 seconds.
Though employing a c u t-o ff tim e is essential to avoid inducing tissue damage
in the animal's paws, i t does d is to rt the dose-response relationship. However,
this d istortion may be compensated fo r by using appropriate m athem atical
models. Such a model was applied to the data to obtain AD^g values and is
described in Appendix B. The Fortran listings fo r the test procedures (run on
the Prime computer operating system) are given in Appendix B.
2.10.2. Paw Pressure Data
A m athematical model was applied to data giving the tim e course of drug
e ffe c t to obtain values fo r peak responses fo r each dose. Comparison
between groups was by unpaired *t’ test. Results were norm ally mean values
o f at least six animals. To obtain a value fo r peak response, a quadratic
equation was f it te d to the data: y = a - b (x-c) , where: a = peak response
score, b = curvature o f line and c = tim e o f 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 s ign ifican tly increased latency. A ll subsequent
antinociceptive data presented represent latencies from mice tested once
only.
Table 2.2. shows the e ffe c t of repeated saline in jection on nociceptive
responses o f mice. There were s ign ificant changes (eg., single in jection 11.7 -
0.73, tr ip le in jection 9.2 - 0.85, t- te s t p<0.05), but these were quan tita tive ly
small.
2.12. E ffect of DFP on Nociception in Mice
Table 2.3. shows tha t DFP at lm g/kg did not exh ib it antinociceptive a c tiv itie s
a t the times studied. However, a t a 2mg/kg dose a 2- to 3-fo ld increase in
hot-p la te response tim e was observed over th is period. A t th is higher dose,
mice exhibited symptoms of anticholinesterase poisoning, such as trem ors and
fasiculations. Mice showed hind-limb abduction and impaired and reduced
ambulation.
When peanut o il was used as the vehicle fo r DFP (Table 2.4.) no a lte ra tion in
hot-pla te response tim e was observed compared w ith saline.
The e ffe c t o f the opioid antagonist, naloxone, and the muscarinic cholinerg ic
antagonist, atropine, on the antinociceptive a c tiv ity o f DFP at 2mg/kg is
shown in Table 2.5.
Table 2.1. E ffect of Repeated Exposure to Antinociceptive Testing
Procedures on Hot-Plate Times of Naive Mice
One exposure
Second exposure (+ 30 mins)
Third exposure (+ 4 hours)
H ot-P la te Latency (Secs)
9.3 - 2.0
17.0 - 3.3
16.8 i 2.0
Each value represents the mean - SEM of 12 observations.
Table 2.2. E ffect of Multiple Saline Injections on Hot-Plate Response Time in Mice
H ot-P la te Latency (Secs)
Number o f Time from last in jection 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 o f observations are given in
parentheses.
Mice receiving two in jections were f irs t in jected 60 minutes before testing,
and those receiving three in jections had the ir f irs t two in jections 60 and 59
minutes before testing.
Table 2.3. E ffect of DFP on Hot-Plate Reaction Times in the Mouse
Saline
DFP (lm g/kg)
DFP (2mg/kg)
H ot-P la te Latency (Secs)
Time a fte r In jection
0.3 hours
11.9 - 1.6
9.7 - 0.8
28.4 - 5.6*
1 hour
13.2 - 1.1
9.4 - 0.7
19.0 - 2.0*
2 hours
12.7 - 1.5
11.8 - 1.2
18.7 - 2.8
4 hours
11.7 - 1.0
12.0 - 0.9
33.0 - 8.7*
Each value represents mean - SEM o f at least 6 observations. Unpaired t -
test vs. saline *p<0.05.
Table 2.4. E ffect of Different Vehicles on the Antinociceptive Activity of DFP
Vehicle used
Peanut O il
Saline
H ot-P la te Latency (Secs)
lm g/kg
9.8 - 1.1 9.2 t 2.3
9.4 - 0.7
2mg/kg
18.6 +4.5
19.0 - 2.0
Each value represents the mean - SEM of 6 observations.
Table 2.5.
Treatm ent
Naloxone
Atropine
Effect of Atropine and Naloxone on 2mg/kg
DFP Induced Antinociception in Mice
H ot-P la te Latency (secs)
Saline 33.9 - 5.9 (13)
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)
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-te s t vs saline *p<0.005 **p<0.001.
Doses o f naloxone between 0.1 and lOmg/kg had no e ffe c t on the hot-p la te
reaction times of mice receiving DFP (2mg/kg). However, when mice
received atropine (1 to 20mg/kg) hot-p la te reaction tim e was s ign ifican tly
reduced. A 20mg/kg, response times were not s ign ifican tly d iffe re n t from
mice receiving saline in jections.
2.13. E ffect of DFP and Drugs Reversing the Effects of DFP on Opioid
Antinociception in Mice
The antinociceptive ac tiv ities of morphine, fentanyl and a lfen tan il are shown
in Table 2.6. A c tiv itie s are represented in terms o f computed ADj-g values as
a measure o f antinociceptive potency.
In mice pre-treated w ith lm g/kg DFP the AD^g values of morphine and
fentanyl were unaltered. However, a tw o-fo ld increase in the antinociceptive
a c tiv ity o f a lfen tan il (as shown by a reduced AD^g), was observed fo llow ing
DFP pre-treatm ent.
The atropine-pralidoxim e mix was administered as a trea tm ent fo r DFP
poisoning. The e ffe c t o f DFP on the antinociceptive ac tiv ities o f fentanyl
and a lfen tan il was unaltered by the atropine-pralidoxim e mix trea tm ent,
however, an increased antinociceptive a c tiv ity o f morphine (denoted by
s ign ifican t decreases in AD^g) was observed by this treatm ent.
Diazepam was used as an additional component in the trea tm ent o f DFP
poisoning. No s ign ificant changes in AD^g values fo r the opioids were
observed fo llow ing additional adm inistration o f diazepam (Table 2.6.).
Table 2.6. E ffect of DFP (Img/kg) and of Drugs Reversing DFP
Poisoning on Opioid-Induced Antinociception in Mice
A D cn Values
A lfe n ta n il
(gg/kg) .
329.7 - 23.3
164.3 - 11.0**
175.0 - 1 5 .0 **
207.3 ± 3 3 .0 **
AD^g values are the mean - SEM (of at least 9 observations). AD^g values
are doses required to increase hot-p la te reaction tim e to 30 seconds and are
calculated from the fu ll dose response curves fo r each agonist. Normal
deviate fo r comparison of means vs. saline **p<0.01; ***p<0.001.
Treatm ent Morphine Fentanyl
(mg/kg) (gg/kg)
Saline 20.7 - 1.9 66.4 - 6.7
DFP 24.7 - 2.5 52.5 - 5.6
DFP + A tropine +
Pralidoxime
12.0-1.0* 54.3 - 5.9
DFP + Atropine +
Pralidoxime + Diazepam
10.7 ± 1.7* 53.3 ± 8.3
2.14. E ffect of Atropine and Pralidoxine and Diazepam on Nociception
As a control, atropine-pralidoxim e mix, and diazepam were administered
alone, to determ ine the ir e ffe c t on mouse hot-p la te latency.
When the atropine-pralidoxim e mix was administered at 1 m inute, w ith saline
given at 0 and 30 minutes, testing at 60 minutes gave a response tim e o f 10.5
- 0.7 seconds (n=12), which is not s ign ifican tly d iffe ren t from saline contro l
values.
The e ffe c t o f diazepam on nociceptive responses is shown in Table 2.7.
Diazepam did not a ffe c t hot-p la te response times, even at doses which
produced clear motor incapacitation.
Using a PEG/saline in jection vehicle o£ in jecting saline i.m . did not give hot
plate reaction times s ign ifican tly d iffe re n t from saline s.c. (Table 2.7.)
2.15 E ffect of Neostigmine and Pyridostigmine on Opioid Antinociception
The e ffe c t o f the quaternary anticholinesterase agents neostigmine and
pyridostigm ine opioid antinociception is shown in Tables 2.8 and 2.9.
Neostigmine did not produce any s ign ifican t potentia tion o f a lfen tan il over
the dose range studied. Pyridostigm ine pre-treatm ent did not s ign ifican tly
a lte r AD^g values fo r the opioids studies, though there was a small
enhancement o f a lfen tan il antinociception (p=0.105).
Table 2.7. E ffect of Intramuscularly-Administered Diazepam
and Injection Vehicle on Hot-Plate Reaction Times in Mice
Treatm ent
Saline
PEG/Saline
Diazepam 6.25 pm ol/kg
Diazepam 12.5 pmol/kg
H ot-P la te Response
Time (Seconds)
8.7 - 1.3 (12)
8.2 - 0.5 (6)
8.7 - 1.6 (6)
10.2 - 2.1 (6)
Values show means - SEM o f hot-p la te response times. Number
observations is shown in parentheses. PEG = polyethylene glycol.
Table 2.8. E ffect of Various Doses of Neostigmine on the
Antinociceptive Activity of Alfentanil
Saline
Neostigmine 50gg/kg
Saline
Neostigmine lOOgg/kg
Saline
Neostigmine 200gg/kg
A lfe n ta n il AD,-g (gg/kg)
143 - 18
146 1 13
178 - 13
170 - 11
155 - 14
128 - 14
Values are mean + SEM o f 6 observations on hot-p la te reactions times.
Contro l (saline) AD^g values were obtained on the same day fo r each dose o f
neostigmine.
Table 2.9. E ffect of Pyridostimine (Img/kg) On Antinociceptive
Potencies of Morphine,Fentany 1 and Alfentanil
Treatm ent
Saline
Pyridostigm ine
Morphine
(mg/kg)
16.8 - 1.3
lm g/kg 16.1 - 1.4
AD^g Values
Fentanyl
(pg/kg)
37.6 - 4.3
36.7 - 2.8
A lfe n ta n il
(pg/kg)
220 - 17.9
172 - 18.9
Values are mean - SEM of 6 observations on hot-p la te reaction tim es.
Control (saline) AD ^q values were obtained on the same day fo r each opioid.
Table 2.10.
Treatm ent
A lfe n ta n il
DFP +
A lfen tan il
Naloxone Antagonism of Antinociception Induced by
Alfentanil With and Without DFP
Naloxone a d 50+s e m Dose Ratio Slope of Apparent pA2 values
(pg/kg) pg/kg pA2 plot (95% confidence in terva
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)
0 0.13 - 0.07 -
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-p la te reaction tim e to 30 seconds.
2.16 Antagonism of Alfentanil, Alone and With DFP, on Naloxone
The e ffe c t o f naloxone antagonism on the antinociceptive a c tiv ity o f
a lfen tan il and the e ffe c t o f naloxone on DFP plus a lfen tan il antinociception
is shown in Table 2.10.
Naloxone antagonism was measured as 'apparent pA£' according to Hayashi
and Takemori (1971). The pA2 value represent the a ff in ity fo r the antagonist
naloxone fo r its receptor. The values obtained fo r 'apparent PA21 were very
s im ilar indicating tha t the compounds were acting through the same
receptors. The slope o f the pA2 p lo t were very s im ila r to the theore tica l
value of -1 fo r com petitive antagonism.
2.17. E ffect of DFP upon Nociceptive Responses in the Rat
Figure 2.3. shows the tim e course fo r the e ffe c t o f DFP adm in istration on the
nociceptive responses o f the ra t in the paw pressure test.
Saline base-line responses remained constant over the tim e studied and w ith
repeated testing. A t lm g/kg, DFP did not a ffe c t the nociceptive response.
However, there were s ign ificant (p<0.001) increases in response thresholds fo r
a ll the tim e points studied fo llow ing DFP at 2mg/kg (Fig 2.3.) A t the
2mg/kg dose DFP produced clear signs o f anticholinesterase poisoning, such
as marked tremors. However, the leg w ithdrawal and struggling responses
were not impaired.
2.18 E ffe c t o f DFP P re-Treatm ent on Opioid Antinociception in Rats
The e ffe c t o f the sub-antinociceptive dose o f DFP (Img/kg) on the
antinociceptive ac tiv ities o f morphine, fentanyl and a lfen tan il are shown in
(Figs. 2.4., 2.5. and 2.6.). DFP pre-treatm ent did not a ffe c t the
antinociceptive a c tiv ity o f morphine, but both fentanyl and a lfen tan il were
s ign ifican tly potentiated at low doses o f these opioids, (p < 0.01, unpaired t -
test).
RESSURE(g)00
0 0 -
00 -
0 0 -
0 0 -
00 -
yy 90 TIME(mins)Pre ao 45 60
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-
1 0 0 -
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 -
10 0 -
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
80 DOSE(|jg/kg)4020
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 o f in jection times and in it ia l doses o f drugs were determined from
p ilo t experiments or were based on available lite ra tu re .
Neostigmine adm in istration (30 minutes before antinociceptive testing) and
in it ia l dose were based on studies o f Lawrence and Livingstone (1979).
Pyridostigm ine has a longer duration of action than neostigmine (B ritish National
Form ulary, 1983) and so fo r these studies pyridostigm ine was in jected one hour
before testing. A high dose of lm g/kg O-D^g = 1.3mg/kg mice; Mestinon data
sheet) was used so tha t any e ffe c t would more like ly be revealed.
Naloxone was administered 30 minutes before testing, a tim e generally given in
the lite ra tu re (eg, Koehn and Karczm ar, 1978).
2.20. E ffect of DFP on Nociceptive Responses in Mice and Rats
Though at lm g/kg DFP did not appear to show any appreciable antinociceptive
a c tiv ity in the mouse hot-p la te test, at 2mg/kg the increase in hot-p la te reaction
tim e was marked. This apparent antinociceptive a c tiv ity o f DFP was long-
lasting. However, at this higher dose mice showed pronounced signs o f
anticholinesterase poisoning, including tremors and fasiculations, decreased
ambulation and apparent sedative ataxia. The nociceptive response in the hot
plate test involves the animal lif t in g and lick ing one or more of its paws. As this
response requires a complex set of co-ordinated non-reflexive movements, i t is
possible tha t the apparent antinociceptive e ffe c t observed fo r DFP a t 2mg/kg
was due to impaired muscular function. This im pairm ent could be o f centra l
origin, eg, disruption of contro l of movement or peripheral orig in, eg, muscular
weakness, or indeed, both.
I t has been reported that in rats at 2mg/kg s.c. DFP caused hind-lim b abduction
(Fernando et al, 1984) and at 2.2mg/kg i.m . the animals appear behaviourally
depressed and le tharg ic (Samson, 1984). Even at doses below lm g/kg, DFP is
reported to produce physical incapacita tion (Harris and S titcher, 1984). I t is not
unreasonable to presume from observations in this study and the available
lite ra tu re , tha t DFP adm in istration could im pair the nociceptive response
w ithout necessarily possessing antinociceptive a c tiv ity . The e ffe c t o f DFP on
the locomotor a c tiv ity of mice and the im plications in nociceptive testing is
given fu ll consideration in Section 3.13.
The data presented in this thesis are consistent w ith the findings o f Cox and Tha
(1972) who did not observe any antinociceptive fo r DFP up to 2mg/kg in the
mouse hot-p late test.
In contrast, studies in the ra t indicate tha t DFP is antinociceptive in this
species. Doses as low as 0.lm g/kg s.c. have been shown to induce antinociception
in the ra t hot-p la te test (Koehn et al, 1980) and 0.5mg/kg s.c. in the ra t ta il f lic k
test (Koehn and Karczm ar, 1978). The results presented in this thesis show tha t
DFP at 2mg/kg produced marked antinociception in the paw pressure test.
Though this antinociceptive dose is much higher than tha t reported by Koehn et
al (1980), this may be a re flec tion on the use o f pressure as the nociceptive
stimulus. The nociceptive response to pressure is believed to be predom inantly
mediated through k -opioid receptors, whilst heat noxia are believed to act
through p-opioid receptors (Tyers, 1980; Upton et al, 1983).
The observation tha t naloxone up to lOmg/kg did not a lter the reaction tim e
fo llow ing adm in istration o f DFP (Table 2.3.) suggests tha t the apparent
antinociceptive a c tiv ity o f DFP. is not mediated through an opioid system.
A tropine, however, did antagonize the increased latencies, and at 20mg/kg
to ta lly reversed DFP's e ffe c t. The apparent antinociceptive a c tiv ity is therefore
only mediated via muscarinic receptors and this contrasts w ith e ffec ts in the ra t
where both opioid and muscarinic receptors mediates its action (Koehn and
Karczm ar, 1978).
Clear species differences emerge, w ith mice being insensitive to an
antinociceptive action o f DFP w h ils t in rats, DFP may product potent
antinociception.
2.21. E ffect of DFP on Morphine Antinociception
DFP had no e ffe c t on morphine antinociception (Table 2.6.) and this contrasts
w ith the findings o f Bhargava and Way (1972). They reported po tentia ting
a c tiv ity o f DFP upon morphine antinociception, though i t should be stressed tha t
these effects, while consistent, fa iled to reach s ta tis tica l significance. The
possibility o f vehicle-re la ted e ffects must be entertained since Bhargava and
Way (1972) used peanut o il fo r DFP adm in istration and 0.9% saline was used in
the experiments described in this thesis. However, though peanut o il has been
shown to induce sedation (Pinsky et al, 1982) no antinociceptive e ffec ts fo r DFP
in th is vehicle could be shown (Table 2.4.) and this possibility can be re a lis tica lly
excluded.
A t a dose of 0.2mg/kg, Saxena (1938) found that DFP did not potentia te morphine
in the ra t ta il c lip test. Though there are many reports o f cholinerg ic drugs
potentia ting opioid antinociception (see Table 1.2.) the available data appears to
m ilita te against a potentia ting e ffe c t o f DFP in morphine-induced
antinociception.
2.22. Effect of DFP on Fentanyl and Alfentanil Antinociception
Though the a c tiv ity of the synthetic opioid analgesic fentanyl was not found to
be potentia ting by DFP in the mouse hot-p late test, the s truc tu ra lly -re la ted
compound a lfen tan il was potentia ted tw o-fo ld (Table 2.6.).
In addition, a lfen tan il antinociception in the ra t paw pressure test appeared to be
potentiated by DFP pre-trea tm ent. Though this potentia ting e ffe c t was only
observed at low doses o f a lfen tan il, i t seems like ly tha t employment o f a c u t-o ff
weight masks potentia tion at higher doses. This problem was also encountered in
the hot-p la te data (see Section 2.11.1).
Fentanyl also appeared to be potentiated in rats by DFP in the same way as
a lfen tan il. So a lfen tan il is potentiated by DFP in both mouse and ra t
antinociceptive test, though fentanyl was only potentiated in the ra t. This could
be a re flec tion o f d iffe ren t nociceptive mechanisms in the ra t compared to those
in mice.
2.23. Proposed Mechanisms fo r DFP Potentia tion o f A lfe n ta n il
Though morphine, fentanyl and a lfen tan il are a ll p rim arily u-opioid agonists,
DFP has been shown in this study to have a d iffe re n tia l potentia ting e ffe c t on
these opioids. There are several ways by which DFP could potentia te the
a c tiv ity o f opioids; these are a lterations in absorption, d is tribu tion, receptor
action, metabolism or excretion.
2.23.1. Absorption
There are a number o f factors which a ffe c t absorption of drugs from s.c.
in jection sites. A major fac to r is cutaneous blood flow and DFP trea tm ent is
believed to decrease c ircu la to ry rate (Ramachandran, 1967). In addition, DFP
dose dependently induces hypothermia (Kenley, e t al, 1982) which would decrease
peripheral blood flow . These factors would tend to reduce drug absorption from
a s.c. site and furtherm ore would not d iscrim inate between the opioids studied.
I t is also probable that drug absorption is not a lim itin g fac to r fo r fen tanyl and
a lfen tan il. Both these drugs are lipid-soluble w ith octanokw ater p a rtition
coeffic ien ts o f 860 and 130 fo r fentanyl and a lfen tan il respective ly (Meuldemans
et al, 1982). Morphine has low lip id so lub ility w ith an octanokw ater p a rtition
coe ffic ien t o f 1.42 (Kaufman et al, 1975). A d iffe re n tia l in te raction between
DFP and a lfen tan il at the site o f in jection is therefore unlike ly.
2.23.2 D is tribution
Many drugs are bound to plasma proteins and may compete fo r the same binding
sites. Such com petition could result in displacement and altered
pharmacokinetic behaviour o f one drug. This type of drug in te raction would be
pa rticu la rly im portant fo r drugs which have high frac tiona l binding (see
MacKichan, 1984 fo r review).
Both fentanyl and a lfen tan il have high frac tiona l binding to blood proteins
(Meuldemans, 1982) as does DFP (M artin, 1985). I t is possible tha t DFP could
displace a lfen tan il from binding sites, allowing more a lfen tan il to enter the
brain. This possib ility was studied and is described in Chapter 5.
2.23.3 Receptor Action
DFP may act at the receptor level to increase the potency o f a lfen tan il. For
example, DFP may be producing opioid receptor super-sensitivity to a lfen tan il.
P ilo t binding studies using a lfen tan il were performed, but were inconsistent.
This is most probably due to the very rapid dissociation from the receptor site
tha t has been reported fo r this ligand (Leysen et al, 1983).
In fac t, an in te raction between DFP at the receptor level would not explain the
d iffe re n tia l action o f DFP on a lfen tan il, since a ll three opioids studied exert
pharmacological e ffec ts through p-opioid receptors (Upton et al, 1983; Leysen et
al, 1983).
2.23.4. Metabolism
In addition to being a potent inh ib ito r o f cholinesterase, DFP may w e ll in h ib it
other enzymes including those involved in drug metabolism (see O’N e ill, 1981).
For example, a s ign ificant inh ib ition o f m itochondrial aldehyde dehydrogenase
occurs fo r at least 85 days fo llow ing adm inistration o f 0.5mg/kg DFP (Messiha,
et al, 1983).
DFP potently inhib its the enzymes hydrolysing substance P (Kato et al, 1980) and
metabolising enkephalins (Snead et al, 1980). A lfen tan il is rapid ly metabolised
fo llow ing i.v. adm inistration, w ith 50-60% metabolised a fte r 4 minutes (M ichie ls
et al, 1981) by N -dealkylation and O-demethylation (Meuldemans, 1980). However
the term ination of a lfentan il's action is predominantly dependent on a
red istribu tion mechanism (Stanski and Hug, 1982). I t is conceivable tha t DFP
could inh ib it the enzymes responsible fo r the metabolism of a lfen tan il, thereby
increasing its antinociceptive a c tiv ity . However, i t must be borne in mind tha t
antinociceptive testing was carried out five minutes a fte r a lfen tan il
adm inistration and over such a short tim e period, DFP would have to be a very
potent inh ib ito r to produce a tw o-fo ld potentia tion o f a lfen tan il.
2.23.5.Excretion
If, as mentioned above, red istribu tion is the main means o f te rm ination o f
action, an in te raction between DFP and a lfen tan il in excretion processes would
probably not a ffe c t a lfen tan il action. Moreover in rats only 0.2% o f the parent
drug is excreted in the urine (Meuldemans, 1980b), and so i t is un like ly tha t
impaired renal excretion would have any e ffe c t on the antinociceptive a c t iv ity
o f a lfen tan il.
2.24. Effect of Atropine and Pralidoxime on DFP Potentiation of Alfentanil
Antinociception
The potentia ting e ffe c t o f DFP on a lfen tan il antinociception may be independent
o f an action on the cholinergic system as the adm inistration o f the m uscarinic
receptor antagonist atropine and pralidoxime to regenerate cholinesterase
enzymes did not a lte r the AD ^q value fo r DFP plus a lfen tan il. However,
pralidoxime is unable to cross the blood-brain barrier, so tha t brain
cholinesterase would remain largely inhib ited by DFP even a fte r pralidoxim e
adm inistration. Though atropine would block muscarinic receptors, i t is possible
tha t an in teraction between DFP and a lfen tan il could involve unprotected
centra l n ico tin ic receptors. Other possibilities regarding the nature o f DFP and
a lfen tan il in teraction are given in Section 4.11.
2.25. E ffe c t o f A trop ine and Pralidoxim e on the A n tinociceptive A c t iv ity o f
Morphine
DFP alone did not a ffe c t the AD^g fo r morphine, but when atropine and
pralidoxime was also administered morphine's antinociceptive a c tiv ity was
enhanced. As mentioned in Section 2.24., the atropine-pralidoxim e mix does not
pro tect centra l n ico tin ic receptors and so i t may be tha t by blocking the
muscarinic receptors w ith atropine, more o f the non-metabolised acetylcholine
would be able to in te rac t w ith n ico tin ic receptors. In other words, the presence
o f a muscarinic antagonist in a cholinesterase inhib ited system could lead to
increased n ico tin ic response. In consideration o f this, i t is worth noting tha t
there have been several studies in mice, ra t and dog showing potent
antinociceptive a c tiv ity o f n ico tin ic agonists (see Table 1.3.).
This in teraction, however, would have to be specific fo r morphine and the
n ico tin ic response since neither fentanyl or a lfen tan il had altered AD^g
fo llow ing atropine and pralidoxime.
An a lte rna tive explanation fo r these findings is tha t morphine may be in te rac ting
w ith atropine. Indeed, i t has been reported tha t in the ra t hot-p la te test,
atropine at 2mg/kg s ign ifican tly potentiated the antinociceptive a c tiv ity o f
morphine (Malec and Langwinski, 1982). This report also notes tha t atropine did
not a lte r the antinociceptive a c tiv ity o f fentanyl or o f pentazocine. The results
of this study showing d iffe re n tia l potentia tion o f morphine antinociception by
atropine are supportive o f the results presented in this thesis.
An additional mechanism for the potentia tion of morphine by atropine which
should be considered is that respiratory alkalosis enhances the percentage of
unionised morphine in the c ircu la tion and thereby increasing the amount entering
the brain (Schulman et al., 1984). I t is possible tha t the additional adm in istra tion
of atropine and pralidoxime produces a degree o f respiratory alkalosis, which
through pharmacokinetic mechanisms potentiates the antinociceptive a c tiv ity o f
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 ho t-p la te la tency
in the mouse. Diazepam has, in fac t, been reported to possess antinociceptive
a c tiv ity 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 potentia te
(M atla and Langwinski, 1982) the antinociceptive a c tiv ity o f opioids including
morphine and fentanyl. In this study, co-adm in istration of diazepam did not
a lte r the AD^g values o f the three opioids studied. I t should be realised,
however, tha t the animals were receiving four d iffe ren t drugs in addition to the
opioid, so tha t any e ffects o f diazepam on antinociceptive ac tiv itie s could be
obscured by non-specific drug in teractions.
2.27. Variation in Antinociceptive Potency of Alfentanil in Mice
The values given fo r AD^g scores fo r a lfen tan il in Tables 2.6. and 2.8. are
d iffe ren t. The orig in of this disparity is not obvious as the animal stra in, test
conditions and experimentor were the same fo r both sets of experiments. Two
differences do exist however, and these are, f irs tly , tha t d iffe re n t a lfen tan il
batches were used and, secondly, these experiments were carried out at d iffe re n t
tim es of the year. Considering the second proposition, i t has been reported tha t
stim ulation-induced antinociception is subject to circadian varia tion (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 varia tion in opioid response as Buckett
(1981) reports tha t stim ulation-induced antinociception as measured by latencies
in mice vary throughout the year. In v itro response of vasa deferentia to
2 5morphine and D -A la , Met enkephalinamide have also been shown to exh ib it
circannual varia tion, w ith a five -fo ld varia tion in potency (De Ceballos and De
Felipe, 1984). The varia tion o f a lfen tan il potency is also considered in Section
5.18. This in form ation described above highlights the importance o f carrying out
appropriate controls in para lle l w ith the experiment.
CHAPTER 3
Locomotor Activity of Mice
Effect 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, locom otor a c tiv ity is probably one o f the most
simple. Its s im p lic ity enables many experiments to be carried out
simultaneously and data to be collected autom atica lly. On the other hand, its
s im p lic ity means tha t only general ambulation is measured and in form ation
on stereotypic resonses (eg, sn iffing and grooming), co-ordination and
muscular r ig id ity cannot be recorded. I t is well known tha t po ten tia lly useful
in form ation goes unrecorded (Krsiak et al, 1970). The in form ation gained
from these locomotor studies should therefore be viewed as only a basic
measure o f a lterations in behaviour and too detailed an in te rp re ta tion o f the
results should be avoided. Nevertheless, w h ils t bearing in mind the
lim ita tions o f these techniques, drug-inducing a ltera tion o f locom otor
function does give an indication of certa in drug-receptor in teractions w ith in
the centra l nervous system. Indeed, i t has been suggested tha t in order to
assess the behavioural e ffects o f a drug, data must be obtained on its
influence on spontaneous m otor a c tiv ity (Robbins, 1977).
Data presented in this thesis shows tha t DFP at 2mg/kg produced apparent
antinociception (Table 2.3.). Though the hot-p la te reaction tim e was
s ign ifican tly increased compared to saline controls, mice exhibited reduced
ambulation and muscular weakness (as evidenced by hind limb abduction). I t is
possible tha t impaired muscular function and co-ordination could delay the
nociceptive end-point (the licking response). Experiments were therefore
devised to assess the e ffects of DFP on spontaneous motor a c tiv ity , and
a c tiv ity fo llow ing opioid adm inistration. The e ffects o f drugs used in
trea ting anticholinesterase poisoning were also assessed fo r e ffec ts on
locomotor a c tiv ity .
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 fo r equ ilibration 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 in jected subcutaneously using the same tim ings to para lle l the
antinociceptive procedures described in Section 2.3.3. Doses o f opioids used
were 20mg/kg morphine, 80ug/kg fentanyl and 400pg/kg a lfen tan il.
3.3. Locomotor Activity Testing
Locomotor a c tiv ity in mice was measured using a system identica l to tha t
described by Francis et al (1983) in which a photobeam m onitoring system was
linked to a m icrocomputer. The apparatus consisted of pairs o f industria l
grade in fra -red source and detector pairs. Three in fra -red ligh t 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 m icrocom puter (BBC B) via an in te rface .
The software employs an in te rrup t routine to assess the state o f the
photocells (on or o ff) every 10ms, and a change in state is recorded as a score.
Locomotor hardware was b u ilt in the B iochem istry Workshops, U nivers ity of
Surrey. O riginal software w ritte n fo r this apparatus was adapted to display
the mean and standard deviation of counts (see Appendix C fo r listing).
Individual mice were placed in each testing box (18.3 x 11cm), allowing five
mice to be tested simultaneously. Locom otor a c tiv ity was measured
im m ediate ly a fte r the last in jection, w ith the exception o f some experiments
where DFP or saline were in jected 1 hour before testing. In jection tim ings
were the same as those described in Section 2.2.3.(a). Testing was m onitored
fo r 15 5-m inute epochs, so tha t changes in a c tiv ity over tim e could be
assessed.
3.6. Statistical Procedures
Locomotor scores were analysed fo llow ing data reduction. Mean locom otor
scores fo r each 5-m inute period were summed over the f irs t 15 and to ta l 75
minute periods. Means fo r the 15 and 75 m inute periods were obtained from
10 or 15 animals recorded on one, two or three separate days.
E ffe c t o f drug trea tm ent was analysed using m ultip le analysis of variance
followed by a posteriori examination using Duncan's M ultip le Range Test
(Duncan, 1955). These s ta tis tica l analyses were carried out using the SPSS
software on the University 's Prime computer (S ta tis tica l Packages fo r the
Social Sciences, Pennsylvania State University, Version M, Release 9.1.).
RESULTS
3.7. E ffect of DFP on Locomotor Activity
DFP at lm g/kg produced a s ign ificant decrease in locomotor a c tiv ity only
over 75 minutes compared to saline (Figure 3.1. and Figure 3.2.). However,
when DFP was in jected 1 hour before testing, DFP s ign ifican tly decreased
exploratory a c tiv ity over the f irs t 15 minutes. This depression o f locom otor
a c tiv ity was fu rthe r decreased fo llow ing DFP 2mg/kg .
3.8. E ffect of Atropine and Pralidoxime on Locomotor Activity
The atropine-pralidoxim e m ix produced hyperlocomotion which was not
decreased in animals pre-treated w ith DFP. The e ffe c t o f DFP alone was
reversed by the atropine-pralidoxim e m ix adm inistration.
3.9. E ffect of Diazepam on Locomotor Activity
Diazepam alone greatly depressed locomotor a c tiv ity and fu rthe r decreased
the hyperlocomotory e ffe c t o f DFP.
3.10. Effect of Various Treatments on Alfentanil Induced Hyperlocomotion
The hyperlocomotion fo llow ing a lfen tan il adm inistration was s ign ifican tly
decreased by DFP (Fig. 3.3.).
Over the f irs t 15 minutes, atropine-pralidoxim e mix adm in istra tion did not
s ign ifican tly a lte r the DFP e ffe c t, but over 75 minutes there was a reversal,
w ith increased locomotor a c tiv ity . The additional in jection o f diazepam
produced a marked decrease in a c tiv ity .
SCORE800 i
700-
600-
500-
400-
300-
2 0 0 -
1 0 0 -
10 15 20 25 30 35 40 45 50 55 6 0 65 70 755
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.
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u t: c.u u O (j oZ c OII (I M| ->•-> Ab
breviations: ALF
=
Alfentanil,
FENT
= Fentanyl,
MORPH
= Morphine,
SAL
= Sa
line
DFP
=
Di-i
sopr
opyl
fluo
roph
osph
ate,
ATR/PRAL
= Atropine
and
Pralidoxime, DIAZ =
Diaz
epam
Arrows
in this
table
indicate
significant
changes
in locomotor
activity
in mi
ce
10000-J
frJRi 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-inducedhyperlocomotion over 15 and 75 minutes
Black histograms: Grey histograms: SAL + ALFENT:DFP + ALFENT:DFP + ATR/PR + ALFENTANIL:
locomotor score over 15 minutes locomotor score over 75 minutes Saline and alfentanil treated. DFP and alfentanil treated.DFP, atropine and pralidoxime and alfentanil treated.
DFP + DIAZ+ A/P + ALF: DFP, diazepam, atropine and
pralidoxime and alfentanil treated.
Values represent mean - S.E.M. o f at least 10 observations.
Saline contro l scores: 15 mins 1442 - 108
(n=5) 75 mins 5630 ^479
3.11. E ffect of Various Treatments on Fentanyl Induced Hyperlocomotion
The hyperlocomotory a c tiv ity o f fentanyl was not s ign ifican tly altered by
adm in istration o f DFP or DFP + atropine/pra lidoxim e over 15 or 75 m inute
periods (Fig. 3.4.). The additional in jection o f diazepam, however,
s ign ifican tly depressed locom otor a c tiv ity scores.
3.12. E ffect of Various Treatments on Morphine Induced Hyperlocomotion
Morphine-induced hyperlocomotion was s ign ifican tly decreased fo llow ing DFP
pretreatm ent, but only over the 75 m inute period (Fig.3.5.). The atropine-
pralidoxime m ix had no e ffe c t on morphine hypolocomotion. Diazepam
produced marked decreases in locom otor scores, and though this was
s ign ificant over the f irs t 15 minutes s ta tis tica l s ign ificant differences were
not observed over the 75 minutes, despite there being nearly a 34-fo ld
d ifference; very high pooled standard deviations existed in these groups.
LOCOMOTOR SCORE 10000-
5000-1
DFP+FEHT DFP+ftTR/PR QFP+DIflZ, +FENT fi/P+FENT
SflLINE+flTR/P+FENT
FENTRNIL SflL+FENT
Fig. 3.4. Effect of DFP on fentany1-inducedhyperlocomotion over 15 and 75 minutes
Black histograms Grey histograms: SAL + FENT:DFP + FENT:DFP + ATR/PR + FENTANYL:
DFP + DIAZ + A/P + FENT:
locomotor score over 15 minutes locomotor score over 75 minutes Saline and fentanyl treated.DFP and fentanyl treated.DFP, atropine and pralidoxime and fentanyl treated.
DFP, diazepam, atropine and pralidoxime and fentanil treated.
Values represent mean t S.E.M. o f at least 10 observations.
Saline contro l scores: 15 mins 1384 - 141
75 mins 5957 - ~531
B$^^/+++////B
LOCOMOTOR SCORE 1000th
5000-
PH
' ••••
SfiL+MORPH DFP+hORPH OFP+HTR/PRtilORPH I HE
DFP+DI.RZ,+ii/P+nuRPH
SRLINE+flTR/P+MmPPm
Fig 3.5. Effect of DFP on morphine-inducedhyperlocomotion over 15 and 75 minutes
Black histograms Grey histograms: SAL + MORPH:DFP + MORPH:DFP + ATR/PR + MORPHINE:
locomotor score over 15 minutes locomotor score over 75 minutes Saline and morphine treated.DFP and morphine treated.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 a t least 10 observations.
Saline contro l scores: 15 mins 1541 - 122
(n=15) 75 mins 6095 - 404
^147901691^8944691576436^1079456^14795
DISCUSSION
3.13. E ffect of DFP on Locomotor Activity
There are several reports in the lite ra tu re describing the e ffec ts o f
anticholinesterase agents in im pairing 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 e t al, 1984;
Wolthuis and Vanworsch, 1984), pyridostigm ine (Harris et al, 1984; Wolthuis
and Vanworsch, 1984), sarin (Landauer and Romano, 1984b), soman (Wolthuis
and Vanworsch, 1984) and tabun (Landauer and Romano, 1984a; Wolthuis and
Vanworsch, 1984). The w e ll documented e ffe c t o f cholinergic agents on
locom otor a c tiv ity has been described as a le rt non-m otile behaviour (ANMB)
(Karczmar, 1977) and is believed to involve a cholinergic-dopam inergic dipole
in n ig rostria ta l and extrapyram idal pathways.
In this study, s ign ificant and marked decreases in locomotor a c tiv ity were
observed fo r DFP at lm g/kg in mice. When administered 1 hour before testing
to paralle l the antinociceptive testing procedures, DFP reduced exploratory
motor a c tiv ity . In in terpre ting the consequences of this data, i t is not
unreasonable to suggest tha t DFP induced 'antinociception' was observed as a
result of impaired motor co-ordination and muscular function ra ther than an
inh ib ito ry 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 a lte r the DFP antinociception but atropine which
reversed the hypolocomotor e ffec ts of DFP (see below), also reversed
antinociceptive a c tiv ity .
3 . 1 4 . Effect of Atropine and Pralidoxime Alone and With DFP on Locomotor Activity
DFP induced reduction o f locom otor a c tiv ity appeared to involve the
cholinergic system as DFP's e ffec ts were com pletely reversed fo llow ing
atropine-pralidoxim e mix adm in istration. When administered alone, atropine
and pralidoxime produced hyperlocomotion. Hyperlocomotion has been
previously reported fo r atropine (Chatervedi, 1984; Seidel et al, 1979) and fo r
another muscarinic antagonist, hyoscine (Sansone, 1983; Kuribara and
Tadokar, 1983).
3.15. Effect of Diazepam Alone and With DFP on Locomotor Activity
Inh ib ition o f locomotor a c tiv ity fo llow ing diazepam adm inistration was
marked and this is in contrast w ith some reports in the lite ra tu re . For
example, i t has a c tiv ity been shown tha t in mice, diazepam up to Im g/kg did
not a ffe c t locomotion (Sansone, 1982). In rats at 2.5mg/kg diazepam enhanced
ambulatory a c tiv ity w hilst at 20mg/kg i t was inhib ited (Matsubara and
Matshushita, 1982). In this study the dose of diazepam used was 1.78mg/kg,
which suggests tha t a marked motor depression was observed, m ice may be
more sensitive than rats to the locomotor depressant action o f diazepam.
When administered a fte r DFP the locomotor a c tiv ity o f diazepam was
decreased s t ill fu rthe r, so tha t the hypolocomotory e ffects o f these drugs
were addictive.
3.16. Effects of DFP on Opioid-Induced Hyperlocomotion
The action o f opioids in mice is to produce a stereotypic running response (eg,
C arro ll and Sharp, 1972) and in this study morphine, fentanyl and a lfen tan il a ll
produced this running response. This hyperlocomotion was reduced fo r a ll
opioids by DFP pretreatm ent. This is consistent w ith the lite ra tu re as DFP
at 2mg/kg has been reported to decrease morphine-induced running in mice
(Su and Loh, 1975). and oxotremorine has also been shown to decrease
morphine hyperlocomotion (Seidel, 1979). I t has been suggested tha t the
augmentation of the opioid-induced running response by cholinergic agents
may be due to an increase in dopamine u tiliza tio n produced by muscarinic
agonists (Seidel, 1979).
The data indicates tha t the e ffe c t o f DFP on opioid induced running is most
pronounced w ith a lfen tan il. However, i f DFP was enhancing the e ffe c t of
a lfen tan il in the same way as tha t observed fo r the antinociceptive a c tiv ity ,
one m ight have expected increased locomotor scores. A possible explanation
o f this apparent anomaly highlights a drawback encountered in this form of
measurement o f locomotor a c tiv ity . Observation o f mice receiving opioids,
especially a lfen tan il, revealed tha t during peak opioid a c tiv ity mice had
running f its ie, periods of intense running a c tiv ity interspersed w ith periods
o f im m ob ility (c f. dopamine catatonia). These periods o f catatonia appeared
w ith greater frequency and duration w ith increasing dose, at 800pg/kg, ha lf
or sometimes all o f the 60 seconds antinociceptive testing period m ice were
catatonic. I f DFP potentiated a lfen tan il, the measured locomotor a c tiv ity
score could have been reduced fo llow ing DFP i f the mice spent a greater
proportion of the ir tim e in a catatonic im m obile state. A c t iv ity is not a
unitary measure and photocell measured locomotor a c tiv ity cannot detect
such stereotyped ac tiv ities such as c irc ling , excessive grooming, lick ing or
b iting or d iffe ren tia te between walking and running ac tiv ities . More
sophisticated a c tiv ity monitors or scored human observation would be
required to iden tify these specific changes in a c tiv ity . So i f DFP potentia ted
a lfentanil's a c tiv ity and produced increased catatonia, this would be re flec ted
in a decreased locomotor a c tiv ity score.
3.17. The Effect of Atropine and Pralidoxime on the Locomotor Activity of
DFP and Opioid-Treated M ice
In DFP and opio id-treated mice the e ffe c t o f the atropine-pralidoxim e mix
trea tm ent on locom otor a c tiv ity was only s ign ificant fo r a lfen tan il over 75
minutes. I t is quite like ly tha t no s ign ificant e ffe c t was observed fo r the
other opioids due to the in te raction o f the four drugs administered and the ir
e ffects on locomotor a c tiv ity . For example, the atropine-pralidoxim e m ix in
addition to possessing hyperlocom otory a c tiv ity (see Section 3.18.), would
tend to antagonize the hypolocomotory e ffects o f DFP. This antagonism of
DFP could therefore decrease the incidence o f a lfen tan il catatonia (see
Section 3.20.). Furtherm ore, atropine is reported to potentia te morphine-
induced c irc ling (Seidel, 1979).
In summary, the combination o f opioid, DFP, and the atropine-pralidoxim e
mix produce a complex e ffe c t on locomotor behaviour. The observed
enhancement of a lfen tan il locomotion w ith atropine-pralidoxim e m ix is like ly
to have been a result o f both a reduced degree o f catatonia and an add itive
cholinergic hyperlocomotion.
3.18. E ffe c t o f Diazepam on Opioid-Induced Locomotion
The large decrease in locomotor score fo llow ing diazepam was common to a ll
three opioids studied. This e ffe c t has been reported previously fo r morphine-
and fentanyl-induced hyperlocomotion (M atla and Langwinski, 1982).
Benzodiazepines are known to have a sedative action and i t could w e ll be this
action on benzodiazepine GABA receptors which produced the decrease in
locom otor score. A lte rna tive ly , diazepam could be acting through another
receptor system; the role of the dopaminergic system has already been
mentioned in its involvement in locomotor a c tiv ity (Section 3.17.) and i t is
possible tha t the observed e ffe c t o f diazepam on locomotion was due to its
action inh ib iting dopamine function (Reubi et al, 1977). I t may also be
possible tha t opioid catalepsy (and hence im m obility ) was enhanced fo llow ing
diazepam, as catalepsy has been shown to involve inh ib ition of the centra l
dopaminergic pathway (Kushinsky and Hornykiew icz, 1972).
3.19. Interpretation of Locomotor Data
Locomotor measurement is a s im plis tic measure o f a ltera tion o f behaviour.
I t is subject to many errors and i t is well known tha t large quantita tive
differences between individual animal a c tiv ity scores are observed (Izazole-
Conde et al, 1983).
Observation of animals during locom otor testing procedures suggested tha t at
least part of the large varia tion o f scores between animals were probably due
to behaviours such as grooming. Mice were occassionally observed to be
grooming d irec tly in the path o f the in fra -red lig h t beam so tha t th e ir rapid
head movements increased the locom otor score.
As mentioned above, production o f catalepsy is produced w ith high doses o f
opioids and would tend to increase varia tion between animals. This form of
error has been reported in the lite ra tu re where alcohol at certa in doses
produces unchanged or increased locomotor a c tiv ity and simultaneously large
increases in im m ob ility (Smoothy and Berry, 1984). Observation revealed tha t
this was due to alcohol's e ffe c t of decreasing non-locomotor a c tiv ity such as
rearing which is not recorded by simple locomotor a c tiv ity monitors. These
lim ita tions notwithstanding, the locomotor a c tiv ity m onitor used in this study
gave a general indication o f the effects o f drugs on locomotor a c tiv ity o f
mice.
CHAPTER 4
Distribution of H Morphine, Fentanyl and Alfentanil
In Mouse Brain: E ffect of DFP Administration
INTRODUCTION
4.1 Distribution Studies
The d iffe re n tia l potentia tion o f a lfen tan il by DFP led to the conclusion tha t
the mechanism probably involved an e ffe c t on pharmacokinetics (Section
2.28) ra ther than altered pharmacodynamic parameters. A simple measure of
d is tribu tion u tilizes radiolabel tracing methodologies, wherein plasma and
brain levels o f a radiolabelled drug are determined. Drug concentration in
contro l and treated animals can then be compared.
A drawback to the method used in this thesis is tha t only to ta l ra d io ac tiv ity
was determined. The presence o f radioactive m etabolites, which may or may
not possess pharmacological a c tiv ity are not distinguished. However, fo r
fentanyl and a lfen tan il the short tim e period between adm in istration o f the
drug and k illing would mean tha t i t is like ly tha t the m a jo rity o f the
rad ioac tiv ity in the brain is the parent drug. For morphine the glucuronide
metabolites do not contribute s ign ifican tly to uptake in to the brain of
labelled morphine (H artv ig 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 m ice (CD-I strain; 25-30g) were used as described in Section
2.2.1. Procedures fo r equ ilibration were also as those previously described.
Radiolabelled morphine and fentanyl were supplied in ethanol, and
radiolabelled a lfen tan il was suplied in w a te r:ace ton itrile 30/70, ethanol 100.
The solvent was removed under a gentle stream o f dry nitrogen at room
temperature.
4.3. Drugs
The drugs used were obtained from sources described in Section 2.4. In
addition, the fo llow ing radiolabelled compounds were used:-
DRUGS
^ H Morphine sulphate
Specific A c t iv ity 20-24 C i/m m ol
Amersham International
3 H Fentanyl c itra te
Specific A c t iv ity 15-18 C i/m m ol
IRE, Fleurus (Belgium)
3 H A lfe n ta n il hydrochloride
Specific A c t iv ity 23.1 C i/m m ol
Janssen Pharmaceuticals
3 H Hexadecane tr it iu m standard Amersham International
Specific A c t iv ity 1.69 qC i/m l
* 3 H A lfen tan il was generously donated by Janssen Pharmaceutical
4.4. Dosing Protocols
Drug adm in istration procedures were carried out by identica l to tha t
described in Section 2.6. fo r DFP and opioid adm in istration.
Mice received either 0.9% w /v saline or DFP (Img/kg) s.c. This was followed
by s.c. in jections o f radiolabelled morphine, fentanyl or a lfen tan il
administered 30, 30 or 33 minutes la te r respectively. Two doses o f each
opioid were studied and each in jection contained 7pCi o f ^H-labelled opioid.
4.5. Distribution Studies Protocol
A t times o f peak opioid antinociceptive a c tiv ity (see Figure 2.1.) (also 1 hour
a fte r DFP or saline adm inistration) animals were k illed by decapitation and
trunk blood collected in to heparinised tubes. The brains were rapid ly
removed and cooled over ice on a moistened f i l te r paper covered pe tri dish.
Brains were then dissected in to eight regions, fo llow ing a protocol s im ila r to
tha t described by Glowinski and Iversen (1966). Each region was weighed and
transferred to glass sc in tilla tion vials to which 1ml o f Soluene-100 (Packard)
was added. Plasma was obtained by centrifugation a t 2500g fo r 10 minutes
and a 20pl o f plasma was solubilized w ith 0.5ml Soluene-100. Brains were le f t
overnight to solubilize. With the larger brain regions, cerebellum and
cortices, solubilization was promoted by warming the vials to 50°C fo r 10 to
30 minutes, and allowing to cool.
4.6. Scintillation Counting
Follow ing solubilization, sc in tilla tion flu id consisting o f 1.5 litre s o f sulphur-
free toluene, 750ml o f synperonic NX PO POP 225mg and PPO 12g (Wood et3
al, 1975) was added to samples. H a c tiv ity was determined in an LKB 1210
U ltrobe ta sc in tilla tion counter. Chemiluminescence was avoided by
acid ify ing the sc in tillan t w ith 10ml glacia l acetic acid per 2.5 litre s of
sc in tillan t.
Presence of solubilised brain tissue has a quenching e ffe c t on detection of
sc in tilla tions. As varying amounts o f brain tissue were present in each v ia l,
i t was im portant to take in to account and compensate fo r 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 o f Soluene-100. To each via l,
10 pi o f hexadecane standard was added, and the vials counted on the 1210
U ltrobeta . Decay tables fo r allowed to ta l a c tiv ity per v ia l to be
calculated. With c.p.m. and R values from these vials, the coe ffic ien ts o f the
data were calculated by applying a Basic regression programme (see Appendix
D (i). This program yielded values fo r AO, A l, A2 and A3 which were
programmed in to the sc in tilla tion counter, enabling rad ioac tiv ity to be given
in d.p.m. The quench curve was also p lotted using the d.p.m. package on a
RackBeta 1215 (see Appendix D (ii)).
A s im ilar procedure was carried out fo r plasma as occasionally samples were
haemolysed. Plasma samples o f varying degree o f haemolysis were counted
w ith 10pi hexadecane standard. The coeffic ien ts were determined as fo r
the brain samples.
RESULTS
4.7. Effect of DFP Pretreatment on Alfentanil Brain and Plasma Levels
Figure 4.1. represents the e ffe c t of DFP on levels in various brain regions o f
a lfen tan il, expressed in d.p.m ./g wet weight o f tissue.
P retreatm ent w ith DFP at Img/kg caused a s ign ificant increase in the levels
in a ll brain regions o f a lfen tan il at 400pg/kg (Figure 4.1.). This increase was
most marked in the hypothalamus w ith a 3.2-fo ld increase in levels compared
to saline controls. There was a marked reduction in plasma a lfen tan il
rad ioac tiv ity (Table 4.1.). A t the 200pg/kg dose o f a lfen tan il, DFP appeared
to l i t t le a ffe c t brain levels. I f brainrplasma ratios are considered (Table
4.2.), DFP pre-trea tm ent produced a s ign ificant e ffe c t in a ll brain regions
and at both doses.
4.8. E ffect of DFP Pretreatment on Morphine and Fentanyl Brain and Plasma
Levels
DFP appeared to have l i t t le e ffe c t on brain levels o f fentanyl or morphine
(Figure 4.2. and 4.3.). There was a slight decrease in morphine levels
fo llow ing DFP but this only reached significance in the hypothalamus at the
higher dose. S im ilarly, DFP did not s ign ifican tly a lte r plasma ra d io a c tiv ity
o f e ither morphine or fentanyl, (Table 4.1.) and so brain:plasma ra tios fo r
these two opioids were very s im ilar in contro l- and DFP-treated animals
(Table 4.2.).
DPM g'"1'{ x10-3)
4 0 0
3 8 0 -
360
340
320
200
180
160
120
100
80 ■
6 0 •
4 0 -
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 -*-) ofalfentanil (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) B2 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 1 0 ~ 3 )
-5 iA B
4 0 -
3 0
25
20
15 -
10 -
X
jL
JL
I1
1I I
I
I 4-X J L
1 X
LL
[I
I
1
JL
X
i l
X
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 R ad ioactiv ity dpm m l" (x 10” )
Opioid
M orph ine, (10mg/kg~ )
Morphine (20mg/kg" )
Fentanyl , (40pg/kg" )
Contro l
197 - 7
118 - 10
7 3 - 7
DFP-Treated
174 - 6 *
97 - 8
90 -13
Fentanyl , (80pg/kg” )
67 - 4 5 7 - 3
A lfe n ta n il . (200pg/kg )
166 - 10 110 ± 12*
A lfe n ta n il (400pg/kg" )
147 +34 9 1 - 6
Each value is the mean o f six observations, t test, DFP vs. contro l * p < 0.05
Table 4.1. E ffe c t of DFP (lm g/kg"^) on Plasma Levels o f H Morphine, Fentanyl and A lfe n ta n il
i—i CL * * * * * * * *, 1cn
c5 ■*
Ll.Q 1
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DISCUSSION
4.9 Opioid Lipophilicity and Access to the CNS
Opioids exert the ir antinociceptive e ffe c t in the centra l nervous system. To
gain access to the ir site o f action, opioids must cross the blood-brain barrier.
Generally speaking, the greater the lip o p h ilic ity o f an opioid (or any other
drug) the greater the ease w ith which i t is able to cross this barrier and enter
the brain (Jacobson, e t al, 1977).
A measure o f lip o p h ilic ity is the octanohwater pa rtition coe ffic ie n t at pH7.4
and the lite ra tu re values fo r the opioids studied are 1.42 fo r morphine
(Kaufman et al, 1975), 130 and 860 fo r a lfen tan il and fentanyl respective ly
(Meuldermans e t al, 1982). These values para lle l the order o f brain:plasma
ratios obtained in this study. Levels o f rad io ac tiv ity in the brain compared
w ith the plasma are highest fo r fentanyl, followed by a lfen tan il and then
morphine. The times fo r measurement o f morphine, fentanyl and a lfen tan il
levels were chosen to correspond to peak antinociception (see Section 2.6.).
These times agree w ith lite ra tu re values fo r these opioids (Gardocki and
Yelnoski, 1963; Johannesson and Becker, 1973). There is also a good
corre lation between levels o f these opioids in the brain and the ir
antinociceptive e ffects (Johannesson and Becker, 1973; Hug and Murphy, 1981).
4.10. E ffect of DFP on Opioid Brain Levels
DFP pretreatm ent produced a disparate e ffe c t on the brain levels and
distribution of radiolabelled morphine, fentanyl and a lfen tan il. The most
marked e ffe c t o f DFP is on the d istribution o f a lfen tan il where plasma levels
are lower and at 400pg/kg a lfen tan il there is a concom itant rise in a lfen tan il
in the brain. This enhanced entry could account fo r the observed increase in
the antinociceptive a c tiv ity o f a lfen tan il fo llow ing DFP (see Section 2.24.).
The absence of an apparent increase in brain levels o f a lfen tan il 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 fo r and contributes to the to ta l
rad ioac tiv ity . A lte rna tive ly , DFP may be a ltering pharm acokinetic
parameters so tha t peak brain a lfen tan il levels could occur earlie r. Peak
opioid receptor occupancy or antinociceptive e ffe c t could persist fo r a short
tim e a fte r peak drug level (see Figure 4.4.)
4.11. Mechanism of Increased Levels of Alfentanil Produced by DFP
There is no lite ra tu re at present describing a potentia tion o f a lfen tan il by
other drugs. However, there are some reports in the lite ra tu re describing the
action of some drugs in increasing the potency o f morphine by a mechanism
o f increasing the plasma and brain levels o f this opioid. For example, a
synergism has been demonstrated between diazepam and morphine (Sanchez
et al, 1982). This appears to be dependent on diazepam com petitive ly
inh ib iting morphine glucuronization and dem ethylation. This results in higher
plasma and hence higher brain levels o f morphine. Brain uptake o f morphine
was shown to be increased fo llow ing adm in istration of the antih istam ine
tripelennam ine (Vadlamani, 1984) possibly by increasing perm eability o f the
blood-brain barrier. By contrast, the potentia tion o f morphine by
physostigmine did not appear to be dependent on a ltera tion in morphine
plasma or brain levels (Bhargava and Way, 1972).
I t is possible tha t DFP could increase a lfentan il's entry into the brain also by
a ffecting the blood-brain barrier. Indeed, DFP has been shown to increase
BRAINALFENTANIL
CONC.
DFP+400pg/kgALFENTANIL
\400pg/kg''
NN
S\
ALFENT/ \\\
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\ \
\ \200yg /kg- / / / \ALFENT-
' y / * / // y * /
\ V ' \ a n ilN Vs \ \ \
✓ ___ ^s \
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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 perm eability of capillaries in the anterior segment o f the eye (von
Sallman and D illon, 1947). Furtherm ore, in considering the mechanism of
potentia tion o f morphine antinociception by the two anticholinesterases,
physostigmine and neostigmine, Rypka and N a vra til (1982) showed tha t the
blood-brain barrier perm eability to acridine orange was markedly increased
fo llow ing adm in istration o f physostigmine and neostigmine. I f however, DFP
exerted this same e ffe c t on blood-brain barrier perm eability, this would be
non-selective and i t m ight be expected tha t a ll three opioids would a ll have
enhanced entry in to the brain. Moreover, as a lfen tan il and fen tanyl are
highly lip id soluble, the blood-brain barrie r would not norm ally l im it the entry
of these drugs into the brain. On the other hand, the low lip id so lub ility o f
morphine accounts fo r the low penetration o f morphine into the brain
(H artv ig et al, 1984). This means tha t an increase in the perm eability o f the
blood-brain barrier would tend to increase the entry o f a low perm eability
agent such as morphine, ra ther than an agent which is highly lip id permeable.
The observation tha t morphine levels are unchanged by DFP tends to m ilita te
against an action on blood-brain barrier perm eability by DFP.
The evidence suggests tha t DFP enhancement o f a lfen tan il entry in to the
brain is probably more like ly to be dependent on pre-blood-brain barrie r
mechanisms. One such candidate fo r this mechanism is a lte ra tion o f
a lfen tan il binding to plasma proteins.
CHAPTER 5
3In Vivo and In Vitro Plasma Protein Binding of H Alfentanil
3and H Fentanyl: Effect of DFP Administration
INTRODUCTION
I A35.1 Plasma Protein Bind[of Drugs
The a b ility of plasma proteins to bind and transport chemicals and drugs has
been known fo r many years (Rosenthal, 1926). The potentia l binding capacity
is enormous w ith an estimated to ta l surface area o f plasma proteins per
person being between 130,000 and 735,000m (Bennhoid, 1967). In practice,
only a frac tion of this area is available as compounds tend to bind to specific
sites (B ickel, 1978).
The binding o f drugs to plasma proteins has been comprehensively reviewed
over the years (Goldstein, 1949; Meyer and Guttman, 1968; Jusko and Gretch,
1976). I t has become apparent tha t the protein binding o f a drug can
influence its d is tribu tion which d irec tly and ind irec tly influences the action
o f the drug by determ ining its ava ilab ility at its sites o f action.
5.2 Plasma Protein Binding of AlfentaniljFentanyl and DFP
Studies in ra t, dog and man have shown tha t fentanyl and a lfen tan il have high
frac tiona l binding to blood proteins (Meuldemans et al, 1982). As only free
unbound drugs may cross the blood-brain barrier increased entry o f drug could
occur if , fo r example, the proportion o f opioid bound to plasma proteins is
altered fo llow ing DFP adm inistration. I t is possible tha t DFP may a lte r
binding of opioids to plasma proteins, as its adm inistration may produce
e ither acidosis or alkalosis by e ither stim ulating respiration at high doses or
depressing i t at low doses (Karczmar, 1967) since fentanyl binding is markedly
affected by a lterations in blood pH (Meuldermans et al, 1982). In addition,
a lte ra tion in pH would a lte r the per cent ionization (ionized drugs cannot pass
through the blood-brain barrier) and this could be im portant fo r fentanyl
where only 9% is unionized at normal pH. Though a lfen tan il would be l i t t le
a ffected as 89% is unionized, change in per cent ionization has been shown to
markedly potentia te the brain uptake o f morphine as shown in a lka lo tic rats
(Schulman et al, 1984). However i t appears tha t altered blood pH is not an
im portant fac to r as o f the three opioids studied a lfen tan il pharmacokinetics
are the most pH-independent.
DFP has also shown to bind, irrevers ib ly , to plasma proteins (Truhaut, 1966)
and so i t is worth enterta in ing the notion tha t DFP competes fo r or inhib its
a lfen tan il binding, thereby increasing the proportion o f free a lfen tan il which
is able to enter the brain. This form of in te raction is well known, and has
also been shown fo r irreversib le anticholinesterase agents; i t has been
suggested tha t the potentia tion o f sodium pentobarbita l anaesthesia by soman
is dependent on com petition fo r plasma protein binding (Clement, 1984). This
mechanism of in teraction is considered in th is section.
5.3 Assessment of Drug Binding to Plasma Proteins
There are two main methods fo r determ ining free drug levels: equilibrium
dialysis and u ltracen trifugation . Equilibrium dialysis is the most used o f
these methods but suffers from a number of methodological drawbacks.
These include Donnan effects, changes in ion concentration which may
compete w ith drugs fo r binding or a lte r protein conform ation (Bowers e t al,
1984). U ltra filtra t io n has the advantage o f being a more rapid procedure
though one drawback is tha t there may be non-specific adsorption o f drug to
the u ltra filtra tio n membrane (Zhirkov and P iotrovski, 1984), however th is
adsorption may be determined and compensated fo r. I t has been concluded
tha t ultracentrifuga'cion is a more sensitive method fo r determ ining small
changes in protein binding (Coyle and Denson, 1984) and be tte r re flec ts the
real values than those obtained by equilibrium dialysis (Tuila, 1984).
I N V I T R O I N V I V O
Mice in jec tedwith DFP or saline
Mice in jec ted with -o p io id
Trunk blood co l lec ted ^
Plasma o b t a i n e d . by centr i fugat ion
Plasma aliquot incubated with
-op io idU ltracentr i fugat ion to obtain unbound
drug f ract ion
^ Liquid scint i l la t ion , 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 fo r equ ilibration 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 o f drug adm inistrations were the same as the previous experiments
(Section 4.4) where DFP or 0.9% w /v saline was given s.c. at tim e 0, fen tanyl
(80pg/kg) at 50 minutes, and a lfen tan il (400pg/kg) was administered at 55
minutes. Each in jection contained 7pCi o f opioid. Trunk blood was
collected fo llow ing 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 la te r. A 250pl a liquot of plasma obtained fo llow ing
centrifugation was incubated fo r 30 minutes at 37°C w ith 25pl ^ H a lfen tan il
or ^ H fentanyl. The incubation medium contained additional cold opioid so
tha t drug concentrations paralleled those fo llow ing a s.c. antinociceptive
dose (Fig 5.1.).
5.8. Ultracentrifugation Protocol
Free drug concentrations were determined by u ltracen trifuga tion o f plasma
samples in an Amicon m icropartition MPS-1 k it (Figure 5.2.). A 200pl aliquot
was pipetted in to the sample reservoir o f the assembled unit. The un it was
centrifuged in a bench top centrifuge at 1500g fo r 10 minutes. A 20pi a liquot
o f the u ltra f ilt ra te (i.e. unbound drug) and 20p i o f the plasma (i.e. to ta l drug)
were added to polythene tubes in duplicate. 3-5mls o f Unisolve-1 sc in tilla tio n
flu id (Koch L ight) was added and tubes were counted in a RackBeta 1215 or
1216.
5.9. Calculation of Free and Bound Drug Concentration
Calculation o f the free and bound drug concentrations were made from data
o f u ltra f ilt ra te and plasma rad ioactiv ities 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 o f
a lfen tan il or fentanyl occurred during the u ltracen trifuga tion process through
adsorption on the membrane or plastic reservoir. A stock of mouse plasma
3 3contaimrKj e ither H a lfen tan il or H fentanyl was aliquoted and
ultracentrifuged as described in Section 5.6. R ad ioactiv ity was determ ined
fo r the stock and fo r u ltra f iltra te s in duplicates. Recovery could then be
determined by comparing rad ioactiv ities in the original stock w ith the
u ltra f ilt ra te .
RESERVOIR ' IC A P ___________________ i
!i ]i !
SAMPLERESERVOIR
CLIP
O R IN G
YMMEMBRANE
MEMBRANESUPPORTBASE
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FILTRATECAP
Fig 5.2. The Amicon Micropartition MPS-1 System
RESULTS
5.11. Recovery Experiments
Recovery experiments fo r a lfen tan il and fentanyl showed tha t the a lfen tan il
u ltra f ilt ra te contained 103 - 11% (n=4) o f the orig inal stock and the fentanyl
u ltra f ilt ra te contained 80.7 - 1.3% (n=6).
5.12. Percentage of Plasma Protein Binding of Fentanyl and Alfentanil
Contro l binding o f a lfen tan il were fa ir ly s im ilar fo llow ing in vivo and in v itro
binding protocols (Table 5.1). The figures fo r fentanyl binding in contro l mice
were quite d iffe ren t, dependent on whether _in vivo or in v itro protocols were
used.
5.13. Effect of DFP on Binding Fentanyl and Alfentanil to Plasma Proteins
Table 5.1. shows the e ffe c t o f DFP (lmg/kg) on the levels o f plasma to ta l and
plasma free a lfen tan il and fentanyl fo llow ing incubation in v itro . The values
presented have been adjusted fo r losses during u ltracen trifuga tion in the in
v itro experiments. There was no a ltera tion in free or bound fentanyl in the
presence o f DFP (Table 5.2.), however, there was a greater than 40% increase
in free a lfen tan il in DFP treated plasma.
The e ffe c t o f DFP on m vivo adm inistration o f a lfen tan il (Table 5.2.) is more
marked w ith nearly a 60% increase in free a lfen tan il. For in vivo binding of
fentanyl, DFP has very l i t t le e ffe c t on free levels (Table 5.2.)
Table 5.1.
Treatment
Saline
DFP
Img/kg
Effect of DFP Treatment on In Vivo and In Vitro
Plasma Levels of Alfentanil
and fentanyl
Alfentanil
ng/ml
In vivo
Fentanil
ng/ml
In v itro In v itroin vivo
Tota l 485 - 25.2 519 - 5 16.87^0.36 13.25 - 0.76
Free 118 - 6.1 140 ± 10 2.01 - 0.14 7.09 - 0.54
Total 286 - 40.2 517 - 9 16.30 - 0.59 12.25 - 1.51
Free 110 - 13.0 195 - 12 1.83 - 0.06 5.63 - 0.68
Values are means ^ SEM of 6 observations.
Table 5.2. E ffect of DFP Treatment on Levels of In Vivo and in Vitro
Free Plasma Levels of Alfentanil and Fentanyl
% Free levels
Treatment Alfentanil Fentanyl
In V itro In Vivo In V itro In Vivo
Saline 24.3 26.9 53.5 11.9
DFP lm g/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; Murachi, 1963;
Truhaut, 1966; Ramachandran, 1967; Hansen et al, 1968; Schuh, 1970; M artin ,
1985). DFP is capable o f combining covalently w ith most proteins (M artin ,
1985) in a general type o f a lky l phosphorylation reaction (Murachi, 1963; see
Figure 5.3.). The percentage of DFP bound to plasma proteins is high
fo llow ing in jection; a dose o f lm g/kg DFP i.v . in mice results in over 95% o f
plasma DFP being bound.
There has been l i t t le work on a ttem pting to iden tify to which protein DFP is
bound. Schuh (1970) however, has indicated that albumin is the main prote in
as the same values fo r 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 a lfen tan il and fentanyl have been shown to have high frac tiona l prote in
binding (Meuldemans et al, 1982). The results in this thesis showing around
75% binding fo r a lfen tan il are consistent w ith those obtained in the
lite ra tu re : fo r ra t, dog and man and the fraction bound is 84, 73 and 92%
respectively (Meuldermans, et al. 1982). Values obtained in this thesis fo r
fentanyl binding vary depending on the method used. Follow ing in v itro
methodology, fentanyl binding was found to be sim ilar to that obtained in ra t,
Fig. 5.3.
DFP Phosphorylation
of Protein
by an
Alkyl Phosphoyrlation
Reaction
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dog and man (Meuldermans, et al, 1982). In vivo binding yields a lower
percentage level which is sim ilar to tha t obtained by Murphy et al (1979) in
the dog. The discrepancy observed in the study presented here between jn
vivo and jn v itro binding could depend on several factors; fentanyl binding is
temperature and pH dependent (H o llt and Teschemacher, 1975) so tha t change
from pH 7.4 to pH 7.55 increases fentanyl binding from 67% to 76%. The
37°C incubation step in the in v itro binding protocol could produce the
observed increase in fentanyl binding compared to the in vivo protocol.
Though s truc tu ra lly s im ilar, these drugs do not apparently bind to the. same
sites. Fentanyl has been shown to bind red blood cells, glycoproteins and
lipoproteins, w h ilst a lfen tan il is predominantly bound to the acute phase
reactant protein a^-acid glycoprotein (a^-AGP) (Meuldemans et al, 1982). In
fac t, Meuldemans et al (1982) have calculated tha t i f a^-AGP were the only
binding protein, i t could account fo r a ll a lfen tan il binding in human
plasma but only about ha lf of fentanyl binding. Volunteer plasma samples
showed a linear relationship between a^-AGP concentration and free drug
frac tion only fo r a lfen tan il (Meuldermans et al, 1982).
5.16. E ffect of DFP on Plasma Protein Binding of Alfentanil
The presence o f DFP decreased the frac tiona l binding o f a lfen tan il to plasma
proteins. As a lfen tan il is highly lipoph ilic , the blood-brain barrier does not
l im it the entry o f free a lfen tan il into the brain. I t is reasonable to suppose,
therefore, tha t by increasing free a lfen tan il increased brain levels and
increased antinociceptive e ffe c t would fo llow . DFP did not a lte r fentanyl
plasma protein binding and so an explanation fo r the observation o f
d iffe re n tia l potentia tion by DFP o f a lfen tan il antinociception could be made
on the basis of DFP specifica lly inh ib iting the binding o f a lfen tan il to plasma
proteins.
Though Schuh (1970) suggested that DFP binds to albumin (see Section 5.12.),
this does not rule out the possib ility that DFP also binds a^-AGP because as
already mentioned, DFP reacts w ith most proteins and w ill phosphorylate
several classes o f proteins when incubated w ith plasma in v itro (Murachi,
1963).
Moreover, i t has been recently shown that albumin potentiates the binding o f
propanolol to a^-AGP (Chauvelot-Moachon e t al, 1985) so tha t i f this held
true fo r a lfen tan il, binding to a^-AGP could be a ffected by DFP
phosphorylation o f albumin.
a-^-AGP is a high a ff in ity low capacity binding protein (Piafsky, 1981;
Mackichan and Zola, 1982), so that i f DFP were to compete fo r and saturate
binding sites on the a-^-AGP molecule this would lead to raised levels o f free
a lfen tan il. Fentanyl, on the other hand, i f displaced from these sites, would
not necessarily result in increased free levels as i t 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 o f drugs as there is only a single binding site (MUller e t al, 1983).
Furthermore, the binding site m ight represent a remote hydrophobic area
w ith in 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 i t is also a hydrophobic region where the DFP molecule
would tend to go due to its lipoph ilic nature (Karczmar, 1967). There is a
good corre lation between displacing potency against ^ H -im ipram ine from
a^-AGP and lipoph ilic parameters for a varie ty o f compounds (MUller et a l,
1983).
5.17. Consequences of Alfentanil Displacement from Plasma Protein
Binding Sites
As the e ffe c t o f a drug appears to be more closely related to the free
unbound levels rather than the to ta l concentration (Lima et al, 1981) the
question arises as to what the consequences o f the displacement o f bound
a lfen tan il by DFP.
The degree of prote in binding o f a lfen tan il lim its its entry in to the brain.
This is re flected in the brainrplasma ratios which average about 0.6 at
400ug/kg (see Table 4.2.). L ite ra tu re values measuring unchanged a lfen tan il
ra ther than to ta l rad io ac tiv ity show braintplasma ratios averaging around 0.13
in the ra t fo r 10 minutes fo llow ing i.v. adm in istration (Michaels et a l, 1981).
I f binding to plasma proteins lim its a lfen tan il entry in to the brain,
displacement by DFP would tend to cause a proportional increase in entry o f
a lfen tan il. In this situation, displacement o f a lfen tan il would not a lte r the
average free drug concentration, though there would be a transient rise in
free drug concentration im m ediate ly fo llow ing displacement (Mackichan,
1984). This is borne out in the data presented in Table 5.1. fo llow ing in vivo
binding as free drug concentrations were l i t t le changed. The jn v itro binding
data shows tha t DFP increases free a lfen tan il concentrations. In vivo this
free a lfen tan il is able to red istribu te in to other tissues including the brain, so
tha t the actual steady state concentration is not altered jn vivo fo llow ing
DFP whilst the average to ta l drug concentration decreases.
5.18. Protein Binding and Relationship to Variation in Alfentanil Potency
As described in Section 2.27. in it ia l studies w ith a lfen tan il showed AD^g
values d iffe ren t from tha t obtained in experiments some months la te r. One
explanation fo r this anomaly was that there is a varia tion in circannual
sens itiv ity to a lfen tan il, however, though this may be true, the data from the
binding experiments which show the relevance o f a lfen tan il binding to its
potency, introduces the possibility tha t varia tion in binding could markedly
a ffe c t potency. If, fo r example, the batch of mice in the in it ia l experiments
were carried out had a low level v ira l in fection, this could greatly increase
a^-AGP levels and hence binding capacity. This could e ffe c tive ly increase
the true AD^-g value.
CHAPTER 6
GENERAL DISCUSSION
6 GENERAL DISCUSSION
In the foregoing chapters an account has been given o f the potentia tion of
a lfen tan il, but not morphine or fentanyl antinociception by DFP pre-trea tm ent in
mice. This e ffe c t was not reversed by drugs used to tre a t DFP poisoning. In
addition, DFP has d iffe re n tia l e ffec ts on opioid-induced running behaviour, w ith
the most marked e ffe c t observed in a lfen tan il-trea ted mice. Further
experiments identified tha t DFP enhanced the entry o f a lfen tan il in to the brain,
w h ils t the d istribu tion o f the other opioids was unaffected. This appeared to be
due to a displacement o f a lfen tan il from plasma protein binding sites, thereby
allowing more drug to enter the centra l nervous system.
The d iffe re n tia l potentia tion o f a lfen tan il may w e ll be dependent on the
observation tha t this opioid p rim arily binds to a^-AGP w hils t fentanyl binds
more than one protein (Meuldermans et al, 1982). Morphine's low frac tio na l
binding means tha t displacement from these sites would not greatly increase free
drugs.
There are im portant im plications to the findings presented here, demonstrating
functiona l increase in antinociceptive potency o f a lfen tan il fo llow ing
displacement from its plasma protein binding sites. In these studies, the data
shows that in mice, 75% of a lfen tan il is bound to plasma protein, w h ils t in
humans the figure is 92% bound (Meuldemans et al, 1982). The higher the
frac tiona l binding the greater the percentage increase in free drug fo llow ing
displacement, thus in humans, potentia tion o f a lfen tan il action could be even
greater than that observed in mice.
A lfe n ta n il binding may vary in patients i f they are not receiving any drug
therapy, as the normal levels o f a^-AGP varies in normal healthy volunteers
from 55 to 140mg/100ml w ith a mean of 66mg/100ml (Piafsky et al, 1978).
Increased levels are probably due to subclin ical or m inor in fections. a^-AGP
levels are also increased as a result o f the physiological stress associated w ith
in flam m ation, cancer (Piafsky, 1980) and surgery (Fremsted e t al, 1978).
This in form ation suggests tha t a lfen tan il potency may be sensitive to
pharmacological and physiological variables, which may be im portan t c lin ica lly .
I t should be borne in mind, however, tha t i t is an assumption to extrapolate the
demonstrated in teraction in an experimental animal to an in te raction in humans.
An assessment o f the potentia l in teractions of a lfen tan il w ith other drugs should
therefore be carried out w ith other drugs and using human plasma.
The property o f the potentia ting a c tiv ity o f DFP does not appear to be
dependent on its anticholinesterase a c tiv ity per se, as the carbamate
anticholinesterases neostigmine and pyridostigm ine, did not a lte r a lfen tan il
antinociception. A plausible in te rpre ta tion o f th is is tha t DFP may produce a
stable phosphorylated plasma protein (which competes fo r a lfen tan il binding) and
this may hold true fo r other organophosphorous compounds including
organophosphates. Given tha t in DFP treated mice, morphine antinociception is
altered fo llow ing atropine and pralidoxime adm in istration, fentanyl appears to be
the opioid of choice and the least like ly to cause in te raction problems w ith
organophosphates or drugs used in the trea tm ent o f anticholinesterase poisoning.
This study has shown the existance and probable mechanisms of DFP potentia tion
o f a lfen tan il antinociception. This is of importance not only as an example o f
the relevance of plasma protein binding on drug e ffe c t, but also in term s of the
possible im plications fo r the c lin ica l use o f a lfen tan il fo llow ing adm in istra tion o f
other drugs. There is clearly scope fo r fu rthe r research in this area.
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aj-PENDIX a
Equation for Unpaired Student’s t-test
t = x
(SEM^2 + (SEM2)2
APPENDIX B
C om puter P rogram fo r A n tin o c ice p tive D ata in Mouse H o t-P la te Test
A set o f data has th ree components, the mean response tim e , the va ria tio n o f
response tim e about the mean, and the e rro r d is tr ib u tio n . For th is data, i t
was found th a t a W eibu ll d is tr ib u tio n could successfu lly be applied to th is la s t
com ponent. The data may also be lin e a rlize d , such th a t log (mean response
tim e ) is linea r in dose. S tra ig h t- lin e regression is ca lcu la ted by a m ethod
s im ila r to the usual least-squares method, using a general procedure ca lled
m axim um like lihood es tim a tion . The sum o f squares is rep laced by the
like lihood fun c tio n , which represents the p ro b a b ility o f obta in ing data under
the model. This model enables p red ic tions to be made on the percentage o f
response tim es th a t exceed 60 seconds. This model also a llows fo r the
inclusion o f zero-dose (ie, saline) data. A fu lle r descrip tion o f th is m odel is
given by K itchen and C row der (1985).
A N A lG fM SCREEN i;A T A FEB S 3 ) : CENSORED RESPONSE H U E S UNDER DRUGS
ANA < O LS U . • >: REFN DATA <FF.R 6 3 M CENSORED RESPONSE T IM E S ..»NOEP DRUGS REAL XS ( .0 >. 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 C R T0 12DATA IX / 6 0 0 . . I W / T O C / I . D - 1 0 /READ C.AfA E T C .READ; 1 . * > I RF>. Iw R , I CHECK
10 C ALL I k R T O l( IR D . IC H E C K )Z F ( N . lE . O ) STOP
20 UR IT E M i PC-1)R E A D H . * ) NOI E <N O .L E . 0 ) GOTO tOMLE F I T BY T R IA L AND ERRORN P -N P R O +2I F ( N P . 0 T . I 5 > STOPno so j«i , isRl.U< 1 , J ) = 0 .
3 0 D L U (2 .* J ) = 1 0 0 .B L U M . N P > * . 01 -C A L L F M IN !C R T 0 1 2 .N P .W 1 ,L L K D » W 2 .W 3 .W 3 ,IW .B L U )
r PARAMETER C O VARIAN C E M ATR IXA - W K 1 )DO 5 0 J s l .N D R G
5 0 B < J ) * W 1 ( J * l >G AM =W 1(NP)W R IT E (1 .9 0 © )R EAD ( I . * > IC O VIF < IC O V . N E .1 ) GOTO 7 0W R IT E ( IW R .9 0 2 ) (W 1 ! J ) , J = 1 »N P ) , LL K DC A L L C R T 0 0 2 ( A .B » G A M .L L K D .W 1 . W 3 » IW .2 . 1 )DO 5 5 J = 1 ,N PC A LL M X S W P 0 (W 3 .IW ■NP * N P »U »P I V » T G L )
5 5 W RITE< 1 ,9 0 .3 ) J . P I V DO 6 0 J - l . N P U 1 ( J ) * - W 3 ! J , J )IF ( W 1 ( J ) . G T . O . ) W 1 ( J )= D S Q R T ( W 1 (J ) )
6 0 C O N TIN UEW R I T E M . 9 0 7 ) (W1 ( J ) . J « I , N P )W R IT E ! IW R .9 0 8 )DO 6 5 J * l . N P
6 5 W R IT E !IW R ,S 0 2 T <U 3 < J . K ) . K = l . U )W R IT E M W R .9 0 6 )DO 6 7 J = 1 • NDRG E D 1 - . 6 9 3 1 5 / B ! J )E D 2 - ( 3 . 4 0 1 2 0 - A ) / B ( J )S D I» E D 1 * W 1 ( J + l ) / B ( J )S 0 2 — < W 3( 1 . 1 ) * 2 . «E D 2« W 3( 1 . J + l ) + E 0 2 *E D 2 *W 3 ( J + l . J + 1 ) ) t F ( S D 2 . G T . O . ) S D 2 -S Q R T ( S D 2 ) /B ( J >
6 7 W R IT E !IW R ,8 0 2 ) E D I* S D 1 . E D 2 .S D 2 C R E S ID U A LS
7 0 W R IT E !1 . 9 0 9 )R E A D M .* ) IR E SI F ! I R E S . E Q . 1 ) C A L L C R T 0 2 2 !A .B .G A M .W Y ,IW R )GOTO 2 0
8 0 1 FOR M AT( 2 0 1 4 )8 0 2 FO R M A T (1 P 8 G 10 . 3 )9 0 ? F O R M A T !/IO H P A R A M E T E R S /!1 P 6 G 1 2 .5 ) )9 0 3 F O R M A T (5 H P IV 0 T . 1 2 . IP G 1 0 . 3 )9 0 4 FORM AT( /4 5 H F I T : ORDER OF REGN ( l- L IN E A R .2 -Q U A D R A T IC .E T C ) )9 0 6 F O R M A T (/4 H E D 5 0 )9 0 7 F O R M A T !/1 0 H S T D E R R O R S / !1 P 8 0 I0 .3 ) >9 0 3 F O R M A T !/27H P A R A M E TE R C O VA RIAN C E M A T R IX )9 0 9 F O R M A T (/9 H R E S ID U A L S )
ENDSU BR O U TIN E IK R T 0 1 ( IR D .IW R )
C A N A L G E S IC SCREEN CENSORED RESPONSE T IM E DATA (F E B 8 3 )C X HAS COLS (DRUG NO. . D O SE. RESPONSE T IM E )
IN TEG E R N O ( 2 0 )R EAL D S ( 2 0 )C O M M O N /R TD A T /Y ( 6 0 0 ) > N .Y M S . YCNSR, X ( 6 0 0 . 1 0 ) . NDRG YUS— 9 9 .Y C N S R -6 0 .D 0 S D IV - 1 0 0 .N * 0R E A D !IR D ,8 0 3 ) NDRGIF ( N D R G .L E .0 ) RETURNDO 3 0 J D R G - l . NDRGR EAD ( IR D .8 0 4 ) NDSREAD < IR D . * ) ( D S ( J ) . J - 1 . N D S)R E A D M R O .* ) ( N D ( .J ) , J * 1 .N D S )IF ! I W R .G T .O ) W R IT E !IW R ,8 0 2 ) ( D S ! J ) , J « 1 . N D S )1 1-N-* 1DO 2 0 J - l . N D S N J - N D ( J )DO 2 0 I - l . N J N -N MX ! N . 1 >*UDRG
20 X ( N ,? ) - n S ! . J ) /D O S D IVR E A D !IR D .# ) ! X ( I * 3 ) . I - I 1 * N >
30 C i-in T IN IIFVC N SR -ALO G ( YCNSR>DO 4 0 1 = 1 ,N Y I = X < 1 .3 )I F ! V I . G T . O. ) Y ( I ) = 01 .00 ( Y I 5
4 0 CONTINUEI F ! I W R . L E . 0 ) RETURN W R IT E !IW R ,9 0 1 >DO 6 0 1 - 1 . N
6 0 W R IT F !IW R .8 0 ? > r X ( I ,..»> . J = 1 ..? ) , Y ! I >RETURN
8 0 2 FORM AT( 1 P 8 G 1 0 . 3■8 0 3 F O R M A T !5 X .1 3 )8 0 4 F O R M A T (J5 X . 13.)9 0 ) F O R M A T !/1 0 H 0 H F C K D ATA)
ENDSU BR O U TIN E C R T0 12 < N P .P A R ,F N , G R ,H . IH , I G R . IW R )
T P A R !. ) *A » B ( D . . . . , B (N D R G ). GAMR EAL B ( 1 5 )DOUBLE P R E C IS IO N F N ,P A R !N P ) . G R (N P ) . H ( I H . I H )C O M M O N /R TD A T /Y ! 6 0 0 ) .N .Y M S ,Y C N S R ,X ( 6 0 0 ,1 0 ) .NDRG A = P A R (1)DO 2 0 J * I ,N D R G
20 B ! J ) * P A R ( J + l )0A M =PAR !N P1CAL t. C R TO O ruA . B .G A M .F N .G R .H . IH , I OR, IW R)RETJIRN END
t IN S E R T IK 'CRTB * IN S E R T FMTNP
SUBROUTINE CRT002 <A,B.GAM.LLKD, Gl.»HL , IH, IGR, IWR)
SUBROUT IN E C R T 0 0 2 !A .6 , G A M .L L K D ,G L »H L , IH , IG R , IW R )M IN U S L O G -L IK D AND D E R IV S TOR L O G -W E IB U LL CENSORED RESPONSE T IM E S
f. M U /S IG MODEL S U P P L IE D BY S /R T N E C R T0 03REAL MU, B ! 1 5 )» GP ! 2 ) . GC ! 2 ) » H P ! 2» 2 ) » H C ! 2» 2 ) . GM! J 3 ) • O S ! 1 5 )»
1 H M <1 5 . 1 5 ) , H S ( 1 5 .1 5 )DOUBLE P R E C IS IO N L L K D ,G L ( I H ) , H L ( I H , I H )C 0 M M O N /R T D A T /Y I6 O 0 ) ,N .Y M S .Y C N S R ,X I6 0 0 ,1 0 ) .NDRG L L K D * 0 .N P -N D R G + 2 I F ( N P , G T . 1 5 ) STOP IF { IG R . I .E . 0 > GOTO 2 0 DO 15 J * 1 , NP G I „ ! J > * 0 .I F ( I G R . L E . l ) GOTO 15 DO 1 0 K = 1 ,N P
1 0 H L ( J . K ) = 0 .15 C O N TIN UE 2 0 DO 4 0 1 *1 ,N
Y I * Y ! I )IF ! Y I , F J 3 . YMS) GOTO 4 0 I D * I F I X ! X ! T » l ) + . 5 >D S * X ( 1 , 2 )C A L L C R T 0 0 3 ( A , B . NDRG. GAM, ID .D S .M U .S IG .G M .G S .H M .H S ,1 5 . IG R )IF IY I . L T . Y C N S R ) C A L L W B L L 0 1 < Y I, M U ,S IG ,P D .C O ,G P .G C ,H P .H C .IG R )IF IY I .G E .Y C N S R ) C A L L W B L L 0 1 < Y I, M U ,S IG .C D .P D ,G C ,G P .H C .H P ,IG R )I.L K D *L L K D --P DI F ( I G R . L E . O ) GOTO 4 0DO 3 5 J * 1 .N PI F ! I G R . L E . 1 ) GOTO 3 5DO 3 0 K * I , JH t L t , K ) * H L ! J . K ) - ! H P ! l . l ) # G M ! J ) + H P < t » 2 ) # G S ! J ) >*G M !K )-G P < 1 ) * H H < J ,K ) -
1 ( H P ( 1 , 2 ) « G M ! J ) + H P ! 2 . 2 ) * G S ! J ) > * G S ! K ) - G P ! 2 ) # H S ! J . K >3 0 H L ( K . J ) * H L ( J . K )3 5 G L ! J ) *G L .! J ) - G P ! t ) « G M !U ) - G P !2 ) * G S < J )4 0 C O N TIN U E
IF ( IW R .L E . O ) RETURN W R ITE < IWR * 9 0 1 )W R IT E ! IW R .8 0 3 ) LLK DW R IT E ! IW R .8 0 2 ) ! G L ! J ) » J * 1 . N P )DO 5 0 J * 1 * NP
5 0 W R IT E !T W R .8 0 2 ) ! H L ! J . K ) . K « 1 . J>RETURN
8 0 1 F O R M A T !2 0 I4 )8 0 2 F O R M A T !1P 8G 10 . 3 )8 0 3 F O R M A T !IP G 1 2 . 5 )9 0 1 F O R M A T !/2 5 H M IN U S L O G -L IK D AND D E R IV S )9 0 6 F O R M A T !2 I 3 .1 P 6 G 1 2 .5 / ! 6 X ,1 P 6 G 1 2 .5 ) )
ENDS U BR O U TIN E C R T 0 0 3 ! A , B »N B • GAM, ID ,D S ,M U .S IG ,G M ,O S .H M .H S ,IH . IG R )
C CENSORED RESPONSE T IM E SC REGN MODEL TYP E M U - A + B ! ID ) * D S !FO R DRUG ID ,D O S E D S ) , SIG *O AM »M UC RETURN M U ,S IG AND D E R IV S WRT < A ,B ( . )» G A M )
R EAL 8 ! N B ) . M U . G M ! I H ) , G S ( I H ) , H M ! I H , I H ) , H S ( I H , I H )B D * B ! ID ) « D SM U *A+BDS IG -G A M *M UI F ! I G R . L E . 0 ) RETURNN P »N B -f2DO 2 0 J - 1 , NPC M ! J ) * 0 .
2 0 G S ! . J ) * 0 .G M (1 ) * 1 .G M !ID + 1 ) * D S O S ( 1 ) *GAM G S (ID + 1 ) *G A M * D S O S ! N P ) *M UI F ! I G R . L E . 1> RETURN HS i 1 , N P ) * 1 .H S 1 N P ,1 ) * I .H S ! ID + 1 »NP)«=DS H S 1 N P ,ID + 1 ) * D S RETURN ENDS U D R O U TIN E C R T 0 2 2 !A ,B ,G A M .O R » IW R )
C 1 0 G -W E IB U L L CENSORED RESPONSE T IM E S ! E X P O N E N TIA L R E S ID U A LSC O R ! 1 ) , , . . . O R !N ) CORRESP TO NON-CENSORED Y 'S
IN TEG E R NW! 6 0 0 )R E A L M U , B ( 1 5 ) » O R !6 0 0 ) ,G M !1 > . O S !1 ) ,H M !1 , 1 ) , H S !1 » 1 )C O M M O N /R TD A T /Y ! 6 0 0 ) . N ,Y M S , YCNSR. X ! 6 0 0 , 1 0 ) , NDRG D ATA E G /O .5 7 7 2 1 5 6 6 4 ? / . P 6 / 1 .2 8 2 5 4 9 8 /I F ! IW R .G T .0 ) W R IT E !IW R ,9 0 1 ) N ,N D R G .Y M S .Y C N S RDO 2 0 1 * 1 . NI D * I F I X ( X ! t . t ) + . 5 >D S - X ! 1 , 2 )C A L L C R T 0 0 3 !A .B .N D R G ,G A M . ID ,D S .M U ,S IG ,C M .O S .H M ,H S ,1 . 0 )Y D * P 6 « ! Y ! I ) - M U ) /S I G O R !I> * E X P !Y O - E G )I F ! I W R . G T . 0 ) W R IT E !IW R .8 0 3 ) I D , D S . M U . Y ! I ) . Y D ,O R ! I )
7.0 C O N TIN UE RETURN
8 0 3 FORM AT! I 3 . 9 F 8 . 4 )9 0 1 FO R M A T !/ ? ! H E X P O N EN TIAL R E S ID U A L S .2 1 4 , F 6 . 1 . F 7 . 4 )
ENDSU BR O U TIN E W Bt.LO l ( Y ,M U . S IG ,P P . CD, G P ,G C , H P . HC, IG R )
C LO G -W E ID U L L D IS T N : P D -LO G D E N S IT Y F N , C D *l O G !1 -D IS T N F N ) . ANDr D E R IV S WRT M U ,S IG
R FAL M U ,G P !2 ) , G C ! 2 ) . H P !2 , 2 ) . H C ( 2 . 2 )DATA E G /O .5 7 7 2 1 5 6 6 4 9 / . P 6 / 1 . 2 8 2 5 4 9 8 /Y D = P 6 * ! Y - M U ) /S IG E Y *E X P (Y P -E G )P D *A LO G ! P 6 /S I G ) + Y D -E G -E Y C D *-E YI F ! IG R . L E . 0 ) RETltRNG P !1 ) * - P 6 * ! 1 . - E Y ) / S I G0 P ! 2 > * - ! ! . + Y 0 * ! 1 . - E Y > ) /S IGG C !1 > *P 6 « E Y /S IGG C !2 > * Y D « E Y /S IGI F ! I G R . L E . t ) RETURNS 0 2 * S IG * S IGH P ! 1 , I> * - P 6 * P 6 * E Y / S 0 2H P ! I , 2 ) * P 6 + ! 1 . - E Y -Y P # E Y ) / $G 2H P !2 » 1 ) « H P !1 , 2 )H P ! 2 , 2 ) * ! 1 . + ! Y D + Y D ) * ! 1 . —E Y ) -Y D * Y D * E Y ) /S G 2H C ( 1 . 1 )» - P 6 * P 6 * E Y /S G 2H C !1 . 2 ) = - P 6 * ( 1 . + Y D )+ E Y /S G 2M C ! 2 , 1 ) * H C ! J , 2 )H C !2 » 2 ) * - ! 2 . + Y D > » F Y *Y D /S G 2RETURNEND
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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 . 8 040 REH T H I S PR0GRAHHE CALCULATES THE C O E F F I C IE N T S OF THE GIVEN DATA5 0 REM B E S T - F I T T I N G THIRD DEGREE P0LYN0HIAL60 REH E=A0+A1*R+A2*R*2+A3*R*3 BY THE HETH0D OF LEAST SQUARES70 DEFINE FILE #1= 'SCOTT",ASC,12071 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 t i l . 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 ti l ,N,A 110 FOR H=1 TO 10 120 S(H)=0 130 NEXT H 140 FOR 1=1 TO N 150 READ t i 1 , 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 K215 PRINT 'R = / , R ( I ) / E = ' , IN T<10A4 * C / A + . 5 ) / 1 0 0220 NEXT I230 D = S ( 2 ) - S ( 1 ) *2 /N240 E = S ( 3 ) - S ( 1 ) * S ( 2 ) / N250 F = S ( 4 ) - S ( 1) * S ( 3 ) / N260 G = S ( 8 ) - S ( 1) * S ( 7 ) / N270 A=EA2 - ( S ( 4 ) - S ( 2 ) " 2 / N ) * D280 B = ( S ( 5 ) - S < 2 ) * S ( 3 ) / N ) * D - E * F290 C = (S < ? ) -S (2 ) *S < 7 ) /N ) *D -E *G295 0 = ( A * ( ( S ( 1 0 ) - S ( 3 > * S ( 7 ) / N ) * D - F * G ) + B * C )300 A 3 = Q / ( A * ( ( S ( 6 ) - S ( 3 ) A2 / N ) * D - F * 2 ) + B "2 )310 A2=(B*A3-C) /A320 A 1 = (G -A 2 * t -A 3 * F ) /D330 A 0 = ( S ( 7 ) - A 1 * S ( 1 ) - A 2 * S ( 2 ) - A 3 * S < 3 ) ) / N335 PRINT340 PRINT 'AO = ' , INT<10 * A 0 + . 5 ) / 1 0 350 PRINT ' A l = ' , I N T ( 1 0 * A 1 + . 5)710 360 PRINT 'A2 = ' , I N T ( 1 0 * A 2 + . 5 ) / 1 0370 PRINT 'A3 = ' , IN T (1 0 * A 3 + . 5 ) / 1 0371 PRINT3.72 FOR i= 1 TO Ni 7 4 Y = A 3 * R ( I ) A3 + A 2 * R ( I ) ‘ 2+A1*R( I ) +A0 376 PRINT 'Y = ' , I N T ( 1 0 " 2 * Y + . 5 ) / 1 0 0 378 NEXT I 400 END
APPENDIX D ( i i )
Quench curve obtained from d.p.m. package on RackBeta 1215 * ’’
Ou6+JL+\ C lEFF1Z RATIO
8947.0 25.06 1.3128019.0 22.46 1.1877492.0 20.99 1.1464523.0 18.27 1.0505985.0 16.76 1.0094275.0 11.97 .8482531.0 7.09 .6721774.0 4.97 .5251743.0 4.88 .5911070.0 3.00 .460
PLOT QUENCH CURVES <R> ->R
IUSY CALCULATING
ISOTOPE 1. UINDOU 1
EFFZ25.0*4 *|
I **»I »*I *
22.50+I *I 0*I «*
2 0 . 00+ *I « +I «*I *0
17.50+ «
I II •I *«
15. 00+ «I ♦«I *I **
12.50+ **
II *I *»
10.00+ *•I •I *•I *
7.50+ ♦*I *0I ♦I +*
3.00+ •#«*«•*
I ••I *10II------♦--------------♦-----------♦-------------♦-------------♦................♦................♦-------------♦............................0
.5 0 0 .A 00 .7 0 0 .0 0 0 .9 0 0 1.000 ' 1.100 1.200 1.300