New and novel uses for paracetamol
Studies on the mechanism of the paracetamolinduced hypothermia and clinical Relevance
Dr Samir S. Ayoub
Paracetamol
• Analgesic and anti-fever drug with weak antiinflammatory effects
• Used clinically for over a century
• Mechanism of pharmacological action still not fully
established
• Cyclooxygenase (COX) enzymes are involved in
production of prostaglandins, which mediate
inflammation, pain and fever
• Suggested to work by inhibition of COX enzymes
activity in the central nervous system (CNS)
Mode of action of NSAIDs
NSAIDs work by inhibition of prostaglandin biosynthesis
by inhibition of COX activity
Inhibition 100
(%)
80
Indomethacin
Aspirin
60
Salicylic acid
40
20
0
0.1
1.0
10
Log concentration (µg/ml)
Sir John Vane, Nobel laureate 1981
100
1000
(Vane, Nature 1971)
Inhibition of COX activity by paracetamol is
tissue selective - in vitro assay
ID50 µgml-1
Synthetase from:
Indomethacin
Sodium aspirin
4 - Acetamidophenol
Dog spleen
0.06
6.6
100.0
Rabbit brain Dog brain
1.3
11.0
14.0
12.5
(Flower and Vane, Nature1972)
Professor Roderick J Flower, FRS
Prostaglandins synthesis
Prostaglandins and NSAIDs
Prostaglandins
Pain
Fever
Inflammation
GI protection
NSAIDs
Analgesic
Antipyretic
Anti-inflammatory
GI damage
Hypothesis
Paracetamol acts by inhibition of
COX activity in central tissues
Paracetamol is centrally acting
• The antipyretic effect of paracetamol was accompanied by
potent reduction in PGE2 levels in the cerebrospinal fluid
(Feldberg et al 1972)
• Reduced spinal cord released of PGE2 in response to
capsaicin (Malmberg & Yaksh, 1994)
• Reduced spinal cord released of PGE2 in the formalin test, but
not the urinary excretion of PGE2, PGF2 & 6-keto-PGF1 (Muth
Selbach et al 1999)
• Reduced the pain threshold in the flexion reflex to
transcutaneous electrical stimulation in man, which is a
model of central nociception (Piletta, et al, 1991)
IC50 values (µg/ml) of NSAIDs on
COX-2 or COX-1 activity in intact cells
Ratio COX-2
COX-1
NSAID
COX-2
COX-1
Tolmetin
7
0.04
175
Aspirin
50
0.3
166
Ibuprofen
15
1
15
Paracetamol (IC30)
20
2.7
7.4
Diclofenac
0.35
0.5
0.7
Naproxen
1.3
2.2
0.6
Celecoxib
0.34
1.2
0.3
Rofecoxib
0.84
63
0.013
Mitchell J et al. (1993) 90(24):11693-7
The Discovery of Cyclooxygenase-3
COX-3, a cyclooxygenase-1 variant inhibited by
acetaminophen and other analgesic/antipyretic
drugs: Cloning, structure, and expression
N.V. Chandrasekharan, Hu Dai, K. Lamar Turepu Roos, Nathan K.Evanson, Joshua Tomsik, Terry S. Elton,
and Daniel L.Simmons*
Department of Chemistry and Biochemistry, E280 Benson Science Building, Bringham Young University, Provo, UT 84602
Communicated by John Vane, William Harvey Foundation, London, United Kingdom, August 5,2002 (received for review April 17, 2002)
 Splice variant of COX-1
 Most abundantly expressed in cerebral
cortex
 Selectively inhibited by paracetamol
Professor Daniel Simmons, Brigham Young University,. Utah, USA
IC 50, (mM)
Drug
Paracetamol
Aminopyrine*
Antipyrine
Dipyrone
Phenacetin
Aspirin
Diclofenac
Ibuprofen
Indomethacin
Caffeine
Thalidomide
COX-1
>1000
>1000
>1000
350
>1000
10
0.035
2.4
0.010
>1000
>1000
COX-2
>1000
>1000
>1000
>1000
>1000
>1000
0.041
5.7
0.66
>1000
>1000
COX-3
460
688
863
52
102
3.1
0.008
0.24
0.016
>1000
>1000
All assays were carried out in the presence of 30mM arachidonic acid.
*4-dimethylaminoantipyrine.
Chandrasekharan et al, 2002
Is the thermoregulatory function of paracetamol mediated
through inhibition of COX-3?
The reduction of basal body temperature with 300mg/kg
paracetamol correlates with reduction of brain PGE2 levels
39.0
200
o
38.0
37.5
37.0
100
36.5
36.0
35.5
0
0
1
2
3
4
5
Time (h)
Paracetamol hypothermia is related brain PGE2 levels
PGE2 (pg/well)
Body Temp ( C)
38.5
COX-3 is constitutively expressed in brain
tissues of mice
Cerebral cortex Mid brain
Brain stem Cerebellum
COX-1 (72KDa)
COX-2 (72KDa)
COX-3 (65KDa)
Time-profile to the effect of 300mg/kg paracetamol on the basal
body temperature in COX-1 & COX-2 genes knock-out mice
COX-1 gene knockout mice
COX-2 gene knockout mice
Paracetamol hypothermia is reduced in COX-1 knockout
mice and is retained in COX-2 knockout mice
The effect of 300mg/kg paracetamol on brain PGE2 levels after
1hr
COX-1 gene knockout mice
COX-2 gene knockout mice
Reduction in the hypothermic effect of paracetamol in
COX-1-/- mice is linked to loss of the effect of paracetamol
on brain PGE
The effect of SC560 & Celecoxib alone and in combination with
paracetamol on the basal body temperature of mice
Inhibition of COX-1 and COX-2 does not cause hypothermia
1. Conclusions
Reduction of basal body temperature by paracetamol confirms
similar findings in humans (Dippel et al, 2003; Tittelboom et al, 1988;
Denes et al, 2002)
The dose-dependency & time-profile of the reduction of
temperature confirms that the effect is related to paracetamol.
Correlation of reduction of body temperature and brain PGE2
confirms that paracetamol targets a central COX enzyme.
The reduction in paracetamol-induced hypothermia and brain
PGE2 in COX-1 knockout mice confirms that the likely target for
paracetamol is a COX-1 variant protein and not COX-1 as the
COX-1 selective inhibitor, SC560, had no effect on temperature.
Paracetamol is converted to N-arachidonylamine
phenolamine (AM404) in the brain by the action of
fatty acid amide hydrolase (FAAH)
Högestätt et al, 2005
The tissues involved in the conversion of
paracetamol to AM404
Fatty acid amido hydrolase
enzyme (FAAH)
CB1 receptor
TRPV1 channel
Mallet et al, 2008
AM404 has analgesic (La Rana et al., 2008 & 2006; Mitchell et al., 2007;
Costa et al., 2006)
and hypothermic actions(Rawl el., 2006)
AM404 activates the CB1 receptor-mediated endocannabinoid
and TRPV1channel systems (Guiffrida et al., 2001; De Petrocellis et al., 2000)
Activation of the brain endocannabionid system
results in hypothermia
Activation of the transient receptor potential
vanilloid-1 (TRPV1) in the brain, also results in
hypothermia
AM404 is able to activate both the endocannabinoid
and TRPV1 systems
Does AM404 mediate the paracetamol-induced
hypothermia through activation of the
endocannabinoid and TRPV1 systems??
Paracetamol-induced hypothermia and
cannabinoids
Cannabinoid-induced hypothermia and COX
Paracetamol-induced hypothermia and TRPV1
TRPV-1-induced hypothermia and COX
AM404 does not induce hypothermia
Inhibition of FAAH does not prevent the induction of
hypothermia with paracetamol
Co-administration of paracetamol and CB1 agonist
produce additive hypothermia
Conclusions
The paracetamol induced hypothermia is not dependent on
the cannabinoid and TRPV1 systems
AM404 does not mediate the paracetamol-induced
hypothermia
A COX-1 variant protein mediates the reduction of body
temperature by paracetamol in normothermic and pyretic
mice
Clinical relevance: therapeutic hypothermia
Following a stroke, cardiac arrest or neurotrauma brain ischemia
leads to significant neuronal cell death leading to long term
disability or death.
Induction of mild, sustained hypothermia is an established
method for the acute management of such patients.
Mechanism through which hypothermia protect the brain include,
reduction in brain metabolic rate, blockade of excitotoxicity
calcium antagonism, preservation of protein synthesis, a decrease
in oedema formation, modulation of the inflammatory response
and modulation of apoptotic cell death.
Most of the damage occurs within the first hour “Golden hour”
Therapeutic hypothermia: the golden hour
Therapeutic hypothermia: Current methods
Current methods used to induce therapeutic-hypothermia can not
be used in the pre-hospital setting, large in size and expensive.
*
*
*
*
*
*
Drug-induced hypothermia: fast onset of action
Combinational
agonist.
hypothermia;
paracetamol
Intravenous formulation
Out-of-hospital use
Induction of hypothermia within minutes
“Inside-out” hypothermia
Easily maintained hypothermia
Cheap
and
cannabinoid
The team
Prof. Gavin Giovannoni
(Consultant Neurologist, Royal
London)
Dr Rupert Pearse (Critical Care
Consultant, Royal London)
Prof. David Baker (Professor of
Neuroimmunology, ICMS)
Dr Samir Ayoub (Pharmacology
lecturer, UEL)