Dr Samir S. Ayoub
New and novel uses for paracetamol
Studies on the mechanism of the paracetamol-induced hypothermia and clinical Relevance
Paracetamol• Analgesic and anti-fever drug with weak anti-inflammatory 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 NSAIDsNSAIDs work by inhibition of prostaglandin biosynthesis
by inhibition of COX activity
Inhibition(%)
Indomethacin
Aspirin
Salicylic acid
Log concentration (µg/ml) (Vane, Nature 1971)
100
80
60
40
20
0
0.1 1.0 10 100 1000
Sir John Vane, Nobel laureate 1981
Inhibition of COX activity by paracetamol is tissue selective - in vitro assay
ID50 µgml-1
Indomethacin 0.06 1.3Sodium aspirin 6.6 11.04 - Acetamidophenol 100.0 14.0
(Flower and Vane, Nature1972)
Synthetase from: Dog spleen Rabbit brain Dog brain
12.5
Professor Roderick J Flower, FRS
Prostaglandins synthesis
Prostaglandins and NSAIDs
Prostaglandins NSAIDs
Pain Analgesic
Fever Antipyretic
Inflammation Anti-inflammatory
GI protection GI damage
Hypothesis
Paracetamol acts by inhibition of COX activity in central tissues
Paracetamol is centrally acting
• 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)
• The antipyretic effect of paracetamol was accompanied by potent reduction in PGE2 levels in the cerebrospinal fluid (Feldberg et al 1972)
COX-2
7
50
15
20
0.35
1.3
0.34
0.84
IC50 values (µg/ml) of NSAIDs on COX-2 or COX-1 activity in intact cells
COX-1
0.04
0.3
1
2.7
0.5
2.2
1.2
63
RatioNSAID
Tolmetin
Aspirin
Ibuprofen
Paracetamol (IC30)
Diclofenac
Naproxen
Celecoxib
Rofecoxib
COX-2COX-1
175
166
15
7.4
0.7
0.6
0.3
0.013
Mitchell J et al. (1993) 90(24):11693-7
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)
The Discovery of Cyclooxygenase-3
Splice variant of COX-1 Most abundantly expressed in cerebral
cortex Selectively inhibited by paracetamol
Professor Daniel Simmons, Brigham Young University,. Utah, USA
IC50, (M)
Drug COX-1 COX-2 COX-3
Paracetamol >1000 >1000 460Aminopyrine* >1000 >1000 688Antipyrine >1000 >1000 863Dipyrone 350 >1000 52Phenacetin >1000 >1000 102Aspirin 10 >1000 3.1Diclofenac 0.035 0.041 0.008Ibuprofen 2.4 5.7 0.24Indomethacin 0.010 0.66 0.016Caffeine >1000 >1000 >1000Thalidomide >1000 >1000 >1000
All assays were carried out in the presence of 30M arachidonic acid.*4-dimethylaminoantipyrine.
Chandrasekharan et al, 2002
Is the thermoregulatory function of paracetamol mediated through inhibition of COX-3?
35.5
36.0
36.5
37.0
37.5
38.0
38.5
39.0B
ody
Tem
p (o
C)
0 1 2 3 4 5
0
100
200
Time (h)
PG
E2 (pg
/well)
The reduction of basal body temperature with 300mg/kg paracetamol correlates with reduction of brain PGE2 levels
Paracetamol hypothermia is related brain PGE2 levels
COX-3 (65KDa)
COX-2 (72KDa)
COX-1 (72KDa)
Cerebral cortex Mid brain Brain stem Cerebellum
COX-3 is constitutively expressed in brain tissues of mice
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
Paracetamol hypothermia is reduced in COX-1 knockout mice and is retained in COX-2 knockout mice
COX-2 gene knockout mice
The effect of 300mg/kg paracetamol on brain PGE2 levels after 1hr
Reduction in the hypothermic effect of paracetamol in COX-1-/- mice is linked to loss of the effect of paracetamol on brain PGE2
COX-1 gene knockout mice COX-2 gene knockout mice
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
Mallet et al, 2008
CB1 receptor
TRPV1 channel
Fatty acid amido hydrolase enzyme (FAAH)
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
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”
Clinical relevance: therapeutic hypothermia
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.
0
10
20
30
40
50
60
Benson(1959)
Bernard(1997)
Zeiner(2000)
Bernard(2002)
HACA(2002)
Oddo(2006)
Normothermia Hypothermia
* * * * * *
Drug-induced hypothermia: fast onset of action
Combinational hypothermia; paracetamol and cannabinoid agonist.
Intravenous formulation
Out-of-hospital use
Induction of hypothermia within minutes
“Inside-out” hypothermia
Easily maintained hypothermia
Cheap
The team
Prof. David Baker (Professor of Neuroimmunology, ICMS)
Prof. Gavin Giovannoni (Consultant Neurologist, Royal London)
Dr Rupert Pearse (Critical Care Consultant, Royal London)
Dr Samir Ayoub (Pharmacology lecturer, UEL)