Dr Samir S. Ayoub

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)