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Dr Samir S. Ayoub

New and novel uses for paracetamol. Studies on the mechanism of the paracetamol -induced hypothermia and clinical Relevance. Dr Samir S. Ayoub. Paracetamol. Analgesic and anti-fever drug with weak anti-inflammatory effects Used clinically for over a century

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Dr Samir S. Ayoub

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  1. New and novel uses for paracetamol Studies on the mechanism of the paracetamol-induced hypothermia and clinical Relevance Dr Samir S. Ayoub

  2. 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)

  3. Mode of action of NSAIDs NSAIDs work by inhibition of prostaglandin biosynthesis by inhibition of COX activity Indomethacin Inhibition (%) 100 80 Aspirin 60 Salicylic acid 40 20 0 0.1 1.0 10 100 1000 (Vane, Nature 1971) Log concentration (µg/ml) Sir John Vane, Nobel laureate 1981

  4. Inhibition of COX activity by paracetamol is tissue selective - in vitro assay ID50 µgml-1 Synthetase from: Dog spleen Rabbit brain Dog brain Indomethacin 0.06 1.3 Sodium aspirin 6.6 11.0 12.5 4 - Acetamidophenol 100.0 14. 0 (Flower and Vane, Nature1972) Professor Roderick J Flower, FRS

  5. Prostaglandins synthesis

  6. Prostaglandins and NSAIDs ProstaglandinsNSAIDs PainAnalgesic FeverAntipyretic InflammationAnti-inflammatory GI protection GI damage

  7. Hypothesis Paracetamol acts by inhibition of COX activity in central tissues

  8. 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(MuthSelbachet 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)

  9. IC50 values (µg/ml) of NSAIDs on COX-2 or COX-1 activity in intact cells COX-2COX-1 NSAID Tolmetin Aspirin Ibuprofen Paracetamol (IC30) Diclofenac Naproxen Celecoxib Rofecoxib COX-2 7 50 15 20 0.35 1.3 0.34 0.84 COX-1 0.04 0.3 1 2.7 0.5 2.2 1.2 63 Ratio 175 166 15 7.4 0.7 0.6 0.3 0.013 Mitchell J et al. (1993) 90(24):11693-7

  10. 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

  11. IC , (mM) 50 Drug COX-1 COX-2 COX-3 Paracetamol >1000 >1000 460 Aminopyrine* >1000 >1000 688 Antipyrine >1000 >1000 863 Dipyrone 350 >1000 52 Phenacetin >1000 >1000 102 Aspirin 10 >1000 3.1 Diclofenac 0.035 0.041 0.008 Ibuprofen 2.4 5.7 0.24 Indomethacin 0.010 0.66 0.016 Caffeine >1000 >1000 >1000 Thalidomide >1000 >1000 >1000 All assays were carried out in the presence of 30mM arachidonic acid. *4-dimethylaminoantipyrine. Chandrasekharan et al, 2002

  12. Is the thermoregulatory function of paracetamol mediated through inhibition of COX-3?

  13. 39.0 200 38.5 C) 38.0 PGE o 37.5 2 37.0 100 Body Temp ( (pg/well) 36.5 36.0 35.5 0 0 1 2 3 4 5 Time (h) The reduction of basal body temperature with 300mg/kg paracetamol correlates with reduction of brain PGE2 levels Paracetamol hypothermia is related brain PGE2 levels

  14. Cerebral cortex Mid brain Brain stem Cerebellum COX-1 (72KDa) COX-2 (72KDa) COX-3 (65KDa) COX-3 is constitutively expressed in brain tissues of mice

  15. 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

  16. 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 PGE2

  17. 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

  18. 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.

  19. Paracetamol is converted to N-arachidonylaminephenolamine (AM404) in the brain by the action of fatty acid amide hydrolase (FAAH) Högestätt et al, 2005

  20. CB1 receptor TRPV1 channel The tissues involved in the conversion of paracetamol to AM404 Fatty acid amido hydrolase enzyme (FAAH) Mallet et al, 2008

  21. 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)

  22. 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??

  23. Paracetamol-induced hypothermia and cannabinoids

  24. Cannabinoid-induced hypothermia and COX

  25. Paracetamol-induced hypothermia and TRPV1

  26. TRPV-1-induced hypothermia and COX

  27. AM404 does not induce hypothermia

  28. Inhibition of FAAH does not prevent the induction of hypothermia with paracetamol

  29. Co-administration of paracetamol and CB1 agonist produce additive hypothermia

  30. 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

  31. 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”

  32. Therapeutic hypothermia: the golden hour

  33. 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.

  34. 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

  35. The team Prof. Gavin Giovannoni (Consultant Neurologist, Royal London) Prof. David Baker (Professor of Neuroimmunology, ICMS) Dr Samir Ayoub (Pharmacology lecturer, UEL) Dr Rupert Pearse (Critical Care Consultant, Royal London)

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