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Investigation of the Mechanisms of Action of the Novel Anticonvulsant

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Investigation of the Mechanisms of Action of the Novel Anticonvulsant Investigation of the mechanisms of action of the novel anticonvulsant topiramate: electrophysiological studies on rat olfactory cortical neurones in vitro and some in vivo rodent models of epilepsy. A thesis submitted in part fulfilment of the University of London for the award of Doctor of Philosophy in Medicine (Pharmacology) by Emilio Russo, Laurea in Chimica e Tecnologia Farmaceutiche Department of Pharmacology The School of Pharmacy 29-39 Brunswick Square London WCIN lAX ProQuest Number: 10104832 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104832 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This thesis describes research conducted in The School of Pharmacy, University of London and The Department of Experimental and Clinical Medicine “G. Salvatore”, Faculty of Medicine and Surgery, University “Magna Graecia” of Catanzaro, Catanzaro, Italy between 5-June-2000 and 16-December-2003 under the supervision of Dr. A. Constanti and Prof. G. De Sarro. I certify that the research described is original and that any parts of the work that have been conducted by collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation, any part of this thesis that has already appeared in publication. Signed.. ................ Abstract Topiramate (TPM; Topamax®) is a novel anticonvulsant that was originally developed as a possible inhibitor of gluconeogenesis, but was found to be a very effective anticonvulsant drug in many animal models. To date, the proposed mechanisms of action of TPM include: inhibition of neuronal Na^ channels, enhancement of G A B A a - receptor mediated effects; inhibition of AMPA/kainate (glutamate) receptors, inhibition of high voltage-activated Ca^^ channels, and inhibition of carbonic anhydrase activity. In the present study, the effects of TPM were investigated in rat olfactory (piriform) cortex neurones in vitro, using an intracellular current/voltage clamp recording technique. Bath-application of TPM induced a slow, dose-dependent and reversible membrane hyperpolarization, accompanied by a decrease in membrane input resistance and inhibition of repetitive action potential firing. Under voltage clamp at -70 mV holding potential (Vh), the TPM response manifested as a slow outward membrane current, developing over 10 min and slowly reversing after washout; the TPM current was partially (-50%) blocked by Ba^^ (a general blocker of conductances), suggesting it was largely carried by ions, but unaffected by Cd^^ (200 pM) or bicuculline (10 pM) indicating that a Ca^^-dependent conductance or G A B A a receptors were not involved, respectively. Current/voltage {I/V) plots (Vh =-70 mV) constructed in the presence and absence of TPM failed to intersect at very negative potentials, suggesting that the TPM current may comprise of a mixture of ionic conductances, or a contribution from some electrogenic pump mechanism. Topiramate (20 pM) also enhanced and prolonged the slow post-stimulus (Ca^^-dependent) afterhyperpolarization (sAHP), that follows a long burst of action potentials (and the underlying slow outward tail current (s Ia h p ) recorded under voltage clamp). We believe this effect was due to a selective enhancement/prolongation of an underlying L-type Ca^^ current that was blocked by nifedipine (20 pM); the modulatory effect of TPM on the sAHP was unlikely to involve an interaction at PKA-dependent phosphorylation sites, since it was unaffected by pre-incubation with forskolin (20 pM), a direct activator of adenylate cyclase (and ultimately PKA). Interestingly, the CA inhibitors acetazolamide (ACTZ, 20 pM) and benzolamide (BZ, 50 pM) both mimicked the membrane effects of TPM, in generating a slow hyperpolarization (slow outward current under voltage clamp) and sAHP enhancement/prolongation. ACTZ and BZ also occluded the effects of TPM in generating the outward current response but were additive in producing the sAHP modulatory effect, suggesting different underlying response mechanisms. In hicarbonate/COz-ffee, HEPES-buffered bathing medium, all the membrane effects of TPM and ACTZ were reproducible, therefore not dependent on CA inhibition. We propose that other molecules possessing the sulpbonamide moiety in their structure might have the same action as TPM on piriform cortical neurones. In a second series of in vivo experiments, we determined the efficacy of TPM in some animal models of epilepsy and also tested whether L-type Ca^^ channel modulators could modify its potency in vivo (as predicted from the in vitro data). The results obtained showed that TPM possesses a wide spectrum of anticonvulsant activity against both convulsive and non-convulsive seizures. In particular, TPM protected against seizures induced by sound stimuli in DBA/2 mice, intracerebroventricular injection of AMPA, subcutaneous administration of 4-aminopyridine or pentylenetetrazole administered intraperitoneally. The co-administration of nifedipine with TPM significantly decreased its anticonvulsant activity only in DBA/2 mice, whereas it did not affect TPM’s anticonvulsant properties in the other animal models. Topiramate was also very effective in two genetic animal models of absence epilepsy (lethargic “/M/z” mice and WAG/Rij rats). However, rather surprisingly, when TPM was co-administered with nifedipine in these models, contrasting results were observed; in WAG/Rij rats, nifedipine antagonized TPM’s anti-absence activity whereas in the Ih/lh mouse model a synergism was found (possibly due to presence of an abnormal L-Ca^^ channel p 4 subunit affecting the normal dihydropyridine-TPM-L-channel interaction). Therefore, an involvement of dihydropyridine-sensitive L-type Ca^^ channels in the anti-absence effects of TPM might also be considered. In conclusion, this work has identified two new novel mechanisms of action for TPM, which are not currently shared by any other marketed anticonvulsant drug and could indicate novel targets for the development of new antiepileptic compounds in the future. Furthermore, the interaction of TPM with L-type Ca^^ channel antagonists analyzed in in vivo animal experimental models of epilepsy support the proposal that a modulation of neuronal L-type Ca^^ channel activity plays an important role in its antiepileptic activity. Finally, our results also predict that nifedipine or other L-type Ca^^ channel antagonists could affect TPM’s anticonvulsant efficacy in human epilepsy, therefore their combination or concomitant administration in therapy should be avoided or at least carefully monitored. ''In searching out the truth be ready for the unexpected, for it is difficult to find and puzzling when you find it”. Heraclitus “La natura è un tempio ove pilastri viventi emettono talvolta parole confuse. L ’uomo la attraversa tra foreste di simboli che lo osservano con sguardi familiari ”. [ “Nature is a temple in which living pillars sometimes emit confused words; man crosses it through forests o f symbols that observe him with familiar glances]. Charles Baudelaire To Antonietta Mazza Acknowledgements After writing so many pages, I must admit that this page has been the most difficult to write. It is very complicated to find the right words to describe how thankful I am to all these persons. Dr. Andy Constanti has been the craftsman of this project. He received me in his small office more than four years ago, when I was only a young man, who could only poorly understand a few things about English life and also pharmacology! I do not know what he originally saw in me, but this thesis is the proof that he believed in me as a scientist. Thanks Andy for being such a good life teacher and above all thanks for being such a good friend. A special thanks also goes to Prof. Giovambattista De Sarro, who allowed me to access his deep knowledge of epilepsy and epilepsy research. I know he believes I can make a good scientist; thanks for the chance, and I will do my best. Oh! Thanks also for all the good wine. I am disappointed that I cannot create new words to thank my parents Francesca and Antonio for modelling me from nothing to what I am today. For their silent guide that left me to make my choices on my own, for teaching me that big results require big efforts, but the same strength has to be used in daily life. I know you will always be there. Lidia, you know how much I love you; thanks for bearing my terrifying moods. Please, love me forever. The most important finding that I have discovered in London is that drinking and thinking are highly connected. No matter what you drink (beer, wine, tea, coffee), it activates some unknown circuitries that make everything easier in life. For this discovery I must thank Ben and Daniele who made my life in London the most intensive and meaningful experience of my life. My return to Italy has not been as easy and predictable. I have been very lucky to meet Dr. Guido, who I will always continue to thank for his unconditional help in everything I have done and I am doing. Mate, keep on cooking as only you know best. This thesis was also finished thanks to the help given by my favourite veterinary Nicola, who represents the best example of ‘Calabrese’ I have ever met. Also, Rita, Carmen, Salvatore and Luca for making my life in Catanzaro not as bad as it might have been. Let’s go on guys! Dear brother, what can I say?; thanks, I will not disappoint you.
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