Russo, Emilio; (2005) 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. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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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 GABAA- receptor mediated effects; inhibition of AMPA/kainate (glutamate) receptors, inhibition of high voltage-activated Ca2+ 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 Ba2+ (a general blocker of K2+ conductances), suggesting it was largely carried by ions, but unaffected by Cd2+ (200 μM) or bicuculline (10 μM) indicating that a Ca2+-dependent conductance or GABAA 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 μM) also enhanced and prolonged the slow post-stimulus (Ca2+-dependent) afterhyperpolarization (sAHP), that follows a long burst of action potentials (and the underlying slow outward tail current (SIAHP) recorded under voltage clamp). We believe this effect was due to a selective enhancement/prolongation of an underlying L-type Ca2+ current that was blocked by nifedipine (20 μM); 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 μM), a direct activator of adenylate cyclase (and ultimately PKA). Interestingly, the CA inhibitors acetazolamide (ACTZ, 20 μM) and benzolamide (BZ, 50 μM) 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 bicarbonate/CO2-free, 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 sulphonamide 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 Ca2+ 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. Topiramate was also very effective in two genetic animal models of absence epilepsy (lethargic "lh/lh" 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 lh/lh mouse model a synergism was found (possibly due to presence of an abnormal L-Ca2+ channel μ4 subunit affecting the normal dihydropyridine-TPM-L-channel interaction). Therefore, an involvement of dihydropyridine-sensitive L-type Ca2+ 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 Ca2+ channel antagonists analyzed in in vivo animal experimental models of epilepsy support the proposal that a modulation of neuronal L-type Ca2+ channel activity plays an important role in its antiepileptic 9-l- activity. Finally, our results also predict that nifedipine or other L-type Ca2+ 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.

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