ABSTRACT
The field of antiepileptic drug development has become dynamic, affording many promising research opportunities. Continued efforts are being made in the development of antiepileptic drugs employing a range of strategies, including modification of the structures of existing drugs, targeting novel molecular substrates and non-mechanism-based drug screening. This research is aimed at conducting anticonvulsant studies on three synthesized dichloro-substituted phenyl propanamides. Isomers of 2,3- (DCP23)- 2,5- (DCP25) and 3,4- (DCP34) Dichloro-substituted Phenyl Propanamides were synthesized from acrylamide and dichloro-substituted anilines. The products were formed by an addition reaction according to Michael’s reaction. The physicochemical properties of the products were determined, and their structures were elucidated using standard analytical procedures; infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy. The test compounds were evaluated for anticonvulsant effect in both acute and chronic animal models, via intraperitoneal (i.p.). In maximal electroshock test (MEST), the percentage protection offered by DCP23, DCP25 and DCP34, at 50 mg/kg, was 71.4%, 57.2% and 42.9% respectively. The middle dose (25 mg/kg) of the compounds offered protection of 42.9% (DCP23), 28.5% (DCP25) and 14.3% (DCP34), while the lowest dose (12.5 mg/kg) offered minimal / no protection. The highest dose (50 mg/kg) of DCP23, DCP25 and DCP34 used in pentylenetetrazole-induced seizure test, offered 66.7%, 66.7 and 0% protections against clonic seizures. Also, DCP23, at doses of 25 and 12.5 mg/kg, produced 16.7% protection while similar doses of DCP25 and DCP34 did not offer any protection. There was statistically significant difference in the mean onset of seizure exhibited by DCP23 at doses of 50 mg/kg (p<0.001) and 25 mg/kg (p<0.001). In 4-aminopyridine-induced seizure test, there was no protection offered by all the tested compounds, but DCP34 at doses of 50 mg/kg and 25 mg/kg
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exhibited statistically (p<0.05) significant difference in the onset of seizures. All test compounds did not offer protection in strychnine-induced seizure test. In Picrotoxin-induced seizure test, DCP23 and DCP25 offered protection against clonic convulsion of 66.7% and 83.3% (50 mg/kg) respectively and 50.0% and 66.7% (25 mg/kg) respectively. There was no protection at 12.5 mg/kg. The medial effective dose (ED50) for DCP23, DCP25 and DCP34 using MEST was found to be 25.12, 39.81 and 44.67 mg/kg respectively, while that of picrotoxin was 35.48 mg/kg (DCP23) and 28.18 mg/kg (DCP25). The median toxic doses (TD50) were 100.0, 100.0 and 104.7 mg/kg for DCP23, DCP25 and DCP34 respectively. The protective index (MEST) was 3.98, 2.51 and 2.33 respectively while that of picrotoxin-induced seizure test was 2.82 (DCP23) and 3.55 (DCP25). In the single oral administration (100 mg/kg) evaluation, DCP23, DCP25 and DCP34 offered 37.5%, 50% and 0.0% protections respectively against tonic hind limb extension (THLE) while a 5-day administration offered higher protection of 50%, 75% and 25% respectively. Co-administration of DCP23 (50 mg/kg), DCP25 (50 mg/kg) and DCP34 (50 mg/kg) each with 5 mg/kg fluphenamic acid, resulted in potentiation as the percentage protection against THLE (MEST) were 100% for DCP23 and DCP25, and 50% for DCP34. When DCP23 and DCP25 at the doses of 25 mg/kg, were coadministered with nickel chloride (5 mg/kg) the percentage protection against PTZ-induced seizure were 66.67% and 33.33% respectively. Similarly, their co-administration produced significant (p<0.05) difference in the mean onset of seizure when compared with the control group. Cyproheptadine at the dose of 4 mg/kg did not affect the anticonvulsant effect of DCP23 (50 mg/kg) and DCP25 (50 mg/kg) against PTZ-induced seizure. DCP23 (50 mg/kg) and DCP25 (50 mg/kg), significantly (p<0.001) decreased the onset of sleep as well as increased the duration of sleep (p<0.05). All the compounds at the dose of 50 mg/kg significantly (p<0.05) reduced the
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severity of seizure episodes induced by kindling. A 28-day sub-chronic study was conducted for DCP25 at doses of 50, 25 and 12.5 mg/kg. The results showed that DCP25 at 50 mg/kg only, caused significant (p<0.05) increase in urea, creatinine and aspartate aminotransferase levels. There was no significant (p<0.05) change in haematological indices, lipid profile parameters as well as other renal and liver function test parameters caused by DCP25. The possible mechanism of action was studied on voltage-gated sodium channels(Nav1.6) at different states of the channel: DCP23 at holding potential of -60 mV, produced concentration-dependent tonic blockade of sodium current of 9.73%, 18.04%, 46.80%, 68.46%, 95.64 and 98.10% at 10μM, 30μM, 60μM, 100μM, 300μM and 600μM respectively. At holding potential of -60 mV, DCP25 at 100μM and 600μM blocked the current by 21.63% and 83.03%; while DCP34 (100μM and 600μM) blocked the current minimally by 3.8% and 16.9%, respectively. DCP23 was further tested at a holding potential of -100 mV at the graded concentration (10μM, 30μM, 60μM, 100μM, 300μM and 600μM) and similarly blocked the sodium currents by 0%, 10%, 28.93%, 50.12%, 88.51% and 90.10% respectively. The IC50 values of DCP23 were 64.76 and 100.37 μM at resting and inactivated states respectively. The activation/inactivation pattern in the presence of DCP23 (100 μM) indicated that there was significant reduction in the elicited current even at depolarized potential where the sodium conductance was found to be highest. The results obtained from this work showed that the compounds possess anticonvulsant effects mediated partly via voltage-gated sodium channel blockade.
TABLE OF CONTENTS
Title page ii Declaration iii Certification iv Acknowledgement v Abstract vii Table of Contents x List of Tables xv List of Figures xvii List of Appendices xviii Abbreviations, Definitions, Glossary and Symbols xix 1.0 INTRODUCTION……………………………………………………….. 1 1.1 Statement of Research Problems……………………………………………..2 1.2 Justification…………………………………………………………………4 1.3 Theoretical Frame Work……………………………………………………5 1.4 Objectives of the Study…………………………………………………….7 1.5 Research Hypothesis………………………………………………………..7 2.0 LITERATURE REVIEW…………………………………………………..8 2.1 Epilepsy………………………………………………………………………8 2.1.1 Neural network oscillations………………………………………………..8 2.1.2 Epileptogenesis and Ictogenesis……………………………………………9 2.2 Ion Channels…………………………………………………………………11 2.3 Action Potential……………………………………………………………..12
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2.4 Current-Voltage Relationship………………………………………………..14 2.5 Voltage-Gated Sodium Channels…………………………………………….14 2.5.1 Nomenclature of voltage-gated sodium channels…………………………..15 2.5.2 Anatomical location of voltage-gated sodium channels……………………15 2.5.3 Structural function of voltage-gated sodium channels………………………16 2.5.4 Sodium channel molecular pharmacology…………………………………..16 2.5.5 Biophysical modulation of voltage-gated sodium channels………………….19 2.5.5.1 State dependent block……………………………………………………..20 2.5.5.2 Use dependent blockade……………………………………………………21 2.5.5.3 Inactivation of Na+ channels……………………………………………….21 2.5.5.4 Fast inactivation…………………………………………………………….21 2.5.5.5 Recovery from inactivation………………………………………………….22 2.5.6 Voltage sensing and voltage-dependent activation…………………………..22 2.5.7 Ion selectivity and conductance……………………………………………….23 2.5.8 Phosphorylation of sodium channels………………………………………….24 2.5.9 Channelopathies of sodium channels…………………………………………25 2.6 Voltage-Gated Potassium Channels (VGKCs)…………………………………26 2.7 Voltage-Gated Calcium Channels (VGCCs)……………………………………28 2.8 Chloride Channels………………………………………………………………30 2.9 Neurotransmitter Mediated Mechanisms………………………………………..31 2.9.1 Gamma amino butyric acid (GABA)…………………………………………..31 2.9.1.1 GABA receptor subtypes…………………………………………………….33 2.9.2 Glycine…………………………………………………………………………34 2.9.3 Glutamate receptors……………………………………………………………35 2.9.3.1 Ionotropic glutamate receptors………………………………………………35
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2.9.3.2 Metabotropic glutamate receptors………………………………………….35 2.10 Neuromodulators (neuropeptide Y and corticotropin-releasing hormone)……36 2.10.1 Neuropeptide Y (NPY)……………………………………………………..36 2.10.2 Neuropeptide corticotrophin-releasing hormone (CRH)……………………37 2.11 Other Modulators……………………………………………………………..37 2.11.1 GABA transporters…………………………………………………………37 2.11.2 Gene expression patterns……………………………………………………37 2.11.3 Gap junction…………………………………………………………………37 2.12 Structure Activity Relationship of Sodium Channel Blockers……………….38 2.13 Michael Reaction………………………………………………………………43 2.14 Pharmacophoric Units for Clinically Available Antiepileptic Agents Acting via Sodium Channels…………………………………………………………………..43 3.0 MATERIALS AND METHODOLOGY…………………………………….45 3.1 Materials, Equipment, Chemicals and Animals……………………………….45 3.1.1 Materials and equipment……………………………………………………..45 3.1.2 Chemicals…………………………………………………………………….45 3.2 Synthesis and Chemical Analysis…………………………………………….46 3.2.1 Synthesis……………………………………………………………………..46 3.2.1.1 Procedure…………………………………………………………………..47 3.2.2 Thin layer chromatography………………………………………………………49 3.2.3 Identification and characterization of the compounds……………………….49 3.3 Study Animals and Cell Lines………………………………………………….50 3.3.1 Animals………………………………………………………………………..50 3.3.2 Cell lines………………………………………………………………………50 3.4 Preparation and Administration of Drugs……………………………………….51
3.5 Toxicity Study…………………………………………………………………. 51
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3.5.1 Acute toxicity study………………………………………………………..51 3.5.2 Sub-chronic toxicity study in rats………………………………………….52 3.5.2.1 Blood analysis…………………………………………………………….52 3.5.3 Determination of median neurotoxicity (TD50)…………………………….53 3.6 Anticonvulsant Studies………………………………………………………53 3.6.1 Maximal electroshock-induced seizure in mice…………………………….53 3.6.2 Pentylenetetrazole-induced seizure test……………………………………54 3.6.3 4- aminopyridine-induced seizure test……………………………………..54 3.6.4 Strychnine –induced seizure test……………………………………………55 3.6.5 Picrotoxin-induced seizure test…………………………………………….55 3.6.6 Pentylenetetrazole-induced kindling……………………………………….55 3.6.7 Determination of median effective dose (ED50)…………………………..56 3.6.8 Tolerance study Using maximal electroshock test………………………..57 3.7 Pharmacological Interaction…………………………………………………57 3.7.1 Effect of fluphenamic acid on anticonvulsant activity of DCP23, DCP25 and DCP34 in mice…………………………………………………………………..57 3.7.2 Effect of nickel chloride on anticonvulsant activity of DCP23, DCP25 and DCP34 in mice……………………………………………………………………………58 3.7.3 Effect of cyproheptadine on anticonvulsant activity of DCP23, DCP25 and DCP34 in mice……………………………………………………………………………58
3.7.4 Diazepam-induced sleeping time in mice…………………………………59
3.8 Actions of DCP23, DCP25 and DCP34 on Voltage-gated (Nav 1.6) Sodium Channels Stably Expressed in Human Embryonic Kidney (HEK Cells 293) 59
4.0 RESULTS……………………………………………………………………..61 4.1 Purification, Identification and Characterization of the Test Compounds…… 61 4.2 Toxicity Study………………………………………………………………….69 4.2.1 Acute toxicity study………………………………………………………….69
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4.2.2 Subchronic Toxicity Study in Rats……………………………………………70 4.3 Anticonvulsant Studies………………………………………………………….75 4.3.1 Maximal electroshock-induced seizure in mice……………………………….75 4.3.2 Pentylenetetrazole-induced seizure in mice……………………………………77 4.3.3 4-Aminopyridine-induced seizure in mice…………………………………….79 4.3.4 Strychnine-induced seizure in mice……………………………………………81 4.3.5 Picrotoxin-induced seizure in mice…………………………………………….83 4.3.6 Pentylenetetrazole-induced kindling in mice………………………………….85 4.3.7 Determination of median effective dose (ED50)………………………………87 4.3.8 Tolerance study using maxmal electroshock test………………………………88 4.4 Pharmacological Interaction……………………………………………………..89 4.4.1 Effect of fluphenamic acid on anticonvulsant activity of DCP23, DCP25 and DCP34 in mice……………………………………………………………………….89 4.4.2 Effect of Nickel Chloride on DCP23 and DCP25 against Pentylenetetrazole-induced Seizure in Mice………………………………………………………………91 4.4.3 Effect of Cyproheptadine on DCP23, DCP25 and DCP34 against Pentylenetetrazole-induced Seizure in Mice………………………………………….93 4.4.4 Effects of DCP23 and DCP25 on Diazepam-induced in Mice…………………95 4.5 Actions of DCP23, DCP25 and DCP34 ON Voltage-Gated Sodium Channels (NaV 1.6)……………………………………………………………………………………98 4.5.1 States Dependent Actions………………………………………………………98 4.5.2 Use Dependent Action…………………………………………………………105 5.0 DISCUSSION……………………………………………………………………107 6.0 SUMMARY, CONCLUSION AND RECOMMENDATION…………………127 6.1 Summary………………………………………………………………………….127 6.2 Conclusion………………………………………………………………………..129
6.3 Recommendation…………………………………………………………………130
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REFERENCE……………………………………………………………………131 APPENDIX………………………………………………………………………147 LIST OF TABLES Table 4.1: Physicochemical Properties of DCP23, DCP25 and DCP34…………61 Table 4.2: Thin Layer Chromatographic Analysis of DCP23, DCP25 and DCP34 using Ethylacetate………………………………………………………………………62 Table 4.3: Pharmacophore Units of the Test Compounds and their Distances…..63 Table 4.4: Infrared Spectra Data for 2,3-, 2,5- and 3,4- Dichloro – 3(aminophenyl) propanamides……………………………………………………………………..64 Table 4.5: H1 and 13C NMR Interpretation of the DCP23 Isomer………………..66 Table 4.6: H1 and 13C NMR Interpretation of the DCP25 Isomer………………..67 Table 4.7: H1 and 13C NMR Interpretation of the DCP34 Isomer………………..68 Table 4.8: LD50 Values of 2,3-, 2,5- and 3,4- Dichloro – 3(aminophenyl) Propanamides in Mice and Rats via Intraperitoneal and Oral Routes…………………………….69 Table 4.9: Effect of 2,5- Dichloro – 3(aminophenyl) Propanamides on Liver Function Parameters after 28-Day Oral Admnistration in Rats…………………………….71 Table 4.10:Effect of 2,5- Dichloro – 3(aminophenyl) Propanamides on Renal Parameters after 28-Day Oral Admnistration in Rats…………………………….72 Table 4.11: Effect of 2,5- Dichloro – 3(aminophenyl) Propanamides on Lipid Parameters after 28-Day Oral Admnistration in Rats…………………………….73 Table 4.12: Effect of 2,5- Dichloro – 3(aminophenyl) Propanamides on Haematological Parameters after 28-Day Oral Admnistration in Rats…………………………….74 Table 4.13: Effect of DCP23, DCP25 and DCP34 Dichloro and Phenytoin on Maximal electroshock-induced Seizures in Mice…………………………………………..76 Table 4.14: Effect of DCP23, DCP25 and DCP34 and Valproate on Pentylenetetrazole-induced Seizures in Mice…………………………………………………………78 Table 4.15: Effect of DCP23, DCP25 and DCP34 and Phenobarbital on 4-aminopyridine-induced Seizures in Mice…………………………………………80 Table 4.16: Effect of DCP23, DCP25 and DCP34 and Phenobarbital on Strychni
CHAPTER ONE
1.0 INTRODUCTION
Epilepsy is defined as a condition characterized by recurrent (two or more) epileptic seizures, unprovoked by any immediate identified cause (Banerjee et al., 2009). It refers to a disorder of brain function characterized by the periodic and unpredictable occurrence of seizures. Epilepsy is currently defined as the occurrence of at least one seizure with an enduring alteration in the brain structure or function that increases the likelihood of future seizures (Gerlach and Krajewski, 2010). Seizures are transient alteration of behavior due to the disordered, synchronous and rhythmic firing of populations of brain neurons (McNamara, 2001). Multiple seizures occurring in 24 h period or an episode of status eilepticus are considered a single event. Individuals who have had only febrile seizures or only neonatal seizures (seizures in the first 30 days of life), and people with acute symptomatic seizures (seizures associated with acute systemic illness, intoxication, substance abuse or withdrawal, or acute neurological insults), and individuals with a single unprovoked seizure, are excluded from this category (Banerjee et al., 2009). Seizures are broadly classified as either generalized or partial depending on whether they involve widespread bilateral cortical regions at the onset or originate from a discrete focal area. This designation is based on both outward symptoms and electroencephalograph (EEG) patterns (Gerlach and Krajewski, 2010). Generalized seizures may be convulsive (tonic myoclonic, tonic clonic, depending on the characteristics of the muscle contraction) or non-convulsive, as in the case of petit mal where the paroxysmal discharge can be accompanied only by suspension of consciousness without motor phenomena (Pevarello et al., 1998). This group of disorders is diverse, and they all
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appear to have in common, the feature of aberrant synchronized discharge of neurons leading to alteration in electroencephalograph (EEG) activity and behavior (Nicholas et al., 2002). The causes of seizures are many and include the full range of neurological diseases, from infection to neoplasm and head injury. In some sub-groups of epilepsy, hereditory is known to be a major contributing factor. These may explain why monotherapy in epilepsy is difficult (Roger and Brians, 2004).
1.1 Statement of Research Problems
Drugs in the Central nervous system (CNS) can be broadly classified according to whether they have a general stimulatory or depressant action, with further sub-division regarding specific actions such as anticonvulsant and psychopharmacological activities (Evans, 1996). Several types of insults such as status epilepticus, hypoxia and trauma are known to alter the normal function of the central nervous system (CNS). Modalities that protect the brain against such insults have been very difficult and challenging. It is important to know that, epilepsy, as one of such CNS disorders, alter the normal function of brain; and its treatment is all about neuroprotection, either to reduce the duration of seizures or to suppress the occurrence of seizures (Arzimanoglous et al., 2002).
The scientific understanding of seizure pathogenesis and propagation is far from complete and the mechanism of action of most available antiepileptic drugs (AEDs) is either unknown or involves multiple interactions (Gerlach and Krajewski, 2010). Epilepsy is one of the most common and widespread neurological disorders. Recent estimates suggest that it accounts for 1% of the global burden of disease and affects over 65 million people; more than 500 million people are indirectly affected by the disease.
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Thus, epilepsy imposes a large economic burden on global health care systems and is a major public health problem in low and middle-income countries (Mbuba and Newton, 2009). About 2 million people in the United States have epilepsy and 3% of persons in the general population will have epilepsy at some point in their lives (Bernard et al., 2003). It has been established that the highest incidence of epilepsy is found in children and elderly (Pevarello et al., 1998).
Typical therapeutic strategy is to optimize the use of a single antiepileptic drug, given that about 60% of patients have become seizure free. As second line approach, concurrent treatment with more than one AED is employed. Unfortunately, only 5% of patients who fail to respond adequately to monotherapy experience long term freedom from seizures using polytherapy (Gerlach and Krajewski, 2010). Nearly 95% of prescriptions written by physicians worldwide for the treatment of epilepsy are from the old AEDS developed prior to 1975. Several new drugs have been approved e.g felbamate, lamotrigine, gabapentine, topiramate, vigabatrin and tiagabine. These drugs have been shown to be effective in reducing seizures in a number of patients, their efficacy does not appear to be superior to that of the drugs developed earlier (Unverferth et al., 1998).
Epilepsy is the most common non-infectious neurologic disease in developing African countries, including Nigeria and it remains a major medical and social problem. In many African countries, people with epilepsy are out-cast as Africans believe that the disease results from visitation of the evil, effect of witch-craft, the revenge of an aggrieved ancestral spirit or consumption of something harmful in utero. Suicide or attempted
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suicide is not uncommon among Nigerians who suffer from epilepsy. The patient with epilepsy is likely to drop out of school, looses his job, finds it impossible to marry, loses his wife or husband, and be tormented to the extent of becoming a vagrant vagabond (Ogunrin, 2006). Untreated epilepsy can lead to impaired intellectual function or death and is typically accompanied with psychosocial prejudices and other psycho pathological consequences such as loss of self-esteem and poor quality of life (Idris et al., 2008c).
1.2 Justification
The need to develop phenytoin-like compounds or compounds with pharmacophore responsible for Na+ channels blockade affinity relies on the need for alternative new drugs because of well established side effects of phenytoin; which include neurologic signs (ataxia, nystagmus, sedation or irritability, orofacial dyskinesia); heamatologic (leucopenia); immunologic (reduction of IgA, lupus syndrome); endocrinologic (hirsutism); and cell growth (gingival hypertrophy) dysfunctions (Vameq et al., 2000).
Most of the current antiepileptic drugs are synthetic and they are still the promising agents employed in controlling seizures among epileptic patients. Therefore, the trend should be continued to source for more promising agents. Synthetic agents are target specific, as molecular structure can be designed based on possible prediction of its pharmacophore. Also, the production of synthetic compounds can be modified to give higher yield as required. Similarly, the side effects of synthetic compounds can be avoided or reduced by optimizing their structural moieties responsible for a particular untoward effect.
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1.3 Theoretical Frame Work
The field of antiepileptic drug development has become dynamic, affording many promising research opportunities (Siddiqui et al., 2009). Continued efforts are being made in the development of antiepileptic drugs employing a range of strategies, including modification of the structure of existing drugs, targeting novel molecular substrates and non-mechanism-based drug screening of compounds in traditional and newer animal models (Gitto et al., 2009). The present AEDs predominantly target voltage gated channels (e.g. alpha-subunits of voltage-gated Na+ channels, T-type voltage-gated Ca2+) or influence GABA-mediated inhibition. Recently identified, new, and potentially interesting molecular targets include KCNQ-type K+ channels, SV2A synaptic vesicle protein, ionotropic and metabotropic glutamate receptors, and gap junctions (Pasquale and Salvatore, 2009).
1.3.1 Pharmacophore model for blockers of sodium channels
Several attempts were made to postulate a general pharmacophore for the different anticonvulsant classes. The various postulated pharmacophore models show no uniform picture. Nevertheless, the presence of at least one aryl unit, one or two electron donor atoms, and/or an NH group in a special spatial arrangement seems to be recommended. Jones and Woodbury defined a model with two electron donors in some proximity to a bulky hydrophobic moiety. Coddling postulated a pharmacophore consisting of a linear arrangement of a rotated phenyl ring, an electron donor atom, and a hydrogen donor site which partially agrees with the model of Jones and Woodbury (Unverferth et al., 1998).
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On the basis of molecular dynamics distance estimations, the suggested pharmacophore model for compounds acting as blockers of the voltage-dependent sodium channel is given as: electron donor D in relatively limited distance ranges of 3.2-5.1 A0 to an aryl ring or other hydrophobic units R and of 3.9-5.5 A0 to a hydrogen bond acceptor/donor (HAD) unit. The distance between R and HAD spans a wider range of 4.2-8.5 A0. The hydrophobic unit R is not oriented in the same plane like the other essential elements. The rings are rotated in relation to the R-D-HAD plane by 10-400 (Unverferth et al., 1998).
Figure 1: Pharmacophore model proposed by Unverferth et al., 1998
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1.4 Aims and Objectives of the Study
This research is aimed at conducting anticonvulsant studies on three synthesized dichloro substituted phenyl propanamides.
The Specific objectives are as follows:
i. To synthesize, identify and characterize the compounds using standard analytical procedures (IR and NMR Spectroscopy)
ii. To determine median lethal dose (LD50), median effective dose (ED50), median toxic dose (TD50) and protective index (PI) of the compounds to ascertain their safety profiles
iii. To establish anticonvulsant activity of the synthesized compounds in mice and rats using both acute and chronic models of convulsion
iv. To conduct subchronic toxicity studies on one representative isomer; aimed at evaluating its effect on renal, hepatic and hematologic indices as well as its effect on lipid profile
v. To determine the action of the synthesized compounds on voltage-gated sodium channels (Nav1.6)
1.5 Hypothesis
Dichloro substituted phenyl propanamide isomers exert their anticonvulsant activity via voltage-gated sodium channels blockade.
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