Thesis ((M. Pharm. (Pharmaceutics)) -- University of Limpopo, 2019 / Epilepsy is the fourth most common neurological disorder after migraine, stroke and Alzheimer’s disease and it affects about fifty million people worldwide. Careful consideration should be taken when deciding to initiate treatment in epilepsy as it should consider the balance between the possibility of further seizures and their associated risks, including the possible risk of sudden expected death, inconvenience and the risks of taking regular medication for each individual. In the early 1980’s, the first-line treatment for epilepsy was polytherapy. This was due to findings that smaller doses of two drugs rather than larger doses of one drug can achieve synergistic effects or less drug toxicity. However, following more trials on the treatment of epilepsy, this was later changed to monotherapy as first-line treatment. Despite the change, patients remain uncontrolled on a single anti-epileptic drug, thus they are initiated on polytherapy, one such combination being carbamazepine in combination with sodium valproate. The use of these in combination has pharmacological threats such as compliance, the control of side effects and the achievement of synergistic effects. The development of a Fixed Dose Combination (FDC) has often been used to resolve pharmacological threats, and this study aims to develop a fixed dose combination tablet of carbamazepine and sodium valproate to resolve the pharmacological threats in epilepsy. Samples of carbamazepine and sodium valproate and a physical mixture (1:1 w/w) of both drugs and excipients were prepared for compatibility with thermal analysis and spectroscopy techniques. Data was analysed by comparing the DSC curves, FTIR spectra, XRPD peaks and TAM analysis of carbamazepine and sodium valproate alone and in their physical mixture (1:1 w/w) and with excipients. Both carbamazepine and sodium valproate were evaluated for flowability using angle of repose, tapped and bulk density, compressibility index and particle size distribution. To formulate the proposed FDC tablet of carbamazepine and sodium valproate, direct compression and wet granulation methods were employed. The tablets were then evaluated for official and non-official post formulation parameters (weight variation, crushing strength, friability, diameter and thickness, and disintegration) according to BP and USP standards. A standardised HPLC method was developed and validated for analytical procedures. Dissolution studies were conducted xiii according to USP methods to verify and quantify the release of the APIs from the FDC tablet. Carbamazepine and sodium valproate were tested for compatibility with excipients using DSC, FTIR, XRPD and TAM analysis. The overall results confirmed that carbamazepine and sodium valproate are compatible, with each other and the excipients used in the study. Powder flow of carbamazepine and sodium valproate was poor, hence they were subjected to granulation prior to compression to improve flowability. The specifications of the fixed-dose combination were developed in accordance with the FDA’s quality by design concept and WHO recommendations. The tablets were subjected to non-official and official pharmacopoeial tests, and passed all the tests. Dissolution studies according to a USP method were conducted to verify and quantify the release of the APIs in the fixed-dose combination. The initial dissolution rate (DRi) of carbamazepine and sodium valproate in the SLS dissolution medium was rapid as required for an immediate release formulation. The study aimed at developing a fixed dose combination of carbamazepine and sodium valproate to try to reduce the burden of taking more than one tablet for epilepsy. Based on the results obtained from preformulation studies to assay of the final product, the study was successful. / Chieta bursary and HWseta

A associação de ácido valpróico/ divalproato de sódio e lamotrigina tem se mostrado eficaz no tratamento das epilepsias refratárias, tendo como limitador ao seu uso os efeitos adversos, principalmente numa população de crianças e adolescentes, onde esses efeitos são maximizados. Este estudo clínico tem como objetivo avaliar as propriedades farmacológicas da associação valproato/divalproato de sódio e lamotrigina em uma população pediátrica refratária ao tratamento medicamentoso usando método de introdução e escalonamento mais lento do que o preconizado, com seguimento prolongado. Para tal, foi estudado um grupo de 51 crianças e adolescentes com epilepsia de difícil controle, com idades de 4 a 16 anos (mediana de 8 anos), sendo 27 (52,9%) meninas. Dezesseis (31,4%) crianças apresentavam epilepsia generalizada; 35 (68,6%), epilepsia parcial. A associação valproato/divalproato de sódio e lamotrigina foi eficaz no primeiro ano de acompanhamento para 39 (76,5 %) pacientes. No segundo ano de tratamento, esta associação foi eficaz para 36 (70,6%) pacientes. Houve melhora dos drop attacks em 22 pacientes (88,5%), mas não houve especificidade em relação à síndrome ou crise epiléptica. Efeitos adversos observados foram rash, em quatro (7,8%) pacientes, com descontinuidade do tratamento, e tremores sutis em seis (11,7%), resolvidos com a diminuição da dose da lamotrigina. A descontinuidade ocorreu em 12 (23,5%) pacientes, sendo que a maior razão foi o rash cutâneo, seguido pela perda da eficácia ao tratamento, em oito (15,7%) pacientes. Concluímos que, com a proposta de introdução mais lenta da lamotrigina, os efeitos adversos são minimizados (principalmente os referentes ao sistema nervoso central), assim como há melhora das crises debilitantes, que comprometem a qualidade de vida desses pacientes, tendo como resultado uma maior adesão ao esquema terapêutico. Além disso, pontuamos que a eficácia terapêutica se mantém com doses mais baixas de lamotrigina, mesmo após o primeiro ano de tratamento / The association lamotrigine and sodium valproate/divalproex sodium has been shown to be effective in the treatment of refractory epilepsy, having as a limiting factor for its use, adverse effects, especially in a population of children and adolescents where these effects are maximized. This clinical study aims to evaluate the pharmacological properties of this association in a pediatric population refractory to medical treatment using a method of introduction and titration slower than the usually recommended, with extended follow-up. For this purpose, we studied a group of 51 children and adolescents, with refractory epilepsy, ranging from 4 to 16 years old (median 8 years), with 27 (52.9%) girls. Sixteen (31.4%) children presented generalized and 35 (69.6%) focal epilepsy. The association sodium valproate/ divalproex sodium and lamotrigine was effective in the first year of followup in 39 (76.5%) patients. In the second year of treatment this combination was effective in 36 (70.6%) patients. An improvement of drop attacks was observed in 22 (88.5%) patients, but there was no specificity as to the epileptic syndrome or seizure type. Adverse effects were rash, leading to discontinuation in four (7.8%), and subtle tremors, that resolved with reduction of the dose of lamotrigine in six (11.7%) patients. In twelve (23.5%) patients treatment was withdrawn, because of rash (7.8%) and loss of efficacy, in eight (15.7%) patients. We concluded that with the proposed slower introduction of lamotrigine, adverse effects are minimized, especially in the central nervous system, as well as better obtained control of debilitating seizures, affecting quality of life, and resulting in better adherence to the therapeutic scheme. Furthermore, we point out that the therapeutic efficacy is maintained with lower doses of lamotrigine, even after the first year of treatment

A associação de ácido valpróico/ divalproato de sódio e lamotrigina tem se mostrado eficaz no tratamento das epilepsias refratárias, tendo como limitador ao seu uso os efeitos adversos, principalmente numa população de crianças e adolescentes, onde esses efeitos são maximizados. Este estudo clínico tem como objetivo avaliar as propriedades farmacológicas da associação valproato/divalproato de sódio e lamotrigina em uma população pediátrica refratária ao tratamento medicamentoso usando método de introdução e escalonamento mais lento do que o preconizado, com seguimento prolongado. Para tal, foi estudado um grupo de 51 crianças e adolescentes com epilepsia de difícil controle, com idades de 4 a 16 anos (mediana de 8 anos), sendo 27 (52,9%) meninas. Dezesseis (31,4%) crianças apresentavam epilepsia generalizada; 35 (68,6%), epilepsia parcial. A associação valproato/divalproato de sódio e lamotrigina foi eficaz no primeiro ano de acompanhamento para 39 (76,5 %) pacientes. No segundo ano de tratamento, esta associação foi eficaz para 36 (70,6%) pacientes. Houve melhora dos drop attacks em 22 pacientes (88,5%), mas não houve especificidade em relação à síndrome ou crise epiléptica. Efeitos adversos observados foram rash, em quatro (7,8%) pacientes, com descontinuidade do tratamento, e tremores sutis em seis (11,7%), resolvidos com a diminuição da dose da lamotrigina. A descontinuidade ocorreu em 12 (23,5%) pacientes, sendo que a maior razão foi o rash cutâneo, seguido pela perda da eficácia ao tratamento, em oito (15,7%) pacientes. Concluímos que, com a proposta de introdução mais lenta da lamotrigina, os efeitos adversos são minimizados (principalmente os referentes ao sistema nervoso central), assim como há melhora das crises debilitantes, que comprometem a qualidade de vida desses pacientes, tendo como resultado uma maior adesão ao esquema terapêutico. Além disso, pontuamos que a eficácia terapêutica se mantém com doses mais baixas de lamotrigina, mesmo após o primeiro ano de tratamento / The association lamotrigine and sodium valproate/divalproex sodium has been shown to be effective in the treatment of refractory epilepsy, having as a limiting factor for its use, adverse effects, especially in a population of children and adolescents where these effects are maximized. This clinical study aims to evaluate the pharmacological properties of this association in a pediatric population refractory to medical treatment using a method of introduction and titration slower than the usually recommended, with extended follow-up. For this purpose, we studied a group of 51 children and adolescents, with refractory epilepsy, ranging from 4 to 16 years old (median 8 years), with 27 (52.9%) girls. Sixteen (31.4%) children presented generalized and 35 (69.6%) focal epilepsy. The association sodium valproate/ divalproex sodium and lamotrigine was effective in the first year of followup in 39 (76.5%) patients. In the second year of treatment this combination was effective in 36 (70.6%) patients. An improvement of drop attacks was observed in 22 (88.5%) patients, but there was no specificity as to the epileptic syndrome or seizure type. Adverse effects were rash, leading to discontinuation in four (7.8%), and subtle tremors, that resolved with reduction of the dose of lamotrigine in six (11.7%) patients. In twelve (23.5%) patients treatment was withdrawn, because of rash (7.8%) and loss of efficacy, in eight (15.7%) patients. We concluded that with the proposed slower introduction of lamotrigine, adverse effects are minimized, especially in the central nervous system, as well as better obtained control of debilitating seizures, affecting quality of life, and resulting in better adherence to the therapeutic scheme. Furthermore, we point out that the therapeutic efficacy is maintained with lower doses of lamotrigine, even after the first year of treatment

Abstract Various genetic aetiologies — including mitochondrial diseases, chromosomal disorders, and other monogenic diseases — are involved in status epilepticus (SE), a common neurologic emergency occurring in children and adults that exhibits high rates of morbidity and mortality. The exact frequency of mitochondrial SE is currently undefined. Furthermore, patients with pathogenic variants of POLG1 encoding mitochondrial DNA polymerase gamma have an increased risk of acute liver failure (ALF) induced by the common antiepileptic drug, valproic acid (VPA), which is problematic due to these patients also often experiencing drug-resistant seizures. Overall, the role of liver transplantation (LT) in VPA-ALF due to mitochondrial disease has been controversial. In the present work, large retrospective cohort studies were conducted for two main purposes: (1) to determine the genetic aetiologies of SE among Finnish paediatric and adult patients by specifically focusing on the common mitochondrial genetic defects associated with an increased risk of SE and (2) to examine whether common POLG1 p.Q1236H and p.E1143G variants are connected to liver or pancreatic toxicity upon exposure to VPA monotherapy. This thesis also describes the characteristics of VPA-ALF associated with the pathogenic POLG1 variant p.W748S and the prognosis of LT in a retrospective case series. Mitochondrial diseases explained 4.5% of SE cases in the study cohort. Patients with mitochondrial SE suffered from refractory SE significantly more often than patients with other forms of genetic or non-genetic SE. Additionally, mortality rates were higher in patients with mitochondrial or chromosomal disorders compared with the other groups, reflecting the severity of the underlying condition and the higher frequency of refractory SE. POLG1 variants p.Q1236H and p.E1143G could not be identified as risk factors for VHT or pancreatic toxicity, implying that VPA treatment might be suitable for patients harbouring these variants when other pathogenic variants are absent. Finally, the homozygous status of the pathogenic POLG1 variant p.W748S and older age of the patient during the presentation of VPA-ALF seem to be associated with higher survival rates following LT, which should be considered in the management of VPA-ALF. / Tiivistelmä Useita perinnöllisiä syitä, kuten mitokondriotauteja, kromosomihäiriöitä ja muita geenimuutoksia on tunnistettu status epilepticuksen (SE) eli pitkittyneen epileptisen kohtauksen taustalla. SE on yleinen neurologinen hätätilanne, johon liittyy merkittävää oheissairastavuutta ja kuolleisuutta sekä lapsilla että aikuisilla. Mitokondriotauteihin liittyvän SE:n tarkkaa esiintyvyyttä ei tiedetä. Potilailla, joilla on patogeenisia variantteja mitokondrioiden DNA-polymeraasia koodaavassa tuman POLG1-geenissä, on todettu kohonnut riski yleisesti käytetyn epilepsialääkkeen valproaatin (VPA) aiheuttaman akuutin maksavaurion kehittymiselle. Tämä tekee lääkehoidon valinnasta ongelmallista, koska näillä potilailla on usein epilepsialääkkeille resistenttejä kohtauksia. Maksansiirron merkitys akuutin maksavaurion hoidossa mitokondriotauteja sairastavilla potilailla on ollut kiistanalainen. Tutkimuksen tavoitteena oli selvittää SE:n perinnöllisiä syitä suomalaisilla lapsi- ja aikuispotilailla retrospektiivisesti kerätyssä laajassa potilasaineistossa. Tutkimuksessa keskityttiin yleisimpiin mitokondriaalisiin perinnöllisiin muutoksiin, joiden on aiemmin todettu liittyvän SE:n lisääntyneeseen riskiin. Tutkimuksen toisena päätavoitteena oli selvittää väestössä yleisten POLG1-geenin muutosten eli varianttien p.Q1236H ja p.E1143G yhteyttä maksatoksisuuteen tai haimatoksisuuteen VPA-monoterapian aikana. Lisäksi tutkittiin VPA:n aiheuttaman maksavaurion kliinisiä erityispiirteitä patogeeniseen POLG1-varianttiin p.W748S liittyen sekä mutaatiostatuksen vaikutusta maksansiirron jälkeiseen ennusteeseen. Mitokondriotaudit selittivät 4,5 % SE-tapauksista tämän väitöskirjatyön potilasaineistossa ja näillä potilailla SE pitkittyi hoitoresistentiksi tai erittäin resistentiksi merkitsevästi muita potilasryhmiä useammin. Kuolleisuus oli suurin potilailla, joilla todettiin mitokondriotauti tai kromosomihäiriö, liittyen todennäköisimmin vakavaan taustasairauteen ja hoitoresistentin SE:n suurempaan esiintyvyyteen. Tutkittuja POLG1-variantteja p.Q1236H ja p.E1143G ei voitu tunnistaa maksa- tai haimatoksisuuden riskitekijöiksi, mikä tarkoittaa, että VPA-hoito voisi sopia näille potilaille, mikäli muita patogeenisiä variantteja ei todeta. Patogeenisen POLG1-variantin p.W748S homotsygoottisuus ja nuoruusikä tai varhainen aikuisikä maksavaurion ajankohtana ovat maksansiirron ennustetta parantavia tekijöitä, mikä tulisi ottaa huomioon hoitopäätöksiä tehtäessä.

Abstract Mitochondrial DNA depletion and deletions are related to mutations in the nuclear genes responsible for replication and maintenance of mitochondrial DNA (mtDNA). The POLG1 gene encodes the enzyme responsible for replication of mtDNA. A particular feature of the POLG1 mutations is an increased risk of acute liver failure (ALF) upon exposure to sodium valproate (VPA), but the pathomechanism is not resolved. The present work studies the molecular genetic aetiology and clinical phenotypes associated with mtDNA depletion and deletion. Another objective was an investigation of clinical phenotypes in POLG1 mutations and disentangling the pathomechanism of VPA-induced ALF in POLG1 mutations. Mitochondrial toxicity of VPA was examined using HepG2 cells as an experimental in vitro model. In this work, mtDNA depletion was associated with severe neonatal-onset encephalopathy. Furthermore, mtDNA depletion was found in muscle dystrophy as a secondary finding to muscle degradation. Multiple mitochondrial DNA deletions were found in two patients with Kearns-Sayre syndrome suggesting a genetic origin of the disease. POLG1 p.R722H mutation has been previously reported as a neutral polymorphism, but we found evidence suggesting that POLG1 p.R722H could be a pathogenic mutation in a homozygous or compound heterozygous state. We identified retrospectively five patients, who required liver transplant after VPA-induced ALF. All five patients harboured POLG1 mutations supporting the evidence of POLG1 mutations as a risk factor for VPA-induced ALF. Previously, patients with POLG1 mutations have been considered unsuitable for liver transplantation, but we found that homozygous POLG1 mutations and adolescent or adult-onset disease predicted a good outcome following liver transplantation. In vitro studies on HepG2 cells showed that VPA disturbs mitochondrial respiration. Our results expand the phenotypes and molecular genetic features in mitochondrial DNA depletion and deletion syndromes. We found evidence that POLG1 mutations are not a contraindication for liver transplantation; rather, mutation status and age at onset affect survival. This finding should be taken in consideration in the treatment of VPA-induced ALF. Furthermore, our findings indicate that sodium valproate is toxic to mitochondria and should be avoided in patients with mitochondrial disease. / Tiivistelmä Mitokondrion DNA:n (mtDNA) kahdentumisesta ja ylläpidosta vastaavien tuman geenien mutaatiot voivat johtaa mtDNA:n määrän vähenemiseen (depleetioon) ja katkoksiin (deleetioihin). MtDNA:n kahdentumisesta vastaavaa entsyymiä koodaa tuman POLG1-geeni. POLG1-mutaatioihin liittyy kohonnut riski sairastua natriumvalproaatin (VPA) aiheuttamaan akuuttiin maksavaurioon. Tutkimuksen tavoitteena oli tutkia mtDNA:n depleetion ja deleetioiden molekyyligeneettistä etiologiaa ja kliinisiä taudinkuvia. Tutkimuksessa selvitettiin myös POLG1-mutaatioihin liittyviä taudinkuvia ja POLG1-mutaatioihin liittyvän akuutin maksavaurion patomekanismia. VPA:n vaikutusta mitokondrioiden toimintaan tutkittiin in vitro HepG2-solumallissa. Tutkimuksessa todettiin mtDNA:n depleetion liittyvän vaikeaan varhain alkavaan aivosairauteen. Depleetio todettiin myös sekundaarisena merosiini-negatiivisessa lihasdystrofiassa. Kahdella Kearns-Sayren syndroomaa sairastavalla potilaalla todettiin multippelit mtDNA:n deleetiot, mikä viittaa syndrooman geneettisen alkuperään. POLG1 p.R722H-mutaatiota on aiemmin pidetty neutraalina polymorfiana, mutta tutkimuksen tulokset viittasivat siihen, että homotsygoottisena tai yhdistelmäheterotsygoottisena mutaatio on tautia aiheuttava. Helsingin yliopistollisen sairaalan elinsiirtorekisteristä tunnistettiin retrospektiivisesti viisi potilasta, jotka olivat saaneet maksansiirteen VPA:n aiheuttaman maksavaurion vuoksi. Kaikilla viidellä potilaalla todettiin POLG1-geenin mutaatio, mikä vahvistaa käsitystä geenin yhteydestä VPA:n aiheuttamaan maksavaurioon. POLG1-mutaatioita on pidetty vasta-aiheena maksansiirrolle, mutta tutkimuksessa todettiin homotsygoottisena esiintyvän POLG1-mutaation ja nuoruusiällä tai varhaisella aikuisiällä alkaneen taudin liittyvän parempaan maksansiirron jälkeiseen ennusteeseen. HepG2-solumallilla tehdyt tutkimukset osoittivat VPA:n haittaavan mitokondrioiden solyhengitystä. Tutkimuksen tulokset tuovat lisätietoa mtDNA:n depleetioon ja deleetioihin liittyvistä taudinkuvista ja molekyyligeneettisestä taustasta. POLG1-mutaatiot eivät ole ehdoton vasta-aihe maksansiirrolle; potilaan geneettinen status ja ikä taudin alkamishetkellä vaikuttavat ennusteeseen, mikä tulisi huomioida potilaiden hoidossa. Tulokset myös osoittivat VPA:n olevan mitokondriotoksinen lääke, jonka käyttöä tulisi välttää mitokondriotautipotilaiden hoidossa.