University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCE PHARMACOKINETIC EVALUATION OF CHITOSAN COATED HYDROXYPROPYLMETHYL CELLULOSE (HPMC) MICROPARTICLES OF LEVODOPA AND CARBIDOPA BY BENEDICTA OBENEWAA DANKYI (10637072) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IN PHARMACOLOGY DEGREE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY JULY, 2019 University of Ghana http://ugspace.ug.edu.gh DECLARATION I, Benedicta Obenewaa Dankyi, hereby declare that this project, aside other cited works, is the outcome of my research carried out under the supervision of Dr. Seth Kwabena Amponsah and Prof. Henry Nettey. This work has not in part or whole been submitted and accepted elsewhere for the award of a degree. …………………… …………………. Benedicta Obenewaa Dankyi Date (Student) …………………… …………………… Dr. Seth Kwabena Amponsah Date (Supervisor) … …27TH FEBRUARY, 2020… Prof. Henry Nettey Date (Co-Supervisor) i University of Ghana http://ugspace.ug.edu.gh ABSTRACT Background: Levodopa, a prodrug of dopamine, remains the gold standard in the treatment of Parkinson’s disease. Current levodopa drugs are formulated in combination with an aromatic amino acid decarboxylase inhibitor, carbidopa, to prevent peripheral metabolism of levodopa. However, chronic use of levodopa is associated with potentially disabling motor complications and side effects, which arise from variable plasma concentration of the drug. Several attempts have been made to improve the formulation and drug delivery of levodopa with the aim of providing constant plasma levels in order to improve the drug’s efficacy. Microparticluate drug delivery systems have shown potentiality to deliver drugs at their target sites over a long of period of time while maintaining constant plasma concentrations. The aim of this study was to formulate and evaluate the pharmacokinetics of chitosan coated hydroxypropylmethyl cellulose (HPMC) microparticles of levodopa and carbidopa using in vitro and in vivo models. Methodology: Microparticles were formulated by encapsulating levodopa/carbidopa powders in HPMC using the spray-drying method. The levodopa microparticles were evaluated for size, drug content, percentage drug loading capacity, encapsulation efficiency and in vitro release profile. For pharmacokinetic evaluation, Sprague Dawley rats were administered either levodopa/carbidopa powder, levodopa/carbidopa microparticles or Sinemet CR (a controlled release formulation of levodopa/carbidopa). Blood samples were collected after predetermined times after the third dose. Plasma was obtained from blood and levodopa levels determined by high performance liquid chromatography. Pharmacokinetic parameters; maximum plasma concentration (Cmax), the time it takes to achieve this peak (Tmax), area under the curve (AUC) and half-life (t1/2) of were estimated from concentration-time curves. Results: The particle size obtained ranged between 0.04 µm to 6 µm with a mean size of 0.2 µm. Of the expected 20% drug loading, the actual drug loading capacity of the microparticles ii University of Ghana http://ugspace.ug.edu.gh was found to be 19.1%, giving an encapsulation efficiency of 95.6%. The in vitro release kinetics showed a controlled and sustained drug release profile of levodopa microparticles with 80% drug release occurring at 12 h. In vivo pharmacokinetic studies showed a kinetic profile of levodopa/carbidopa microparticles as compared to the conventional control release formulation. The AUC (704.5 ± 85.37), and Cmax (262.4 µg/mL) of levodopa/carbidopa microparticles were relatively higher than Sinemet CR (AUC 252.7 ± 33.88 and Cmax 128.8 µg/mL). Conclusion: Findings from the study suggest that levodopa/carbidopa microparticles may give adequate levels of levodopa plasma concentration over a period of time. iii University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to my family for their encouragement and support throughout this study. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I am thankful to the Almighty God for his mercies, divine favor, guidance and protection. I wish to express heartfelt gratitude to my supervisors, Dr. Seth Amponsah and Prof. Henry Nettey. Their resources helped lessen the financial burden and their constant dedication, guidance and motivation kept me on my toes. I also appreciate their intuitive reviews and evaluations that shaped and enhanced the quality of this study. A profound thanks to Dr. Grace Lovia Allotey-Babington for her immense support. God bless you all. A special appreciation goes to Lt. Obeng Jnr., Mr. Martin Akandawen, miss Stephanie Osafo Kissi, Mr. George Marfo, Mr. Ekow Addai, Miss Agyeiwaa Christabel, my lecturers, the entire staff of the Department of Pharmacology and Toxicology, University of Ghana School of Pharmacy and to Mr. Sasu Clement for his technical assistance. v University of Ghana http://ugspace.ug.edu.gh Table of Contents DECLARATION ...................................................................................................................... i ABSTRACT ............................................................................................................................. ii DEDICATION ........................................................................................................................ iv LIST OF FIGURES ................................................................................................................. x LIST OF TABLES .................................................................................................................. xi LIST OF ABBREVIATIONS .............................................................................................. xii CHAPTER ONE ...................................................................................................................... 1 1.0 BACKGROUND ................................................................................................................. 1 1.1 PROBLEM STATEMENT .................................................................................................. 5 1.2 JUSTIFICATION ................................................................................................................ 7 1.3 AIM ...................................................................................................................................... 8 1.4 SPECIFIC OBJECTIVES .................................................................................................... 9 CHAPTER TWO ................................................................................................................... 10 2.0 LITERATURE REVIEW .................................................................................................. 10 2.1 PARKINSON’S DISEASE ................................................................................................ 10 2.1.1 Background and History ............................................................................................ 10 2.1.2 Epidemiology ............................................................................................................. 12 2.1.3 Comorbidity ............................................................................................................... 13 2.1.4 Etiology ...................................................................................................................... 14 2.1.5 Pathophysiology ......................................................................................................... 16 2.1.6 Clinical Presentation .................................................................................................. 18 vi University of Ghana http://ugspace.ug.edu.gh 2.1.7 Diagnosis .................................................................................................................... 21 2.1.8 Treatment ................................................................................................................... 22 2.2 LEVODOPA ...................................................................................................................... 23 2.2.1 Introduction ................................................................................................................ 23 2.2.2 Pharmacokinetics of levodopa/carbidopa .................................................................. 26 2.2.3 Pharmacodynamical Changes in the body with the Progression of PD ..................... 28 2.2.4 Levodopa drug delivery systems ................................................................................ 29 2.3 MICROPARTICLES ......................................................................................................... 34 2.3.1 Introduction ................................................................................................................ 34 2.3.2 Techniques of Microparticle preparation ................................................................... 35 2.3.3 Materials used in the Preparation of Levodopa Microparticles ................................. 36 2.3.4 Evaluation/Characterization of Microparticles .......................................................... 40 CHAPTER THREE ............................................................................................................... 43 3.0 MATERIALS AND METHODS ....................................................................................... 43 3.1 STUDY DESIGN............................................................................................................... 43 3.1.1 Study Location ........................................................................................................... 43 3.2 PREPARATION AND CHARACTERIZATION OF LEVODOPA MICROPARTICLES .................................................................................................................................................. 43 3.2.1 Preparation of microparticles ..................................................................................... 44 3.2.2 Particle Size Determination ........................................................................................ 44 3.2.3 Content Analysis ........................................................................................................ 45 3.2.4 Encapsulation Efficiency ........................................................................................... 45 3.2.5 In Vitro Drug Release Test ......................................................................................... 45 vii University of Ghana http://ugspace.ug.edu.gh 3.3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) FOR LEVODOPA LEVELS ................................................................................................................................... 46 3.3.1 Preparation of Stock and Working Solutions. ............................................................ 46 3.4 IN VIVO PHARMACOKINETIC EVALUATION OF LEVODOPA MICROPARTICLES ............................................................................................................... 47 3.4.1 Animal Acquisition and Housing ............................................................................... 47 3.4.2 Animal Groupings for Pharmacokinetic Study .......................................................... 47 3.4.3 Drug Administration and Sample Collection ............................................................. 47 3.4.4 Determination of Plasma Levodopa Levels ............................................................... 48 3.5 DATA ANALYSIS ............................................................................................................ 49 CHAPTER FOUR .................................................................................................................. 51 4.0 RESULTS .......................................................................................................................... 51 4.1 CHARACTERIZATION OF LEVODOPA MICROPARTICLES ................................... 51 4.1.1 Particle Size Distribution ........................................................................................... 51 4.1.2 Drug Content Analysis ............................................................................................... 51 4.1.3 Percent Drug Loading ................................................................................................ 53 4.1.4 Encapsulation Efficiency ........................................................................................... 53 4.2 IN VITRO RELEASE OF LEVODOPA FROM MICROPARTICLES ............................ 54 4.3 IN VIVO PHARMACOKINETIC EVALUATION OF LEVODOPA MICROPARTICLES ............................................................................................................... 55 4.3.1 Concentration-time curves for 3 treatment groups of Sprague-Dawley (SD) rats ..... 55 viii University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE ................................................................................................................... 59 5.0 DISCUSSION .................................................................................................................... 59 5.1 CONCLUSION .................................................................................................................. 65 5.2 RECOMMENDATIONS ................................................................................................... 65 REFERENCES ....................................................................................................................... 66 APPENDICES ........................................................................................................................ 95 ix University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 2.1 : A diagram of the brain showing the parts affected by Parkinson's Disease ......... 17 Figure 2.2: Schematic representation of Levodopa and dopamine synthesis pathway (Hadad et al., 2018). ................................................................................................................................. 25 Figure 2.3 : Levodopa metabolism in the periphery and the CNS (Palma et al., 2013). ......... 26 Figure 2.4: Schematic representation of preparation of microparticles by spray drying method (Agnihotri et al., 2004). ........................................................................................................... 36 Figure 2.5 : Chitosan microparticles prepared by spray drying and crosslinked with glutaraldehyde (He et al., 1998) .............................................................................................. 39 Figure 4.1: Results showing the particle size distribution of the formulated levodopa microparticles.. ......................................................................................................................... 51 Figure 4.2: Levodopa standard curve showing the equation of the line (y = 2.1383x + 1.4208) and the correlation coefficient (R2 = 0.9958) .......................................................................... 52 Figure 4.3: Graph showing the average cumulative percentage release of levodopa against time. .................................................................................................................................................. 54 Figure 4.4: Concentration-time curves for the 3 treatment groups of SD rats ......................... 56 x University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 4.1: Concentrations obtained from the content analysis of levodopa. The test was done in triplicate giving an average levodopa concentration of 478.84 µg/ml. ............................... 52 Table 4.2: Showing data for the in vitro release of levodopa from the formulated microparticles .................................................................................................................................................. 55 Table 4.3: Pharmacokinetic parameters of rats in the 3 treatment groups ............................... 57 xi University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS ANOVA Analysis of Variance AUC Area Under the Curve BBB Blood Brain Barrier CD Carbidopa Cmax Maximum Plasma Drug Concentration CNS Central Nervous System COMT Catechol-O-Methyl Transferase CR Controlled Release EDTA Ethylenediaminetetraacetic Acid GPi Internal Globus Pallidus HCL Hydrochloric acid HPLC High Performance Liquid Chromatography HPMC Hydroxypropylmethyl Cellulose Ke Elimination Rate Constant LBs Lewy Bodies LD Levodopa LRRK2 Leucine-rich Repeat Kinase 2 gene xii University of Ghana http://ugspace.ug.edu.gh MAO-B Monoamine Oxidase-B MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MP Microparticles PD Parkinson's Disease PRKN Parkin gene PINK1 PTEN-Induced Putative Kinase 1 gene PBS Phosphate Buffered Saline SD Sprague-Dawley SNCA Alpha Synuclein SNpr Substantia Nigra pars reticulata STDEV Standard Deviation Tmax Time to achieve maximum plasma drug concentration xiii University of Ghana http://ugspace.ug.edu.gh xiv University of Ghana http://ugspace.ug.edu.gh xv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 BACKGROUND Parkinson’s disease (PD) as defined by Khor and Hsu, (2007) is a progressive neurodegenerative disorder of the extrapyramidal nervous system, which affects the individual’s mobility and ability to control the skeletal muscular system. The disease mostly presents with tremor (shaking), rigidity, and bradykinesia (slowed movements), with postural instability appearing in some patients as the disease progresses (Kouli et al., 2018). It was first medically described by the English scientist, James Parkinson in 1817. In a paper he wrote referring to the disease as the Shaking Palsy, he described it as “Involuntary tremulous motion, with lessened muscular power, in parts not in action and even when supported; with a propensity to bend the trunk forward and to pass from a walking to a running pace: the senses and intellects being uninjured” (Parkinson, 1817). Many researchers and clinicians from the 19th century to the present day have attempted to further characterize and decipher the nature of the disease, yielding phenomenal discoveries in therapeutics (Goldman and Goetz, 2007). After Alzheimer’s disease, PD is considered the most common neurodegenerative disorder which usually affects the elderly and the middle aged (Cole and Murphy, 2002). The disease prevalence ranges from 100 - 200 per 100,000 people, and the annual incidence is estimated to be 15 per 100,000 according to Tysnes and Storstein, (2017). A survey done by Chen et al., in 2001 projects that, with an ageing population, both the prevalence and incidence of Parkinson’s disease are expected to rise by more than 30% by the year 2030 which will increase the economic and social burden. PD is pathologically characterized by the loss of dopamine producing neurons in the basal ganglia of the brain. The low dopamine levels in the brain leads to fundamental motor 1 University of Ghana http://ugspace.ug.edu.gh symptoms such as: bradykinesia (slowed movements), distal tremor (shaking) and muscle rigidity (stiffness). Although, neurodegeneration is not limited to only the nigral dopaminergic neurons but also involves cells located in other regions of the neural network. This often leads to the non-motor features associated with PD and is in many cases evident in PD patients even years or decades before the motor symptoms set in (Kouli et al., 2018). Comorbidities known to occur with PD include sleep disturbances, depression, dementia, falls and fractures and impulse control disorders as reported in several articles in literature (Suzuki et al., 2011; Dissanayaka et al., 2011; Aarsland and Kurz, 2010; Duncan et al., 2012 and Djamshidan et al., 2011). The manner of the causation of the disease is generally unknown, however, it is postulated that both genetic and environmental factors may be involved (Nord, 2017). With age being the biggest risk factor, the median age for the onset of PD is reported to be 60 years (Lees et al., 2009) with higher prevalence recorded in Europe, North America, and South America compared with African, Asian and Arabic countries (Kalia and Lang, 2015). Environmental influences such as smoking, caffeine consumption, and pesticide exposure have been hypothesized to alter the risk of Parkinson’s disease development, however the role of these factors still remains unclear (Kouli et al., 2018). Due to the idiopathic nature of the disease, treatment of PD focuses on reducing symptoms while improving the overall quality of life of the patient (Horstink et al., 2006). Treatment is symptomatic and this may be achieved through drugs that are metabolized to dopamine, that activate the dopamine receptor, or that prevent the breakdown of endogenous dopamine (Zahoor et al., 2018). Drugs used in the management of PD include Catechol-O-Methyl transferase (COMT) inhibitors, example entacapone; dopamine agonists, example ropinirole; monoamine oxidase B inhibitors, example selegiline; and dopamine precursor, levodopa 2 University of Ghana http://ugspace.ug.edu.gh (Korczyn, 2004). Currently, the most effective treatment, and also considered gold standard in PD therapy is levodopa-based preparations (Gunay et al., 2016). Levodopa, a precursor of dopamine, is able to cross the blood brain barrier (BBB) as opposed to dopamine itself which is unable to cross the BBB. After absorption and transport across the BBB, levodopa is converted into the neurotransmitter dopamine by dopa decarboxylase thus restoring the level of dopamine in the depleted striatum (Zahoor et al., 2018; Kouli et al., 2018). The challenge with the use of levodopa is its peripheral (gut wall, liver and kidney) decarboxylation to dopamine (Waller and Sampson, 2018) that is, the conversion of levodopa to dopamine outside the CNS by dopa decarboxylase. This often leads to untoward effects such as nausea, vomiting, cardiac arrhythmias, and hypotension. Additionally, the amount of plasma levodopa decreases (as a result of the short half-life of levodopa), and an attempt to increase drug dose often leads to an increase in peripheral adverse effects (Brasnjevic et al., 2009). For this reason, levodopa is co- administered with a peripheral dopa decarboxylase inhibitor, Carbidopa. Carbidopa prevents the peripheral conversion of levodopa to dopamine and as such, controls levodopa-induced peripheral adverse reactions, and increases levels of levodopa in the brain (Afroz and Bach, 2014). Carbidopa does not cross the blood brain barrier (BBB) hence selectively prevents the formation of dopamine in peripheral tissues. The current and most frequently prescribed combination drugs available on the market for the management of PD is levodopa/carbidopa with the trade name SINEMETâ. The compound is available in several formulations, including immediate release preparations, modified release preparations, which is useful for controlling symptoms overnight and limiting early morning 3 University of Ghana http://ugspace.ug.edu.gh symptoms, as well as suspensions, which can be useful for patients that have swallowing difficulties (Oertel, 2017; Koller and Pahwa, 1994; MacMahon et al., 1990). Although the combination therapy of levodopa/carbidopa seems effective, studies show that there are a few drawbacks with its prolonged use and can result in significant motor complications such as dyskinesias (abnormal involuntary hyperkinetic movements) and severe on-off motor fluctuations (Salat and Tolosa, 2013). These potentially disabling motor complications can be associated with variable drug absorption and transit across the BBB, and fluctuating plasma concentrations of levodopa due to its short half-life (Senek et al., 2017). Recent studies have tried novel ways to maintain a near constant plasma concentration of levodopa in order to maximize therapeutic effect. Some of these new approaches include controlled release formulations and improved drug delivery systems of levodopa (Goole et al., 2007; Di Stefano et al., 2009; Ngwuluka et al., 2010; LeWitt, 2015). Studies have reported some success with these approaches that seek to improve the clinical efficacy and side effect profiles of levodopa. One such approach is formulating levodopa using microparticles, characteristically free flowing powders consisting of proteins or biodegradable polymers, which deliver the drug to target site with specificity and in a controlled manner (Sahil et al., 2010). Microparticles have gained increasing popularity in medical technology due to such advantages as the ability to target the release of the encapsulated material, improve bioavailability of the drug, provide controlled and sustained release profiles, improved efficiency in treatment and better patient compliance (Nagpal et al., 2016). Microparticles can be made of polymers, which act as carriers in forming efficient drug delivery systems. The polymers are responsible for microparticle stability, drug loading 4 University of Ghana http://ugspace.ug.edu.gh capacity and tunable properties (Saikia et al., 2015). Chitosan, a natural cationic polymer is a hydrophilic biodegradable polysaccharide which has received increasing attention in research mainly due to its low toxicity, biodegradable and target specific delivery properties (Saikia et al., 2015). The current study, therefore, seeks to formulate and evaluate the pharmacokinetics of chitosan microparticles of levodopa and carbidopa. 1.1 PROBLEM STATEMENT Parkinson’s disease (PD) is the second most common neurodegenerative disorder, being a notable cause of neurological disability (Goole and Amighi, 2009). Unfortunately, PD has no cure and this may be due to its idiopathic nature. As such, the current therapies in use can neither slowdown nor stop the progression of the disease (Singh et al., 2007). Treatment is therefore aimed at relieving symptoms (while minimizing adverse effects of drugs) and improving quality of life of patients. Of all the drugs available for the management of PD, none surpasses the clinical efficacy of dopamine’s biological precursor, levodopa (LeWitt, 2008). Although levodopa is regarded as the gold standard, the motor complications and side effects that comes with its prolonged use is potentially disabling (Gunay et al, 2016). Patients initially respond well to treatment; however, over time, the beneficial effects are associated with complications such as motor fluctuations and levodopa-induced dyskinesias (Muller, 2012). Several attempts have been made to improve the clinical efficacy of levodopa. Dopamine agonists may slowdown the occurrence of motor complications by allowing the use of lower doses of levodopa, and ensuring long-lasting receptor stimulation. However, dopamine agonists may even worsen the adverse effects compared to using levodopa alone (Stowe et al., 2008). Also, with the combination of levodopa/carbidopa with COMT inhibitors and/or MAO- 5 University of Ghana http://ugspace.ug.edu.gh B inhibitors, although there is improved levodopa bioavailability, plasma concentrations still fluctuate (Stocchi and Olanow, 2004; Pfeiffer, 2006). Other levodopa treatment strategies to minimize motor complications have been developed several years ago, which included intravenous infusion of levodopa. However, it was found that levodopa was irritating to the veins and therefore could not be administered continuously for more than 7-10 days via the intravenous route (Shoulson et al., 1975; Hardie et al., 1984). Intraduodenal levodopa infusion has also been proven to be a successful therapeutic approach for PD (Kurlan et al., 1988). However, the most frequently reported complications include dislocation of the distal part of the tube from the duodenum into the stomach which leads to sudden failure of clinical response with recurring motor fluctuations (Nyholm et al., 2005). It is therefore apparent that oral levodopa/carbidopa formulations provide safer profiles as compared to the other treatment strategies. The development of oral controlled release formulations aims to minimize levodopa-associated dyskinesias and motor fluctuations (caused by pulsatile stimulation of dopaminergic receptors which occurs as a result of the short acting levodopa), by providing more continuous dopaminergic stimulation (Olanow et al., 2006). The most current and effective drug treatment for PD is Sinemet CR, an oral formulation of controlled release levodopa/carbidopa. Pharmacokinetic studies show that steady state levodopa plasma levels do not fluctuate as much with Sinemet CR compared to the immediate release, Sinemet. However, the bioavailability of Sinemet CR is about 30% less than Sinemet (Yeh et al., 1989). Additionally, Sinemet CR is absorbed slowly, and although the absorption occurs over an extended period, there is a late onset of drug action. Clinically, it is difficult to use Sinemet CR in patients with advanced PD since the late onset to clinical benefit could worsen their motor complications (Contin et al., 1996). These drawbacks of Sinemet CR have 6 University of Ghana http://ugspace.ug.edu.gh been suggested to be a result of delayed gastric emptying and erratic absorption which occurs across the intestinal lumen (Salat and Tolosa, 2013). Clearly, patients are still left with unmet clinical needs and new oral levodopa formulations are needed to overcome the irregular absorption and variable plasma concentrations of levodopa. 1.2 JUSTIFICATION The oral route is undoubtedly the most preferred route of drug administration. However, extensive first pass metabolism and narrow therapeutic window associated with the oral route limits the therapeutic benefits of many drugs. Therefore, current research has tried to explore new strategies to improve oral drug delivery. An intelligent approach to improve the bioavailability and pharmacokinetic properties of drugs is through the use of microparticles as carriers for controlled release drug delivery (Kumar et al., 2011). It has been established in literature that microparticles, because of their distinctive properties such as small particle size, drug loading capacity, entrapment efficiency and controlled release profiles are useful drug delivery systems that are successful in encapsulating both water insoluble and sparingly-soluble agents to improve their efficacy (Bale et al., 2016). Polymer based delivery systems have been shown to improve the pharmacokinetics of drugs, decrease side effects and increase efficacy (Saikia et al., 2015). One unique polymer for oral drug delivery: pH sensitive and a reactive primary amine group, is chitosan (Sahil et al., 2010). Chitosan, a natural polymer, has remarkable physicochemical (cationic polyamine, easily modified chemically and reactive side groups) and biological (biocompatible, non-toxic and biodegradable) properties (Sandford, 1990). This makes chitosan a promising candidate for drug delivery in the gastrointestinal tract (Saikia et al., 2015). Additionally, chitosan consists of several positively charged groups which readily interact with the negatively charged mucous 7 University of Ghana http://ugspace.ug.edu.gh membrane, thereby increasing adhesion to the mucosal surface, and thus improves contact time for drug absorption (Saikia et al., 2015). With the current increase in the use of polymer based microparticulate delivery systems, there is the need for extensive studies into chitosan as a way of improving oral drug delivery of levodopa. Levodopa has a short half-life (Goole et al., 2009), hence the need to develop controlled release formulation in order to reduce fluctuations and improve clinical efficacy. Carbidopa on its own has little pharmacological activity, but it is combined with levodopa as a peripheral decarboxylase inhibitor to inhibit the breakdown of levodopa in the periphery. This increases the levels of levodopa reaching the brain and reduces its peripheral side effects (Safavi et al., 2007). There is currently no known study that has evaluated the pharmacokinetics of microparticles of levodopa for oral drug delivery. If pharmacokinetic studies in the current study show improved bioavailability and constant plasma concentration with levodopa microparticles, this would be of great clinical relevance, as it will address one shortfall in PD therapy. Furthermore, this novel therapeutic approach, if this shows stable plasma levodopa levels, could be incorporated into standard treatment guidelines for PD. Finally, for patient care, this novel approach may be one that will improve the quality of life of PD patients. Therefore, the current study seeks to employ the use of chitosan microparticles for controlled release formulation of levodopa and carbidopa in a mucoadhesive oral drug delivery approach. 1.3 AIM To formulate and evaluate the pharmacokinetics and biodistribution of chitosan microparticles of levodopa and carbidopa using in vitro and in vivo models. 8 University of Ghana http://ugspace.ug.edu.gh 1.4 SPECIFIC OBJECTIVES 1. To formulate microparticles of levodopa and carbidopa by encapsulating drugs in hydroxypropylmethyl cellulose (HPMC), coating with chitosan and evaluating microparticles for size, drug content and encapsulation efficiency and in vitro release characteristics. 2. To estimate the pharmacokinetic profile of the formulated levodopa microparticles in an animal model. 9 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 PARKINSON’S DISEASE 2.1.1 Background and History Parkinson’s disease (PD) is a neurodegenerative disorder characterized by four fundamental motor features: resting tremor, bradykinesia, rigidity and postural instability (Hughes et al., 1992). PD affects a notable percentage of the adult population (Wickremaratchi et al., 2009; McCrone et al., 2011). It was first medically described by the English Scientist, James Parkinson in 1817. In a paper he wrote referring to the disease as the Shaking Palsy, he described the clinical signs and symptoms as “Involuntary tremulous motion, with lessened muscular power, in parts not in action and even when supported; with a propensity to bend the trunk forward and to pass from a walking to a running pace: the senses and intellects being uninjured” (Parkinson, 1817). Parkinson in his paper (An Essay on the Shaking Palsy) perceptively describes the features of the disease as “insidious onset and slow progression, presence of rest tremor, flexed posture and festinating gait”. He observed that some patients had more advanced states with increased immobility and dependence, as well as sleep disturbances and altered bodily functions such as with the bowels, slowed speech and difficulty swallowing. Although Parkinson found that the symptoms presented with the disease had similar features with other neurological diseases published in medical literature, he truly believed that the ‘shaking palsy’ was unique and warranted further study. A seminal figure in the history of Parkinson’s disease was the French physician, Jean-Martin Charcot (1825-1893). Charcot acknowledged that Parkinson would always be recognized for 10 University of Ghana http://ugspace.ug.edu.gh the first description of this condition but noting errors in Parkinson’s generality, he sought to add his own contributions (Goetz, 1986, 2002). His elaboration of motor and non-motor features of the disorder, distinction of the disease from other similar disorders, advocacy of clinicopathological studies and of course, coining the eponym Parkinson’s Disease remain his legacy. Treatment used in Charcot’s time did not reveal significant advances in efficacy. Although medications such as strychnine, belladonna and opium were tried with limited benefit, hyoscyamine, an anticholinergic agent, provided some palliative action. Charcot’s contribution to PD include not only his studies but also that of his pupils. Pupils of Charcot such as Ordenstein wrote his medical thesis on Parkinson’s disease (1867) and introduced belladonna as a treatment. Eduard Brissaud (1852-1909) described midbrain lesions in Parkinson’s disease which set the stage for future investigations of phenotypes and clinicopathological correlates of neurological diseases. Although it has been proposed that PD emerged as a result of the industrial revolution, there is some evidence that a disease known as “kampavata,” consisting of shaking (kampa) and lack of muscular movement (vata), existed in the ancient Indian medical system, Ayurveda, as long as 4500 years ago (Manyam, 1990). The Mucuna Puriens plant was used in ancient times to treat symptoms, and was later discovered to contain levodopa (Katzenschlager et al., 2004). Many researchers and clinicians from the 19th century to the present day have attempted to further characterize and decipher the nature of the disease, and have yielded incredible discoveries and advances in medical and surgical therapeutics (Goldman and Goetz, 2007). 11 University of Ghana http://ugspace.ug.edu.gh 2.1.2 Epidemiology 2.1.2.1 Global prevalence of Parkinson’s disease Parkinson’s disease, is the second most common neurodegenerative disease and affects over 1% of the population above age 55 and nearly 3 % of the population over age 70 (Samii et al., 2004). In Africa, prevalence rate ranges from 7/100,000 in Ethiopia to 67/100,000 in Nigeria, according to studies done by Williams et al., (2018). The low prevalence rate in Africa is reported to be attributed to the youthfulness of the African population, paucity of published reports and cultural perception of neurologic disorders (Akinyemi and Okubadejo, 2010). The mean age at which PD usually presents is 60 years, with about 80 % of individuals developing the disorder between the ages of 40 and 70 years. Such cases where individuals develop symptom onset before age 40, is classified as young onset PD and this occurs in about 5 % of the PD cases (Yao et al., 2013). 2.1.2.2 Socio-demographic factors It has been reported that PD affects more men than women with about 50% cases occurring more in men, however the reasons for this disparity remains unclear (Hirtz et al., 2007). Although PD affects people worldwide, studies have shown that incidence rate is higher in the developed countries. Increase in the use of pesticides has also been found to be associated with the increased risk of acquiring PD among people living in the rural areas. However, these perceivable risks are not fully characterized and age continues to be the major risk factor for PD (Yao et al., 2013). 12 University of Ghana http://ugspace.ug.edu.gh 2.1.3 Comorbidity 2.1.3.1 Depression PD patients usually experience frustration, demoralisation, grief, and embarrassment as normal psychological reactions to the disease. Depression in PD is as common as the motor symptoms and involves more prevalent mood changes that generally resemble idiopathic forms of depression (Jankovic, 2008). There is convincing evidence that depression in PD is an inherent part of the disorder and not just a reaction to disability. Drugs used to treat psychologic disorders and other anti-Parkinsonian agents may aggravate depression in PD (Jankovic, 2008; Murray et al., 2012). 2.1.3.2 Anxiety About 40 % of patients with PD usually experience anxiety disorders in the form of agitation, panic and social phobia. This may occur in isolation or together with depression or cognitive impairment (Jankovic, 2008). Drug induced motor fluctuations can aggravate the problem by instigating anxiety during the off periods which in extreme cases may resemble anger attacks. In general, the anxiety syndromes in Parkinson’s disease mimic those in idiopathic conditions and frequently co-occur with depression (Jankovic, 2008). 2.1.3.3 Cognitive impairment Nearly all PD patients experience some level of cognitive impairment. This usually occurs in the advanced stages of the disease and often presents as a result of the dysfunction of the frontal lobe. Common cognitive disorders include difficulty with long-term planning, memorizing or retrieving new information, mental flexibility and word fluency. However, language and simple mathematical skills are relatively spared, unlike in patients with Alzheimer’s disease (Ayano, 2016). 13 University of Ghana http://ugspace.ug.edu.gh 2.1.3.4 Psychosis About 20% of PD patients receiving long term treatment with dopaminergic agents such as bromocriptine, levodopa and pramipexole, usually experience psychotic symptoms. More often than not, the psychotic symptoms present as chronic hallucinations and delusions. Up to about 50% of patients experience these psychotic symptoms over the course of their illness. Psychosis most commonly occurs in patients who have corresponding cognitive impairment (Shergill et al., 1998; Friedman, 2010). 2.1.4 Etiology Parkinson’s disease is dependent on a number of factors, with both genetic and environmental factors playing a major role. Age remains the biggest risk factor for PD, with a mean onset age of 60 years (Lees et al., 2009). 2.1.4.1 Pesticides, herbicides and heavy metals It was discovered in 1983, that several people after the intravenous administration of drugs contaminated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), exhibited typical signs of PD. Following this, there were findings that MPTP selectively damages dopaminergic cells in the substantia nigra (Langston et al., 1983) and this lead to the hypothesis that exposure to environmental toxins might be associated to the risk of PD. MPTP as well as certain herbicides and pesticides have been found to selectively inhibit Complex-I and induce depletion of dopamine in animal studies (Betarbet et al., 2000). In a study done by Greenamyre and Hastings, (2004) complex I defects in the substantia nigra was found in patients with irregular PD. It has been hypothesized that welding and exposure to heavy metals such as iron, manganese, copper, lead, aluminium or zinc leads to accumulation of these heavy metals in the substantia nigra and increasing oxidative stress which in turn leads 14 University of Ghana http://ugspace.ug.edu.gh to the degeneration of cells (Lai et al., 2002). However, some investigations have been done and the report suggests that the relation between metal exposure and risk of PD is uncertain (Lai et al., 2002; Jankovic, 2005). 2.1.4.2 Cigarette Smoking Out of the many risk factors studied for PD, cigarette smoking is one of the few for which very consistent results have been obtained. Interestingly, epidemiological studies have shown a reduced risk of PD among cigarette smokers. The reasons underlying this associated reduced risk are not fully understood. In order to explain this possible neuroprotective effect of smoking, several mechanisms have been propounded. Activation of nicotinic acetylcholine receptors on dopaminergic neurons by nicotine or selective agonists has been shown to be neuroprotective in experimental models of PD (Bordia et al., 2015; Srinivasan et al., 2016). Nevertheless, nicotine can also stimulate the release of dopamine, which is involved in the reward mechanisms; it is therefore difficult to confirm whether smoking prevents PD or whether PD helps prevent the habitual use of cigarettes. As a result of the reduction in dopamine in PD, patients may be immune to addictive behaviours, and thus less likely to smoke. This hypothesis is supported by the fact that patients with prodromal PD were able to give up smoking much easier than controls, suggesting this association could be due to the diminished responsiveness to nicotine (Ritz et al., 2014). 2.1.4.3 Caffeine Caffeine as a risk factor for PD has also been studied widely and it has been reported that coffee drinkers have a diminished risk of developing the disease. Caffeine is an adenosine A2A receptor antagonist, which is believed to be protective in PD (Ross et al., 2000) and has been shown to be neuroprotective in a mouse model of PD (Chen et al., 2001). Studies have already 15 University of Ghana http://ugspace.ug.edu.gh reported that there is a 25% risk reduction in developing PD among coffee drinkers (Noyce et al., 2012). Several other studies also investigated the risk of developing PD among coffee drinkers and non-coffee drinkers and published that coffee drinkers showed a reduced risk of developing PD (Ross et al., 2000; Ascherio et al., 2001; Benedetti et al., 2000). 2.1.4.4 Genetics Evidences identified that about 15% of individuals living with PD have a very close relative who also has the disease (Samii et al., 2004). Not less than 5% of people have also been shown to have developed the disease as a result of gene mutation (Lesage and Brice, 2009). Mutations in specific genes have been identified by scientists to have undeniably caused PD. The specific genes that have been implicated code for such proteins as leucine-rich repeat kinase 2 (LRRK2 or dardarin), DJ-1, PTEN-induced putative kinase 1 (PINK1), alpha-synuclein (SNCA), and parkin (PRKN) (Davie, 2008; Lesage and Brice, 2009). More often than not, people having these mutations end up developing PD. Mutations in the gene that code for leucine-rich repeat kinase 2 (LRRK2) is however the most common cause of familial and sporadic PD. It is still inconclusive, the mechanism by which LRRK2 is implicated in the pathogenesis of PD but it is somewhat proposed that mutations in this gene leads to an over activation of its kinase activity by which the pathogenesis of the disease might be associated (Greggio et al., 2006). 2.1.5 Pathophysiology Until the early 20th century, the pathology of PD remained incompletely understood. It was the German pathologist Frederick Lewy who in 1912, reported “neuronal cytoplasmic inclusions in a variety of brain regions”. Another scientist, Tretiakoff, in 1919, found out that the loss of neurons in the substantia nigra pars compacta (SNc) of the brain was the most critical abnormality in PD (a diagram of the brain and parts affected by PD is shown in figure 2.1 below). The importance of dopamine and its depletion from the basal ganglia was discovered 16 University of Ghana http://ugspace.ug.edu.gh by investigators in the late 1950s to be the key to deciphering the pathophysiology of PD (Hornykiewicz, 2006). Even though the cause of the disease is still unclear, striking advances have been made to possibly explain the underlying mechanisms (Jankovic and Sherer, 2014). The presence of Lewy bodies (LBs) and the loss of dopamine producing neurons have been hypothesized to be the neuropathological hallmarks of PD (Braak and Braak, 2000; Kovari et al., 2009). Figure 2.1 : A diagram of the brain showing the parts affected by Parkinson's Disease (Source: Designua/ Shutterstock.com) 17 University of Ghana http://ugspace.ug.edu.gh The loss of dopaminergic function in PD patients is suggested to result from the progressive degeneration of dopamine producing neurons in the SNc which project to the striatum via the nigrostriatal dopamine pathway. Motor activity in the extrapyramidal system is influenced by two types of dopamine receptors, D1 and D2 which are responsible for excitatory and inhibitory reactions respectively. The extrapyramidal system includes the pars reticulata portion of the substantia nigra (SNpr), and the basal ganglia which involves the internal globus pallidal segment (GPi) of the ventral striatum. The loss of striatal dopamine eventually results in increased activity in the GPi/SNpr circuits which leads to the dysfunction of gamma aminobutyric acid (GABA). The inhibition of GABA subsequently inhibits the thalamus and causes a reduction in the ability of the thalamus to activate the frontal cortex which gives rise to decreased motor activity as presented by PD (DeMaagd and Philip, 2015). The other hallmark of the disease is the presence of LBs seen in the histopathological studies of PD brain. These LBs are described as intracellular cytoplasmic aggregates composed of proteins, lipids, and other materials (Braak et al., 2003; Del and Braak, 2012). Excessive production of misfolded forms of ubiquitin proteins which are involved in protein recycling has been found to be the precursor for the formation of these LBs. The formation of the LBs causes lesion patterns in the brain and is believed to be the cause of neurodegeneration in such parts of the brain. Lesions in the dorsal nucleus, medulla, and pons are claimed to be the cause of the early olfactory and rapid eye movement features of PD. The common motor features of PD are also thought to be caused by lesions in the nigrostriatal region during later stages of the disease (Braak et al., 2004). 2.1.6 Clinical Presentation In up to about 90% of patients, PD may begin in an insidious manner such as difficulty in getting out of a chair. Sometimes diagnosis is delayed as the non-motor symptoms preceding 18 University of Ghana http://ugspace.ug.edu.gh the disease may be misinterpreted as related to normal ageing. The non-motor features (autonomic dysfunction, sleep disorders, olfactory dysfunction, fatigue) usually sets in at the early phase of the disease and can last for roughly 4 to 6 years on average (Meissner, 2012; Postuma et al., 2012). Thermoregulatory dysfunction, neuropsychiatric symptoms and other clinical signs may occur as the disease progresses. For some patients, neuropathic and nociceptive pain may occur at either the early or later stages of the disease (Postuma et al., 2012; Maetzler and Hausdorff, 2012) The pre-motor or prodromal phase of PD may start as early as 12-14 years before diagnosis (Postuma et al., 2012). There is however a great deal of evidence supporting the fact that the disease may begin in the peripheral autonomic nervous system and/or the olfactory bulb with the pathology then spreading through the CNS affecting the lower brainstem structures before involving the substantia nigra (Katzenschlager et al., 2008). This may thus explain the presence of non-motor symptoms in PD patients before motor symptoms begin. These non-motor symptoms contribute significantly to disability and poor quality of life and thus predict admission to care homes (Noyce et al., 2012). The clinical features historically associated with PD are the triad of motor symptoms, namely tremor, rigidity and bradykinesia, with postural instability often appearing as the disease progresses (Kouli et al., 2018). Some relationship has been shown between the frequency of motor presentations and the onset of the disease, with tremor specifically, being as twice as common in patients above 64 years than in patients younger than the age of 45 (Suchowersky et al., 2006). 2.1.6.1 Tremor In about two-thirds of PD patients, tremor occurs as the initial symptom. Deuschi et al., defined tremor as “rhythmical, involuntary, oscillatory movement of a body part produced by 19 University of Ghana http://ugspace.ug.edu.gh alternating or synchronous contractions of antagonist muscles” (Deuschi et al., 1998). Tremor normally begins in a mild and recurrent fashion and usually occurs even at rest. By the position that underlines the tremor, it can be classified as either rest tremor, that is, tremor occurring in a body part while there is no voluntarily activated muscle contraction, or action tremor, thus involving voluntary muscle contractions and occurring during movements (Deuschi et al., 1998; Bhidayasiri, 2005). 2.1.6.2 Bradykinesia Bradykinesia, a cardinal motor feature of PD, has been defined by Grabli et al., as “a reduction in the speed, gait and amplitude of a repetitive action involving voluntary movements” (Grabli et al., 2012). Difficulty getting started or initiating movements together with a slow, shuffling gait are common clinical presentations associated with bradykinesia. Patients experiencing bradykinesia may try to overcome it by increasing their walking speed with small, rapid steps in an effort to “catch up” with their displaced centre of gravity (Garcia et al., 2011; Hallett, 2012; Xia and Mao, 2012). It is often difficult for such patients with bradykinesia to turn or enter through a narrow door when confronted with the need to, because of the immobility that is associated with bradykinesia (Mendoca, 2008). Typically, in the advanced states, some patients also experience episodes of “freezing” as an acute manifestation of PD (Xia and Mao, 2012). 2.1.6.3 Rigidity The third most common feature of PD is rigidity, which presents as worsened muscle tone or heightened resistance to a dormant range of motion. This is best described as tension in the muscle, which displays small jerks or a ratchet-like quality when moved passively. Besides the limbs, rigidity of PD can affect other body parts, such as the face, which can display a “masked” look (hypomimia) (Jankovic, 2008; Reichmann, 2010; Xia and Mao, 2012). 20 University of Ghana http://ugspace.ug.edu.gh 2.1.6.4 Postural instability As the PD advances, a fourth clinical feature known as postural instability can manifest. This symptom has a many different underlying causes related to other motor symptoms, such as rigidity and neurodegeneration in the hypothalamic brainstem or peripheral nervous system. Because of the associated loss of balance and the risk of falls, postural instability is considered a seriously disabling feature of PD (Kang et al., 2012; Doherty et al., 2011). 2.1.7 Diagnosis There are no definitive tests to confirm the diagnosis of PD. The cardinal motor features of PD- described as the “classical triad”- include a 4-Hz to 6-Hz resting tremor, “cogwheel” rigidity, and bradykinesia are often reported as the first clinical findings of the disease. A fourth feature, postural instability, occurs usually within five years of diagnosis and it affects approximately 50% of PD patients ( Baumann,2012; Berardelli et al., 2013; Reichmann, 2010) The fact that clinical motor features may not present until approximately 50 to 80% of dopaminergic neurons are lost poses a great challenge in PD diagnosis and quite unfortunately, at this point, significant disease progression may have already started (Berg, 2012; Postuma et al., 2012). There is therefore the need to identify subtle motor features that can easily go unrecognised, such as diminished ability to swing the arms or the jerking motions (Lee et al., 2013; Tolosa et al., 2009). The presence of non-motor comorbidities, including depression, anxiety, fatigue, constipation, anosmia and sleep disorders, which the clinician may not recognise as being associated with PD, further complicates early diagnosis (Schrag et al., 2015, Kang et al., 2012; Irano et al., 2007). Early identification of these symptoms and their association with PD may enable an 21 University of Ghana http://ugspace.ug.edu.gh earlier diagnosis. Investigators however, continue to search for biomarkers that may allow a more expeditious diagnosis since the onset of motor features is the point at which PD is usually diagnosed and treatment is initiated (Chahine et al., 2014; Sharma et al., 2013). Patients may have a life expectancy similar to that of unaffected individuals once the diagnosis of PD has been confirmed (Grosset et al., 2010; Suchowersky et al., 2006). 2.1.8 Treatment The successful management and treatment of PD remains still remains elusive despite the fact that was discovered almost two centuries ago (Ngwuluka et al., 2010). The principal aim in PD management is to treat the motor and non-motor symptoms with the goal of enhancing the quality of life of patients (George and Ashok, 2015). 2.1.8.1 History of the treatment of PD The first drugs to have been used in the symptomatic therapy of PD were anticholinergics (Brocks, 1999; Schapira, 2005). The discovery of the loss of dopamine in specific areas of the brain in the 1960s, opened the door to modern treatment of PD (Korczyn, 2004). Later on, in 1961, it was discovered that dopamine is unable to cross the blood-brain barrier and this led to the trial studies of its precursor, levodopa (Birkmayer and Hornykiewicz, 1961). Although the modern history of levodopa unfolds from the 19th century, it would be interesting to note that ancient Indian Ayurvedic physicians used the seeds of Mucuna Pruriens, which later proved to contain 4 to 6% of levodopa, to treat symptoms of PD as early as 300 BC (Gourie-Devi et al., 1991; Ovallath and Deepa, 2013). Several drugs have been used for the treatment of PD and these include: levodopa, Catechol- O-Methyl transferase (COMT) inhibitors (example entacapone), dopamine agonists (example 22 University of Ghana http://ugspace.ug.edu.gh apomorphine), Monoamine Oxidase-B inhibitors (example selegiline) (Korczyn, 2004). The most effective of these agents is levodopa, although the oldest, and is considered the gold standard for treatment of PD. 2.2 LEVODOPA 2.2.1 Introduction Levodopa is chemically known as L-3,4-di-hydroxyphenyl-alanine, a neutral, large amino acid that is synthesized from L-thyroxine by hydroxylation. The schematic representation of the biosynthesis of levodopa is shown in figure 2.2 below. Levodopa undergoes decarboxylation by L-aromatic amino acid decarboxylase (nonspecific enzyme widely distributed in liver, kidney, gut, lungs and brain) to dopamine, a neurotransmitter that cannot pass through the BBB (Holtz, 1993). The active metabolite of levodopa, dopamine, is responsible for the control of symptoms of Parkinson’s disease; however, it is not able to traverse the formidable brain barrier. Levodopa therefore acts as a prodrug for dopamine since it is able cross the blood brain barrier (via large neutral amino acid transporters) (Khor and Hsu, 2007). Levodopa is required by all PD patients because of its better tolerability, low cost and ease of administration. However, the therapeutic efficacy of levodopa was found to be significantly reduced because of its extensive metabolism which lead to a reduction in the bioavailability of the drug (Fahn, 2008; Muzzi et al., 2008). The active metabolite of levodopa (dopamine) was observed to generate adverse effects such as cardiac arrhythmias, vomiting and nausea (Muzzi et al., 2008). In general, approximately 95% of levodopa is metabolized in peripheral tissues (GIT, liver and plasma) whereas only 1% of the ingested dose penetrates the CNS for the treatment of PD (Muzzi et al., 2008; Jankovic, 2002; Kostoff and Briggs, 2008). Metabolism of levodopa both in the periphery and the central nervous system is shown in figure 2.3. 23 University of Ghana http://ugspace.ug.edu.gh It has been observed that levodopa absorption is highly affected by irregular gastric emptying time, metabolism and competition with aromatic amino acids for absorption and transport. Also, its half-life is relatively short, just about 50 minutes and the time to reach its maximum peak in plasma is also about 1.4 hours (Seeberger and Hauser, 2007). Owing to poor bioavailability of levodopa (33%), further attempts were made to improve the efficacy of oral formulations by increasing levodopa dosage and number of times the dose is given (Nutt et al., 2000; LeWitt and Nyholm, 2004). Unfortunately, these did not reduce the side effects emanating from the extensive metabolism of levodopa but rather exacerbated it (Stocchi, 2003). Administration of a dopa decarboxylase inhibitor together with levodopa was found to enhance the availability of levodopa in the brain by a ten-fold (Cedarbaum, 1987). However, these dopa decarboxylase inhibitors (benserazide, carbidopa) are not able to cross the blood-brain barrier so their effect is mainly in the peripheral tissues thereby reducing the extensive metabolism of levodopa and subsequently slowing the rate at which levodopa is cleared from plasma. Also, since the peripheral conversion of levodopa to dopamine is reduced, the adverse effects caused by dopamine in the periphery (nausea, vomiting, anorexia) is also ameliorated. Furthermore, administration of levodopa with dopa decarboxylase inhibitors prolongs the half-life of levodopa to approximately 90 minutes and reduces the required dosage by 60 to 80% (Janckovic, 2002). In 1975, the combination of levodopa with either of the dopa decarboxylase inhibitors (carbidopa, benserazide) was made commercially available (Tolosa et al., 1998) and till date, the approved drug by the FDA for the treatment of PD is a combination of levodopa and carbidopa. 24 University of Ghana http://ugspace.ug.edu.gh Figure 2.2: Schematic representation of Levodopa and dopamine synthesis pathway (Hadad et al., 2018). 25 University of Ghana http://ugspace.ug.edu.gh Figure 2.3 : Levodopa metabolism in the periphery and the CNS (Palma et al., 2013). 2.2.2 Pharmacokinetics of levodopa/carbidopa 2.2.2.1 Levodopa Levodopa is absorbed via L-neutral amino acid transport system. With the administration of levodopa orally, it is practically completely absorbed, with just about 2% of the drug appearing in faeces (Morgan et al., 1971). In spite of this, when levodopa is administered without carbidopa, only about 30% of the initially administered dose reaches systemic circulation, but with the concomitant administration of dopa decarboxylase inhibitors, the bioavailability increases by two to three-fold (Nutt, 1984). At steady states, levodopa has a high volume of distribution and low plasma protein binding (Hinterberger and Andrews, 1972). 26 University of Ghana http://ugspace.ug.edu.gh Metabolism of levodopa is via four pathways (decarboxylation by aromatic amino acid decarboxylase, 3-O-methylation by Catechol-O-methyltransferase, transamination by tyrosine aminotransferases, and oxidation by tyrosinase). The decarboxylation pathway is the major metabolic pathway for levodopa (Khor and Hsu, 2007). Dopamine, which is the first decarboxylation product may be further metabolised to form 3,4-dihydroxyphenyl acetic acid, homovanillic acid, and to a lesser extent, norepinephrine and vanillinemandelic acid (Khor and Hsu, 2007). Levodopa is mainly excreted via urine. When given alone, levodopa has a plasma half-life of about 50 minutes but this may be increased to 90 minutes when co-administered with carbidopa. 2.2.2.2 Carbidopa About 40 to 70 % of the dose of orally administered carbidopa is absorbed (Vickers et al., 1974). Carbidopa is absorbed quite slowly than levodopa, and takes approximately 2 hours (for an immediate release formulation) or close to 2.8 hours (for a controlled release formulation) to peak in plasma. The bioavailability of Carbidopa is not affected by the co-administration of Levodopa (Vickers et al., 1974). However, carbidopa is peripherally acting and therefore not able to cross the blood-brain barrier even at high doses (Lotti and Porter, 1970; Porter, 1973). Also Carbidopa is not highly protein bound. The percentage plasma protein binding is 36±1.6%. Carbidopa undergoes metabolism to form four metabolites, 2-methyl-3-methoxy-4-hydroxy- phenylpropionic acid, 2-methyl-3,4-dihydroxy-phemnylpropionic acid, 3-hydroxy-a-methyl- phenylpropionic acid, and 3,4-didydroxy-phenylacetone. There has not yet been any published information on the potential pharmacological activities of these metabolites (Khor and Hsu, 2007). Carbidopa is mainly excreted through urine and has a half-life of about 2 hours when administered with Levodopa. A similar half-life of 2.08 hours was observed when carbidopa 27 University of Ghana http://ugspace.ug.edu.gh is given alone (Vickers et al., 1974). 2.2.3 Pharmacodynamical Changes in the body with the Progression of PD Studies have shown that although PD is initiated by loss of dopaminergic nigral neurons, there are still sufficient neurons to maintain constant striatal dopamine concentrations and continuous activation of the striatal dopamine receptors for normal or close to normal basal ganglia function in the early stage of the disease (Pfeiffer, 2005; Olanow et al., 2006). The dopaminergic neurons convert the administered levodopa to dopamine and can still store, release, re-uptake, recycle or auto regulate large amounts of dopamine to maintain constant dopamine concentrations. Furthermore, it has been proposed that dopaminergic neurons are able to buffer the fluctuation of the plasma levels by masking the pulsatile stimulation of the striatal receptors (Olanow, 2004). However, the pulsatile stimulation becomes magnified as the loss of dopaminergic neurons progresses because the buffering and autoregulation capacity lessens, leading to motor complications such as dyskinesia and motor fluctuations (Olanow et al., 2006). The conversion of administered levodopa then takes place at the non-dopaminergic sites such as glial cells, serotonergic neurons and non-aminergic interneurons (Melamed et al., 2000). These sites do not store dopamine, but rather convert levodopa to dopamine and release it to the striatum. Lack of storage capacity forces the striatal dopamine receptors to depend almost entirely on the peripheral availability of levodopa, which in turn is limited by its short half-life (Olanow, 2004). Hence constant striatal dopamine concentration and continuous activation of the striatal dopamine receptors are impaired and replaced by pulsatile stimulation determined by the pharmacokinetics of levodopa, leading to motor complications (dyskinesia and motor fluctuations) experienced by PD patients. Motor fluctuations consist of ‘on’ periods in which 28 University of Ghana http://ugspace.ug.edu.gh therapeutic response and good antiparkinsonian effect are experienced, and ‘off’ periods during which patients experience crippling Parkinsonism resulting from the absence of or low levodopa concentrations in the plasma (Olanow, 2004). Therefore, while combination therapy seemed to solve most of the difficulties initially facing LD therapy, the major drawbacks of levodopa therapy as the disease progresses still persists. The development of drug related fluctuations emphasizes the need for improving drug delivery (LeWitt, 2015). 2.2.4 Levodopa drug delivery systems 2.2.4.1 Immediate release oral drug delivery The first immediate drug release systems for levodopa were tablets composed of levodopa in combination with carbidopa and registered under the trade name SinemetÒ (Koller et al., 1999). Administration of levodopa with carbidopa controlled the concentration of dopamine at appropriate levels with reduced side effects (Sagar and Symth, 2000). This was proposedly achieved through the diminished conversion of levodopa to dopamine in the peripheral tissues, which also permitted lower effective doses of levodopa for the treatment of PD (Sagar and Symth, 2000). The combination, Sinemet, could reduce the peripheral metabolism of levodopa and adverse effects (nausea and vomiting) but was ineffective in controlling dyskinesia and motor fluctuations which accompanied long term use of levodopa (Olanow, 2004). Generally, Sinemet and Madopar (combination of levodopa with benserazide) produce fluctuations of levodopa plasma levels that either exceed safe therapeutic concentrations or fall below the minimum effective concentrations (Sagar and Symth, 2000). However, the plasma fluctuations depend not only on levodopa’s biological half-life but also on frequency of administration and release rate from the drug delivery systems (Sagar and Symth, 2000). In 29 University of Ghana http://ugspace.ug.edu.gh order to block the second metabolic pathway of levodopa and improve its half-life, a COMT inhibitor, entacapone was added to make a triple combination and was sold under the name Stalevo. Even though entacapone increases the plasma level of levodopa by 35% and the half-life from 1.5 (with carbidopa) to 2.4 hours (Hsu and Han, 2006), entacapone has also been observed to increase dopaminergic side effects such as dyskinesias, therefore necessitating levodopa dose reduction (Hauser, 2004). In another study, it was shown that Stalevo could not improve dyskinesias and motor fluctuations mainly because of its inability to provide constant therapeutic plasma concentrations (Olanow and Stocchi, 2004). With the aim of providing continuous activation of striatal dopamine receptors so as to reduce the incidence of dyskinesias, Smith and co-workers in their study suggested the frequency of dosing of Stalevo be increased to four times daily (Smith et al., 2005). However, frequency of dosing could certainly instigate patient non-compliance, which in return would not achieve the aim of increased dosing. Although certain combinations can enhance the pharmacokinetic profile of immediate release levodopa systems, plasma fluctuations and dyskinesia remain a major set-back along with decreased patient compliance which comes increasing frequency of dosing. 2.2.4.2 Levodopa as liquid formulation In order to enable rapid onset of action, liquid levodopa formulations were introduced. However, their effects were found to be short acting. Patients were observed to benefit from liquid levodopa formulation within 5 min, with the duration of the effect persisting for 1 to 2 hours (Stacy, 2000). Levodopa liquid formulations are therefore administered to minimize delay in the ‘on’ effect, which has been proven to be increased by controlled release formulations (Adler, 2002). 30 University of Ghana http://ugspace.ug.edu.gh Unlike conventional formulations, the pharmacokinetic profiles of liquid LD formulations are not affected by the gastric emptying rate. Liquid formulations allow for a rapid gastric transit as the gastric emptying interval does not affect their absorption (Woitalla et al., 2006). Thus, levodopa liquid formulations may allow more precise dose titration throughout the day and may therefore prove to be useful particularly in patients with prolonged gastric transit time, difficulties in swallowing, advanced motor fluctuations and also during the ‘off’ effect period (Metman, 2002; Weintraub et al., 2008). It has been observed however, that, despite liquid levodopa formulations being independent to gastric emptying rate, there is still pulsatile drug delivery and the desired constant delivery is still not achieved (Obering et al., 2006). Furthermore, liquid formulations are cumbersome as their therapeutic effects are short-lived, thus requiring patients to take an hourly or bihourly dose, which makes them considerably prone to non-compliance. Thus, patients cannot rely entirely on liquid levodopa because the frequent dosing does not consistently reduce motor fluctuations (Obering et al., 2006). 2.2.4.4 Delivery of levodopa by infusions Infusions for the delivery of levodopa were introduced with the intention of achieving constant plasma concentrations, which would in turn produce continuous dopaminergic stimulation of the dopamine receptors. Duodopa was developed as levodopa/carbidopa enteral gel with a portable pump and intestinal tube (LeWitt and Nyholm, 2004; Nyholm et al., 2005a). In a study that compared intraduodenal infusion as monotherapy with individual combinations of conventional pharmacotherapy, there was significantly improved motor performance with the infusion (Nyholm et al., 2005a). Nyholm et al in another study stated that in addition to improvement of motor performance, a 24 hour intraduodenal infusion improved sleep in advanced PD patients without clinically relevant tolerance or side effects. Hence patients were 31 University of Ghana http://ugspace.ug.edu.gh found to have had improved quality of life when placed on intraduodenal infusion (Nyholm et al., 2005a; Mouradian, 2005). Thus far, levodopa duodenal infusion has been found to produce continuous plasma concentrations of levodopa, and reduced dyskinesias and ‘off time’ in clinical studies (Antonini, 2007). However, the method is invasive and adverse events associated with the use of the small tube and/or surgical procedure limits the use of intraduodenal levodopa infusion. The most frequently reported complications include dislocation of the distal part of the tube from the duodenum into the stomach which can lead to sudden deterioration of treatment response with recurring motor fluctuations (Nyholm et al., 2005). 2.2.4.5 Nasal delivery of levodopa The nasal route has been explored for drug delivery because of its large surface area enhanced by the presence of microvilli that cover the highly vascularized epithelial cells (Brime et al., 2000). Additionally, it is easily accessible and avoids first-pass metabolism. Brime et al., (2000) fabricated levodopa-loaded gelatin microspheres by emulsification solvent extraction technique for delivery by means of the trans nasal route. The bioadhesive properties of gelatin facilitated prolongation of the contact between the microspheres and the nasal mucosa, thereby enhancing absorption. However, nasal drug delivery system is an immediate release system and thus would not provide constant and sustained delivery of levodopa for the achievement of continuous dopaminergic stimulation (Ngwuluka et al., 2010). 2.2.4.6 Controlled release drug delivery The main aim of the development of controlled release delivery systems was to address the difficulties accompanied with traditional methods of administration. This drug delivery system 32 University of Ghana http://ugspace.ug.edu.gh employs devices such as polymer-based disks, rods, pellets or microparticles that encapsulate drug and release it at controlled rates for reasonably long periods of time. The controlled release system offers several potential advantages over traditional methods of administration (Kim and Pack, 2006). First, drug release rates can be modified to the needs of a specific application; for example, providing a constant rate of delivery or pulsatile release. Second, provision of protection of drugs that are otherwise rapidly destroyed by the body (example proteins) and finally, improve patient comfort and compliance by replacing frequent doses with infrequent ones. For example, replacing daily doses of injection with once per month injection or replacing four times daily oral doses with once daily dose (Kim and Pack, 2006). While a variety of devices have been used for controlled release drug delivery, biodegradable polymer microspheres have proven to be the most common types with several advantages (Kim and Pack, 2006). One of the approaches used to solve the problem of motor fluctuations encountered with levodopa was to reduce the interval between levodopa doses through the administration of controlled release formulations (Barone, 2003). Thus, controlled release levodopa formulations were developed with the hope that they would further prolong the half-life of levodopa and stabilize plasma levels, thereby decreasing the development of motor complications (LeWitt et al., 1989; Hammerstad et al., 1994) 2.2.4.7 Biodegradable microspheres as a drug delivery system for levodopa Microspheres are controlled release drug delivery systems which have been applied in many instances: hormone therapy, chemotherapy, cardiovascular diseases, neurological disorders, ocular drug delivery, and protein and vaccine deliveries (Benoit et al., 2000; Hickey et al., 33 University of Ghana http://ugspace.ug.edu.gh 2002). Microspheres are known to modulate the release profile of drugs and their absorption characteristics (Vasir et al., 2003). It has been shown that with the use of microspheres as a drug delivery system for levodopa, the dosage size, frequency of administration, systemic side effects and dose-dumping decreased, while the drug could be released continuously (Hickey et al., 2002). This ultimately enhances patient compliance. Microspheres have been fabricated from a variety of biodegradable polymers, which include gelatin, albumin, polyanhydrides, polyorthoesters, polyesters and polysaccharides (Herrero-Vanrell and Refojo, 2001). The use of microspheres as drug delivery agents for levodopa and carbidopa is another approach for improving the bioavailability and subsequent clinical response of levodopa (Arica et al., 2005). Arica and co-workers prepared levodopa and carbidopa microspheres by a solvent-evaporation technique using biodegradable polymers, namely poly (DL-lactide) and poly(DL-lactide-co-glycolide). Both in vitro and in vivo studies were performed. Reduced rotational behaviour was observed in the rats, indicating that levodopa/carbidopa-loaded microspheres exerted functional effects on the striatal dopamine receptors (Arica et al., 2005). The bioavailability of levodopa was improved because the levodopa/carbidopa microparticles released the drug directly on the striatal dopamine receptors, thereby reducing wide distribution of side effects (Arica et al., 2005). 2.3 MICROPARTICLES 2.3.1 Introduction Microspheres are characteristically free flowing powders consisting of proteins or synthetic polymers having a particle size ranging from 1-1000µm. They are sometimes referred to as microparticles (Sahil et al., 2010). Undoubtedly, oral route is the most preferred for taking 34 University of Ghana http://ugspace.ug.edu.gh medications. However, the short half-life and limited absorption via a specific segment of intestine restricts the therapeutic potential of many drugs. In order to improve the bioavailability and pharmacokinetics profile, it is only rational to release the drug in a controlled and site specific manner. Such is by using microparticles as drug carriers. Microparticulate drug delivery system has been shown to be a reliable means to deliver drugs to the target site with specificity, if modified, and to maintain the desired concentration at the site of interest (Sahil et al., 2010). Hence they play a key role at aiming at improving the bioavailability of conventional drugs and minimizing their adverse effects (Gattani, 2010). There are several opportunities to control characteristics of drug administration and improve the therapeutic efficacy of many drugs due to the variety of techniques for the preparation of microparticles (Sahil et al., 2010). 2.3.2 Techniques of Microparticle preparation The method by which drugs are entrapped in a polymeric matrix or shell is known as microencapsulation (Kumar et al., 2011). In order to select a method for microencapsulation, the factors to consider are: high yield and drug encapsulation efficiency, integrity of the stability and biological activity of the drug during the microencapsulation process, and microparticles should not exhibit aggregation or adherence. Microencapsulation methods include: hot-melt encapsulation, phase/wet inversion, coacervation, solvent removal, polymerization, solvent evaporation, and spray drying (Subedi et al., 2016). Among these, spray drying method is the commonest and most feasible technique which is extensively studied for the preparation of microparticles. 2.3.2.1 Spray Drying Method Spray drying is a famous method used in the production of agglomerates, granules or powders from the mixture of drug and excipient solutions as well as suspensions (Agnihotri et al., 2004). 35 University of Ghana http://ugspace.ug.edu.gh The method is based on drying of atomized droplets in a stream of hot air. In this method, the polymer is first dissolved in aqueous solution, drug is then dissolved or dispersed in the solution and then a suitable cross-linking agent is added. This solution is then atomized into a hot stream of air (He et al., 1999). Atomization leads to the formation of small droplets, from which solvent evaporates instantaneously leading to the formation of free flowing particles as shown in Figure 2.4. The instrument settings such as inlet temperature, rate of feed flow, spray air flow and aspirator flow can together influence the product parameters such as particle size, yield, temperature load and content of residual solvents (Giunchedi, 1995). Significant advantages of using this technique include high encapsulation efficiencies and no residual surfactant. Figure 2.4: Schematic representation of preparation of microparticles by spray drying method (Agnihotri et al., 2004). 2.3.3 Materials used in the Preparation of Levodopa Microparticles There is diversity of agents that can be used in the preparation of microparticles. Below are the examples of raw materials that have been used in the preparation of levodopa microparticles. 36 University of Ghana http://ugspace.ug.edu.gh They are: levodopa powder, glutaraldehyde, sodium bisulphite, chitosan, and hydroxypropyl methyl cellulose (HPMC). 2.3.3.1 Glutaraldehyde Glutaraldehyde is a clear, colorless to pale straw-coloured, pungent oily liquid that is soluble in water, alcohol, as well as inorganic solvents. Glutaraldehyde has had great success because of its commercial availability and low cost in addition to its high reactivity. It reacts rapidly with amine groups at around neutral pH (Okuda et al., 1991) and is more efficient than other aldehydes in generating thermally and chemically stable crosslinks (Nimni et al., 1987). Glutaraldehyde acts as a crosslinking agent in the formulation of microparticles. Acting as a crosslinking agent, glutaraldehyde controls diffusion of drugs from the polymer matrix. Drug release in general is affected by the molecular weight and concentration of the polymer, the crosslinking agent and its concentration, variables like stirring speed, additives, and drug-polymer ratio. Amount of glutaraldehyde used in microparticle formulation affects the release of the drug from the microparticle, the encapsulation efficiency and particle size. The higher the amount of crosslinking agent used, the slower the release rate of the drug which is essential in controlled release formulations (Thanoo et al., 1992). 2.3.3.2 Hydroxypropyl methyl cellulose (HPMC) Hypromellose is the simple name for hydroxypropyl methyl cellulose which is used as a polymer in a controlled-delivery drug in oral-medicaments (deSilver and Oliver, 2005). It is an odorless, tasteless granular or fibrous powder which is creamy white or white in colour and soluble in cold water. HPMC is a semi-synthetic, viscoelastic, non-ionic hydrophilic polymer which is used extensively in various formulations of controlled release dosage form. It exhibits pH independent drug release profile (Rell et al., 2009). 37 University of Ghana http://ugspace.ug.edu.gh Extensive use of HPMC in controlled drug delivery system has been realized because they offer unique properties so far not been attained by any other polymers. The rate of the drug release from a matrix product depends on the relaxation of the polymer chains, which overall displays sustained release characteristics (Williams et al., 2011). When HPMC is used as a polymeric matrix for the controlled released dosage form, the release of the drug takes place at a steady state rather than the controlled released dosage form which has been coated with normal coating ingredient. This results in uniform drug release with very lower chances of adverse effects like dose dumping (Zajic and Buckton, 1990). HPMC is used as a coating polymer as well as a matrix former in various drug deliveries. Due to its gel forming nature, it can be well accepted in controlled release drug delivery (Tamasree et al., 2016). 2.3.3.3 Chitosan Chitosan is a hydrophilic, biocompatible and biodegradable polymer. It is commercially available in a range of molecular weights, degrees of deacetylation and types of salts (such as glutamate, hydrochloride and lactate). Chitosan has –OH and –NH2 groups that allow hydrogen bonding. Due to its linear molecule, it expresses a sufficient chain flexibility, the conformation of which is highly dependent on ionic strength. These properties are considered essential for mucoadhesion (Peppas and Buri, 1985; Robinson and Mlynek, 1995). Mucoadhesion can be defined as the state in which two materials, at least one of which is biological in nature, are held together for a prolonged time period by means of interfacial forces (Kaurav et al., 2012). Mucoadhesive microparticles made from the naturally occurring biodegradable polymers like chitosan have attracted considerable attention for several years in sustained drug delivery (Huang et al., 2003). They have advantages such as absorption and enhanced bioavailability of drugs as a result of a high surface to volume ratio, a much more 38 University of Ghana http://ugspace.ug.edu.gh intimate contact with mucus layer, and specific targeting of drugs to the absorption site (Raval et al., 2010). Furthermore, the cationic polyelectrolyte nature of chitosan provides a strong electrostatic interaction with mucus or a negatively charged mucosal surface. The importance of this mucoadhesive property of chitosan has been demonstrated in earlier work by Lehr et al., (1992), Illum et al., (1994), Lueβen et al., (1994) Fiebrig et al., (1995); Aspden et al., (1996). More specifically, chitosan has been used as a delivery vehicle for nasal and oral delivery of peptide drugs, in order to improve drug absorption (Illum et al., 1994; Lueβen et al., 1996, 1997). Mucoadhesive tablets containing chitosan, have been developed by Takayama et al., (1990), Miyazaki et al., (1994) and Nakayama et al., (1994). These tablets have both adhesive and sustained release characters. Reports suggest that coating of liposomes with chitosan improved their adsorption to mucosal surfaces (Takeuchi et al., 1996). Below is an image of chitosan microparticles prepared by the spray-drying method (figure 2.5). Figure 2.5: Chitosan microparticles prepared by spray drying and crosslinked with glutaraldehyde (He et al., 1999) 39 University of Ghana http://ugspace.ug.edu.gh 2.3.4 Evaluation/Characterization of Microparticles The essential characterization parameters for microparticles are as follows: 2.3.4.1 Particle size The mean particle size and the width of particle size distribution are important characterisation parameters as they govern the saturation solubility, dissolution velocity, physical stability and even biological performance of microparticles. Particle size can influence the biopharmaceutical properties of microparticles, their biodistribution and the particle content uptake (Shah et al., 2014). The most common method used to measure particle size of microparticles is dynamic light scattering. The principle of dynamic light scattering is based on the theory that particle size can be determined by measuring the random changes in the intensity of light scattered from a suspension or solution. Small particles in suspension undergo random thermal motion known as Brownian motion (Burgess et al., 2004). This random motion is measured to calculate particles size. The Malvern Nano Zetasizer is an instrument used to measure particle size. 2.3.4.2 Encapsulation efficiency Encapsulation efficiency is the percentage of drug that is successfully entrapped into the microparticle. Encapsulation efficiency is calculated as a ratio of the actual drug content to the theoretical drug content expressed as a percentage (El-Say, 2016). It has been observed and widely accepted that an increase in the polymer concentration increases the entrapment of the drug inside the microparticle (Subedi et al., 2016). Also, Banerjee et al. observed that incorporation of optimized amount of cross-linking agents like glutaraldehyde in the formulation process can improve the entrapment efficiency (Banerjee et al., 2010). A high 40 University of Ghana http://ugspace.ug.edu.gh entrapment efficiency shows better control of drug release from the matrix system (Ranjha et al., 2010). 2.3.4.3 Drug loading capacity The drug loading capacity is the amount of drug loaded per unit weight of the microparticle, indicating the percentage of mass of the microparticle that is due to the encapsulated drug. Drug loading can be done by two methods, that is, during the preparation of microparticles (incorporation) and after the formation of particles (incubation) (Padalkar et al., 2011). In these systems, drug is physically embedded into the matrix in the case of incorporation method or adsorbed onto the surface, in the incubation method. Maximum drug loading can be achieved by incorporating the drug during the formation of microparticles (Agnihotri et al., 2004). Drug loading serves as a complementary characteristic to encapsulation efficiency hence a high drug loading capacity is indicative of a high encapsulation efficiency (Vyslouzil et al., 2014). 2.3.4.4 In Vitro release studies The in vitro release studies aid in understanding the behaviour of the delivery system in terms of drug release and their efficacy. Since microsphere is a heterogeneous system, the drug release from the polymer takes place through a diffusion process, in an in vitro environment. The release of the drug is determined by the extent of degradation of polymeric microsphere. There are several apparatus of varying designs that have been used to study the in vitro release characteristics of microparticles (Jain, 2004). The various methods include Beaker method which involves the adhesion of dosage form of the microparticles at the bottom of a beaker containing the medium and stirring uniformly using the overhead stirrer, Interface diffusion system, which uses four compartments (each representing the oral cavity, buccal membrane, body fluids and protein binding respectively) 41 University of Ghana http://ugspace.ug.edu.gh containing different medium (Venkatesh, 1989). There is also the Modified Keshiary Chien Cell apparatus which contains distilled water as the dissolution medium and uses a trans membrane drug delivery system placed in a glass tube and fitted at the bottom of the medium (Save and Venkatachalam, 1994). Lastly, the Dissolution apparatus method, which is the method used in the current study for investigating the release profile of the prepared microparticles. In this method, a weighed quantity of the microsphere is placed in a dissolution basket, which is immersed in a larger volume of continuous phase acceptor fluid (phosphate buffered saline). The compartment is stirred and the drug which diffuses out of the microspheres into the continuous phase is periodically sampled and assayed (Lopez et al., 1998). 42 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS Levodopa and carbidopa powders were purchased from Sigma-Aldrich (USA). Hydroxypropylmethyl cellulose (HPMC) was a gift from Ernest Chemist Limited (Ghana), glutaraldehyde was purchased from Amresco, Inc. VWR International (USA), sodium bisulfite and all other reagents used for experiments were of analytical grade and purchased from approved suppliers. 3.1 STUDY DESIGN The study was an experimental design using in vitro assays and animal models. 3.1.1 Study Location The formulation aspect of the study (preparation and characterization of microparticles) was done at the Pharmaceutics Research Laboratory of the School of Pharmacy, University of Ghana. The Pharmacokinetic aspect of this study was done at the Animal Experimentation Unit of the School of Biomedical and Allied Health Sciences, University of Ghana, Korle-Bu, and drug levels in plasma was done at the Mycotoxin and Food Analysis Laboratory, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. 3.2 PREPARATION AND CHARACTERIZATION OF LEVODOPA MICROPARTICLES The preparation and characterization of levodopa microparticles was done according to methods described by Nettey et al., (2017) with minor modifications. Carbidopa microparticles were also prepared to be administered in combination with levodopa microparticles for the pharmacokinetic studies. 43 University of Ghana http://ugspace.ug.edu.gh 3.2.1 Preparation of microparticles Microparticles of levodopa/carbidopa were prepared using the spray drying technique. In order to make a 20% drug loading capacity of the drug-polymer solution, a 1- part drug solution was added to 4 parts of the polymer solution. The polymer (HPMC) solution was prepared by weighing 10 g of the HPMC powder into 200 mL of distilled water. A 2.5 g of levodopa/carbidopa powder was weighed into 100 mL of distilled water, followed by the dropwise addition of 32% hydrochloric acid (HCl) until the drug was completely dissolved in the solution. The levodopa/carbidopa solution was slowly added to the HPMC solution with stirring. This was followed by the addition of distilled water to make a final volume of 400 mL in order to dilute the viscous solution. The homogenous mixture obtained was stirred on the magnetic stir plate for 1 h, after which 200 mL glutaraldehyde (50%) solution was added to cross-link the HPMC molecules. This was left to stir for another 1 h. In order to quench the cross-linking, 2 mL of a 1% sodium bisulfite solution was added to the mixture and stirring continued for 1 h. After, a 75 mg of chitosan powder was weighed and dissolved in 50 mL of distilled water with the dropwise addition of HCl. The chitosan solution was then added to the drug-HPMC solution in order to further coat the drug and the polymer. The solution was left to stir on the magnetic stir plate for 1 h. The resulting solution was fed into the Bilon-6000Y laboratory spray-dryer and levodopa/carbidopa microparticles obtained. 3.2.2 Particle Size Determination One milligram (1 mg) of the levodopa microparticles was weighed and suspended in 20 mL of distilled water. The suspension was further diluted by adding more of the distilled water and transferring the suspension into a cuvette. The sample in the cuvette was placed into the cell holder of the Malvern Nano Zetasizer, where it was illuminated with a laser. The intensity 44 University of Ghana http://ugspace.ug.edu.gh fluctuations in the scattered light was analysed and used to calculate the size of microparticles by an inbuilt digital correlator. 3.2.3 Content Analysis In order to determine the actual amount of drug contained in the microparticles, 25 mg of levodopa microparticles (weighed in triplicate) was crushed in a mortar, and suspended in 10 mL of distilled water. The suspension was transferred into Eppendorf tubes, and centrifuged at 3000 rpm for 10 min. A portion of the supernatant was pipetted into new Eppendorf tubes and 20 µL was injected into a High Performance Liquid Chromatography (HPLC) system as described subsequently for the analysis of the drug content. The samples were analysed against a standard plot to calculate the drug content of the microparticles. 3.2.4 Encapsulation Efficiency Encapsulation efficiency of microparticles was measured to determine how effective the HPMC polymer was able to encapsulate the drug. From the actual drug content determined initially, the encapsulation efficiency was calculated as the ratio of actual drug concentration to the expected drug concentration. Mathematically this can be represented as: %Encapsulation Efficiency = !"#$%& ()$* "+,"-,#)%#.+, x 100 /01-"#-( ()$* "+,"-,#)%#.+, The concentration of levodopa was determined using HPLC as described subsequently. 3.2.5 In Vitro Drug Release Test Samples (75 mg) of levodopa microparticles were weighed and transferred into empty size 0 gelatin capsule shells. The filled capsules were placed in baskets of USP Dissolution Apparatus 1, and each of the outer flasks were filled with 500 mL of phosphate buffered saline (PBS), of pH 7.4. The baskets were allowed to rotate at 100 rpm (and at a temperature of 37°C). At 0, 45 University of Ghana http://ugspace.ug.edu.gh 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h, 5 mL of the samples were drawn from the flask and replaced with an equal amount of PBS. The amount of drug released was determined using HPLC as described below. The release experiment was performed in triplicate. 3.3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) FOR LEVODOPA LEVELS For content analysis and release studies, samples were analysed using Agilent Technologies system (Jos. Hansen, Hamburg/Germany) with the following chromatographic conditions: Mobile Phase comprised of 0.01 M Phosphate buffer (adjusted to a pH of 2.68 with hydrochloric acid) and Methanol (20:80 v/v). An injection volume of 20 µL, a flow rate of 1.0 ml/min for 10 min at wavelength of 280 nm were used. A Vertex Plus C18, 150 X 4 mm column was used for all analysis. 3.3.1 Preparation of Stock and Working Solutions. A stock standard solution (1 mg/mL) of levodopa was prepared by accurately weighing 50 mg of levodopa and transferring into a 50 mL volumetric flask. To that, 30 mL of deionised water was added followed by dropwise addition of HCl to dissolve the drug completely. Then the volume was made up to the 50 mL mark with more deionised water. Further working standard solution of levodopa was prepared by making two-fold serial dilutions with concentrations 1.25, 5, 12.5, 25 and 50 µg/mL. Twenty microliters (20 µL) of the standard working solutions were injected and the retention time and peak area were determined. Standard plots were made from the HPLC values and the equation of the calibration curve obtained. The sample solutions were also analysed by injecting 20 µL of the sample solution into the HPLC system and peak area determined for each sample. The amount of levodopa in microparticles was calculated by substituting the peak areas determined from the HPLC analysis into the equation of the line obtained from the calibration curve (y = mx + c) as shown in Appendix B. 46 University of Ghana http://ugspace.ug.edu.gh 3.4 IN VIVO PHARMACOKINETIC EVALUATION OF LEVODOPA MICROPARTICLES 3.4.1 Animal Acquisition and Housing Male Sprague-Dawley (SD) rats (Hsd; SD strain), weighing 150-200 g and 6-8 weeks old, were obtained from the Department of Animal Experimentation, School of Biomedical and Allied Health Sciences, University of Ghana, Korle-Bu. The animals were housed in stainless steel cages. Each rat occupied a minimum space of 2 cubic feet (61 cm x 31 cm) with soft wood shavings as bedding for their comfort. They were fed with normal pellet diet (AGRIMAT Kumasi), given water ad libitum, and maintained under optimal laboratory conditions (temperature 25 ± 1°C, relative humidity 60-70%, and 12-hour light-dark cycle). All feeding and water troughs were cleaned regularly to prevent contamination. The animals were acclimatized to this environment for two weeks before experimentation. All animal procedures and techniques used in this study were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (N.R.C., 2010). 3.4.2 Animal Groupings for Pharmacokinetic Study By simple random sampling, SD rats were put into 3 groups consisting of 5 rats each. Group 1 received levodopa/carbidopa (Sinemet CR), Group 2 received levodopa/carbidopa powder and animals in Group 3 were given microparticles of levodopa/carbidopa. Levodopa and carbidopa microparticles were combined in a 1:4 ratio of carbidopa to levodopa. 3.4.3 Drug Administration and Sample Collection For pharmacokinetic studies, carbidopa and levodopa microparticles were combined (in a 1:4 ratio respectively before administration). Animals were fasted overnight prior to administration of treatments. A dose of (5/20 mg/kg) of carbidopa/levodopa of the respective formulations 47 University of Ghana http://ugspace.ug.edu.gh was given via the oral route to rats in each group. Treatments were administered every 12 h. After administration of the dose on the second day, rat tails were snipped and blood samples collected into Ethylenediaminetetraacetic acid (EDTA) tubes at time intervals of 0.25, 0.5, 1, 2, 4, and 12 h. A solution of 25% sodium metabisulfite in water was made and added in a ratio of 1: 10 (v/v) to rat blood samples collected to minimize oxidation of the levodopa in blood. Blood samples were centrifuged at 10,000 rpm for 5 min to separate plasma and stored at - 20°C until analysis. 3.4.4 Determination of Plasma Levodopa Levels Levodopa in plasma was analysed using HPLC as described by Elbarbry et al., (2018) with minor modifications. 3.4.4.1 Chromatographic Conditions The Cecil Adept HPLC system (Cecil Instrumentation Services Ltd., Cambridge, UK) consisting of an auto-sampler, a binary pump and a model Diode-array detector was used. The chromatographic analysis was performed using the Zorbax C18 column (4.6 mm x 300 mm) with a particle size of 5 µm and column temperature of 40 °C. The mobile phase consisted of 20 mM phosphate buffer (pH 2.5) and methanol HPLC grade (95:5, v/v). This was run at a flow rate of 1 mL/min for 10 min. Levodopa’s absorbance was detected at 230 nm. 3.4.4.2 Preparation of Stock and Working Solutions Stock solution of levodopa was prepared fresh everyday by dissolving the analytical standard in 1 M perchloric acid at a concentration of 4.6 mg/mL. Working solutions were obtained by serial dilution of stock solution with methanol containing 0.05% v/v perchloric acid at concentrations of 46, 460, 920, and 1840 µg/mL. A stock of the internal standard (IS) solution was prepared by dissolving 20 mg of methyl dopa in 15 mL of water to obtain a concentration 48 University of Ghana http://ugspace.ug.edu.gh of 1.3 mg/mL. Working internal standard solution was prepared by diluting 1 µL of the methyl dopa standard in 99 µL of methanol containing 0.05% perchloric acid to obtain a final volume of 100 µL. All stock and working standards were stored at -20°C until analysis using HPLC. Standard plots were made by correlating peak ratios of the analytical and internal standards to the known standard concentrations and a calibration equation obtained. Calibration curve showing equation of the line and the coefficient of correlation is shown in Appendix D. 3.4.4.3 Sample Preparation Levodopa in plasma was extracted using protein precipitation method with perchloric acid as the precipitating agent. To 50 µL of rat plasma, 100 µL of the working internal standard solution and 25 µL of 0.5 M perchloric acid were added. The mixture was vortexed for 2 min. After, centrifugation was done at 10,000 rpm and the supernatant transferred into an auto- sampler vial. An injection of 20 µL of the supernatant was made directly into the analytical column for immediate HPLC analysis. Quantitation of the unknowns (levodopa levels in plasma) was by interpolation from the weighted linear regression line of the ratios of the peak areas of the analytical and internal standard. The study protocol was approved by the Ethics and Protocol Review Committee for College of Health Sciences, University of Ghana, with a Protocol Identification Number: CHS-Et/M.8 - 5.14/2018 – 2019. 3.5 DATA ANALYSIS Statistical test of significance was taken as p < 0.05 and performed on all continuous data using unpaired t-test for two independent sample means, and one-way analysis of variance (ANOVA) for comparison of more than two independent sample means. All tests were expressed as mean and standard deviation from mean (STDEV). 49 University of Ghana http://ugspace.ug.edu.gh Pharmacokinetic parameters for levodopa in the 3 groups were determined by non- compartmental analysis. The maximum plasma drug concentration (Cmax) and the time to achieve this peak (Tmax) of levodopa were extrapolated from concentration-time curves. The elimination rate constant (Ke) was determined by linear regression analysis of the terminal- linear part of the log plasma concentration-time curves. Area under the concentration-time curve (AUC) was calculated by the linear trapezoidal rule. 50 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS 4.1 CHARACTERIZATION OF LEVODOPA MICROPARTICLES 4.1.1 Particle Size Distribution The particle size of the prepared levodopa microparticles was determined using Malvern Nano Zetasizer. The size distribution of levodopa microparticles ranged between 0.04 µm to 6 µm with a mean size of 0.2 µm as shown in Figure 4.1. Figure 4.1: Results showing the particle size distribution of the formulated levodopa microparticles 4.1.2 Drug Content Analysis Content analysis was done to determine the actual amount of levodopa in the microparticles. HPLC was used to analyse the levodopa standard solutions prepared. The peak areas generated from the chromatogram were plotted against the various concentrations of the standard solutions and equation of the straight line (y =mx + c) obtained, this is shown in Figure 4.2. 51 University of Ghana http://ugspace.ug.edu.gh For the content analysis test, peak areas that were integrated from the HPLC analysis were substituted into the equation of the straight line obtained from the standard curve. This was used to calculate for levodopa concentrations (shown in Table 4.1). Levodopa Standard Curve 250 200 150 100 Y = 2.138*X + 1.421 50 R2=0.9958 0 0 20 40 60 80 100 120 LD Conc (ug/ml) Figure 4.2: Levodopa standard curve showing the equation of the line (y = 2.1383x + 1.4208) and the correlation coefficient (R2 = 0.9958) Table 4.1: Concentrations obtained from the content analysis of levodopa Levodopa Peak Area Concentration Average microparticles (µg/mL) Concentration (µg/mL) ± STDEV Run 1 1082 505.34 478.84 ± 29.03 Run 2 959 447.82 Run 3 1035 483.36 Tests were done in triplicate giving an average levodopa concentration of 478.84 µg/mL. 52 Peak Area University of Ghana http://ugspace.ug.edu.gh 4.1.3 Percent Drug Loading Results obtained from the drug content analysis translated to 478.84 µg mass of the drug in every mL of solution. Therefore, for the 10 mL of distilled water that was used for content analysis, the mass of microparticle contained in that was 4788.4 µg. In order to calculate for the actual drug loading, the actual mass of the drug was compared to the total drug-polymer weight of 25 mg. All microgram units were converted to milligrams and the values substituted into the equation below: %Drug Loading = 345637 8399 :; <=6> x 100 5:537 <=6>?@:7A8B= CBD>E5 = F.HII 8> x 100 JK 8> = 19.15 % Therefore, out of the expected 20% drug loading, the actual drug loading was 19.15%. 4.1.4 Encapsulation Efficiency From the drug content analysis, the encapsulation efficiency was determined by the percentage of the ratio of the actual levodopa concentration to the expected levodopa concentration. This was calculated as follows: % Encapsulation efficiency = 𝒂𝒄𝒕𝒖𝒂𝒍 𝒅𝒓𝒖𝒈 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒙 𝟏𝟎𝟎 𝒕𝒉𝒆𝒐𝒓𝒆𝒕𝒊𝒄𝒂𝒍 𝒅𝒓𝒖𝒈 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 Amount of microparticles weighed for content analysis = 25 mg With the expected 20% drug loading, expected amount of levodopa contained in the 25 mg = J\ x 25 mg = 5 mg ]\\ Volume of water used to dissolve the microparticles = 10 mL 53 University of Ghana http://ugspace.ug.edu.gh Expected concentration of levodopa = %c+$,# +d ()$* = K c* = 0.5 mg/mL e+&$c- +d f%#-) (.gg+&e-( ., ]\ ci = 500 µg/mL % Encapsulation Efficiency = FHI.IF x 100 = 95.6% K\\ Therefore, for an expected concentration of 500 µg/ml, the actual concentration of levodopa in the microparticles was 478.84 µg/mL, indicating an encapsulation efficiency of 95.6% for the prepared levodopa microparticles. 4.2 IN VITRO RELEASE OF LEVODOPA FROM MICROPARTICLES In vitro release studies of levodopa microparticles was performed in phosphate buffered saline (pH 6.8) at 37°C using the Dissolution Apparatus 1 equipment. The dissolution studies revealed that in vitro drug release of the levodopa microparticles was sustained with maximum release occurring at 12 h followed by a decrease in release-time points as shown in Figure 4.3. Results of the in vitro drug release profile of levodopa microparticles is summarised in Table 4.2. In vitro Levodopa Release Study 100 80 60 40 20 0 0 4 8 12 16 20 24 Time (h) Figure 4.3: The average cumulative percentage release of levodopa against time 54 AV Cum %Drug Release University of Ghana http://ugspace.ug.edu.gh Table 4.2: In vitro release of levodopa from the formulated microparticles Time (h) Average Cumulative % Drug Release ± STDEV 0 2.98 ± 0.94 0.25 4.07 ± 1.18 0.5 11.4 ± 3.04 1 23.71 ± 4.24 2 37.51 ± 4.52 4 49.07 ± 2.10 8 66.22 ± 1.62 12 78.87 ± 4.15 24 57.33 ± 3.37 4.3 IN VIVO PHARMACOKINETIC EVALUATION OF LEVODOPA MICROPARTICLES 4.3.1 Concentration-time curves for 3 treatment groups of Sprague-Dawley (SD) rats The concentration-time curves for SD rats in the 3 groups; Group 1 administered Sinemet CR, Group 2 administered levodopa/carbidopa powder and Group 3 administered microparticles of levodopa/carbidopa are shown in Figure 4.4. From the graph, it can be observed that the concentration-time curve of rats administered microparticles of levodopa/carbidopa had the highest peak. 55 University of Ghana http://ugspace.ug.edu.gh Figure 4.4: Concentration-time curves for the 3 treatment groups of SD rats 4.3.2 Pharmacokinetic parameters of rats in the 3 treatment groups 4.3.2.1 One-way ANOVA among 3 treatment groups The pharmacokinetic parameters of SD rats in the 3 treatment groups obtained from the concentration-time curve are summarized in Table 4.3. Analysis of pharmacokinetic parameters with one-way ANOVA showed that Tmax, Cmax, AUC0→∞, Ke and t1/2 varied significantly (p < 0.05) between the 3 treatment groups. 56 University of Ghana http://ugspace.ug.edu.gh Table 4.3: Pharmacokinetic parameters of SD rats in the 3 treatment groups Treatment Group [Mean (STDEV)] PK parameter Sinemet CR LD+CD powder LD+CD MP p-value Tmax 0.5 0.67 (0.17) 1 0.027 Cmax 128.8 (45.38) 46.0 (6.57) 262.35 (22.43) 0.0058 AUC0→∞ 355 (36.28) 107.85 (4.75) 749.77 (70.8) 0.0002 Ke 0.11 (0.05) 0.44 (0.08) 0.20 (0.05) 0.016 t1/2 6.3 (1.97) 1.66 (0.27) 4.0 (1.07) 0.0304 LD+CD powder = levodopa plus carbidopa powder; LD+CD MP = levodopa plus carbidopa microparticles 4.3.2.2 Post hoc analysis with Tukey’s multiple comparison Time to reach peak concentration (Tmax) A comparison between Sinemet CR and levodopa/carbidopa powder showed that the time to reach peak concentration after administration (Tmax) did not differ significantly (p = 0.48). A comparison of Tmax between levodopa/carbidopa powder and levodopa/carbidopa microparticles also showed no statistically significant difference (p = 0.10). However, Tmax was found to differ significantly (p = 0.024) between levodopa/carbidopa microparticles and Sinemet CR. 57 University of Ghana http://ugspace.ug.edu.gh Peak levodopa concentration (Cmax) A comparison between Sinemet CR and levodopa/carbidopa powder showed that the peak concentration after administration (Cmax) did not differ significantly (p = 0.196). However, a comparison of Cmax between levodopa/carbidopa microparticles and levodopa/carbidopa powder showed a statistically significant difference (p = 0.005) between these two. Also, Cmax was found to differ significantly (p = 0.04) between levodopa/carbidopa microparticles and Sinemet CR. Area under the concentration-time curve (AUC) A comparison between Sinemet CR and levodopa/carbidopa powder showed a statistically significant difference (p = 0.0208) in the area under the concentration time curves (AUC) obtained after administration. A comparison of AUC between levodopa/carbidopa microparticles and Sinemet CR also showed a statistically significant difference (p = 0.0022) between these two. The difference between the AUC values obtained for levodopa/carbidopa microparticles and levodopa/carbidopa powder was also statistically significant (p = 0.0002). Elimination half-life (t1/2) A comparison between the elimination half-lives of Sinemet CR and levodopa/carbidopa powder was found to differ significantly (p = 0.0448). However, a comparison between the half-life of Sinemet CR and levodopa/carbidopa microparticles showed no significant difference (p = 0.224). Also, half-life of levodopa/carbidopa microparticles did not differ found significantly (p = 0.4600) from the that of the levodopa/carbidopa powder. The Tukey’s multiple comparison table has been shown in Appendix E. 58 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION Particle size influences many properties of particulate materials and is a valuable indicator of quality and performance. In the pharmaceutical industry, the size of active ingredients influences some vital drug properties including content uniformity, dissolution and absorption rates (PSA Guidebook, 2016). For these reasons, it is important to measure and control the particle size distribution of drugs (microparticlulate in some instances) in drug delivery. The size of the microparticles influences the biodistribution and particle content uptake of the drug and thus influences its pharmacokinetic properties in the body. In the current study, microparticles were measured for their average particle size using dynamic light scattering. Results from dynamic light scattering are typically reported as an intensity distribution which quantifies distribution width. From the results obtained, the size of the prepared microparticles ranged between 0.04 µm to 6 µm with a mean size of 0.2 µm. The small size of the microparticles could be accounted for by the process of fabrication and also the formulation parameters. The spray drying method has traditionally been used to produce particles with a narrow size distribution usually ranging from 0.1 to 10 µm. It is known that, the manufacturing parameters (nozzle used, pump rate and compressed spray air flow) affects the particle size of the resultant microspheres (Kumar et al., 2011). Smaller nozzle diameter as well as increased air flow rate produces microparticles with smaller sizes. A small size of microparticles confers the extra advantage of a large surface area to volume ratio. Thus, for a given rate of drug diffusion through the microparticle, the rate of flux of drug out of the microsphere, per mass of formulation, will increase with decreasing particle size. In addition to a large surface area to volume ratio, water penetration into smaller particles may also be quicker (due to shorter 59 University of Ghana http://ugspace.ug.edu.gh distance from the surface to the centre of the particle) hence ensuring a more efficient drug release (Kim and Pack, 2006). The percentage drug loading of the levodopa microparticles was measured to determine the percentage of the actual mass of active ingredient. With a 19.1% drug loading, the actual drug content of the microparticles was 478.84 µg/mL out of the expected 500 µg/mL, giving an encapsulation efficiency of 95.6%. The very high drug content and encapsulation efficiency calculated gives an indication of how effective the HPMC polymer was in entrapping the drug which leads to prolonged release. These findings are consistent with work done by Choudhary et al., (2010) in which preparative variables for floating microspheres of levodopa/carbidopa were evaluated and optimized as a gastro retentive drug delivery approach. Results from the study clearly indicate that a high drug load (resulting from increasing encapsulation efficiency) exhibited prolonged drug release for about 10 hours and remained buoyant for more than 12 hours (Choudhary et al., 2010). Arica et al., also prepared and evaluated injectable biodegradable carbidopa/levodopa microspheres for the intracerebral treatment of Parkinson’s disease. Likewise, optimization of formulation variables was done and increasing entrapment efficiency increased drug yield leading to an extended duration of drug release (Arica et al., 2005). The in vitro orelease study was performed in phosphate buffer, pH 6.8 and at 37 C to mimic physiologic conditions. The release of the drug from the levodopa microparticles was controlled, showing a steady increase in drug release with increasing time for the first 12 h followed by decreasing release with time. The increase in drug release was sustained over a long period with 78% release of levodopa occurring at 12 h. The release profile showed better sustained release characteristics rather than the usual initial burst effect associated with smaller microparticles (Bozdag et al., 2001). This could be as a result of the ‘incorporation’ technique 60 University of Ghana http://ugspace.ug.edu.gh (combining both the drug and the polymer before spraying into microparticles) used in drug loading, which yielded a high drug loading capacity and therefore was able to entrap most of the drug in the polymer. The high entrapment effect prevented the adsorption of drug to the surface of microparticles thus prevented a burst release and ensured a more controlled drug release from the microparticles. However, the decrease in release after 12 h could result from the degradation of levodopa after its release into the dissolution medium. This result is similar to some others from literature. Pappert et al., (1996) investigated the environmental factors that promote levodopa stability in solution and confirmed that levodopa in solution is an unstable compound and degrades naturally over time. In a plot of levodopa levels against time, the graph showed decreasing levels of levodopa with increasing time when the solution was kept at room temperature. In the current study, the samples were kept at room temperature during the experiment before refrigerating overnight prior to analysis. Therefore, the degradation process could have already started in the dissolution medium at room temperature before refrigeration. Autoxidative reaction of levodopa in solution is catalysed by oxygen, a reaction which is faster at higher pH (Umek et al., 2018). A study done by Di Stefano et al., (2006) supports the claim that levodopa is unstable and undergoes chemical hydrolysis at higher pH while it has good stability at low pH. D’Aurizio et al., in 2011 performed an in vitro release study of microparticles of a prodrug of levodopa in a dissolution medium at a low pH and found that the stability of the prodrug was ensured after its release into the dissolution medium. The in vitro release kinetics from the investigation showed increasing percentage levodopa release with increasing time. The pH of the phosphate buffer saline which was used as the dissolution medium in the present study was (6.8) which is quite high, and this could have also promoted the degradation of levodopa microparticles in the dissolution medium. The degradation lead to the decrease in the percentage release of the levodopa with time hence the descending release- time points observed in the in vitro release graph. 61 University of Ghana http://ugspace.ug.edu.gh Furthermore, the drawbacks with the conventional oral controlled release formulation are erratic absorption and latency to onset of action which was attributed to the irregular gastric emptying of the gastrointestinal tract (Salat and Tolosa, 2013). The present study used a gastro- retentive drug delivery approach (mucoadhesion by coating microparticles with chitosan) in the formulation of oral controlled release levodopa microparticles. As part of the study, the pharmacokinetic characteristics of the formulated microparticles in SD rats was evaluated. From the concentration-time curves, it was evident that the highest plasma levodopa levels (Cmax) was achieved with the microparticles, compared to the conventional controlled release form (Sinemet CR) and the pure levodopa powder. These results are similar to other results published in literature on the comparison of microparticles with traditional dosage forms (Nettey et al., 2017; LeWitt, 2015). The very small size of the levodopa microparticles enhance substantially the extent of absorption from the site of administration into circulation due to the large surface area to volume ratio (Nettey et al., 2017). Coating microparticles with chitosan may have also enabled the microparticles to adhere to the intestinal mucosal surface thereby enhancing contact time for absorption and resulting in a higher plasma concentration-time profile for the levodopa/carbidopa microparticles (Raval et al., 2010). The peak plasma concentration (Cmax) of a drug is often related to the intensity of pharmacological response and should ideally be above minimum effective concentration but less than what would lead to adverse drug reactions (Brahmankar et al., 2009). Since efficacy studies have not yet been done to ascertain the minimum and maximum effective concentrations of the levodopa/carbidopa microparticles, the high C max may be desirable and cause improvement in the pharmacological benefit of levodopa or disadvantageous and may lead to adverse effects like dyskinesia (Nyholm et al., 2012). 62 University of Ghana http://ugspace.ug.edu.gh The AUC of a plasma drug concentration versus time plot reflects the total drug exposure after administration (DiPiro et al., 2005). The AUC was found to be highest with the levodopa/carbidopa microparticles followed by Sinemet CR and levodopa/carbidopa powder. There was a significant difference between the AUCs of the microparticles and the conventional formulation. Also, the microparticles had, a two-fold increase in AUC0→∞ compared to Sinemet CR. This high AUC of the microparticles could be attributed to the small particle size of the microparticles and the mucoadhesive property conferred by the chitosan which resulted in enhanced and efficient absorption across the intestinal lumen. Furthermore, because bioavailability describes the extent of drug eventually reaching the systemic circulation, comparison of the AUCs of the various formulations may give an indication of bioavailabilities (DiPrio et al., 2005). Thus, it can be inferred from the results that levodopa/carbidopa microparticles attained greatest bioavailability than the levodopa/carbidopa powder and conventional controlled release product on the market (Sinemet CR). The time to achieve peak concentration in plasma after drug administration (Tmax) was also evaluated. This parameter is of particular importance in assessing the time to reach peak concentration in circulation. A comparison between Sinemet CR and levodopa/carbidopa powder showed no significant difference in the time to reach peak concentration after administration. However, there was a significant difference between the Tmax of levodopa/carbidopa microparticles and Sinemet CR. The mean Cmax for Sinemet CR, was reached at 0.5 h (Tmax), whereas for levodopa/carbidopa microparticles, a mean Cmax was reached after 1 h. From the concentration-time graph, it could be observed that at time 0.5 h, the plasma levels of microparticles of levodopa/carbidopa was higher than that of both Sinemet CR and levodopa/carbidopa powders. This means that although levodopa/carbidopa microparticles had a longer T max, the plasma concentrations at the various time points before 63 University of Ghana http://ugspace.ug.edu.gh C max was attained were relatively high. This initial rapid absorption and high plasma levels of levodopa/carbidopa microparticles could be advantageous in achieving an early onset of drug action even before peak plasma concentration is reached. The elimination half-lives of the various formulations were also assessed. This is the time it takes for the drug concentration in the blood to decrease to half the original concentration (Anderson, 2005). This parameter gives an indication of how the drug is cleared from the plasma. It also suggests whether accumulation of the drug will occur under a multiple dosage regimen, and it is an essential parameter when it comes to deciding on the appropriate dosing interval (Buclin et al., 2009). A comparison of the (t½) of Sinemet CR and levodopa/carbidopa powder showed a significant difference. This difference could be due to the fact that Sinemet CR has been designed to release levodopa/carbidopa in plasma over a period of time (controlled release) whereas levodopa/carbidopa powder has not undergone any modifications to retard its stay in plasma. This resulted in Sinemet CR having a longer half-life than levodopa/carbidopa powder. Comparing the half-life of Sinemet CR and levodopa/carbidopa microparticles, it was observed that the half-life of Sinemet CR was longer than levodopa/carbidopa microparticles Sinemet CR, which is an already established drug, most likely has undergone many formulation modifications in order to prolong its release as compared to the experimental levodopa/carbidopa microparticles, and this may be the reason for Sinemet CR having a longer half-life than the levodopa/carbidopa microparticles. Again, although levodopa/carbidopa microparticles had a larger AUC than Sinemet CR, the decline in the concentration-time curves was sharper, with levodopa/carbidopa microparticles. This means that levodopa/carbidopa microparticles were eliminated faster than the Sinemet CR and resulted in Sinemet CR having a longer half-life than the levodopa/carbidopa microparticles. However, the difference in half- life between these two formulations is not statistically significant. 64 University of Ghana http://ugspace.ug.edu.gh 5.1 CONCLUSION Comparing AUC and the Cmax, the formulated levodopa/carbidopa microparticles had better profiles compared to levodopa/carbidopa powder Sinemet CR (the current controlled release product on the market). The mucoadhesive drug delivery approach used in the formulation of the levodopa/carbidopa microparticles may have aided in overcoming the irregular gastric emptying associated with oral administration as this is seen in the enhanced absorption of levodopa/carbidopa microparticles in the current study. 5.2 RECOMMENDATIONS Based on half-life of levodopa/carbidopa microparticles, additional studies should be done to further sustain the release of levodopa/carbidopa microparticles by optimizing the formulation variables in order to prolong the half-life. Also, further studies should be done to ascertain the biodistribution of levodopa/carbidopa microparticles in the brain. This will be essential in knowing the levels of levodopa reaching the brain. 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 94 University of Ghana http://ugspace.ug.edu.gh APPENDICES APPENDIX A Calculation of Animal Equivalent Dose (AED) of Levodopa/Carbidopa Human dose = 50/200 mg b.i.d (carbidopa/levodopa) Average human weight = 60 kg Dosage carbidopa = 50 mg/60 kg = 0.83 mg/kg Dosage levodopa = 200 mg/60 kg = 3.33 mg/kg AED = Human dose x Km ratio Km = correction factor = body weight of species/body surface area = 6.2 AED carbidopa = 0.83 mg/kg x 6.2 = 5.146 mg/kg AED levodopa = 3.33 mg/kg x 6.2 = 20.646 mg/kg Animal equivalent dose = 5/20 mg/kg APPENDIX B Calculation for concentrations obtained from content analysis of levodopa Interpolation from calibration equation (y = 2.1383x + 1.4208) y-intercept = 1.4208 95 University of Ghana http://ugspace.ug.edu.gh slope = 2.1383 y = peak areas obtained from HPLC x = concentration of levodopa concentration of levodopa (x) = (y- 1.4208)/ 2.1383 From the triplicate test done, Run 1, Peak area = 1082 Concentration of levodopa = (1082- 1.4208)/ 2.1383 = 505.34 µg/mL Run 2, Peak area = 959 Concentration of levodopa = (959- 1.4208)/ 2.1383 = 447.822 µg/mL Run 3, Peak area = 1035 Concentration of levodopa = (1035- 1.4208)/ 2.1383 = 483.36 µg/mL APPENDIX C Calculation for average percentage cumulative release of levodopa % Average Cumulative Release = (Average concentration of levodopa released into the dissolution medium/ expected concentration) X 100 Expected concentration = (actual amount of levodopa in the weighed 75 mg of microparticles used for in vitro release test)/ volume of dissolution medium 96 University of Ghana http://ugspace.ug.edu.gh Expected concentration = 15 mg/ 500 mL = 0.03 mg/mL =30 µg/mL Average concentration of levodopa released = average concentrations interpolated from the calibration equation At time 0 % Average cumulative release = (0.89/30) x 100 = 2.98 % At time 0.25 h, % Average cumulative release = (1.22/ 30) x 100 = 4.07 % Time 0.5 h, % Average cumulative release = (3.42/ 30) x 100 = 11.40 % Time 1 h, % Average cumulative release = (7.11/30) x 100 = 23.71 % Time 2 h, % Average cumulative release = (11.25/30) X 100 = 37.51 % Time 4 h, % Average cumulative release = (14.72/30) x 100 = 49.07 % Time 8 h, % Average cumulative release = (19.86/30) x 100 = 66.22 % 97 University of Ghana http://ugspace.ug.edu.gh Time 12 h, % Average cumulative release = (23.66/30) x 100 = 78.87 % Time 24 h, % Average cumulative release = (17.2/30) x 100 = 57.33 % APPENDIX D Calculation of concentrations of Standard Levodopa for pharmacokinetic analysis 46 mg of levodopa powder in 10 mL of water = 4.6 mg/ mL = 4600 µg/mL stock standard Add 5 µL of stock standard to 495 µL of methanol containing 0.05 % perchloric acid C 1 V 1 = C 2 V 2 C 2 = (4600 x 5 µL)/ (500 µL) = 46 µg/mL Add 50 µL of stock standard to 450 µL of methanol containing 0.05 % perchloric acid = 460 µg/mL Add 100 µL of stock standard to 400 µL of methanol containing 0.05 % perchloric acid = 920 µg/mL Add 200 µL of stock standard to 300 µL of methanol containing 0.05% perchloric acid = 1840 µg/mL 98 University of Ghana http://ugspace.ug.edu.gh Preparation of calibration standards for injection into HPLC Addition of working analytical standards to internal standards: 50 µL of levodopa standard 1 (46 µg/mL) + 100 µL of dilute methyldopa solution 50 µL of levodopa standard 2 (460 µg/mL) + 100 µL of dilute methyldopa solution 50 µL of levodopa standard 3 (920 µg/mL) + 100 µL of dilute methyldopa solution 50 µL of levodopa standard 4 (1840 µg/mL) + 100 µL of dilute methyldopa solution 20 µL each out of the final volumes were injected into HPLC Chromatograms generated from the HPLC analysis of the calibration standards (A) 99 University of Ghana http://ugspace.ug.edu.gh (B) (C) 100 University of Ghana http://ugspace.ug.edu.gh (D) Chromatograms showing the peak areas of (a) calibration standard 1, (b) standard 2, (c) standard 3 and (d) standard 4. 101 University of Ghana http://ugspace.ug.edu.gh Calibration curve for levodopa levels determination Levodopa Analysis 1.4 1.2 y = 0.0006x + 0.0356 1 R² = 0.99945 0.8 0.6 0.4 0.2 0 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Concentration (µg/mL) APPENDIX E Post Hoc Analysis with Tukey’s Multiple Comparison Time to reach peak concentration (Tmax) ANOVA summary F 7.0 P value 0.0270 P value summary * Significant diff. among means (P < 0.05)? Yes R square 0.70 Tukey's multiple comparisons test Mean 95.00% CI Signific Summ Adjusted P Diff. of diff. ant? ary Value Sinemet CR (µg/ml) vs. LD+CD Powder (µg/ml) -0.17 -0.58 to 0.25 No ns 0.4827 Sinemet CR (µg/ml) vs. LD+CD -0.92 to - Microparticles (µg/ml) -0.50 0.082 Yes * 0.0242 LD+CD Powder (µg/ml) vs. LD+CD Microparticles (µg/ml) -0.33 -0.75 to 0.084 No ns 0.1089 102 Peak ratios of levodopa to methyldopa University of Ghana http://ugspace.ug.edu.gh Peak levodopa concentration (Cmax) ANOVA summary F 14 P value 0.0058 P value summary ** Significant diff. among means (P < 0.05)? Yes R square 0.82 Tukey's multiple comparisons test Mean 95.00% CI Signific Summ Adjusted P Diff. of diff. ant? ary Value Sinemet CR (µg/ml) vs. LD+CD Powder (µg/ml) 83 -45 to 211 No ns 0.1964 Sinemet CR (µg/ml) vs. LD+CD Microparticles (µg/ml) -134 -261 to -5.7 Yes * 0.0423 LD+CD Powder (µg/ml) vs. LD+CD Microparticles (µg/ml) -216 -344 to -88 Yes ** 0.0049 Area under the concentration-time curve (AUC) ANOVA summary F 50 P value 0.0002 P value summary *** Significant diff. among means (P < 0.05)? Yes R square 0.94 Tukey's multiple comparisons test Mean 95.00% CI Signific Summ Adjusted P Diff. of diff. ant? ary Value Sinemet CR (µg/ml) vs. LD+CD Powder (µg/ml) 248 48 to 447 Yes * 0.0208 Sinemet CR (µg/ml) vs. LD+CD Microparticles (µg/ml) -394 -594 to -195 Yes ** 0.0022 LD+CD Powder (µg/ml) vs. LD+CD Microparticles (µg/ml) -642 -842 to -442 Yes *** 0.0002 103 University of Ghana http://ugspace.ug.edu.gh Elimination half-life (t1/2) ANOVA summary F 5.0 P value 0.0518 P value summary ns Significant diff. among means (P < 0.05)? No R square 0.63 Tukey's multiple comparisons test Mean 95.00% CI Signific Summ Adjusted P Diff. of diff. ant? ary Value Sinemet CR (µg/ml) vs. LD+CD Powder (µg/ml) 5.8 0.16 to 11 Yes * 0.0448 Sinemet CR (µg/ml) vs. LD+CD Microparticles (µg/ml) 3.5 -2.2 to 9.1 No ns 0.2224 LD+CD Powder (µg/ml) vs. LD+CD Microparticles (µg/ml) -2.3 -8.0 to 3.3 No ns 0.4600 104