Novel sustained release formulation of leveodopa-dendrimer conjugate

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Mulla, Nihal S.
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Fibrillation of alpha synuclein (AS) leading to the formation of Lew body has been ubiquitously reported in many geriatrics disorders such as Parkinson's disease (PD), Alzheimer's disease, and other neurodegenerative disorders. Therefore, any substance inhibiting or reducing fibrillation of alpha synuclein is expected to potentially alleviate these disease symptoms. Levodopa is the current drug of choice for the treatment of PD. The effectiveness of any investigative PD drug is compared with that of levodopa. However, its dose needs to increase with chronic use and there are some side effects like nausea and vomiting are associated with the metabolic products of levodopa such as3-ortho methyl dopa and dopamine. It has also been reported that the fluctuating levels of levodopa is responsible for the wearing off effect. The best way to solve these problems was to deliver a sustained amount of levodopa to the brain so that a constant level of dopamine is maintained at the dopamine receptor sites. Therefore, in this study a novel sustained release formulation of levodopa was prepared which efficacy was evaluated by measuring the extent/amount of AS fibrillation. In vitro fibrillation of AS was induced by shaking its solution (37OC, pH=7.4) at 600 rpm. The fibrillation of AS was characterized quantitatively by using Thioflavin t-based fluorescence and Fourier Transform Infrared (FTIR) spectroscopy, and qualitatively by using light microscopy. Thioflavin t assay was chosen due its property to specifically bind to amyloid fibrils and as a result exhibits a higher fluorescence at excitation and emission wavelengths of 440 nm and 490 nm respectively. FTIR technique is also a sensitive method to evaluate formation of alpha synuclein fibrils. There is a characteristic increase in the secondary structure of alpha synuclein due to a change in the arrangement of AS structure from natively unstructured to a highly oriv dered structure after fibrillation. Light microscopy was used to qualitatively observe for the morphology of fibrils formed. Thus any drug which could inhibit the formation of fibrils would reduce the Thioflavin t fluorescence and also reduce the secondary structure formation observed using FTIR. A novel sustained release formulation of levodopa was prepared by conjugating it with PAMAM dendrimer which was further incorporated in a nanoparticle. To prepare the conjugate levodopa (1mg/ml) was added to dendrimer solution (1mg/ml) at alkaline pH. They were allowed to react for 45 minutes with constant stirring and then lyophilized. The conjugate between levodopa and PAMAM dendrimer was characterized using FTIR, UV spectroscopy and Differential scanning Calorimetry (DSC). The effect of this conjugate on the AS fibrillation was observed using Thioflavin t assay technique, FTIR and light microscopy. There was significant difference between the inhibition of AS fibrillation caused due to levodopa-dendrimer conjugate and inhibition caused due to levodopa or dendrimer alone (p<0.05). The reason behind higher inhibition of AS fibrillation by the conjugate might be due to a greater binding between the conjugate and AS. The conjugate was further used to prepare surface modified solid lipid nanoparticles. The conjugate was added to the lipid phase made up of Glyceryl monooleate (GMO) and oleic acid. Tween 80 was used as the emulsifying agent having a HLB value of around 13. Tween 80 was used due to its reported property of increasing brain targeting. Nanoparticles were prepared by ultra-sonication to reduce particle size. The nanoparticles were characterized by particle size and surface charge analyzer, DSC and FTIR. In vitro drug conjugate release was observed at pH 7.4. The developed delivery systems showed high encapsulation efficiency and were able to sustain the release of the drug for 5 days. The nanoparticles showed burst release by releasing about 40-50% of drug conjugate by the end of 24 hrs. Nanoparticles existed in the particle size range of approximately 230-240 nm having a net negative charge of around -25 mV. The negative charge developed might be due to the presence of sodium tri-polyphosphate. The effectiveness of the drug loaded nanoparticles against AS fibrillation was studied and characterized using Thioflavin t fluorescence spectroscopy, FTIR and light microscopy. The nanoparticles were found to inhibit AS fibrillation by about 90%. The conjugate inhibited the AS fibrillation better than nanoparticles but this was not a major concern since the nanoparticles would sustain the release of drug conjugate in vivo and the levodopadendrimer conjugate would get eliminated before nanoparticles by the body’s elimination mechanism. To summarize, the conjugate showed better inhibition of AS fibrillation compared to levodopa or dendrimer alone and the drug loaded nanoparticles showed sustained release for 5 days.
Creighton University
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