Development of Polymer-based Sol-Gel Therapeutics for Parkinson's Disease

Abstract

Neurodegeneration in Parkinson’s disease is associated with the formation of neuronal cytoplasmic inclusion bodies, termed Lewy Bodies, which are comprised mainly of the pre-synaptic protein α-synuclein. Aggregation of α-synuclein and subsequent formation of Lewy Bodies is associated with raised intraneuronal Ca (II) and Cu (I/II) levels. In alignment with this notion, post-mortem studies of Parkinson’s disease brain tissue have demonstrated the sparing of neurons that express the Ca (II) buffering protein, Calbindin-D28k. As a result, the upregulation of endogenous metal binding proteins may serve as a potential target to inhibit α-synuclein aggregation. Recent in vitro studies displayed that the vitamin-D analogue Calcipotriol and glucocorticoid Dexamethasone can potently induce the calcium and copper buffering proteins Calbindin-D28k and Metallothionein, respectively. As a result, Calcipotriol and Dexamethasone may serve as a potential therapeutic option for Parkinson’s disease. The treatment of central nervous system disorders is often challenging due to the delivery of therapeutics to the central nervous system being hindered by the blood brain barrier and interactions with the periphery. As a result, considerable attention has been garnered by the nose to brain drug delivery route as it bypasses the blood brain barrier and periphery, thereby mitigating pharmacokinetic challenges faced with conventional delivery routes. A major limitation faced with the nose to brain delivery method is the removal of therapeutics such as sprays from the nasal mucosa due to the mucociliary clearance and lack of mucosal adherent properties in the therapeutic. This problem faced by conventional delivery vehicles is overcome by a new delivery vehicle, termed a soluble gel, which possesses muco-adhesive, thermo-responsive and sustained drug release properties. Hence it was hypothesized that the addition of Calcipotriol and Dexamethasone into the soluble gel formulation may upregulate Calbindin-D28k and Metallothionein in vitro, respectively. This study was divided into two objectives. The first objective involved formulating soluble gels containing Dexamethasone (DXN) or Calcipotriol (CP) which possessed optimal properties for nose to brain delivery. The second objective involved the soluble gels formulated as part of the first objective being applied to SH-SY5Y human neuroblastoma cells, in order to determine whether cytoplasmic Calbindin-D28K and Metallothionein could be upregulated. The gelation temperature and viscosity of the soluble gels were the two parameters which were evaluated for each soluble gel formulation, which were measured by a rheometer. The ranges deemed acceptable for nose to brain delivery for gelation temperature was between 25-34℃ and for viscosity was between 0.4-1 Pa.s at 34℃. The thermo-responsive and muco-adhesive agents employed for the soluble gels were Poloxomer-407 and Carbomer-934P, respectively. It was determined that a soluble gel with Poloxamer-407 15.5% w/w + Carbomer-934P 0.3% w/w + Dexamethasone 0.05% (P15.5/C0.3/DXN) had a gelation temperature of 28.156℃ and viscosity of 0.653 Pa.s at 34℃, which were both between the acceptable ranges for nose to brain delivery. As a result, P15.5/C0.3/DXN underwent a Dexamethasone drug release study involving the use of a Franz drug diffusion apparatus and high-performance liquid chromatography. P15.5/C0.3/DXN displayed sustained release of Dexamethasone over a 72-hour period. The same process was conducted for the soluble gels containing Calcipotriol. Two Calcipotriol containing soluble gels were formulated, which comprised of Poloxamer-407 15% w/w + Carbomer-934P 0.3% w/w + Calcipotriol 0.005% w/w (P15/C0.3/CP0.5) and Poloxamer-407 15.5% w/w + Carbomer-934P 0.3% w/w + Calcipotriol 0.0005% w/w (P15.5/C0.3/CP). P15/C0.3/CP0.5 had a gelation temperature of 28.517℃ and viscosity of 0.574 Pa.s at 34℃, which were both between the acceptable ranges for nose to brain delivery. P15.5/C0.3/CP had a gelation temperature of 28.530℃ and viscosity of 0.494 Pa.s at 34℃, which were also both between the acceptable ranges for nose to brain delivery. No Calcipotriol drug release data was obtained for P15/C0.3/CP0.5 and P15.5/C0.3/CP due to low detection sensitivity. P15.5/C0.3/DXN, P15/C0.3/CP0.5 and P15.5/C0.3/CP were then applied to SH-SY5Y human neuroblastoma cells as part of objective 2. It was determined that the application of these formulations significantly upregulated cytoplasmic Calbindin-D28k and Metallothionein expression. The application of P15.5/C0.3/DXN for 20-minutes demonstrated approximately a 1.871-fold increase in Metallothionein expression compared to the control cells. The application of P15/C0.3/CP0.5 and P15.5/C0.3/CP for 30-minutes and 1-hour displayed approximately a 2-fold increase in Calbindin-D28K expression compared to the control cells. In summary, these findings suggest that the intranasal delivery of soluble gels with active drug components may serve as a potential treatment for Parkinson’s disease by inhibiting metal ion dependent α-synuclein aggregation and subsequent Lewy Body formation.