A Stable Micellar Formulation of RAD001 for Intracerebroventricular Delivery and the Treatment of Alzheimer's Disease and Other Neurological Disorders

Abstract

A large body of evidence, replicated in many mouse models of Alzheimer's disease (AD), supports the therapeutic efficacy of the oral mammalian target of rapamycin inhibitors (mTOR-Is). Our preliminary data show that intracerebroventricular (ICV) administration of everolimus (RAD001) soon after clinical onset greatly diminished cognitive impairment and the intracellular beta amyloid and neurofibrillary tangle load. However, RAD001 shows >90% degradation after 7 days in solution at body temperature, thus hampering the development of proper therapeutic regimens for patients. To overcome such a drawback, we developed a stable, liquid formulation of mTOR-Is by loading RAD001 into distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) micelles using the thin layer evaporation method. The formulation showed efficient encapsulation of RAD001 and a homogeneous colloidal size and stabilised RAD001, with over 95% of activity preserved after 14 days at 37 °C with a total decay only occurring after 98 days. RAD001-loaded DSPE-PEG2000 micelles were unchanged when stored at 4 and 25 °C over the time period investigated. The obtained formulation may represent a suitable platform for expedited clinical translation and effective therapeutic regimens in AD and other neurological diseases.

Keywords: Alzheimer’s disease; DSPE-PEG2000 micelles; drug stabilisation; intracerebroventricular; mTOR; mTOR-I; micellar liquid formulation; neurodegeneration; neurological disorders; rapalog.

Figure 1

RAD001-loaded micelles show homogeneous and consistent colloidal sizes. Size distributions of Ev-mic obtained by photocorrelation spectroscopy in physiological solution diluted in ultrapure water at 25 °C.

Figure 2

Micelles show higher stability in physiological solution. Size distributions of Ev-mic obtained by photocorrelation spectroscopy in (a) physiological solution and (b) 10 mM PBS pH 7.4 before and after 21 days at 37 °C.

Figure 3

RAD001 loaded in micelles shows higher stability in physiological solution. RAD001 stability in micelles in 10 mM PBS pH 7.4 and physiological solution over time at 37 °C.

Figure 4

Micelles stabilise RAD001 in solution. RAD001 decay in 10% v/v DMSO/physiological solution (vehicle) (grey) and in micelles (black) at 37 °C, 25 °C, and 4 °C. After 15 days at 37 °C, over 95% of the Ev-mic activity was still maintained, while Ev-sol decayed completely.

Figure 5

Micelle safety in vitro. HeLa and SH-SY5Y cells were challenged for (A) 24 h and (B) 48 h with increasing concentrations of blank DSPE-PEG2000 micelles and analysed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Cells were seeded in a 96-well plate at concentrations of 3000 or 6000 cells/well, respectively, and were maintained in culture medium (100 µL) for 24 h before the treatment.

Figure 6

Micelles extend the activity of RAD001 in vitro. Ev-sol and Ev-mic activity on (A) HeLa and (B) SH-SY5Y cells was measured using the MTT assay. HeLa and SH-SY5Y cells were seeded into a 96-well plate at concentrations of 3000 cells/well and 6000 cells/well, respectively, and were maintained in culture medium (100 µL) for 24 h before the treatment. Ev-sol and Ev-mic were used fresh and after incubation at 37 °C, 25 °C, and 4 °C for 7, 14, 35, 50, and 77 days. Untreated, DMSO-treated (Veh), and blank micelle-treated (Empty mic) cells were employed as negative controls, while fresh RAD001 solution-treated cells (Ev-sol fresh) were used as positive controls. All treatments were performed at the same equivalent concentration of RAD001 (5 nM). Data are representative of three independent experiments. * p < 0.001, ** p < 0.01.

Figure 7

Hypothetical explanation of the prolonged recovery observed in the AD mouse model treated with ICV everolimus [30]. On the left, a cognitively impaired 3xTg-AD mouse. On the right, a cognitively recovered 3xTg-AD mouse. On the upper part of the figure, the in vitro production of βA-specific Tregs is described: (A) in vitro conditioning of DCs with βA and a rapalog [8,9,10,11,12,13], (B) injection of conditioned DCs into the bloodstream, (C) conditioned DCs migrate to regional lymph nodes; (D) infusion into the bloodstream of specific Tregs into 3xTg-AD mouse; (E) the infusion of βA-specific Tregs leads to improved βA tolerance and cognitive recovery [46] (green arrow), but not to βA load reduction (red arrow). On the bottom part of the figure, the in vivo approach and the hypothetical explanation of its rapid and prolonged efficacy are described [30,47]. (AB) The rapalog-loaded osmotic pump is implanted into the lateral ventricle, likely leading to conditioning of CNS-patrolling DCs; (C) conditioned DCs migrate to regional lymph nodes; (D) the treatment might lead to in vivo βA-specific Treg production that could lead to both βA tolerance [46] (green arrow) and to reduction of the βA load, promoting autophagy (F). The combined effect could explain the results observed.