Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the formation of extracellular amyloid plaques, intracellular neurofibrillary tangles (NFTs) as a result of tau hyperphosphorylation [1], loss of cholinergic neurons in the basal forebrain [2], mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP) genes [3] microglial activation alongside inflammation, oxidative damage, iron dysregulation, and cholesterol metabolism [4]. A study conducted by Pivovarora et al. inferred insulin resistance and reduced insulin signaling in the brain as one of the critical factors in AD pathology, owing to the inhibition of IDE-dependent beta-amyloid protein breakdown [5]. According to estimates, 40 million individuals worldwide have dementia, and it is likely to double every 20 years until about 2050 [6].
Donepezil hydrochloride (DPL), galantamine, and rivastigmine are acetylcholinesterase inhibitors (AChEIs) approved for AD treatment [7]. Besides, Memantine is an FDA-approved noncompetitive N-methyl d-aspartate (NMDA) receptor antagonist, which reduces NMDA-mediated ion flow and pathologically elevated glutamate levels and alleviates neuronal dysfunction [8]. DPL and memantine combinatorial therapy were also approved in 2014. Emerging disease-modifying therapies (DMTs) that interfere in the underlying pathophysiological mechanisms of the disease process that lead to cell death captivates a new area of drug research and development in AD which includes BACE inhibitors, γ secretase inhibitors, α secretase modulators, Aβ aggregation inhibitors, Kinase inhibitors, Tau aggregation inhibitors, Microtubule stabilizers, immunotherapy, etc. [2, 9]. Cognitive impairment is reduced temporarily by existing treatments. The failure to traverse the blood-brain barrier, systemic side effects that restrict dosage, and complex dosing schedules are typical obstacles associated with current medications that ultimately reduce patient adherence and result in the discontinuation of therapy.
Oral administration for brain disorders is ineffective, requires a high dose, and has systemic side effects. Intranasal (IN) delivery is a promising alternative as it avoids first-pass metabolism, bypasses the blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier, and is extensively vascularized, allowing for higher drug uptake in the brain [10]. It utilizes the olfactory neuron and trigeminal pathway for drug transport directly to the brain [11] (Fig. 1), reducing the frequency and dosage. The nasal route is achieving more focus as an alternative to the parenteral route by needless avoidance; it represents the most direct method of non-invasive entry into the brain.
Drug delivery approaches to target the brain have been researched to maximize drug effectiveness, minimize degradation and loss, and reduce peripheral and systemic side effects. Approaches include chemical modifications such as prodrug, P-glycoprotein inhibition, physiological such as receptor-mediated transcytosis, and biological methods such as conjugation of drugs with antibodies, use of genomics and non-invasive techniques for direct delivery of drugs to the brain [12]. Researchers have prioritized the emergence of Nano drug delivery techniques lately, with a particular focus on lipid-based systems like Nano emulsions, SLNs, and NLCs to surmount the obstacle of BBB, attain brain targeting, ameliorate the systemic adverse effects and achieve sustainable treatment with desired therapeutic effect. These systems can efficiently deliver hydrophobic molecules and protect them from being eliminated in the nasal cavity. Scientists are also exploring the use of biomolecules, thermosensitive polymers, and mucoadhesive polymers to enhance the therapeutic potential of these lipid-based Nano systems [13].
The present research centers on developing nanostructured lipid carriers (NLCs) for drug delivery to the brain for addressing AD. The higher drug loading and entrapment of NLCs are attributed to the presence of nano-oil sections in the solid lipid matrix and the uneven distribution of liquid lipids in the solid lipid's crystal defects [14]. Including liquid lipids also maintains sub-saturation conditions of solid lipids, preventing crystallinity and polymorphic changes and providing long-term stability to the formulation. Improved brain accessibility, controlled drug release, efficient penetration into tiny capillaries and cellular uptake owing to their small size and lipophilic nature, site-specific targetability, avoidance of first-pass metabolism, and defense against P-gp efflux transporters are other significant attributes of NLCs [15]. Many strategies, such as the development of Duloxetine-loaded NLCs for depression [16], Carbamazepine loaded NLCs for Epilepsy [15], Nimodipine loaded Lactoferrin customized NLCs for ischemic stroke [17], artemether-loaded-NLCs-for-cerebral malaria [18], Curcumin loaded NLCs for brain tumor [19], Asenapine loaded NLCs for schizophrenia [20], Salvianolic acid and Bacicap loaded NLCs modified with transferrin receptor monoclonal antibody (OX26) for cerebral reperfusion injury [21] and Glial cell line-derived neurotrophic factor loaded NLCs modified with Trans activator of transcription (TAT) peptide for Parkinson [22] have been brought out which have corroborated the advantages of brain delivery of NLCs over conventional dosage forms.
DPL was chosen as a drug candidate for the current study due to its high efficacy towards AD and superior anti-Alzheimer activity than other AChE inhibitors. However, it has certain limitations, such as extensive plasma protein binding (96%) owing to long elimination half-life (70hrs), which in turn causes dose-related toxicity (gastrointestinal hemorrhage, bronchoconstriction, vagotonic effects, bradycardia, and hypotension.) [23], limited brain accessibility, first-pass metabolism, and serving as a substrate for P-gp at clinically relevant doses [24]. Based on the above findings, i.e., the challenges faced by existing formulations [tablets (oral), solutions (IV)], the molecule's superior clinical profile, and high lipophilicity, it is a suitable candidate to be formulated into a nanostructured lipid carrier (NLC). Thus, the study aims to be performed, which widens the scope of research on DPL as an intranasal NLC formulation in AD. Embelin (EMB), a naturally occurring substance found in Embelia ribes Burm fruits, has been reported to have a significant role in suppressing symptoms of AD in preclinical investigations by inhibiting AchE, BChE, BACE-1 as per the in silico studies and enzyme inhibition assays [25, 26] and elevating the expression of scavenger enzymes (SOD1 and CAT), reducing oxidative stress and lipid peroxidation, promoting neurogenesis, and contributing to synaptic plasticity (BDNF-CREB levels) [26]. Recent research has also demonstrated that EMB can ameliorate scopolamine-induced amnesia and reverse STZ-induced memory impairment in rats [26, 27]. EMB has also been reported to increase P-gp activity in LS-180 cells, an efflux pump that removes amyloid- from the AD brain [28]. These studies furnished excellent results stipulating EMB as a potent molecule for AD. Moreover, its physicochemical properties make it a good candidate for formulation into an NLC. It has been contemplated that the combination therapy of DPL and EMB could be highly effective, possess lesser side effects, and provide a synergistic effect by a multitargeted mechanistic approach.
The current work aims to prepare DPL-EMB NLCs with an emphasis on achieving optimum drug loading, safer nasal administration, efficient neuronal/cell uptake, better brain accessibility, sustained release, and desired therapeutic effect. The in-silico studies were performed to determine the target proteins/sites and stipulate the possible mechanism of action of the DPL-EMB combination. Cell line studies were further conducted to assess the drug cytotoxicity and determine the combination ratio with maximum effective synergism. DPL-EMB NLCs were synthesized via a hot emulsification sonication technique and were optimized by implementing a Central composite rotatable design (CCRD). The physicochemical characteristics of combinatorial NLCs were ascertained through the utilization of Differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). NLCs were further assessed for particle size, particle-size distribution, zeta potential, drug entrapment, drug loading, in vitro release, and ex vivo permeation in the goat-excised nasal mucosa. Moreover, histopathological studies, DPPH assay, HET-CAM assay, and cellular uptake studies have also been performed.