Intranasal therapeutic strategies for management of Alzheimer's disease.

http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, 2014; 22(4): 279–294 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.876644

REVIEW ARTICLE

Intranasal therapeutic strategies for management of Alzheimer’s disease Department of Pharmaceutics, J.S.S. College of Pharmacy, Udhagamandalam, Tamil Nadu, India

Abstract

Keywords

Alzheimer’s disease (AD) is a chronic and progressive age-related irreversible neurodegenerative disorder that represents 70% of all dementia with 35 million cases worldwide. Successful treatment strategies for AD have so far been limited, and present therapy is based on cholinergic replacement therapy and inhibiting glutamate excitotoxicity. In this context, role of neuroprotective drugs has generated considerable interest in management of AD. Recently, direct intranasal (IN) delivery of drug moieties to the central nervous system (CNS) has emerged as a therapeutically viable alternative to oral and parenteral routes. IN delivery bypasses the blood–brain barrier by delivering and targeting drugs to the CNS along the olfactory and trigeminal neural pathways which are in direct contact with both the environment and the CNS. In an attempt to understand how neurotherapeutics/nanoparticulate delivery systems can be transported from the nose to the CNS, the present review sets out to discuss the mechanism of transport from nose to brain. The aim of this review is to discuss and summarize the latest findings of some of the major studies on IN drug delivery in AD models, with a focus on the potential efficacy of neuroprotective treatments.

Alzheimer’s disease, dementia, intranasal, nanoparticles, neuroprotective

Introduction Alzheimer’s disease (AD) is a chronic and progressive agerelated neurodegenerative disorder that represents 70% of all dementia with 36 million cases worldwide and projections suggest that these may increase to 115 million by 2050 [1,2]. It is characterized by accumulation of b-amyloid (Ab), senile plaques, neurofibrillary tangles (NFTs), cognitive and memory impairments; eventually progressing to physical impairment and death [3]. Cerebrovascular pathology, including cerebral amyloid angiopathy is observed in AD patients [4]. The number of people suffering from AD has been increasing exponentially with one new case every 4 min. The cognitive decline associated with AD drastically affects the social and behavioural skills of the people living with the disease. Notwithstanding the social impact, however, AD also imparts great financial burden on patients, families and communities as a whole [5]. As such, the disease poses heavy economic and societal burden, with associated annual cost of care over $100 billion [6]. There are several limitations associated with present therapy and intranasal (IN) strategy seems to be promising route for delivery of drugs to brain. In an attempt to *These authors have contributed equally to this work. Address for correspondence: Sumeet Sood and K. Gowthamarajan, Department of Pharmaceutics, J.S.S. College of Pharmacy, Udhagamandalam, Tamil Nadu 643001, India. Tel: +91 423 2443393, +91 9443089812. Fax: +91 423 2442937. E-mail: sumeetsood@rediff mail.com (S. Sood); [email protected] (K. Gowthamarajan)

History Received 20 September 2013 Revised 8 December 2013 Accepted 14 December 2013 Published online 9 January 2014

understand how neurotherapeutics/nanoparticulate delivery systems can be transported from the nose to central nervous system (CNS), the relevant mechanisms of nose to brain transport are discussed. The overall aim of this review is to discuss and summarize the latest findings of some of the major studies that convincingly show the transport of drugs into the CNS following IN route in AD models, with a focus on the potential efficacy of neuroprotective treatments.

Genetics of AD On the basis of genetic etiology, AD can be classified as familial AD (FAD) and sporadic AD (SAD). The inheritance of AD has been explained by genetic analysis of some rare cases of early onset FAD [7]. FAD represents 51% of all AD cases and is caused by mutations in app, ps1 and ps2 genes responsible for expression of amyloid precursor protein (APP), presenilin-1 and -2 (PS-1 and PS-2) proteins. They are located on chromosome 21, 14 and 1, respectively [8]. SAD occurs at 465 years of age and represents 99% of AD cases. In majority of these cases, there were no mutations in genes involved in FAD [9]. Olson and co-workers have found a gene on chromosome 20p to be involved in SAD [10,11]. Mutations and polymorphism in multiple genes contribute to SAD pathogenesis along with non-genetic factors [7]. Elevated Ab levels in brain generate neurotoxic Ab oligomers which may disrupt normal brain function or aggregate to form plaques which represent a major pathological step in progression of AD. In addition, polymorphism in genes for apolipoprotein E (apoE), a-2 microglobulin, very low

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Sumeet Sood*, Kunal Jain*, and K. Gowthamarajan

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density lipoprotein receptor and low density lipoprotein receptor-related protein are involved in AD pathogenesis [12]. Presence of apoE "4 allele has been linked to increased risk of AD. apoE increases Ab aggregation in CNS. Patients carrying "4 allele have minimal inhibitory effect of apoE leading to higher deposition of Ab [13].


Amyloid cascade hypothesis Amyloid cascade hypothesis of AD states that formation of Ab is a key step in initiation of AD further leading to formation of senile plaques and subsequent hyperphosphorylation of tau resulting in formation of NFTs. APP, a key molecule involved in AD, is a type I mammalian transmembrane protein encoded by single gene on chromosome 21q21 [7,14]. APP can be cleaved by two proteolytic processing pathways- non-amyloidogenic and amyloidogenic pathway [15] as shown in Figure 1. In non-amyloidogenic pathway, APP is cleaved by a-secretase at lys16-leu17 bond within Ab region to yield extracellular large fragment called soluble APPa (sAPPa) and C83 fragment. C83 can be processed by g-secretase to produce a small 3 kDa peptide p3. sAPPa has several neuroprotective properties [16]. Stimulating a-cleavage of APP leads to decrease in formation of Ab [17]. In amyloidogenic pathway, APP is cleaved by b-secretase to yield soluble APPb (sAPPb) and a membrane bound fragment of 99 amino acids (C99). C99 is further hydrolysed to produce Ab peptide with varying number of amino acids (Ab1-40 and Ab1-42). Ab is a highly insoluble protein with b-pleated-sheet conformation and is deposited extracellularly in the form of 5–10 nm wide straight fibrils [18]. Two enzymes capable of b-cleavage have been identified-BACE-1 and BACE-2 [19]. BACE-1 (also called Asp2 or memapsin 2) is a transmembrane aspartyl protease [20]. It is a key rate limiting enzyme in proteolytic processing of APP to form Ab [21]. BACE-2 is a homologue of BACE-1 (55% homology) having similar substrate specificity but there is no evidence of it being involved in the cleavage of APP and AD pathology. PS-1 and PS-2 are catalytic components of g-secretase [22]. Their mutation results in the enhanced Ab42 level and is involved in FAD. They are membrane proteins comprising

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463 and 448 amino acids [23–25]. Apart from senile plaques, AD is associated with NFT which are intraneuronal aggregates of hyperphosphorylated tau, a microtubule-associated protein. They are found in neurons with trace amounts in nonneuronal cells. They play an important role in polymerization and stabilization of microtubules and transport of organelles. Tau proteins bind microtubules through microtubule-binding domains [26]. Hyperphosphorylation of tau causes destabilization of microtubules by aggregation of tau proteins into paired helical filaments, impairs axonal transport and forms NFTs leading to death of neurons. Hyperphosphorylation of tau proteins may be due to increase in kinase activity or decrease in phosphatase activity. Glycogen synthase kinase 3 (GSK3) is one of the kinases implicated in AD [27]. Tau pathology commences in the entorhinal/hippocampal region and spreads into different cortical regions [28].

Cholinergic hypothesis Cholinergic deficits have been found to contribute significantly to neuropsychiatric manifestations of AD. Degeneration of cholinergic neurons of basal forebrain is considered as one of the earliest pathological events in AD [29,30]. AD is associated with decrease in choline acetyl transferase (ChAT) activity, high affinity choline uptake, acetylcholine (ACh) release and muscarinic and nicotinic ACh receptor binding. These cholinergic deficits correlate with cognitive impairment in AD and non-cognitive behavioural disturbances [31].

Insulin signalling in AD Insulin, a peptide hormone, is produced by beta cells of pancreas and is involved in regulation of carbohydrate and fat metabolism in the body. It is essential for energy metabolism in brain tissues. It was believed that insulin acts only on peripheral tissues and has no effect on brain. However, the discovery of insulin receptors (IRs) in rodent and human brains confirmed that brain was not insulin-independent as thought earlier [32–34]. IRs are present in hippocampus and cerebral cortex and play an important role in learning and memory [35]. Insulin is transported to brain via receptormediated saturable transport mechanism and is highest in olfactory bulb followed by pons, medulla and hypothalamus.

Figure 1. Amyloid cascade hypothesis of AD. APP can undergo proteolytic processing by two pathways- non-amyloidogenic and amyloidogenic pathway. In non-amyloidogenic pathway APP is cleaved by a- and g-secretase to form sAPPa and p3. Amyloidogenic pathway involves b- and g-secretase to form sAPPb and amyloid b (Ab).


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Dysregulation of brain IRs has been associated with pathogenesis of AD [36,37]. AD is associated with disturbed cerebral glucoregulation [38], reduced brain insulin receptor activity along with the lower CSF insulin level, peripheral hyperinsulinemia [39] and attenuated insulin and insulin-like growth factor expression [40] suggesting that impaired brain insulin signaling plays a critical role in the loss of memory functions. Insulin has been found to improve memory functions particularly hippocampal-dependent memory in animals and humans. Peripheral insulin resistance is considered as an important etiological factor in AD and is associated with reduced basal and insulin-induced activation of IRs. It promotes AD by increase in Ab in brain, phosphorylation of tau, oxidative stress (OS), neuroinflammation, reactive advanced glycation endproducts, dyslipidemia and apoptosis. In addition, brain itself becomes resistant to insulin that promotes the progression of disease [41]. Diabetes has also been found to be a significant risk factor in AD. People with diabetes are 41.5-fold more likely to develop AD [42]. Both diseases have similar metabolic abnormalities like disordered glucose metabolism, abnormal insulin receptor signaling and insulin resistance, OS and structural abnormalities in proteins and Ab deposits. Microvascular damage in diabetic polyneuritis with CNS changes and cognitive impairment due to systemic OS constitute the two major hypotheses of link of diabetes and AD [43]. Recently, experimental induction of type-2 diabetes in the senescence-accelerated mouse prone-8 (SAMP8) model of accelerated ageing triggered Alzheimerlike pathology and memory deficits. Diabetic SAMP8 mice exhibited increased cerebral Ab, dysregulated tauphosphorylating GSK3b, reduced synaptophysin immunoreactivity, and displayed memory deficits, indicating Alzheimer-like changes [44].

Drugs for AD Currently approved drugs for treating the cognitive impairments in AD are based on neurotransmitter or enzyme replacement/modulation. Acetylcholinesterase inhibitors (AChEIs) are the main class of drugs used in AD. Tacrine was the first AChEI approved by the US Food and Drug Administration (FDA) for the treatment of AD. However, it was withdrawn due to side effects like hepatotoxicity. Donepezil, galantamine and rivastigmine are secondgeneration AChEIs [45,46]. AChEIs are associated with gastrointestinal adverse effects like nausea and vomiting that most commonly lead to discontinuation of treatment [47]. Tacrine and physostigmine have low oral bioavailability (BA) [48,49]. Another approach to the treatment of AD is to block glutamatergic neurotransmission. Glutamate excitoxicity mediated through excessive activation of N-methylD-aspartate (NMDA) receptors is believed to play a role in the neuronal dysfunction and loss observed in AD [50]. Memantine, approved in 2004 by FDA for the treatment of moderate to severe AD, is a non-competitive NMDA-receptor (NMDAR) antagonist with moderate affinity that appears to be able to protect neurons without interference with physiological NMDAR activation [51]. These treatment

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strategies provide only moderate benefit and thus there is an apparent need for improved therapies such as Ab, secretase or tau inhibitors and vaccine therapy.

Alternative treatment strategies OS and Ab are considered as a major etiological and pathological factor in the initiation and promotion of neurodegeneration in AD. OS can cause cellular damage and subsequent cell death because the reactive oxygen species oxidize vital cellular components such as lipids, proteins and DNA. Antioxidants and natural products of widely varying chemical structures have been investigated as potential therapeutic agents [52–55]. The cause of such OS includes transition metals, such as iron and copper, which are found in high concentrations in the brains of AD patients and accumulate specifically in the pathological lesions; and are viewed as key contributors to the altered redox state. Likewise, the aggregation and toxicity of Ab is dependent upon transition metals. As such, chelating agents that selectively bind to and remove and/or ‘‘redox silence’’ transition metals have long been considered as attractive therapies for AD [56,57]. Free heme, a powerful pro-oxidant containing iron, which occurs in brains of AD patients has been found to inactivate, via OS, human brain muscarinic ACh receptors involved in memory and learning [58]. Heme is synthesized in all nucleated cells, including brain cells by ferrochelatase located in the inner membrane of mitochondria which then reaches cytosol to form regulatory heme [59]. Regulatory heme controls the mitochondrialcytosolic distribution of d-aminolevulinic acid synthase, iron uptake, specific neuronal signaling pathways and transcription factors [60]. Heme metabolism is altered in AD brain and heme binds with Ab forming Ab-heme complex [61]. Sequestration of heme by Ab causes its deficiency in nerve cells thus disturbing normal biochemical processes. This results in consequences similar to AD cytopathologies [59]. Ab, to some extent, exerts its neurotoxic effects through numerous secondary pathways, including tau hyperphosphorylation and NFTs formation, oxidation, inflammation, demyelination and excitotoxicity [62]. These processes are mediated by nuclear factor kB (NF-kB), GSK3, peroxisome proliferator-activated receptor-g (PPAR-g), etc. and are potential targets for neuroprotective therapies. NF-kB is a transcription factor located within cytoplasm and has role in regulation of cytokine production. When activated, NF-kB increases transcription of different inflammatory mediators. PPAR-g is a ligand-dependent nuclear hormone receptor transcription factor; it regulates inflammatory responses in different organ systems, including CNS. Activation of PPAR-g suppresses Ab-mediated induction of microglial cells from producing pro-inflammatory cytokines. It also inhibits NFkB-mediated inflammatory pathways by reducing its nuclear translocation [14]. However, therapeutic use of most of these compounds is limited since they do not cross the blood–brain barrier (BBB).

Challenges imposed at BBB Conventional drug delivery methods fail to deliver a number of neuropharmaceutical agents like anti-oxidants, natural


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products to the CNS efficiently for treatment of AD [63]. The action of therapeutic agents depends on their ability to be delivered into the brain. The BBB, which segregates the brain interstitial fluid from the circulating blood, consists of the endothelial tight junctions (zonulae occludens) of the cerebral microvasculature [64]. Furthermore, brain is protected by blood–cerebrospinal fluid barrier (BCSFB) which is an active interface between the blood and cerebrospinal fluid that runs in the subarachnoid space surrounding the brain. This barrier is located at the choroid plexus, and is created of epithelial cells held together at their apices by tight junctions [65]. The BBB and BCSFB provide the brain with essential nutrients such as glucose or iron through glucose transporter 1 and transferrin receptor, respectively, and also serves to protect the brain and spinal cord from a variety of pathogens and toxic substances in the blood that could damage neurological functions, and maintain proper homeostasis [66,67]. In addition to its decreased paracellular permeability and low rate of pinocytosis, the BBB also expresses a high number of drug transporters such as P-glycoprotein (P-gp) which further restrict the transport of various endogenous and exogenous substances from systemic circulation into the brain that would otherwise be predicted to cross the BBB based on molecular weight and lipophilicity considerations [68]. Thus, BBB and BCSFB represent a significant barrier for in vivo therapeutic drug delivery into the CNS for various disease treatments. Many different strategies have been used to obtain CNS targeting of systematically administered drugs. These strategies range from invasive and dangerous approaches such as intracerebral implants, intraparenchymal, intraventricular, intrathecal delivery (BBB disruption) to the non-invasive techniques involving the modification of the physicochemical properties of the drug, pro-drug approach, chimeric peptides and conjugation of a drug with antibodies or ligands [69].

IN delivery-challenges and possibilities IN delivery of drug moieties has seriously emerged as a therapeutically viable alternate route to oral and parenteral routes. It is a non-invasive method for bypassing the BBB, allowing direct drug delivery to the brain via the olfactory region and respiratory epithelium since the olfactory nerve cells and trigeminal nerves are in direct contact with both the environment and the CNS [70,71]. Thus, this route has potential for treating neurologic disorders in mice, rats, primates and humans [72–76]. It provides several advantages over other routes of drug administrations. These include fast onset of action, avoidance of the intestinal and hepatic presystemic disposition, reduction of systemic exposure and side effects with direct delivery and targeting to the brain and CSF, practicality and ease of administration and better patient compliance. Some of the limitations associated with this route are mucociliary clearance of nasally applied drugs and poor nasal permeability [77]. Alternative approaches have been adopted to overcome these problems such as the use of mucoadhesive formulations or chemical penetration enhancers. Colloidal

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drug delivery systems are also a promising approach for enhanced and controlled nasal drug delivery. The promising perspective on the use of nanocarriers for IN delivery is their nano size (10–1000 nm) which are able to pass through various physiological and biological barriers and hence facilitate rapid transport of neurotherapeutics through the nasal mucosal barrier [78,79]. Furthermore, nanoparticles can protect the entrapped drugs from degradation thereby increasing the drug concentration in the brain. Studies have disclosed some essential prerequisites for IN drug delivery systems. The delivery systems ideally should have a longer residence time of drugs administered to nasal cavity in order to overcome the nasal mucociliary clearance [80]. The obstacles imposed by brain protective mechanisms and limitations related to nasal administration has increased an interest in developing strategies to overcome them when brain drug exposure is required. Of these strategies, the most often studied is the nanoparticulate system employing mucoadhesive polymers for nose to brain delivery [81–83]. These systems increase the nasal absorption of drugs by prolonging the drug retention time in mucosa and by opening up the tight junctions between the epithelial cells [84]. Protection of the encapsulated drug from enzymes and chemical degradation and/or efflux back into the nasal cavity by inhibiting the P-gp efflux proteins, preventing drug irritant effects, controlled drug release and obtaining desired blood concentrations are possible within these particulate systems. They also enhance the stability of drugs or pro-drugs in physiological fluids. Direct transport of drug to the brain may, therefore, lead to the administration of lower doses and in return reduce their side effects [85,86]. Mucoadhesive polymers such as degradable starch, dextran, hyaluronic acid, chitosan, MCC, HPC, HPMC, CarbopolÕ , wax-like maize starch, PLA and PLGA have been used to improve the drug absorption through nasal mucosa. These polymers enhance the drug residence time and promote the paracellular absorption of most hydrophilic and macromolecular drugs in the nasal cavity whereas reducing their ciliary clearance. Most of these polymers are generally recognized as safe pharmaceutical excipients and are not absorbed, so they would not cause any systemic toxicity and damage to the nasal mucosa [87]. The process of mucoadhesion occurs following IN administration, with the polymer interacting with mucin secreted by the submucosal glands [88]. The sequential events that occur during mucoadhesion include hydration and swelling of the polymer followed by an intimate contact between the polymer and nasal mucosa. The swelled mucoadhesive polymer then interacts with the biological tissue (including the mucus). The polymer chains penetrating into the tissue crevices can prevent the ciliary movement, which enhances the residence time of the drugs intranasally [89]. Factors influencing mucoadhesion are: Factors associated with the polymer are molecular weight, free chain length and cross-link density. Environmental factors include hydration, pH and swelling. Physiological factors are mucociliary clearance, mucus turnover and any presence of ailment.

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These factors have been explained in detail in various publications [80,90].


Pathways of transport from nose to brain The precise mechanisms and pathways by which neurotherapeutics/nanoparticulate delivery systems can be transported from the nasal epithelium to various regions of the CNS have been described in this section. Various routes for nose to brain drug delivery are shown schematically in Figure 2. IN route presents the delivery of neurotherapeutics or nanoparticles via olfactory and trigeminal nerve pathways in the nasal epithelium to the olfactory bulb and brainstem, respectively, with further entry to distant widespread sites within the CNS [77,91]. Transport of drugs across the epithelial barriers (olfactory of respiratory) in the nasal passages may occur either by intracellular or extracellular pathways. The first step in intracellular transport across the olfactory and respiratory epithelia includes endocytosis into olfactory sensory neurons and trigeminal ganglion cells, respectively [92]. This pathway is also called as intraneuronal transport and is very slow and agents may take 24 h to reach CNS after nasal administration [93]. This is followed by intracellular transport to olfactory bulb and brain stem. Transcytosis or transcellular transport of drug into lamina propria is a common pathway in transport across both epithelial barriers. Transcytosis involves the permeation of lipid soluble molecules across the apical cell membrane, intracellular space and basolateral membrane either by passive diffusion or receptor-mediated endocytosis (active transport). Extracellular transport across olfactory and respiratory epithelia takes place by paracellular diffusion to lamina propria. It has been estimated it takes 0.73–2.3 h for diffusion to olfactory bulb along olfactory associated extracellular pathway and 17–56 h for diffusion to brain stem along


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trigeminal associated extracellular pathway [92]. Paracellular route is associated with tight junctions, intracellular space, gaps or pores present between the epithelial cells. Hayashi et al. [94] reported the pore size of nasal membrane to be ˚ and McMartin et al. [95] investigated that substance 3.9–8.4 A less than 1000 Da (without enhancers) would have easy permeation through the nasal mucosa. This route constitutes an important pathway for the absorption of polar or hydrophilic substances, peptides and proteins. These molecules diffuse slowly from nasal membrane into the blood stream, later into the olfactory mucosa and finally transported into CNS. This pathway is less efficient with respect to transcellular pathway and is strongly dependent on drug molecular weight and size [96]. Moreover, this mechanism is quite fast and responsible for transport of low molecular weight drugs to CNS within minutes of administration [97]. The drugs may also be transported by rapid extracellular delivery through intercellular clefts in the olfactory and respiratory epithelium and extracellular transport along the olfactory and trigeminal neural pathway to reach the CSF and brain. One the drug reaches lamina propria it may undergo different fates and include (1) absorption into olfactory blood vessels and further transport to systemic circulation; (2) drugs may enter nasal lymphatic vessels and enter deep cervical lymph vessels; (3) the drug if escapes transport to systemic circulation or lymphatic transport may enter cranial compartments associated with olfactory nerve bundles by extracellular diffusion. The average distance of olfactory pathway and trigeminal pathway to CNS targets in an adult rat is 4–5 and 20 mm, respectively [92]. The clearance of nasally administered drugs, transport to CNS and subsequent elimination is governed by various physicochemical properties of drug-like lipophilicity, molecular weight and presence of specific

Figure 2. Pathways for delivery of neurotherapeutics/nanoparticulate delivery systems to brain. Drug or nanoparticles may be transported across olfactory or respiratory epithelium along both the olfactory and trigeminal nerve pathways to the brain and upper spinal cord. EC, endocytosis; IC, intracellular; TC, transcellular; PC, paracellular transport.


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receptors on olfactory neurons and also the characteristics of drug formulation [98,99]. The nasally administered drugs may be cleared from nasal cavity by mucociliary clearance. The drug reaching blood circulation may be cleared via normal clearance mechanisms or enter brain by crossing BBB and BCSFB. The drug may also be eliminated through CSF into blood [77]. The transport of drug may encompass one or combination of pathways as discussed above. The transmucosal transport of nanoparticles from nose to brain may take place by through olfactory neurons via various endocytic pathways of sustentacular or neuronal cells in the olfactory epithelium [100]. This has been confirmed by novel spectroscopy and microscopy techniques like electron energy loss spectroscopy and energy filtering transmission electron microscopy which have provided new insights into endocytosis and cellular mechanisms for transmucosal delivery of nanoparticles [101]. Confocal microscopy revealed that fluorescent labelled polystyrene nanoparticles of diameter in the range of 20–200 nm were involved with clathrin-coated pits whereas nanoparticles of size in the range of 200– 1000 nm were found to be involved with caveole-mediated endocytosis. The authors incubated these nanoparticles with murine melanoma cells (B16-F10) and cells were treated with selective endocytic inhibitors in order to reveal the pathways associated with nanoparticles uptake [102]. Other factors that influence the internalization pathway of nanoparticles is their surface charge, cell type and concentration of particles applied to the cells [103,104]. Thus, the endocytic trafficking of nanoparticles is dependent on both of its size and surface characteristics which could be used as a means of improving their cellular uptake into olfactory epithelial cells. The challenge now is to improve the uptake of drug-loaded nanoparticles from nose to brain in order to achieve sufficient therapeutic levels in the target brain regions of humans. The higher localization of drug in brain is necessary to target the receptors for obtaining the therapeutic efficacy of drug for the management of CNS diseases such as Parkinson’s, schizophrenia, Alzheimer’s, brain tumors, meningitis and migraine thereby reducing the drug delivery to non-targeted sites.

Safety considerations for nasal formulations One of the key considerations for development of nasal dosage forms is their safety and toxicological assessment. The extended contact of formulations with nasal mucosa may lead to irritation, tissue damage, epithelial/sub epithelial toxicity or ciliotoxicity and may result in environment suitable for microbial growth [90]. In addition, IN drug formulation should not damage the primary olfactory nerves and the sense of smell. Among various mucoadhesive polymers evaluated for nasal safety studies, chitosan [105,106] and carbopol 974 P [107] have been found to be suitable for nasal administration. Cytotoxic and ciliotoxicity studies are among common techniques to determine the safety of formulations or excipients. The measurement of ciliary beat frequency is another accurate and reproducible technique to study effects on ciliated epithelium and is a good in vitro screening method for studying toxicity [108,109]. These studies offer preliminary data on toxicity profile of an

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excipients or formulation. Further to achieve accurate and reliable results on potential toxicity, long-term studies in animals and humans need to be carried out.

Strategies to enhance nasal drug absorption IN route has been investigated for topical, systemic or CNS delivery of drugs. Some of the strategies investigated for enhancing the absorption and thereby the BA of nasally administered drugs includes the use of pro-drugs, absorption enhancers, enzymatic inhibitors and particulate drug delivery systems such as microparticles, nanoparticles and nanoemulsion (Figure 3). However, for treatment of neurological disorders like AD, it is necessary to achieve higher drug concentration in CNS [110,111].

IN delivery strategies for AD There has been a growing interest in IN strategy for AD as evidenced from number of research papers published recently. AChEIs, natural anti-oxidants, insulin, nerve growth factor (NGF), peptides and several other molecules have been investigated as summarized in this section. IN delivery for treatment of AD and other CNS disorders was first proposed and claimed by Frey in 1989 [112]. He and co-workers first discovered and patented the use of IN delivery to bypass the BBB and target therapeutics to the brain, including neuroprotective factors and growth factors such as NGF, FGF2(bFGF), IGF-I, insulin, CNTF, BDNF, EPO, NT-4, etc. He also invented and patented the IN insulin treatment for AD and, along with his collaborators, the IN deferoxamine (DFO) treatment for AD [74,113–118]. Thus, IN route is a promising approach for delivery of current drugs and alternative treatment molecules in AD and overcome limitations of oral dosing. Huperizin A Huperizin A (Hup A), an unsaturated sesquiterpene alkaloid, extracted from a club moss (Huperzia serrata) is a powerful and reversible inhibitor of AChE. It easily penetrates the BBB and is a promising therapeutic agent for AD. However, it influences peripheral cholinergic system leading to side effects and alternate delivery systems are required to specifically target the drug to brain. To overcome these limitations Zhao et al. [119] have investigated Hup A nasal delivery by means of in situ gel of gellan gum. Hydrogel-based systems have been used to modulate drug release and prevent the rapid clearance of the drug formulation from the nasal cavity. The authors studied the brain uptake of Hup A after IN administration of in situ gel to rats and compared the pharmacokinetic parameters with intravenous (IV) and peroral route. The results indicated that concentration of the drug after 6 h in the cerebrum, hippocampus, cerebellum, left olfactory bulb and right olfactory bulb were 1.5, 1.3, 1.0, 1.2 and 1.0 times of those after IV administration, and 2.7, 2.2, 1.9, 3.1 and 2.6 times of those after oral administration. The AUC brain0!6h/AUCplasma0!6h of drug in the cerebrum, hippocampus and left olfactory bulb following the IN administration was significantly higher than the IV administration. The results revealed that IN route is a viable option for improving the brain-targeting efficiency of Hup A and



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Figure 3. Strategies to enhance nasal drug absorption.

also to reduce the side effects to peripheral tissues. This is the first report of IN delivery of Hup A and represents a promising approach for management of AD. Among the various nasal formulations studied for CNS delivery in AD, nanoparticles have been found to improve drug transport across the epithelium due to the small particle size and the large total surface area [120]. Tacrine Peroral administration of tacrine, a potent, centrally active, reversible AChEI is associated with low BA due to an extended hepatic metabolism, short elimination half-life and hepatotoxicity. Jogani et al. [121] have investigated direct nose-to-brain delivery of tacrine to improve its BA, avoid first-pass effect and minimize hepatotoxicity. Tacrine was labelled with 99mTc (technetium) and administered in BALB/c mice intranasally and intravenously. Intranasally administered tacrine was transported quickly to brain (Tmax 60 min) compared with IV administration (Tmax 120 min). The drug targeting efficiency and drug transport efficiency following IN administration was found to be 207.23% and 51.75%, respectively. The results showed tacrine was directly transported to brain from nasal cavity resulting in higher BA of drug with reduced distribution to non-target sites. Same authors also reported mucoadhesive microemulsion for targeting tacrine to the brain after IN administration in mice. Mucoadhesive microemulsion of tacrine showed fold-fold higher BA in brain compared with drug solution.

Rat brain scintigraphy studies were carried out using 99 m Tc as a marker. The results revealed higher uptake in brain after IN administration. Pharmacodynamic studies of the developed formulations were carried out in the scopolamineinduced amnesia model in mice and showed faster regain of memory loss in the mucoadhesive microemulsion-treated group. Their results suggest a possible role of IN tacrine delivery in treating Alzheimer’s patients [122]. Luppi et al. [123] prepared and evaluated the albumin nanoparticles carrying beta cyclodextrins and its hydrophilic derivatives for nasal delivery of tacrine hydrochloride. Bovine serum albumin nanoparticles were prepared by the coacervation method, followed by thermal cross-linking. The prepared nanoparticles had a mean size and polydispersity lower than 300 and 0.33 nm, respectively. The authors also reported that the presence of the different beta cyclodextrins in the polymeric network affected drug loading and could differently modulate nanoparticle mucoadhesiveness and drug permeation behaviour through the nasal mucosa. The ex vivo drug permeation studies across sheep nasal mucosa indicated that tacrine standard solution showed 100% drug permeation in 100 min whereas nanoparticles exhibited sustained permeation profiles and in particular nanoparticles containing the different cyclodextrins revealed enhanced drug permeation with respect to nanoparticles based on albumin alone. The lower permeation and better mucoadhesion of nanoparticles in comparison to drug solution is suitable for improvement of nasal drug BA. A decrease in lag time and an increase in the flux was observed and correlated to beta cyclodextrins

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ability to interact with the lipophilic components of biological membranes changing their permeability. Though tacrine has been discontinued due to hepatotoxicity IN administration could be a useful strategy to minimize its distribution to non-target sites and re-introduce the drug as a new treatment strategy.

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dose level to compare the BA in two dosing routes. The emesis studies revealed significant decrease in emesis or retching by IN route with only 3 observed events compared with 34 events with oral administration. The IN route not only reduced emesis but also increased Cmax more than 4-fold with AUC0-60 and AUC0-120 of 200% and 128%, respectively, compared with oral dosing.


Galantamine Galantamine is an AChEI which has been associated with dose-limiting GI-mediated side effects such as nausea and vomiting, the most common adverse events leading to discontinuation of treatment [124]. However, IN administration of galantamine hydrobromide salt is limited due to its poor aqueous solubility. Leonard et al. [125] investigated different techniques like the addition of co-solvents, cyclodextrins and counter-ion exchange in order to enhance solubility of galantamine. Among these, replacement of bromide with lactate resulted in 12-fold increase in drug solubility. Galantamine lactate formulations showed a better permeation across epithelial membrane than hydrobromide salt. In vivo pharmacokinetic studies revealed that IN galantamine had comparable blood levels compared with oral route. The authors further studied IN formulations of galantamine containing methylated-b-cyclodextrin as stabilizer, L-a-phosphatidylcholine didecanoyl as lipid surfactant and disodium edetate as a chelator [126]. An in vitro tissue model EpiAirwayÔ system consisting of human upper airway epithelia was used to assess permeation and toxicity of the developed formulation. The presence of three permeation enhancers resulted in about three-fold greater permeation of galantamine HBr. MTT and LDH assays showed that cell viability was high and formulations were non-toxic to the membrane. Transepithelial electrical resistance (TER) measurements showed dramatic decrease in TER in formulations containing permeation enhancers which is related to opening of tight junctions. Pharmacokinetic studies of the galantamine formulations were carried out in Sprague-Dawley rats by IN route at dose of 1.75 mg/kg. IN galantamine HBr dosed alone or in the presence of permeation enhancers had absolute BA of 22% and 41%, respectively, resulting in 86% increase in BA with permeation enhancers. The addition of permeation enhancers to IN formulation resulted in 78% increase in BA compared with oral dosing. The in vitro–in vivo correlation between in vitro permeation and in vivo availability suggested that in vitro data are reasonably predictive of in vivo behaviour (R2 ¼ 0.986). Authors further carried out series of experiments using design of experiment to optimize concentration of selected penetration enhancers and their effect on permeation of galantamine lactate, a more soluble salt of galantamine. The optimized formulation of galantamine lactate had four-fold increase in permeation of galantamine in the in vitro model. Galantamine lactate formulations exhibited similar results in term of cell viability, toxicity and TER measurements. The in vivo emetic response of galantamine lactate formulations was studied in ferrets. Ferrets were dosed orally or nasally at dose of 20 mg/kg and emesis or retching was observed for 4 h after dosing. A separate pharmacokinetic study was conducted at the same

Rivastigmine Rivastigmine is an another AChEI approved by US FDA for treatment of AD. Arumugam et al. [127] have investigated multilamellar liposomes for IN delivery of rivastigmine using soy lecithin and cholesterol by the lipid layer hydration method. The developed liposomes had particle size of 10.0 ± 2.8 mm with encapsulation efficiency of 80.0 ± 5.0%. The in vitro release studies showed an initial burst release followed by a log phase with 56.0 ± 2.3% release in 6 h. Pharmacokinetic studies in Wistar rats revealed five-fold higher AUC (36.13 ± 1.87 mg min ml1) for IN liposomes compared with oral-treated group (6.58 ± 0.26 mg min ml1) and three-fold higher value compared with free drug administered intranasally (12.99 ± 0.87 mg min ml1). IN liposomal formulation had Cmax of 0.60 mg ml1 which was 1.7-fold and 10-fold higher than intranasally administered free drug (0.35 mg ml1) and oral administration (0.06 mg ml1), respectively. Nasally administered free drug reached peak within 5 min compared with IN liposomal formulation which had Tmax of 45 min. The authors suggested that free drug reached systemic circulation rapidly via nasal route whereas liposomal formulations accumulated in nasal mucosa and released the drug slowly delaying Tmax. The concentration of rivastigmine in brain was 5.6 times higher following IN administration compared with oral administration. IN liposomes had 20-fold and 3.2-fold higher AUC in brain compared with free drug orally and intranasally, respectively. IN free drug attained Tmax in 15 min whereas IN liposomes reached Tmax in 60 min. Yang and co-workers have investigated tissue distribution and pharmacodynamics of rivastigmine after IN and IV administration at dose of 2 mg/kg. The drug was rapidly and completely absorbed into systemic circulation followed by rapid decline in plasma concentration. IN administration showed higher concentration in CNS and longer inhibition of AChE and BuChE compared with IV route. The inhibitory action on these two enzymes was more pronounced in CNS than peripheral tissues [128]. Fazil et al. [129] investigated rivastigmine loaded chitosan (CS) nanoparticles for IN delivery. The brain/blood ratio of rivastigmine for different formulations were 0.235, 0.790 and 1.712 of rivastigmine (IV), rivastigmine (IN) and rivastigmine nanoparticles (IN), respectively, at 30 min indicative of direct nose to brain transport bypassing the BBB. The brain concentration achieved from IN administration of nanoparticles was significantly higher than those achieved after IV and IN administration of drug solution. The higher drug transport efficiency (355 ± 13.52%) and direct transport percentage (71.80 ± 6.71%) were found with rivastigmine loaded chitosan nanoparticles as compared with other formulation. Thus, IN administration of AChEIs could overcome limitations of oral therapy such as nausea and vomiting.


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Physostigmine Physostigmine, an AChEI, and arecoline, a muscarinin agonist, have shown to improve Alzheimer presenile dementia when administered parenterally. Both the compounds undergo extensive first pass metabolism and are ineffective orally. IN delivery of these drugs was investigated as an alternative route for parenteral delivery. The nasal BA of physostigmine was 100% compared with IV administration and that of arecoline was 85% compared with intramuscular administration [130]. NXX-066, a physostigmine analogue, is a potent inhibitor of AChE. It is well-absorbed orally but oral BA is poor due to its presystemic metabolism. Dahlin and Bjork have studied uptake of NXX-066 into CSF after IN and IV administration in Sprague-Dawley rats. It was absorbed rapidly and completely into systemic circulation after nasal administration with Tmax of 1.5 min and 100% BA which was similar to nasally administered physostigmine as reported earlier. Tmax value for NXX-066 was lesser than physostigmine which had Tmax value of 6.5 ± 1.7 min. Authors suggested this difference could be because of difference in log p value of two drugs. However, the concentration of drug in CSF was very low after IN and IV administration indicating that uptake into CSF was not enhanced by nasal administration [131]. The transport of drugs to CNS via nasal administration may be significant for poorly soluble drugs and insignificant for drugs which are completely and rapidly absorbed into systemic circulation. Ratio of AUCBrain:AUCPlasma closer to one for a nasally administered indicates negligible drug targeting efficiency [132]. Quercetin Several studies have suggested that free radicals play a crucial role in the progression of AD [133]. Quercetin, a flavonoid, is one of the most prominent dietary antioxidant. It is claimed to improve learning, memory ability and reduce the incidence of certain age-related neurological disorders, including macular degeneration and dementia [134]. However, its therapeutic efficacy has been hampered by poor absorption, rapid metabolism and limited ability to cross the BBB [135]. Tong-Un et al. [136] have evaluated the effects of nasally administered quercetin liposomes on cognition and biochemical alterations in the ethylcholine aziridinium (AF64A) model of AD in rats. Rats were treated with quercetin liposomes, containing 0.5 mg of quercetin in 20 mL via IN route once daily two weeks before and one week after AF64A administration. Cognitive function was assessed seven days after AF64A administration by Morris water maze test. Animals treated with quercetin liposomes showed significant decrease in acquisition time, increase in retention time and decrease in AChE activity compared with negative control. The authors suggested cognitive enhancing effects of quercetin may be attributed to its antioxidant property and inhibition of AChE in hippocampus. Biochemical estimation of OS markers revealed decrease in lipid peroxidation and increase in the level of antioxidant enzymes superoxide dismutase and glutathione peroxidase. In an another study, same authors reported IN administration of quercetin liposomes significantly increased the survival of neurons and

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cholinergic neurons density in hippocampus of the AF64A model of AD [137]. This suggests that in addition to neuroprotective effect quercetin has neurotrophic effect and stimulates neurogenesis in hippocampus. Curcumin Curcumin (diferuloyl methane) is a phenolic phytochemical obtained from rhizome of herb Curcuma longa L commonly known as turmeric [138]. It has been found to exert beneficial effects on experimental models of AD [139]. In vitro studies have shown that curcumin inhibits Ab aggregation and Abinduced inflammation [140–142]. Oral administration of curcumin in animal models of AD has been found to inhibit Ab deposition, Ab oligomerization and tau phosphorylation in brain [140,143,144]. Curcumin has been found to decrease Ab-related inflammation and Ab burden in APP transgenic mice [143]. It also enhances activity of glutathione-Stransferase and inhibits NF-kb. Activation of NF-kb increases the transcription of various inflammatory mediators [14,145,146]. Furthermore, curcumin has been found to improve memory and cognitive deficits in rats [35]. In spite of being a ‘‘wonder molecule’’ the therapeutic efficacy of curcumin is limited by poor aqueous solubility, chemical instability in alkaline medium, rapid metabolism and poor absorption from gastrointestinal tract [147]. Chen et al. [148] have investigated curcumin thermosensitive hydrogel for IN delivery. The hydrogel was composed of Pluronic F127 and Poloxamer 188 had short gelation time, longer mucociliary transport time and prolonged residence in nasal cavity of rats. Nasal mucociliary toxicity studies of the developed formulations revealed that they did not cause any toxicity and integrity of mucocilia was maintained up to 14 days. In vitro release studies revealed that hydrogel release was diffusion controlled by the dialysis bag technique and dissolution controlled by the membraneless method. In vivo pharmacokinetic studies revealed that drugtargeting efficiency for drug after IN administration in cerebrum, cerebellum, hippocampus and olfactory bulb were 1.82, 2.05, 2.07 and 1.51 times compared with IV administration of curcumin solution, respectively. There was significant increase in distribution of curcumin into cerebellum and hippocampus. Recently, we have investigated curcumin mucoadhesive nanoemulsions for IN delivery [83]. The formulations were subjected to in vitro cytotoxicity using SK-N-SH cell line and nasal ciliotoxicity studies. The developed formulations did not show any toxicity and were safe for IN delivery for brain targeting. In vitro diffusion studies revealed that nanoemulsions had a significantly higher release compared to drug solution. Ex vivo diffusion studies were carried out using sheep nasal mucosa fixed onto Franz diffusion cells. Mucoadhesive nanoemulsion showed higher flux and permeation across sheep nasal mucosa. Insulin Increasing the level of insulin in CSF may help to improve memory deficits associated with AD [115]. IV insulin administration in AD patients has been shown to improve performance hippocampus-dependent verbal memory task [149]. However, this route is limited by peripheral side effects


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like hypoglycemia and high dose required for achieving sufficient concentration in CSF due to restrictive barriers at BBB. Any therapeutic strategies which can overcome CNS insulin deficiency and resistance might be useful in improving cognitive deficits in AD. IN administration of insulin is a promising approach to overcome the limitations associated with conventional delivery systems [150]. Clinical studies have shown IN insulin exerts rapid effects on EEG parameters. These changes were comparable to those induced by IV bolus injections of the hormone, indicating a significant amount of the applied dose reaches the brain in a functionally active state upon IN administration [151]. IN administration of insulin in humans was first studied by Benedict et al. [152]. Delayed recall of words was significantly improved after eight weeks of IN insulin (4 40 IU/d) administration. Moreover subjects showed marked improvement in mood and self-confidence with reduced anger. These benefits were observed without any systemic side effects which could be of relevance in AD patients. A recent clinical trial on IN insulin has shown to improve memory, attention and functioning in patients with the disease. AD was also characterized by reduced uptake and utilization of glucose in the brain that has been documented by positron emission tomography with fludeoxyglucose F 18 [153]. Reger et al. [72,154] assessed the acute effects of different doses of IN insulin (10, 20, 40 and 60 IU) on hippocampusdependent memory function in memory impaired subjects (early stage AD) in comparison to age-matched healthy controls. IN insulin treatment produced significant memory improvement, with the patients showing maximal benefits at dose of 20 IU. In order to further examine the therapeutic potential of IN insulin in individuals with early stage AD or amnestic MCI, Craft and coworkers treated patients with daily insulin doses for 21 days and tested their memory performance before and after treatment [149]. Insulin-treated AD and MCI subjects displayed greater memory savings than placebo-assigned subjects. These results indicate a beneficial effect of IN insulin on memory preferentially with regard to hippocampus-dependent declarative memory functions. IN insulin has also been found to prevent cognitive decline, cerebral atrophy and white matter changes in the brain [155]. Recently, IN insulin was found to enhance brain energy levels in humans, including ATP and phosphocreatine [156]. Deferoxamine DFO, the natural prototype iron chelator/radical scavenger, has been clinically applied to slow down the progression of the cognitive decline associated with iron-induced AD and also used for the treatment of chronic Fe overload [157]. DFO binds iron with extremely high affinity and IN DFO has been shown to treat and prevent stroke damage after middle cerebral artery occlusion in rats [158]. IN administration of 10% DFO (2.4 mg) three times a week for five months in P301L tau transgenic mice was shown to improve performance in radial arm water maze test. DFO was found to phosphorylate GSK3b and stabilized HIF-1a [159]. Recently, Guo et al. [160] used APP and PS-1 double transgenic mice as a model system to investigate the effects and potential

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mechanisms of i.n administration of DFO on iron-induced abnormal tau phosphorylation. It was shown that high-dose iron treatment markedly increased the levels of tau phosphorylation at a variety of serine and threonine residues, whereas highly induced tau phosphorylation was abolished by IN administration of DFO in APP/PS1 transgenic mice. The results also indicated that DFO IN administration decreases Fe-induced activities of cyclin-dependent kinase 5 (CDK5) and GSK3b. This in turn suppresses tau phosphorylation in neurons and inhibits the formation of intracellular NFTs in the cortical and hippocampal regions of the brain in AD thus improving the deficits of spatial learning and memory in diseased patients. The authors concluded that IN DFO treatment shows its antagonistic effects on iron-induced cognitive dysfunctions and tau phosphorylation via CDK5 and GSK3b pathways. These findings suggest that IN DFO treatment is a potential therapeutic approach against AD neuropathology in the brain. In their previous work, they had reported that chelation of iron by IN administration of DFO (200 mg/kg once every other day for three months) reduced neuritic plaque formation, inhibited iron-induced amyloidogenic APP processing and reversed behavioural alterations in the mouse model of AD (APP/PS 1 double transgenic mice) [160]. They also found that DFO treatment reduced the expression and phosphorylation of APP protein by shifting the processing of APP to the non-amyloidogenic pathway, and the reduction was accompanied by attenuating the Ab burden, and then significantly promoted memory retention in APP/PS1 mice. The effects of DFO on iron-induced amyloidogenic APP cleavage were further confirmed in vitro. These findings suggest that i.n DFO treatment reverses Fe-induced memory defects and inhibits amyloidogenic APP processing in APP/ PS1 mice. Angiotensin receptor blocker The Renin-angiotensin system in the brain has been implicated in pathogenesis of cognitive decline. Danielyan et al. found that IN administration of losartan, an angiotensin receptor blocker, at sub-antihypertensive dose (10 mg/kg every other day for two months) exhibited neuroprotective effect in the APP/PS1 transgenic mouse model. There was 3.7-fold reduction in Ab plaques, decreased interleukin-12 (IL-12) p40/p70, IL-1beta, decreased granulocytemacrophage colony-stimulating factor and increased IL-10 in mice treated with IN losartan compared with vehicletreated group. The authors concluded that losartan had direct anti-inflammatory and neuroprotective effect in CNS at concentration below than that would cause hypotensive reaction in AD patients [161]. Nerve growth factor Another strategy of treatment involves the delivery of neurotrophic factors such as NGF to brain via nasal route [162]. NGF is the most important target derived trophic factor for basal forebrain cholinergic neurons (BFCN) [163]. It increases the synthesis of ChAT and prevents BFCN atrophy associated with ageing and AD [164–166]. Deprivation of NGF causes progressive neurodegeneration



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in AD11 mice by BFCN atrophy and accumulation of tau and Ab [167–170]. The efficacy of NGF has been evaluated by intracerebroventricular (i.c.v.) infusion in animal models. The clinical trials conducted involved infusion of NGF in brain parenchyma or by ex vivo gene therapy. But these strategies are invasive and require surgery. Thus, IN delivery of NGF is a promising non-invasive technique [171,172]. Frey and co-workers demonstrated non invasive delivery of unconjugated NGF for first time in rats [71]. IN administration of 125I-labelled NGF in rats showed rapid appearance in olfactory bulb within 20 min of administration. The authors suggested that 125I-NGF was transported via intercellular clefts in the olfactory epithelium and extracellular transport along the olfactory neural pathway rather than uptake by olfactory neurons and subsequent intracellular axonal transport. In an another study, same authors reported significant delivery of recombinant human NGF (rhNGF) to olfactory bulbs and other regions of brain upon IN administration [173]. IN administration of NGF was found to decrease cholinergic deficits, phopshorylated tau and Ab in AD11 mice. Neurodegeneration induced by anti-NGF antibodies has been found to be reversed by nasal delivery of NGF [174]. The IN administration improves the transport of NGF to brain and prevents potent nociceptive actions in animals and humans. Capsoni et al. also studied the form of NGF mutated at R100 called ‘‘painless’’ hNGFER100 to overcome limitations of NGF due to its potent nociceptive action [175]. The mutant showed neurotrophic and anti-amyloidogenic activity in neuronal culture and a reduced nociceptive activity in vivo. Its IN administration in App X PS1 mice prevented the progress of neurodegeneration and behavioural deficits. Human acidic fibroblast growth factor (haFGF), also called as FGF1, has been investigated and found to exert beneficial effects in the mouse model of AD. haFGF, a neurotrophin-like growth factor, plays a significant role in development, differentiation and regeneration of brain neurons. It regulates expression of NMDAR and fibroblast growth factor receptor and exerts neuroprotective effect against glutamate excitotoxicity [176]. haFGF is also involved in regulation of synaptic plasticity and processes attributed to learning and memory by improving cholinergic nerve functions [177]. However, its transport to brain is limited by BBB and BCSF barrier. Lou et al. [178] have investigated a novel technique of delivering haFGF14-154 to brain by fusing it with transactivator of transcription protein transduction domain (TAT-PTD), a cell penetrating peptide. haFGF14-154 (300 mg/ kg), TAT-haFGF14-154 (35, 100 and 300 mg/kg) and recombinant human b-NGF (250 mg/kg) were administered intranasally in SAMP8 and senescence-accelerated resistant mouse prone-1 (SAMR1) mice. TAT-PTD significantly increased the concentration of haFGF in brain and TAT-haFGF14-154 was more effective than haFGF14-154 at the same dose (300 mg/kg). The learning and memory abilities of mice were improved as seen in Morris water maze test and step down test. TAT-haFGF14-154 showed a dose-dependent increase in the level of ACh and ChAT and decreased AChE activity which was significant compared with haFGF14-154 or NGF-treated group. It showed a dosedependent decrease in number of Ab1-42 plaques in

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hippocampus and cortex. The level of Ab soluble forms such as 4G8, 6E10 and A11-positive oligomers were also decreased. TAT-haFGF14-154 significantly decreased level of apoptotic neurons as determined by TUNEL assay. TAThaFGF14-154 was found to ameliorate OS impairment in brain and serum. The level of superoxide dismutase and glutathione was significantly increased and nitric oxide synthase activity was decreased compared with the model group. Similar results were found in the serum, but there was no difference among three doses of TAT-haFGF14-154. High dose TAT-haFGF14-154-treated group showed significant effect compared with haFGF14-154 or NGF-treated group. Peptide Vasoactive intestinal peptide (VIP) is a major neuropeptide has been found to be neuroprotective and play important role in acquisition of learning and memory. Blockade of VIP or reduced expression in experimental animals have been associated with impairment of learning and memory. Its expression is significantly decreased in cortex of aged animals [179–181]. Gozes et al. synthesized a potent lipohilic analogue of VIP [stearyl-norleucinel7] VIP ([St-Nle17]VIP) that has been found to exhibit neuroprotection in AD models. It was found to completely prevent Ab-induced cell death in rat cerebral cortical cultures with several folds greater potency than VIP exhibiting maximal potency at 1014 M. The ability of [St-Nle17]VIP to improve learning and memory capacities was tested in cholinotoxin AF64A-treated animals by Morris water maze test. Daily i.c.v. injections of [St-Nle17]VIP completely prevented the learning impairment in animals treated with the cholinergic blocker. The authors also studied the effect of intranasally administered [St-Nle17]VIP on learning and memory. IN administration significantly improved performance of animal in Morris water maze test compared with animals treated with AF64A alone [182]. Recently, Odorranalectin (OL) from the skin secretions of the amphibian Odorrana grahami, a novel non-immunogenic small peptide (MW 1.7 kDa) was investigated as a model ligand to enhance the binding of cubosomes (Cubs) to nasal mucosa and improve drug brain uptake via IN route. S14GHN, a derivative of Humanin (HN) with substitution of glycine for serine 14, a novel 24-amino acid peptide was selected as a model drug in a study done by Wu et al. [183]. Polyethylene glycol (PEG)-ylated Cubs were prepared by dilution-sonication. Coumarin-6, a lipophilic fluorescent probe with high sensitivity, was incorporated into Cubs to investigate the brain distribution of OL-Cubs in vivo. S14GHN-loaded Cubs were prepared by the reverse phase evaporation method. OL-modified Cubs were prepared by adding biotinylated OL into streptavidin-coated Cubs. Cubs were found to have average particle size of 177.3 nm with zeta potential of 16.7 mV. OL-modified Cubs, OL50-Cubs and OL150-Cubs, had average particle size of 175.5 and 187.7 nm and zeta potential of 19.4 and 19.9 mV, respectively, suggesting that conjugation had no significant effect on properties of Cubs. Incorporation of S14G-HN lead to decrease in particle size and zeta potential which was attributed to embedding of hydrophobic peptide chain of


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S14G-HN and maleimide-PEG-oleate that changed the curvature of lipid membrane. The systemic absorption and brain delivery of 6-Coumarin-loaded Cubs and OL-Cubs (OL50-Cubs and OL150-Cubs) was carried out after IN administration in conscious rats. Both Cubs and OL-Cubs exhibited similar plasma concentration–time profiles however OL Cubs had faster Tmax (51 h) compared with unmodified Cubs which had Tmax of 2 h. Authors suggested that the presence of OL on surface of Cubs increased transport of drug to blood. The drug targeting efficacy of OL50-Cubs and OL150-Cubs was significantly higher in olfactory bulb, cerebrum and cerebellum compared with unmodified Cubs. This might be attributed to close contact of OL-conjugated Cubs with mucosal cells thus leading to higher penetration with rapid extracellular transport. The transport of coumarin to brain was faster and higher when OL density on Cubs was higher. Furthermore, neuroprotective effect of the unmodified and OL-modified S14GHN-loaded Cubs was tested in Ab2535-treated rats following i.n administration. S14GHN-loaded Cubs showed improvement in spatial memory in dosedependent manner in Morris water maze test. The escape latency was shortened on day 2, 3 and 4 in animals treated with 7.5 mg/kg of S14GHN-loaded Cubs whereas 2.5 mg/kg was not effective. OL-modified S14GHN-loaded Cubs showed significant improvement at dose of 2.5 mg/kg. The animals spent more time in quadrant containing the platform in animals treated with 7.5 mg/kg of S14GHN-loaded Cubs and 2.5 mg/kg of OL-modified S14GHN-loaded Cubs. AChE activity determination suggested that S14GHN-loaded Cubs alleviated reduction in the enzyme level in a dosedependent manner. OL functionalization further enhanced the therapeutic effects of S14G-HN-loaded Cubs and resulted in increased cholinergic activity. Hormone Melatonin, an indole amide neurohormone, secreted by pineal gland has been found to protect neurons against Ab toxicity and inhibit the progressive formation of b-sheets and amyloid fibrils [184,185]. However, it has been found to have low oral BA, short biological half-life (45 min) and erratic pharmacokinetic profile. Jayachandra Babu et al. [186] have studied IN transport of melatonin using polymeric gel suspensions prepared with carbopol, carboxymethyl cellulose (CMC) and PEG400. In vitro permeation of formulations was studied using EpiAiwayÔ tissue model confirmed significantly higher permeation of polymeric suspensions. The concentration of melatonin in olfactory bulbs after IN administration were 9.22-, 6.77- and 4.04-fold higher for carbopol, CMC and PEG400, respectively, than that of IV melatonin in rats. Immunization Vaccination with Ab1-42 has been found to prevent Ab accumulation and clearance of pre-existing amyloid plaques. Several studies have been carried out to study active and passive immunization using anti-Ab antibodies [187,188]. Cattepoel et al. [189] studied immunization of APP transgenic mice with single-chain variable fragment (scFv) derived from full IgG antibody raised against C-terminus of Ab. scFv was found to enter brain after IN application and

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bind to amyloid plaques in cortex and hippocampus of APP transgenic mice. It was also found to inhibit Ab fibril formation and Ab-mediated neurotoxicity. Chronic IN administration of scFv was found to reduce congophilic amyloid angiopathy (CAA) and Ab plaques in cortex of APPswe/ PS1dE9 mice. The authors suggested that reduction of CAA and plaque pathology was associated with a redistribution of brain Ab from the insoluble fraction to the soluble peptide pool.

Future perspectives Apart from AChEIs, many attempts have been made to investigate the several neuroprotective strategies like insulin, NGF and peptides. Since AD is a multifactorial disease involving various hypothesis and mechanisms further understanding of the disease is required to design novel therapies involving IN administration. Despite of progress made in area of IN delivery to target drugs to brain and AD, many of these therapies are still under preclinical stage. With increase in number of ageing population, it is predicted that more number of elderly will have AD in decades to come. There is a need to develop formulations that can specifically deliver a drug to intended target site within brain rather than throughout the CNS. In future, novel pathways will be elucidated to determine how precisely drugs are transported upon IN administration and this knowledge can be utilized to improve therapeutic outcomes in AD.

Conclusion So far the treatment strategies for AD have been limited and it remains among leading cause of death worldwide. IN administration of present drugs, neuroprotective molecules or other alternatives has potential to modify their pharmacokinetics and biodistribution which may prove beneficial for the treatment of AD as highlighted in this review. Advances in formulation research and development have provided methods for delivering molecules that were not considered as candidates for IN delivery. In the future, nasally administered preparations with particulate systems of many antiAlzheimer’s drugs are expected to take their place on pharmacy shelves.

Declaration of interest Mr Sumeet Sood is grateful to Department of Science and Technology (DST), New Delhi, India for award of INSPIRE Fellowship (IF10316, DST/INSPIRE Fellowship/2010/160). Mr Kunal Jain wish to express his gratitude to Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial assistance in the form of Senior Research Fellowship (File No:8/484 (0006)/2012-EMR-I). The authors report no conflict of interest.

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