Müge İpek Konaklı1, Özüm Atasoy2, Oytun Erbaş1,3

1Institute of Experimental Medicine, Gebze-Kocaeli, Turkey
2Department of Radiation Oncology, Kartal Dr. Lütfi Kırdar City Hospital, Istanbul Turkey
3Department of Physiology, Medical Faculty of Demiroğlu Bilim University, Istanbul, Turkey

Keywords: Alzheimer's disease, erythropoietin, exosome, insulin, intranasal administration, mesenchymal stem cells, rifampicin

Abstract

Alzheimer's disease (AD) is a neurodegenerative brain disease which does not currently have a fully known treatment. Research over the past 30 years has provided numerous treatment options to correct the underlying neurodegenerative pathology. The drugs investigated in these studies focus on Amyloid beta (Aβ) muscle acceleration, which prevents the accumulation of amyloid aggregates. Despite many critical discoveries, failures in late-phase clinical trials indicated that targeting Aβ pathology alone is not effective in altering disease progression. These discouraged researchers were prompted to search for new approaches, one of which was intranasal release methods. In this review, intranasal drug administration for the treatment of AD and the effects of insulin, erythropoietin, exosome, mesenchymal stem cells, and rifampicin administered by this route on AD will be discussed. It is foreseen that definitive treatment possibilities can be developed by further investigating this method.

Alzheimer’s disease (AD) generally affects individuals over age 65. With the growing elderly population, prevalence of the disease has increased.[1] While this has led to increased efforts to develop treatment, there is no definitive treatment of the disease.[2,3] More than 200 therapeutic agents have been evaluated as a result of studies, and no new drug has been approved by the FDA for the treatment of the disease since 2003.[4,5] Many speculations have been made regarding the failure of the applied treatments. The most important of these are the wrong choice of main treatment goals and misinterpretation of AD pathophysiology.[6]

Research suggests that the characteristic amyloid plaques and tau nodes in the brain are not a cause but an effect. Alzheimer's disease is a result of neuroinflammation and brain wound healing gone awry.[7] This neurologic circumstance results in cognitive and behavioral disorders. Conventional treatment strategies, such as acetylcholinesterase inhibitor drugs, are often ineffective due to their poor solubility, low bioavailability, and inability to permeate the blood-brain barrier.[8] These limitations associated with current therapy draw attention to the intranasal strategy. This strategy seems to be a promising route for the delivery of drugs to the brain.[9] Recent studies examine direct intranasal delivery of drug groups to the central nervous system (CNS) rather than oral or parenteral routes.[9]

Intranasal Drug AsmInIstratIon In the Treatment of AlzheImer's Disease

Intranasal drug administration has emerged as an alternative to oral and parenteral routes. Since the olfactory nerve cells and trigeminal nerves are in direct contact with both the peripheral and the CNS, intranasally administration is a non-invasive method for allowing drugs to pass the blood brain barrier (BBB).[9] Accordingly, drugs can directly pass from the olfactory region and respiratory epithelium to the brain.[10,11] Thus, the treatment of neurological disorders is targeted.[12] Advantages compared to other drug administration routes include rapid onset of action, avoidance of the presystemic metabolism of the intestines and liver, reduced systemic exposure, direct administration to the brain and cerebrospinal fluid (CSF), ease of application, and better patient compliance.[9] Limitations of this route include weak nasal permeability and mucociliary clearance.[13] Studies show that drugs administered into the nasal cavity must have a more prolonged residence time to overcome nasal mucociliary clearance.[14]

Transport of drugs across the nasal barriers occurs intracellularly or extracellularly. The first step in intranasal transport is endocytosis to olfactory sensory neurons and then trigeminal ganglion cells respectively.[15] This pathway is referred to as intraneuronal transport. Since it is very slow, it may take 24 hours after nasal administration for agents to reach the CNS.[16] Then, intracellular transport to the olfactory bulb and the brain stem occurs. Transcytosis occurs by passive diffusion or by receptor-mediated endocytosis throughout the intracellular space and basolateral membrane. Diffusion into the olfactory bulb throughout the extracellular pathway associated with olfaction takes about 0.73-2.3 hours. Diffusion into the brain stem takes approximately 17-56 hours.[15] Substances acquired through this pathway are distributed through the nasal membranes to the blood, then to the olfactory mucosa and eventually to the CNS. This pathway is less effective than the transcellular route and is dependent on the molecular weight and size of the drug.[17] However, this mechanism is faster and allows drugs with low molecular weight to be delivered to the CNS in a few minutes.[18]

Nasal drugs can be cleared in the nasal cavity thanks to mucociliary clearance. The drug that enters blood circulation can be cleared with normal clearance mechanisms or may pass the BBB into the brain. The challenging aspect of application is ensuring adequate therapeutic levels of drug delivery to target areas of the brain. The drug’s localization in the brain must target necessary receptors for managing CNS diseases such as Parkinson's disease, schizophrenia, AD, brain tumors, meningitis, and migraine.[9]

This method, proposed by Frey in 1989 for the treatment of AD and other CNS disorders,[19] is a promising approach for the delivery of existing drugs and alternative therapy molecules in AD.[9]

Intranasally AdmInIstered Agents In AlzheImer's Treatment

Insulin

The use of intranasal insulin in AD has been investigated. Studies have proven that intranasally administered insulin is rapidly distributed throughout the brain at the cribriform plate level and reverses learning and memory loss in the AD mouse model. Intranasal insulin poorly enters the bloodstream and has no peripheral metabolic effects. Protein kinase-C (PKC) inhibition occurs as a result of uptake from the cribriform plate into the brain. Thus, cellular pathway inhibitors play a role in the transport of insulin in the blood-brain barrier. These results suggest that the intranasal route is an effective means of delivering insulin to the brain.[20]

Patients with AD have reduced insulin receptor sensitivity in the brain.[21,22] Insulin has important functions in the CNS. Insulin receptors in the brain are found in the olfactory bulb, hypothalamus, hippocampus, cerebral cortex, and cerebellum.[23] Insulin signaling contributes to synaptogenesis and synaptic remodeling.[24] Acute uptake of insulin into the hippocampus improves spatial memory in a dependent manner on phosphatidylinositol 3-kinase (PI3K) by modulating glucose utilization.[25]

Intranasal administration or perfusion of insulin to healthy individuals improves cognitive function.[26,27] Intranasal insulin administration before anesthesia prevented AD-like tau hyperphosphorylation in a widely used transgenic mouse model of AD.[28] Tau hyperphosphorylation, a pathological feature of AD, increases with anesthetic exposure.[29] This causes significant learning and memory impairments in elderly rodents.[30,31]

Enhanced brain insulin signal enhances memory in cognitively healthy people, indicating its neuroprotective features. These results indicate that increasing brain insulin concentrations in AD patients could prevent or slow down the development of this debilitating disease.[20]

In a clinical study on patients with AD,[32] patients given placebo or 20 or 40 IU insulin intranasally for four months and a positron emission tomography was performed before and after patients received intranasal insulin. Alzheimer's Disease Rating Scale was used to evaluate results, which showed that there was an improvement in delayed memory and cognitive function in the insulin group.

A single dose of intranasal insulin acutely improved memory in older adults with AD.[12] Insulin was effective in improving verbal memory test performance in the AD group. However, among these groups, only women with the Apolipoprotein-e4 (APO-e4) allele were unaffected by administration of high doses of insulin.[32] In another study, APO-e4 positive AD patients were less responsive to insulin. Given that APO-e4 carriers represent 40-65% of the late-onset AD population, it is suggested that intranasal insulin may have limited therapeutic use.[33] Although the studies conducted so far are promising, more clinical studies are needed. Little is known about the mechanism by which intranasally administered insulin reaches the brain. Some hypotheses regarding intranasal delivery of insulin to the brain include transport along axon bundles of olfactory receptor cells on the roof of the nasal cavity, transport along the trigeminal nerve pathway, and via the rostral migration flow.[34] Recent studies have shown that the rapid distribution of intranasally administered molecules via the olfactory tract or trigeminal pathway involves the bulk flow within the perivascular space of cerebral blood vessels.[35] All of these studies support that intranasally administered insulin is carried

along the nasal epithelium. Ongoing studies are examining the regional distribution and course of action of administered insulin to investigate the effects on cellular mechanisms of brain intake and cognition in rodent models of AD.[20]

Erythropoietin

Erythropoietin (EPO) is a hematopoietic growth factor[36] and a glycoprotein cytokine involved in the regulation of erythropoiesis. It also has biological functions such as non-hematopoietic neuroprotection and neurogenesis.[37] For these reasons, EPO has begun to be used as a therapeutic agent for neurodegeneration.[36]

The use of EPO in clinical applications has not yielded positive results. This is due to the erythropoietic effect of EPO.[38] The use of recombinant human EPO (rh-EPO) in neurological diseases increases hematocrit and blood viscosity. This condition causes cardiovascular diseases such as infarction or stroke. To solve this problem, EPO derivatives lacking erythropoietic activity but preserve neuroprotective activity against neuronal injury have begun to be used.[39] Neuro-EPO was developed as a non-erythropoietic derivative of EPO with chemical modifications.[37]

Neuro-EPO is a recombinant human glycoprotein produced in Chinese hamster ovary cells and contains low sialic acid. Its most important feature is that it lacks erythropoietic activity while exhibiting high neuroprotective properties.[40] Neuro-EPO is rapidly degraded in the liver and is therefore more suitable for intranasal administration.[41] Thanks to the intranasal administration, it quickly reaches the brain and does not stimulate erythropoiesis after acute treatments.[40] The neuroprotective effects of Neuro-EPO, such as improving of cognitive function after stroke, have been studied in animal models of stroke.[40] In one study, Neuro- EPO was intranasally administered in doses of 62, 125 and 250 μg/kg in non-transgenic Alzheimer rodent models. Results of the study indicated that intranasal administration of Neuro- EPO prevented Ab25-35 toxicity in the rodent hippocampus and showed a strong potential protection against amyloid toxicity. Apart from a few differences depending on dosage, Neuro- EPO showed a positive effect in the treatment of memory loss. Doses such as 125 and 250 μg/kg were effective in most of the tests. However, the dose of 125 μg/kg showed a prolonged effect lasting up to 22 days.[42] Erythropoietin directly affects synaptic plasticity, especially in the hippocampus.[43,44] It has been determined that EPO increases primary hippocampal neuronal networks. That is, EPO improves hippocampus-derived memory by enhancing plasticity, synaptic connections, and neuronal networks.[44] Erythropoietin has a protective effect against inflammatory pathologies. TNFa and IL-1b released by A25-35 toxicity are blocked by Neuro-EPO. This neuroprotective ability of EPO is due to its ability to provide extrinsic cell homeostasis, by modulating microglial activation and controlling cytokine release.[45] Different forms of EPO show different affinities in activating EPO receptors. Higher concentrations are needed for the low sialic acid form of EPO to activate EPO receptors.[45] Another important feature of Neuro-EPO is that it can produced to be biologically similar to endogenous EPO without chemically modification, therefore causing less side effects in long-term use.[42]

Erythropoietin selectively induces the synthesis of the neuroglobin protein in damaged regions, providing angiogenesis and protection of the vascular endothelium. Lost functions in damaged regions of the brain are preserved by ensuring homeostasis.[42]

Intranasally EPO is currently being tested for the treatment of stroke.[40,46] Studies on intranasal EPO applications in neurodegenerative pathologies are ongoing.[42]

Exosomes

Microglial cells are the brain’s macrophages. Microglial cells contribute to inflammation by producing interleukin in inflammatory diseases such as Alzheimer's. Therefore, it makes sense to target microglial cells for anti-inflammatory treatments.[47] No specific strategy for targeting brain microglial cells is in practice.[48] Drugs that target inflammatory cells without damaging normal tissues have been developed, but they do not cross the BBB.[49-51] Therefore, exosomes have been used to deliver anti-inflammatory drugs to the brain via a non-invasive intranasal route.[48] One of these anti-inflammatory drugs is curcumin, a natural polyphenol found in Curcuma longa (turmeric) rhizomes. Curcumin is anti- inflammatory, antineoplastic, antioxidant, and chemopreventive.[52-54]

Since curcumin has poor solubility and poor bioavailability, it poses problems in clinical application.[55] To overcome this, exosomes have been used as nanoparticle drug carriers. Exosomes are 30-100 nm sized nanoparticles that can be secreted into the extracellular environment. Curcumin was loaded into exosomes at 22°C and then subjected to sucrose gradient centrifugation. Thus, curcumin was encapsulated to exosomes, increasing its in vitro solubility, stability, and bioavailability.[56] In order to demonstrate the therapeutic use of curcumin with exosomes (Exo-cur), mice in a lipopolysaccharide (LPS)- induced inflammatory state were used. Curcumin was loaded into exosomes and administered intranasally. The presence of curcumin in brain lysates was identified using high performance liquid chromatography one hour after Exo-cur application. The presence of curcumin in brain lysates was observed for up to 12 hours. This proves that microglial cells are selectively targeted by exosomes. Subsequently, a decrease in the number of activated inflammatory microglial cells was observed in the brains of LPS-treated mice. Since exosomes can be delivered to the brain in a rapid and selective manner, they are regarded as a vehicle of drug delivery.[47]

Studies have shown that brain microglial cells are targeted by exosomes, but do not address the mechanism of this selectivity. It is unknown whether exosomes enter recipient cells through the endosomal route,[57] or whether exosomes derived from T cells use the endosomal route for entry into microglial cells. The degree of exosomal entry into various regions of the brain and spinal cord, and whether or not glial cell populations like astrocytes are targeted by exosomes are also unclear. It is also unknown whether or not exosome uptake by microglial cells in the brain depends on inflammation, or whether it increases parallel to inflammation. When these questions are answered, it is thought that the applicability of intranasal exosome technology to neurological diseases will become clear.[47]

Mesenchymal Stem Cells

Alzheimer's disease is a disease of complex mechanisms that does not originate from a single cause. Therefore, approaches to target the complex pathology of the disease are needed. It is thought that stem cells applications will provide multi-targeted therapies required for the solution of the disease.[58] The most important issue before starting stem cell therapy is selecting the correct stem cell. Mesenchymal stem cells (MSC) can be obtained from many types of cells and have ease of application. For these reasons, it is the most frequently studied type of stem cell.[59]

Mesenchymal stem cells are neuroprotective stem cells with anti-amyloidogenic activities.[60] Mesenchymal stem cells has a therapeutic effect on AD. Mesenchymal stem cells administration demonstrated anti-inflammatory and anti-amyloid properties in animal models of AD, resulting in improved memory.[59,60] Therefore, it is also promising for the treatment of various neurological diseases.[61]

Mesenchymal stem cells therapeutically restore degenerated neurons, provide neuroprotection through secretory factors, exert immunomodulatory effects on cells responsible for disease development, and proliferate endogenous cells.[62] However, implantation risks limit its clinical usage. These limitations include: the process of invasive cell isolation, loss of potency, limited lifetime, large-scale expansion cost[63] and a low possibility of uncontrolled cell proliferation.[63,64]

It has been stated that MSC act through paracrine mechanisms by releasing bioactive components when exposed to an injured environment, rather than through direct engraftment. Although studies have demonstrated the neuroprotective and anti-inflammatory effects of MSC in various disease models,[63,64] this was not observed in AD mouse models.[58] Studies have demonstrated that secretomes derived from MSC in vitro in an AD environment (MSC-CS) fully replicate multiple neuro-reparative activities in implanted mice.[58]

In transgenic mice with AD aged 22 to 25 months, repeated intranasal administration of MSC-CS resulted in a decrease in cortical and hippocampal plaque burden, the number of surrounding activated glial cells, and expression of the phagocytic marker CD68. However, the ability of MSC-CS to reduce the level of AbO suggests that MSCs have strong therapeutic potential.[65,66] Mesenchymal stem cells-CS also reduces hippocampal atrophy and neuronal damage in the brains of transgenic mice.

Long-term MSC-CS treatment has yielded positive results.[58] This is attributed to the self- renewing, neuroprotective, and regenerative abilities of MSCs.[67] Mesenchymal stem cells treatment eliminates brain atrophy by plaque reduction and by decreasing in amyloid beta oligomer (AbO) concentrations. Thus, a preclinical, non-invasive, and continuous treatment model has been to reverse functional and structural damage in elderly AD mice.[58]

Rifampicin

Most studies up to now have investigate methods to eliminate the pathogenesis of neurodegenerative dementia such as cerebral accumulation of amyloid oligomers and tauopathy to treat AD.[68,69] Clinical studies have found that these treatments do not provide particularly good results in the cognitive levels of patients.[70,71] Such negative consequences have led to the introduction of rifampicin, an antibiotic that can be taken orally, has few side effects, and can reduce neurotoxic oligomers to a large extent, as a new method of preventing the disease.[72]

Rifampicin has been shown to have preventive effects against AD under certain conditions.[73] Therefore, preventive treatment should be initiated before the start of memory problems.[74]

Among patients without memory decline, having AD-type hypometabolism is sufficient reason to begin preventive therapy. Rifampicin has been used as a preventive treatment. Its effective dosage was considered to be 450 mg/day or higher (e.g. 600 mg/day). Rifampicin treatment must be continued for at least 12 months to achieve positive results in the elderly. It is thought that the memory impairment can be prevented if rifampicin is used continuously for several years.[73]

Orally administered rifampicin reduces amyloid beta (Ab) and tau pathologies in mice. On the other hand, long-term use is not preferred because it has adverse effects such as liver damage. This situation has prompted scientists to seek a safer route of rifampicin administration. Therefore, the therapeutic efficacy and safety of rifampicin was evaluated by administering rifampicin treatment for one month by oral, intranasal, and subcutaneous routes to transgenic AD mouse models. The results of the experiment indicated that intranasal or subcutaneous administration was better than oral administration at improving memory. In addition, a decrease was observed in neuropathologies such as Ab oligomer accumulation, abnormal phosphorylation of tau, and loss of synapses. Pharmacokinetic studies show that the highest rifampicin level in the brain is achieved with the intranasal rifampicin administration strategy. Due to its ease of administration and non-invasive nature, intranasal administration is a reasonable option for long-term rifampicin dosages. The inexpensive availability of rifampicin also increases the drug’s potential for long-term use.[75]

Many attempted methods to treat AD have failed. This has steered researchers towards different approaches. Intranasal administration provides direct delivery to the CNS unlike oral and parenteral routes. Since the olfactory nerve cells and trigeminal nerves are in direct contact with both the environment and the CNS, it is a promising non-invasive method to cross the blood brain barrier. In this manner, direct drug passage from the olfactory region and respiratory epithelium to the brain can be achieved, therefore allowing the treatment of the neurological disorder, AD. Advantages compared to other drug administration routes include rapid onset of action, avoidance of the presystemic metabolism of the intestines and liver, reduced systemic exposure, direct administration to the brain and CSF, ease of application, and better patient compliance. The limitations of this route are mucociliary clearance and poor nasal permeability of intranasally administered drugs. Many agents are applied or attempted to be applied with this route. Studies on mouse models have demonstrated that the intranasal administration of substances such as insulin, erythropoietin, exosomes, MSC, and rifampicin is applicable for the treatment of AD. Clarifying the uncertainties in treatment doses and mechanism of action will help to evaluate the applicability of these agents in AD treatment.

Conflict of Interest

The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Financial Disclosure

The authors received no financial support for the research and/or authorship of this article.

References

  1. Kingston A, Comas-Herrera A, Jagger C; MODEM project. Forecasting the care needs of the older population in England over the next 20 years: estimates from the Population Ageing and Care Simulation (PACSim) modelling study. Lancet Public Health 2018;3:e447-e455.
  2. Scheltens P, Blennow K, Breteler MM, de Strooper B, Frisoni GB, Salloway S, et al. Alzheimer’s disease. Lancet 2016;388:505-17.
  3. Cummings J, Lee G, Ritter A, Sabbagh M, Zhong K. Alzheimer’s disease drug development pipeline: 2019. Alzheimers Dement (N Y) 2019;5:272-93.
  4. Anderson RM, Hadjichrysanthou C, Evans S, Wong MM. Why do so many clinical trials of therapies for Alzheimer’s disease fail? Lancet 2017;390:2327-9.
  5. Atri A. Current and future treatments in Alzheimer’s disease. Semin Neurol 2019;39:227-40.
  6. Gauthier S, Albert M, Fox N, Goedert M, Kivipelto M, Mestre-Ferrandiz J, et al. Why has therapy development for dementia failed in the last two decades? Alzheimers Dement 2016;12:60-4.
  7. Lehrer S, Rheinstein PH. Nasal steroids as a possible treatment for Alzheimer’s disease. Discov Med 2017;24:147-52.
  8. Yin F, Sancheti H, Patil I, Cadenas E. Energy metabolism and inflammation in brain aging and Alzheimer’s disease. Free Radic Biol Med 2016;100:108-22.
  9. Sood S, Jain K, Gowthamarajan K. Intranasal therapeutic strategies for management of Alzheimer’s disease. J Drug Target 2014;22:279-94.
  10. Illum L. Nasal drug delivery--possibilities, problems and solutions. J Control Release 2003;87:187-98.
  11. Frey W, Liu J, Chen X. Delivery of 125I-NGF tothebrainviatheolfactoryroute. Drug Deliv 1997;4:87-92.
  12. Reger MA, Watson GS, Frey WH 2nd, Baker LD, Cholerton B, Keeling ML, et al. Effects of intranasal insulin on cognition in memory-impaired older adults: modulation by APOE genotype. Neurobiol Aging 2006;27:451-8.
  13. Illum L. Transport of drugs from the nasal cavity to the central nervous system. Eur J Pharm Sci 2000;11:1-18.
  14. Ugwoke MI, Verbeke N, Kinget R. The biopharmaceutical aspects of nasal mucoadhesive drug delivery. J Pharm Pharmacol 2001;53:3-21.
  15. Lochhead JJ, Thorne RG. Intranasal delivery of biologics to the central nervous system. Adv Drug Deliv Rev 2012;64:614-28.
  16. Kristensson K, Olsson Y. Uptake of exogenous proteins in mouse olfactory cells. Acta Neuropathol 1971;19:145-54.
  17. Dalpiaz A, Contado C, Vighi E, et al. Preparation and characterization of particulate drug delivery systems for brain targeting. In: Ravikumar MNV, editor. Handbook of particulate drug delivery. California: American Scientific Publishers; 2008. p. 95-119.
  18. Chou K-J, Donovan MD. Lidocaine distribution into the CNS following nasal and arterial delivery: a comparison of local sampling and microdialysis techniques. Int J Pharm 1998;171:53-61.
  19. Frey WH, II. Method for administering neurologic agents to the brain. US Patent 5624898. Available at: https://patents.google.com/patent/US5624898A/en [April 29, 1997]
  20. Salameh TS, Bullock KM, Hujoel IA, Niehoff ML, Wolden-Hanson T, Kim J, et al. Central Nervous System Delivery of Intranasal Insulin: Mechanisms of Uptake and Effects on Cognition. J Alzheimers Dis 2015;47:715-28.
  21. Rivera EJ, Goldin A, Fulmer N, Tavares R, Wands JR, de la Monte SM. Insulin and insulin-like growth factor expression and function deteriorate with progression of Alzheimer’s disease: link to brain reductions in acetylcholine. J Alzheimers Dis 2005;8:247-68.
  22. Talbot K, Wang HY, Kazi H, Han LY, Bakshi KP, Stucky A, et al. Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest 2012;122:1316-38.
  23. Plum L, Schubert M, Brüning JC. The role of insulin receptor signaling in the brain. Trends Endocrinol Metab 2005;16:59-65.
  24. Abbott MA, Wells DG, Fallon JR. The insulin receptor tyrosine kinase substrate p58/53 and the insulin receptor are components of CNS synapses. J Neurosci 1999;19:7300-8.
  25. McNay EC, Ong CT, McCrimmon RJ, Cresswell J, Bogan JS, Sherwin RS. Hippocampal memory processes are modulated by insulin and high-fat- induced insulin resistance. Neurobiol Learn Mem 2010;93:546-53.
  26. Benedict C, Hallschmid M, Hatke A, Schultes B, Fehm HL, Born J, et al. Intranasal insulin improves memory in humans. Psychoneuroendocrinology 2004;29:1326-34.
  27. Kern W, Peters A, Fruehwald-Schultes B, Deininger E, Born J, Fehm HL. Improving influence of insulin on cognitive functions in humans. Neuroendocrinology 2001;74:270-80.
  28. Chen Y, Run X, Liang Z, Zhao Y, Dai CL, Iqbal K, et al. Intranasal insulin prevents anesthesia-induced hyperphosphorylation of tau in 3xTg-AD mice. Front Aging Neurosci 2014;6:100.
  29. Dong Y, Wu X, Xu Z, Zhang Y, Xie Z. Anesthetic isoflurane increases phosphorylated tau levels mediated by caspase activation and Ab generation. PLoS One 2012;7:e39386.
  30. Shen X, Dong Y, Xu Z, Wang H, Miao C, Soriano SG, et al. Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. Anesthesiology 2013;118:502-15.
  31. Bianchi SL, Tran T, Liu C, Lin S, Li Y, Keller JM, et al. Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics. Neurobiol Aging 2008;29:1002-10.
  32. Claxton A, Baker LD, Wilkinson CW, Trittschuh EH, Chapman D, Watson GS, et al. Sex and ApoE genotype differences in treatment response to two doses of intranasal insulin in adults with mild cognitive impairment or Alzheimer’s disease. J Alzheimers Dis 2013;35:789-97.
  33. Rosenbloom MH, Barclay TR, Pyle M, Owens BL, Cagan AB, Anderson CP, et al. A single-dose pilot trial of intranasal rapid-acting insulin in apolipoprotein E4 carriers with mild-moderate Alzheimer’s disease. CNS Drugs 2014;28:1185-9.
  34. Lochhead JJ, Thorne RG. Intranasal delivery of biologics to the central nervous system. Adv Drug Deliv Rev 2012;64:614-28.
  35. Lochhead JJ, Wolak DJ, Pizzo ME, Thorne RG. Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration. J Cereb Blood Flow Metab 2015;35:371-81.
  36. Sun J, Martin JM, Vanderpoel V, Sumbria RK. The promises and challenges of erythropoietin for treatment of Alzheimer’s disease. Neuromolecular Med 2019;21:12-24.
  37. Rama R, Garzón F, Rodríguez-Cruz Y, Maurice T, García-Rodríguez JC. Neuroprotective effect of Neuro-EPO in neurodegenerative diseases: “Alea jacta est”. Neural Regen Res 2019;14:1519-21.
  38. Ehrenreich H, Weissenborn K, Prange H, Schneider D, Weimar C, Wartenberg K, et al. Recombinant human erythropoietin in the treatment of acute ischemic stroke. Stroke 2009;40:e647-56.
  39. Leist M, Ghezzi P, Grasso G, Bianchi R, Villa P, Fratelli M, et al. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science 2004;305:239-42.
  40. Rodríguez Cruz Y, Mengana Támos Y, Muñoz Cernuda A, Subirós Martines N, González-Quevedo A, Sosa Testé I, et al. Treatment with nasal neuro- EPO improves the neurological, cognitive, and histological state in a gerbil model of focal ischemia. ScientificWorldJournal 2010;10:2288-300.
  41. Garcia-Rodriguez JC, Sosa-Teste I. The nasal route as a potential pathway for delivery of erythropoietin in the treatment of acute ischemic stroke in humans. Scientific World Journal 2009;9:970-81.
  42. Maurice T, Mustafa MH, Desrumaux C, Keller E, Naert G, de la C García-Barceló M, et al. Intranasal formulation of erythropoietin (EPO) showed potent protective activity against amyloid toxicity in the Ab25-35 non-transgenic mouse model of Alzheimer’s disease. J Psychopharmacol 2013;27:1044-57.
  43. Adamcio B, Sargin D, Stradomska A, Medrihan L, Gertler C, Theis F, et al. Erythropoietin enhances hippocampal long-term potentiation and memory. BMC Biol 2008;6:37.
  44. Kamal A, Al Shaibani T, Ramakers G. Erythropoietin decreases the excitatory neurotransmitter release probability and enhances synaptic plasticity in mice hippocampal slices. Brain Res 2011;1410:33-7.
  45. Maiese K, Li F, Chong ZZ. Erythropoietin in the brain: can the promise to protect be fulfilled? Trends Pharmacol Sci 2004;25:577-83.
  46. Fletcher L, Kohli S, Sprague SM, Scranton RA, Lipton SA, Parra A, et al. Intranasal delivery of erythropoietin plus insulin-like growth factor-I for acute neuroprotection in stroke. Laboratory investigation. J Neurosurg 2009;111:164-70.
  47. Lakhal S, Wood MJ. Intranasal exosomes for treatment of neuroinflammation? Prospects and limitations. Mol Ther 2011;19:1754-6.
  48. Zhuang X, Xiang X, Grizzle W, Sun D, Zhang S, Axtell RC, et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti- inflammatory drugs from the nasal region to the brain. Mol Ther 2011;19:1769-79.
  49. Potschka H. Targeting the brain--surmounting or bypassing the blood-brain barrier. Handb Exp Pharmacol 2010;197:411-31.
  50. Gabathuler R. Blood-brain barrier transport of drugs for the treatment of brain diseases. CNS Neurol Disord Drug Targets 2009;8:195-204.
  51. Bidros DS, Vogelbaum MA. Novel drug delivery strategies in neuro-oncology. Neurotherapeutics 2009;6:539-46.
  52. Ravindran J, Prasad S, Aggarwal BB. Curcumin and cancer cells: how many ways can curry kill tumor cells selectively? AAPS J 2009;11:495-510.
  53. Anand P, Thomas SG, Kunnumakkara AB, Sundaram C, Harikumar KB, Sung B, et al. Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature. Biochem Pharmacol 2008;76:1590-611.
  54. Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol 2009;41:40-59.
  55. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Mol Pharm 2007;4:807-18.
  56. Sun D, Zhuang X, Xiang X, Liu Y, Zhang S, Liu C, et al. A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol Ther 2010;18:1606-14.
  57. Record M, Subra C, Silvente-Poirot S, Poirot M. Exosomes as intercellular signalosomes and pharmacological effectors. Biochem Pharmacol 2011;81:1171-82.
  58. Santamaria G, Brandi E, Vitola P, Grandi F, Ferrara G, Pischiutta F, et al. Intranasal delivery of mesenchymal stem cell secretome repairs the brain of Alzheimer’s mice. Cell Death Differ 2020.
  59. Duncan T, Valenzuela M. Alzheimer’s disease, dementia, and stem cell therapy. Stem Cell Res Ther 2017;8:111.
  60. FanX,SunD,TangX,CaiY,YinZQ,XuH.Stem- cell challenges in the treatment of Alzheimer’s disease: a long way from bench to bedside. Med Res Rev 2014;34:957-78.
  61. Laroni A, Novi G, Kerlero de Rosbo N, Uccelli A. Towards clinical application of mesenchymal stem cells for treatment of neurological diseases of the central nervous system. J Neuroimmune Pharmacol 2013;8:1062-76.
  62. Bali P, Lahiri DK, Banik A, Nehru B, Anand A. Potential for Stem Cells Therapy in Alzheimer’s Disease: Do Neurotrophic Factors Play Critical Role? Curr Alzheimer Res 2017;14:208-20.
  63. Abbasi-Malati Z, Roushandeh AM, Kuwahara Y, Roudkenar MH. Mesenchymal stem cells on horizon: A new arsenal of therapeutic agents. Stem Cell Rev Rep 2018;14:484-99.
  64. Teixeira FG, Carvalho MM, Sousa N, Salgado AJ. Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration? Cell Mol Life Sci 2013;70:3871-82.
  65. Forloni G, Artuso V, La Vitola P, Balducci C. Oligomeropathies and pathogenesis of Alzheimer and Parkinson’s diseases. Mov Disord 2016;31:771-81.
  66. Mucke L, Selkoe DJ. Neurotoxicity of amyloid b-protein: synaptic and network dysfunction. Cold Spring Harb Perspect Med 2012;2:a006338.
  67. Sadatpoor SO, Salehi Z, Rahban D, Salimi A. Manipulated Mesenchymal Stem Cells Applications in Neurodegenerative Diseases. Int J Stem Cells 2020;13:24-45.
  68. Benilova I, Karran E, De Strooper B. The toxic Ab oligomer and Alzheimer’s disease: an emperor in need of clothes. Nat Neurosci 2012;15:349-57.
  69. Viola KL, Klein WL. Amyloid b oligomers in Alzheimer’s disease pathogenesis, treatment, and diagnosis. Acta Neuropathol 2015;129:183-206.
  70. Imbimbo BP, Giardina GA. g-secretase inhibitors and modulators for the treatment of Alzheimer’s disease: disappointments and hopes. Curr Top Med Chem 2011;11:1555-70.
  71. Panza F, Logroscino G, Imbimbo BP, Solfrizzi V. Is there still any hope for amyloid-based immunotherapy for Alzheimer’s disease? Curr Opin Psychiatry 2014;27:128-37.
  72. Umeda T, Ono K, Sakai A, Yamashita M, Mizuguchi M, Klein WL, et al. Rifampicin is a candidate preventive medicine against amyloid-b and tau oligomers. Brain 2016;139:1568-86.
  73. Iizuka T, Morimoto K, Sasaki Y, Kameyama M, Kurashima A, Hayasaka K, et al. Preventive effect of rifampicin on alzheimer disease needs at least 450 mg daily for 1 year: An FDG-PET follow-up Ssy. Dement Geriatr Cogn Dis Extra 2017;7:204- 14.
  74. Jack CR Jr, Knopman DS, Jagust WJ, Shaw LM, Aisen PS, Weiner MW, et al. Hypothetical model of dynamic biomarkers of the Alzheimer’s pathological cascade. Lancet Neurol 2010;9:119-28.
  75. Umeda T, Tanaka A, Sakai A, Yamamoto A, Sakane T, Tomiyama T. Intranasal rifampicin for Alzheimer’s disease prevention. Alzheimers Dement (N Y) 2018;4:304-13.