Keywords

6.1 Introduction

Stroke is the second leading cause of death and third main reason for severe disabilities worldwide [1, 2]. Over 80% of stroke cases present as ischemic stroke [3]. Targeting the highly dynamic events that occur during ischemic stroke in the relatively inaccessible brain microenvironment remains a challenge.

After decades of research, a variety of peptides, including erythropoietin (EPO ), interleukin 4 (IL-4), and transforming growth factor (TGF)-β1, have emerged as effective therapeutic agents to treat brain diseases, such as neurodegeneration, pain, psychiatric disorders and stroke [4]. However, some peptides cannot pass the blood brain barrier due to their high molecular weight, thereby requiring some form of invasive delivery to access the brain [5]. Recently, intranasal delivery has gained attention as a novel non-invasive drug delivery route. In this chapter, we will define intranasal delivery, describe its possible mechanisms, and discuss its advantages and disadvantages as a delivery method of therapeutic peptides. Some peptides that have shown protection against stroke through intranasal delivery along with the regimens that have been used, will also be covered in this chapter.

6.2 Definition and Mechanisms of Intranasal Delivery of Therapeutic Peptides

Intranasal delivery is an alternative drug delivery strategy used to treat brain injuries, such as stroke [6]. Intranasal administration circumvents the blood brain barrier (BBB ) and provides a direct and rapid route for drugs to enter the brain and cerebrospinal fluid (CSF ) through the olfactory epithelium [7]. Because of the large absorptive surface area of the nasal cavity (~160 cm2) [8], along with its high vascularization and porous epithelium, drugs or treatment agents delivered through the intranasal route can enter the brain within minutes [9]. Chemicals, peptides, genes, and cells have been successfully delivered to the brain by intranasal delivery to achieve neuroprotection [10].

After intranasal administration of [125I]-labeled proteins to rats and monkeys, radiolabeling has been shown to extend from the olfactory and trigeminal nerve components in the nasal epithelium to the olfactory bulb and brainstem, respectively. The signals from these [125I]-labeled proteins can spread to other central nervous system (CNS ) areas from these initial sites [11, 12]. The olfactory region is adjacent to the CSF flow tracts around the olfactory lobe [13]. Therefore, intranasal administration could lead to direct delivery into the CSF.

Two possible mechanistic routes have been suggested to underlie direct brain delivery after intranasal administration: (1) extracellular routes and (2) intracellular routes [7]. The extracellular route is a rapid pathway by which the drug is absorbed across olfactory epithelial cells, either by transcellular or paracellular mechanisms. Then the agent can be taken up into the CNS directly [7], which explains why the delivery of intranasal drugs to the brain occurs within minutes [14]. On the other hand, during the intracellular route, drugs are internalized into primary neurons of the olfactory epithelium by endocytosis, which includes pinocytotic mechanisms, followed by intracellular axonal transport to the olfactory bulb [7]. This intracellular pathway may take hours for drugs to be dispersed within the brain [15]. The delivery route and mechanisms of intranasal drug absorption is illustrated in Fig. 6.1.

Fig. 6.1
figure 1

Intranasal delivery pathways across the olfactory epithelium are depicted. Potential transport routes are shown in red. Some agents may be delivered via an intracellular pathway from the olfactory epithelium to the olfactory bulb by olfactory sensory neurons by endocytic, including pinocytotic, mechanisms, followed by intracellular axonal transport to the olfactory bulb. Others agents may pass the olfactory epithelial barrier by paracellular or transcellular transport to reach the lamina propria, and then distribute to the brain in multiple ways: (1) via entry into olfactory blood vessels and the systemic circulation; (2) by absorption into olfactory lymphatic vessels, which leads to the deep cervical lymph nodes of the neck; (3) by diffusion to the subarachnoid space with CSF , or. (4) entry into channels created by olfactory enshreathing cells surrounding the olfactory nerves, where they can access the olfactory bulbs. Subsequently, intranasally delivered drugs are transported from the olfactory bulb to the CSF surrounding the whole brain

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6.3 Advantages of Intranasal Delivery of Therapeutic Peptides

Compared to conventional delivery methods, intranasal delivery of therapeutic peptides possesses the following advantages; (1) Safe and less invasive [16] compared to local delivery into the brain, which usually requires intraventricular and parenchymal injections [17, 18]; (2) easier passage into the brain [16]; (3) less requirement for the high diffusibility of therapeutic agents [16]; (4) lower dosage can be used with more reliable pharmacological profiles due to less drug metabolism and degradation along the delivery routes [19, 20]; (5) faster onset of action [21]; (6) less systemic side effects due to the absence of intestinal absorption and degradation by the enterohepatic cycle as well as general distribution [9, 22]; (7) large molecules can gain entry into the brain which is not easily attainable via the oral route [23]; and (8) less requirement for sterile preparation [24].

6.4 Peptides that Have Been Administered Intranasally to Treat Stroke

Recently, there has been increased focus on specific peptides as neuroprotective agents in experimental models of cerebral ischemic stroke. For example, numerous studies have shown that EPO administered subcutaneously, intraperitoneally, or through intra-cerebroventricular (ICV ) injection, is effective against ischemic stroke [25,26,27]. Subsequently, intranasal administration of EPO, as well as other peptides, has shown efficacy in experimental models of ischemic stroke. The protective effects of these peptides mainly fall into four categories: (1) inhibition of neuronal apoptosis, as observed with EPO treatment [28]; (2) amelioration of inflammatory responses, as observed with Tat-NEMO-binding domain (Tat-NBD ), a stable-mutant form of plasminogen activator inhibitor-I (CAPI) [20, 29]; (3) promotion of progenitor cell proliferation and brain repair mechanisms, as detected with insulin-like growth factor-I (IGF-1 ), TGF-β1, and TGF-α; [30,31,32,33] and (4) improvement in white matter function by preventing demyelination and promoting axon sprouting, as observed with tissue plasminogen activator (tPA ) and Apo-transferrin (aTf ) [31, 34]. Table 6.1 summarizes the neuroprotective effects of intranasal administration of these peptides against cerebral ischemic stroke .

Table 6.1 Neuroprotective effect of intranasal delivery of various peptides against stroke
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In addition to the above-mentioned peptides, there are numerous peptides that have shown to be effective regulators of ischemic brain injury through ICV or subcutaneous administration, such as IL-4 and IL-33 [41, 42]. The efficacy of intranasal delivery of these peptides against ischemic stroke has not been examined. However, they are promising candidates for this new route of administration . Thus further studies investigating the efficacy of intranasal delivery of these two peptides against ischemic stroke is warranted.

6.5 Administration Regimens of Intranasal Delivery in the Treatment of Stroke

One of the advantages of intranasal delivery is that it reduces the effective dosage compared to systemic administration. However, there are still tremendous differences regarding the dosages reported by different studies for different peptides. In addition, some studies also report multiple intranasal peptide administrations or combination of different peptides. In Table 6.2, we summarize the intranasal infusion protocols used to administer various peptides and their corresponding experimental ischemic animal model.

Table 6.2 Administration regimens of intranasal delivery of therapeutic peptides
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6.6 Disadvantages and Limitations of Intranasal Delivery of Therapeutic Drugs

Despite the well-known advantages of intranasal drug delivery as described above, intranasal delivery of therapeutic drugs also carries some disadvantages: (1) Potentially rapid degradation of the drug due to active mucociliary clearance and the presence of nasal cytochrome P450/peptidases/proteases. This disadvantage can be overcome by covalently binding the drug to polyethylene glycol (PEG ), also known as PEGylation [48]. PEGylation has been used to enhance drug delivery of BDNF into the brain, where it reduced systemic BDNF clearance and facilitated neuroprotection [49, 50]. Intranasal delivery of PEGylated-TGF-α induced similar effects to intracranial delivery of TGF-α, such as inducing neural stem cells in the ependymal layer and progeny in the subventricular zone to proliferate, migrate to the lesion area, and differentiate into context-appropriate neurons [51]. However, intranasal infusion of un-PEGylated TGF-α failed to produce similar effects, which may be attributed to its vulnerability to degradation [33]. (2) Although intranasal delivery can bypass the BBB , the major disadvantage of this route is the limited absorption across the nasal epithelium. This has restricted its application to particularly potent substances. Hyaluronidase, an enzyme that binds to hyaluronic acid, can reduce the viscosity of hyaluronic acid or tissue [52]. Therefore, application of hyaluronidase has become a routine procedure used to facilitate intranasal drug delivery. Using 100 U hyaluronidase 30 min before apelin-13 administration increased its tissue permeability. It helped to reduce microglia activation, decreased the levels of inflammatory cytokines or chemokines and inhibited neuronal apoptosis [35]. However, there is also evidence that without hyaluronidase, apelin-13 alone can produce similar protective effects [53,54,55]. Therefore, further studies should be carried out to determine whether there is actual benefit to applying hyaluronidase. (3) Because of the size of the nasal cavity, there is a limit to the drug volume that can be delivered, which ranges from 25 to 200 μL. Indeed, in order to avoid discomfort, 2 μL drops are typically infused into one nasal cavity at a time, alternating between nostrils every 2 min, which takes over 20 min to complete drug administration.

6.7 Future Directions and Concluding Remarks

As a non-invasive approach , intranasal delivery of therapeutic peptides has numerous advantages, including easier passage to the brain, reduced dosage, and faster onset of action. Thus it is emerging as a promising alternative route for drug administration in the treatment of cerebral ischemic stroke. Multiple therapeutic peptides, including EPO , tPA , TGF-β1 have been shown to protect against stroke when administered intranasally. However, there are a lot of unknowns pertaining to the delivery details, such as the concentration that is needed, the number of injections required, and the times at which the drug should be delivered. Further investigation into intranasal delivery of therapeutic peptides as a neuroprotective strategy may lead to an easily accessible stroke treatment strategy, and hence better recovery after stroke.