Drug Delivery and Translational Research (2022) 12:735–757

https://doi.org/10.1007/s13346-020-00891-5

ORIGINAL ARTICLE

Intranasal drug delivery: opportunities and toxicologic challenges

during drug development
Lea-Adriana Keller1,2 · Olivia Merkel2 · Andreas Popp1

Accepted: 21 December 2020 / Published online: 25 January 2021

© The Author(s) 2021

Abstract
Over the past 10 years, the interest in intranasal drug delivery in pharmaceutical R&D has increased. This review article
summarises information on intranasal administration for local and systemic delivery, as well as for CNS indications. Nasal
delivery offers many advantages over standard systemic delivery systems, such as its non-invasive character, a fast onset
of action and in many cases reduced side effects due to a more targeted delivery. There are still formulation limitations and
toxicological aspects to be optimised. Intranasal drug delivery in the field of drug development is an interesting delivery
route for the treatment of neurological disorders. Systemic approaches often fail to efficiently supply the CNS with drugs.
This review paper describes the anatomical, histological and physiological basis and summarises currently approved drugs
for administration via intranasal delivery. Further, the review focuses on toxicological considerations of intranasally applied
compounds and discusses formulation aspects that need to be considered for drug development.

Keywords Intranasal drug delivery · Olfactory pathways · Nose-to-brain-delivery · Nasal toxicity

Introduction Frey II proposed a patent for a nasal drug delivery method

to treat neurological disorders in the brain [1]. Subsequently,
Nasal administration has been used for therapeutic reasons there was an increased interest in nasal delivery especially
for centuries. As the respiratory tract is a primary contact for the growing field of nose-to-brain-delivery (ntb).
zone for the environment, it represents a gateway, not only Ntb delivery, in contrast to systemic delivery, presents a
for infectious particles such as bacteria and viruses but promising alternative enabling the delivery of therapeutic
also for potential treatments. In the past century, the use of drugs to the CNS, while bypassing the blood-brain barrier
intranasally (IN) administered drugs was mainly restricted to (BBB). Compared with conventional drug delivery
treating topical symptoms of seasonal rhinitis or infectious approaches, ntb delivery represents a non-invasive method,
diseases of the respiratory tract, for example. At the end of to access the CNS directly through the olfactory or trigeminal
the twentieth century, the nasal delivery route became more nerves. Each drug formulation favours different transport
prominent as an alternative route to treat systemic symptoms mechanisms, including intracellular and extracellular
such as in cardiovascular indications. The possibility to pathways, from the nasal cavity to the higher brain regions.
deliver drugs to the central nervous system (CNS) through During drug formulation development, the influence of
nasal pathways remained unexplored until in 1991. William absorption into the blood circulation, lymphatic systems
and into the cerebrospinal fluids has to be considered. For
several CNS disorders, specific and efficient therapeutic
* Olivia Merkel
[email protected] proteins already exist, and for others, new biologics need to
be developed. What they have in common is their potential
Lea-Adriana Keller
[email protected] to improve therapy outcomes and to reduce side effects
compared with currently approved systemic medications
1
Preclinical Safety, AbbVie Deutschland GmbH & Co. KG, or compared with the systemic doses currently necessary if
Knollstrasse, 67061 Ludwigshafen, Germany delivery to their site of action is enhanced. While there are a
2
Department of Pharmacy, Pharmaceutical Technology number of approved drug formulations for local and systemic
and Biopharmacy, Ludwig-Maximilians-University, indications, the development of nasal drug formulations for
Butenandtstraße 5-13, 81337 Munich, Germany

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736 Drug Delivery and Translational Research (2022) 12:735–757

CNS delivery is still a challenge. This review paper not only 80–90%, while in rodents, it only covers 50% of the nasal
discusses the possibilities and advantages of intranasal drug cavity (Fig. 1). The respiratory region is a pseudostratified
delivery (INDD) but also highlights its current limitations and columnar epithelium and is the most vascular region. It
and toxicological considerations. Anatomical, histological, consists of mainly four cell types: goblet cells which secrete
physiological and pathological information about the nose, mucin for the mucus layer, ciliated, non-ciliated columnar
together with data on drugs and their formulations, was and basal cells. The third region is the olfactory region
gathered, to further discuss their influence on INDD and drug which in humans covers ~10 cm2 of the nasal cavity [3–6].
development. The future perspectives for the establishment In humans, the nasal cavity is connected to the paranasal
of intranasal drugs for local, systemic and CNS indications sinuses. The paranasal sinuses are four paired spaces named
are highlighted in this review paper. after the bone in which they are located (frontal sinus,
sphenoid sinus, ethmoid air cells and maxillary sinus). Even
though the function of the paranasal sinuses is unclear,
Anatomy of the nose they support functions such as reducing head-weight,
cleaning and humidifying inhaled air and improving the
The nasal anatomy resonance of sound and speech [7, 8]. Besides differences
in the surface area of the olfactory epithelium between
The nose is a structurally and functionally complex organ preclinical animal models and humans, there are also other
and hosts one of the 5 main senses. Besides filtration, translational limitations, which should be considered. The
humidification and temperature control, the olfaction is accessibility of the olfactory epithelium of rodents and
an important function of the nose, not only for animals humans, for example, is different, as the nasal cavity of
to detect food, predators and mates, but also for humans rodents is narrower and thus less accessible than in humans
[2, 3]. The principal structure of the nose is in general [9, 10]. In humans, the collection of olfactory mucosa can
comparable between rodents, which are commonly used be carried out by an ear, nose and throat surgeon using
as laboratory animals, and humans. The nasal cavity a local anaesthetic, while in rats, the animal has to be
is divided into two areas which reach from the nostrils euthanised before removing the nasal bone to access to
towards the nasopharynx. They can be separated into three the olfactory epithelium [10]. More recently, new surgical
regions: the vestibular region, the respiratory region and methods for olfactory epithelium biopsies in rats without
the olfactory region. The vestibular region in humans euthanasia have been described [9]. As shown in Fig. 1,
spans over ~ 0,6 cm2 and is located at the nostril opening. other anatomical factors such as the bend from the nostrils
This area contains nasal hairs, squamous epithelial cells into the nasal cavity, the length and volume, the structure of
and few if any ciliated cells. The respiratory area covers the conchae and the presence of a septal window can cause
with 150 cm2 the lateral part of the nose and represents the differences in nasal uptake and absorption between species.
largest area. In humans, the respiratory region makes up to Further physiological conditions of the nose, dosage form

Fig. 1 Nasal cavity of humans (left) and rodents (right), showing the (OE). Rodents have the maxilloturbinates and nasoturbinates in the
different regions within the nose. Starting with the squamous mucosa RE and the ethmoturbinates in the OE. The olfactory bulb (OB) is
(SM) right at the nostril openings. The respiratory epithelium (RE) in close proximity to the cribriform plate and connected to the OE
covers the main part of the nasal cavity in humans. Humans have via the axons of the sensory neurons projecting towards the brain.
three turbinates (T), the inferior turbinate, the middle turbinate as a The rodent nasal cavity further has a predominant vomeronasal organ
part of the RE and the superior turbinate in the olfactory epithelium (VNO) which also takes part in the olfaction of specific compounds

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factors, administration and sampling techniques as well as dendritic extension to the mucosal surface, where cilia carry
choice of administration device should be considered during olfactory receptors (ORs) [16]. From the cell body of OSN,
species selection, as they can limit the translation towards an axon reaches through the cribriform plate of the ethmoid
the human situation [11–13]. bone (which separates the nasal cavity from the brain)
directly to the olfactory bulb (OB). The axons are enclosed
by interconnecting olfactory ensheating cells, which are
The olfactory region additionally covered by layers of neural fibroblasts (ONFs).
The ONF layers form the perineural sheath. Together with the
The olfactory epithelium (OE) is the first contact zone of OEC, the ONF encompass the OSN axons along the olfactory
environmental information to the body, situated in the upper nerve until they reach the OB [17].
part of the nasal cavity, called the olfactory cleft. While in The specialised ORs represent the first part in the
humans this region is only ~10% of the area, in rodents which signalling pathway of the olfactory system. They possess
are mainly used for IN administration studies, the olfactory the enormous discrimination power of this system through
region can make up to 50% of the total area [6, 14, 15]. It which humans can distinguish between thousands of
contains several cell types including tubular Bowman’s different odours. As they belong to the large gene family
glands which secrete a mucus layer that covers the epithelium. of the G-protein coupled receptors (GPCRs), odorant
Horizontal basal cells (HBCs) and globose basal cells (GBCs) receptors (ORs) contain the GPCR characteristic seven-
are located close to the lamina propria and act as progenitor transmembrane domain (7TMD) structure [18]. The
cells for the other cell types. Further, the OE encloses olfactory specificity of the ORs towards molecules is based on their
sensory neurons (OSNs), also called olfactory receptor extensive sequence diversity within the transmembrane
neurons (ORNs), which are surrounded by supporting cells domains, where the ligand binds. The consensus view is that
(Fig. 2). The OSNs are unmyelinated, bipolar cells, having a one OSN expresses one OR gene, but this rule may not be

Fig. 2 Structure and composition of the olfactory mucosa. In the ONF form the olfactory nerve bundles (ONBs) in the lamina propria.
posterior part of the nasal cavity, the olfactory mucosa, together with The OE further consists of sustentacular (SUS) and mucus producing
the olfactory epithelium (OE) and the lamina propria (LP), repre- Bowman’s glands (BGs). In the middle part of the OE are the imma-
sents the first contact zone of environmental cues towards the human ture ORN (ORNi). The OE is surrounded by a layer of immature
body. Within the OE, the mature olfactory sensory neurons (OSNm) basal cells, the globose (GBC) and horizontal basal cells (HBCs).
are projecting their axons towards the olfactory bulb (OB), where The lamina propria (LP) is seperated from the OE by a basal lamina
they form glomeruli with the dendrites of mitral cells. The axons of (BL). Further, the LP also contains blood vessels (BVs) and lym-
OSNms are enclosed by olfactory ensheating cells (OECs) and olfac- phatic vessels (LVs)
tory nerve fibroblasts (ONFs). The axons together with the OEC and

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conclusive [19, 20]. Nevertheless, the observations revealed the OB. Many studies have been carried out to investigate
a mechanism of receptor gene choice, in which a cell selects the underlying hierarchy of cues, but the axonal wiring is
one allele but can switch at low frequencies, as long as a a complex process which cannot be explained by simple
functional receptor is expressed [21, 22]. Each OSN extends single gradients. There are, however, different factors and
a single dendrite to the surface of the epithelium. From there approaches discussed which may also play a role in the
cilia, which are enriched with OR extend to contact odorants axonal targeting to the OB. The role of these factors in the
in the air. The OR interacts with its ligand, which results signal transduction cascade can be studied using knockout
in the activation of heterotrimeric G-proteins, G-protein α experiments. The knock-out of adenylyl cyclase 3 (AC3)
subunit (Gαolf) and G beta-gamma complex (Gβγ). The for example resulted in disturbed axon wiring [28]. The
activated Gαolf then activates type III adenylyl cyclase, formation of ectopic glomeruli was seen when the tested
which catalyses the production of cyclic AMP (cAMP) OSNs were in an AC3-deficient background. Further, the
from ATP. The increase of the cAMP-level opens the cyclic formation of glomeruli was impaired even when the axons
nucleotide-gated (CNG) channel, which leads to an influx of were shown to project to the correct position [28]. Other
sodium and calcium ions, and hence to the depolarisation of approaches studied the influence of the OR sequence itself
the neuron. The initial depolarisation is amplified through on axonal wiring [20]. Other important factors for axonal
the opening of other channels and chloride efflux [23]. The wiring are the cell adhesion and cell surface molecules.
OR interacts with its ligand and the signal is transmitted Along the dorsal-ventrolateral axis, the segregation is
along the axons towards the OB. The OSNs which express established by neurons expressing the receptor Robo2
the same OR, all send their axons to a distinct area, called and the axon guidance cues Slit1. While Slit1 is highly
glomerulus, in the OB [23]. Thus, the identity of the OSN expressed in the ventral bulb, the receptor Robo2 shows
through the expression of a specific OR seems to play a role the highest expression pattern in dorsomedial OSNs [29,
for the axonal wiring to the OB. 30]. Additionally, the chemorepellent Semaphorin3F,
released by dorsal axon terminals of OSNs, and its
receptor Neuropilin2, being expressed in high levels by
The axonal wiring towards the olfactory bulb the ventral OSNs in the OE, influence together with Slit1
and Robo2 the positioning of OSN projections along the
The first site for processing the olfactory information is the dorsal-ventral axis [23]. For the medial-lateral axis, the
OB, a forebrain structure, where the axons of each olfactory picture is not as clear as for the dorsal-ventral axis. It was
sensory neuron with the same identity meet. For a long time, shown that insulin-like growth factor (IGF) plays a crucial
the question on how the axonal connection of 1000 different role in the innervation of either lateral or medial regions in
types of odorant receptors is organised, in such a precise the OB. IGF can act as a direct chemoattractant for OSN
way, kept the field of olfactory research busy [24]. growth cones in cell culture, but the existence of IGF1 and
The mammalian OB has a simple cortical structure with 2 is not sufficient to solely explain the wiring of axons to
thousands of signal-processing modules called “glomeruli.” either the lateral or medial OB [31]. Along the third axis,
Those glomeruli represent the olfactory sensory convergence the anterior-posterior axis, the OR influences itself. There
centre, where all inputs of odorant receptors, belonging to is one plausible model, which explains the restriction of
the same type, transmit their either signals to mitral or tufted OSN with a specific receptor to a specific glomerulus along
cells. After the first axonal wiring to the mitral cell, which the anterior-posterior axis. The intrinsic activity of OSNs
is processed by the local neuronal circuits, that mediate seems to be essential for the guidance along this axis. One
synaptic interactions, the information is sent to the olfactory important factor is cAMP which can modulate the growth
cortex [25]. The glomeruli of the OB represent a map of the cone’s response to axon guidance cues. Interestingly, the
axons of OSNs and therefore their OR. Hence, studies have transcription level of specific genes such as Neuropilin1
shown that each odour has a specific characteristic pattern is correlated with the cAMP-mediated level. High levels
of glomerular activity [25, 26]. This relationship of odorant of the Neuropilin1 receptor lead to an axon projection
receptor pattern in OB and OE is called zone-to-zone into the posterior OB, while low levels result in a more
projection. The OB has four zones (zone I, II, III, IV) lining anterior projection [32]. Additionally, the cell adhesion
from dorsomedial to ventrolateral parts of the OE. One molecules Kirrel2 and 3 as well as the repulsive Ephrin A5
striking finding was that glomeruli which are located next and its receptor act in a complementary pattern in OSNs
to each other respond to molecules with similar structures [22]. Hence, the different pattern of expression of each of
[24, 25]. It is now clear that the zones overlap and do not those genes results in a specific pattern that determines
have a sharp boarder to each other [27]. the identity of the OR and the convergence of the same
Another aspect is how this map is maintained and how OR type to one glomerulus. The model suggests that the
OSN axons target and converge in the appropriate region of sensitivity of OSNs to cues positions the OSN axon to a

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glomerulus. These three mechanisms for axon guidance several amygdaloid nuclei (A) and the entorhinal cortex
show how complex the organisation of the olfactory (EC), posterior [30, 40]. The olfactory cortex represents
system is. Especially the mechanism of convergence of not only a complex system of interconnections between
randomly spread OSN expressing the same type of OR cortex regions and the OB but also has intercortical
reaching to the same glomeruli provides more detailed connections to higher brain regions such as the thalamus,
knowledge of the signalling-network. hypothalamus, neocortex and hippocampus. The axonal
wiring during development is even more complicated
From the olfactory bulb to higher CNS regions than the axonal wiring within the higher CNS and
towards the OB. Since the intercortical communication
The OB consists of five layers, which are important for between the areas within the cortex are more complex
further signal transduction after the first excitatory afferent than the communication within the OB, the process of
projection from the OSN axon to the primary dendritic axon wiring to the areas of the brain is still a field of
terminals of mitral/tufted cells within the glomerular layer research.
[33, 34]. Mitral and tufted cells both act as efferent neurons
of the OB. This afferent excitatory synapse between
OSN and mitral/tufted cells transduces its signal via the
neurotransmitter glutamate towards different regions of Olfactory epithelium uptake pathways
the olfactory cortex (OC). Mitral cells (MCs) and tufted and mechanisms
cells (TCs) transmit temporally distinct information
to different OC targets. While TCs were described to It has been known since the nineteenth century that
project densely to focal targets only in anterior areas of transport mechanisms and flows between the different
the OC, individual MCs dispersedly project to all OC spaces within the nose exist [41]. But these transport
areas [35–37]. The activation of mitral/tufted cells results mechanisms remained unexplored until increasing
in a feedback inhibition involving lateral inhibition via numbers of neurological diseases pushed the field of
dendrodendritic (GABAergic signal) reciprocal synapses nose-to-brain (ntb) drug delivery forward. Only a few
with periglomerular cells, acting as interneurons. These decades ago, in 1991, William Frey II proposed a patent
mechanisms are important for the tuning of the incoming for a nasal drug delivery method to treat neurological
signal and lead to a sharpened specificity between high disorders in the brain [1]. Since then, many exciting basic
and less-activated mitral/tufted cells [36, 38]. Besides the preclinical and applied clinical studies were performed
periglomerular cells, the glomerular layer also receives showing the great potential of nose-to-brain delivery
signals from higher parts of the CNS via centrifugal methods to treat brain diseases such as Alzheimer’s
afferent fibres, using a wide range of neurotransmitters. disease [42–45]. Nevertheless, the exact mechanisms
The external plexiform layer contains the somata of tufted through which molecules get transported from the
cells, but also primary and secondary dendrites of mitral/ nasal mucosa towards the CNS are still unclear but will
tufted cells, as well as the apical part of granule cells. The benefit from further drug delivery approaches using IN
mitral cell layer is a thin layer, mainly consisting of the administration.
somata of mitral cells and axons of tufted and granule cells The IN administration approaches are mainly focused
together with the centrifugal fibres. The granule cell layer on two morphological structures, the olfactory and the
is mainly consisting of the interneuron’s granule cells, trigeminal nerve. The pathways used by IN administered
being important for the inhibitory circuit to mitral cells. substances include intracellular, paracellular and
They also receive input from the anterior olfactory nucleus, transcellular mechanisms (Fig. 3) for the transport along
olfactory cortex, cells of the diagonal band, locus ceruleus the olfactory and trigeminal nerves towards the CNS. The
and raphe nucleus, via afferent centrifugal fibres [33, 39]. intracellular olfactory nerve pathway uses pinocytosis
Interestingly, unlike other sensory systems, the and endocytosis to take up the substance into the OSN
olfactory pathway does not pass the thalamus before [15]. Subsequently, the substance is transported along the
reaching the cortical regions. From the OB, the axons OSN axon to the OB. Once the molecules are delivered to
of mitral and tufted cells form the lateral olfactory tract the origins of the nerves in the cerebrum and pons, they
transmitting the signals directly to the olfactory cortex. can disperse throughout the brain. Another pathway is
The olfactory cortex is a structurally distinct cortical the olfactory epithelium pathway, where the substance is
region on the ventral surface of the forebrain, which is absorbed into the lamina propria and further enters the
innervated and composed of several subregions from CNS by using the gaps surrounding the olfactory nerve
anterior, starting with the anterior olfactory nucleus tract [46, 47]. From the lamina propria, the substance can
(AON), the olfactory tubercle, the piriform cortex (PC), be absorbed by local blood vessels or lymphatic vessels,

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Fig. 3 There are three different pathways through which a substance From there, the substance can (1) be absorbed by local blood vessels
can pass the olfactory epithelium (OE). The substance can bind to a (BVs) reaching the circulation or (2) be absorbed by lymphatic ves-
receptor and be internalised by for example receptor-mediated endo- sels and be drained into the deep cervical lymph nodes of the neck.
cytosis. Then, it travels through the olfactory sensory neuron (OSN) (3) The substance can use perineural spaces between the olfactory
towards the olfactory bulb (OB). Another possibility is that the sub- ensheating cells and olfactory nerve fibroblasts to travel associated to
stance uses leaky passages within the OE and travels paracellular into the olfactory nerves to the OB. After passing the cribriform plate, the
the lamina propria. Third, the substance can also be transported tran- substance can theoretically also reach the cerebrospinal fluid and dis-
scellular through the sustentacular cells (SUS) to the lamina propria. tribute through the different brain regions

but most of the substances are translocated through the 1995, experiments were published which indicated that
perineural space, between olfactory ensheathing cells in contrast to HRP alone, WGA-HRP shows an increased
and olfactory nerve fibroblasts, by bulk flow. This space uptake through receptor-mediated endocytosis [56]. Even
leads to the subarachnoid space of the brain from where though pinocytosis seems to be more likely than receptor-
the substance can further distribute [15]. mediated endocytosis, further approaches, besides WGA-
HRP, indicate specific receptor expression in the olfactory
Intracellular pathways mucosa, as well as in the OB. One early discovered example
is the receptor-mediated uptake of the brain-derived
The intracellular pathways are used by a wide range of neurotrophic factor (BDNF) [57]. Recently, many studies
different molecules. Apart from the widely examined peptide strongly support the view that metabolic receptors are
insulin (5.8 kDa) [48], also gold particle (50 nm) [49, 50] expressed in the olfactory system and that hormones such
and aluminium salts [51] are known to endocytose. The as insulin, leptin and orexin can bind to those receptors on
classic model molecule for studying intracellular pathways the olfactory mucosa [58–62].
of the olfactory system is wheat germ agglutinin (WGA) However, vir uses can use receptor-mediated
conjugated with horseradish peroxidase (HRP) with a size of endocytosis to enter the olfactory epithelium, too. Many
80 kDa. In the late twentieth century, several studies showed influenza virus-subtypes, the herpesvirus and poliovirus,
that HRP uses pinocytosis to enter OSN [52–55]. Later, in use different receptors on the cilia surface to enter an

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olfactory sensory neuron. By 1912, studies discovered the Extracellular pathways

ability of the poliovirus to infect the CNS via entering the
ORN [63]. In the 1930s, this hypothesis was supported The extracellular pathways start with either the transcellular
by studies which finally resulted in the chemical uptake into supporting cells or paracellular diffusion through
cauterisation of the OE of Canadian school children with leaky parts of the nasal epithelium. Even though the epithelial
zinc sulphate to prevent the disease during a poliomyelitis cells are connected via tight junction (TJ) and express
epidemic [64–66]. Even though the poliovirus receptor proteins such as zona occludens (ZO)-1, 2, 3, occludins and
was identified in the 1990s, the amount of studies claudin-1, 3, 4, 5, 19, it is known that intercellular clefts
focussing on the expression of the receptor on the OE exist [87, 88]. The nasal epithelium undergoes a constant
surface is still rare [67–69]. Influenza subtypes can turnover with an average lifespan of OSN of 30–60 days until
however also use structures on the cell surface to enter they are mined by apoptosis [89–91]. As long as the OSN
the olfactory epithelium. They bind to glycans with is not fully replaced and the TJ are not functional, there are
terminal sialic acid linked to galactose (SAα2,6Ga in channels remaining through which also proteins and peptides
nasal epithelium) to enter OSN [70–72]. Further, there such as insulin (5.8 kDa), IGF-1 (7.5 kDa), albumin (65 kDa)
is strong support that the herpesviruses can use heparan and even stem cells can reach the CNS [92–96]. There are
sulphate and nectin-1 on the cell surface to enter the signalling mechanisms and formulation approaches to
olfactory neuroepithelium [73–75]. The latest example increase TJ permeability to improve the passage of drugs in
for a virus using the nose as an entry to the human body the nasal epithelium. Besides various signalling regulators
is the SARS-COV-2 virus. Recent studies showed that the such as protein kinases, mitogen activated protein kinase,
nasal and olfactory epithelium expresses the obligatory myosin light chain kinase and others [97], also surfactants
receptor, angiotensin-converting enzyme II (ACE2) and such as bile salts and cationic polymers such as the chitin
the priming protease TMPRSS2. Interestingly, loss of derivate chitosan are known to increase the permeability of
sense of smell is an early marker for a SARS-COV-2 TJs and absorption of drugs [98].
infection. It is proposed that the damage of sustentacular From the lamina propria, the substance travels three
cells which express, as non-neuronal cells, ACE2 leads alongside different ways to further distribute. First, it can
to the olfactory deficits in COVID-19 patients [76–79]. be absorbed by local blood vessels and enter the systemic
After internalisation of a molecule, drugs, viruses or circulation. Although the nasal epithelium, in particular the
other substances, the cargo containing vesicles (endosomes) respiratory parts, is rich of vasculature, IN insulin studies
traffic down the soma. Some pass the Golgi apparatus, while support the view that the absorption in local vessels and
others are directly transported down the axons towards the systemic distribution after IN administration is no significant
OB. Intracellular axonal transport was visualised by using factor in the nose [44, 99]. Further, substances and molecules
labelled tracers, showing that anterograde and retrograde can be absorbed from the submucosa into the lymphatic
transport mechanisms are present [55, 80]. The vesicles have system. The lymphatic vessels in the nose drain towards
to cover an intracellular distance within the olfactory nerve the deep cervical lymph nodes of the neck [100, 101]. The
of about ~4 mm and about ~20 mm [81] within the trigeminal major aspect of intranasally administered molecules seems
nerve in rats to reach the CNS. Neuronal transport is discussed to be extracellular diffusion and convection into perineural
to be a rather slow process. Crowe et al. state that the olfactory and perivascular spaces, having a connection to cranial
nerve axonal transport alone should only take 45 min for compartments. As mentioned previously, the intracellular
an intranasally administered drug to reach the brain [15]; axonal transport is a rather slow movement with calculated
however, other processes such as endocytosis and exocytosis times of 0.74 (fast)–2.7 h (slow) for the distance of ~4 mm
are not included in these calculations and thus prolong the within the olfactory nerve and 3.7 (fast)–13 h (slow)
time in practice. This is based on velocity values measured for ~20 mm within the trigeminal nerve. Instead, extracellular
in an ex vivo study using WGA-HRP and olfactory C-fibres. diffusion along the olfactory and trigeminal nerve, as it takes
Two values were estimated, a slow rate with 36 mm per day, place in the perineural space, is slightly faster. Lochhead and
and a fast transport mechanism with 130 mm per day [80]. The Throne calculated times of 0.73–2.3 h for the olfactory nerve,
fast velocity rate has been supported by further studies [53, using a simplified version for only one dimension (distance)
81]. As previously mentioned, some molecules and viruses of Fick’s second law [15, 81]. These velocities, however,
can be endocytosed and translocated along the trigeminal do not match experimental data, as they predict slower
nerve, which innervates the nasal cavity with two of its three transport. IN studies using [125I]-labelled IGF-1 suggest that
main nerve divisions (V1 ophthalmic nerve; V2, the maxillary the rapid distribution towards the CNS (~30 min) is rather
nerve) [81, 82]. Besides WGA-HRP, IN administration studies due to extracellular convection than diffusion, or intracellular
also showed that IGF-1 and the herpes simplex virus can use transport [92]. One mechanism which could explain the
the trigeminal nerve pathway to reach the CNS [83–86]. rapid rates is bulk flow in the perivascular spaces in the nose.

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Many arteries along the olfactory nerve supply the axons with period from 2000 to 2010 (ClinicalTrial.gov) (Fig. 4). This
nutrients. The systolic waves result in high-pressure waves increase in number of clinical studies is further higher than
within the perivascular space. The so-called perivascular pump for other administration routes. There are around 18 times
is suggested to be responsible for the rapid distribution of fluids more completed intravenous interventional studies listed
and molecules towards the brain [102–104]. Calculation of at ClinicalTrial.gov from 2000 to 2020, compared with
the time needed for intranasally applied [125I]-labelled IGF-1 completed intranasal interventional studies; however, the
with a velocity of ~200 μm/min resulted in a predicted time of number of published interventional intravenous studies,
0.33 h for IGF-1 to reach the brainstem via perivascular bulk using the same filters, only increased twofold in the past
flow [81]. These are predictions and do not fully represent decade.
the situation in vivo, but the predicted values support the Even though IN delivery is usually associated with locally
hypothesis that extracellular transport is faster and probably acting drugs, also systemic effects and treatment of CNS
the most prominent pathway for drugs to reach the CNS. disorders are increasingly focussed upon by preclinical
research and the pharmaceutical industry [14, 47, 105,
106]. The nasal route offers many advantages over common
Current status of approved drugs for nasal routes such as the oral route, as it is non-invasive and easily
application accessible for the administration of drugs. Further, IN
administered systemically acting drugs are rapidly absorbed
The IN-delivery route has gained more interest in the and exhibit a fast onset of action. The rapid absorption is
delivery of various drugs and treatments. In the past due to the previously mentioned rich vasculature in the
decade, the amount of interventional IN studies, including submucosa. The avoidance of the metabolic first pass
patients of all age groups, all sex and overall clinical effect can lead to a higher bioavailability [14, 106, 107].
phases increased around threefold compared with the time It is also known for drugs intended to reach the brain that

Fig. 4 Number of clinical trial papers published at ClinicalTrial.gov ria (differentiated after age groups (child, adult, older adult), all sex);
using intranasal administration. Filters used contained the recruitment study results (all); study phase (early phase 1, phase 1, phase 2, phase
status (recruiting, active (not recruiting), completed); eligibility crite- 3, phase 4)

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the side effects are in many cases reduced, because nasal century. Levocabastine is an H1-receptor–selective drug
drug delivery leads to a lower systemic exposure and a with high potency. It belongs to the second generation of
more efficient targeting [108]. One of the most prominent histamine H1-receptor antagonists. Those second-generation
arguments supporting the increasing interest in IN drug drugs show an improved benefit/risk profile without CNS
delivery is the possibility to bypass the BBB. The BBB is a sedative effect when administered locally instead of orally
limiting factor for treating neurological CNS disorders [47, [115, 116]. Another group of topically administered drugs
109]. There are many additional limitations and aspects for the treatment of seasonal rhinitis is the steroidal anti-
which have to be considered for the nasal application of inflammatory agents, namely corticosteroids. Corticosteroids
drugs and biopharmaceuticals. While small, lipophilic drugs bind to a single glucocorticoid receptor, localised in the
(MW < 1 kDa) are well absorbed; the nasal mucosa exhibits target cell. Through transactivation or transrepression, anti-
poor permeability for big hydrophilic drugs (MW > 1 kDa) inflammatory effects are generated. Those effects include
such as peptides and proteins. Additionally, processes influencing the release of cytokines and mediators as well
such as the mucociliary clearance, enzymatic degradation as a reduced inflammatory cell recruitment within the nose
and a low drug retention time limit the efficiency of drug [117]. Many approved corticosteroids are on the market,
delivery via IN delivery systems. Many technologies and such as budesonide, flunisolide, fluticasone, mometasone
approaches which aim to decrease these limiting effects are and triamcinolone. Even though corticosteroids are regarded
under investigation [106, 107]. Nasal device technologies to be more potent and efficient than antihistamines, no
are under optimisation for precise targeting of the location significant differences have been found for these two groups
of action and therapeutic effect. In the following sections, regarding the benefit/risk profile [113].
the terms local delivery, systemic delivery and CNS delivery
are used as terms for the distribution route of a substance or
drug after IN application, but not as terms for the indication. Intranasal application used for systemic delivery

Intranasal application used for local delivery Apart from IN administration for local delivery of drugs,
which aims to achieve local effects, IN delivery systems also
Nasal delivery of locally acting drugs is commonly aim to result in systemic exposure to achieve a wide range
administered as liquid formulations via nasal sprays/aerosols of therapeutic effects. IN application for systemic exposure
pumps [110]. This delivery system is convenient as it has an and thus systemic delivery is under investigation in a wide
additional humidifying effect and offers the possibility to range of indications such as migraine, headache, infection
reach a wide drug distribution within the nasal cavity, as it is prevention, pain management, hormone replacement therapy,
required for local and systemic applications [111]. However, smoking cessation and emergency therapy, like for epileptic
main limitations of water-based formulations and long- seizures [9, 98]. These approaches are due to advantages of IN
term usage is their microbial stability and the presence of application for systemic delivery. The nasal mucosa offers a
preservatives, which can lead to irritation and allergic effects relatively large surface area for drug absorption; however, the
[110, 112]. Approaches are constantly developing delivery individual absorption and bioavailability values really depend
technologies, which are increasing the retention time on the on the compound, the drug formulation, the species tested in
mucosal surface and are reducing the variability within the and the delivery device itself. Costatino et al. summarises
patient’s individual administration procedure, for example that for low-molecular weight drugs, the bioavailability after
the patients head position. Besides others nasal pressurised IN administration is relatively high and the variability low,
metered-dose inhalers, containing filters are used to apply while for high-molecular drugs, the bioavailability is low and
budesonide to the anterior parts of the nasal cavity where the the variability high, compared with injections [114, 118]. For
non-ciliated regions are the major site of deposition [112]. systemic delivery after IN administration, the high vascularity
Popular drugs for nasal delivery are antihistamines and within the nasal cavity is a particular advantage. Further
corticosteroids being the first-line medications to treat advantages are a rapid drug onset, no first-pass metabolism
seasonal rhinitis and nasal congestion based on allergic and non-invasiveness to maximise patient comfort and
reactions or infections [14]. Topically acting drugs offer compliance [107, 119]. Historically, as it is for local delivery
advantages in these indications since they have a rapid onset. also for systemic delivery, most approved intranasal drug
Further, antihistamines and corticosteroids have as topically products are delivered through nasal sprays/aerosols devices
acting drugs a low systemic bioavailability and hence lead to [14, 112, 120]. For systemic delivery indications however, one
less adverse side effects and no CNS effects. Compared with aim of nasal device investigations is to prolong the retention
oral application, the IN administration of these drugs requires time of the drug on the nasal mucosa. Therefore, to mention a
lower doses [113, 114]. The role of histamines as mediators few approaches, nasal gels, microemulsions/suspensions and
for allergic rhinitis has been known since the early twentieth microspheres are under development [112].

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744 Drug Delivery and Translational Research (2022) 12:735–757

Feasibility of systemic delivery via IN application is treatment for antiepileptic medication [114, 127, 128].
proven for drugs used for cardiovascular indications, as For the application via the IN route, both indications
some popular examples such as propranolol, nifedipine benefit from the rapid onset and improved bioavailability,
and nitroglycerin showed in the clinic [114]. Intranasally compared with the oral route and easy handling, which
administered propranolol was shown to improve the exercise is in particular relevant for caregivers in pre-clinical
tolerance of patients suffering from angina pectoris [121]. situations [129, 130].
Further, clinical studies were performed which showed that The replacement of hormones with an IN delivered
IN administration and systemic delivery of cardiovascular drug is an important indication for IN application with
drugs are suitable alternatives because they result in a more systemic delivery. One example is the aqueous formulated
rapid onset of action compared with the commonly paren- 17-β-estradiol (Aerodiol®), which is used as an oestrogen
teral administered formulations [121, 122]. therapy to reduce menopause symptoms in women. Studies
Apart from achieving systemic effects, delivery via the nose have shown that IN compared with oral and transdermal
is also used to target the CNS after reaching systemic exposure. administration resulted in similar area under the plasma
One example for CNS targeting via systemic delivery after IN concentration time curve (AUC) up to 24 h, efficacy and
application is the administration of analgesics. frequency of adverse effects [131–134]. Furthermore,
IN analgesics are used for indications such as headache biomacromolecules such as peptides and proteins are
and migraine treatments, as well as cancer pain. The main marketed for hormone replacement therapies. For instance,
advantage of IN analgesics is bypassing the metabolic first- nasal sprays containing salmon calcitonin (Miacalcin®,
pass effect which leads to an increased bioavailability and a Novartis; Fortical®, Unigene) are available on the market to
rapid onset. In particular, morphine, used for breakthrough treat osteoporosis; desmopressin (Desmospray®, Ferring) is
pain in chronic cancer patients, underlies an extensive first- used IN as a antidiuretic hormone to treat diabetes insipidus,
pass effect, when given orally; thus, IN delivery offers enuresis, haemophilia A and the von Willenbrand’s disease
rapid and effective relief [123, 124]. Morphine is a polar, (type I), and gonadotropin-releasing hormone analogs
hydrophilic, low molecular weight drug. Nasal administration such as busrelin (Suprecur®, Sanofi-Aventis) and nafarelin
of morphine without any absorption enhancers results in 10% (Synarel®, Pharmacia) are used to reduce the testosterone
bioavailability, while the latter can be increased up to 80% and oestrogen levels in blood of patients suffering of prostate
for morphine in humans using chitosan-based formulations cancer and endometriosis [105, 135–140]. None of these
[123]. The same is true for migraine and headache treatments products contain any absorption enhancers, even though
with IN drugs such as butorphanol, fentanyl, sumatriptan systemic bioavailability of peptides and proteins after IN
and zolmitriptan [105, 114]. The approved intranasal application is usually below 1%. However, these mentioned
drug products of these small molecules do not contain peptide-based products are effective as they are potent and
nasal absorption enhancers because the drug molecules therapeutic even at low systemic levels [125].
are characterised by high permeability and low molecular IN delivery is in general increasingly being used as a
weight (< 1 kDa) which allows them to reach therapeutic delivery route for small but also for biomacromolecules
levels in the systemic circulation. For example, the marketed treating systemic indications. Nevertheless, limitations such
drug formulation of butorphanol tartrate is delivered as an as the fast-mucosal clearance and low absorption capacity
aqueous solution nasal spray containing sodium chloride, of the nasal mucosa for non-aqueous formulations have to
citric acid and benzethonium chloride (pH 5.0) [105, 125]. be resolved by improving the composition of excipients and
Other examples for IN application of systemic acting small absorption enhancers within the formulation.
molecules are analgesics with anti-inflammatory effects such
as indomethacin and ketorolac [14]. Intranasal application used for CNS delivery
Feasibility of IN dr ug delivery with systemic
dispersion for sedative agents and in emergency Oral drug delivery is the most common way to administer
situations such as epileptic seizures (benzodiazepines) a drug to humans. However, for treatment of neurological
or for opioid overdoses (naloxone) is under investigation. disorders or diseases, oral delivery methods often fail to
Nyxoid® is for example the nasal formulation of deliver drugs efficiently to the central nervous system
naloxone approved in Europe. The formulation contains (CNS). Barriers exist in the brain, most importantly the
naloxone hydrochloride dihydrate, trisodium citrate BBB, which protect the CNS from pathogens, neurotoxic
dihydrate, sodium chloride, hydrochloric acid, sodium molecules and other potentially harmful substances.
hydroxide and purified water [126]. The benzodiazepine The BBB is an essential interface between CNS and
midazolam is used intranasally for both indications. In periphery and is composed of endothelial cells, which
clinical trials, its use has not only been described as a are tightly connected to each other by TJs and adherents
sedative agent but also as an emergency pre-hospital junctions (AJs) [119, 141–143]. Another factor reducing

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Drug Delivery and Translational Research (2022) 12:735–757 745

the amount of substance reaching the CNS via the oxytocin known for its positive effects on social behaviour
systemic route is the presence of multidrug efflux protein and autism [161–163] as well as orexin-A improving the
transporters. The active P-glycoprotein efflux pumps are CNS hypocretin signalling and olfaction and thus offering
found at the BBB luminal side and reduce the amount a possible treatment for narcolepsy [164–166]. Another
of drug exposure in the CNS, by expelling them back potential agent for nasal drug delivery is leptin for treating
into the bloodstream [142, 144]. Studies showed that the obesity and sleep disorders. While systemic and peripheral
olfactory system contains such efflux transporter [145], administration of leptin failed to lead to a positive effect,
too. However, only small drug molecules (< 500 Da) preclinical IN leptin studies showed a reduced appetite and
of high lipophilicity are able to cross the BBB which resulting weight loss [167–169].
accounts only for < 1% of macromolecules and only An example for illustrating the feasibility of IN
2% of small molecules [108, 141, 146]. In the context application of drugs targeting the CNS via olfactory-
of treating neurological disorders such as AD, the IN associated pathways is insulin. Insulin is an important
administration of drugs gains more and more interest, regulator for the energy metabolism in the CNS. Insulin-
because it bypasses the BBB and the systemic first- sensitive glucose transporters transport insulin across
pass effect and is therefore a promising approach as an the BBB, while insulin receptors are widely expressed in
efficient drug delivery route [47, 119]. different regions of the brain, with highest concentration in
As described previously, a direct path from the OE to the the OB, cerebral cortex, hypothalamus, hippocampus and
CNS exists. This path is in association with intracellular and cerebellum [170, 171]. The perturbation of its function in
extracellular pathways, involving transcellular, paracellular the CNS and the shift towards lower insulin concentration in
and extracellular transport mechanisms through which the brain (or CSF) to the periphery are shown to contribute
drugs can be transported from the nasal mucosa to the CNS to the formation of cognitive deficits and Alzheimer’s
alongside the olfactory and trigeminal nerve fibres. Studies disease. In addition, peripheral abnormalities such as
showed that not only small molecules but also peptides and hyperglycaemia and the diabetic state are improving the risk
proteins use these pathways to directly reach the OB, being to develop AD [42, 172–174]. This suggests that patients
a forebrain structure. From the OB, they can also either suffering from diabetes are more likely to develop AD. In
disperse extracellularly or they are further transported turn, many studies, preclinical and clinical, show the ability
intracellularly towards higher brain regions [81, 146]. of IN insulin to improve cognition and memory in age-
Regardless of the administration route, achieving constant related cognitive deficits [43, 175–178]. Still, no approved
and targeted delivery in the brain parenchyma is still formulation for IN administration of insulin is on the market.
challenging because substances have to travel long distances RNA therapeutics have also to be mentioned and are in
within the brain (~ mm) to reach their target [147, 148]. the focus for IN delivery to treat neurodegenerative diseases.
Transport within the extracellular space plays a critical There is an increasing number of publications in the last
role for the diffusion of drugs, as it makes up ~ 20% of the decade investigating the potential of siRNA and anti-sense-
brain volume [148, 149]. No matter if a drug or substance oligonucleotides for such approaches, as it was shown that,
reaches the brain after intranasal delivery, crossing the BBB for example, siRNAs travel along the olfactory nerve after
or direct infusion into the brain, the distribution within the IN delivery [48, 179–182].
microenvironment involves extracellular diffusion [148]. Another exciting new application for IN application is
Brain distribution can be facilitated by nanotechnology the delivery of stem cells along the olfactory tract towards
[150]. Studies have shown that nanotherapeutics and the CNS. In 2009, a group of scientists first suggested that
nanomaterials improve the biodistribution of drugs in the fluorescently labelled rat mesenchymal stem cells (MSCs)
brain for more efficient treatment of glioblastoma, not and human glioma cells intranasally applied to naive mice
only via convection-enhanced delivery [151] but also via and rats use the olfactory-associated neuronal pathways to
intranasal delivery [152]. reach different regions in the CNS [183]. Later, the potential
Chemotherapeutic agents are in general considered for of IN applied neuronal stem cells/progenitor cells carrying
INDD systems [152, 153]. The main advantage of the IN bioactive gene products to target intracerebral glioma was
route is the chance of reduced side effects in other organ elucidated [184]. Currently, the number of pre-clinical studies
systems [146, 154]. Different approaches showed that using neuronal stem cells, progenitor cells or mesenchymal
chemotherapeutic agents for treating brain tumours such as stem cells to investigate their potential to treat brain tumours
perillyl alcohol, methotrexate and telomerase inhibitors can or neurodegenerative diseases is rising. These approaches
be delivered via the nasal route and are effective alternative seem to offer a safe and efficient alternative to the surgical
strategies [155–160]. injection or intravascular administration [185–188].
Further, several peptides and proteins are under The approval of drug formulations for CNS delivery
investigation for ntb delivery. Prominent examples include through olfactory tract–associated pathways is, however,

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746 Drug Delivery and Translational Research (2022) 12:735–757

depending on appropriate nasal devices, which are mucosa. For this review, only intrinsic biologic factors are
increasing the drug distribution and absorption through considered. Those biological factors are affecting the drug
the olfactory epithelium. The distribution pattern of nasal absorption in the nasal mucosa and therefore influence the
formulations strongly depends on the particle size, which toxicologic profile of the final drug product. The nasal blood
in turn is affected by the administration device and the flow regulates important conditions in the nose such as
physiochemical properties of the formulation. The viscosity temperature or humidification of inhaled air. There is a range
of the formulation influences the droplet size of a nasal of drugs that are known to influence the blood flow, such as
spray and thus its deposition site [189]. Higher viscosity vasomotors. Oxymetazoline, which is used as a decongestant
of nasal formulations enhances the absorption through for allergies and colds, was shown to decrease the blood
the nasal mucosa because of prolonged retention time. flow within the nose as a vasoconstrictor [195–197]. Further,
Conversely, it reduces systemic delivery because of slower IN corticosteroids are also vasoconstrictors leading to relief
diffusion [190]. In this context, great efforts are made to in patients with seasonal rhinitis. Rare side effects such as
develop new delivery strategies such as nanotechnology- nose bleeding and the very rare occurrence of nasal septal
based approaches and devices such as bi-directional nasal perforation were observed [113, 115, 198, 199]. In contrast,
insufflators which facilitate the distribution to the posterior other drugs increase the blood flow in the nose, for example
part of the nose and minimise lung deposition [112, histamine, albuterol, isoproterenol and fenoterol [113, 118].
191]. A recently published review from Rabiee et al. [45] Another biologic factor which should be considered
summarises and discusses existing approaches using natural regarding toxicologic and safety issues is the enzymatic
and polymeric nanoparticles for ntb delivery. The authors activity in the nasal mucosa. As the nasal mucosa is a direct
conclude that polymeric nanoparticles are promising carriers contact zone towards environmental keys, it also represents a
for ntb delivery of drugs against Alzheimer’s disease. This barrier towards harmful substances and xenobiotics. Hence,
conclusion, however, may be also applicable for other CNS there are also defensive enzymes present that metabolise
indications. substances and drugs. To date, it is known that the nasal
mucosa has a wide spectrum of xenobiotic-metabolizing
enzymes, comprising enzymes belonging to the P450-
Toxicological challenges of intranasal dependent metabolism pathway (e.g. P450 monoxygenase),
application Phase I enzymes (flavin monooxygenases, aldehyde
dehydrogenases, epoxide hydrolases, carboxylesterases,
Safety is a key issue when designing an effective and etc.) and Phase II enzymes (glucuronyl and sulphate
safe drug formulation, for IN administration. During the transferases, glutathione transferase) [118, 200, 201]. It
development process, safety consideration not only of the can be assumed that these enzymes are also metabolizing
drug itself but also of the active ingredients and excipients intranasally administered small-molecule drugs such as
within the formulation must be considered. Absorption opioids, histamines, corticosteroids and more [118, 202].
enhancers are necessary for large molecules such as peptides Not only enzymes protect the nose and upper airways
and proteins. They increase the bioavailability of the drug from potentially harmful substances and xenobiotics,
following IN administration by improving the permeability but also the nasal mucociliary system represents a major
of the nasal mucosa. Other excipients act as mucoadhesives part of defence mechanisms within the nose. The mucus
and prolong the contact time with the nasal mucosa. Because layer covers the nasal epithelium and transports particles
of their own safety profile and the increased local exposure through ciliary beating towards the nasopharynx. The
time of the drug, the excipients can significantly decrease ciliary beating frequency (CBF) is under cellular control,
the safety of the final drug product [192–194]. Also, the regulated by temperature, intracellular Ca2+, cAMP and
toxicological considerations must be discussed regarding extracellular ATP level. Other physiological functions of
local, systemic, CNS and pulmonary effects of the drug the nasal mucosa include its water-holding capacity and its
formulation. responsibility for the efficient heat transfer within the airway.
Further, it exhibits surface electrical activity [193]. Thus,
Local side effects the impairment of these systems can lead to longer contact
times of formulations, physiologic impairment and damage
The local tolerability of a drug product depends on of the mucosa and the nasal epithelium.
many different factors and differs between individuals. The human nasal mucosa has an average physiologic pH
Environmental cues such as temperature and humidification, of 6.3 and is therefore slightly acidic. The maintenance of the
psychologic factors but also individual physiological factors pH in the mucus ensures the function of the ciliary clearance
such as infections, pre-existing illness or allergies influence [203]. Therefore, the pH of nasal formulations should be
the local interactions between drug product and nasal within a pH range of 4.5 to 6.5 to avoid nasal irritation [118].

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Drug Delivery and Translational Research (2022) 12:735–757 747

Not only the pH but also the osmolarity has an influence BKC in aqueous formulation in vivo has been considered as
on the ciliary beat and can therefore contribute to local safe [194, 213, 216]. The European Medicines Agency (EMA)
toxicological considerations [118, 190]. Many substances summarises that the average nasal use of BKC in medicine
are however influencing the mucociliary clearance (MCC) products is between 0.02 and 0.33 mg/mL and that preclinical
through either stimulation or inhibition. Instead of stimulation data show a time- and concentration0dependent toxic effect
effects, the inhibitory effects are the main cause of adverse on cilia in vitro and in vivo in rats. Further, they state that it
side effects such as nasal dryness, irritation, sneezing, nasal is not possible to recommend any safety limit for the general
itching but also rhinitis medicamentosa and congestion. It population of patients [217].
is worth to mention that MCC and CBF effects are usually Besides preservatives, also penetration enhancers
evaluated in vitro. Those in vitro tests do not allow predictions are used in nasal formulations. They are improving the
about ultimate effects in vivo, as in vitro tests show effects on bioavailability and transport of compounds across the
MCC and CBF, whereas in vivo the same compounds often nasal epithelium and mucosa. Wanted effects of enhancers
do not lead to detectable side effects [204]. In general, it was include opening of TJs, alteration of the mucus layer and
shown for many compounds that the inhibitory effect on the inhibition of proteolytic enzymes. In turn, those functions
mucosal clearance and CBF is dose and time dependent. For have a disruptive character and can thus lead to adverse side
example, the α-adrenergic receptor agonists oxymetazoline effects, which can be additive [192, 194, 204, 216]. It is
and xylometazoline showed inhibitory effects for human nasal essential to consider that many substances and compounds
mucosa in vitro in a dose-dependent manner [205, 206]. Many act as irritants to the nasal mucosa but are non-damaging.
corticosteroids and anti-histamines are also influencing the The local effects of a formulation are always an interplay
MCC and CBF in in vitro studies but at the same time show between drug and excipients. Further, the testing procedure
no adverse effects in vivo [194, 207–209]. The mucociliary such as dose, time and in vitro test system as well as animal
effect of drugs is however only one aspect. Excipients are species has to be evaluated carefully before concluding
used not only to improve the drug transport and bioavailability about safety issues.
through the nasal mucosa and epithelia but also to protect the
drug product from microbial contamination and degradation.
Those enhancers and preservatives have to be considered and Systemic and CNS side effects
evaluated in toxicological examinations. A prominent example
for the toxicologic relevance of preservatives is benzalkonium One of the main advantages using INDD compared with
chloride (BKC), which is used for cosmetics and in several other administration routes such as oral or intravenous
nasal formulations. BKC showed in different animal models application is the bypassing of the metabolic first-pass
such as chicken embryo tracheas, rat and guinea pig tracheal effect and the reduced risk of systemic adverse effects.
tissue the inhibitory effect on CBF. This effect is dose and time Intranasal 17 b-estradiol, marketed as AERODIOL, is an
dependent with ciliostasis and ciliotoxicity as the ultimate example for the possible superiority of IN drug delivery
response [210, 211]. In vivo histological examinations over oral drug delivery. Several clinical studies showed that
in rats showed that BKC can also provoke nasal lesions. AERODIOL leads to less systemic adverse side effects, such
Concentrations of 0.05 and 0.10 w/v % BKC administered as mastalgia and breakthrough bleeding, compared with
into the nasal cavity of rats led to histopathological findings oral or transdermal delivery, while exhibiting at least the
such as epithelial desquamation, degeneration, oedema or same efficiency [132–134, 218]. The same was observed
neutrophilic cellular infiltration in the anterior parts of the for intranasally delivered benzodiazepines such as diazepam
nasal mucosa [212]. Further studies support the toxic effect of and midazolam, which are used to treat seizures and epilepsy
BKC on the nasal mucosa in vivo [213]. For example, in one in emergency situations besides other indications. Major
study, 10 µl of nasal steroid formulations was administered observed systemic side effects include not only sedation,
twice daily to rats for 21 days, either with or without BKC drowsiness, sleepiness or amnesia, but also respiratory
(310 or 220 µg/ml). In the nasal cavities of rats receiving a depression is a potential side effect [219]. However, clinical
formulation containing BKC, a range of alterations including and pre-hospital studies are supporting the view that IN
reduced epithelial cell high, pleomorphism of individual benzodiazepines are as safe or safer than oral, rectal or
epithelial cells, reduced number of cilia and goblet cells intravenous administration [220, 221]. Many systemic
associated with a loss of mucus covering the epithelial cell side effects and effects on the CNS are the result from the
layer was observed [214]. Further, in in vitro studies, the ability of the substance to reach the blood circulation and
toxic and CBF inhibitory effects on human nasal mucosa traverse the BBB. In conventional epilepsy treatments, drug
were observed as well [207, 215]. Nevertheless, the safety resistance to anti-epileptic drugs can occur when drug does
concern about BKC remains controversial, as there are studies not pass the BBB sufficiently, as a result from improper
reporting no toxic effect of BKC in vivo. However, the use of dosing and wrong drug choice [222]. These complications,

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748 Drug Delivery and Translational Research (2022) 12:735–757

as well as drug-associated toxicities, can be overcome by delivery of benzodiazepines such as midazolam and diazepam
suitable drug delivery systems. Nanotechnology-based is safer with regard to respiratory depression compared with
systems are a rising technology for improving ntb delivery. oral or intravenous administration. It is judged to be as safe
They facilitate a more targeted and efficient brain delivery as rectal administration [219, 233, 234]. Another example
and reduce side effects at the same time [223]. The advantage is the neurohormone oxytocin, which is used intravenously
of using nanotechnology-based drug delivery systems has for labour induction, for abortions or for the control of post-
been shown for different CNS indications, such as epilepsy, partum bleeding. Even though oxytocin is described as a
psychosis-related disorders and glioma [152, 223–227]. relatively safe medication, rare cases of treatment-induced
T h e n a s a l ly a d m i n i st e r e d s y m p a t h o m i m et i c severe adverse and life-threatening side effects such as
oxymetazoline is used not only as a topical treatment for pulmonary hypertension and pulmonary oedema were
rhinitis but also as an anaesthetic and for the treatment reported [235–238]. Intranasally delivered oxytocin is under
of epistaxis. Oxymetazoline is a potent peripheral alpha investigation to treat psychiatric disorders. There are a number
adrenergic 1 and 2 agonist, but when it reaches the of IN oxytocin studies in humans; however, adverse events
systemic blood circulation, it can also stimulate central are not described according to a standardised scheme and are
alpha 2 adrenoreceptors. Hence, adverse systemic therefore inconsistently reported [161–163, 239, 240]. Since
effects include, amongst others, vasoconstriction and IN delivery is not a popular administration route, studies
sympathetic effects such as fast, irregular or pounding comparing effects on safety of IN vs IV administration of
heartbeat, headache, dizziness, drowsiness, high blood oxytocin are rare. Literature reviews that report safety data
pressure, nervousness and trembling [197, 228, 229]. of IN oxytocin administration have only mentioned mild
These effects can cause hypertension, tachycardia and respiratory effects such as asthma attacks [241, 242].
peripheral vasoconstriction. However, the adverse side Pulmonary effects can be an important reason for a
effect profile of intranasal oxymetazoline is not unique drug formulation to get delayed market approval or even
and aligned with the side effects of sympathomimetics in withdrawn after approval. This was a lesson learned for the
general, regardless of the administration route. Further, field of pulmonary drug delivery. Exubera® was the first
those side effects are in particular relevant in paediatric approved formulation of inhalable insulin from Pfizer Labs
medicine and for patients with underlying medical (New York, NY), which reached the US market in 2006.
conditions [228–232]. It was used to treat type 1 and 2 diabetes in non-smokers
Intranasally administered drugs and formulations show without pulmonary diseases [243]. Apart from economic
an overall better systemic tolerability compared with other reasons for withdrawing Exbuera®, it showed pulmonary
administration routes such as intravenous or oral application. toxicity issues [244]. During clinical use, symptoms
This is mainly due to bypassing of the metabolic fist-pass such as non-progressive dry cough were observed, and
effect. Further, systemic adverse effects observed in the pulmonary function test became necessary, as pulmonary
clinic, such as drug resistance to anti-epileptic drugs, function parameters decreased during long-term use [243,
depend on the properties of the drug itself, wrong handling 245]. Further, there were some cases of previous smokers
or overdosing but not on the administration route [222]. treated with Exbuera® who developed lung cancer. Insulin
is a growth factor, and inhalation of insulin could lead to a
secondary activation of pro-proliferative insulin-like growth
Pulmonary effects factor (IGF-1) pathway [246]. There were, however, too
few cases to determine whether these lung cancer cases
Drug or substance-induced respiratory and pulmonary were associated with the inhaled insulin formulation [243].
problems are intensively described in clinical and histological Since, 2014, the company Mannkind Cooperation received
observations. They range from mild effects such as coughing the US market approval for Afrezza, an inhaled insulin with
or breathing problems during sleep to severe effects such improved PK/PD properties, but the pulmonary toxicity
as pulmonary toxicity, infections, pneumonia and acidosis. issue could not be ruled out after all [247].
Over 1300 substances and drugs are listed to affect the It is important to consider that not only the drug itself
respiratory tract (www.pneum otox.com). In this review but also other components of a drug formulation can result
article, we will only focus on drugs inducing adverse in adverse pulmonary and respiratory effects. In a dose- and
respiratory effects after IN administration. As described concentration-dependent manner, benzalkonium chloride
above, benzodiazepines are applied intranasally to treat showed that its target organ is the lung. It induces lung
emergency seizure events in paediatric populations. One irritation, inflammation and alveolar damage after inhalation
known adverse side effect is treatment-induced respiratory and can lead to pulmonary oedema and pneumonia after
depression. This side effect is, however, independent of the oral or intravenous administration in rats [248]. Indeed,
administration route. In fact, collected data suggests that IN benzalkonium chloride used as a preservative in nasal sprays

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Drug Delivery and Translational Research (2022) 12:735–757 749

in low concentrations of 0.007–0.01% is considered to be the guidance document includes histological assessments
safe regarding pulmonary effects [249]. of local tissue (lung, nasal mucosa, bronchi) and potentially
affected brain areas [254].
Salminen, Wiles and Stevens summarised in a review the
Regulatory considerations nonclinical requirements for 505(b)(2) NDAs; however, they
conclude that the nonclinical requirements are highly drug
Obtaining regulatory approval of a candidate to market product–dependent [255]. For example, the question whether
is the end of a long drug development procedure, but additional studies are needed for efficacy evaluation depends
regulatory aspects should be aware from the beginning. To on the product’s use. For OINDPs for systemic use, it may
get approval of any new drug product in the USA requires be possible to show efficacy of the respiratory route via
safety, efficacy and quality considerations. This information bioequivalence pharmacokinetics and relative bioavailability
will be submitted as a new drug application (NDA) to the studies. For local use however, the efficacy cannot be derived
FDA. However, at the moment, orally inhaled and/or nasal from previous studies of different purpose [253].
drug products (OINDP) are most frequently discussed
for repurposing of an already approved product, as it was Clinical considerations and human factors
previously described for several examples like inhaled
insulin (Exbuera®). Therefore, this section focuses on In the clinical area, the safety information from systemic
special safety, efficacy and quality considerations for the exposure can be reused for already approved active
respiratory delivery regarding requirements of the US Food pharmaceutical ingredients (APIs) and for excipients
and Drug Administration (FDA). Further, biologics are (inactive ingredients), but not if the drug is intended to be
not separately mentioned as those considerations apply to used for local exposure. Then, new safety information have
biologicals as well as to small-molecule drug products. to be generated; this is also true for formulations which use
There are three different regulatory pathways to get a new non-approved excipients [253]. Typically, Phase III clinical
drug product approved by the FDA, 505 (b)(1), 505(b)(2) for trials are required for a 505(b)(2) application as the efficacy
NDAs and 505(j) abbreviated NDAs (ANDAs). Repurposed of the nasal administration route has to be demonstrated.
drugs using a new administration route like respiratory One of the biggest hurdles for nasal drug delivery devices
delivery can be qualified for the regulatory procedure is the interaction interface of the patient with the device.
505(b)(2). This can be beneficial, since some safety and This can influence the performance and efficacy and can
efficacy data could be used from previously approved drugs lead to ineffective or unsafe use. Hence, the FDA is working
[250–252]. Even though preclinical studies and systemic on guidelines for human factors engineering (HFE) and
safety could be justified by data of previous studies of usability engineering (UE) [256–258].
approved drugs, there are still additional information
required from new preclinical and clinical studies [253]. CMC considerations

Preclinical considerations In the area of Chemistry, Manufacturing and Controls

(CMC), there is a substantial package of information
The FDA published a guidance document summarizing required, containing a quality control (QC) program, as
preclinical studies, which could be required for a well as ‘product characterisation’ tests. The QC program
reformulated drug product and alternate administration for OINDPs includes for example recommendations to
routes. In general, for all drug product reformulations and test delivered dose uniformity, aerodynamic particle size
for all drug products with new routes of administration, the distribution and extractables of nasal sprays, metered
recommendations outlined in ICH M3 (R2) and ICH S9 must dose inhalers (MDI) and dry powder inhalers (DPI)
be followed. The study design requirements for preclinical [259, 260]. The recommendation is that the applicant
studies for IN delivery should be similar to inhalation should develop a list of critical quality attributes early
studies for new formulations. These studies include a short- in the device development. However, the CQAs, which
term study (2–4 weeks) in two species (at least one non- are physical, chemical, biological or microbiological
rodent) and then followed by a chronic study with up to 6 properties or characteristics, should be in line with the
months in the most appropriate species. If in the chronic desired product quality outlined in ICHQ8(R2) [260].
inhalation study no toxicity resulting in proliferative or The CMC area is for OINDPs a special challenge as
preneoplastic changes is observed and when adequate local the device performance is not fixed, also for approved
airway exposure by the oral route in previous carcinogenicity nasal delivery devices. The performance depends on
studies was reached, then no additional carcinogenicity the formulation and the patient’s interaction and has to
study is required for the inhalation route. For local safety, therefore be critically evaluated.

13
750 Drug Delivery and Translational Research (2022) 12:735–757

Conclusion Author contributions This review was written through contribution

of all authors. All authors have given approval to the final version of
the manuscript.
To date, IN drug formulations are mainly approved
and used for local and systemic indications, not only Funding Open Access funding enabled and organized by Projekt
for seasonal rhinitis and pain management but also DEAL. AbbVie sponsored and funded the study; contributed to the
for emergency situations such as epileptic seizures in design; participated in the collection, analysis and interpretation of
data and in writing, reviewing and approval of the final publication.
paediatric population. The interest in INDD is however
growing for CNS indications as in neurodegenerative
diseases and other types of CNS disorders, because Compliance with ethical standards
the BBB is limiting the bioavailability of drugs after
Competing interests AbbVie sponsored and funded the study;
systemic delivery. Furthermore, vaccine development will contributed to the design; participated in the collection, analysis,
increasingly include nasal delivery, as the current COVID- and interpretation of data, and in writing, reviewing, and approval
19 pandemic, resulting in pulmonary disorders, highlights. of the final publication. Lea-Andriana Keller and Andreas Popp are
employees of AbbVie and may own AbbVie Stock. Olivia Merkel is
Apart from the advantages of the non-invasive use and the
a Professor at the Ludwig-Maximilians-University and Lea-Adriana
possibility of more targeted dosing to reduce systemic Keller’s doctoral adviser.
side effects, there are still limitations and toxicological
considerations regarding formulation aspects. There is a Ethical approval and consent to participate No animal or human stud-
ies were carried out by the authors for this article.
need for more basic research to understand the anatomy
and physiology, but also pathologic mechanisms and
their influence on each other. Elucidating the pathways Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
and distribution routes that individual compounds take
tion, distribution and reproduction in any medium or format, as long
after IN administration is crucial to develop suitable as you give appropriate credit to the original author(s) and the source,
drug delivery systems for IN approaches. There are more provide a link to the Creative Commons licence, and indicate if changes
experimental approaches and studies needed, combining were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
imaging methods in vivo (live and retrospective), with
otherwise in a credit line to the material. If material is not included in
quantitative analysis of compound levels in the respective the article’s Creative Commons licence and your intended use is not
tissue or target organ. The influence of the formulation permitted by statutory regulation or exceeds the permitted use, you will
composition on the drug distribution routes and pathways need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
has to be further studied. Formulation properties and nasal
device technologies will be in focus for the optimisation for
targeted delivery regarding how to favour for example ntb
delivery or pulmonary delivery over systemic absorption. References
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