Chemical modification of drug molecules as strategy to reduce interactions with mucus

    Chemical modification of drug molecules as strategy to reduce interactions with mucus Francisca Ara´ujo, Cl´audia Martins, Cl´audia Azevedo, Bruno Sarmento PII: DOI: Reference:

S0169-409X(17)30201-6 doi:10.1016/j.addr.2017.09.020 ADR 13190

To appear in:

Advanced Drug Delivery Reviews

Received date: Revised date: Accepted date:

26 July 2017 9 September 2017 25 September 2017

Please cite this article as: Francisca Ara´ ujo, Cl´ audia Martins, Cl´ audia Azevedo, Bruno Sarmento, Chemical modification of drug molecules as strategy to reduce interactions with mucus, Advanced Drug Delivery Reviews (2017), doi:10.1016/j.addr.2017.09.020

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ACCEPTED MANUSCRIPT Chemical modification of drug molecules as strategy to reduce interactions with mucus

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Francisca Araújo1,2, Cláudia Martins1,2*, Cláudia Azevedo1,2, 3*, Bruno Sarmento1,2,4

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*Equal contribution

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1- INEB - Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-393 Porto, Portugal

2 - i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo

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Allen 208, 4200-393 Porto, Portugal

3 - ICBAS - Instituto Ciências Biomédicas Abel Salazar, Universidade do Porto, R. Jorge de

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Viterbo Ferreira 228, 4150-180 Porto, Portugal

4 - CESPU - Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da

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Saúde, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal

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*Corresponding author: Francisca Araújo, i3S, Instituto de Investigação e Inovação em Saúde, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal, [email protected],

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Phone: +351 220408800

ACCEPTED MANUSCRIPT Abstract Many drug molecules possess inadequate physical-chemical characteristics that prevent to

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surpass the viscous mucus layer present in the surface of mucosal tissues. Due to mucus

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protective role and its fast turnover, these drug molecules end up being removed from the

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body before being absorbed and, thus, before exerting any physiologic affect. Envisaging a better pharmacokinetics profile, chemical modifications, to render drug a more

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mucopenetrating character, have been introduced to drug molecules backbone towards more effective therapies. Mucus penetration increases when drug molecules are provided with net-

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neutral charge, when they are conjugated with mucolytic agents and through modifications that makes them resistant to enzymes present in mucus, with the overall increase of their

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hydrophilicity and the decrease of their molecular weight. All of these characteristics act as a

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whole and influence each other so they must be well thought when drug molecules are being

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designed for mucosal delivery.

Keywords: mucus; mucopenetration; drug delivery; chemical modification; charge; mucolytic

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agents; chemical stability, hydrophilicity, molecular weight

ACCEPTED MANUSCRIPT 1. Introduction Mucosal tissues are naturally found in body cavities, in many regions including gastrointestinal

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(GI) and respiratory tract, eyes and reproductive organs. Due to their extension and easy

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accessibility, mucosal drug delivery is highly pursued offering several advantages in

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comparison with other invasive delivery routes. Mucosal delivery may promote systemic drug absorption, but it is special desired to target local disorders. This makes possible to reduce drugs systemic effect, thereby, diminish possible side effects, and, also, increase drugs

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distribution at the target place [1].

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Nevertheless, mucosal tissues are covered by a viscous hydrophilic layer – the mucus layer, which acts as a barrier and highly impairs drugs absorption. The presence of this mucus turns

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drug delivery through mucosal tissues challenging [2]. Most of drug molecules do not possess

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adequate characteristics that enable them to pass through mucus, getting stuck into its mesh, and eventually being removed from the body. In order to improve pharmacokinetics and

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facilitate localized delivery to mucosal tissues, chemical modifications have been introduced to drug molecules towards more effective therapies. These modifications aim to render drugs a

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more mucopenetrating profile, i.e. changing drugs so that they have proper features to penetrate mucus, with minimal adhesion to it, so they can reach epithelial cells and improve their absorption. These modifications must be well thought out during drug molecules design to prevent the loss of their biological activity. In this review, the main strategies to modify drug molecules, in order to overcome their interactions with mucus and thus increase their bioavailability and efficacy, will be discussed.

2. Mucus Mucus is an adherent sheet of a highly hydrated fluid, viscoelastic and adhesive, that covers all the mucosal tissues [3]. The majority of mucus composition is water (90% or more) and mucin glycoproteins (1-5%) although other components such as electrolytes, cells or cell debris,

ACCEPTED MANUSCRIPT lipids, soluble proteins, enzymes and various immune factors are also present [4, 5]. These constituents are interdependent and if the balance between them does not exist, it gives rise

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to adverse effects on the physical properties of mucus which can seriously contribute to

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disease conditions [6].

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Mucus acts mainly as a physical barrier against chemical and biological insult from the external environment, protecting primarily the layer of the underlying tissue, the epithelium. Its protection occurs through natural lubrication of mucosal surfaces, opposing shear damaging,

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and trapping and removing foreign particulates [7, 8]. Due to its important protection role, mucus is continuously secreted and spread throughout the entire mucosa. Mucus blanket is

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determined by the balance between the rate of secretion and the rate of degradation and shedding, in a balance between the protective capability and absorption rate [9, 10]. This

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balance needs to be closely regulated to ensure an efficient clearance with adhesive properties

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despite shearing forces due to swallowing, peristalsis, blinking, and copulation [6]. Most of the

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mucus is secreted into the GI and respiratory tracts, being later digested and re-cycled. The remaining is shed in feces, sputum, saliva, and nasal secretions, reproductive tract secretions and tears [10].

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Mucus is organized into mucin fibers that are crosslinked, bundled, and entangled with each other and have a diameter of 3–10 nm [11]. They are secreted by both goblet cells and the seromucinous glands of the lamina propria at the apical epithelium [5]. Mucin glycoproteins have a large molecular weight, ranging from 0.2 to 3 million Dalton [4] and are classified into secreted mucins (up to several µm long), which contribute to the viscosity of the mucus, and membrane-associated mucins (100–500 nm in length), that serve for cell adhesion, pathogen binding, and signal transduction [8, 12, 13]. These two types of mucins are especially important in the gastrointestinal and cervicovaginal tracts due to their excellent lubrication properties [6].

ACCEPTED MANUSCRIPT Mucins consist of a linear protein backbone, with repeated hydrophilic structures of 8-169 amino acids rich in proline, serine and threonine, also known as PTS domains [13]. To these

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specific PTS domains, carbohydrate side-chains are linked mainly by O-glycosylation. The non-

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PTS regions are hydrophobic domains that have cysteine residues and allow for hydrophobic

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interactions [14, 15]. When in aqueous media, each mucin monomer may be linked by cysteine bridges to several other monomers, forming a randomly organized mucin fiber mesh, with variable porosity of diameters between 50 and 1800 nm (Figure 1) [6, 15, 16]. Due to free

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carboxylate and sulfonate groups found in the glycosylated PTS domains, as well as the high

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hydrogen bonding interactions [17].

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sialic acid content, mucus exhibits an overall negative charge capable of electrostatic and

Figure 1. Architecture of the mucus network. Mucin fibers form a network of about 50–1800 nm mesh size. The fibers consist of proline, serine, and threonine-rich regions (PTS domains), covered with carbohydrates, and linked by cysteine-rich domains. Reprinted with permission from [8].

ACCEPTED MANUSCRIPT Mucus enables the exchange of nutrients, water, gases, odorants, hormones and gametes. However, due to its mesh-like structure, it creates a steric and potentially highly adhesive

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barrier to the transport of drugs to the underlying mucosal surface or, additionally, impair the

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distribution of drugs through all the mucosa [18-20]. Drug molecules that are prevented to

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penetrate mucus, may adhere to mucus instead, through non-covalent interactions that include electrostatic (e.g. ionic interactions and hydrogen bonding) and hydrophobic interactions. When this happens, drugs will be rapidly trapped and removed due to mucus fast

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turnover and clearance, usually measured from minutes to few hours [18, 19]. Besides the physical barrier, mucus also plays important homeostatic functions, namely in

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regulating water balance, ion transport and acting as a buffer barrier to pH oscillations [8, 21]. According to different anatomic parts of the body (Figure 2) and the physiophathological

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conditions, those factors change, highly influencing and significantly varying mucus rheology

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and properties. Understanding mucus characteristics, such as composition, thickness, viscosity

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and turnover, and how its properties change in different mucosal surfaces or in different conditions (e.g., higher viscosity/lower clearance of sputum in cystic fibrosis (CF) patients, cervicovaginal dryness in menopausal women), is essential to design drug molecules that can

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efficiently cross the mucus layer and reach epithelial cells to be absorbed [3]. Penetrating deep into the mucus barrier, without compromising its protective properties, can lead to improved prophylactic and therapeutic treatments through mucosal routes [2].

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Figure 2. Composition of barriers of (A) eye, (B) bronchial tract, (C) gastrointestinal tract and (D) vagina. (A) The conjunctival barrier consists of a stratified columnar epithelium and the

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tear film composed of lipid layer (LL), an aqueous mucin layer with proteins (AL) and the mucin layer linked to the glycocalyx of the corneal epithelium cells (ML). (B) A simple columnar

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epithelium is present in the upper respiratory tract with mucus composed of a periciliary sol

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layer (SL) with low viscosity, where the kinocilia of the respiratory cells beat, and a gel layer (GL) with higher viscosity. (C) The gastrointestinal barrier consists of a simple columnar

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epithelium and a mucus layer with two strata. Microvilli (MV) at the apical surface of the enterocytes are covered by a glycoprotein layer, termed glycocalyx. This layer leads to a firmly

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attached stratified mucus layer (FML) and a loosely attached mucus layer (LML). (D) In the vagina, a squamous non-keratinized epithelium is covered by a mucus layer organized in two strata: a firmly attached mucus layer (FML), and a loosely attached one (LML). All epithelia reside on a basal lamina (BM). Adapted from [8].

3- Strategies to reduce the interaction between drug molecules and mucus Similarly to what happens to foreign particulates and bacteria or other pathogens, after drug molecules being administered through mucosae, are trapped in the mucus and washed away, unless they have certain features. By changing characteristics of these drug molecules, in order to reduce their interactions with mucus and thus, decrease their mucoadhesion, it is possible

ACCEPTED MANUSCRIPT to render to these molecules properties that help them to penetrate mucus and reach epithelial cells before being removed. To penetrate mucus, drug molecules must be small

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enough to avoid steric obstruction and have a hydrophilic, net-neutral surface to avoid

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adhesion [2]. In addition, to reach the mucopenetrating profile of drugs, the introduction of

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strategies to improve their resistance to enzymes located in the mucus layer, or their conjugation with agents capable of disrupting the mesh-like structure of mucus, could be considered a plus. In the following sections, drugs-related parameters such as molecular

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weight, hydrophilicity, charge, conjugation with mucolytic agents and chemical stability, will be addressed. All of these characteristics act together, contributing as a whole to the

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mucopenetration behavior of drug molecules.

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3.1. Molecular weight

It was previously described that mucins entangle with each other, forming a mucin fiber mesh

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with variable porosity [6, 15]. Thus, drug molecules should have adequate molecular weight (MW) that allows them to pass through the mucin fiber mesh without interacting with mucus.

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MW is therefore a parameter strongly related with the way drug molecules interact with biological matrices, such as mucus, defining their absorption profiles. However, the most common drug molecules can cross the mucus layer without hindrance since its mesh size is at least 100 times higher than these drugs [22]. Typically, these are small uncharged molecules, with size below 10 nm [8]. At this length scale macromolecules such as proteins (<10 nm), have negligible affinity to mucus constituents due to resistance to their Brownian diffusion similar to water [23, 24]. Likewise, when length scales increased to the dimensions of small capsid viruses, their diffusion rates were also comparable with water. As example, Norwalk virus with 38 nm and human papilloma virus (HPV) with 55 nm were both observed to diffuse in human cervical mucus, as rapidly as they do in water [23], and Polio virus with 28 nm rapidly diffuse through intestinal mucus [25]. At length scales up to 55 nm, the microviscosity of virus

ACCEPTED MANUSCRIPT particles remains similar to the viscosity of water [2, 6]. When the size increases from 30–60 nm as in the case of antibodies, they can cross cervicovaginal mucus matrices only slowly

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hindered by low-affinity bonds with mucins [6, 24]. Molecules with size of 180 nm, such as

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herpes simplex viruses, are slowed, and for sizes above 200 nm, viscosity is greatly increased

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[2]. Thus, generally decreasing the size contributes to enhanced diffusion rates of drug molecules as they have less mucoadhesive properties and less points of interaction with mucus [26, 27] and can be further transported across smaller channels of the heterogeneous mesh

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formed by mucins [20] (Figure 3A).

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Figure 3. Represents permeation via the pores in the mucus gel. (A) Demonstrates small molecules can permeate whereas large molecules are retarded. (B) Interacting molecules are represented in purple, non-interacting molecules are in blue. The blue molecules pass through mucus if they are small enough whereas even small purple molecules are retarded due to interaction with the mucus. (C) Molecules with surface mucolytic agents can enlarge the pores of mucus and consequently permeate the mucus barrier. Molecules with red surface and nobs are mucolytic particles. Adapted from [4].

ACCEPTED MANUSCRIPT 3.2. Hydrophilicity Mucus is mainly composed by water and thus, it possesses mainly a hydrophilic character. Due

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to this feature, drug molecules with hydrophobic nature tend to bind to mucus through their

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hydrophobic residues [28]. Thus, hydrophilic-lipophilic balance is an essential parameter to

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overcome certain barriers in the body. As an example, Christopher Lipinski noticed that oral bioavailability is poor when the log P value of drugs is higher than 5 [29]. For a better understanding of the chemical nature of mucus, Figure 4 shows the hydrophilic (glycosylated

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regions of mucins) and the hydrophobic domains (non-glycosylated protein) as well as some

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interaction points with drugs.

Figure 4: Illustration of the glycosylated and non-glycosylated domains structure of the mucus. When the two domains are linked a monomer is formed, which linked with S-S bond form a

ACCEPTED MANUSCRIPT dimer. In large scale it is possible to obtain a mucin gel through hydrophobic association.

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Adapted from [17, 30].

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By changing molecules character to a more hydrophilic one, they are not able to form such

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strong adhesive bonds with mucus and therefore their interactions are diminished and molecules are free to penetrate through the mucus layer (Figure 3B). Thus, hydrophilicity is an important characteristic for drugs to behave as mucopenetrating molecules.

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It is known that different amino acids present different hydrophilic-lipophilic balance. When drug molecules are peptides or proteins, an approach that can be adopted is to use site-

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directed mutagenesis to make lipophilic to hydrophilic mutations on the protein surface [31]. However, the hydrophobic residues that are usually targeted for replacement are not often

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found on the protein surface. Moreover, it needs to be stressed that every variation may

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of proteins activity.

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modify proteins activity and the benefit of modifications should be evaluated to the detriment

Nevertheless, mucosal delivery is not only greatly limited by the mucus layer but also by epithelial cells. This worsens even more the mucosal absorption of drugs since to overcome

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both the mucus and epithelium barriers, opposite surface properties of drug molecules are required. Hydrophilic properties are necessary for mucus permeation, while hydrophobic ones are preferable for epithelium internalization.

3.3 – Charge One of the desired characteristics to obtain drugs with a mucopenetrating profile is an overall net-neutral charge [32]. It is well known that charged molecules are able to interact with the negatively-charged sialic acid residues of mucins [33]. In particular, positively charged drug molecules will bind to mucus and negatively charged ones will be repulsed. Desai et al. tested the diffusion of compounds with different charges in a model of porcine mucus. They found

ACCEPTED MANUSCRIPT that

the

positively

charged

compounds

(NAD,

5-hydroxy-L-tryptophan

and

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hydroxytryptamin) presented higher retarded diffusion across the mucus layer in comparison

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to the non-charged compounds (phloroglucinol, phenolphthalein diphosphate and

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charged compounds with the negatively charged mucus.

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cyanocobalamine) [34]. This retardation was attributed to the interaction of the positively

The charge of drug molecules is highly dependent on both the pH of local mucus and drug pKa (Figure 5) [35]. These two properties are able to induce oscillations in the degree of ionization

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of drugs, hence tuning their charge, and consequently increasing or decreasing the penetration of drug molecules through mucus [36]. For example, acidic drugs with low pKa values are more

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prone to be protonated and neutrally-charged at low mucus pH values, while basic drugs with high pKa values are more prone to be deprotonated and neutrally-charged at high mucus pH

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values.

Figure 5. Representation of the influence of local mucus pH and drug pKa on the ionization degree, and consequently the charge, of drugs. (A) pH values of different types of mucus. Adapted from [3]. (B) An overall net-neutral charge favors the permeation of drug molecules through mucus, since it avoids their interaction with the negatively-charged sialic acid residues

ACCEPTED MANUSCRIPT of mucins in the mucus layer. The charge of drugs is highly dependent on their ionization

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degree, which is regulated by both the pH of local mucus and drug pKa. Adapted from [37].

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In order to achieve a neutral charge, drugs can be modified with neutrally-charged ligands

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which are strong enough to affect the overall charge of the drug, or charged ligands that can balance the overall charge of the complex resulting in a near-neutral molecule. The neutral-charged polysaccharide dextran is known to be very well compacted due to the

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existence of branching and specific glycosidic linkages in its structure. A branched structure is a desirable feature since it provides a more accurate masking of drugs surfaces, the so-called

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“umbrella-like effect” [38]. Besides the favorable neutral charge, dextran is also not available to interact with mucins due to physical reasons, since it is not able to contract in response to

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ionic strength variations [39, 40]. Another polysaccharide, the Streptococcus thermophilus

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the requirements [40].

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exopolysaccharide, is also neutrally-charged and does not interact with mucins, hence fulfilling

N-(2-hydroxypropyl) methacrylamide copolymer (HPMA) is a linear hydrophilic polymer with a neutral charge, biodegradable and non-immunogenic [41, 42]. Tao et al. synthesized

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polyHPMA terminated with a functional group consisting on thiazolidine-2-thione which may establish linkages with amine residues of protein drugs by covalent amide bonding [43]. In other study, the same authors used reversible addition-fragmentation chain transfer (RAFT) polymerization to produce a thio-reactive HPMA with a midchain functionality. This polymer may be conjugated with protein drugs by interacting with free cysteine residues of the therapeutic molecules [44]. Recent studies on the nanotechnology field reported the use of HPMA polymeric coatings as a way to diminish the adhesive interactions of substances with mucus in order to attain a fast mucus penetration [45, 46]. This concept corroborates the benefits of a possible chemical modification of drugs with HPMA, known to be easily tunable in relation to its physiochemical properties by manipulating the monomers [46]. This may be a

ACCEPTED MANUSCRIPT good option to obtain HPMA-derived charged ligands that can provide an overall neutral charge to the drug.

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Due to the key role of the neutral charge of drugs avoiding of their interaction with mucus,

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there are already numerous patents which emphasize this issue. These patents state charge

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modifying methods as a way to provide an unionized or neutral charge profile to therapeutic compounds as interferon beta [47], parathyroid hormone peptide (PTH) or PTH analogs [48], and glucose-regulating peptides [49], in order to improve their transmucosal delivery.

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Other strategies found in the literature could be explored to provide a neutral charge to drugs in order to facilitate their penetration through the mucus layer [50, 51]. Wang et al.

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conjugated the zwitterionic polymer poly(carboxybetaine methacrylate) (PCBMA) with doxorubicin. Although the zwitterionic polymer presented a net-neutral charge due to the

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equal number of anions and cations, the prominent positive charge of the drug could change

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the overall charge of the complex. Thus, the authors introduced a negatively-charged group in

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the polymer, to neutralize the positive charge associated with the drug [50, 52]. In another work, Herr et al. chemically modified an angiotensin II receptor antagonist drug, the BMS183920, by masking its acidic tetrazole N–H bond, which means that it was protected by a

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chemical group capable of being removed in physiological conditions. This resulted on a neutrally-charged pro-drug, with a proved increase in its transport ability across biomembranes [51]. Chemical modifications of drugs with most of the above mentioned conjugates are not only associated with charge alterations, but also with improvements on the stability, resistance to enzymatic degradation and water solubility of the molecules [52, 53]. However, this approach must be well designed to keep the efficacy of drug molecules unchanged. Still, and despite the increasing acceptance of the paradigm, there is an obvious lack of information regarding the modified-neutral charge of drugs as a way to reduce their interactions with mucus.

ACCEPTED MANUSCRIPT 3.4 – Conjugation with mucolytic agents The dense network of mucus may lead to trapping phenomena of drug molecules. A possible

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strategy to avoid this type of interactions is their conjugation with mucolytic or mucus-clearing

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that allows them to reach the epithelial membrane (Figure 3C).

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agents, in order to disrupt the mucus layer and render drugs with a mucopenetrating profile

Mucolytic agents affect the three-dimensional mesh structure of mucus by reducing its viscosity [54]. Based on their mechanism of action, the most studied mucolytic agents can be

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divided into three main classes: proteases, which act on mucin glycoproteins by cleaving their amino acid sequences; disulfide-reducing agents, which act on mucoproteins by converting

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their disulfide bonds (S-S) into sulfhydryl bonds (-SH) that are no longer available to participate in cross-links; and detergents, which affect the internal non-covalent linkages of mucus [55,

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56].

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Papain is known to cleave mucoglycoprotein substructures in the mucus layer, hence allowing

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its proteolytic digestion [57, 58]. This enzyme is a naturally occurring endolytic cysteine protease in Carica papaya [57, 59]. Papain is one of the components of the pharmaceutical product Clear-ease® due to the ability of this enzyme to thin mucus secretions, which are

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usually associated with inflammatory scenarios [60]. Grabovac et al. tested an orally administered formulation of low molecular weight heparin conjugated with papain [61]. It was suggested that the linkage between the negatively-charged heparin and the positively-charged papain occurred through an electrostatic interaction. Moreover, other authors demonstrated that the α-helix content of papain significantly increases in the presence of heparin, and this is related with a consequent increase in the affinity of the enzyme to the substrate [62]. In vivo studies in rats showed a remarkable increase in the absorption rate of heparin when combined with papain, in comparison to the administration of the free drug [61]. Pronase, another enzymatic mucolytic agent, consists on a protease which is able to disrupt the mucin matrix and is purified from the extracellular fluid of Streptomyces griseus [63].

ACCEPTED MANUSCRIPT Orally-administered pronase is commonly used as a premedication for endoscopy, since it is known to remove the gastric mucus [64]. Liu et al. investigated pronase-enhanced antibiotics

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on the eradication of H. pylori [65]. Since this bacteria is located in the deep gastric mucus

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layer or on epithelial cell surfaces, the antibiotics should have a mucopenetrating capacity in

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order to reach these sites. The removal of the mucus layer by pronase enabled the reduction of antibiotics dosage required to achieve considerable concentrations of drug in the deep gastric mucous layer, in comparison to the administration of a formulation containing only

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antibiotics. In the same context, Gotoh et al. demonstrated that the combination of pronase with a triple therapy of lansoprazole, amoxicillin and metronidazole used against H. pylori

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improved the cure rate by more than 15% [66].

Trypsin and bromelain have also been conjugated with drugs to achieve the same effect. Both

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were found to significantly increase the permeability of low molecular size drug-like molecules

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(low-molecular size marker fluorescein) in the small intestine, and bromelain caused this effect

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for high molecular size drug-like molecules (high-molecular size marker FITC-dextran) as well [67]. Clinical experience already stated bromelain, a group of sulfur-containing proteolytic enzymes mostly found in the pineapple stem [68], as a mixture capable of solubilizing mucus

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before the acquisition of X-ray images of the uterus, and in the treatment of sinusitis and asthma [69, 70].

In a nanotechnology-based approach, Samaridou et al. used the mucolytic enzymes trypsin, papain and bromelain to functionalize nanoparticles (NPs) surfaces, and they concluded that papain- and bromelain-functionalized NPs exhibited the highest permeation rate in mucus. This work could support new enzymatic-based chemical modification approaches to reduce the interactions of drugs with mucus [71]. The disulfide-reducing agent N-acetylcysteine (NAC) is already known to be enrolled in the treatment of pathologies like CF and chronic obstructive pulmonary disease (COPD), providing the liquification of accumulated mucus [72]. This molecule is associated with low toxicity and

ACCEPTED MANUSCRIPT no local irritation as documented by years of clinical experience [73]. In the field of GI tract, Bernkop-Schnürch et al. proved the efficacy of the conjugation of NAC with perorally

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administered protein drugs on the permeability of native mucus from porcine small intestine

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[74]. Using 2% of NAC (w/v), the mucus penetration of large sized drugs (66 kDa) and small

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sized drugs (6.5 kDa) significantly increased by 50% and 300%, respectively. In the nasal mucosa, Matsuyama et al. demonstrated that the conjugation of intranasal administered salmon calcitonin with NAC provided an increase in the drug bioavailability from 7% to 16%

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[73]. The same authors also tested this combination in a powder-like formulation, and the results reinforced the increase in the salmon calcitonin bioavailability when conjugated with

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NAC [75]. This data indicates the importance of the reduction of the mucus layer by NAC in the nasal absorption of salmon calcitonin. Another example of a disulfide-reducing agent is the

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dithiothreitol, whose mucolysis ability in the ileum and colon mucus layer was successfully

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demonstrated in a study conducted by Ferry et al. [76]. However, the mucolytic potential of

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the dithiothreitol is usually associated with irritation phenomena, which is the reason to avoid the use of this agent in the clinical practice [77]. Saponins are natural detergents with proven mucolytic activity [78] consisting on triterpenoid

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glycosides derived from Quillaja saponaria [79]. Saponins detergent properties are due to both their water- and fat-soluble components [80]. Recchia et al. conjugated a semisynthetic saponin, DS-1, with two aminoglycoside antibiotics, gentamicin and tobramycin [81]. Then, an in vivo study in rats with nasally, ocularly, and rectally administered formulations was performed. They observed that DS-1 worked very well as a transmucosal delivery agent, since significant transport of the drugs across mucous membranes was only possible in the presence of this saponin. Bile salts are also reported to cause the depletion of mucus, hence facilitating the permeation of the mucosa [82]. Sodium taurodihydrofusidate (TDHF), a bile salt analogue, was studied as a possible conjugate to intranasally administered insulin, in a work conducted in vivo [83]. This bile salt-like compound demonstrated to cause a significant increase in the

ACCEPTED MANUSCRIPT intranasal absorption of the drug. Besides the evidences that detergent-based mucolytic agents can improve the mucopenetrating features of drugs, there is a lack of information

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regarding this conjugation yet.

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Other promising mucolytic agents that can be explored to this purpose are the glycosidases

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and deoxyribonucleases (DNAses). Glycosidases catalyze the hydrolysis of glycosidic bonds within mucus glycoproteins. Although some glycosidases such as α-amylase showed only a slightly mucus clearing capacity [84], bacteria-born glycosidases seem to have a highly

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mucolytic effect [85]. DNA is associated with an increase in mucus viscosity, being mainly released by disintegrating neutrophils during inflammatory responses, and this is a typical case

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in diseases such as CF [86]. In these situations, the mucolytic agent DNase could be a good choice. Yang et al. showed the benefits of conjugate DNase with the antibiotic ciprofloxacin in

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an antipseudomonal therapy [87]. Using an artificial sputum model, they verified that the

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mucolytic activity of DNase provided a higher penetration of ciprofloxacin into the mucus.

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Despite the great potential of mucolytic agents on the chemical modification of drugs to reduce their interactions with mucus, this strategy has to be carefully planned. Severe damages or complete degradation of the mucus are not acceptable because there is a need to

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preserve the protective function of the mucus and the integrity of the overall mucosa [88].

3.5 – Chemical stability The mucus layer contains a variety of enzymes which establish interactions with drug molecules in order to start their degradation process. In order to be absorbed in their active form, drug molecules must cross the mucus and avoid their degradation by the enzymes that are present in this layer. These enzymes are mainly proteases, but other digestive enzymes are also present in mucus [17, 22, 89]. Within the proteases group, exopeptidases, as carboxypeptidases and aminopeptidases, and endopeptidases, as cysteine, asparagine, and serine proteases, are the most known ones [90]. In the specific case of the nasal mucosa, the

ACCEPTED MANUSCRIPT monoxygenase system based on the cytochrome-P450 also plays a key role on enzymatic degradation processes [90, 91].

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Therefore, the enzymatic environment of mucus leads to a pre-systemic degradation of drugs

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and a consequent decrease in their diffusion though this layer. A possible strategy to reduce

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this kind of interaction between drugs and mucus is the conjugation of specific enzyme inhibitors with the therapeutic molecules. The inhibitor may use different mechanisms to hinder enzymatic activity, including changes in the conformation of the drugs to make them

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less attractive to the enzymes, and/or direct changes in the conformation of the enzymes to make them less capable of capturing drugs [92].

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Diethylene triamine pentaacetic acid (DTPA) is a synthetic polyamino-carboxylic acid and a chelating agent which can act on metal cations, as Ca2+ or Zn2+, through its amines and

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carboxylates [93, 94]. DTPA is known to be associated with the inhibition of proteases activity,

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since it is capable of removing essential metal ions from the structure of these enzymes [95].

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Su et al. studied the effect of the conjugation of insulin with this chelating agent in relation to the resistance of the therapeutic compound against degradation by proteolytic enzymes in the mucus layer of the GI tract, using freshly isolated proximal intestinal segments from rats

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(Figure 6) [96]. After 2.5 h, free-insulin was rapidly degraded by the enzymes (about 90% of degradation), and the complex insulin-DTPA presented only around 30% of degradation. This result emphasizes the protective role of DTPA on insulin in terms of enzymatic degradation. Still within the chelating agents field, dissodium EDTA also demonstrated to be effective in providing protection against enzymatic digestion to peptidic drugs [97].

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Figure 6. Analysis of the effect of DTPA on the enzymatic degradation of insulin. Adapted from

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[96].

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Amastatin and bestatin are inhibitors of a variety of aminopeptidases. Amastatin inhibits the aminopeptidase A and N, whereas bestatin inhibits the aminopeptidase B, N and leucine

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aminopeptidase [98-101]. O’hagan et al. demonstrated that the compound amastatin conjugated with the biosynthetic human growth hormone (hGH) enabled an enhancement on

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the nasal absorption of the drug [102]. However, data from the same study demonstrated that bestatin is not as efficient as amastatin to be conjugated with hGH for the above mentioned purpose. Alpha-aminoboronic acid derivatives are also known to be potent and reversible inhibitors of aminopeptidases [103]. Hussain et al. tested the effect of the conjugation of the alpha-aminoboronic acid derivatives boroleucine, borovaline, and boroalanine on the prevention of leucine-enkephalin degradation by nasal mucosa aminopeptidases [104]. They found that boroleucine is the most appropriated inhibitor of these enzymes, hence performing the most effective protective action on the drug. Moreover, they found that boroleucine is far more accurate in aminopeptidases inhibition than bestatin. In addition, aprotinin, a reversible serine protease inhibitor, was appointed by Yamamoto et al. as a compound able to hinder the

ACCEPTED MANUSCRIPT enzymatic digestion of insulin and proinsulin in homogenates of albino rabbit buccal mucosa [105].

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Bacitracin is an antibiotic synthesized by some species of bacilli and consists on a complex

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mixture of branched cyclic dodecylpeptides [106]. This antibiotic is able to inhibit the

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aminopeptidase N [107]. Raehs et al. conjugated bacitracin with luteinizing-hormone-releasing hormone (LH-RH) and an agonist of LH-RH, and tested the nasal absorption of the drugs [108]. They concluded that bacitracin is a potent enzyme inhibitor since they found a significant

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increase in the serum levels of both drugs in comparison to the administration of drugs without the antibiotic.

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Some enzyme inhibitors based on polymeric materials can also be used. The well-known PEGylation process is associated with the avoidance of enzymatic degradation in general, and

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thus it may be applied to protect drugs from mucus-related enzymes. After intranasal

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administration in type 2 diabetic mice, PEGylated GLP-1 demonstrated to have an increased

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half-life of at least 2.4-fold compared to the non-PEGylated GLP-1, resulting in a better hypoglycaemic effect of the drug [109]. Another well-known process, the palmitoylation, which consists on the covalent attachment of

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fatty acids moieties to proteins, may also be applied to protect drugs from enzymatic degradation in the mucus layer. Muranishi et al. stated that, besides increasing the lipophilicity of insulin, chemical modification of this drug by palmitoylation was related to an increase in its stability against enzymatic degradation in the intestinal mucosa, which includes the mucus layer [110]. There are other strategies that aroused interest in the possibility of reducing this type of interactions between drugs and mucus. For example, Merks et al. noted that cyclodextrins, namely the α- and β-cyclodextrin, decreased the metabolic polypeptide degradation related to insulin in the nasal mucosa, where is located the mucus layer, hence providing an increase on the nasal absorption of this drug [111]. Bile salts as sodium glycocholate and the sulfhydryl

ACCEPTED MANUSCRIPT complexing agent p-chloromercuri-phenylsulfonate (pCMPS) also proved to be useful to prevent the degradation of peptide and protein drugs [105]. Finally, the substitution of D-form

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for L-form aminoacids in the structure of the drugs may be a possibility to increase their

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resistance to proteases hydrolysis [112, 113]. This process must be well designed though, since

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the changes of the enantiomeric form of aminoacids in the structure of the drugs may also affect their hydrophilicity. Regarding the study conducted by Chen et al., who used an amphipathic α-helical peptide, the replacement of L-analogs with D-analogs in its structure led

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to the disruption of the hydrophobic face of the amphipathic α-helix and a consequent decrease in the hydrophobicity of the nonpolar face of this helix [114].

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The conjugation of drugs with enzyme inhibitors opened new doors to different interventions that could be applied to diminish the interactions of the therapeutic molecules with mucus.

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However, once again, this strategy has to be carefully planned. The enzyme inhibitors may be

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toxic due to the inhibition of the physiological digestive enzymes, and the resulting feedback

4. Conclusion

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can lead to an increased secretion of mucus-associated enzymes [101].

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The mucus layer that naturally covers mucosal tissues is vital for protection against foreign particulates or pathogens, but also for the normal regulation of several physiological processes. Nonetheless, mucus also highly impairs the absorption of drug molecules administered through mucosal routes, being a physical barrier to overcome in order to reach the absorptive cells of epithelium. The knowledge and understanding of mucus characteristics is important in the design of drug molecules able to penetrate mucus while maintaining its native barrier function. In this paper, several strategies of chemical modification of drug molecules, as a strategy to reduce interactions with mucus, were described, addressing charge, conjugation with mucolytic agents, hydrophilicity, stability and molecular weight features. By way of conclusion, mucus penetration increase when drug molecules have neutral charge, with

ACCEPTED MANUSCRIPT the increase of their hydrophilicity, with the decrease of their molecular weight, upon conjugation with mucolytic agents and through modifications that makes them stable in the

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presence of enzymes present in mucus. Nevertheless, all of these characteristics act together

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and influence each other so all of them should be considered while designing drug molecules

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for mucosal delivery.

Acknowledgments

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This article is a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020

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Partnership Agreement, through the European Regional Development Fund (ERDF). This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the

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COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI),

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Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/

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Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274) and through project PTDC/BBB-NAN/3249/2014. Francisca Araújo is receiving a scholarship from the project

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PTDC/BBB-NAN/3249/2014. Cláudia Azevedo would like to thank to FCT for funding the PhD scholarship (SFRH/BD/117598/2016).

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Graphical abstract