International Journal of Peptide Research and Therapeutics https://doi.org/10.1007/s10989-018-9704-y
Thiolated Polymers: Pharmaceutical Tool in Nasal Drug Delivery of Proteins and Peptides Ashish Jain1 · Pooja Hurkat1 · Anki Jain1 · Ankit Jain3 · Abhishek Jain2 · Sanjay K. Jain1 Accepted: 4 April 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract Although bioavailability of peptides administered through nasal route is still under 1% because of low membrane permeability, a short local residence time and a high metabolic turnover in the nasal epithelium but the richly supplied vascular nature of the nasal mucosa coupled with its high drug permeation makes the nasal route of administration attractive for many drugs including proteins and peptides. Thiolated polymers (thiomers) which are also recognized as mucoadhesive polymers, discovered so far, attach itself to mucus membrane by covalent and non-covalent binding such as electrostatic forces and physical mechanism. These new generation polymers are capable of forming covalent bonds. Thiomers are of two types firstly anionic thiolated polymers having carboxylic acid groups as anionic substructures and cationic thiomers are mainly based on chitosan. Thiomers are hydrophilic molecules exhibiting free thiol groups responsible for numerous qualities of well-established polymeric excipients such as poly(acrylic acid) and chitosan. Thiomers possess cohesive, mucoadhesive, enzyme inhibitory and permeation enhancing properties. Thiomers based microparticles, microspheres, nanoparticles and gels for nasal drug delivery systems have shown promosing potential. Thiomers matrix-tablets are found to be stable under all storage conditions. On the basis of various properties, thiomers represent a promising new generation of multifunctional polymer for protein delivery through nasal route. Keywords Thiomers · Protein delivery · Nasal delivery · Carbopol-cysteine · Mucoadhesion · Chitosan
Introduction Conventionally, the nasal route of delivery has been used for delivery of drugs for the treatment of local diseases such as nasal allergy, nasal congestion and nasal infections. It has been shown that the nasal route can be exploited for the systemic delivery of drugs such as small molecular weight polar drugs, peptides and proteins that are not easily administered via other than injection, or where a rapid onset of action is required (Illum 2003). It has been considered that nasal mucosa can also be an administration route to achieve faster and higher level of drug absorption. The highly vascular * Sanjay K. Jain
[email protected] 1
Pharmaceutics Research Projects Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Vishwavidyalaya, Sagar, MP 470003, India
2
Sagar Institute of Pharmaceutical Sciences, Sagar, MP, India
3
Institute of Pharmaceutical Research, GLA University, Mathura, UP 281 406, India
nature of the nasal mucosa coupled with its high drug permeation makes the nasal route of administration an attractive passage for many drugs including proteins and peptides. Structural features of different sections of nasal cavity and their relative impact on permeability are listed in Table 1. Advances in biotechnology unfolded the applicability of large number of proteins and peptide drugs in the treatment for various diseases. It is known that these drugs are unsuitable for oral administration because they are significantly degraded in the gastrointestinal tract or considerably metabolized by first pass effect in the liver (Jain et al. 2013). At present most of these extraordinary pharmacological potential therapeutic agents (mostly peptides and proteins) have to be administered via parenteral routes, which are inconvenient because of pain, risk and fear being associated with this type of administration (Ramaprasad et al. 1996). Hence noninvasive- mode of drug administration is highly in demand. In order to provide a sufficiently high bioavailability with non-invasive peptide delivery system various hurdles have to be overcome (Bernkop-Schnürch et al.
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13 Anterior part of nasal cavity and narrowest part of the nasal cavity with nasal hairs (vibrissae) and surface area of 0.6 cm2 Nasal portion is covered by a stratified squamous keratinized epithelial with sebaceous gland It is the area between nasal vestibule and respiratory region. Transepithelial region, stratified squamous cells are present in anterior region whereas pseudostratifiedcolumnar cells with microvilli are present in posterior region. It is narrowest region of nasal cavity It is largest part of nasal cavity and is also known as conchae. Pseudostratified ciliated columnar cells with microvilli (300 per cell) and large surface area receive maximum nasal secretions because of the presence of seromucus glands, nasolacrimal duct and goblet cells Richly supplied with blood for heating and humidification of inspired air. There is also presence of paranasal sinuses It is located in the roof of the nasal cavity. Specialized ciliated olfactory nerve cells for smell perception receives ophthalmic and maxillary divisions of trigeminal nerve. It has neuroepithelium Neuroepithelium is the cavity part of the CNS and is directly exposed the external environment - It has pseudostratified cells Upper part contains ciliated cells and lower part contains squamous epithelium
Nasal vestibule
Nasopharnyx
Olfactory region
Respiratory region (inferior turbinate middle turbinate superior turbinate)
Atrium
Structural features
Region
Table 1 Structural features of different sections of nasal cavity and their relative impact on permeability
Upper part contains ciliated cells and lower part contains squamous epithelium
Direct access to cerebrospinal fluid
Most permeable region because of large surface area and rich vasculature Drug delivery is very good in this region - It consists pseudo stratified columnar epithelial, globet cells, basal cells, mucous and serous glands - Microvilli are important to enhance the respiratory surface area
Less permeable as it has small surface area and stratified cells are present in anterior region
Least permeable because of the presence of keratinized cellswhich offerhigh resistance against toxic environment
Permeability
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2004a). It includes diffusion barrier, being based on the mucus gel layer covering mucosal membranes, which has to be surpassed in order to reach the absorption site (Bernkop-Schnürch and Fragner 1996) and enzymatic barrier secreted and membrane bound peptidases (BernkopSchnürch 1998). Moreover after reaching at the absorption membrane inintact form therapeutic peptides have to permeate membrane barrier in order to reach the systemic circulation (Bernkop-Schnürch and Clausen 2002). Therefore, number of attempts have been made to overcome these barriers which include the use of enzyme inhibitor (Aungst et al. 1996), permeation enhancer (BernkopSchnürch and Walker 2001) and multifunctional polymers (Bernkop-Schnürch et al. 2003a). Recently, it has been shown that polymers with thiol groups provide much higher adhesive properties than the polymers generally considered to be muco-adhesive. The enhancement of muco-adhesion can be explained by the formation of covalent bonds between the polymer and the mucus layer which are stronger than non-covalent bonds (Sreenivas and Pai 2008). Among these multifunctional polymers, thiolated polymers designated as thiomers are the most promising for non-invasive peptide delivery. Due to the immobilization of thiol groups on well-established multifunctional polymers such as poly(acrylates) or chitosan, their enzyme inhibitory, permeation enhancing and mucoadhesive properties can be strongly improved (Bernkop-Schnürch et al. 1999a, 2001a). Most of the peptides and proteins are potent and specific in their physiological activities and have become drugs of choice for the treatment of numerous diseases as a result of their incredible selectivity and their ability to provide effective and potent action (Morishita and Peppas 2006). The development of the protein drug delivery has been the major issue for pharmaceutical and biomedical research because the success of a protein as a therapeutic agent depends on the development of a formulation in which structure and activity during preparation and delivery as well as during shipping and long-term storage is maintained (Allison et al. 1996; Jorgensen et al. 2006; Wang 1999). Despite rapid progress in the large-scale manufacture of therapeutic proteins, the convenient and effective delivery of these drugs to the body remains a major challenge. Proteinous drugs represent a significant part of the pharmaceuticals coming in the market every year and are now widely used to treat or relieve symptoms related to many metabolic and oncologic diseases (Sarmento 2010). Proteins are the engines of life that perform essential functions inside cells, such as enzyme catalysis, signal transduction, gene regulation and maintaining a fine balance between cell survival and programmed death (Gu et al. 2011). Consequently, intracellular delivery of functional proteins has significant therapeutic implications in biological
applications, including disease therapies, vaccination, and imaging (Biswas et al. 2011; Jain et al. 2013). Expectations concerning the delivery of therapeutic proteins have been limited by their fragile structure and the frequent administrations (Giteau et al. 2008; Lam et al. 2000; Sinha and Trehan 2003; Yang and Cleland 1997). The oral route will no longer be the exclusive route of administration; other mucosal routes such as the nasal (Pringels et al. 2006), pulmonary (Chen et al. 2002; Desai et al. 2002; Johannson et al. 2002), ocular (Chiou and Li 1993; Soni et al. 1998; Srinivasan and Jain 1998; Yamamoto et al. 1989), buccal (Veuillez et al. 2001), and transdermal routes (Cevc 1996) may hold the key to the ultimate success of peptide and protein drugs in therapeutics (Lee 1991). Increased knowledge and advancements in biotechnological and pharmaceutical applications makes us able to produce large quantities of proteins (Sinha and Trehan 2003). Although, these new pharmaceuticals showed high therapeutic promise, the systemic application of proteins to the body quickly became a large hurdle due to the sensitivity of these molecules. The stabilization of proteins in delivery devices and the design of appropriate protein carriers are the major research issues in protein delivery. Among them, polymeric nanoparticles and microspheres have shown a certain degree of success for the delivery of proteins to the systemic circulation and to the immune system (Vila et al. 2002). However, protein stability still remains one of the most important barriers for their successful incorporation in biodegradable drug delivery formulations (Cuifang 2007).
Nasal Route for Protein Delivery Besides inconvenient intravenous/subcutaneous route used to deliver peptides to the human body, the nasal route seems to be one of the most feasible alternative ways for peptide delivery. Peptides administered through nasal route are rapidly absorbed due to high vascularity and large surface area of the nasal cavity. After permeation into the nasal blood vessels, peptides are transported immediately to their site of action, avoiding a first-pass metabolism. The nasal delivery of most of the peptides representing polar and hydrophilic macromolecules, has a great challenge. Consequently, only few nasal formulations containing peptides are on the market like nasal sprays of desmopressin and calcitonin. Generally peptides are showing 1% or less bioavailability of when administered to the nasal cavity (Illum 1996). This low bioavailability is the result of different nasal barriers, namely the enzymatic barrier (I), the absorption barrier (II) and the mucociliary clearance (III). To overcome the enzymatic barrier, the coadministration of enzyme inhibitors like amastatin (O’Hagan and Illum 1990) was used, but most of these auxiliary agents are toxic if absorbed systemically. The
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absorption barrier is overcome bychemically modification of peptides (Hashimoto and Tatsumi 1989). The most common method to improve nasal peptide absorption is the use of permeation enhancers. Many permeation promoters cause unfortunately significant damage to the nasal mucosa when used in very effective concentrations (Hinchcliffe and Illum 1999).The mucociliary clearance rate can be decreased by the use of mucoadhesive polymers. Chitosan has shown in different studies to prolong the residence time of nasal drug delivery systems at the site of drug absorption (Soane et al. 1999, 2001). Additionally, chitosan improves the absorption of peptides by transiently opening the tight junctions (Illum 1998). Accordingly chitosan represents a promising polymer in nasal peptide delivery. To attain faster and higher level of drug absorption, the nasal mucosa has to be considered as an administration site (Fig. 1). The nasal route is attractive for many drugs including proteins and peptides due to the highly vascular nature of the nasal mucosa and high drug permeation (Arora et al. 2002).
Thiomers In 1990s, a unique generation of mucoadhesive thiolated polymers (thiomers) was introduced (Perrone et al. 2017). Recently, a new generation of mucoadhesive polymers has been introduced in drug delivery system. Mucoadhesive polymers, discovered so far, attach itself to mucus membrane by non-covalent binding such as electrostatic forces (hydrogen bonding, ionic interaction, vander waal forces) (Calceti et al. 2004) and physical mechanism (mucus interpenetration, chain entanglement), these new generation polymers are capable of forming covalent bonds. The disulphide bond representing the most commonly encountered bridging structure in biological system has thereby been discovered for the covalent adhesion of polymers to the epithelial mucus layer (Leitner et al. 2004). This new generation of mucoadhesive polymers is generated by immobilization of thiol bearing Fig. 1 Nasal route for drug delivery (a nasal vestibule, b palate, c inferior turbinate, d middle turbinate, e superior turbinate (olfactory mucosa), and f nasopharynx)
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ligands on the polymeric backbone of well-established mucoadhesive polymers (Bernkop-Schnürch et al. 1999b). Based on thiol/disulphide exchange reactions and oxidation process thiolated polymers, or thiomers, have been shown to interact with cysteine-rich subdomains of mucus glycoproteins, thereby forming disulphide bonds (Fig. 2). These covalent bonds are stronger and are unaffected by parameters such as ionic strength and pH (Synder et al. 1983).
Types of Thiomers Anionic Thiomers Anionic thiolated polymers generated thus far all exhibit carboxylic acid groups as anionic substructures. These carboxylic acid groups offer the advantage, that sulfhydryl moieties can be easily attached to such polymers via the formation of amide bonds. Appropriate ligands are overall cysteine (Bernkop-Schnürch et al. 1999a), cysteamine (Bernkop-Schnürch et al. 2001b) and homocysteine (Bernkop-Schnürch et al. 2004a). The formation of amide bonds can be mediated by carbodiimides. An unintended
Fig. 2 Interact with cysteine-rich subdomains of mucus glycoproteins; forming disulphide bonds (mechanism of mucoadhesion of thiomer)
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oxidation of thiol groups during synthesis can be avoided by performing the reaction under inert conditions. Alternatively, the synthesis can be performed at a pH 5 to eliminate the formation of disulfide bonds because at this condition the concentration of thiolate-anions, representing the reactive form for oxidation of thiol groups, is low. In addition, disulfide bonds formed during synthesis can be cleaved thereafter by the addition of reducing agents such as dithiothreitol or N aBH4. The total amount of immobilised, reduced and oxidized thiol groups can be determined by reducing first of all the entire amount of oxidised thiol groups with NaBH4 followed by quantifying the thiol groups with Ellman’s reagent. Skipping the reduction process allows the determination of the ratio of oxidized to reduced thiol groups. The chemical structures of anionic thiolated polymers are shown in Fig. 3 (Bernkop-Schnürch et al. 2004b).
Cationic Thiomers Cationic thiomers are mainly based on chitosan (Fig. 4). The primary amino group at the 2-position of the glucosamine subunits of this polymer is the main target for the immobilisation of thiol groups. In the case of amide bonds the carboxylic acid group of the ligands cysteine and thioglycolic acid react with the primary amino group of chitosan mediated for instance by carbodiimides (Bernkop-Schnürch et al. 1999b). The formation of disulfide bonds by air oxidation during the synthesis can be avoided as described above. In the case of amidine bonds 2-iminothiolane is used as coupling reagent (Bernkop-Schnürch et al. 2003b). It offers the advantage of a simple one step coupling reaction. In addition, the thiol group of their agent is protected towards oxidation because of the chemical structure of the reagent. Table 2 shows the drug encapsulated various thiolated delivery systems.
Fig. 3 Chemical structures of anionic thiomers
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Characteristics of Thiomers Thiol groups of thiolated polymers are responsible to improve various features of polymers. Thiomers have been characterized on following parameters. Thiol Group Content Determination The thiol groups provide much higher adhesive properties than general muco-adhesive polymers. The thiol group content on the polymer may be determined by iodometry procedure using 1 M HCl, 0.1 N iodine, 0.1 N sodium thiosulphate solution and Starch is used as an indicator. (Barbarić et al. 2007.) Aydogmus et al. (2016) developed liquid chromatography (LC) coupled to an electrochemical detector was applied for the determination of small thiols such as cysteine (Cys), glutathione (GSH) and dithiothreitol (DDT) and the free thiol content in a protein. Cohesive Properties
Fig. 4 Chemical structures of cationic thiomers
Thiomers are capable of forming intra and inter chain disulfide bonds within polymeric network leading to strongly improved cohesive properties and stability of drug delivery systems such as matrix tablets (Guggi et al. 2004). Thiolated chitosans in addition to their strong mucoadhesive and permeation enhancing properties also show excellent cohesive properties.
Table 2 Drug encapsulated various thiolated delivery systems Thiolated drug delivery system
Drug
Purpose
References
Thiolated chitosan nanoparticles Thiolated polycarbophil
Enhance the bioavailability Enhance the permeation and improve the stability Enhance the bioavailability Insulin delivery through nasal route Treatment of depression For alleviation of pain For the nasal administration
Shahnaz et al. (2012) Vetter et al. (2010)
Thiolated chitosan microparticles Thiolated microspheres Thiolated chitosan nanoparticles Thiolated chitosan nanoparticles Thiolated poly(acrylic acid) microparticles Thiolated gellan gum Thiomers Nanoparticles of thiolated chitosan
Leuprolide Phosphorothioate antisense oligonucleotide Insulin Insulin Selegiline hydrochloride Tizanidine HCl Exenatide Dimenhydrinate Leu-enkephalin Zolmitriptan
Conjugated thiomers Thiomeric microparticles
Apomorphine Insulin
For the nasal administration For the nasal administration Brain targeting via intranasal drug delivery Nasal delivery of drug Safe nasal insulin delivery system leading to improved absorption Nose-to-brain drug delivery
Dopamine Chitosan (CS), glycol chitosan (GCS) and corresponding thiomer-based nanoparticles Preactivated thiolated xanthan gum 2-Mercaptonicotinic acid Nasal drug delivery
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Krauland et al. (2006) Nema et al. (2013) Singh et al. (2016) Misra (2002) Millotti et al. (2014) Mahajan et al. (2009) Bernkop-Schnurch et al. (2006) Sunena et al. (2016) Netsomboon et al. (2016) Deutel et al. (2016) Gioia et al. (2015) Menzel et al. (2017)
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Mucoadhesive Properties
Disulfide Bond Formation
Mucoadhesive drug delivery systems should provide close, prolonged contact of the drug with the mucosa. Thiomers show the strongest mucoadhesive properties of all so far tested polymeric excipients due to the formation of disulfide bonds with mucous glycoproteins (Leitner et al. 2003a). Nema et al. (2013) determined mucoadhesion using a strip of nasal mucosa was mounted on a glass slide and placed nonthiolated and thiolated microspheres on it then washed out microspheres was determined to calculate percent mucoadhesion.
Disulfide bonds in proteins are formed between the thiol groups of cysteine residues by the process of oxidative folding. The disulphide bond representing the most commonly encountered bridging structure in biological system. To cleave all the disulfide bonds to free thiol groups of thiomer is hydrated with iodine and pH was adjusted to acidic range. Then treat with solution of sodium borohydride. The thiomer before and after reduction of thiol groups is subtracted to estimate disulfide content (Leitner et al. 2003b).
Enzyme Inhibitory Properties
This is enlargement of volume of thiomers in presence of liquid medium. Briefly swelling behavior may be determined by compresing the thiomers into a disc and dipped in demineralized water (Hanif et al. 2015). Swelling behavior of mucoadhesive polymer has a great impact on their adhesive and cohesive properties.
Most of non- invasively administered drugs such as therapeutic peptides were degraded on the mucosa by membrane bound enzymes. Because of the capability to bind Z n 2+ ions via thiol groups, thiomers are potent inhibitors of most membrane bound and secreted zinc dependent enzymes. So, thiomers can significantly improve the bioavailability of noninvasively administered drugs. To evaluate the potential of polycarbophil cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin from enzymatic degradation by the intestinal mucosa a study was carried out and results showed that PCP-cys had a significantly greater inhibitory effect than PCP on the amino peptidase N activity towards both substrates. Zinc-dependent proteases such as aminopeptidases and carboxypeptidases are inhibited by thiomers that is highly beneficial for the oral administration of peptide and protein drugs. The underlying mechanism is based on the capability of thiomers to bind zinc ions (Bernkop-Schnürch et al. 2001a; Samuel et al. 2010). Permeation Enhancing Properties Thiolated polymers have been confirmed to show a powerful permeation enhancing effect for the paracellular transfer of drugs. In comparison to other permeation enhancers, thiolated polymers offer the advantage of not being absorbed from the mucosal membrane. therefore the permeation enhancing property can be maintained for a longer period of time. The mechanism may be responsible for this improved permeation enhancement has been attributed to the inhibition of the protein, tyrosine phosphatase which might be involved in the opening and closing process of the tight junctions. Thiolated polymers also exhibit permeation enhancing effect for the paracellular uptake of drugs. The effect is based on a glutathione mediated opening of the tight junction (Kast and Bernkop-Schnürch 2002; Samuel et al. 2010).
Swelling Behavior
Stability of Thiomers Thiomers show comparatively low stability in solutions, as they are subject to oxidation of thiol at pH ≥ 6 unless sealed under inert conditions (Bernkop-Schnürch et al. 2003c). Thiomers when stored in the form of a powder, a decrease in free thiol groups was observed only after storage at 20 °C and 70% RH. Thiomers were found to be stable under all storage conditions when compressed into matrix-tablets (Bernkop-Schnürch et al. 2002).
Stability of Peptides Incorporated into Thiomers As most of the peptide drugs bear thiol and/or disulfide bonds in their chemical structure, thiol/disulfide exchange reactions with thiomers cannot be excluded a priori. Studies investigating such peptide–thiomer interactions, however, revealed that they take place only to a very limited extent. Moreover, for many therapeutic peptides such interactions can be excluded completely. Although generalizations must always be viewed with great caution, thiol/disulfide exchange reactions do not seem to take place if at least one of following demands are fulfilled: • Solid delivery systems with no or comparatively low
water content are generated.
• The pH of the thiomers is < 5 leading to a marginal ratio
of thiolate anions, which are the functional groups being liable for thiol/disulfide interactions and oxidation. • The thiol/disulfide moieties of the therapeutic peptide being embedded in an anionic thiomer are neighboured by non-ionic or anionic amino acids.
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• The thiol/disulfide moieties of the therapeutic peptide
being embedded in a cationic thiomer are neighboured by non-ionic or cationic amino acids (Bernkop-Schnürch and Thaler 2000).
Role of Thiomers as Mucoadhesive Nasal Drug Delivery The nasal route represents an attractive alternative to parenteral delivery of therapeutic peptides such as calcitonin, insulin, desmopressin, buserelin and octreotide. However, bioavailability of nasally administered peptides often do not exceed 1% due to low membrane permeability, a short local residence time at the site of absorption and a high metabolic turnover in the nasal epithelium (Illum 2003). The three major strategies to increase the bioavailability of intranasally administered peptide drugs are (i) the use of permeation enhancers, (ii) incorporation of enzyme inhibitors and (iii) increasing local drug residence time using mucoadhesive polymers (Ugwoke et al. 2001). The ability of synthetic or biological macromolecules to adhere to mucosal tissues is known as mucoadhesion which offers several advantages due to (I) localization at a given target site, (II) a extended residence time at the site of drug absorption, and (III) an intensified contact with the mucosa increasing the drug concentration gradient (Lehr 1996). Therefore, the uptake and subsequently the bioavailability of the drug may be increased and as a result the patient compliance improved. Therefore, numerous natural and synthetic polymers have been discovered as mucoadhesive excipients. Their mucoadhesive properties can be explained due to the interaction with the glycoproteins of the mucus mainly based on non-covalent bonds such as hydrogen bonds, ionic interactions and van der Waal’s forces (Calceti et al. 2004). It could be revealed that polymers with thiol groups provide much higher adhesive properties than generally considered to be mucoadhesive polymers (Bernkop-Schnürch et al. 1999a). The formation of covalent bonds between the polymer and the mucus layer, which are stronger than noncovalent bonds is responsible for enhancement of mucoadhesion. It is supposed that these thiomers, are interact with cysteine rich subdomains of mucus glycoproteins via disulfide exchange reactions (Synder et al. 1983; Kast and Bernkop-Schnürch 2002). The thiolated polymers designated as thiomers are the most promising for non-invasive peptide delivery. Ugwoke et al. (2001) reported that polymers with thiol groups provide much higher adhesive properties than polymers generally considered to be mucoadhesive. These thiomers are supposed to interact with cysteine rich subdomains of mucus glycoproteins via disulfide exchange reactions (BernkopSchnürch et al. 1999a, 2004b; Lehr 1996).
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Nasal Drug Delivery Systems Based on Thiomers Microparticles Microparticles display per se a prolonged residence time on mucosal membranes compared to single-unit dosage forms (Rozier et al. 1989). This residence time at mucosal membranes is even further improved when they exhibit mucoadhesive properties. Due to the immobilisation of thiol groups on microparticles the mucoadhesive properties are additionally improved (Edsman et al. 1998). Microparticles based on poly(acrylic acid) or chitosan are lack of strong cohesive properties. Consequently they disintegrate rapidly and cannot control the release of the embedded peptide drug. Chitosan microparticles can be stabilized by addition of multivalent anions, but as a consequence mucoadhesion decreases. The use of multifunctional polymers like PAA450-Cys for microparticle preparation led to particles with highly improved cohesive properties (Krauland and Bernkop-Schnürch 2004). They are stabilized by the formation of intramolecular disulfide bonds within the microparticles during the preparation process. Consequently a controlled drug release out of such microparticles can be achieved. The release of the peptide drug can be prolonged by the addition of hydrophobic excipients like Eudragit RS to the polymer. Disintegration studies showed a stability of these thiomeric microparticles over 24 h, whereas particles comprising unmodified poly(acrylic acid) disintegrated within minutes. Microparticles display per se a prolonged residence time on mucosal membranes compared to single-unit dosage forms (Coupe et al. 1991). This residence time on mucosal membranes is even further improved when they exhibit mucoadhesive properties. Due to the immobilisation of thiol groups on microparticles the mucoadhesive properties are additionally improved. PAA450-Cys microparticles, for instance, were almost 15-times more mucoadhesive on the intestinal mucosa than unmodified polymer particles (Krauland and Bernkop-Schnürch 2004). Nema et al. (2013) investigated the potential of developed thiolated microspheres for insulin delivery through nasal route. In that study cysteine was immobilized on carbopol using EDAC. They found that Thiolated microspheres bearing insulin showed better reduction in blood glucose level in comparison to nonthiolated microspheres as 31.23 ± 2.12 and 75.25 ± 0.93% blood glucose of initial blood glucose level was observed at 6 h after nasal delivery of thiolated and nonthiolated microspheres, in streptozotocine induced diabetic rabbits. Bernkop-Schnürch et al. (2003c) prepared thiolated polyacrylate microparticles for the nasal delivery of hGH.
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The intranasal administration of this microparticulate formulation to rats resulted in a relative bioavailability of 8.11 ± 2.15% that represents a threefold improvement compared to microparticles comprising the corresponding unmodified polymer (Bernkop-Schnürch et al. 2003a). When the maximum plasma concentration of hGH following nasal administration of the thiomer microparticles and the thiomer gel formulation were compared, a sixfold higher uptake is achieved with the microparticulate formulation.
Nanoparticles Wang et al. (2009) investigated chitosan-N-acetyl-l-cysteine (chitosan-NAC) nanoparticles as a potential carrier system for the nasal delivery of insulin. They observed mucoadhesive properties, which were evaluated by measuring the in vitro absorbed mass of mucin, of chitosan-NAC nanoparticles were > 1.8-fold that of unmodified chitosan nanoparticles and intranasal administration of chitosan-NAC nanoparticles in rats enhanced the absorption of insulin by the nasal mucosa compared with unmodified chitosan nanoparticles and control insulin solution. It is concluded that the novel thiolated chitosan nanoparticles represent a promising vehicle for nasal insulin administration. Sunena et al. (2016) prepared nanoparticles of mucoadhesive thiolated chitosan polymer. controlled drug release was observed on ex vivo drug permeation studies andthey conclude that the thiolated chitosan based nanoparticles severves a potential drug delivery tool for nasal delivery.
Gels Mucoadhesive gels are valuable for nasal, intraoral, vaginal and ocular delivery. The use of thiomers in gel formulations has to be found not only in their mucoadhesive but also in their in situ gelling properties. According to the previous study it was found that thiolated polycarbophil might be a promising excipient for nasal administration of Leucineenkephalin to get sustained release (Bernkop-Schnürch et al. 2006). Leitner et al. (2004) developed a nasal gel formulation for systemic delivery of hGH and found a significantly higher and prolonged nasal bioavailability of hGH which was incorporated in the thiomer gel formulation. Thiolated polymers have been developed to get strong permeation enhancing effect for the uptake of drugs from the nasal mucosa. In a study the enzymatic degradation of Leu-enkephalin on bovine nasal mucosa was analyzed on thiolated polycarbophil for the nasal administration of Leu-enkephalin and it was found that degradation process is lowered in presence of thiolated polycarbophil. Diffusion studies showed Sustained release of Leu-enkephalin from thiolated polycarbophil gel. Hence, thiolated polycarbophil
might be a hopeful excipient for nasal administration of Leu-enkephalin. Recently, the potential of a thiomer gel formulation could be demonstrated by in vivo studies. Leitner et al. developed a nasal gel formulation for systemic delivery of hGH. The efficacy of a mucoadhesive gel formulation being based on unmodified polycarbophil and polycarbophil–cysteine was compared in rats. A significantly higher and prolonged nasal bioavailability of drug, which was incorporated in the thiomer gel formulation was observed Utilizing the thiomer gel formulation an absolute nasal bioavailability of 2.75 ± 0.37% was achieved (Bernkop-Schnürch and Thaler 2000). As thiomers exhibit also a strong permeation enhancing effect, however, it is difficult to attribute this improved in vivo efficacy exclusively to the improved mucoadhesive properties. These results are in good agreement, where nasal particulate dosage forms have been shown to give an improved bioavailability of the delivered drugs compared with solutions, mainly due to their ability to reside longer in the nasal cavity before being cleared by the mucociliary clearance system. Thiomers seem to be capable of combining most of these strategies. Therefore, the suitability of thiomers as multifunctional vehicles for systemic nasal peptide delivery was evaluated in vivo. As model drug, human growth hormone (hGH), a protein drug of 191 amino acids (22 kDa) was utilised, which is used to treat short stature in children. Currently, hGH has to be administered by daily injections, which are difficult and painful resulting in low patient acceptance (Laursen et al. 1996). A nasal delivery system for hGH would therefore be highly appreciable. For the in vivo study, an aqueous nasal gel formulation was developed consisting of PCP-Cys, glutathione and hGH in a final concentration of 0.3, 0.5 and 0.6% (m/v), respectively. As controls a 0.3% (m/v) PCP gel and physiological saline were prepared containing the same amount of hGH. These formulations were administered to conscious rats and the hGH plasma level was monitored via ELISA as a function of time. The PCP-Cys/glutathione/ hGH nasal gel delivery system resulted in a significantly higher hGH plasma concentration compared to both controls with an absolute bioavailability of 2.75 ± 0.37%. Furthermore, in contrast to the controls the thiomer gel delivery system was able to prolong the efficacy of hGH. In recent studies that demonstrated thiolation of chitosan resulted higher mucoadhesive and permeation enhancing properties (Bernkop-Schnürch et al. 2003c, 2004b) compared to unmodified chitosan. By the combination of thiolated chitosan in particular—chitosan-4-thiobutylamidine conjugate (chitosan–TBA) with the permeation mediator reduced glutathione, the permeation enhancing effect of this chitosan derivative could be further improved. The usefulness of chitosan–TBA for the oral administration of peptide drugs has already been demonstrated in various
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in vivo studies (Guggi et al. 2003a, b; Krauland et al. 2006), but its effectiveness in nasal peptide delivery was not evaluated. Hence chitosan–TBA bearing insulin was prepared and evaluated for its nasal mucoadhesion and enhanced for nasal permeation. It was observed that the chitosan–TBA/glutathione system is a useful vehicle for the nasal administration of therapeutic peptide drugs.
Activated Thiomers/Conjugated Thiomers Netsomboon et al. (2016) were synthesized poly(acrylic acid) thiomers by using different molecular weights of the polymer backbone for the intranasal administration of apomorphine. They considered that high degree of preactivation (poly(acrylic acid)-cysteine-2-mercaptonicotinic acid) could be as hopeful excipient for nasal delivery of drug.
Conclusion The delivery of therapeutic proteins/peptides has been limited by their fragile structure and the frequent administration. Hence, an alternative therapy to present conventional therapy is desired. Therefore number of approaches has been envisaged to stabilize proteins in delivery devices as well as in biological fluids and to design suitable protein carriers for effective delivery to target organs. The immobilization of thiolated materials on polymeric compounds is responsible for improvement in their mucoadhesive, enzyme inhibitory and permeation enhancing properties. It has been presented that polymers with thiol groups provide much higher adhesive properties than the polymers generally considered to be muco-adhesive by the formation of covalent bonds between the polymer and the mucus layer which are stronger than non-covalent bonds. In comparison to noninvasive peptide delivery systems comprising unthiolated multifunctional polymers, the efficacy of delivery systems comprising the corresponding thiolated version is therefore significantly higher. Administration of proteins/peptides through nasal route using thiolated polymer resulted in a significantly higher effect than comprising nonthiolated polymer. According to the results, thiomers seem to represent a promising new generation of multifunctional polymers for noninvasive peptide delivery.
Compliance with Ethical Standards Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Human and Animal Rights Manuscript does not report any results on human experimentation (institutional and national).
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International Journal of Peptide Research and Therapeutics
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