Thiolated Polycarbophil as an Adjuvant for Permeation Enhancement in Nasal Delivery of Antisense Oligonucleotides

doi:10.1002/jps.21887

Journal of Pharmaceutical Sciences

Volume 99, Issue 3, March 2010, Pages 1427-1439

https://doi.org/10.1002/jps.21887 Get rights and content

Abstract

The purpose of this study was to investigate the effect of thiolated polycarbophil as an adjuvant to enhance the permeation and improve the stability of a phosphorothioate antisense oligonucleotide (PTO‐ODN) on the nasal mucosa. Polycarbophil‐cysteine (PCP‐Cys) was synthesized by the covalent attachment of L‐cysteine to the polymeric backbone. Cytotoxicity tests were examined on human nasal epithelial cells from surgery of nasal polyps confirmed by histological studies. Deoxyribonuclease I activity in respiratory region of the porcine nasal cavity was analyzed by an enzymatic assay. The enzymatic degradation of PTO‐ODNs on freshly excised porcine nasal mucosa was analyzed and protection of PCP‐cysteine toward DNase I degradation was evaluated. Permeation studies were performed in Ussing‐type diffusion chambers. PCP‐Cys/GSH did not arise a remarkable mortal effect. Porcine respiratory mucosa was shown to possess nuclease activity corresponding to 0.69 Kunitz units/mL. PTO‐ODNs were degraded by incubation with nasal mucosa. In the presence of 0.45% thiolated polycarbophil and 0.5% glutathione (GSH), this degradation process could be lowered. In the presence of thiolated polycarbophil and GSH the uptake of PTO‐ODNs from the nasal mucosa was 1.7‐fold improved. According to these results thiolated polycarbophil/GSH might be a promising excipient for nasal administration of PTO‐ODNs. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1427–1439, 2010

Section snippets

INTRODUCTION

The nasal route of administration has intrigued researchers for more than six decades and has received widespread attention due to several advantages. First, it features a noninvasive mode, which can contribute to enhanced patient compliance. Second, it provides rapid onset of action. Third, first pass effect through the liver and metabolism in the gastrointestinal tract (GIT) is avoided and it provides a relatively large and well vascularized surface area for absorption.¹ Hence, the nasal

Materials

PCP (Noveon™ AA1) was obtained from Noveon Pharma GmbH & Co. KG (Raubling, Germany). 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDAC), L‐cysteine free base, 2‐iminothiolane HCl (Traut's reagent), Ellman's reagent (DTNB, 5,5′‐dithiobis(2‐nitrobenzoic acid)), 2,4,6‐trinitrobenzenesulfonic acid (TNBS), L‐GSH reduced form (GSH), and N‐(2‐hydroxyethyl)piperazine‐N′‐(2‐ethanesulfonic acid) (HEPES) were obtained from Sigma–Aldrich (Vienna, Austria).

A 16‐mer phosphorothioate antisense

Characterization of the PCP‐Cys Conjugate

The covalent attachment of cysteine to PCP was achieved by the formation of amide bonds between the primary amino group of the amino acid and the carboxylic acid group of the polymer as shown in Figure 1. The amount of covalently attached cysteine on the thiomer is crucial for its permeation‐enhancing properties.⁶ The PCP‐Cys conjugate used in this study displayed 520.36 ± 86.97 µmol thiol groups per gram polymer. Furthermore, 187.46 ± 51.83 µmol disulfide bonds per gram polymer could be

DISCUSSION

Most permeation enhancers cause severe damage to the nasal epithelium irrespective to the mechanism of absorption. Cytotoxicity tests and histological examination are regarded as a useful tool for determining the viability of tissues. In this study neither a long permeation period nor the thiolated polymer had a strong mortal effect on the mucosa. Histological studies provided evidence for tissue viability over at least 3 h after sample preparation. Hence, toxic effects of PCP‐Cys on the mucosa

CONCLUSION

Nasal administration of phosphorothioate antisense ODNs could be an alternative opportunity to injectable administration for the treatment of several diseases. Therefore, the mucosal barrier in the nasal cavity has to be overcome. Within this study anionogenic thiolated PCP was shown to enhance the transport of PTO‐ODN across porcine respiratory mucosa of the nasal cavity. The results from the permeation studies in combination with the histological studies also showed that PCP‐Cys did not

Acknowledgements

The authors wish to thank Prof. G. Klima and Mr. Haring from the Department for Anatomy, Histology and Embryology, Medical University of Innsbruck for accomplishment of the histology studies and for the microscopic images. The authors wish to thank Prof. A. Neher from the Department of Otorhinolaryngology, Medical University of Innsbruck for allocation of the human nasal tissue samples.

REFERENCES (38)

S. Agrawal
Antisense oligonucleotides: Towards clinical trials
Trends Biotechnol
(1996)
M. Gonzalez Ferreiro et al.
Characterization of complexes of an antisense oligonucleotide with protamine and poly‐L‐lysine salts
J Control Release
(2001)
A.E. Clausen et al.
In vitro evaluation of the permeation enhancing effect of thiolated polycarbophil
J Pharm Sci
(2000)
A. Bernkop‐Schnürch et al.
Synthesis and characterisation of mucoadhesive thiolated polymers
Int J Pharm
(2000)
V.M. Leitner et al.
Thiolated polymers: Evidence for the formation of disulphide bonds with mucus glycoproteins
Eur J Pharm Biopharm
(2003)
R.G. Riley et al.
The gastrointestinal transit profile of ¹⁴C‐labelled poly(acrylic acid): An in vivo study
Biomaterials
(2001)
G.L. Ellman
A colorimetric method for determining low concentrations of mercaptans
Arch Biochem Biophys
(1958)
A.F. Habeeb
A sensitive method for localization of disulfide containing peptides in column effluents
Anal Biochem
(1973)
F. Föger et al.
Inhibition of malarial topoisomerase II in Plasmodium falciparum by antisense nanoparticles
Int J Pharm
(2006)
S. Glorieux et al.
In vitro culture of porcine respiratory nasal mucosa explants for studying the interaction of porcine viruses with the respiratory tract
J Virol Methods
(2007)

M.C. Schmidt et al.

Validation of excised bovine nasal mucosa as in vitro model to study drug transport and metabolic pathways in nasal epithelium

J Pharm Sci

(2000)

N. Dias et al.

Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl‐2 antisense oligonucleotides

Eur J Pharm Biopharm

(2002)

E. Bechgaard

The viability of isolated rabbit nasal mucosa in the Ussing chamber and the permeability of insulin across the membrane

Int J Pharm

(1992)

M.A. Wheatley

Nasal drug delivery: An in vitro characterization of transepithelial electrical properties and fluxes in the presence or absence of enhancers

J Control Release

(1988)

C. Wadell et al.

Nasal drug delivery—Evaluation of an in vitro model using porcine nasal mucosa

Eur J Pharm Sci

(1999)

A. Bernkop‐Schnürch et al.

Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N

J Pharm Sci

(2001)

K. Takakura et al.

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite

Arch Biochem Biophys

(1999)

K. Nakamura et al.

The enhancing effect of nasal absorption of FITC‐dextran 4,400 by beta‐sitosterol beta‐D‐glucoside in rabbits

J Control Release

(2002)

V.M. Leitner et al.

Nasal delivery of human growth hormone: In vitro and in vivo evaluation of a thiomer/glutathione microparticulate delivery system

J Control Release

(2004)

Cited by (24)

Development of chitosan based β-carotene mucoadhesive formulation for skin cancer treatment
2023, International Journal of Biological Macromolecules
Mucopermeating nanoformulations can enhance mucosal penetration of poorly soluble drugs at their target site. In this work, thiolated chitosan (TCS)-lithocholic acid (LA) nanomicelles loaded with β-carotene, a safe phytochemical with anticancer properties, were designed to improve the pharmaceutical and pharmacological drug profile. The TCS-LA nanomicelles were characterized by FTIR to confirm the presence of the thiol group that favors skin adhesion, and to corroborate the conjugation of hydrophobic LA with hydrophilic CS to form an amphiphilic polymer derivative. Their crystalline nature and thermal behavior were investigated by XRD and DSC analyses, respectively. According to DLS and TEM, their average size was <300 nm, and their surface charge was +27.0 mV. β-carotene entrapment and loading efficiencies were 64 % and 58 %, respectively. In vitro mucoadhesion and ex vivo mucopenetration analyses further corroborated the potential of the nanoformulation to deliver the drug in a sustained manner under conditions mimicking cancer micro-environment. Anticancer studies in mice demonstrated that the loaded nanomicelles delayed skin cancer growth, as revealed by both morphological and biochemical parameters. Based on the results obtained herein, it can be concluded that drug-loaded TCS-LA is a novel, stable, effective and safe mucoadhesive formulation of β-carotene for the potential treatment of skin cancer.
Mucoadhesion as a strategy to enhance the direct nose-to-brain drug delivery
2021, Direct Nose-to-Brain Drug Delivery: Mechanism, Technological Advances, Applications, and Regulatory Updates
It is well apprehended that the nose-to-brain drug delivery system enables both small and large molecules to bypass the BBB via the nerves of the nasal cavity, i.e., the olfactory and trigeminal nerves. However, mucociliary clearance greatly limits the residence time of substances administered inside the nasal cavity. As a consequence, pharmaceutical scientists have now opted for the mucoadhesion-based drug delivery system wherein one can achieve prolonged, controlled retention of the drug at the site of application. This chapter provides an overview of recent advancements made in intranasal mucoadhesive polymers and their application in formulating the nose-to-brain drug delivery system. This section sheds some light on the underlying critical aspect and surface chemistry involved in mucoadhesion and summarizes the evidence supporting the development and therapeutic outcomes of mucoadhesive nose-to-brain drug delivery systems.
Biomolecule-assisted synthesis of Ag/reduced graphene oxide nanocomposite with excellent electrocatalytic and antibacterial performance
2017, Materials Science and Engineering C
Citation Excerpt :
Zhang and coworkers prepared CuS/rGO nanocomposites with enhanced photocatalytic performance in the presence of Cys [28]. Furthermore, thiol-functions of Cys could react with the outer cell surface and improve absorptive endocytosis [29], as well as could increase the electroactivity of graphene sheets [30]. To the best of our knowledge, there is a gap in scientific literatures regarding the synthesis of metal-graphene nanocomposites in the presence of cysteine.
In this work, an environmentally friendly method was applied for the synthesis of aqueous suspension of l-cysteine modified Ag nanoparticles (NPs)-decorated reduced graphene oxide (rGO) nanocomposite. l-cysteine played a triple role as reducing agent, stabilizer and linker of Ag NPs onto the surface of rGO. The resultant nanocomposite was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction studies (XRD), zeta potential, Raman spectroscopy, scanning electron microscopy (SEM) and energy dispersive analysis of X-ray (EDX). Meanwhile, minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC), agar well diffusion and cyclic voltammetry (CV) techniques were used for the investigation of antibacterial and electrocatalytic behaviors of the nanocomposite, respectively. The obtained nanocomposite showed not only enhanced electrocatalytic activity for glucose but also excellent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).
Next generation of buccadhesive excipient: Preactivated carboxymethyl cellulose
2016, International Journal of Pharmaceutics
Citation Excerpt :
Vetter et al. (2010) referred those thiol groups to form a mixed disulfide with Cys 215. Cys 215 inactivates protein tyrosine phosphatase (PTP) reducing tightness of the tight junctions (Vetter et al., 2010). Furthermore, explanation could be given that preactivated carboxymethyl cellulose is able to transform oxidized glutathione (GSSG) to reduced glutathione (GSH).
Assessment of preactivated carboxymethyl cellulose as potential excipient for buccal drug delivery.
Firstly, carboxymethyl cellulose (CMC) and cysteine (SH) were covalently coupled via amide bond formation to obtain thiolated carboxymethyl cellulose (CMC–SH). Further, preactivated carboxymethyl cellulose (CMC-S-S-MNA) was obtained by preactivation with 2-mercaptonicotinic acid (MNA). Sulforhodamine 101 (SRH101) was used as a model drug for permeation study through buccal mucosa. CMC-S-S-MNA was evaluated with respect to mucoadhesive and permeation enhancing effect and cytotoxicity.
Thiolated carboxymethyl cellulose exhibited a total amount of 112.46 ± 0.46 thiol groups. CMC-S-S-MNA exhibited around 50% of preactivated thiol groups. The preactivated polymer showed no toxic effect. Furthermore, compared to unmodified CMC, CMC-S-S-MNA revealed 3.0-fold improved mucoadhesive properties according to the rotating cylinder method and 8.8-fold enhancement in mucoadhesiveness by tensile assay, respectively.
Preactivated carboxymethyl cellulose fulfills the requirements as potential excipient of being mucoadhesive and permeation enhancing for the buccal drug delivery.
Chitosan in nasal delivery systems for therapeutic drugs
2014, Journal of Controlled Release
There is an obvious need for efficient and safe nasal absorption enhancers for the development of therapeutically efficacious nasal products for small hydrophilic drugs, peptides, proteins, nucleic acids and polysaccharides, which do not easily cross mucosal membranes, including the nasal. Recent years have seen the development of a range of nasal absorption enhancer systems such as CriticalSorb® (based on Solutol HS15) (Critical Pharmaceuticals Ltd), Chisys® based on chitosan (Archimedes Pharma Ltd) and Intravail® based on alkylsaccharides (Aegis Therapeutics Inc.), that is presently being tested in clinical trials for a range of drugs. So far, none of these absorption enhancers have been used in a marketed nasal product. The present review discusses the evaluation of chitosan and chitosan derivatives as nasal absorption enhancers, for a range of drugs and in a range of formulations such as solutions, gels and nanoparticles and finds that chitosan and its derivatives are able to efficiently improve the nasal bioavailability. The revirtew also questions whether chitosan nanoparticles for systemic drug delivery provide any real improvement over simpler chitosan formulations. Furthermore, the review also evaluates the use of chitosan formulations for the improvement of transport of drugs directly from the nasal cavity to the brain, based on its mucoadhesive characteristics and its ability to open tight junctions in the olfactory and respiratory epithelia. It is found that the use of chitosan nanoparticles greatly increases the transport of drugs from nose to brain over and above the effect of simpler chitosan formulations.
Thiolated chitosan nanoparticles for the nasal administration of leuprolide: Bioavailability and pharmacokinetic characterization
2012, International Journal of Pharmaceutics
Citation Excerpt :
Subsequently, the supernatant was extracted with acetonitrile and analyzed via HPLC as described above (Iqbal. et al., 2011). The viability of the porcine respiratory mucosa of the nasal cavity was evaluated by an embedding treatment as previously described by our research group (Vetter et al., 2010). Human nasal epithelial cells for cultures were harvested under the middle turbinate from healthy volunteer with nasal brushing (Toskala et al., 2005).
The purpose of this study was to develop thiolated nanoparticles to enhance the bioavailability for the nasal application of leuprolide. Thiolated chitosan–thioglycolic acid (chitosan–TGA) and unmodified chitosan nanoparticles (NPs) were developed via ionic gelation with tripolyphosphate (TPP). Leuprolide was incorporated during the formulation process of NPs. The thiolated (chitosan–TGA) NPs had a mean size of 252 ± 82 nm, a zeta potential of +10.9 ± 4 mV, and payload of leuprolide was 12 ± 2.8. Sustained release of leuprolide from thiolated NPs was demonstrated over 6 h, which might be attributed to inter- and/or intramolecular disulfide formation within the NPs network. Ciliary beat frequency (CBF) study demonstrated that thiolated NPs can be considered as suitable additives for nasal drug delivery systems. Compared to leuprolide solution, unmodified NPs and thiolated NPs provoked increased leuprolide transport through porcine nasal mucosa by 2.0 and 5.2 folds, respectively. The results of a pharmacokinetic study in male Sprague–Dawley rats showed improved transport of leuprolide from thiolated NPs as compared to leuprolide solution. Thiolated NPs had a 6.9-fold increase in area under the curve, more than 4-fold increase in elimination half-life, and a ∼3.8-fold increase in maximum plasma concentration compared to nasal solution alone. The relative nasal bioavailability (versus s.c. injection) of leuprolide thiolated NPs calculated on the basis of AUC_(0–6) was about 19.6% as compared to leuprolide solution 2.8%. The enhanced bioavailability of leuprolide is likely due to facilitated transport by thiolated NPs rather than improved release.

View all citing articles on Scopus

View full text

Research ArticlesThiolated Polycarbophil as an Adjuvant for Permeation Enhancement in Nasal Delivery of Antisense Oligonucleotides

Abstract

Section snippets

INTRODUCTION

Materials

Characterization of the PCP‐Cys Conjugate

DISCUSSION

CONCLUSION

Acknowledgements

Trends Biotechnol

J Control Release

J Pharm Sci

Int J Pharm

Eur J Pharm Biopharm

Biomaterials

Arch Biochem Biophys

Anal Biochem

Int J Pharm

J Virol Methods

J Pharm Sci

Eur J Pharm Biopharm

Int J Pharm

J Control Release

Eur J Pharm Sci

J Pharm Sci

Arch Biochem Biophys

J Control Release

J Control Release

Research Articles
Thiolated Polycarbophil as an Adjuvant for Permeation Enhancement in Nasal Delivery of Antisense Oligonucleotides