Elsevier

Journal of Chromatography A

Volume 1351, 18 July 2014, Pages 70-81
Journal of Chromatography A

Mucin-based stationary phases as tool for the characterization of drug–mucus interaction

https://doi.org/10.1016/j.chroma.2014.05.031Get rights and content

Highlights

  • Development of Mucin based affinity chromatography stationary phases.

  • Study of the influence of the immobilization chemistry on retention.

  • Investigation of the influence of mobile phase composition on the retention process by experimental design.

  • Screening of 41 pharmaceutically active ingredients for their interaction with mucin.

Abstract

Mucin glycoproteins belong to a class of high molecular weight, heavy glycosylated, proteins that together with water, salts and lipids constitute mucous secretions. Particular disease states (e.g. obstructive chronic bronchitis and ovarian tumor) are known to modify the composition and the thickness of those barriers. Therefore, it is important to address whether the absorption of potential drug candidates to be administered is influenced by the presence of interaction with this class of proteins. Typically, the methods adopted to characterize drug–protein interaction are dialysis, ultrafiltration and gel filtration. Besides these, bio-affinity chromatographic methods have demonstrated to be valuable tools offering the advantageous characteristics such as simplicity, efficiency, high-throughput capability and robustness. The present contribution reports on the synthesis and analytical characterization of a new chromatographic stationary phase based on covalently immobilized mucin and explores the use of LC-UV affinity zonal chromatography as a tool to screen drugs for their affinity to mucin. A series of different binding chemistries for the covalent linkage of mucin to silica-based supports as well as distinct immobilization protocols (static and dynamic) have been evaluated in order to optimize surface coverage. Resultant stationary phases have been characterized chromatographically by studying the effect of mobile phase and analyte structure on the distribution and retention of test compounds. As conclusive study, we report the evaluation of the retention characteristics of 41 drug-like compounds (having heterogeneous chemical properties) for their interaction with this novel stationary phase.

Introduction

Mucus is a viscous adherent secretion composed of a water mixture of proteins, lipids, immunoglobulins and salts, covering the mucosa surfaces that are directly or indirectly in contact to the external environment. Its physiological functions are manifold and comprise maintaining the hydration state, lubrication, and cytoprotection of mucous tissues in districts such as the respiratory, gastrointestinal, reproductive and oculo-rhino-otolaryngeal tracts [1], [2]. Interaction between drugs and mucus represents a potential cause of reduced availability of drugs administered via oral, nasal, inhalatory, intraocular, intravaginal and rectal route. Molecules that show low affinity to the mucus layer are more readily available for absorption and transport through underlying epithelial cells. They are easily distributed at the site of application or systemically. The mucous clearance depends on the district (the rate of the elimination process varies from about 8 min in the nose to about 48 h in the colon [3]). In general, however, mucus is continuously secreted and sequentially shed or digested. Therefore, a long permanence in this secretion is unfavorable unless the target of the therapy is the mucus itself (e.g. mucolytic agents).

The thickness, mechanical properties, and chemical constituents of the mucus layer vary among tissues, depending on its function and/or as consequence of a disease state (e.g. chronic obstructive pulmonary disease). Irrespective of its origin the mucus is composed of water (content of 90–95% by weight), glycoprotein and lipids (0.5–5%), mineral salts (0.5–1%) and free proteins [1], [4]. The main protein component of the mucus, responsible for its characteristic visco-elastic properties, is the family of mucin glycoproteins (2–5% of the whole mucus content).

Mucins are macromolecules with molecular weight ranging between 0.5 and 30 MDa composed of approximately 10–30% by weight of proteins and 70–80% of oligosaccharides. The peptide sequences of these proteins are characterized by the presence of regions rich in serine and threonine that constitute sites of covalent attachment of a diverse array of glycans (from 4 to 15 units [5], many of which are sialylated or sulfated) via O-glycosidic bonds [5], [6], [7]. The glycan chains are clustered in specific domains and confer important structural and biological properties to the protein, including protease resistance, pathogen sequestration, and ion and water binding. In addition to the glycosylated domains the secreted mucins possess cysteine-rich areas at their N- and C-termini that are responsible for disulfide-mediated polymer formation [8]. As consequence, mucin momomers in solution form extended linear structures (5 nm in diameter and 100–5000 nm in length for pig gastric mucin [9]) which further aggregate in clusters of 10 or more units at low pH (typically below 5) through hydrophobic interactions [10].

Mucin, besides constituting a physical gel network important for the mucous characteristics, has been reported as molecular barrier that limits the diffusion of compounds such as aminoglycoside antibiotics [11], [12], β-lactam antibiotics [11], ergopeptides [13] and testosterone [14]. Previous methods to study the drug-mucus binding were based on diffusion [15] or diafiltration (or continuous ultrafiltration) [16]. However, such approaches are performed with different protein solutions in every experiment and require relatively long experimental time. Such conditions limit the applicability of these methodologies for the application to extended compound screenings.

The use of chromatography to investigate the binding of drugs to target proteins has been extensively studied and within the possible approaches high-performance affinity chromatography (HPAC) has attracted recent interest [17], [18], [19], [20], [21]. Typically, in affinity chromatography the targeted protein is adsorbed or chemically immobilized onto a solid support and used as a stationary phase. The retention characteristics presented by these chromatographic columns with respect to the injected solutes originates from the same types of specific interactions that are found in biological systems. HPAC has been used to study a variety of serum proteins (such as human serum albumin, bovine serum albumin, α1-acid glycoprotein) [22], membrane and transmembrane receptors [23] as well as cell line [24]. An extended overview of investigations in this field can be found in a recent review [25].

Two approaches are typically adopted in this technique: zonal elution or frontal analysis [18]. In the first approach, a narrow plug of solute is injected onto the stationary phase and the sample is then eluted with mobile phase. Solute's elution times or volumes are monitored along with a void volume marker. Resultant retention factors increase with increasing binding strength between injected solutes and immobilized protein and thus can be correlated with their binding constants. An alternative to this is represented by frontal analysis where instead the analyte is continuously applied. Once all binding sites of the stationary phase are saturated the solute breaks through and a more or less sharp sigmoidal breakthrough curve is monitored. Its inflection point or first central moment of the elution profile can be used to determine the binding constant and the number of active sites present on the surface of the stationary phase. In the present study, we evaluated the possibility of using zonal elution affinity chromatography to study the interactions of ligands with mucin (see Fig. 1 for a schematic representation). To accomplish the aim, different protein immobilization chemistries have been evaluated using model compounds with known mucin affinity constants. These results have been compared to commercially available protein-type reference columns (Bovine Serum Albumin, BSA, and α1-Acid Glycoprotein, AGP). In order to further characterize the retention principles, we investigated the effect of mobile phase variation using a Design of Experiment approach [26] finding conditions suitable for LC-MS analysis. As conclusive part of the study we characterized the retention behavior of 41 active pharmaceutical drug formulations under the optimized mobile phase condition.

Section snippets

Reagents and materials

Mucin from porcine stomach (type II), α1-acid glycoprotein from human plasma and the glycoprotein detection kit were obtained from Sigma Aldrich (Stockholm, Sweden). Saliva Orthana was a kind gift from A/S Orthana Kemisk Fabrik (Kastrup, Denmark). Chiralpak AGP and Chiralpak BSA were obtained from Chiral Technologies Europe (Strasbourg, France). Kromasil 300 Å silica gel was a kind gift from AkzoNobel Separation Products (Bohus, Sweden). Empty HPLC-cartridges with steel frits were obtained from

Porcine mucin preparation

The first step in the development of our stationary phase was the purification of the affinity ligand. Commercially available crude form of gastric porcine mucin is characterized by a wide molecular weight distribution and is often contaminated with other proteoglycans, DNA and cellular debris. Therefore, we decided to adopt a purification step previously described in literature based on dissolution, precipitation and pelleting prior to protein utilization [16], [27].

Immobilization chemistry

In order to create a

Concluding remarks

The present contribution describes the development of novel silica-based mucin stationary phases. Except for one case the relative retention properties of the stationary phases did not appear to be significantly influenced by the choice of the immobilization chemistry. The investigation of mobile phase effects on the retention process showed that the interaction of the solutes with the mucin phase is dependent on pH, % of isopropanol and concentration of buffer ions.

As conclusive study we

Acknowledgements

This work was financially supported by the University of Vienna through the interdisciplinary doctoral program “Initiativkolleg Functional Molecules” (IK I041-N), by AstraZeneca and by the TI-COAST project HYPERformance Liquid Chromatography of the University of Amsterdam. The authors would like to thank Dr. Bäckström and Dr. Klarqvist from AstraZeneca (Medicinal Chemistry, RIA, Mölndal) and BS Johansson (University of Gothenburg) for their assistance and valuable discussions.

References (50)

  • K. Khanvilkar et al.

    Adv. Drug Deliver. Rev.

    (2001)
  • R.A. Cone

    Adv. Drug Deliver. Rev.

    (2009)
  • S.K. Lai et al.

    Adv. Drug Deliver. Rev.

    (2009)
  • M.J. Rathbone et al.

    Int. J. Pharm.

    (1991)
  • M.D.G. Oates et al.

    Carbohydrate Res.

    (1974)
  • R. Bansil et al.

    Curr. Opin. Colloid Interface Sci.

    (2006)
  • J.K. Sheehan et al.

    J. Biol. Chem.

    (2004)
  • J.-J. Niibuchi et al.

    Int. J. Pharmaceut.

    (1986)
  • J.M. Franz et al.

    Int. J. Pharmaceut.

    (1980)
  • I. Komiya et al.

    Int. J. Pharmaceut.

    (1980)
  • P.G. Bhat et al.

    Int. J. Pharmaceut.

    (1995)
  • P.G. Bhat et al.

    Int. J. Pharmaceut.

    (1996)
  • D.S. Hage et al.

    J. Chromatogr. B, Biomed. Sci. Appl.

    (1997)
  • D.S. Hage

    J. Chromatogr. B

    (2002)
  • G. Massolini et al.

    Biochem. Pharmacol.

    (1993)
  • E. Calleri et al.

    J. Pharmaceut. Biomed. Anal.

    (2011)
  • S. Wang et al.

    J. Chromatogr. A

    (2010)
  • D.B. Hibbert

    J. Chromatogr. B

    (2012)
  • K. Jumel et al.

    Int. J. Biol. Macromol.

    (1996)
  • H. Xuan et al.

    Anal. Biochem.

    (2005)
  • J. Haginaka et al.

    J. Chromatogr. A

    (1997)
  • J. Haginaka et al.

    J. Chromatogr. A

    (1994)
  • J. Hermansson

    J. Chromatogr.

    (1983)
  • J.E. Sandoval

    J. Chromatogr. A

    (1999)
  • P.F. Ruhn et al.

    J. Chromatogr. A

    (1994)
  • Cited by (17)

    • Purified mucins in drug delivery research

      2021, Advanced Drug Delivery Reviews
      Citation Excerpt :

      Finally, Gargano et al. (2014) used a chromatographic method to screen a panel of 41 different drug-like molecules with a broad range of chemical properties for their capability to bind to mucins. In their approach, the authors immobilized commercial PGM onto the surface of silica-based resins which they used as stationary phases in liquid chromatography; then, they determined binding affinities of the probed molecules based on their retention characteristics [213]. Of course, all those techniques require a surface bound mucin film, which is stable during the binding test.

    • Mucin binding to therapeutic molecules: The case of antimicrobial agents used in cystic fibrosis

      2019, International Journal of Pharmaceutics
      Citation Excerpt :

      Inhaled tobramycin associated with aztreonam lysine is usually the first line of treatment for early infections. The affinity of some drugs for mucin has been previously studied using spectroscopic methods by Pontremoli et al. (2015), by chromatographic methods by Gargano et al. (2014), whereas Barbero et al have investigated the interactions of both carbon nanotubes (CNTs) and gold nanoparticles (GNPs) with mucin (Barbero et al., 2018, 2016). Starting from the work of Gargano et al., in our previous paper (Caron et al., 2015) we tried to highlight the balance of the intermolecular forces governing the interaction and we concluded that hydrogen bonding donor (HBD) properties of solutes favor the interaction with mucin.

    • Selective permeability of mucus barriers

      2018, Current Opinion in Biotechnology
      Citation Excerpt :

      The example of the ER peptide begins to illustrate the importance of hydrophobic interactions, which occur in mucus primarily with hydrophobic domains of mucins or mucus-associated lipids [4]. As with net charge, correlations between mucus binding and quantitative estimates of hydrophobicity of small molecules such as the octanol–water partition coefficient are substantial but far from perfect [4,31•] and we expect that spatial arrangements of proximal charge and other parameters such as double bonds or aromaticity for π–π bonding come into play. For nanoparticles, any exposed hydrophobic surface typically means trapping: synthetic polystyrene [24] or metal oxide nanoparticles [32] and single-walled carbon nanotubes [32] are immobilized in mucus likely due to polyvalent hydrophobic interactions.

    • The effect of 2-hydroxypropyl-β-cyclodextrin on the intestinal permeation through mucus

      2018, European Journal of Pharmaceutical Sciences
      Citation Excerpt :

      Moreover, Wuyts et al. demonstrated compatibility of mucus overlaid Caco-2 cells with complex media such as human intestinal fluids of the fasted state (FaHIF), which are normally detrimental to this cell type (Wuyts et al., 2015). Cell-free models on the other hand are useful to specifically assess the permeation through mucus without the confounding involvement of cellular transport (e.g. transporter involvement, metabolism) (Gargano et al., 2014; Legen and Kristl, 2001; Sanders et al., 2000). Sanders et al. developed a method in which a cystic fibrosis sputum sample was positioned between two half chambers.

    • Mucin-drugs interaction: The case of theophylline, prednisolone and cephalexin

      2015, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      Many studies highlighted that no definitive picture of the nature of the molecular interactions between drug molecules and mucus components can be drawn.6,7 Mucin–drugs complex has been studied by different methods: (a) chromatography using biomimetic stationary phases8 and novel stationary phase based on covalently immobilized mucin9 and (b) ultrafiltration isothermal titration micro-calorimetry (ITC) and transmission electron microscopy (TEM).10 In our knowledge very little literature is available on the spectrophotometrical study of drug/mucin interaction.11

    View all citing articles on Scopus
    View full text