Anti-Inflammatory Activity of Chitosan and 5-Amino Salicylic Acid
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pharmaceutics Article Anti-Inflammatory Activity of Chitosan and 5-Amino Salicylic Acid Combinations in Experimental Colitis Henusha D. Jhundoo 1 , Tobias Siefen 1 , Alfred Liang 2, Christoph Schmidt 3, John Lokhnauth 4, Arnaud Béduneau 5 , Yann Pellequer 5, Crilles Casper Larsen 2 and Alf Lamprecht 1,5,* 1 Department of Pharmaceutics, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany; [email protected] (H.D.J.); [email protected] (T.S.) 2 Ferring Pharmaceuticals Inc., Parsippany, NJ 07054, USA; [email protected] (A.L.); [email protected] (C.C.L.) 3 Bayer Consumer Care AG, 4052 Basel, Switzerland; [email protected] 4 Vytaderm, Fair Lawn, NJ 07410, USA; [email protected] 5 PEPITE (EA4267), University of Bourgogne/Franche-Comté, 25000 Besançon, France; [email protected] (A.B.); [email protected] (Y.P.) * Correspondence: [email protected] Received: 5 October 2020; Accepted: 22 October 2020; Published: 29 October 2020 Abstract: Chitosan is used in various drug delivery approaches as a pharmaceutical excipient. Although its potential as an immunomodulatory agent has been reported, its use in this capacity has not been fully explored. The efficacy of chitosan as an active pharmacological agent, particularly in anti-inflammatory therapy in inflammatory bowel diseases (IBD), was investigated in this study. The potential impact of the molecular weight (MW) and degree of deacetylation (DD) of chitosan was investigated together with 5-amino salicylic acid (5-ASA) for its efficacy in a combination anti-inflammatory therapy in murine experimental colitis. Such a combination would potentially be developed into novel dual strategies whereby chitosan acts as a mucoadhesive excipient as well as provide an additional anti-inflammatory benefit. Chitosan grades with different MW and DD were administered intrarectally alone or in combination with 5-ASA to colitis mice for 3 days. Myeloperoxidase (MPO) and alkaline phosphatase (ALP) activity and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and nuclear factor kappa-B (NF-κB) levels were assessed from the colon. Intrarectal treatment of colitis with 30 mg/kg chitosan alone and with 30 mg/kg 5-ASA for 3 days led to a significant decrease in MPO, ALP, TNF-α, IL-6, IL-1β and NF-κB in colitis mice compared to untreated mice. Surprisingly, the efficacy of chitosan as an anti-inflammatory polymer was relatively independent from its structural properties, namely DD and MW. However, combinations of chitosan with 5-ASA showed a significant pharmacological improvement, whereby the additive anti-inflammatory efficacy observed shows the possibility of finetuning chitosan by combining it with anti-inflammatory agents to optimize its anti-inflammatory potential. Keywords: chitosan; IBD; colitis; inflammation; RAW 264.7 macrophages; 5-amino salicylic acid 1. Introduction Inflammatory bowel disease (IBD) is characterized by recurrent episodes of inflammation in the gastrointestinal tract (GIT) with periods of remission and symptoms such as abdominal pain, diarrhoea, rectal bleeding and weight loss. In the treatment of IBD, which is classified into two distinct idiopathic inflammatory diseases, namely ulcerative colitis and Crohn’s disease, anti-inflammatory therapy tends to target known inflammatory mechanisms since the etiology of the disease remains Pharmaceutics 2020, 12, 1038; doi:10.3390/pharmaceutics12111038 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 1038 2 of 16 unclear [1,2]. Conventional IBD therapy involves the use of drugs such as anti-inflammatory and immunosuppressant agents such as aminosalicylates (5-amino salicylic acid, 5-ASA), cyclosporine, corticosteroids, thiopurines and sulfasalazine and newer therapies such as anti-TNF-α monoclonal antibodies. However, a high incidence of adverse effects is observed in patients treated with conventional IBD immune suppressive drugs and, besides, antibody-based therapy is expensive and is reserved for later lines when patients show no response to drugs such as salicylates [3,4]. Although 5-ASA has been developed using various formulation strategies that enhance its delivery to the rectal or colonic site of inflammation in colitis, e.g., rectal enemas, foam gels or as controlled release preparations or as prodrugs, anti-inflammatory therapy in IBD still remains a challenge and has to date not been optimized for every patient. The incidence of treatment-emergent adverse events with 5-ASA therapy in IBD shows that alternative therapies are required for certain patients [5–7]. While most approaches focus on the design of delivery systems that specifically release the drug to the site of inflamed tissue by selecting excipients to optimize the delivery, the potential anti-inflammatory effect of excipients as single agents or in combination with other drugs has been hardly analyzed to date. This could be of major benefit in therapy since the known active pharmaceutical ingredient could be complemented by an excipient that not only fulfils formulation-related functions but also provides a direct therapeutic effect itself. One of the first examples for such therapeutic strategy is the use of chitosan. Interestingly, the anti-inflammatory effects of chitosan and its oligosaccharides have been reported in numerous studies [8–24]. Additionally, chitosan has been found to exhibit noteworthy biological effects, namely anti-microbial, hypocholesterolemic, immune-boosting and anti-tumor properties [25,26]. It has been suggested that the biological activities of chitosan may depend on the molecular weight (MW) and the degree of deacetylation (DD) of the parent chitosan material and these properties are inherently affected by the distribution pattern of d- glucosamine (GlcN) and β-1,4-linked N-acetyl-d-glucosamine (GlcNAc) along the oligomeric chain of the parent chitosan [26–29]. The anti-inflammatory effects of chitosan (ChS) and its oligosaccharides were reported a few years ago [8–10]. Although chitosan-based pellets showed enhanced efficacy in the treatment of experimental colitis [11] and that chitosan has a protective effect against intestinal inflammation [8], a detailed characterization of the polymer properties such as molecular weight (MW) and the degree of deacetylation (DD) and their potential influence on the therapeutic effect is missing. In order to investigate the relationship between structure and anti-inflammatory properties of chitosan in a systemic way, defined fractions of chitosan with different MW and DD were studied for their therapeutic benefit in experimental murine colitis. As mentioned earlier, preliminary findings indicated a certain limitation of anti-inflammatory effects for chitosan alone and therefore the potential therapeutic use of chitosan in IBD would be surely preferable in combination with a well-established conventional drug. Accordingly, the focus of this study was therefore to elucidate the pharmacological anti-inflammatory effect of chitosan and chitosan and 5-ASA combinations in a murine experimental colitis model. 2. Materials and Methods 2.1. Materials 5-Amino salicylic acid (5-ASA) was obtained from Sigma-Aldrich (Deisenhofen, Germany). The chitosan derivatives with varying viscosity and degree of deacetylation were purchased from Heppe Medical Chitosan GmbH (Halle, Germany). The MW, viscosity and DD of each chitosan grade are listed in Table S1 (Supplementary File). All tested chitosan grades were free of endotoxin. Lipopolysaccharides (LPS) derived from Escherichia coli 0111:B4 were purchased from Sigma-Aldrich (Taufkirchen, Germany). All other reagents for cell culture were purchased from Biochrom GmbH (Berlin, Germany). All other chemicals used were obtained from Sigma Aldrich (Taufkirchen, Germany). Pharmaceutics 2020, 12, 1038 3 of 16 2.2. Animal Treatment Animal experiments were conducted using male Swiss/CD-1 mice (4–6 weeks old, average weight = 25 g) purchased from Janvier (Saint-Berthevin, France). All animal experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (N01-OD-4-2139 Task Order #188, 2011, Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, US). Experiments were performed at the University of Burgundy/Franche-Comté in Besançon, France in compliance with French legislation on animal experimentation under experimentation authorization no. A-25-48. The 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis model was used as it enables colitis induction at an exact predetermined location [12]. All mice (n = 5) were acclimatized to laboratory conditions for one week preceding the start of the experiments with food and water ad libitum. Food was withdrawn from the animals 24 h before the start of the experiment, although water was provided. The mice were lightly narcotized using isoflurane prior to intrarectal catheterization (4 cm) to insert 100 µL of TNBS in 50% ethanol at a dose of 90 mg/kg body weight. The mice were kept for 24 h without any treatment to allow the full development of colitis. Subsequently, the mice were treated with 30 mg/kg body weight of chitosan solutions made up using different grades of chitosan. The treatment was also administered using intrarectal catheterization (4 cm) in a volume of 100 µL for three consecutive days to ensure a full compound deposition at the site of inflammation. The control group received saline solution for three consecutive days after induction of colitis with TNBS. The mice