Dual action tofacitinib-loaded PLGA nanoparticles alleviate colitis in an IBD mouse model

Abstract

Inflammatory bowel disease (IBD) affects over 7 million people worldwide and significant side effects are associated with current therapies such as tofacitinib citrate (TFC), which is linked to increased risks of malignancy and congestive heart issues. To mitigate these systemic adverse effects, localised drug delivery via nano-sized carriers to inflamed gut tissues represents a promising approach. Herein, we aimed to optimise the synthesis of nanoparticles (NPs) using a low molecular weight grade of Poly(lactic-co-glycolic acid) (PLGA) 50:50 loaded with TFC. This approach leverages the dual anti-inflammatory action of TFC and the local production of anti-inflammatory short-chain fatty acids from the degradation of PLGA by colonic gut microbiota. NPs were produced by nanoprecipitation and characterised for their drug release profile in vitro. The efficacy of the enhanced PLGA-TFC NPs was then tested in a C57BL/6 DSS colitis mouse model. The release profile of TFC from the enhanced PLGA NPs showed a 40% burst release within the first hour, followed by up to 80% drug release in the colonic environment. Notably, the degradation of PLGA by colonic gut microbiota did not significantly influence TFC release. In the mouse model, neither PLGA NPs alone nor TFC alone showed significant effects on weight loss compared to the TFC-loaded PLGA NPs, emphasising the enhanced efficacy potential of the combined formulation. Altogether, these results suggest a promising role of NP delivery systems in enhancing TFC efficacy, marking a significant step towards reducing dosage and associated side effects in IBD treatment. This study underscores the potential of PLGA-TFC NPs in providing targeted and effective therapy for IBD.

Introduction

Inflammatory bowel disease (IBD) is an umbrella term used to describe two inflammatory conditions of the gastrointestinal (GI) tract, ulcerative colitis (UC) and Crohn’s disease (CD) affecting over 7 million individuals globally [1]. The limited efficacy of current IBD treatments, often accompanied by significant side effect profiles, has prompted the exploration of novel therapeutic modalities [2, 3]. In recent years, this pursuit has led to the emergence of novel anti-inflammatory drugs, such as the Janus kinase (JAK) inhibitors. JAK inhibitors modulate intracellular signal transductions critical to the progression of immune and inflammatory responses thereby preventing the release of inflammatory proteins including over 50 soluble factors [4, 5]. This mechanism of action contrasts significantly with existing therapies on the market, which primarily target specific extracellular cytokines, such as TNF-α inhibitors. However, the clinical application of these therapies has been marred by significant side effects, including an elevated risk of malignancies, cardiovascular disorders, and venous thromboembolism [6, 7]. Tofacitinib (TFC), a recently marketed JAK inhibitors for the treatment of mild to severe UC, has garnered attention due to its safety concerns, leading to regulatory interventions such as an United States Food and Drug Administration (FDA) black box warning and a Medicines and Healthcare products Regulatory Agency (MHRA) black triangle warning [8, 9]. Current usage guidelines advise against its administration in specific patient groups, such as those aged 65 years or older, individuals with a history of long-term smoking, and patients with cardiovascular disease or malignancy risk factors, unless no suitable alternatives are available [9]. Reducing adverse effects associated with JAK inhibitors could serve as a strategic approach for the cautious reintroduction of these drugs to a broader patient demographic.

The current marketed dosage form of TFC is available as both a twice-daily immediate release tablets and a once-daily modified release tablets ensuring more convenient dosing for patients [6, 10, 11], however no colonic targeted formulations are available.Local drug delivery of UC treatments via colonic targeting aims to reduce systemic side effects by enabling high drug concentrations at the site of inflammation [12, 13]. This approach bypasses absorption in the upper GI tract and minimises systemic drug exposure, which typically occurs within one hour of administration with immediate release formulations [14]. Various strategies have been demonstrated to achieve colonic drug delivery, including colon-targeted formulations and azo-bonded prodrugs of TFC [15, 16]. Work by Yadav et al. [16] was particularly promising, they showed that ileocolonic-targeted TFC capsules decreased systemic drug exposure, increased colonic tissue exposure and reduced the levels of the pro-inflammatory cytokine IL-6 in an LPS-induced acute rat model of inflammation [16]. Altogether these results suggest that a reduction in side effects by local drug delivery is possible. As such, a drug delivery system specifically designed to minimise systemic side effects associated with TFC while enhancing its local tissue concentrations, is highly needed [17]. One approach to reduce TFC-associated systemic side effects by is through inflammation-targeted colonic drug delivery.

This method may enable more precise delivery to the inflamed tissue, characterised by the “leaky gut” phenomenon due to gaps in the tight junctions of the epithelial cells. Such a system could employ negatively charged synthetic nanoparticles (NPs) to pass through the leaky gut and target the positively charged inflamed areas [18,19,20]. In this study, we aim to load TFC into poly(lactic-co-glycolic acid) (PLGA) NPs to achieve this targeted delivery [21,22,23].PLGA is a biocompatible and biodegradable polymer composed of repeating units of lactic acid and glycolic acid monomers. PLGA’s versatility in drug delivery arises from its ability to be tailored with different ratios of lactic acid (LA) to glycolic acid (GA), which influence its hydrolytic degradation kinetics [24]. In vivo, the ester bonds in PLGA are hydrolysed into lactic acid and glycolic acid, which are naturally occurring and exit the body by Krebs cycle metabolism as carbon dioxide and water [25]. This unique feature makes PLGA an attractive choice for controlled drug release applications, and it has been approved for pharmaceutical applications by the FDA and the European Medicines Agency (EMA), one of the most notable marketed products being the Zoladex Depot® [26].

PLGA has been extensively used for the formulation of NPs and investigated for the oral drug delivery of several compounds [27,28,29,30]. Apart from its established role as a drug carrier, PLGA has recently gained attention for its own impact on the gut microbiome and colonic health. Recently, it has been shown that a low molecular weight (MW) grade (2000–2500 g/mol) of PLGA is metabolised into lactate which is a precursor in the production of short-chain fatty acids (SCFAs) by the human colonic microbiota and found to reduce the expression of inflammatory markers (Interleukin(IL)-8 and IL-10) in a colonic in vitro model of inflammation [31].

Indeed, SCFAs (i.e. propionate and butyrate) mainly originate from bacterial fermentation of dietary fibre in the colon and are involved in maintaining epithelial barrier function, suppressing colitis, and protecting against immune disorders [32,33,34]. While PLGA is susceptible to hydrolysis, a studies have shown that PEGylated or hyaluronic acid functionalised PLGA nanoparticles can maintain stability within the upper GI environment for colonic targeting [35, 36]. Given the potential benefits of encapsulating TFC to target inflammation in the inflamed leaky gut and of its local colonic delivery (to reduce systemic side effects), we hypothesised that encapsulating TFC within low molecular weight PLGA nanoparticles would be a promising dual-action strategy to treat colitis. TFC’s role would be to reduce inflammation, while PLGA would form the nanoparticle carrier and its metabolism in the colon would result in the formation of the beneficial SCFAs. To test this hypothesis, PLGA particles loaded with TFC were produced, characterised in vitro, and then evaluated in a C57BL/6 DSS colitis mouse model. In this paper, we report on the preparation and physico-chemical characterisation of blank and of drug-loaded PLGA particles, and on their in vivo action in a colitis mouse model.

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Materials

Resomer® Condensate RG 50:50 MN 2300 (PLGA, acid terminated, 50:50, Mw 2000 – 2500 g/mol) AND Resomer® R 202 H, (PLA, acid terminated Mw 10,000—18,000 g/mol) were purchased from Evonik Industries (Essen, Germany). Acetone, Bryant and Burkey broth medium, lipase from porcine pancreas, glycolic acid, poloxamer 407 (Kolliphor® P 407), potassium phosphate monobasic, hexadecyltrimethylammonium bromide, dianisidine dihydrochloride and human neutrophil MPO were purchased from Merck Life Science (Gillingham, UK). Anaerogen packs, purified pepsin (derived from porcine stomach mucosa with an activity of 2,000—2,400 units/mg), sodium chloride, phosphoric acid, HPLC-grade acetonitrile, lecithin, N, N-Dimethylacetamide (DMA), Dextran Sulphate Sodium (DSS) and SYBR Green PCR master mix were purchased from Fisher Scientific (Loughborough, UK). Sodium taurocholate and TFC (MW: 504.49 g/mol) were purchased from Cambridge Bioscience Ltd. (Cambridge, UK). Sodium hydroxide pellets were purchased from VWR International (Pennsylvania, USA). Hydrochloric acid was purchased from LP Chemicals Ltd. (Winsford, UK). Nucleospin RNA II kit was purchased from Macherey–Nagel (Hoerdt, France). Where used, water was of HPLC-grade and obtained via an ELGA HPLC water purification system (ELGA LabWater, High Wycombe, UK).

Seegobin, N., McCoubrey, L.E., Vignal, C. et al. Dual action tofacitinib-loaded PLGA nanoparticles alleviate colitis in an IBD mouse model. Drug Deliv. and Transl. Res. (2024). https://doi.org/10.1007/s13346-024-01736-1

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