Development of mucoadhesive 3D-printed Carbopol/Eudragit/SNAC tablets for the oral delivery of enoxaparin: In vitro and ex vivo evaluation

3D-printed dosage forms comprised of Carbopol and Eudragit were fabricated through semi-solid extrusion, combining Enoxaparin (Enox) and the permeation enhancer SNAC in a single-step process without subsequent post-processing. Inks were characterized using rheology and Fourier-transform infrared (FTIR) spectroscopy. The stability of Enox in the fabricated dosage forms was assessed by means of Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) analysis. In vitro release studies revealed the release of Enox in a sustained manner, whereas ex vivo experiments demonstrated the mucoadhesive properties of the 3D-printed dosage forms and their ability to enhance Enox permeability across intestinal mucosa. Cellular assays (CCK-8 assay) revealed a dose- and time-dependent response following incubation with the 3D-printed dosage forms. The encapsulation of SNAC in the 3D-printed dosage forms demonstrated their capacity to increase the transcellularly transport of macromolecule across Caco-2 monolayer in a reversible manner, as confirmed by Transepithelial Resistance (TEER) measurements.

Introduction

Low molecular weight heparins (LMWHs) are derived from unfractionated heparin (UFH) through chemical and enzymatic breakdown processes and are widely utilized due to their reduced adverse effects and superior antithrombotic effectiveness compared to heparin (Lanke et al., 2009, Tang et al., 2020). Enox is a LMWH used to prevent and treat thrombotic disorders for both preventing and treating deep vein thrombosis (DVT) and pulmonary embolism. The mechanism of the anticoagulant effect is attributed to Enox binding to antithrombin (AT), catalyzing the indirect inhibition of factor Xa (FXa) and, to a lesser extent, factor IIa (FIIa) (Patriota et al., 2021). The current form of injectable aqueous solution for intravenous and subcutaneous administration limits its usage due to the discomfort and inconvenience associated with injections. Moreover, its brief duration of effectiveness requires frequent administration, affecting patient adherence when compared to non-invasive alternatives. Therefore, there are several efforts to deliver Enox via different routes of administration e.g. oral, nasal and pulmonary in an attempt to match the efficacy of injections, ensuring ease of use and safety (Lee et al., 2007). However, therapeutic strategies relying on heparin face limitations since its absorption in the intestine is hindered by its high molecular weight, hydrophilic characteristics and negative charged structure and to their vulnerability to physiological challenges, like first-pass metabolism and degradation in the gastrointestinal (GI) tract by proteolytic enzymes leading to ineffective absorption (Maretti et al., 2021, Patriota et al., 2021).

Creating an oral Enox formulation is crucial for extended anticoagulant therapy in chronic conditions, mitigating the challenges of daily injections of out-patient treatment, minimizing side effects, and enhancing patient adherence to treatment. This could enhance acceptance and compliance compared to injectable forms, particularly for patients preferring oral treatment, potentially enabling earlier initiation of these agents in disease management. Strategies involving formulation, such as enteric coatings (i.e. Eudragit L100), the use of microparticles, nanoparticles, lipid-drug conjugates and co-administration with absorption enhancers have been investigated to address the obstacle of delivering LMWHs orally (Lamprecht et al., 2007, Li et al., 2014, Paliwal et al., 2011, Tang et al., 2020, Twarog et al., 2022). Hydrophobic alteration of LMWH proves effective in improving its oral absorption. Utilizing solid lipid nanoparticles (SLNs) loaded with LMWH-saturated fatty acid conjugates (e.g., stearic, palmitic, and myristic acid) significantly enhanced bioavailability (p <0.05) compared to unmodified LMWH-loaded SLNs (Paliwal et al., 2011). Polymeric nanoparticles offer benefits such as safeguarding against acidic denaturation and enzymatic degradation, amplifying contact and absorption area, improving permeability through intestinal membranes, and enabling controlled release (Patriota et al., 2021).

Permeation enhancers such as N-[8-(2-hydroxybenzoyl)amino] caprylate (SNAC), a derivative of a minor fatty acid, have been extensively investigated for over two decades in oral formulations aiming to improve peptide permeability. The mechanism involves enhancing the flow of peptides that are poorly absorbed, achieved by modifying either the transcellular route through plasma membrane disruption or the paracellular route by adjusting tight junctions (Twarog et al., 2022). In particular, a weak and reversible association was observed between the lipophilic SNAC and API (Active Pharmaceutical Ingredient), which facilitates the transit across the intestinal barrier and easily disintegration upon entering the bloodstream due to simple dilution (Arbit et al., 2006). Recent research indicates that incorporating a carrier molecule such as SNAC may enhance the gastrointestinal absorption of both UFH and LMWH, achieving sufficient levels for preventing and treating venous thromboembolic disease in both animal and human subjects (Arbit et al., 2006).

The Eligen® technology utilizes low molecular weight substances (referred to as drug delivery agents or carriers) that have a mild, non-covalent interaction with insulin. This interaction enhances insulin’s lipophilicity, thereby improving its capacity to traverse the gastrointestinal epithelium and enter the bloodstream. The effectiveness of this technology has been showcased in numerous clinical trials encompassing both healthy volunteers and individuals with diabetes (Malkov et al., 2005). The initial oral protein delivery formulation, Rybelsus® (Novo-Nordisk, Bagsvaerd, Denmark), gained approval from the Food and Drug Administration (FDA) in 2019, marking a milestone as the first oral peptide treatment for type 2 diabetes (Rasmussen, 2020). This formulation co-formulates oral semaglutide with the absorption enhancer SNAC to aid glycemic control. SNAC functions by increasing local pH, preserving semaglutide from enzymatic degradation, and encouraging its absorption through the gastric epithelium in a concentration-dependent manner (Rasmussen, 2020). Furthermore, it promotes semaglutide absorption across the gastric epithelium, with the extent depending on its concentration, temporarily and reversibly impacting transcellular pathways. Semaglutide absorption primarily concentrates in a specific region, contingent on the proximity of macromolecule and permeation enhancer (Rasmussen, 2020).

The literature on orally administered macromolecules using 3D printing technology is limited and focused mainly on fused filament fabrication. Previous studies have been investigated the encapsulation of a macromolecular dye in a discontinuous manner and the development of a 3D-printed osmotic capsule loaded with octreotide, which releases API under pressures higher than 300 mbar (Berg et al., 2021, Eleftheriadis et al., 2020). In another study, an intestinal administration device (IAD) was developed using fused deposition modeling on which mini tablets for orally co-administered peptide MEDI7219 and a permeation enhancer were loaded, achieved the drug release at a specific point in the intestine due to IAD design (Berg et al., 2023). Our previous work, it was the first attempt for the direct encapsulation of octreotide in 3D printing inks, followed by additional processing to make the 3D-printed dosage forms easier to be handled (Chatzitaki et al., 2023).

The objective of this study was to develop 3D-printed orally administered dosage forms, co-administering LMWH and SNAC in a single step and an eco-friendly process. This method was designed to avoid the use of organic solvents, high-energy homogenization, and heat to ensure minimal harm to the drug candidate. Subsequently, the Enox-loaded dosage forms were assessed for their physicochemical properties and further analyzed for drug release profiles, cytotoxicity, and intestinal permeability.

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Materials

Carbopol 974P NF and Eudragit® L100 (Mw of 135 kDa, apparent viscosity of 50–200 mPa s) were supplied by Lubrizol (Brussels, Belgium) and Evonik (Essen, Germany), respectively. α-Lactose monohydrate (DCL21TM) was obtained from DMV International (Veghel, Netherlands). Enoxaparin (Enox, Clexane ® 4000 IU/0.4 mL, average molecular weight 4500 Da) was purchased by Sanofi Aventis (Athens, Greece).

Aikaterini-Theodora Chatzitaki, Michaela Patila, Stamatis Haralampos, Ioannis S. Vizirianakis, Eleni A. Rekka, Dimitrios Tzetzis, Apostolos Spyros, Constantinos K. Zacharis, Christos Ritzoulis, Dimitrios G. Fatouros, Development of mucoadhesive 3D-printed Carbopol/Eudragit/SNAC tablets for the oral delivery of enoxaparin: In vitro and ex vivo evaluation, International Journal of Pharmaceutics, 2024, 124627, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2024.124627.

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