(M1030-05-30) Development of Nanosuspension Loaded Dissolving Microneedles for Delivery of a Novel Combination of Drugs for Treatment of Rheumatoid Arthritis

Purpose: Rheumatoid Arthritis (RA) causes joint inflammation and severe pain due to increased proinflammatory factors. The current line of therapies relies on pain reduction via the use of NSAIDs, DMRADs, and recently approved products based on TNF-α antibodies that are administered via an invasive parenteral route. The missing piece of the puzzle can be dual-targeting the inflammatory pathway, which blocks the NF-Kappa B promoter activity in the nucleus via selective BRD4 inhibition via use of proteolysis-targeted chimera (PROTAC), which is a bifunctional large molecule based on a combination of BRD4 inhibitor and E3 ubiquitin ligase with a linker, i.e., ARV-825(ARV) in combination with an FDA approved anti-inflammatory molecule, edaravone (EDA). The solubility constraints of drugs set a challenge to enhance their effect via transdermal route, hence, ArVEDA (ARV-825 and EDA) would be administered in the form of nanosuspension loaded in dissolving microneedles (MNs). The double-edged sword of efficacy and the need for multiple painful injections is mitigated using novel techniques to formulate painless needle patch delivery systems. Microneedle-based drug delivery allows for improved patient compliance and enhances drug bioavailability by delivering drugs locally without any environmental barriers. Therefore, the rationale for this current study is to evaluate the effectiveness of the combination of ARV with Edaravone on TNF-α and inflammatory marker levels for treating RA. To further enhance availability at the desired site while making the therapy more patient-centric, microneedle formulation strategy is considered in our research.

Methods: The MNs were prepared via solvent casting with custom-made molds 6x6 Array, H=1,000µm, Base=250µm, Pitch=1,000µm and Pyramidal in shape obtained from Micropoint Technologies. MNs were loaded with drugs in form of nanosuspension prepared via dual centrifugation using Netzsch Delvita^®1. The significant components of research studies: A) Screening water-soluble polymers (PVA, Pullulan (PL), Petox at 25, 35, 45%) with suitable mechanical properties for MN formation. B) Screening stabilizers (Kolliphor® P407, Cavasol™ W7 HP, Polysorbate 80, Kolliphor® TPGS, Lecithin, petox-oleic acid polymer) to prepare ArVEDA nanosuspension. C) Optimizing ArVEDA-NS loading into MNs. The rheological properties of polymeric solutions were analyzed via Discovery Hybrid Rheometer (DHR-2), and then the mechanical properties of prepared MNs were tested by TA. XT plus Texture Analyzer. The prepared NS batches were analyzed for Particle size using malvern zetasizer. Further, ArVEDA-NS loaded MNs were characterized for insertion using Parafilm® M. Morphological changes were imaged (stereomicroscope) using Rhodamine B dye. Drug release was optimized via the novel dissolution method using 3D printed mold as skin model. Permeability studies using rat skin (Franz cells) are underway. Cytotoxicity of drugs alone/combination were assessed as shown in Fig 1. Anti-inflammatory properties were evaluated on lipopolysaccharide (LPS) model, assessing morphological changes (calcein-DAPI) and LPS effects (CD11b/DAPI immunofluorescence staining, complete cytokine screening of IL-1, IL-6, and TNF-α are under investigation.

Results: Screening water-soluble polymers for dissolving microneedles based on insertion properties led to selecting pullulan for further microneedle development. Fig 1 shows the rheological studies correlated polymer concentrations with microneedle strength, wherein PL enabled effective insertion up to 800 μm at 25-45% concentrations. Moreover, morphological studies for the dimensions of 1000μm microneedles were studied via stereomicroscopy imaging of the Rhodamine B loaded dye shown in fig 2(A). Consequently, PL was investigated for drug loading into MNs due to its best insertion characteristics. In terms of loading of drugs in form of nanosuspension, from the different stabilizers screened, a novel oleic acid conjugated polymer PeTOx provided nanosuspensions with uniform dispersion of nanoformulation in size 296±5.44nm with 0.144 PDI shown in fig 2(B). Nanoformulations prepared via wet media milling were optimized with 10% EDA and 0.2% ARV-825(ArVEDA-NS), which were further freeze dried with 5% mannitol. The in vitro dissolution studies for ArVEDA-NS MNs with the novel ex vivo mold attained >80% drug release within 48 hrs. as depicted in fig 2(C). The in vitro morphological studies with both macrophage cells confirmed the LPS-induced stimulation with 1µg/ml within 24 and 48hrs. To further understand the anti-inflammatory properties of combination cell viability was confirmed with at different concentrations of ARV, EDA and ArVEDA. Fig 3(B) illustrates cell viability studies confirming >80% of cells were viable with the ARV-825(25 and 50 nm) and EDA (5 and 10µM).

Conclusion: The present research aims to tackle rheumatoid arthritis pain by targeting nuclear and cytosolic inflammatory pathways using a novel ArVEDA drug combination. It focuses on developing a formulation technology to effectively deliver ArVEDA-NS via patient compliant, pain free approach via skin in form of microneedles. Findings indicate ArVEDA-NS loaded microneedles potentially provide minimally invasive, targeted therapy overcoming limitations of existing treatments. Future work will assess in vivo efficacy and explore synergistic effects.


Figure 1. Polymer screening using rheological and mechanical properties analysis of water-soluble polymers such as PVA, PeTOx, and Pullulan at 25,35 and 45% concentrations (A) graphs showing the trends in the viscosity of polymers with different concentrations (B) The changes in MN insertion properties are shown which were conducted on parafilm M as ex vivo skin model.


Figure 2. ArVEDA-NS and ArVEDA-NS loaded MNs characterization (A) morphological studies of MNs prepared via stereomicroscope imaging for rhodamine b, NS and polymers alone MNs(B) shows the particle size distribution of NS prepared for combination of drugs using wet media milling with uniform dispersion at 296±5.44nm, with 0.144 PDI(C) In vitro dissolution studies for ArVEDA-NS loaded MNs with both drugs release more than 50% within 24hrs.


Figure 3. Rheumatoid Arthritis Pathophysiology with rationale of combination of BRD4 PROTAC and edaravone for targeted anti-inflammatory properties(3A) Graphical representation of challenges in delivering ArVEDA through skin (3B) graphs showing the in vitro cell viability testing on RAW 264.7 and HEK cell line confirming safety of the corresponding drug concentrations.