Targeted Rheumatoid Arthritis Therapy with HA-Coated Leflunomide NLCs

Abstract

Systemic rheumatoid arthritis treatment by disease modifying anti-rheumatics has been associated with numerous side effects. Herein an attempt to develop hyaluronic acid (HA) coated leflunomide (LEF) loaded nanostructured lipid carriers (NLCs) that can passively and actively target inflamed rheumatic joints following oral administration and hence decrease systemic side effects, improve LEF anti-arthritic potential. In vitro characterization of NLCs including colloidal characteristics, entrapment efficiency, drug loading, FTIR, DSC and in vitro drug release were carried out. Also, in vivo evaluation by studying LEF-NLCs pharmacokinetics, anti-arthritic effects, hepatotoxicity and nephrotoxicity.

The particle size of NLCs was 284.9 nm with 96.89% entrapment efficiency. The particles had a spherical shape and provided sustained release for about 30 days. DSC results revealed the presence of LEF in amorphous form. No chemical interactions were observed in FTIR spectra. In vivo studies revealed the ability of NLCs to increase LEF bioavailability compared to LEF suspension. Histopathological examinations revealed improved joint healing with no signs of hepatotoxicity. Furthermore, NLCs were able to reduce TNF α serum level.

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The superiority of HA coated NLCs may be attributed to their ability to target inflamed rheumatic joints passively and actively through targeting CD44 receptors that are overexpressed in rheumatic joints.

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease that can affect joints causing severe disability. Over the years, treatment strategy has been shifted from symptoms treatment (steroids and NSAIDs) to suppression of RA progression by (Disease modifying anti-rheumatics (DMARDs), biologic drugs and gene therapy). One of the DMARDs is leflunomide (LEF) that is considered efficient to turn aside RA progression.

LEF is a prodrug that is converted to active metabolite teriflunomide upon administration. Teriflunomide acts by inhibiting dihydroorotate dehydrogenase enzyme that is responsible for de novo pyrimidine biosynthesis and this will eventually regulate RA autoimmune reactions. Systemic LEF administration has been accompanied by several side effects like diarrhea, esophagitis and colitis. Furthermore, LEF can cause more serious side effects as it can also affect the liver and the lungs.

Nanocarriers can be used to minimize side effects related to systemic drug administration. Nanocarriers can selectively accumulate in the inflamed arthritic region by passive targeting. A recent study revealed that passive targeting of inflammatory sites may involve a new retention mechanism that resembles the classical enhanced permeability and retention (EPR). “ELVIS” stands for extravasation through the leaky vasculature and subsequent inflammatory cell-mediated sequestration.

Besides passive targeting, nanocarriers can also target the joints by active targeting mechanism through the modification of the nanocarrier’s surface with ligands that can target specific receptors that are usually overexpressed in inflamed joints. Thus, active targeting strategies will be able to improve treatment outcomes, decrease systemic sides and eventually increase patient compliance.

Various types of cells are activated in inflamed synovial regions, such as macrophages, vascular endothelial cells and fibroblast-like synoviocytes. CD44, folate, scavenger receptor type A and notch receptors are overexpressed on activated macrophages Hyaluronic acid (HA) is a naturally occurring glucosaminoglycan that can target CD44 receptors that are overexpressed on inflamed joint cells like macrophages.

The aim of the current study is nanoencapsulation LEF in nanocarriers that can selectively target inflamed rheumatic joints following oral administration. Besides promoting joint healing and reducing undesirable side effects associated with systemic LEF administration. To achieve this, LEF loaded nanostructured lipid carriers (NLCs) were prepared. And to ensure passive synergistic and/or active targeting, LEF-NLCs were further coated with HA. LEF loaded HA coated NLCs were orally administered to healthy and RA-induced rats. The influence of NLCs on LEF pharmacokinetic profile and anti-arthritic effects of the prepared NLCs were investigated.

Materials and Methods

Materials

Key materials included Precirol ATO 5, caproyl 90, LEF, teriflunomide, high molecular weight HA, and other analytical grade chemicals.

Preparation of HA Coated LEF Loaded NLCs

LEF loaded NLCs were prepared using the hot melt emulsification method, followed by coating with HA to target CD44 receptors overexpressed in inflamed rheumatic joints.

In Vitro and In Vivo Evaluations

Evaluations included particle size analysis, entrapment efficiency, morphological examination, FTIR, DSC, in vitro drug release, pharmacokinetic analysis in healthy rats, and the assessment of anti-arthritic effects, hepatotoxicity, and nephrotoxicity in an adjuvant-induced arthritis rat model.

Results

Colloidal Characteristics and Morphology

Characteristics of HA Coated LEF Loaded NLCs

Pharmacokinetic Parameters in Rats

TNFα Serum Levels and Joint Diameter Changes

In vitro release studies showed sustained LEF release over 30 days. Pharmacokinetic analysis revealed increased LEF bioavailability with HA coated NLCs compared to LEF suspension. Anti-arthritic effects were significantly enhanced, with reduced TNF α serum levels and improved joint healing observed.

Adult male Sprague Dawley rats weighing (170 ± 20 g) were maintained at 25 ± 2ºC and 50-60% RH. A 12-h dark/light cycle was maintained throughout the study. Rats had free access to a standard pellet diet and distilled water and libitum. All experimental procedures in this study were conducted according to the published guidelines of the Ethical Committee for Care and Use of Laboratory Animals (Faculty of Pharmacy Alexandria University). Rats were divided into four groups (n=4). Adjuvant-induced rheumatoid arthritis rat model (AA) was prepared by injecting 200 µl of complete Freund's adjuvant in the right knee of each rat, while the left knee was kept as control.

On the fourth day of induction, treatment was started and extended for 14 days. During the treatment period, rats administered an oral daily dose equivalent to 10 mg LEF per kg body weight. The first group administered LEF suspension while the second group administered HA coated LEF-NLCs. The third and fourth groups were positive and negative control groups, respectively.

Discussion

The study demonstrated the effectiveness of HA coated LEF loaded NLCs in targeting inflamed rheumatic joints, thereby improving LEF's pharmacokinetic profile and anti-arthritic effects while minimizing systemic side effects. The use of HA for active targeting to CD44 receptors contributed to the NLCs' enhanced therapeutic efficacy.

Conclusion

HA coated LEF loaded NLCs represent a promising strategy for targeted rheumatoid arthritis treatment, offering a significant reduction in systemic side effects and improved therapeutic outcomes. Further research is warranted to optimize this delivery system for clinical application.