Nanocrystal-Polymer Particles: Extended Delivery Carriers for Osteoarthritis Treatment.

An efficient treatment for osteoarthritis (OA) can benefit from the local release of a high therapeutic dose over an extended period of time. Such a treatment will minimize systemic side effects and avoid the inconvenience of frequent injections. To this aim, nanocrystal-polymer particles (NPPs) are developed by combining the advantages of nanotechnology and microparticles. Nanocrystals are produced by wet milling kartogenin (KGN), which is known to promote chondrogenesis and to foster chondroprotection. A fluorescent biodegradable polymer is synthesized for intravital particle tracking. Polymer microparticles with 320 nm embedded KGN nanocrystals (KGN-NPPs) show a high drug loading of 31.5% (w/w) and an extended drug release of 62% over 3 months. In vitro, these particles do not alter mitochondrial activity in cultured human OA synoviocytes. In vivo, KGN-NPPs demonstrate higher bioactivity than a KGN solution in a murine mechanistic OA model based on histological assessment (Osteoarthritis Research Society International score), epiphyseal thickness (microcomputed tomography), OA biomarkers (e.g., vascular endothelial growth factor, Adamts5), and prolonged intra-articular persistence (fluorescence analysis). This work provides proof-of-concept of a novel and innovative extended drug delivery system with the potential to treat human OA.

Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens.

Candida albicans is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC50s ≤ 32 µg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato (Solanum lycopersicum) and exhibited high levels of fungistatic activity against Candida species (MIC50s ≤ 1 µg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated C. albicans cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole-genome sequencing identified 2 nonsynonymous mutations in ERG6 (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent.

Recent advances in intra-articular drug delivery systems for osteoarthritis therapy.

Osteoarthritis (OA) is the most common degenerative disease of the joint. Despite many reports and numerous clinical trials, OA is not entirely understood, and there is no effective treatment available for this disease. To satisfy this unmet medical need, drug delivery systems (DDSs) containing disease-modifying OA drugs (DMOADs) for intra-articular (IA) administration are required to improve the health of OA patients. DDSs should provide controlled and/or sustained drug release, enabling long-term treatment with a reduced number of injections. This paper reviews the role and interaction among different tissues involved in OA and summarizes recent clinical trials and research on DDSs, focusing on small-molecule delivery. To achieve an ideal treatment, various key criteria have been identified to design and develop an IA DDS matching the clinical needs.

Nanocrystals of a potent p38 MAPK inhibitor embedded in microparticles: Therapeutic effects in inflammatory and mechanistic murine models of osteoarthritis.

This study aimed to formulate nanocrystal-polymer particles (NPPs) containing the potent p38α/ß MAPK inhibitor PH-797804 (PH-NPPs) and to test their extended-release properties over months in comparison to those of conventional PH microparticles for the intra-articular treatment of inflammatory and mechanistic murine models mirroring aspects of human osteoarthritis (OA). The steps of the study were (i) to formulate PH nanocrystals (wet milling), (ii) to encapsulate a high payload of PH nanocrystals in fluorescent particles (spray drying), (iii) to assess in vitro drug release, (iv) to evaluate PH-NPP toxicity to human OA synoviocytes (MTT test), (v) to investigate the in vivo bioactivity of the particles in mice in an inflammatory antigen-induced arthritis (AIA) model (using histology and RT-qPCR) and (vi) to investigate the in vivo bioactivity of the particles in the OA model obtained by mechanistic surgical destabilization of the medial meniscus (DMM) (using histology, micro-CT, and multiplex ELISA). The PH nanocrystals stabilized with vitamin E TPGS had a monomodal size distribution. The PH-NPPs had a mean diameter of 14.2 µm and drug loading of ~31.5% (w/w), and ~20% of the PH was released over 3 months. The NPPs did not exhibit toxicity to cultured human OA synoviocytes at 100 × IC50. Finally, in vivo studies showed good retention of PH-NPPs in the joint and adjacent tissues for up to 2 months, and the PH-NPPs exhibited good functional relevance by significantly reducing inflammation and joint destruction and by inhibiting several biomarkers (e.g., IL-1ß). In conclusion, local treatment with PH-NPPs, used as an extended-release drug delivery system, improved inflammation and joint degradation in two distinct mouse models, indicating treatment potential for human OA.

Self-assembled thermoresponsive nanostructures of hyaluronic acid conjugates for osteoarthritis therapy.

Under pathological conditions, joints and skin are often affected by an imbalance in the breakdown and production of hyaluronic acid (HA). The unique biochemical and biomechanical properties provided by HA must be restored for the long-term lubrication and cushioning effects. To overcome the inconvenience of repeated injections and the rapid degradation of exogenous HA treatments, HA is conjugated to a thermosensitive polymer, enabling the spontaneous formation of nanoparticles (HA Nano) at body temperature. Three HA Nano preparations are tested for their injectability, sensitivity to enzymatic degradation and cytocompatibility. One of them is delivered via subcutaneous and intra-articular injections to healthy mice and tested in a murine osteoarthritis (OA) model. It is found to be biocompatible, to offer a prolonged residence time at the injection site, have the ability to protect cartilage, to reduce pro-inflammatory cytokines and to preserve epiphysis thickness. In this study, HA Nano spontaneously forms nanoparticles at body temperature in vivo and is a promising candidate for the next generation of the sustainable/long-lasting treatment of OA and potentially also dermatological conditions.

Effect of particle size on the biodistribution of nano- and microparticles following intra-articular injection in mice.

Intra-articular (IA) injection of extended drug release forms based on biodegradable microparticles holds promise for the treatment of joint diseases. However, the fate of microparticles following intra-articular injection is controversial and has not been thoroughly investigated. The aim of this work was therefore to evaluate the biodistribution of fluorescent poly(lactic acid) particles of different sizes after IA injection in arthritic or healthy mice. Regardless of the inflammatory status of the joint, 300 nm-nanoparticles leaked from the joint. Due to inflammation and related increase of vascular permeability, 3 µm-microparticles that were retained in the non-inflamed synovial membrane leaked from the inflamed joint. Complete retention of 10 µm-microparticles was observed independently of the joint inflammatory status. Embedding particles in a hyaluronic acid gel prolonged the retention of the formulations only in inflamed joints. Depending on particle's size, formulations were preferentially eliminated by blood vessels or lymphatic pathways. Poly(lactic acid) particles of 3 µm were biocompatible and retained in knee joints at least for 6 weeks. This work highlights the need to deliver hyaluronic acid-embedded particles of at least 3 µm to guarantee their retention in inflamed joints. These results will contribute to the rational design of long-lasting formulations to treat acute and chronic joint diseases.

Intra-articular bioactivity of a p38 MAPK inhibitor and development of an extended-release system.

In the treatment of arthritic diseases, oral or systemic administration of anti-inflammatory substances, such as p38 MAPK inhibitors, is hampered by numerous side effects. To overcome them, formulations of rapid and extended drug delivery systems were studied in intra-articular administration. For the first time, VX-745, a highly selective p38 MAPK inhibitor, demonstrated in vivo bioactivity, similar to dexamethasone activity, following intra-articular administration in an antigen-induced arthritic (AIA) mouse model. The in vitro bioactivity of VX-745 was also shown on synoviocytes, reducing the IL-6 concentration. Process and formulation parameters (i.e., polymer concentration, aqueous/organic phase ratio, emulsification speed and process, and evaporation pressure) and particle characterisation (i.e., drug loading, size of particle, and surface aspect) were extensively examined to produce optimised formulations. Indeed, a burst release provides a rapid saturation of intracellular p38 MAPK to relieve patients from pain and inflammation. Then, drug diffusion would be sufficient to maintain an effective dose over 2-3 months. This study confirms the effectiveness of encapsulated p38 MAPK inhibitors in extended drug delivery systems and seems to be a promising strategy for intra-articular treatment.