Prospective Evaluation of Local Sustained Release of Celecoxib in Dogs with Low Back Pain.

Back pain affects millions globally and in 40% of the cases is attributed to intervertebral disc degeneration. Oral analgesics are associated with adverse systemic side-effects and insufficient pain relief. Local drug delivery mitigates systemic effects and accomplishes higher local dosing. Clinical efficacy of intradiscally injected celecoxib (CXB)-loaded polyesteramide microspheres (PEAMs) was studied in a randomized prospective double-blinded placebo controlled veterinary study. Client-owned dog patients suffering from back pain were treated with CXB-loaded (n = 20) or unloaded PEAMs ("placebo") (n = 10) and evaluated by clinical examination, gait analysis, owners' questionnaires, and MRI at 6 and 12 weeks follow-up. At 6 and 12 weeks, CXB-treated dogs experienced significantly less pain interference with their daily life activities compared to placebo. The risk ratio for treatment success was 1.90 (95% C.I. 1.24-2.91, p = 0.023) at week 6 and 1.95 (95% C.I. 1.10-3.45, p = 0.036) at week 12. The beneficial effects of CXB-PEAMs were more pronounced for the subpopulation of male dogs and those with no Modic changes in MRI at inclusion in the study; disc protrusion did not affect the outcome. It remains to be determined whether intradiscal injection of CXB-PEAMs, in addition to analgesic properties, has the ability to halt the degenerative process in the long term or restore the disc.

Intra-Articular Slow-Release Triamcinolone Acetonide from Polyesteramide Microspheres as a Treatment for Osteoarthritis.

Osteoarthritis (OA) is a common cause of pain and disability. Local corticosteroid injections are effective in treating OA pain and inflammation but are short-acting. Prolonged intra-articular (IA) corticosteroid exposure may even lead to cartilage deterioration. The aim of this prospective study was to assess safety and provide proof-of-concept of IA-applied biodegradable polyesteramide-based microspheres (PEAMs) gradually releasing triamcinolone acetonide (TA). Mimicking continuous exposure associated with local drug delivery in canine articular chondrocytes cultured in the continuous presence of TA tissue regeneration was not affected, whereas intermittent exposure reduced proteoglycan production. In this respect, TA-PEAMs administered IA in a proof-of-concept study in 12 client-owned dogs with established OA also showed safety by radiographic examination, without changes in OA severity and in glycosaminoglycan synovial fluid levels. Treatment also resulted in clinical improvement in 10 out of 11 dogs during the two-month follow-up period, which persisted in 6 out of 10 dogs after 6 months, based on objective gait analysis and owner questionnaires. Synovial prostaglandin E2, a pro-inflammatory marker, was decreased two months after treatment. This study showed safety and proof-of-concept of IA-administered TA-PEAMs in dogs with OA, as a first step towards translation into the veterinary and human clinic.

Local controlled release of corticosteroids extends surgically induced joint instability by inhibiting tissue healing.

BACKGROUND AND PURPOSE: Corticosteroids are intra-articularly injected to relieve pain in joints with osteoarthritis (OA) or acute tissue damage such as ligament or tendon tears, despite its unverified contraindication in unstable joints. Biomaterial-based sustained delivery may prolong reduction of inflammatory pain, while avoiding harmful peak drug concentrations. EXPERIMENTAL APPROACH: The applicability of prolonged corticosteroid exposure was examined in a rat model of anterior cruciate ligament and medial meniscus transection (ACLT + pMMx) with ensuing degenerative changes. KEY RESULTS: Intra-articular injection of a bolus of the corticosteroid triamcinolone acetonide (TAA) resulted in enhanced joint instability in 50% of the joints, but neither instability-induced OA cartilage degeneration, synovitis, nor the OA-related bone phenotype was affected. However, biomaterial microsphere-based extended TAA release enhanced instability in 94% of the animals and induced dystrophic calcification and exacerbation of cartilage degeneration. In healthy joints, injection with TAA releasing microspheres had no effect at all. In vitro, TAA inhibited cell migration out of joint tissue explants, suggesting inhibited tissue healing in vivo as mechanisms for enhanced instability and subsequent cartilage degeneration. CONCLUSIONS AND IMPLICATIONS: We conclude that short-term TAA exposure has minor effects on surgically induced unstable joints, but its extended presence is detrimental by extending instability and associated joint degeneration through compromised healing. This supports a contraindication of prolonged corticosteroid exposure in tissue damage-associated joint instability, but not of brief exposure.

Safety of intradiscal delivery of triamcinolone acetonide by a poly(esteramide) microsphere platform in a large animal model of intervertebral disc degeneration.

BACKGROUND CONTEXT: Local corticosteroids have been used to relieve symptoms of chronic low back pain, although treatment effects have been shown to wear off relatively fast. Prolonging corticosteroid presence by controlled release from biomaterials may allow for longer pain relief while circumventing adverse effects such as high bolus dosages. PURPOSE: The purpose of this study was to evaluate the safety and efficacy of intradiscal controlled release of triamcinolone acetonide (TAA) by poly(esteramide) microspheres in a canine degenerated intervertebral disc (IVD) model. STUDY DESIGN: In a preclinical experimental large animal model, the effect of prolonged glucocorticoid exposure on disc degeneration was evaluated. METHODS: Degeneration was accelerated by nucleotomy of lumbar IVDs of Beagle dogs. After 4 weeks, microspheres loaded with 8.4 µg TAA, and 0.84mg TAA were administered to the degenerated IVDs by intradiscal injection (n=6 per group). Empty microspheres (n=6) and all adjacent non-nucleotomized noninjected IVDs were included as controls (n=24). Immediately prior to TAA administration and after 12 weeks, magnetic resonance imaging was performed. Degenerative changes were evaluated by disc height index, Pfirrmann grading, T1ρ and T2 mapping values, postmortem CT scans, macroscopic and microscopic grading, and biochemical/immunohistochemical analysis of inflammation and extracellular matrix content. In addition, nerve growth factor (NGF) protein expression, a biomarker for pain, was scored in nucleus pulposus (NP) tissues. The study was funded by a research grant from Health Holland (1.3million euros = 1.5million US dollars). RESULTS: Macroscopic evaluation and CT images postmortem were consistent with mild disc degeneration. Other abnormalities were not observed. Nucleotomy-induced degeneration and inflammation was mild, reflected by moderate Pfirrmann grades and PGE2 levels. Regardless of TAA dosage, local sustained delivery did not affect disc height index nor Pfirrmann grading, T1ρ and T2 mapping values, PGE2 tissue levels, collagen, GAG, and DNA content. However, the low dosage of TAA microspheres significantly reduced NGF immunopositivity in degenerated NP tissue. CONCLUSIONS: This is the first in vivo application in a preclinical large animal model of a controlled release formulation of corticosteroids in mild IVD degeneration. Sustained release of TAA locally in the IVD appeared safe and reduced NGF expression, suggesting its potential applicability for pain relief, although beneficial effects were absent on tissue degeneration. CLINICAL SIGNIFICANCE: The present platform seems to be promising in extending the local controlled delivery of TAA with the potency to provide long-standing analgesia in the subset of LBP patients suffering from discogenic pain.

Intra-articular injection of triamcinolone acetonide releasing biomaterial microspheres inhibits pain and inflammation in an acute arthritis model.

Inflammation of the synovium and joint capsule is a main driver of pain in an osteoarthritic (OA) joint. Triamcinolone acetonide (TAA) is a classical corticosteroid that reduces synovitis and alleviates pain, albeit transiently. Biomaterial-based local TAA release may prolong the suppression of pain without the need for multiple injections. Polylactic-co-glycolic acid (PLGA) formulations of TAA prolong OA pain relief to a limited extent. A novel polyesteramide (PEA) microsphere platform allows for extended release in the OA joint for over 3 months. To evaluate their effect on pain and inflammation, TAA-loaded microspheres were intra-articularly delivered to the knee joint in a rat model of acute arthritis induced by intra-articular injection of streptococcal cell wall peptidoglycan-polysaccharide (PGPS) and subsequent flare-ups by intravenous PGPS injections. PEA-loaded microspheres were benchmarked with TAA-loaded PLGA microspheres and bolus TAA injection. TAA treatments were injected intra-articularly before the first induced flare-up. TAA-loaded PEA and PLGA microspheres reduced joint swelling and signs of pain-like behavior over the entire study period, as assessed by weight bearing and referred mechanical hypersensitivity, whereas bolus suspension was effective for a shorter time period. TAA-loaded PEA microspheres reduced lameness to a greater extent than TAA-loaded PLGA microspheres. In conclusion, a single intra-articular injection of TAA-loaded PEA microspheres reduced joint swelling and induced longer pain relief compared to bolus injection. Hence relief of inflammation and pain by PEA-based delivery of TAA may prove to be effective and durable.

Applicability of a Modified Rat Model of Acute Arthritis for Long-Term Testing of Drug Delivery Systems.

Episodes of inflammation and pain are predominant features of arthritic joint diseases. Drug delivery systems (DDS) could reduce inflammation and pain long-term without chances of infection upon multiple injections. To allow for long-term evaluation of DDS, we modified a previously published acute arthritis model by extending follow-up periods between flare-ups. Unilateral synovial inflammation of the knee was induced by intra-articular injection of streptococcal cell wall peptidoglycan polysaccharide (PGPS), and flare-ups were induced by intravenous PGPS injections every 4 weeks for a total duration of 84 days. In PGPS-reactivated animals, joint swelling, pain behavior, post mortem synovitis, and osteophyte formation were notable features. Hepatitis, splenitis and inflammation of non-primed joints were observed as systemic side effects. To test the applicability of the modified arthritis model for long-term testing of DDS, the duration of anti-inflammatory and analgesic effects of a corticosteroid released from two different polymer-based platforms was evaluated. The current modified arthritis model has good applicability for testing of DDS for a prolonged period of time. Furthermore, the novel autoregulatory polyesteramide (PEA) microsphere platform releasing triamcinolone acetonide (TAA) was benchmarked against poly lactic-co-glycolic acid (PLGA) and reduced joint swelling and pain behavior more potently compared to TAA-loaded PLGA microspheres.

Evaluation of polyesteramide (PEA) and polyester (PLGA) microspheres as intravitreal drug delivery systems in albino rats.

PURPOSE: To study the suitability of injectable microspheres based on poly(ester amide) (PEA) or poly lactic-co-glycolic acid (PLGA) as potential vehicles for intravitreal drug delivery in rat eyes. Dexamethasone-loaded PEA microspheres (PEA + DEX) were also evaluated. METHODS: Forty male Sprague Dawley rats were divided into four groups that received different intravitreally injected microspheres: PEA group (n = 12); PLGA group (n = 12); PEA + DEX group (n = 8); and control group (no injection, n = 8). Electroretinography (ERG), fundus autofluorescence (FAF), and spectral domain optical coherence tomography (sdOCT) were performed at baseline, weeks 1 and 2, and months 1, 2, and 3 after intravitreal injection. Eyes were histologically examined using light microscopy and transmission electron microscopy at the end of the in vivo study. RESULTS: There were no statistically significant changes in ERG among the groups. Abnormal FAF pattern and abnormal deposits in OCT were observed after injection but almost completely disappeared between week 2 and month 3 in all injected groups. GFAP staining showed that Müller glia cell activation was most pronounced in PLGA-injected eyes. Increased cell death was not observed by TUNEL staining at month 1. In electron microscopy at month 3, the remnants of microparticles were found in the retinal cells of all injected groups, and loss of plasma membrane was seen in the PLGA group. CONCLUSIONS: Although morphological changes such as mild glial activation and material remnants were observed histologically 1 month and 3 months after injection in all injected groups, minor cell damage was noted only in the PLGA group at 3 months after injection. No evidence of functional abnormality relative to untreated eyes could be detected by ERG 3 months after injection in all groups. Changes observed in in vivo imaging such as OCT and FAF disappeared after 3 months in almost all cases.

Safety of intradiscal injection and biocompatibility of polyester amide microspheres in a canine model predisposed to intervertebral disc degeneration.

Repair of degenerated intervertebral discs (IVD) might be established via intradiscal delivery of biologic therapies. Polyester amide polymers (PEA) were evaluated for in vitro cytotoxicity and in vivo biocompatibility, and thereafter intradiscal application of PEA microspheres (PEAMs) in a canine model predisposed to IVD degeneration at long-term (6 months) follow-up. PEA extracts did not induce cytotoxicity in mouse fibroblast cells (microscopy and XTT assay), while a slight foreign body reaction was demonstrated by histopathology after intramuscular implantation in rabbits. Intradiscal injection of a volume of 40 µL through 26 and 27G needles induced no degenerative changes in acanine model susceptible to IVD disease. Although sham-injected IVDs showed increased CAV1 expression compared with noninjected IVDs, which may indicate increased cell senescence, these findings were not supported by immunohistochemistry, biomolecular analysis of genes related to apoptosis, biochemical and histopathological results. PEAM-injected IVDs showed significantly higher BAX/BCL2 ratio vs sham-injected IVDs suggestive of an anti-apoptotic effect of the PEAMs. These findings were not supported by other analyses (clinical signs, disc height index, T2 values, biomolecular and biochemical analyses, and IVD histopathology). PEAs showed a good cytocompatibility and biocompatibility. PEAMs are considered safe sustained release systems for intradiscal delivery of biological treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 707-714, 2017.

Celecoxib-loaded PEA microspheres as an auto regulatory drug-delivery system after intra-articular injection.

In this study, we investigated the potential of celecoxib-loaded polyester amide (PEA) microspheres as an auto-regulating drug delivery system for the treatment of pain associated with knee osteoarthritis (OA). Celecoxib release from PEA microspheres and inflammation responsive release of a small molecule from PEA was investigated in vitro. Inflammation responsive release of a small molecule from PEA was observed when PEA was exposed to cell lysates obtained from a neutrophil-like Hl-60 cell line. Following a short initial burst release of ~15% of the total drug load in the first days, celecoxib was slowly released throughout a period of >80days. To investigate biocompatibility and degradation behavior in vivo, celecoxib-loaded PEA microspheres were injected in OA-induced (ACLT+pMMx) or contralateral healthy knee joints of male Lewis rats. Bioactivity of celecoxib from loaded PEA microspheres was confirmed by PGE2 measurements in total rat knee homogenates. Intra-articular biocompatibility was demonstrated histologically, where no cartilage damage or synovial thickening and necrosis were observed after intra-articular injections with PEA microspheres. Degradation of PEA microspheres was significantly higher in OA induced knees compared to contralateral healthy knee joints, while loading the PEA microspheres with celecoxib significantly inhibited degradation, indicating a drug delivery system with auto regulatory behavior. In conclusion, this study suggests the potential of celecoxib-loaded PEA microspheres to be used as a safe drug delivery system with auto regulatory behavior for treatment of pain associated with OA of the knee.

CXCL1 microspheres: a novel tool to stimulate arteriogenesis.

CONTEXT: After arterial occlusion, diametrical growth of pre-existing natural bypasses around the obstruction, i.e. arteriogenesis, is the body's main coping mechanism. We have shown before that continuous infusion of chemokine (C-X-C motif) ligand 1 (CXCL1) promotes arteriogenesis in a rodent hind limb ischemia model. OBJECTIVE: For clinical translation of these positive results, we developed a new administration strategy of local and sustained delivery. Here, we investigate the therapeutic potential of CXCL1 in a drug delivery system based on microspheres. MATERIALS AND METHODS: We generated poly(ester amide) (PEA) microspheres loaded with CXCL1 and evaluated them in vitro for cellular toxicity and chemokine release characteristics. In vivo, murine femoral arteries were ligated and CXCL1 was administered either intra-arterially via osmopump or intramuscularly encapsulated in biodegradable microspheres. Perfusion recovery was measured with Laser-Doppler. RESULTS: The developed microspheres were not cytotoxic and displayed a sustained chemokine release up to 28 d in vitro. The amount of released CXCL1 was 100-fold higher than levels in native ligated hind limb. Also, the CXCL1-loaded microspheres significantly enhanced perfusion recovery at day 7 after ligation compared with both saline and non-loaded conditions (55.4 ± 5.0% CXCL1-loaded microspheres versus 43.1 ± 4.5% non-loaded microspheres; n = 8-9; p < 0.05). On day 21 after ligation, the CXCL1-loaded microspheres performed even better than continuous CXCL1 administration (102.1 ± 4.4% CXCL1-loaded microspheres versus 85.7 ± 4.8% CXCL1 osmopump; n = 9; p < 0.05). CONCLUSION: Our results demonstrate a proof of concept that sustained, local delivery of CXCL1 encapsulated in PEA microspheres provides a new tool to stimulate arteriogenesis in vivo.

Novel biodegradable polyesteramide microspheres for controlled drug delivery in Ophthalmology.

Most of the posterior segment diseases are chronic and multifactorial and require long-term intraocular medication. Conventional treatments of these pathologies consist of successive intraocular injections, which are associated with adverse effects. Successful therapy requires the development of new drug delivery systems able to release the active substance for a long term with a single administration. The present work involves the description of a new generation of microspheres based on poly(ester amide)s (PEA), which are novel polymers with improved biodegradability, processability and good thermal and mechanical properties. We report on the preparation of the PEA polymer, PEA microspheres (PEA Ms) and their characterization. PEA Ms (~15µm) were loaded with a lipophilic drug (dexamethasone) (181.0±2.4µg DX/mg Ms). The in vitro release profile of the drug showed a constant delivery for at least 90days. Based on the data from a performed in vitro release study, a kinetic ocular model to predict in vivo drug concentrations in a rabbit vitreous was built. According to the pharmacokinetic simulations, intravitreal injection of dexamethasone loaded PEA microspheres would provide release of the drug in rabbit eyes up to 3months. Cytotoxicity studies in macrophages and retinal pigment epithelial cells revealed a good in vitro tolerance of the microsystems. After sterilization, PEA Ms were administered in vivo by subtenon and intravitreal injections in male Sprague-Dawley rats and the location of the microspheres in rat eyes was monitored. We conclude that PEA Ms provide an alternative delivery system for controlling the delivery of drugs to the eye, allowing a novel generation of microsphere design.

Controlling the kinetic chain length of the crosslinks in photo-polymerized biodegradable networks.

Biodegradable polymer networks were prepared by photo-initiated radical polymerization of methacrylate functionalized poly(D,L-lactide) oligomers. The kinetic chains formed in this radical polymerization are the multifunctional crosslinks of the networks. These chains are carbon-carbon chains that remain after degradation. If their molecular weight is too high these poly(methacrylic acid) chains can not be excreted by the kidneys. The effect of the photo-initiator concentration and the addition of 2-mercaptoethanol as a chain transfer agent on the molecular weight of the kinetic chains was investigated. It was found that both increasing the initiator concentration and adding 2-mercaptoethanol decrease the kinetic chain length. However, the effect of adding 2-mercaptoethanol was much larger. Some network properties such as the glass transition temperature and the swelling ratio in acetone are affected when the kinetic chain length is decreased.

Photo-crosslinked networks prepared from fumaric acid monoethyl ester-functionalized poly(D,L-lactic acid) oligomers and N-vinyl-2-pyrrolidone for the controlled and sustained release of proteins.

Photo-crosslinked networks were prepared from fumaric acid monoethyl ester-functionalized poly(D,L-lactic acid) oligomers and N-vinyl-2-pyrrolidone. Two model proteins, lysozyme and albumin, were incorporated into the network films as solid particles and their release behavior was studied. By varying the NVP content and macromer molecular weight the degradation behavior and protein release profiles of the prepared networks could be tuned. The more hydrophilic and less densely crosslinked networks released albumin and lysozyme at a faster rate. Although active lysozyme was released from the networks over the complete release period, lysozyme release was often incomplete. This was most likely caused by electrostatic and/or hydrophobic interactions between the protein and the degrading polymer network.

Photo-crosslinked biodegradable hydrogels prepared from fumaric acid monoethyl ester-functionalized oligomers for protein delivery.

Photo-crosslinkable, fumaric acid monoethyl ester-functionalized triblock oligomers are synthesized and copolymerized with N-vinyl-2-pyrrolidone to form biodegradable photo-crosslinked hydrogels. Poly(ethylene glycol) is used as the middle hydrophilic segment and the hydrophobic segments are based on D,L-lactide, trimethylene carbonate or a mixture of these monomers. Two model proteins, lysozyme and albumin, are incorporated in the hydrogels and their release is studied. The composition of the hydrophobic segments could be used to tune degradation behavior and release rates. Careful optimization of photo-polymerization conditions is needed to limit conjugation of proteins to the hydrogels and protein denaturation.

Fine-tuning DNA/albumin polyelectrolyte interactions to produce the efficient transfection agent cBSA-147.

We present the preparation and isolation of different chemically modified BSA species with varying numbers of primary amino groups at the surface. Highly cationic albumin proteins with increased numbers of amino groups were achieved and complex formation with plasmid DNA was carefully investigated. We compare the transfection results, polyelectrolyte complexes morphologies with their impact on complex stabilities, cytotoxicities and DNA accessibility. This knowledge-driven approach led to the identification of the efficient non-viral DNA delivery agent cBSA-147, which showed high transfection efficacies and stability.

Novel fluorescent core-shell nanocontainers for cell membrane transport.

The synthesis and characterization of novel core-shell macromolecules consisting of a fluorescent perylene-3,4,9,10-tetracarboxdiimide chromophore in the center surrounded by a hydrophobic polyphenylene shell as a first and a flexible hydrophilic polymer shell as a second layer was presented. Following this strategy, several macromolecules bearing varying polymer chain lengths, different polymer shell densities, and increasing numbers of positive and negative charges were achieved. Because all of these macromolecules reveal a good water solubility, their ability to cross cellular membranes was investigated. In this way, a qualitative relationship between the molecular architecture of these macromolecules and the biological response was established.