J Control Release. 2013 Apr 28;167(2):120-9.

An intra-articular salmon calcitonin-based nanocomplex reduces experimental inflammatory arthritis.

Ryan SM, McMorrow J, Umerska A, Patel HB, Kornerup KN, Tajber L, Murphy EP, Perretti M, Corrigan OI, Brayden DJ.

UCD School of Veterinary Medicine and Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

 

Abstract

Prolonged inappropriate inflammatory responses contribute to the pathogenesis of rheumatoid arthritis (RA) and to aspects of osteoarthritis (OA). The orphan nuclear receptor, NR4A2, is a key regulator and potential biomarker for inflammation and represents a potentially valuable therapeutic target. Both salmon calcitonin (sCT) and hyaluronic acid (HA) attenuated activated mRNA expression of NR4A1, NR4A2, NR4A3, and matrix metalloproteinases (MMPs) 1, 3 and 13 in three human cell lines: SW1353 chondrocytes, U937 and THP-1 monocytes. Ad-mixtures of sCT and HA further down-regulated expression of NR4A2 compared to either agent alone at specific concentrations, hence the rationale for their formulation in nanocomplexes (NPs) using chitosan. The sCT released from NP stimulated cAMP production in human T47D breast cancer cells expressing sCT receptors. When NP were injected by the intra-articular (I.A.) route to the mouse knee during on-going inflammatory arthritis of the K/BxN serum transfer model, joint inflammation was reduced together with NR4A2 expression, and local bone architecture was preserved. These data highlight remarkable anti-inflammatory effects of sCT and HA at the level of reducing NR4A2 mRNA expression in vitro. Combining them in NP elicits anti-arthritic effects in vivo following I.A. delivery. Copyright © 2013 Elsevier B.V.

PMID: 23391443

 

Supplement

This is one of the most important papers to emerge from this lab in the past decade and there is an interesting story to it.  The reason for its high impact are several fold. First, it addresses an area a real unmet medical need, the fact that apart from oral NSAIDS, intra-articular therapies for osteoarthritic joints are restricted to steroids and hyaluronic acid (HA) lubricants.  Most reviews have concluded however, that the benefits of either agent on pain scores are moderate and temporary and do  not address the underlying condition (1), with cost-benefit issues for HA and some potential for systemic side effects in the case of steroids.  We know that we need new disease-modifying drugs for osteoarthritis, but are there agents out there that can be repurposed and combined with existing agents in a proper controlled release nanoparticle implant rather than leaking away out of the joint as current therapies do?  The second reason for the significant impact was that several strands came together and it involved combining disciplines, getting training in the animal model, and designing an ‘all-or-nothing’ mouse study with some confidence of success, based on very solid in vitro cell data.

The sequence was as follows: my group was trying to develop an oral formulation of the anti-osteoporotic peptide, salmon calcitonin (sCT), and we noticed that it was being touted in the literature for having lesser-known anti-inflammatory actions and indeed was in an oral clinical trial for that condition.  Could we repurpose sCT for intra-articular delivery in osteoarthritis?   Next, my colleague in the University College Dublin Vet School, Dr.  Evelyn Murphy, is a renowned expert on the orphan receptor family, NR4A, which is a potential biomarker of inflammation.  Two postdocs combined to insert sCT in established assays to knock down mRNA for three NR4A receptors, as well as for a host of metalloproteinases linked to inflammation in several human macrophage models.  Then we asked the question, ‘What if we combined sCT with HA?’  The in vitro results showed a synergy in the knock-down of inflammatory marker mRNA, which was a novel mechanism for each agent and a discovery in itself, but it created additional excitement as to what we could do next.  I had a collaboration with Trinity College’s School of Pharmacy and Dr.  Lidia Tajber is able to make nanocomplexes of various molecules using chitosan and HA by electrostatic interaction; she made sCT-HA chitosan nanocomplexes (Figure 1) and characterised them, and we demonstrated that the released sCT was bioactive on cells.

 

fig dbFigure 1. Hyaluronic acid was combined with chitosan (CL113) to create a composite, which surrounded salmon calcitonin electrostatically to form a stable nanocomplex.

 

The next piece in the jigsaw was to twig that inflammation is a very important part of osteoarthritis and not just rheumatoid arthritis and that we should test the nanocomplexes in an inflammatory model.  What would happen if we injected them into the joints of an arthritic animal model?  We scoured the literature and focussed on an acute serum transfer murine model of inflammatory arthritis, the K/BxN (2). It would be really novel to generate efficacy in this new model rather than the collagen-induced chronic model, which is very time consuming and can give unreliable efficacy data.   How do you set up the K/BxN mouse and do the key study in a reasonable time frame?  In London, the leading pharmacologist, Prof.  Mauro Perretti at the William Harvey Institute, agreed to host our postdoc, Dr.  Sinead Ryan, on a Science Foundation Ireland (SFI) Travel Grant to work on the established K/BxN mouse model in his lab.  Crucially, Perretti had a supply of potent serum, which meant reliable induction of joint inflammation. This was a better idea than setting it up ourselves, which could have taken over a year to get ethical approval, a licence and then to establish the model.  Data from a single injection of the nanocomplexes to the joint of K/BxN mice in Prof. Perretti’s lab showed dramatic reductions in swelling and improvement in joint histology, on a par with the gold standard dexamethasone steroid injection.  We filed a patent in 2012 and wrote the JCR paper in 2013.  The paper has weaknesses in that we have yet to label the particles to show how long they reside in the joint, nor have we optimised them in terms of composition ratios.  However, we are confident that the nanoparticle controlled release concept is robust, as within a year of our paper, another group independently showed that nanocomplexes of sCT conjugated to HA could reduce joint-associated inflammation and damage in a different animal model (3).  We are currently planning a study to inject the nanocomplexes into the joints of a lipopolysaccharide equine model (4), as a large animal model of inflammation would be more appropriate to examine this therapeutic concept, which can potentially benefit human and animal health. A long-lasting safe and efficacious joint injection that is not based on steroids would be a significant advance on current systems.

 

References

  1. Ayhan E, Kesmezacar H, Akgun I. 2014 Intraarticular injections (corticosteroid, hyaluronic acid, platelet rich plasma) for the knee osteoarthritis. World J Orthop. 5:351-361
  2. Christianson CA, Corr M, Yaksh TL, Svensson CI 2012 K/BxN serum transfer arthritis as a model of inflammatory joint pain. In:  Pain Research:  Methods and Protocols, Methods in Molecular Biology, 851: 249-260. Ed. Luo, D. Z. Springer, New York.
  3. Mero A, Campisi M, Favero M, Barbera C, Secchieri C, Dayer JM, Goldring MB, Goldring SR, Pasut G. 2014 A hyaluronic acid-salmon calcitonin conjugate for the local treatment of osteoarthritis: chondro-protective effect in a rabbit model of early OA. J Control Release 187:30-38.
  4. de Grauw JC, van de Lest CH, Brama PA, Rambags BP, van Weeren PR 2009 In vivo effects of meloxicam on inflammatory mediators, MMP activity and cartilage biomarkers in equine joints with acute synovitis. Equine Vet J. 41:693-699.

 

Acknowledgments This study was funded by SFI’s Strategic Research Cluster Grant 07/SRC/B1154 (Irish Drug Delivery Network), by an SFI Short Term Travel Fellowship Grant 07/SRC/B1154 STTF 09, and by The William Harvey Research Foundation.

Contact

David Brayden, Ph.D.

Professor of Advanced Drug Delivery

Room 214, Veterinary Sciences Centre

University College Dublin

Belfield, Dublin 4, Ireland

Tel:  +3531 7166013

http://www.ucd.ie/vetmed/staff/veterinarysciences/davidbrayden/

 

 

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