A series of studies were carried out in Farber disease (OMIM #228000) cells and mice to evaluate the feasibility of enzyme replacement therapy (ERT) for this disorder. Media from Chinese hamster ovary (CHO) cells overexpressing human recombinant acid ceramidase (rhAC) was used to treat fibroblasts from a Farber disease patient, leading to significantly reduced ceramide. We also found that chondrocytes from Farber disease mice had a markedly abnormal chondrogenic phenotype, and this was corrected by rhAC as well. Acute dosing of rhAC in Farber mice confirmed the enzyme's bioactivity in vivo, and showed that it could be safely administered at doses up to 50 mg/kg. These studies also revealed little or no re-accumulation of ceramide in tissues for at least 7 days after enzyme administration. Once weekly administration of rhAC moderately improved survival of the mice, which could be enhanced by starting enzyme administration at an earlier age (3 days vs. 3 weeks). Repeat administration of the enzyme also led to normalization of spleen size, significantly reduced plasma levels of monocyte chemoattractant protein 1 (MCP-1), reduced infiltration of macrophages into liver and spleen, and significantly reduced ceramide and sphingosine in tissues. Overall, we conclude that ERT should be further developed for this debilitating and life-threatening disorder.

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ∼2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

As seen in other MPS animal models, subQ PPS treatment reduced plasma cytokine levels and macrophage infiltration in systemic tissues. ICV administration did not elicit these systemic effects. SubQ PPS administration also significantly impacted brain neuropathology, inflammation, and behavior. The effect of early subQ treatment was more significant than dose. Surprisingly, ICV PPS treatment had intermediate effects on most of these brain markers, perhaps due to the limited dose and/or duration of treatment. Consistent with these neuropathological findings, we also observed significant improvements in the hyperactivity/anxiety and learning behaviors of the MPS IIIA mice treated with early subQ PPS.

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Background The mucopolysaccharidoses (MPS) comprise a group of lysosomal storage disorders (LSDs) resulting from inherited deficiencies of 11 specific glycosaminoglycan (GAG) degrading enzymes. In certain types of MPS (those without significant CNS involvement), bone and joint disease and progressively worsening contractures have the greatest negative influence on patients' quality of life, and lead to severe disability. We have previously shown that GAG storage leads directly to activation of the TLR4/TNF-alpha inflammatory pathway, and that inflammation is a major contributor to bone and connective tissue pathology in MPS[1]. This pattern of inflammation resembles that occurring in arthritis, and we feel that the naturally occurring MPS animal models provide excellent systems in which to study disease pathogenesis and treatment. By using TNF-alpha blocking agents, we were able to show a decrease in inflammatory cytokines and an associated improvement in bone and joint changes in MPS animals[2]. Currently, treatment approaches available for MPS patients do not prevent worsening of joint pathology. Objectives We recently identified an FDA-approved drug, pentosan polysulphate (SP-54, PPS), which resulted in reduction in inflammatory cytokine levels and remarkable improvements in bone and joint disease, as well as overall physical functioning, in a rat model of MPS type VI. Our goal was to assess dose-related effects of the medication to further establish the basis for clinical trials. Methods We previously reported the effects of daily oral PPS treatment in MPS VI rats. Here we compare results of using oral dosing vs. weekly subcutaneous (sc) injections. Four groups of MPS VI rats were treated with weekly sc PPS at human equivalent doses of 1, 2 and 4 mg/kg. Results The most efficacious sc PPS dose was 2 mg/kg. Treatment of all MPS VI rats with weekly sc PPS for up to 6 months was safely tolerated. Conclusions We propose that weekly subcutaneous injections of PPS will be efficacious and safe in MPS patients with joint disease, and a phase 1/2 clinical study is planned. Further assessment of the role of inflammation in joint pathology associated with MPS, along with exploration of MPS joint disease as a type of arthritis model are also topics of our group's continuing research. References Simonaro C, et al., Mechanism of glycosamionglycan-mediated bone and joint disease. Am J Path. 2008;172 (1): 112-22. Eliyahu E, et al., Anti-TNF-alpha therapy enhances the effects of enzyme replacement therapy in rats with mucopolysaccharidosis type VI. PLoS One. 2011;6(8):e22447. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.3340