Michael Frohbergh

Dose responsive effects of subcutaneous pentosan polysulfate injection in mucopolysaccharidosis type VI rats and comparison to oral treatment.

Background We previously demonstrated the benefits of daily, oral pentosan polysulfate (PPS) treatment in a rat model of mucopolysaccharidosis (MPS) type VI. Herein we compare these effects to once weekly, subcutaneous (sc) injection. The bioavailability of injected PPS is greater than oral, suggesting better delivery to difficult tissues such as bone and cartilage. Injected PPS also effectively treats osteoarthritis in animals, and has shown success in osteoarthritis patients. Methodology/Principal Findings One-month-old MPS VI rats were given once weekly sc injections of PPS (1, 2 and 4 mg/kg, human equivalent dose (HED)), or daily oral PPS (4 mg/kg HED) for 6 months. Serum inflammatory markers and total glycosaminoglycans (GAGs) were measured, as were several histological, morphological and functional endpoints. Overall, weekly sc PPS injections led to similar or greater therapeutic effects as daily oral administration. Common findings between the two treatment approaches included reduced serum inflammatory markers, improved dentition and skull lengths, reduced tracheal deformities, and improved mobility. Enhanced effects of sc treatment included GAG reduction in urine and tissues, greater endurance on a rotarod, and better improvements in articular cartilage and bone in some dose groups. Optimal therapeutic effects were observed at 2 mg/kg, sc. No drug-related increases in liver enzymes, coagulation factor abnormalities or other adverse effects were identified following 6 months of sc PPS administration. Conclusions Once weekly sc administration of PPS in MPS VI rats led to equal or better therapeutic effects than daily oral administration, including a surprising reduction in urine and tissue GAGs. No adverse effects from sc PPS administration were observed over the 6-month study period.

The molecular medicine of acid ceramidase.

Abstract Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is the lipid hydrolase responsible for the degradation of ceramide into sphingosine and free fatty acids within lysosomes. The enzymatic activity was first identified over four decades ago and is deficient in two rare inherited disorders, Farber lipogranulomatosis (Farber disease) and spinal muscular atrophy with myoclonic epilepsy (SMA-PME). Importantly, AC not only hydrolyzes ceramide into sphingosine within acidic compartments, but also can synthesize ceramide from sphingosine at neutral pH, suggesting that the enzyme may have diverse functions depending on its subcellular location and the local pH. Within cells, AC exists in a complex with other lipid hydrolases and requires a polypeptide cofactor (saposin D) for full hydrolytic activity. Recent studies also have shown that AC is overexpressed in several human cancers, and that inhibition of this enzyme may be a useful cancer drug target. Aberrant AC activity has also been described in several other common diseases. The cDNA and gene (ASAH1) encoding AC have been isolated, several mouse models of AC deficiency have been constructed, and the recombinant enzyme is currently being manufactured for the treatment of Farber disease and SMA-PME. Current information concerning the biology of this enzyme and its role in human disease is reviewed within.

Pentosan Polysulfate: Oral Versus Subcutaneous Injection in Mucopolysaccharidosis Type I Dogs

Background We previously demonstrated the therapeutic benefits of pentosan polysulfate (PPS) in a rat model of mucopolysaccharidosis (MPS) type VI. Reduction of inflammation, reduction of glycosaminoglycan (GAG) storage, and improvement in the skeletal phenotype were shown. Herein, we evaluate the long-term safety and therapeutic effects of PPS in a large animal model of a different MPS type, MPS I dogs. We focused on the arterial phenotype since this is one of the most consistent and clinically significant features of the model. Methodology/Principal Findings MPS I dogs were treated with daily oral or biweekly subcutaneous (subQ) PPS at a human equivalent dose of 1.6 mg/kg for 17 and 12 months, respectively. Safety parameters were assessed at 6 months and at the end of the study. Following treatment, cytokine and GAG levels were determined in fluids and tissues. Assessments of the aorta and carotid arteries also were performed. No drug-related increases in liver enzymes, coagulation factors, or other adverse effects were observed. Significantly reduced IL-8 and TNF-alpha were found in urine and cerebrospinal fluid (CSF). GAG reduction was observed in urine and tissues. Increases in the luminal openings and reduction of the intimal media thickening occurred in the carotids and aortas of PPS-treated animals, along with a reduction of storage vacuoles. These results were correlated with a reduction of GAG storage, reduction of clusterin 1 staining, and improved elastin integrity. No significant changes in the spines of the treated animals were observed. Conclusions PPS treatment led to reductions of pro-inflammatory cytokines and GAG storage in urine and tissues of MPS I dogs, which were most evident after subQ administration. SubQ administration also led to significant cytokine reductions in the CSF. Both treatment groups exhibited markedly reduced carotid and aortic inflammation, increased vessel integrity, and improved histopathology. We conclude that PPS may be a safe and useful therapy for MPS I, either as an adjunct or as a stand-alone treatment that reduces inflammation and GAG storage.

Acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model

OBJECTIVE The overall aim of this study was to evaluate how supplementation of chondrocyte media with recombinant acid ceramidase (rhAC) influenced cartilage repair in a rat osteochondral defect model. METHODS Primary chondrocytes were grown as monolayers in polystyrene culture dishes with and without rhAC (added once at the time of cell plating) for 7 days, and then seeded onto Bio-Gide® collagen scaffolds and grown for an additional 3 days. The scaffolds were then introduced into osteochondral defects created in Sprague-Dawley rat trochlea by a microdrilling procedure. Analysis was performed 6 weeks post-surgery macroscopically, by micro-CT, histologically, and by immunohistochemistry. RESULTS Treatment with rhAC led to increased cell numbers and glycosaminoglycan (GAG) production (∼2 and 3-fold, respectively) following 7 days of expansion in vitro. Gene expression of collagen 2, aggrecan and Sox-9 also was significantly elevated. After seeding onto Bio-Gide®, more rhAC treated cells were evident within 4 h. At 6 weeks post-surgery, defects containing rhAC-treated cells exhibited more soft tissue formation at the articular surface, as evidenced by microCT, as well as histological evidence of enhanced cartilage repair. Notably, collagen 2 immunostaining revealed greater surface expression in animals receiving rhAC treated cells as well. Collagen 10 staining was not enhanced. CONCLUSION The results further demonstrate the positive effects of rhAC treatment on chondrocyte growth and phenotype in vitro, and reveal for the first time the in vivo effects of the treated cells on cartilage repair.

Biomimetic Scaffolds for Craniofacial Bone Tissue Engineering: Understanding the Role of the Periosteum in Regeneration

The role of the periosteum in bone tissue engineering is a new and exciting development. Although its regenerative capacity is known and its role in initiating wound healing is well-documented, a complete understanding of the underlying mechanisms and specific cues that cause healing induction is still unknown. Recently, a number of different studies have begun to explore how stimulating periosteal recruitment is involved in regeneration. In this chapter we review the importance of the periosteum as well as a number of different materials used to activate and initiate the healing process indicative of the periosteum. Our own work has focused on using electrospun chitosan/hydroxyapatite composite scaffolds in order to integrate the native periosteal tissue with our material and instigate the healing process in critical size calvarial bone defects. Critical size defects remain elusive and problematic in the clinic to date and tissue engineering is a promising candidate to alleviate such problems. In this chapter we will briefly review our material and its ability to induce osseointegration, osteoinduction and support the formation of new, mineralized tissue in a murine model. This material, along with others, reflect promising and auspicious developments in musculoskeletal tissue engineering and are helping to pave the way in understanding how the periosteum is involved in wound healing.

A63: Treatment of Arthritis in Animal Models of the Mucopolysaccharidoses Using a Novel Anti-Inflammatory Drug, Pentosan Polysulfate

The mucopolysaccharidoses (MPS) comprise a group of 11 lysosomal storage disorders (LSDs) due to inherited deficiencies of glycosaminoglycan (GAG) degrading enzymes. Naturally occurring animal models exist for most of these disorders, providing excellent systems in which to study disease pathogenesis and treatment. We have previously shown that GAG storage in MPS leads to activation of the TLR4/TNF‐alpha inflammatory pathway, and that inflammation is a major contributor to the degenerative cartilage disease occurring in MPS patients. The pattern of inflammatory changes in MPS closely resembles that occurring in arthritis, and the MPS animals also represent excellent, naturally occurring genetic models of arthritis. Various treatment approaches have been investigated for MPS, and we recently identified one FDA‐approved drug, pentosan polysulfate (PPS), that resulted in remarkable clinical improvements in a rat model of MPS type VI (Schuchman et al., PLoS One, 2013).