purified ProCTSK 개발 및 Pycnodysostosis 마우스 모델과 섬유아세포를 이용하여 시행한 in vitro, in vivo 실험을 근거로 한 치료 효과 분석
Development of purified ProCTSK and investigation of its effectiveness through in vitro and in vivo studies using skin fibroblasts and CTSK knock out mouse model for pycnodysostosis
Abstract
Pycnodysostosis is a rare autosomal recessive bone disorder resulting from osteoclast dysfunction with an estimated prevalence of 1 to 1.7 per million. The disorder is caused by mutation in cathepsin K (CTSK), which is a lysosomal cysteine protease that is highly expressed in osteoclasts. CTSK is involved in the degradation of bone matrix proteins, type I and type II collagen, osteopontin, and osteonectin at a low pH. Patients usually present with short stature, an increase in the bone density of long bones, pathological fractures with poor healing, stubby hands and feet with dystrophic nails, unossified fontanels, and obtuse mandibular angle. Frequent fractures, craniosynostosis, respiratory-sleep problems, and dental problems and their treatments may cause significant suffer to the patients. However, no specific treatment for this disorder has been described, and only conservative management for each symptom can be considered. In this study, we tried to develop new treatment drug for pycnodysostosis. Purified ProCTSK was activated in acidic condition and activated ProCTSK had enzymatic activity. We confirmed that purified ProCTSK was able to degrade not only human but also mouse type I collagen. ProCTSK entered into patients skin fibroblast and could be activated in cells. This was a clue that ProCTSK was located lysosomes after uptake. In addition, ProCTSK entered into cells as a dose dependent manner. As shown in skin fibroblast, ProCTSK was able to enter into osteoclasts treated with siRNA, and activated there, where are the main target of pycnodysostosis. From these results, we expected that ProCTSK would be able to show an efficacy on in vivo studies. We generated CTSK knock out mouse model, and urine CTX-I was determined as a biomarker. Micro computed tomography, bone strength, and biodistribution were evaluated. Based on these results, ProCTSK replacement tended to improve urine CTX-I and bone status, however, there was no significant effectiveness in pycnodysostosis model mice although we observed ProCTSK was able to distribute to the bones and remain for twenty four hours. This is first trial to develop new treatment for pycnodysostosis, and this trial can be considered in other diseases such as osteopetrosis. Further studies with different formulation, dosage and frequency to overcome short half-life and bone targeting effectiveness are planned.