Research data for Development of a translational strategy for using TIMP-3 to inhibit aggrecanase activity in osteoarthritis

These data files contain the raw data from the ELISAs used to measure the concentration of the LAP-TIMP-3 protein in mouse sera after injection of the proteins either intravenously (Pk data i.v) or intraperitoneally (Pk data i.p). There is also a file that contains the raw data, again from ELISAs, measuring the levels of LAP-TIMP-3 proteins after injection of varying concentrations of the LAP-TIMP-3 NoCLV protein (Pk dosage data).Access to these ELISA data will allow readers to perform the pharmacokinetic analyses as described in the published work.Article abstractObjectiveTherapeutic potential of selective aggrecanase inhibition in osteoarthritis (OA) was previously demonstrated using a variant of endogenous tissue inhibitor of metalloproteinase-3 (TIMP-3); however, this relied on transgenic mice overexpressing TIMP-3. Here, we develop a translational approach for harnessing the aggrecanase-selective inhibitory activity of TIMP-3 using the latency associated peptide (LAP) technology.MethodsWe successfully produced and purified recombinant LAP-TIMP-3 fusion proteins and determined the pharmacokinetics of these proteins in vivo following systemic injection. Surgical and non-surgical mouse models of OA were used to establish the therapeutic potential of these proteins in reducing aggrecanase activity in mouse joints affected by OA.ResultsThe presence of the LAP conferred favourable TIMP-3 pharmacokinetics, with effective delivery of LAP-TIMP-3 to knee joints after systemic injection. We find that LAP-TIMP-3 also effectively reduced aggrecanase activity in OA-affected joints, both in spontaneously-occurring OA and in the destabilisation of the medial meniscus (DMM) model of OA. We also found that reductions in aggrecanase activity in articular cartilage correlated with improved disease scores, but only in earlier stages of disease.ConclusionsThis study describes the potential of LAP-TIMP-3 as a therapeutic agent in OA, showing delivery to the cartilage of joints affected by OA after systemic administration and lower levels of the neoepitope of aggrecan in articular cartilage in mild disease (mean difference versus vehicle control for LAP-TIMP-3: 535 [95% CI: 336, 733] and for LAP-mutTIMP-3: 522 [95% CI: 323, 720] arbitrary units). These first in vivo data will inform further explorations into dose optimization and timing.

本批数据文件包含酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）的原始检测数据，用于测定经静脉注射（药代动力学数据i.v.）或腹腔注射（药代动力学数据i.p.）后，小鼠血清中LAP-TIMP-3蛋白的浓度。另有一份文件同样包含ELISA原始数据，用于检测注射不同浓度的LAP-TIMP-3 NoCLV蛋白后，LAP-TIMP-3蛋白的表达水平（药代动力学剂量数据）。获取本批ELISA数据可使读者开展已发表研究中所述的药代动力学分析。

论文摘要

研究目的
既往通过过表达金属蛋白酶组织抑制因子-3（tissue inhibitor of metalloproteinase-3, TIMP-3）的转基因小鼠，证实了选择性聚集蛋白聚糖酶（aggrecanase）抑制用于骨关节炎（osteoarthritis, OA）治疗的潜力，但该方法依赖于过表达TIMP-3的转基因小鼠。本研究借助潜伏相关肽（latency associated peptide, LAP）技术，开发了一种可利用TIMP-3聚集蛋白聚糖酶选择性抑制活性的转化研究策略。

研究方法
本研究成功制备并纯化了重组LAP-TIMP-3融合蛋白，并测定了该蛋白经全身注射后的体内（in vivo）药代动力学特征。本研究采用手术性及非手术性骨关节炎小鼠模型，验证了该蛋白在减轻OA受累小鼠关节内聚集蛋白聚糖酶活性方面的治疗潜力。

研究结果
LAP的存在可赋予TIMP-3良好的药代动力学特性，使LAP-TIMP-3经全身注射后可有效递送至膝关节。研究发现，LAP-TIMP-3可有效降低OA受累关节内的聚集蛋白聚糖酶活性，无论是自发性骨关节炎还是内侧半月板失稳（destabilisation of the medial meniscus, DMM）模型骨关节炎均有效。此外，本研究观察到关节软骨（articular cartilage）内聚集蛋白聚糖酶活性的降低与疾病评分改善呈正相关，但该相关性仅存在于疾病早期阶段。

研究结论
本研究阐明了LAP-TIMP-3作为OA治疗剂的潜力：经全身给药后，该蛋白可递送至OA受累关节的软骨组织，且轻度OA模型的关节软骨内聚集蛋白聚糖新表位（neoepitope）水平显著降低（LAP-TIMP-3组与溶剂对照组（vehicle control）的平均差值为535 [95%置信区间（95% confidence interval, 95% CI）：336, 733]，LAP-mutTIMP-3组为522 [95%置信区间：323, 720]，单位为任意单位（arbitrary units））。这批首次发表的体内数据将为后续剂量优化与给药时机探索提供参考。