Identification of unapproved orthopedic regenerative medicine: Usefulness of the Act on Safety of Regenerative Medicine.

In Japan, the Act on Safety of Regenerative Medicine regulates unapproved regenerative medicine. Other nations market regenerative medicine products, bypassing regulatory approval. To identify unapproved orthopedic regenerative medicine, we have used data based on the Act. Platelet-rich plasma was often used. The common target was the knee. Prices averaged $2,490.

Remote audit practice for inspection of structural and equipment standards for cell processing facilities under the act on the safety of regenerative medicine in Japan.

Introduction: The Act on the Safety of Regenerative Medicine enforced in Japan in 2014, regulates the manufacture of cellular processed products. However, with regards to the manufacturing facilities at medical institutions, only the submission of necessary documents is required for a license, and the need for third-party inspection has been highlighted. Remote activities are becoming more prominent with the spread of the Severe Acute Respiratory Syndrome Coronavirus 2 infection; therefore, the current assessment of compliance with structural facility standards was conducted remotely. Methods: The entire process, including start-up meetings, preparation of the survey schedule, submission and review of preliminary materials, audits, and reporting of results, was conducted via e-mail and web conferencing systems. The survey was conducted remotely, to minimize the risk of contamination of the cell processing facility (CPF) and reduce the burden on surveyors, while contributing to the establishment of suitable structural facilities by identifying and highlighting the areas or items that were considered to be non-compliant with the regulations. The series of audits were completed in ten weeks, with a period of six weeks between the start-up meeting and the audit implementation. The audit was completed in approximately 3 h on the day of the inspection. Results: The audit results were delivered in the report, with four items requiring improvement and several other recommended items listed as non-conformities. Conclusions: We believe that this remote method allows the effective inspection of regenerative medicine manufacturing facilities and assessment of more cell culture processing facilities than the current in-person audit method, with limited human resources.

Safety and immunogenicity of BK-SE36/CpG malaria vaccine in healthy Burkinabe adults and children: a phase 1b randomised, controlled, double-blinded, age de-escalation trial.

Background: BK-SE36/CpG is a recombinant blood-stage malaria vaccine candidate based on the N-terminal Plasmodium falciparum serine repeat antigen5 (SE36), adsorbed to aluminium hydroxide gel and reconstituted, prior to administration, with synthetic oligodeoxynucleotides bearing CpG motifs. In healthy Japanese adult males, BK-SE36/CpG was well tolerated. This study assessed its safety and immunogenicity in healthy malaria-exposed African adults and children. Methods: A double-blind, randomised, controlled, age de-escalating clinical trial was conducted in an urban area of Ouagadougou, Burkina Faso. Healthy participants (n=135) aged 21-45 years (Cohort 1), 5-10 years (Cohort 2) and 12-24 months (Cohort 3) were randomised to receive three vaccine doses (Day 0, 28 and 112) of BK-SE36/CpG or rabies vaccine by intramuscular injection. Results: One hundred thirty-four of 135 (99.2%) subjects received all three scheduled vaccine doses. Vaccinations were well tolerated with no related Grade 3 (severe) adverse events (AEs). Pain/limitation of limb movement, headache in adults and fever in younger children (all mild to moderate in intensity) were the most frequently observed local and systemic AEs. Eighty-three of BK-SE36/CpG (91%) recipients and 37 of control subjects (84%) had Grade 1/2 events within 28 days post vaccination. Events considered by the investigator to be vaccine related were experienced by 38% and 14% of subjects in BK-SE36/CpG and control arms, respectively. Throughout the trial, six Grade 3 events (in 4 subjects), not related to vaccination, were recorded in the BK-SE36/CpG arm: 5 events (in 3 subjects) within 28 days of vaccination. All serious adverse events (SAEs) (n=5) were due to severe malaria (52-226 days post vaccination) and not related to vaccination. In all cohorts, BK-SE36/CpG arm had higher antibody titres after Dose 3 than after Dose 2. Younger cohorts had stronger immune responses (12-24-month-old > 5-10 years-old > 21-45 years-old). Sera predominantly reacted to peptides that lie in intrinsically unstructured regions of SE36. In the control arm, there were no marked fold changes in antibody titres and participants' sera reacted poorly to all peptides spanning SE36. Conclusion: BK-SE36/CpG was well-tolerated and immunogenic. These results pave the way for further proof-of-concept studies to demonstrate vaccine efficacy. Clinical trial registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1921, PACTR201701001921166.

Homoharringtonine is a transdermal granular permeation enhancer.

Although modulation of claudin-1-based tight junction (TJ) in stratum granulosum is an option for transdermal absorption of drugs, granular permeation enhancers have never been developed. We previously found that homoharringtonine (HHT), a natural alkanoid, weakened intestinal epithelial barrier with changing expression and cellular localization of TJ components such as claudin-1 and claudin-4. In the present study, we investigated whether HHT is an epidermal granular permeation enhancer. Treatment of normal human epidermal keratinocytes (NHEK) cells with HHT decreased claudin-1 and claudin-4 but not zonula occludens-1 and E-cadherin. HHT lowered TJ-integrity in NHEK cells, accompanied by permeation-enhancement of dextran (4 kDa) in a dose-dependent manner. Transdermal treatment of mice with HHT weakened epidermal barrier. HHT treatment enhanced transdermal absorption of dextran with a molecular mass of up to 10 kDa. Together, HHT may be a transdermal absorption enhancer.

A phase I dose-escalation, safety/tolerability, and preliminary efficacy study of the intratumoral administration of GEN0101 in patients with advanced melanoma.

Despite recent advance in immunotherapy agents, safe new therapies that enhance the effects of immune checkpoint inhibitors are still required to develop. We previously demonstrated that hemagglutinating virus of Japan-envelope (HVJ-E) induced not only direct tumor cell death but also antitumor immunity through the activation of T and natural killer (NK) cells, thereafter, developed a manufacturing process of HVJ-E (GEN0101) for clinical use. We here performed a phase Ia clinical trial of intratumoral GEN0101 administration in six patients with stage IIIC or IV malignant melanoma. The primary aim was to evaluate the safety and tolerability of GEN0101, and the secondary aim was to examine the objective tumor response. Patients were separated into two groups (n = 3 each) and received a low dose of 30,000 and high dose of 60,000 mNAU of GEN0101. All patients completed a two-week follow-up evaluation without severe adverse events. The overall response rate was 33% (2 of 6), with 2 partial responses in the high-dose group and 2 with stable disease, and 2 with progressive disease in the low-dose group. Local complete or partial responses were observed in 11 of 18 (61%) target lesions. One patient demonstrated shrinkage of lung metastases after the treatment. The activity of NK cells and interferon-γ levels were increased in the circulation, indicating augmentation of antitumor immunity by GEN0101. This trial showed not only the safety and tolerability but also the significant antitumor effect of GEN0101, suggesting that GEN0101 might be a promising new drug for patients with advanced melanoma.

Impact of surgical resection on survival in patients with metastatic soft tissue sarcoma and comparison between synchronous and metachronous metastases.

BACKGROUND: The role of local surgery in patients with metastatic soft tissue sarcoma (STS) remains unknown. The study aims to assess the clinical outcomes and impact of surgical resection on survival in patients with metastatic STS and elucidate the survival differences between synchronous and metachronous metastatic groups. METHODS: Among the 272 patients with STS treated between 2000 and 2018, 84 with synchronous or metachronous metastasis were included. Associations between overall survival and primary tumor resection and metastasectomy were assessed using multivariate Cox regression analyses to adjust for baseline differences between surgically and non-surgically treated patients. Propensity score matching was applied to compare synchronous and metachronous metastasis. RESULTS: Among the 84 patients included, 69 (82%) and 41 (49%) underwent primary tumor resection and metastasectomy, respectively. The 2- and 5-year overall survivals of all patients after first detection of metastasis were 51.1% and 24.4%, respectively. Multivariate analysis showed that size <8 cm, grade <3, and number of metastases <4 were associated with longer overall survival. After adjusting for baseline demographic and tumor characteristics, primary tumor resection and metastasectomy still had favorable effects on survival. Tumor subtypes, grade, and number of metastases differed significantly between synchronous and metachronous groups. However, after adjusting for these valuables, both groups exhibited comparable survival. CONCLUSIONS: Approximately one fourth of the patients with metastatic STS survived for >5 years. Our results showed that surgical resection of primary tumors or metastatic lesions had favorable impact on survival even after adjusting for patient backgrounds, with comparable survival observed between those with synchronous and metachronous metastases.

First-in-human randomised trial and follow-up study of Plasmodium falciparum blood-stage malaria vaccine BK-SE36 with CpG-ODN(K3).

BACKGROUND: BK-SE36 is blood-stage malaria vaccine candidate that is undergoing clinical trials. Here, the safety and immunogenicity of BK-SE36 with a novel adjuvant, CpG-ODN(K3) (thus, BK-SE36/CpG) was assessed in a phase 1a trial in Japan. METHODS: An investigator-initiated, randomised, single-blind, placebo-controlled, dose-escalation study was conducted at Osaka University Hospital with 26 healthy malaria naïve Japanese male adults. The trial was conducted in two stages: Stage/Group 1, half-dose (n = 7 for BK-SE36/CpG and n = 3 for control) and Stage/Group 2, full-dose (n = 11 for BK-SE36/CpG and n = 5 for control). There were two intramuscular vaccinations 21 days apart for both half-dose (0.5 ml: 50 µg SE36 + 500 µg aluminum + 500 µg K3) and full-dose (1.0 ml: 100 µg SE36 + 1000 µg aluminum + 1000 µg K3). A one-year follow-up was done to monitor changes in autoimmune markers and vaccine-induced antibody response. RESULTS: BK-SE36/CpG was well tolerated. Vaccination site reactions were similar to those observed with BK-SE36. During the trial and follow-up period, no subject had clinical evidence of autoimmune disease. The full-dose group had significantly higher titres than the half-dose group (Student's t-test, p = 0.002) at 21 days post-second vaccination. Antibody titres remained above baseline values during 12 months of follow-up. The vaccine induced antibody was mostly composed of IgG1 and IgM, and recognised epitopes close to the polyserine region located in the middle of SE36. CONCLUSIONS: BK-SE36/CpG has an acceptable safety profile. Use of CpG-ODN(K3) greatly enhanced immunogenicity in malaria naïve Japanese adults when compared to BK-SE36 alone. The utility of BK-SE36/CpG is currently under evaluation in a malaria endemic setting in West Africa. TRIAL REGISTRATION: JMACCT Clinical Trial Registry JMA-IIA00109.

Clinical Outcomes of Osteoarticular Extracorporeal Irradiated Autograft for Malignant Bone Tumor.

METHODS: We retrospectively reviewed 33 patients who underwent osteoarticular ECIA after bone tumor resection from 1988 to 2014. We investigated complications, radiographic changes by the International Society of Limb Salvage graft evaluation criteria, and functional outcomes according to the Musculoskeletal Tumor Society scoring system. RESULTS: Fifteen patients were reoperated upon due to infection (n = 9), protruding fixation implant (n = 4), or fracture of the grafted bone (n = 2). The average radiographic evaluation score was 66.4%, and the median functional score was 23 (77%). The radiographic score for the proximal humerus or proximal tibia was lower than that for the other locations. The functional score was not different among the autograft sites but was related to the radiographic score. CONCLUSION: Although osteoarticular ECIA is one of the reasonable surgical options for patients with tumors for which reliable prostheses are not available, we do not recommend osteoarticular ECIA as a routine procedure because of high complication rate.

Intratumoral injection of hemagglutinating virus of Japan-envelope vector yielded an antitumor effect for advanced melanoma: a phase I/IIa clinical study.

Hemagglutinating virus of Japan (HVJ; Sendai virus) is an RNA virus that has cell fusion activity. HVJ-envelope (HVJ-E) is a UV-irradiated HVJ particle that loses viral replication and protein synthesis activity but retains cell fusion activity. We recently reported that HVJ-E has antitumor effects on several types of tumors. Here, we describe the results of a first-in-human phase I/IIa study in patients with advanced melanoma, receiving intratumoral administration of HVJ-E. The primary aim was to evaluate the safety and tolerability of HVJ-E, and the secondary aim was to examine the objective tumor response and antitumor immunity. Six patients with stage IIIC or IV progressive malignant melanoma with skin or lymph metastasis were enrolled. Patients were separated into two groups (n = 3 each) and received low and high doses of HVJ-E. Five of the six patients completed 4 weeks of follow-up evaluation; one patient discontinued treatment owing to progressive disease. Complete or partial responses were observed in 3 of 6 (50%) injected target lesions, 7 of 15 (47%) noninjected target lesions, and 10 of 21 (48%) target lesions. Induction of antitumor immunity was observed: activation of natural killer cells, a marked increase in interferon-γ levels in the peripheral blood, and infiltration of cytotoxic T cells into both injected and noninjected tumor lesions. Thus, intratumoral injection of HVJ-E in advanced melanoma patients showed safety and tolerability with local regression of the tumor mediated by antitumor immunity. The results suggest that HVJ-E might be a new treatment approach in patients with advanced melanoma.

Pediatric drug development in Japan: Current issues and perspectives.

The number of clinical trials in Japan that aim to obtain regulatory approval for new drugs and devices has increased for adults, but not children. The following reasons have been proposed for this discrepancy: the wide range of ages from newborns to adolescents, requirements for many drug formulations, the difficulties associated with obtaining consent, and less profit for companies. The processes required to obtain regulatory approval for drugs and devices, particularly in the pediatric field, differ among Japan, Europe, and the United States (US). While clinical trials are not necessarily required for the development of new drugs or obtaining additional indications in Japan, laws in Europe and the US require clinical trials on children for newly developed drugs; however, pharmaceutical companies are entitled to a 6-mo extension for a patent when pediatric data are added to the attached documents for clinical trials. We herein discuss the current status of and issues associated with pediatric drug development, including clinical trials, in Japan as well as future perspectives.

Author Correction: Establishment of a novel human CIC-DUX4 sarcoma cell line, Kitra-SRS, with autocrine IGF-1R activation and metastatic potential to the lungs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Eribulin Suppresses Clear Cell Sarcoma Growth by Inhibiting Cell Proliferation and Inducing Melanocytic Differentiation Both Directly and Via Vascular Remodeling.

Clear cell sarcoma (CCS) is a rare but chemotherapy-resistant and often fatal high-grade soft-tissue sarcoma (STS) characterized by melanocytic differentiation under control of microphthalmia-associated transcription factor (MITF). Eribulin mesilate (eribulin) is a mechanistically unique microtubule inhibitor commonly used for STS treatment, particularly liposarcoma and leiomyosarcoma. In this study, we examined the antitumor efficacy of eribulin on four human CCS cell lines and two mouse xenograft models. Eribulin inhibited CCS cell proliferation by inducing cell-cycle arrest and apoptosis, shrunk CCS xenograft tumors, and increased tumor vessel density. Eribulin induced MITF protein upregulation and stimulated tumor cell melanocytic differentiation through ERK1/2 inactivation (a MITF negative regulator) in vitro and in vivo Moreover, tumor reoxygenation, probably caused by eribulin-induced vascular remodeling, attenuated cell growth and inhibited ERK1/2 activity, thereby upregulating MITF expression and promoting melanocytic differentiation. Finally, downregulation of MITF protein levels modestly debilitated the antiproliferative effect of eribulin on CCS cells. Taken together, eribulin suppresses CCS through inhibition of cell proliferation and promotion of tumor differentiation by acting both directly on tumor cells and indirectly through tumor reoxygenation.

Establishment of a novel human CIC-DUX4 sarcoma cell line, Kitra-SRS, with autocrine IGF-1R activation and metastatic potential to the lungs.

Approximately 60-70% of EWSR1-negative small blue round cell sarcomas harbour a rearrangement of CIC, most commonly CIC-DUX4. CIC-DUX4 sarcoma (CDS) is an aggressive and often fatal high-grade sarcoma appearing predominantly in children and young adults. Although cell lines and their xenograft models are essential tools for basic research and development of antitumour drugs, few cell lines currently exist for CDS. We successfully established a novel human CDS cell line designated Kitra-SRS and developed orthotopic tumour xenografts in nude mice. The CIC-DUX4 fusion gene in Kitra-SRS cells was generated by t(12;19) complex chromosomal rearrangements with an insertion of a chromosome segment including a DUX4 pseudogene component. Kitra-SRS xenografts were histologically similar to the original tumour and exhibited metastatic potential to the lungs. Kitra-SRS cells displayed autocrine activation of the insulin-like growth factor 1 (IGF-1)/IGF-1 receptor (IGF-1R) pathway. Accordingly, treatment with the IGF-1R inhibitor, linsitinib, attenuated Kitra-SRS cell growth and IGF-1-induced activation of IGF-1R/AKT signalling both in vitro and in vivo. Furthermore, upon screening 1134 FDA-approved drugs, the responses of Kitra-SRS cells to anticancer drugs appeared to reflect those of the primary tumour. Our model will be a useful modality for investigating the molecular pathology and therapy of CDS.

Transplantation of autologous bone marrow-derived mesenchymal stem cells under arthroscopic surgery with microfracture versus microfracture alone for articular cartilage lesions in the knee: A multicenter prospective randomized control clinical trial.

INTRODUCTION: To investigate the efficacy of the transplantation of autologous bone marrow-derived mesenchymal stem cells (BMSCs) under arthroscopy with microfracture (MFX) compared with microfracture alone. METHODS: Eleven patients with a symptomatic articular cartilage defect of the knee were included in the study. They were randomized to receive BMSCs with MFX (cell-T group, n=7) or MFX alone (control group, n=4). Clinical results were evaluated using International Knee Documentation committee (IKDC) knee evaluation questionnaires and the Knee Injury and Osteoarthritis Outcome Score (KOOS) before and 48 weeks after surgery. Quantitative and qualitative assessments of repair tissue were carried out at 48 weeks by T2 mapping of magnetic resonance images (MRIs) and the magnetic resonance observation of cartilage repair tissue (MOCART) scoring system with follow-up MRI. RESULTS: No significant differences between preoperative and postoperative IKDC and KOOS were observed in the cell-T or control group. However, forty-eight weeks after surgery, the cell-T group showed a trend for a greater KOOS QOL score compared with the control group (79.4 vs. 39.1, respectively; P=0.07). The T2 value did not differ significantly between the two groups, but the mean MOCART score was significantly higher in the cell-T group than in the control group (P=0.02). CONCLUSIONS: Compared with MFX alone, BMSC transplantation with MFX resulted in better postoperative healing of the cartilage and subchondral bone as determined by the MOCART score. Clinically, BMSC transplantation with MFX gave a higher KOOS QOL score after 48 weeks.

Three-Dimensional Corrective Osteotomy for Malunited Fractures of the Upper Extremity Using Patient-Matched Instruments: A Prospective, Multicenter, Open-Label, Single-Arm Trial.

BACKGROUND: Medical image processing has facilitated simulation of 3-dimensional (3-D) corrective osteotomy, and 3-D rapid prototyping technology has further enabled the manufacturing of patient-matched surgical guides and implants (patient-matched instruments, or PMIs). However, 3-D corrective osteotomy using these technologies has not been the standard procedure. We aimed to prospectively verify the efficacy and safety of PMIs in corrective osteotomy for deformities of the upper extremity. METHODS: We enrolled 16 patients with a total of 17 bone deformities in the upper extremity. Eight patients had distal radial malunion; 5, distal humeral malunion; and 3, forearm diaphyseal malunion. All cases underwent 3-D corrective osteotomy with PMIs. The primary end point was the residual maximum deformity angle (MDA), which was calculated from 2 deformity angles-1 on the anteroposterior and 1 on the lateral postoperative radiograph. Secondary end points included the deformity angle on radiographs, 3-D error between the preoperative planning model and the postoperative result, range of motion, grip strength, pain measured with a visual analog scale (VAS), patient satisfaction, and Disabilities of the Arm, Shoulder and Hand (DASH) score. RESULTS: The average MDA significantly improved from 25.5° preoperatively to 3.3° at the final follow-up (p < 0.001). The angular deformity was within 5° in all cases, except for 1 with distal radial malunion who had a higher angle on the anteroposterior radiograph. The error between the correction seen on the postoperative 3-D bone model and the planned correction was <1° and <1 mm. Flexion and extension of the wrist and pronation of the forearm of the patients treated for distal radial malunion improved significantly, and pronation improved for those treated for forearm diaphyseal malunion. The average VAS score, grip strength, and DASH score significantly improved as well. Of the 16 patients, 15 were very satisfied or satisfied with the outcomes. CONCLUSIONS: Corrective osteotomy using PMIs achieved accurate correction and good functional recovery in the upper extremity. Although our study was limited to cases without any deformity on the contralateral side, 3-D corrective osteotomy using PMIs resolved treatment challenges for complex deformities in upper extremities. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Impact of non-thermal plasma surface modification on porous calcium hydroxyapatite ceramics for bone regeneration.

In the physiochemical sciences, plasma is used to describe an ionized gas. Previous studies have implicated plasma surface treatment in the enhancement of hydrophilicity of implanted musculoskeletal reconstructive materials. Hydroxyapatite (HA) ceramics, widely used in bone tissue regeneration, have made great advancements to skeletal surgery. In the present study, we investigate the impact of low-pressure plasma on the interconnected porous calcium hydroxyapatite (IP-CHA) both in vitro and in vivo. Our results indicate that dielectric barrier discharge (DBD) plasma, when used with oxygen, can augment the hydrophilicity of non-porous HA surfaces and the osteoconductivity of the IP-CHA disc via increased water penetration of inner porous structures, as demonstrated through microfocus computed tomography (µCT) assay. In vivo implantation of plasma-treated IP-CHA displayed superior bone ingrowth than untreated IP-CHA. Though plasma-treated IP-CHA did not alter osteoblast cell proliferation, it accelerated osteogenic differentiation of seeded marrow mesenchymal stem cells. In vitro X-ray photoelectron spectroscopy (XPS) revealed that this plasma treatment increases levels of oxygen, rather than nitrogen, on the plasma-treated IP-CHA surface. These findings suggest that plasma treatment, an easy and simple processing, can significantly improve the osteoconductive potential of commonly used artificial bones such as IP-CHA. Further optimization of plasma treatment and longer-term follow-up of in vivo application are required toward its clinical application.

Time-Dependent Recovery of Human Synovial Membrane Mesenchymal Stem Cell Function After High-Dose Steroid Therapy: Case Report and Laboratory Study.

BACKGROUND: The use of mesenchymal stem cells from various tissue sources to repair injured tissues has been explored over the past decade in large preclinical models and is now moving into the clinic. PURPOSE: To report the case of a patient who exhibited compromised mesenchymal stem cell (MSC) function shortly after use of high-dose steroid to treat Bell's palsy, who recovered 7 weeks after therapy. STUDY DESIGN: Case report and controlled laboratory study. METHODS: A patient enrolled in a first-in-human clinical trial for autologous implantation of a scaffold-free tissue engineered construct (TEC) derived from synovial MSCs for chondral lesion repair had a week of high-dose steroid therapy for Bell's palsy. Synovial tissue was harvested for MSC preparation after a 3-week recovery period and again at 7 weeks after therapy. RESULTS: The MSC proliferation rates and cell surface marker expression profiles from the 3-week sample met conditions for further processing. However, the cells failed to generate a functional TEC. In contrast, MSCs harvested at 7 weeks after steroid therapy were functional in this regard. Further in vitro studies with MSCs and steroids indicated that the effect of in vivo steroids was likely a direct effect of the drug on the MSCs. CONCLUSION: This case suggests that MSCs are transiently compromised after high-dose steroid therapy and that careful consideration regarding timing of MSC harvest is critical. CLINICAL RELEVANCE: The drug profiles of MSC donors and recipients must be carefully monitored to optimize opportunities to successfully repair damaged tissues.

ONO-1301 Enhances in vitro Osteoblast Differentiation and in vivo Bone Formation Induced by Bone Morphogenetic Protein.

STUDY DESIGN: In vitro and in vivo assessment of osteogenic effect by prostacyclin agonist (ONO-1301). OBJECTIVE: The aim of this study was to investigate the effects of ONO-1301 on in vitro osteoblastic differentiation and in vivo bone formation induced by bone morphogenetic protein (BMP). SUMMARY OF BACKGROUND DATA: Among prostaglandins (PGs), PGE2 is the most abundant in bone tissue and its effects on bone formation have been well studied. PGI2 (prostacyclin) is the second most abundant PG in bone tissue and plays important roles in hemodynamics. However, the effects of PGI2 on osteoblast differentiation and bone regeneration have not been elucidated. METHODS: The effects of PGI2 agonist (ONO-1301), with and without recombinant human (rh) BMP-2, on osteoblastic differentiation and cell proliferation were investigated in vitro using alkaline phosphatase (ALP) and WST-1 assays. Murine primary osteoblasts and cell lines (ST2, MC3T3-E1, C2C12, and CH310T1/2) were used for the study. The effects of ONO-1301 on rhBMP-2 induced bone formation were investigated in a mouse model of muscle pouch transplantation (ectopic model) and in a rat model of spinal fusion (orthotopic model). RESULTS: ONO-1301 significantly increased ALP activity in the primary osteoblasts and ST2 cells. In addition, cotreatment with ONO-1301 and rhBMP-2 significantly increased ALP activity in the primary osteoblasts, as well as in ST2 and MC3T3-E1 cells. Cell proliferation was not affected by both ONO-1301 and ONO-1301 as well as rhBMP-2. In the ectopic model, ONO-1301 significantly increased the volume of ectopic bone whose formation was induced by BMP. In addition, in the orthotopic model, ONO-1301 significantly increased bone volume and fusion rate. CONCLUSION: This study has demonstrated that the PG IP agonist ONO-1301 improves in vitro BMP-2 induced osteoblast differentiation and in vivo ectopic and orthotopic bone formation. The results suggest that ONO-1301 has a potential clinical application as an enhancer of BMP-induced bone formation. LEVEL OF EVIDENCE: N/A.

Trabectedin is a promising antitumor agent potentially inducing melanocytic differentiation for clear cell sarcoma.

Clear cell sarcoma is an aggressive soft tissue sarcoma and highly resistant to conventional chemotherapy and radiation therapy. This devastating disease is defined by EWSR1-ATF1 fusion gene resulting from chromosomal translocation t(12;22)(q13;q12) and characterized by melanocytic differentiation. A marine-derived antineoplastic agent, trabectedin, inhibits the growth of myxoid liposarcoma and Ewing sarcoma by causing adipogenic differentiation and neural differentiation, respectively. In this study, we examined the antitumor effects and mechanism of action of trabectedin on human clear cell sarcoma cell lines. We showed that trabectedin decreased the cell proliferation of five clear cell sarcoma cell lines in a dose-dependent manner in vitro and reduced tumor growth of two mouse xenograft models. Flow cytometry and immunoblot analyses in vitro and immunohistochemical analysis in vivo revealed that trabectedin-induced G2/M cell cycle arrest and apoptosis. Furthermore, trabectedin increased the expression of melanocytic differentiation markers along with downregulation of ERK activity in vitro and the rate of melanin-positive cells in vivo. These results suggest that trabectedin has potent antitumor activity against clear cell sarcoma cells by inducing cell cycle arrest, apoptosis, and, in part, by promoting melanocytic differentiation through inactivation of ERK signaling. Our present study indicates that trabectedin is a promising differentiation-inducing agent for clear cell sarcoma.

Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma.

BACKGROUND: The prognosis of synovial sarcoma (SS), an aggressive soft tissue sarcoma, remains poor. We previously reported that c-MET or platelet-derived growth factor receptor α (PDGFRα) signalling pathway is related to SS progression based upon the findings of phospho-receptor tyrosine kinase (RTK) arrays. TAS-115 is a novel c-MET/ vascular endothelial growth factor receptor-targeting tyrosine kinase inhibitor that has been shown to inhibit multiple RTKs. Here we aimed to investigate the therapeutic potential of TAS-115 against SS. METHODS: We first evaluated which signalling pathway was relevant to the viability of three human SS cell lines: Yamato-SS, SYO-1 and HS-SY-II. Next, we assessed the anticancer activity and mechanism of action of TAS-115 in these SS cell lines. Finally, we compared the ability of TAS-115 to inhibit c-MET and PDGFRα phosphorylation with that of pazopanib. RESULTS: We classified the SS cell lines as c-MET-dependent or PDGFRα-dependent based upon the differences in the signalling pathway relevant for growth and/or survival. We also found that c-MET and PDGFRα were the primary activators of both phosphatidylinositol 3-kinase/AKT and mitogen-activated protein kinase pathways in c-MET-dependent and PDGFRα-dependent SS cells, respectively. TAS-115 treatment blocked the phosphorylation of PDGFRα as well as that of c-MET and their downstream effectors, leading to marked growth inhibition in both types of SS cell lines in in vitro and in vivo studies. Furthermore, PDGFRα phosphorylation, on at least four representative autophosphorylation sites, was impeded by TAS-115 equivalently to pazopanib. CONCLUSIONS: These experimental results have demonstrated the significance of c-MET and PDGFRα signalling for growth and/or survival of SS tumours. TAS-115 monotherapy may benefit SS patients whose tumours are dependent upon either c-MET or PDGFRα signalling by functioning as a multiple tyrosine kinase inhibitor to suppress c-MET as well as PDGFRα pathways.