Potential for Drug Repositioning of Midazolam as an Inhibitor of Inflammatory Bone Resorption.

Drug repositioning is a method for exploring new effects of existing drugs, the safety and pharmacokinetics of which have been confirmed in humans. Here, we demonstrate the potential drug repositioning of midazolam (MDZ), which is used for intravenous sedation, as an inhibitor of inflammatory bone resorption. We cultured a mouse macrophage-like cell line with or without MDZ and evaluated its effects on the induction of differentiation of these cells into osteoclasts. For in vivo investigations, we administered lipopolysaccharide (LPS) together with MDZ (LPS+MDZ) to the parietal region of mice and evaluated the results based on the percentage of bone resorption and calvaria volume. Furthermore, we examined the effects of MDZ on the production of reactive oxygen species (ROS) in cells and on its signaling pathway. MDZ inhibited osteoclast differentiation and bone resorption activity. In animal studies, the LPS+MDZ group showed a decreasing trend associated with the rate of bone resorption. In addition, the bone matrix volume in the LPS+MDZ group was slightly higher than in the LPS only group. MDZ inhibited osteoclast differentiation by decreasing ROS production and thereby negatively regulating the p38 mitogen-activated protein kinase pathway. Thus, we propose that MDZ could potentially be used for treating inflammatory bone resorption, for example, in periodontal disease.

Clearance of heparan sulfate in the brain prevents neurodegeneration and neurocognitive impairment in MPS II mice.

Mucopolysaccharidosis II (MPS II), a lysosomal storage disease caused by mutations in iduronate-2-sulfatase (IDS), is characterized by a wide variety of somatic and neurologic symptoms. The currently approved intravenous enzyme replacement therapy with recombinant IDS (idursulfase) is ineffective for CNS manifestations due to its inability to cross the blood-brain barrier (BBB). Here, we demonstrate that the clearance of heparan sulfate (HS) deposited in the brain by a BBB-penetrable antibody-enzyme fusion protein prevents neurodegeneration and neurocognitive dysfunctions in MPS II mice. The fusion protein pabinafusp alfa was chronically administered intravenously to MPS II mice. The drug reduced HS and attenuated histopathological changes in the brain, as well as in peripheral tissues. The loss of spatial learning abilities was completely suppressed by pabinafusp alfa, but not by idursulfase, indicating an association between HS deposition in the brain, neurodegeneration, and CNS manifestations in these mice. Furthermore, HS concentrations in the brain and reduction thereof by pabinafusp alpha correlated with those in the cerebrospinal fluid (CSF). Thus, repeated intravenous administration of pabinafusp alfa to MPS II mice decreased HS deposition in the brain, leading to prevention of neurodegeneration and maintenance of neurocognitive function, which may be predicted from HS concentrations in CSF.

Pathogenic Roles of Heparan Sulfate and Its Use as a Biomarker in Mucopolysaccharidoses.

Heparan sulfate (HS) is an essential glycosaminoglycan (GAG) as a component of proteoglycans, which are present on the cell surface and in the extracellular matrix. HS-containing proteoglycans not only function as structural constituents of the basal lamina but also play versatile roles in various physiological processes, including cell signaling and organ development. Thus, inherited mutations of genes associated with the biosynthesis or degradation of HS can cause various diseases, particularly those involving the bones and central nervous system (CNS). Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders involving GAG accumulation throughout the body caused by a deficiency of GAG-degrading enzymes. GAGs are stored differently in different types of MPSs. Particularly, HS deposition is observed in patients with MPS types I, II, III, and VII, all which involve progressive neuropathy with multiple CNS system symptoms. While therapies are available for certain symptoms in some types of MPSs, significant unmet medical needs remain, such as neurocognitive impairment. This review presents recent knowledge on the pathophysiological roles of HS focusing on the pathogenesis of MPSs. We also discuss the possible use and significance of HS as a biomarker for disease severity and therapeutic response in MPSs.

Usability of detecting delivery errors during treatment of prostate VMAT with a gantry-mounted transmission detector.

Volumetric-modulated arc therapy (VMAT) requires highly accurate control of multileaf collimator (MLC) movement, rotation speed of linear accelerator gantry, and monitor units during irradiation. Pretreatment validation and monitoring of these factors during irradiation are necessary for appropriate VMAT treatment. Recently, a gantry mounted transmission detector "Delta4 Discover® (D4D)" was developed to detect errors in delivering doses and dose distribution immediately after treatment. In this study, the performance of D4D was evaluated. Simulation plans, in which the MLC position was displaced by 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mm from the clinically used original plans, were created for ten patients who received VMAT treatment for prostate cancer. Dose deviation (DD), distance-to-agreement (DTA), and gamma index analysis (GA) for each plan were evaluated by D4D. These results were compared to the results (DD, DTA and GA) measured by Delta4 Phantom + (D4P). We compared the deviations between the planned and measured values of the MLC stop positions A-side and B-side in five clinical cases of prostate VMAT during treatment and measured the GA values. For D4D, when the acceptable errors for DD, DTA, and GA were determined to be ≤3%, ≤2 mm, and ≤3%/2 mm, respectively, the minimum detectable errors in the MLC position were 2.0, 1.5, and 1.5 mm based on DD, DTA, and GA respectively. The corresponding minimum detectable MLC position errors were 2.0, 1.0, and 1.5 mm, respectively, for D4P. The deviation between the planned and measured position of MLC stopping point of prostate VMAT during treatment was stable at an average of -0.09 ± 0.05 mm, and all GA values were above 99.86%. In terms of delivering doses and dose distribution of VMAT, error detectability of D4D was comparable to that of D4P. The transmission-type detector "D4D" is thus suitable for detecting delivery errors during irradiation.

Pharmacokinetics and pharmacodynamics of JR-051, a biosimilar of agalsidase beta, in healthy adults and patients with Fabry disease: Phase I and II/III clinical studies.

Fabry disease is a rare X-linked lysosomal disease, in which mutations in the gene encoding α-galactosidase A result in progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs including the skin, kidney, and heart, often leading to life-threatening conditions. Enzyme replacement therapy is currently the standard therapy for the disease, to which two α-galactosidase A formulations have been approved: agalsidase α (Replagal®, Shire) and agalsidase ß (Fabrazyme®, Sanofi). We have recently developed a biosimilar of agalsidase ß, JR-051, and investigated its pharmacokinetics and pharmacodynamics to assess its bioequivalence to agalsidase ß. In a randomized phase I study, healthy adult male volunteers were treated with JR-051 or agalsidase ß and the pharmacokinetics of the drugs were compared. The ratio of geometric means (90% confidence interval [CI]) of the AUC0-24 and Cmax for JR-051 over agalsidase ß were 0.91 (0.8294, 1.0082) and 0.90 (0.7992, 1.0125), respectively. In a 52-week, single-arm, phase II/III study, patients with Fabry disease switched therapy from agalsidase ß to JR-051 to evaluate its pharmacodynamics. The mean (95% CI) plasma GL-3 concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.03 (0.91, 1.15) and 0.96 (0.86, 1.06), respectively, which were within the pre-determined bioequivalence acceptance range (0.70, 1.43). The mean (95% CI) plasma globotriaosylsphingosine (lyso-GL-3) concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.07 (0.92, 1.23) and 1.13 (1.03, 1.22), respectively. Estimated glomerular filtration rate and left ventricular mass index, as renal and cardiac function indicators, showed no notable changes from baseline throughout the study period, and no new safety concerns were identified. In conclusion, these studies demonstrated bioequivalence of JR-051 to agalsidase ß in terms of its pharmacokinetics and pharmacodynamics. JR-051 offers a potential new treatment option for patients with Fabry disease.

A Blood-Brain-Barrier-Penetrating Anti-human Transferrin Receptor Antibody Fusion Protein for Neuronopathic Mucopolysaccharidosis II.

Mucopolysaccharidosis II (MPS II) is an X-linked recessive lysosomal storage disease caused by mutations in the iduronate-2-sulfatase (IDS) gene. Since IDS catalyzes the degradation of glycosaminoglycans (GAGs), deficiency in this enzyme leads to accumulation of GAGs in most cells in all tissues and organs, resulting in severe somatic and neurological disorders. Although enzyme replacement therapy with human IDS (hIDS) has been used for the treatment of MPS II, this therapy is not effective for defects in the CNS mainly because the enzyme cannot cross the blood-brain barrier (BBB). Here, we developed a BBB-penetrating fusion protein, JR-141, which consists of an anti-human transferrin receptor (hTfR) antibody and intact hIDS. The TfR-mediated incorporation of JR-141 was confirmed by using human fibroblasts in vitro. When administrated intravenously to hTfR knockin mice or monkeys, JR-141, but not naked hIDS, was detected in the brain. In addition, the intravenous administration of JR-141 reduced the accumulation of GAGs both in the peripheral tissues and in the brain of hTfR knockin mice lacking Ids, an animal model of MPS II. These data provide a proof of concept for the translation of JR-141 to clinical study for the treatment of patients with MPS II with CNS disorders.

Potential for Drug Repositioning of Midazolam for Dentin Regeneration.

Drug repositioning promises the advantages of reducing costs and expediting approvalschedules. An induction of the anesthetic and sedative drug; midazolam (MDZ), regulatesinhibitory neurotransmitters in the vertebrate nervous system. In this study we show the potentialfor drug repositioning of MDZ for dentin regeneration. A porcine dental pulp-derived cell line(PPU-7) that we established was cultured in MDZ-only, the combination of MDZ with bonemorphogenetic protein 2, and the combination of MDZ with transforming growth factor-beta 1. Thedifferentiation of PPU-7 into odontoblasts was investigated at the cell biological and genetic level.Mineralized nodules formed in PPU-7 were characterized at the protein and crystal engineeringlevels. The MDZ-only treatment enhanced the alkaline phosphatase activity and mRNA levels ofodontoblast differentiation marker genes, and precipitated nodule formation containing a dentinspecificprotein (dentin phosphoprotein). The nodules consisted of randomly orientedhydroxyapatite nanorods and nanoparticles. The morphology, orientation, and chemicalcomposition of the hydroxyapatite crystals were similar to those of hydroxyapatite that hadtransformed from amorphous calcium phosphate nanoparticles, as well as the hydroxyapatite inhuman molar dentin. Our investigation showed that a combination of MDZ and PPU-7 cellspossesses high potential of drug repositioning for dentin regeneration.

Evaluation of cerebrospinal fluid heparan sulfate as a biomarker of neuropathology in a murine model of mucopolysaccharidosis type II using high-sensitivity LC/MS/MS.

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes glycosaminoglycans (GAGs) including heparan sulfate (HS) and dermatan sulfate (DS). GAG accumulation leads to severe neurological and somatic impairments. At present, the most common treatment for MPS II is intravenous enzyme replacement therapy; however, the inability of recombinant IDS to cross the blood-brain barrier (BBB) restricts therapeutic efficacy for neurological manifestations. We recently developed a BBB-penetrating IDS fusion protein, JR-141, and demonstrated its ability to reduce GAG accumulation in the brain of human transferrin receptor knock-in and Ids knock-out mice (TFRC-KI/Ids-KO), an animal model of MPS II, following intravenous administration. Given the impossibility of measuring GAG accumulation in the brains of human patients with MPS II, we hypothesized that GAG content in the cerebrospinal fluid (CSF) might serve as an indicator of brain GAG burden. To test this hypothesis, we optimized a high-sensitivity method for quantifying HS and DS in low-volume samples by combining acidic methanolysis and liquid chromatography-tandem mass spectrometry (LC/MS/MS). We employed this method to quantify HS and DS in samples from TFRC-KI/Ids-KO mice and revealed that HS but not DS accumulated in the central nerve system (CNS). Moreover, concentrations of HS in CSF correlated with those in brain. Finally, intravenous treatment with JR-141 reduced levels of HS in the CSF and brain in TFRC-KI/Ids-KO mice. These results suggest that CSF HS content may be a useful biomarker for evaluating the brain GAG accumulation and the therapeutic efficacy of drugs in patients with MPS II.

Non-clinical evaluation of JR-051 as a biosimilar to agalsidase beta for the treatment of Fabry disease.

Fabry disease (FD) is an X-linked lysosomal storage disease. It is caused by deficiency of the enzyme α-galactosidase A (α-Gal A), which leads to excessive deposition of neutral glycosphingolipids, especially globotriaosylceramide (GL-3), in cells throughout the body. Progressive accumulation of GL-3 causes life-threatening complications in several tissues and organs, including the vasculature, heart, and kidney. Currently available enzyme replacement therapy for FD employs recombinant α-Gal A in two formulations, namely agalsidase alfa and agalsidase beta. Here, we evaluated JR-051 as a biosimilar to agalsidase beta in a non-clinical study. JR-051 was shown to have identical primary and similar higher-order structures to agalsidase beta. Mannose-6-phosphate content was higher in JR-051 than in agalsidase beta, which probably accounts for a slightly better uptake into fibroblasts in vitro. In spite of these differences in in vitro biological features, pharmacokinetic profiles of the two compounds in mice, rats, and monkeys were similar. The ability to reduce GL-3 accumulation in the kidney, heart, skin, liver, spleen, and plasma of Gla-knockout mice, a model of FD, was not different between JR-051 and agalsidase beta. Furthermore, we identified no safety concerns regarding JR-051 in a 13-week evaluation using cynomolgus monkeys. These findings indicate that JR-051 is similar to agalsidase beta in terms of physicochemical and biological properties.

Effects of Er:YAG and Diode Laser Irradiation on Dental Pulp Cells and Tissues.

Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers-two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.

TGF-ß and Physiological Root Resorption of Deciduous Teeth.

The present study was performed to examine how transforming growth factor ß (TGF-ß) in root-surrounding tissues on deciduous teeth regulates the differentiation induction into odontoclasts during physiological root resorption. We prepared root-surrounding tissues with (R) or without (N) physiological root resorption scraped off at three regions (R1-R3 or N1-N3) from the cervical area to the apical area of the tooth and measured both TGF-ß and the tartrate-resistant acid phosphatase (TRAP) activities. The TGF-ß activity level was increased in N1-N3, whereas the TRAP activity was increased in R2 and R3. In vitro experiments for the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that proteins from N1-N3 and R1-R3 enhanced the TRAP activity in RAW264 cells. A genetic study indicated that the mRNA levels of TGF-ß1 in N1 and N2 were significantly increased, and corresponded with levels of osteoprotegerin (OPG). In contrast, the expression level of RANKL was increased in R2 and R3. Our findings suggest that TGF-ß is closely related to the regulation of OPG induction and RANKL-mediated odontoclast differentiation depending on the timing of RANKL and OPG mRNA expression in the root-surrounding tissues of deciduous teeth during physiological root resorption.

Hyaluronan and chondroitin sulfate in chicken-vegetable bone broth delay osteoporosis progression.

Bone broth has recently gained worldwide recognition as a superfood that supplements several nutrients lacking in modern human diets; however, little is known of its efficacy on osteoporosis. Therefore, we aimed to identify the components of chicken-vegetable bone broth (CVBB) that are associated with osteoporosis prevention and verified the efficacy of these components using in vivo studies. In biochemical and cell biological experiments, CVBB was fractionated using ion exchange chromatography (IEC), and the effect of each IEC fraction on osteoclast differentiation was evaluated based on tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and quantitative polymerase chain reaction analysis using mouse macrophage-like cells (RAW264 cell). In animal experiments, an ovariectomized (OVX) rat model was generated, followed by whole bone broth (OVX/CVBB) or IEC fraction (OVX/CVBB-Ext) administration and bone structural parameter characterization of OVX rat tibia based on micro-CT. Four CVBB fractions were obtained using IEC, and the fraction containing both hyaluronan and chondroitin sulfate (CVBB-Ext) led to the maximum inhibition of RAW264 cell differentiation. CVBB-Ext downregulated the expression of osteoclast differentiation marker genes. In animal experiments, the OVX group showed a clear decrease in bone density compared to that in the Sham operation group. The OVX/CVBB and OVX/CVBB-Ext groups showed increased bone mineral density and bone volume/tissue volume values compared to those in the OVX/control group. These results suggested that CVBB and CVBB-Ext slowed osteoporosis progression. Therefore, we conclude that hyaluronan and chondroitin sulfate in CVBB are key substances that impede osteoporosis progression. PRACTICAL APPLICATION: This study provides practical information on the effects of bone broth ingredients on osteoporosis to expand the current knowledge on the efficacy of bone broth, which is a widely consumed food. These results may help in the future development of bone broth as a dietary supplement for managing osteoporosis.

Synergistic effect of FGF-2 and TGF-ß1 on the mineralization of human umbilical cord perivascular cells.

OBJECTIVE: Human umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) on HUCPVC mineralization. DESIGN: We prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-ß1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. RESULTS: FGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. CONCLUSION: We found that FGF-2 synergizes with TGF-ß1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

Enzyme replacement with transferrin receptor-targeted α-L-iduronidase rescues brain pathology in mucopolysaccharidosis I mice.

Mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by dysfunction of α-L-iduronidase (IDUA), is characterized by the deposition of dermatan sulfate (DS) and heparan sulfate (HS) throughout the body, which causes several somatic and central nervous symptoms. Although enzyme-replacement therapy (ERT) is currently available to treat MPS I, it does not alleviate central nervous disorders, as it cannot penetrate the blood-brain barrier. Here we evaluate the brain delivery, efficacy, and safety of JR-171, a fusion protein comprising humanized anti-human transferrin receptor antibody Fab and IDUA, using monkeys and MPS I mice. Intravenously administered JR-171 was distributed in major organs, including the brain, and reduced DS and HS concentrations in the central nervous system and peripheral tissues. JR-171 exerted similar effects on peripheral disorders similar to conventional ERT and further reversed brain pathology in MPS I mice. We found that JR-171 improved spatial learning ability, which was seen to deteriorate in the vehicle-treated mice. Further, no safety concerns were noted in repeat-dose toxicity studies in monkeys. This study provides nonclinical evidence that JR-171 might potentially prevent and even improve disease conditions in patients with neuronopathic MPS I without serious safety concerns.

Antitubercular activity of disulfiram, an antialcoholism drug, against multidrug- and extensively drug-resistant Mycobacterium tuberculosis isolates.

The antimycobacterial activities of disulfiram (DSF) and diethyldithiocarbamate (DDC) against multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB) clinical isolates were evaluated in vitro. Both DSF and DDC exhibited potent antitubercular activities against 42 clinical isolates of M. tuberculosis, including MDR/XDR-TB strains. Moreover, DSF showed remarkable bactericidal activity ex vivo and in vivo. Therefore, DSF might be a drug repurposed for the treatment of MDR/XDR-TB.

[Pharmacological property, mechanism of action and clinical study results of Pabinafusp Alfa (Genetical Recombination) (IZCARGO® I.V. Infusion 10†mg) as the therapeutic for Mucopolysaccharidosis type-II (Hunter syndrome)].

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease with the accumulation of glycosaminoglycans in tissues and organs throughout the body caused by dysfunction or loss of iduronate-2-sulfatase (IDS), resulting in somatic and central nervous system (CNS) disorders. Although enzyme replacement therapy (ERT) with recombinant human IDS is the current first-line therapy for MPS II, it is not effective for the CNS because intravenously administered enzyme cannot cross the blood-brain barrier (BBB) and thereby does not reach the brain parenchyma. Pabinafusp alfa, approved in March 2021 in Japan, is a recombinant fusion protein composed of human IDS and humanized anti-human transferrin receptor (hTfR) antibody, utilizing the BBB-penetrating technology "J-Brain Cargo®" established by JCR Pharmaceuticals. Nonclinical studies showed that pabinafusp alfa was distributed in the brain of hTfR knock-in mice and monkeys after intravenous administration, and dose-dependently decreased heparan sulfate (HS) glycosaminoglycan deposited in major organs including the brain of MPS II mice. Pabinafusp alfa also suppressed neurodegeneration in cerebellum and hippocampus, leading to the maintenance of spatial learning ability. Phase II/III clinical study conducted in Japan showed that pabinafusp alfa decreased HS concentration in the cerebrospinal fluid, which serves as an efficacy biomarker for central nervous symptoms, and improved or stabilized the developmental age of the patients. Moreover, pabinafusp alfa exerted comparable effects to current ERT in terms of improvement of somatic manifestations. Therefore, pabinafusp alfa is a promising therapeutic option as a BBB-penetrating enzyme for the treatment of patients with neuronopathic MPS II.

Dose-dependent effects of a brain-penetrating iduronate-2-sulfatase on neurobehavioral impairments in mucopolysaccharidosis II mice.

Deposition of heparan sulfate (HS) in the brain of patients with mucopolysaccharidosis II (MPS II) is believed to be the leading cause of neurodegeneration, resulting in several neurological signs and symptoms, including neurocognitive impairment. We recently showed that pabinafusp alfa, a blood-brain-barrier-penetrating fusion protein consisting of iduronate-2-sulfatase and anti-human transferrin receptor antibody, stabilized learning ability by preventing the deposition of HS in the CNS of MPS II mice. We further examined the dose-dependent effect of pabinafusp alfa on neurological function in relation to its HS-reducing efficacy in a mouse model of MPS II. Long-term intravenous treatment with low (0.1 mg/kg), middle (0.5 mg/kg), and high (2.0 mg/kg) doses of the drug dose-dependently decreased HS concentration in the brain and cerebrospinal fluid (CSF). A comparable dose-dependent effect in the prevention of neuronal damage in the CNS, and dose-dependent improvements in neurobehavioral performance tests, such as gait analysis, pole test, Y maze, and Morris water maze, were also observed. Notably, the water maze test performance was inversely correlated with the HS levels in the brain and CSF. This study provides nonclinical evidence substantiating a quantitative dose-dependent relationship between HS reduction in the CNS and neurological improvements in MPS II.

Repurposing MDZ as a tool for tissue regeneration in dental cells.

BACKGROUND: Several recent studies have focused on the utility of drug repurposing to expand clinical application of approved therapeutics. Here, we investigate the efficacy of midazolam (MDZ) and cytokines for regenerating calcified tissue, using immortalized porcine dental pulp (PPU7) and mouse skeletal muscle derived myoblast (C2C12) cells, with the goal of repurposing MDZ as a new treatment to facilitate calcified tissue regeneration. HIGHLIGHTS: We noted that PPU7 and C2C12 cells cultured with various MDZ regimens displayed increased bone morphogenic protein (BMP-2), transforming growth factor beta (TGF-ß), and alkaline phosphatase activity. These increases were highest in PPU7 cells cultured with MDZ alone, and in C2C12 cells cultured with MDZ and BMP-2. PPU7 cells cultured under these conditions demonstrated markedly elevated expression of odontoblastic gene markers, indicating their likely differentiation into odontoblasts. Expression levels of osteoblastic gene markers also increased in C2C12 cells, suggesting that MDZ potentiates the effect of BMP-2, inducing osteoblast differentiation in these cells. Newly formed calcified deposits in both PPU7 and C2C12 cells were identified as hydroxyapatite via crystallographic and crystal engineering analyses. CONCLUSION: MDZ increases ALP activity, inducing expression of specific marker genes for both odontoblasts and osteoblasts while promoting hydroxyapatite production in both PPU7 and C2C12 cells. These responses were cell type specific. MDZ treatment alone could induce these changes in PPU7 cells, but C2C12 cell differentiation required BMP-2 addition.

Response of TGF-ß isoforms in epithelial-mesenchymal transition of enamel epithelial cells.

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.

Characterization of bioactive substances involved in the induction of bone augmentation using demineralized bone sheets.

PURPOSE: To investigate the bone augmentation ability of demineralized bone sheets mixed with allogeneic bone with protein fractions containing bioactive substances and the interaction between coexisting bioactive substances and proteins. METHODS: Four types of demineralized bone sheets mixed with allogeneic bone in the presence or absence of bone proteins were created. Transplantation experiments using each demineralized bone sheet were performed in rats, and their ability to induce bone augmentation was analysed by microcomputed tomography images. Bioactive substances in bone proteins were isolated by heparin affinity chromatography and detected by the measurement of alkaline phosphatase activity in human periodontal ligament cells and dual luciferase assays. Noncollagenous proteins (NCPs) coexisting with the bioactive substances were identified by mass spectrometry, and their interaction with bioactive substances was investigated by in vitro binding experiments. RESULTS: Demineralized bone sheets containing bone proteins possessed the ability to induce bone augmentation. Bone proteins were isolated into five fractions by heparin affinity chromatography, and transforming growth factor-beta (TGF-ß) was detected in the third fraction (Hep-c). Dentin matrix protein 1 (DMP1), matrix extracellular phosphoglycoprotein (MEPE), and biglycan (BGN) also coexisted in Hep-c, and the binding of these proteins to TGF-ß increased TGF-ß activity by approximately 14.7% to 32.7%. CONCLUSIONS: Demineralized bone sheets are capable of inducing bone augmentation, and this ability is mainly due to TGF-ß in the bone protein mixed with the sheets. The activity of TGF-ß is maintained when binding to bone NCPs such as DMP1, MEPE, and BGN in the sheets.