Effects of NRF2 polymorphisms on safety and efficacy of bardoxolone methyl: subanalysis of TSUBAKI study.

BACKGROUND: In the TSUBAKI study, bardoxolone methyl significantly increased measured and estimated glomerular filtration rates (GFR) in patients with multiple forms of chronic kidney disease (CKD), including Japanese patients with type 2 diabetes and stage 3-4 CKD. Since bardoxolone methyl targets the nuclear factor erythroid 2-related factor 2 pathway, this exploratory analysis of the TSUBAKI study investigated the impact of the regulatory single nucleotide polymorphism, rs6721961, on the effects of bardoxolone methyl. METHODS: Japanese patients aged 20-79 years with type 2 diabetes and stage 3-4 CKD were randomized to bardoxolone methyl 5-15 mg/day (titrated as tolerated) or placebo for 16 weeks. Genotype frequency, clinical characteristics, renal function, and adverse events were primarily assessed. RESULTS: Of 104 patients (bardoxolone methyl n = 55, placebo n = 49); 57% were genotype C/C, 32% C/A and 12% A/A. The frequency of the A/A genotype was higher among patients with diabetic kidney disease than in the general Japanese population (~ 5%). Measured and estimated GFRs increased from baseline in all genotypes receiving bardoxolone methyl. There were no significant differences between genotypes for safety parameters, including blood pressure, bodyweight, and levels of B-type natriuretic peptide, or in the type and frequency of adverse events, suggesting that the efficacy and safety of bardoxolone methyl are unaffected by the rs6721961 polymorphism-617 (C→A) genotype. CONCLUSIONS: Our approach of combining genome analysis with clinical trials for an investigational drug provides important and useful clues for exploring the efficacy and safety of the drug. TRIAL REGISTRATION: ClinicalTrials.gov; NCT02316821.

[Challenges in kidney disease: therapeutic potential of bardoxolone methyl].

Kidney diseases are highly prevalent worldwide and significantly reduce the quality of life in such patients, creating an urgent need for effective therapeutic options. Oxidative stress and inflammation are thought to be related to the progression of kidney diseases, and the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) system is known to a key factor for anti-oxidative, anti-inflammatory properties, which is being widely investigated. Bardoxolone methyl, known to be a potent activator of this Keap1-Nrf2 pathway, has demonstrated increases in glomerular filtration rate (GFR) in several clinical studies, suggesting its therapeutic potential for kidney diseases. This review summarizes available knowledge on the therapeutic potential of bardoxolone methyl for treatment of kidney diseases, with history and current situation of its developmental activities.

Blockade of the interaction between BMP9 and endoglin on erythroid progenitors promotes erythropoiesis in mice.

Bone morphogenetic protein-9 (BMP9), a member of the transforming growth factor ß (TGFß) superfamily, plays important roles in the development and maintenance of various cell lineages via complexes of type I and type II TGFß receptors. Endoglin is a coreceptor for several TGFß family members, including BMP9, which is highly expressed in a particular stage of differentiation in erythroid cells as well as in endothelial cells. Although the importance of the interaction between BMP9 and endoglin for endothelial development has been reported, the contribution of BMP9 to endoglin-expressing erythroid cells remains to be clarified. To address this point, we prepared an anti-BMP9 antibody that blocks the BMP9-endoglin interaction. Of note, challenge with the antibody promotes erythropoiesis in wild-type mice but not in a mouse model of renal anemia in which erythropoietin (EPO) production in the kidneys is genetically ablated. While endoglin-positive erythroid progenitors are mainly maintained as progenitors when bone marrow-derived lineage-negative and cKit-positive cells are cultured in the presence of EPO and stem cell factor, the erythroid-biased accumulation of progenitors is impeded by the presence of BMP9. Our findings uncover an unrecognized role for BMP9 in attenuating erythroid differentiation via its interaction with endoglin on erythroid progenitors.

Bardoxolone methyl: drug development for diabetic kidney disease.

Bardoxolone methyl activates the Keap1/Nrf2 system that plays an important role in defense responses against oxidative stress. Importantly, bardoxolone methyl has demonstrated increases in estimated glomerular filtration rate (eGFR) in patients with diabetic kidney disease (DKD) in clinical studies. However, an overseas Phase 3 study of bardoxolone methyl in patients with stage G4 DKD was prematurely terminated due to an increased risk for heart failure, which was considered to have been caused by early-onset fluid overload. Subsequently, a Japanese Phase 2 study demonstrated, for the first time, that bardoxolone methyl directly improves GFR, which is a true indicator of kidney function, using the inulin clearance method. In Japan, bardoxolone methyl was designated for the treatment of DKD under the Priority Review and Designation (SAKIGAKE Designation) System established by the Ministry of Health, Labour and Welfare. A Japanese Phase 3 study, with endpoints such as a ≥ 30% decrease in eGFR, is currently ongoing to assess the efficacy and safety of bardoxolone methyl in more than 1,000 patients with stages G3 and G4 DKD who have no identified risk factors.

Nrf2 activator for the treatment of kidney diseases.

Kidney diseases are highly prevalent worldwide, and significantly reduce the quality of life of patients, creating an urgent need for effective therapeutic modalities. Despite this significant unmet medical need, none of the drugs launched to date have demonstrated promising potential to cure kidney diseases. This is likely due to the structural complexity of the kidney as well as difficulties in setting appropriate endpoints for clinical trials and identifying appropriate therapeutic targets. Recently, an alternative endpoint for clinical trials (i.e., a 30% or 40% reduction in estimated glomerular filtration rate [eGFR] from baseline following 2-3 years of observation) has been considered in the United States, European Union, and Japan, and is expected to contribute to the progress of drug development for kidney diseases. Further, oxidative stress and inflammation are currently thought to be key factors in the progression of kidney diseases, prompting more research on drugs targeting the mechanisms related to these factors for treatment. The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) system has drawn much attention in recent years for its anti-oxidative and anti-inflammatory properties, and its pharmacological potential for treatment of kidney diseases is being widely investigated in both clinical and non-clinical studies. This review summarizes the current issues in the treatment of kidney diseases, including clinical endpoints, Nrf2 activators as treatment options, and perspectives on pharmaceutical applications of Nrf2 activators.

The soluble form of BMPRIB is a novel therapeutic candidate for treating bone related disorders.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the TGF-beta superfamily. Recently, several soluble BMP receptors, such as ActRIIA-Fc, ActRIIB-Fc, and ALK1-Fc, are undergoing clinical trials. Both BMPRIA and BMPRIB are type I BMP receptors, and while BMPRIA-Fc has been reported to have bone-increasing properties, there have been no investigations concerning the biological functions of BMPRIB-Fc. Therefore, comparing the effects of BMPRIA-Fc and BMPRIB-Fc in vivo should be helpful in revealing the differences in biological function between BMPRIA and BMPRIB, and would also aid in the evaluation of BMPRIB-Fc as a therapeutic agent. Here, we produced Tg chimeras in which BMPRIA-Fc and BMPRIB-Fc proteins circulated at high concentrations (36.8-121.4 µg/mL). Both Tg chimeras showed a significant increase of bone volume and strength. Using histological analysis, adenoma of the glandular stomach was observed only in BMPRIA-Fc chimeras suggesting the tumorigenic activity of this protein. Administration of recombinant BMPRIB-Fc protein to normal mice also increased bone volumes. Finally, treatment with BMPRIB-Fc decreased the area of osteolytic regions in a mouse model of breast cancer metastasis. In conclusion, our data suggest that BMPRIB-Fc can be used for the treatment of bone-related disorders with a lower risk than BMPRIA-Fc.

Identification of osteoblast stimulating factor 5 as a negative regulator in the B-lymphopoietic niche.

Recent studies have revealed the crucial role of the niche which supports B-lymphocyte differentiation from hematopoietic stem cells. In this study, we aimed to identify a novel regulator of B lymphopoiesis secreted in the specific niche using the signal sequence trap method. Among the identified proteins from MS5 stromal cells, expression of pleiotrophin, placental proliferin 2, and osteoblast stimulating factor 5 (OSF-5) was dominantly high in several stromal cell lines. We found that OSF-5 suppressed early B lymphopoiesis in transgenic mice producing the target protein. The number of pre-B and immature B cells was reduced by more than half compared with control in the transgenic mice. In vitro studies showed that a secreted variant of OSF-5 inhibited the proliferation and colony formation of pre-B cells, whereas cell-intrinsic form had no influence on B lymphopoiesis. The main components of the B-lymphopoietic niche, osteoblasts in mice and mesenchymal cells in humans, are primary producers of OSF-5. These results define a novel mechanism of B lymphopoiesis in bone marrow. In the specific niche, B-lymphocyte differentiation is fine-tuned by negative regulators as well as supportive factors.

Estrogen-inducible sFRP5 inhibits early B-lymphopoiesis in vivo, but not during pregnancy.

Mammals have evolved to protect their offspring during early fetal development. Elaborated mechanisms induce tolerance in the maternal immune system for the fetus. Female hormones, mainly estrogen, play a role in suppressing maternal lymphopoiesis. However, the molecular mechanisms involved in the maternal immune tolerance are largely unknown. Here, we show that estrogen-induced soluble Frizzled-related proteins (sFRPs), and particularly sFRP5, suppress B-lymphopoiesis in vivo in transgenic mice. Mice overexpressing sFRP5 had fewer B-lymphocytes in the peripheral blood and spleen. High levels of sFRP5 inhibited early B-cell differentiation in the bone marrow (BM), resulting in the accumulation of cells with a common lymphoid progenitor (CLP) phenotype. Conversely, sFRP5 deficiency reduced the number of hematopoietic stem cells (HSCs) and primitive lymphoid progenitors in the BM, particularly when estrogen was administered. Furthermore, a significant reduction in CLPs and B-lineage-committed progenitors was observed in the BM of sfrp5-null pregnant females. We concluded that, although high sFRP5 expression inhibits B-lymphopoiesis in vivo, physiologically, it contributes to the preservation of very primitive lymphopoietic progenitors, including HSCs, under high estrogen levels. Thus, sFRP5 regulates early lympho-hematopoiesis in the maternal BM, but the maternal-fetal immune tolerance still involves other molecular mechanisms that remain to be uncovered.

Adult-specific systemic over-expression reveals novel in vivo effects of the soluble forms of ActRIIA, ActRIIB and BMPRII.

Bone morphogenetic proteins (BMPs)/growth differentiation factors (GDFs), which belong to the TGF-beta superfamily, are pleiotropic factors that play a role in regulating the embryonic development and postnatal homeostasis of various organs and tissues by controlling cellular differentiation, proliferation and apoptosis. Conventional transgenic and knockout (KO) mouse approaches have provided only limited information regarding the in vivo functions of BMP signaling in adult animals due to the effects on prenatal development and the difficulty in manipulating multiligand signals simultaneously. We recently produced transgenic chimeric mice(Tg chimeras) in which the soluble IgG1-Fc fusion protein of three BMP type II receptors (ActRIIA, ActRIIB, BMPRII) was highly circulated (281-709 µg/ml), specifically in adult mouse blood. Since each BMP receptor can bind to multiple BMP ligands, these Tg chimeras should be useful to investigate the effects of trapping multiple BMP ligands. Remarkably, some phenotypes were unexpected based on previous studies, such as KO mouse analyses, presumably representing the effects of the multiple ligand trapping. These phenotypes included increased red blood cells (RBCs) and decreased viability in adults. In a further study, we focused on the phenotype of increased RBCs and found that extramedullary hematopoiesis in the spleen, not in the bone marrow, was increased using histological and flow cytometric analyses. Although it remains to be elucidated whether the transgene products affect the tissues directly or indirectly, our data provide novel and important insight into the biological functions of the soluble IgG1-Fc fusion protein of three BMP type II receptors in adults, and our approach should have broad applications to research on other ligand receptor families and studies involving mouse models.

R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling.

In mammals, female development has traditionally been considered a default process in the absence of the testis-determining gene, Sry. Recently, it has been documented that the gene for R-spondin1 (RSPO1), a novel class of soluble activator for Wnt/beta-catenin signaling, is mutated in two Italian families with female-to-male (XX) sex reversal. To elucidate the role of Rspo1 as a candidate female-determining gene in a mouse model, we generated Rspo1-null (Rspo1(-/-)) mice and found that Rspo1(-/-) XX mice displayed masculinized features including pseudohermaphroditism in genital ducts, depletion of fetal oocytes, male-specific coelomic vessel formation and ectopic testosterone production in the ovaries. Thus, although Rspo1 is required to fully suppress the male differentiation program and to maintain germ cell survival during the development of XX gonads, the loss of its activity has proved to be insufficient to cause complete XX sex reversal in mice. Interestingly, these partial sex-reversed phenotypes of Rspo1(-/-) XX mice recapitulated those of previously described Wnt-4(-/-) XX mice. In accordance with this finding, the expression of Wnt-4 and its downstream genes was deregulated in early Rspo1(-/-) XX gonads, suggesting that Rspo1 may participate in suppressing the male pathway in the absence of Sry and maintaining oocyte survival through positively regulating Wnt-4 signaling.