Reduction in the magnitude of serum potassium elevation in combination therapy with esaxerenone (CS‐3150) and sodium–glucose cotransporter 2 inhibitor in patients with diabetic kidney disease: Subanalysis of two phase III studies

Aims/Introduction: We evaluated the effect of co-administration of esaxerenone and a sodium–glucose cotransporter 2 (SGLT2) inhibitor on the magnitude of serum potassium elevation in Japanese patients with diabetic kidney disease. Materials and Methods: We carried out a prespecified subanalysis of data from two phase III studies: a multicenter, randomized, double-blind, placebo-controlled trial in patients with type 2 diabetes and microalbuminuria (J308); and a multicenter, single-arm, open-label trial in patients with type 2 diabetes and macroalbuminuria (J309). Changes in serum potassium levels during the studies and other measures were evaluated according to SGLT2 inhibitor use. Results: In both studies, time-course changes in serum potassium levels, and incidence rates of serum potassium elevation were lower in patients with co-administration of SGLT2 inhibitor in both the placebo and esaxerenone groups than those without the inhibitor. In contrast, time-course changes and mean percentage changes from baseline in urinary albumin-to-creatinine ratio, the proportion of patients with albuminuria remission and time-course changes in blood pressure did not change with or without SGLT2 inhibitor, whereas the albumin-to-creatinine ratio and blood pressure were reduced with esaxerenone. The blood glucose-lowering effect of SGLT2 inhibitor was not affected by esaxerenone. Conclusions: In Japanese patients with type 2 diabetes and albuminuria treated with esaxerenone, concomitant use of SGLT2 inhibitor reduced the magnitude of serum potassium elevation without any change of its antihypertensive and albuminuria-suppressing effects. Co-administration of esaxerenone and SGLT2 inhibitor might be a beneficial treatment option for patients with diabetic kidney disease

High concentrations of L-ascorbic acid specifically inhibit the growth of human leukemic cells via downregulation of HIF-1α transcription.

We examined the antileukemic effects of high concentrations of L-ascorbic acid (high AA) on human leukemic cells. In vitro, high AA markedly induced apoptosis in various leukemic cell lines by generating hydrogen peroxide (H2O2) but not in normal hematopoietic stem/progenitor cells. High AA significantly repressed leukemic cell proliferation as well as neoangiogenesis in immunodeficient mice. We then noted that in leukemic cells, HIF-1α transcription was strongly suppressed by high AA and correlated with the transcription of VEGF. Our data indicate that exposure to high AA markedly increased the intracellular AA content of leukemic cells and inhibited the nuclear translocation of NF-κB, which mediates expression of HIF-1α. We next generated K562 cells that overexpressed HIF-1α (K562-HIF1α cells) and assessed the mechanistic relationship between inhibition of HIF-1α transcription and the antileukemic effect of high AA. The ability of high AA to induce apoptosis was significantly lower in K562-HIF1α cells than in K562 cells in vitro. We found that expression of HIF-1α-regulated antiapoptotic proteins of the Bcl-2 family, such as Mcl-1, Bcl-xL, and Bcl-2, was significantly suppressed by high AA in K562 cells, but was sustained at higher levels in K562-HIF1α cells, regardless of high AA exposure. Moreover, repression of cell proliferation and neoangiogenesis by high AA was completely abrogated in mice receiving transplants of K562-HIF1α cells. These results indicate that, along with H2O2 generation, downregulation of HIF-1α transcription plays a crucial role in growth inhibition of human leukemic cells by high AA

In vitro effects of AA on human leukemic and CB-CD34⁺ cells, relative to catalase activity.

<p>A) Cell viability assay of various leukemic cell lines and 2 independent isolates of CB-CD34⁺ cells. Cells were treated with different concentrations of AA for 1 h, and then washed, cultured, and analyzed after 72 h. The viability of all cell lines reduced significantly in the presence of 280 and 2800 µM AA (*<i>P</i><0.0001, as compared with vehicle), but this finding was not obtained for CB-CD34⁺ cells (<i>P</i>>0.05). The values represent the mean ± SD values of quadruplicate samples. B) Flow cytometric measurement of apoptosis of HL60 cells. Cells were treated with vehicle or AA for 1 h, and then washed, cultured, and analyzed after 18 h. Representative profiles are shown. The annexin V⁺ propidium iodide (PI)⁺ cell fraction indicates apoptotic cells. Note that AA-induced apoptosis was almost completely abrogated by the addition of catalase. C) Intracellular catalase activity. Leukemic cells generally expressed lower catalase activities than did CB-CD34⁺ isolates (*<i>P</i><0.001, as compared with each cell line). The values represent the mean ± SD values of quadruplicate samples. D) Histochemical analysis demonstrated lower catalase activity in HL60 cells than in CB-CD34⁺ cells. The bars indicate 50 μm.</p

Differences in NF-κB activation and intracellular AA content between human leukemic and CB-CD34⁺ cells in the presence of high AA.

<p>A) Western blotting analysis of p-IκB in HL60 cells. Cells were treated with the vehicle or with high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There was a significant difference in the expression levels (*<i>P</i><0.001). Values represent the mean ± SD of triplicate samples. B) Immunocytochemical (left) and Western blotting (right) analyses of NF-κB in CB-CD34⁺ and HL60 cells. Cells were treated with vehicle or high AA for 1 h, then washed, cultured, and analyzed after 24 h. Note that translocation of NF-κB into the nucleus was markedly decreased in high AA-treated HL60 cells. Green and blue signals represent NF-κB and DAPI, respectively. Bars indicate 20 μm. There were significant differences in the expression levels (*<i>P</i><0.001, **<i>P</i><0.0001). The values represent the mean ± SD values of triplicate samples. C) Intracellular AA content of human leukemic cells and 2 different isolates of CB-CD34⁺ cells. Cells were treated with high AA for 1 h, washed in PBS, and analyzed immediately. There were significant differences in the content between leukemic and CB-CD34⁺ cells. *<i>P</i><0.001, as compared with CB-CD34⁺ cells (1) or (2). The values are mean ± SD values of triplicate samples.</p

In vivo effects of high AA on progression of leukemia.

<p>A) High AA or the vehicle was injected intravenously for 6 days with a rest period of 2 days between 3 daily injections of mice transplanted with HL60 cells. Compared with vehicle (blue line), high AA (red line) significantly inhibited tumor growth (*<i>P</i><0.01). The values represent the mean ± SD values of 5 mice. B) Appearance of mice treated with vehicle (left) and high AA (right), 4 days after the final injection. C) Representative macroscopic appearance of tumors of mice treated with the vehicle (left) and high AA (right). Note that the tumors of high AA-treated mice were smaller and less erythematous than those of vehicle-treated mice. D) Immunohistochemical analysis of tumor neoangiogenesis in mice treated with the vehicle (left) and high AA (right). The green and blue signals represent CD31 and 4′,6-diamidino-2-phenylindole (DAPI), respectively. The bars indicate 100 μm.</p

Relationship between antileukemic effects of high AA and HIF-1α expression.

<p>A) Quantitative real-time PCR analysis of <i>HIF-1α</i> mRNA expression in K562 and K562-HIF1α cells. Cells were treated with the vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. After high AA exposure, <i>HIF-1α</i> mRNA expression significantly reduced in K562 (*<i>P</i><0.01), but not in K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 24 h. High AA exposure significantly reduced the HIF-1α protein level in both types of cells. However, the HIF-1α protein level in K562-HIF1α cells was significantly higher than that in K562 cells after vehicle or high AA exposure. *<i>P</i><0.01, **<i>P</i><0.0001, ***<i>P</i><0.00001. The values represent the mean ± SD values of triplicate samples. C) Flow cytometric measurement of apoptosis of K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured in the medium, and analyzed after 18 h. There was a significant difference in the number of apoptotic (annexin V⁺ propidium iodide (PI)⁺) cells between high AA-treated K562 and K562-HIF1α cells (*<i>P</i><0. 001). The values represent the mean ± SD values of triplicate samples. D) Flow cytometric measurement of cleaved caspase-3 expressed by K562 and K562-HIF1α cells. Cells were treated with vehicle (gray lines) or high AA (black lines) for 1 h, washed, cultured, and analyzed after 24 h. Activation of caspase-3 by high AA was lower in K562-HIF1α than in K562 cells. E) Western blotting analysis of Mcl-1, Bcl-x_L, and Bcl-2 in K562 and K562-HIF1α cells. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.05) and between the vehicle-treated and high AA-treated K562 cells (**<i>P</i><0.0001). There was no significant difference between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05). The values represent the mean ± SD values of triplicate samples. F) Western blotting analysis of Sp1, Sp3, Sp4, and VEGF. Cells were treated with vehicle or high AA for 1 h, washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels of these molecules between the vehicle-treated K562 and K562-HIF1α cells (*<i>P</i><0.01, ** <i>P</i><0.0001). There were significant differences in the expression levels of Sp1, Sp3, and Sp4 between the vehicle-treated and high AA-treated K562 or K562-HIF1α cells (^†<i>P</i><0.01, ^††<i>P</i><0.001, ^†††<i>P</i><0.0001). There was a significant difference in the expression level of VEGF between the vehicle-treated and high AA-treated K562 (^†††<i>P</i><0.0001), but not between the vehicle-treated and high AA-treated K562-HIF1α cells (<i>P</i>>0.05).</p

Expression of angiogenesis-related molecules in human leukemic and CB-CD34⁺ cells exposed to the vehicle or to high AA.

<p>A) Quantitative real-time PCR (qRT-PCR) analysis of <i>HIF-1α</i> mRNA in CB-CD34⁺ and HL60 cells. The cells were treated with vehicle or high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There were no significant differences in the expression levels for the 2 conditions (<i>P</i>>0.05) in CB-CD34⁺ cells. In contrast, there were significant differences in the expression levels between the 2 conditions (*<i>P</i><0.0001) in HL60 cells. The values represent the mean ± SD values of triplicate samples. B) Western blotting analysis of HIF-1α in CB-CD34⁺ and HL60 cells. The cells were treated with vehicle or high AA for 1 h, and then washed, cultured, and analyzed after 24 h. There were significant differences in the expression levels (*<i>P</i><0.01, **<i>P</i><0.0005). The values are mean ± SD values of triplicate samples. C) Sequential analysis of qRT-PCR results of <i>HIF-1α</i> and <i>VEGF</i> mRNA in HL60 cells. The cells were treated with high AA for 1 h, and then washed, cultured, and analyzed after 1, 3, 22, and 26 h. The expression of <i>VEGF</i> mRNA reduced along with that of <i>HIF-1α</i> over time. Compared with the expression levels at 0 h, there were significant differences in the expression levels (*<i>P</i><0.01, **<i>P</i><0.001, ***<i>P</i><0.0001). The values represent the mean ± SD values of triplicate samples.</p

Effects of high AA on tumor growth in the presence or absence of overexpression of <b><i>HIF-1α</i></b><b>.</b>

<p>A) Immunohistochemical analysis of tumor neoangiogenesis in vehicle-treated (left) and high AA-treated (right) mice transplanted with K562 (upper column) or K562-HIF1α cells (lower column). The green and blue signals represent CD31 and DAPI, respectively. The bars indicate 100 μm. Note that administration of high AA suppressed tumor neoangiogenesis in mice transplanted with K562 cells, but not in mice transplanted with K562-HIF1α cells. B) In the xenogeneic transplant model, high AA or vehicle was injected for 5 days. Administration of high AA significantly inhibited tumor growth of K562 cells (*<i>P</i><0.05) but not of K562-HIF1α cells (<i>P</i>>0.05). Tumor growth rate was estimated using the following equation: tumor volume on day 4 after high AA treatment/tumor volume just before high AA treatment. The values represent the mean ± SD values for 4 mice.</p