Anti-FGF-23 neutralizing antibodies ameliorate muscle weakness and decreased spontaneous movement of Hyp mice.

Fibroblast growth factor 23 (FGF-23) plays causative roles in the development of several hypophosphatemic rickets/osteomalacia such as X-linked hypophosphatemic rickets/osteomalacia (XLH) and tumor-induced rickets/osteomalacia. Patients with hypophosphatemic rickets/osteomalacia often complain of muscle weakness and bone pain that severely affect daily activities of these patients. The purpose of this study was to examine whether anti-FGF-23 antibodies, which have been shown to improve hypophosphatemia and rachitic changes of juvenile Hyp mice in a murine model of XLH, also ameliorate hypophosphatemic osteomalacia and affect muscle force and spontaneous motor activity in adult Hyp mice. Repeated injections of anti-FGF-23 antibodies increased serum phosphate and 1,25-dihydroxyvitmain D levels and enhanced mineralization of osteoid in adult Hyp mice, whereas bone length did not change. We found that grip strength was weaker and that spontaneous movement was less in adult Hyp mice than in wild-type mice. In addition, FGF-23 antibodies increased grip strength and spontaneous movement. These results suggest that the inhibition of excess FGF-23 action not only ameliorates hypophosphatemia and impaired mineralization of bone but also improves muscle weakness and daily activities of patients with FGF-23-related hypophosphatemic rickets/osteomalacia.

Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects.

BACKGROUND: Alpha-Klotho (alphaKl) regulates mineral metabolism such as calcium ion (Ca(2+)) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type alphaKl has been demonstrated, less is known regarding the physiological importance of soluble-type alphaKl (salphaKl) in circulation. OBJECTIVES: The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum salphaKl, and (2) to determine reference values for salphaKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. RESULTS: We successively developed an ELISA to measure serum salphaKl in healthy volunteers (n=142, males 66) of ages (61.1+/-18.5year). The levels (mean+/-SD) in these healthy control adults were as follows: total calcium (Ca; 9.46+/-0.41mg/dL), Pi (3.63+/-0.51mg/dL), blood urea nitrogen (BUN; 15.7+/-4.3mg/dL), creatinine (Cre; 0.69+/-0.14mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8+/-17.7pg/mL), intact parathyroid hormone (iPTH; 49.2+/-20.6pg/mL), calcitonin (26.0+/-12.3pg/mL) and intact fibroblast growth factor (FGF23; 43.8+/-17.6pg/mL). Serum levels of salphaKl ranged from 239 to 1266pg/mL (mean+/-SD; 562+/-146pg/mL) in normal adults. Although salphaKl levels were not modified by gender or indices of mineral metabolism, salphaKl levels were inversely related to Cre and age. However, salphaKl levels in normal children (n=39, males 23, mean+/-SD; 7.1+/-4.8years) were significantly higher (mean+/-SD; 952+/-282pg/mL) than those in adults (mean+/-SD; 562+/-146, P<0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that salphaKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum salphaKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of alpha-klotho gene. In this patient, salphaKl level was notably lower than those of age-matched controls. CONCLUSION: We established a detection system to measure human serum salphaKl for the first time. Age, Ca and Pi seem to influence serum salphaKl levels in a normal population. This detection system should be an excellent tool for investigating salphaKl functions in mineral metabolism.

Therapeutic effects of anti-FGF23 antibodies in hypophosphatemic rickets/osteomalacia.

X-linked hypophosphatemia (XLH), characterized by renal phosphate wasting, is the most common cause of vitamin D-resistant rickets. It has been postulated that some phosphaturic factor plays a causative role in XLH and its murine homolog, the Hyp mouse. Fibroblast growth factor 23 (FGF23) is a physiological phosphaturic factor; its circulatory level is known to be high in most patients with XLH and Hyp mice, suggesting its pathophysiological role in this disease. To test this hypothesis, we treated Hyp mice with anti-FGF23 antibodies to inhibit endogenous FGF23 action. A single injection of the antibodies corrected the hypophosphatemia and inappropriately normal serum 1,25-dihydroxyvitamin D. These effects were accompanied by increased expressions of type IIa sodium-phosphate cotransporter and 25-hydroxyvitamin-D-1alpha-hydroxylase and a suppressed expression of 24-hydroxylase in the kidney. Repeated injections during the growth period ameliorated the rachitic bone phenotypes typically observed in Hyp mice, such as impaired longitudinal elongation, defective mineralization, and abnormal cartilage development. Thus, these results indicate that excess actions of FGF23 underlie hypophosphatemic rickets in Hyp mice and suggest a novel therapeutic potential of the FGF23 antibodies for XLH.

Herbal medicine Shakuyaku-kanzo-to reduces paclitaxel-induced painful peripheral neuropathy in mice.

OBJECTIVES: Paclitaxel is widely used in cancer chemotherapy for the treatment of solid tumors such as breast, ovarian and lung cancer. However, it sometimes induces moderate to severe muscle pain, and impairs the patients' quality of life. An appropriate method for relieving this pain is not well established. Shakuyaku-kanzo-to, a herbal medicine, is known to relieve menstrual pain, muscle spasm, and muscle pain, and its effectiveness is expected. To ascertain the effectiveness of Shakuyaku-kanzo-to on paclitaxel-induced pain, we investigated the effects of Shakuyaku-kanzo-to and its constituent herbal medicines in a mouse model. METHODS: Seven-week-old male ddY mice were used. To make a mouse model of paclitaxel-induced pain, different single, intraperitoneally injected doses of this drug were tested in various groups of mice, and the optimal dose was determined. To estimate the effects of Shakuyaku-kanzo-to, the constituent herbal medicines Shakuyaku and Kanzo, and loxoprofen sodium as a non-steroidal anti-inflammatory drug on paclitaxel-induced pain, mechanical allodynia and hyperalgesia of the hind paw were assessed. RESULTS: Paclitaxel administered at a dose of 10mg/kg or more produced allodynia and hyperalgesia; the time courses were similar to those of pain after paclitaxel administration in cancer patients. Shakuyaku-kanzo-to significantly relieved the allodynia and hyperalgesia induced by paclitaxel (10mg/kg). Shakuyaku and Kanzo inhibited the allodynia and hyperalgesia to some extent, but not significantly, while loxoprofen sodium was without effects. CONCLUSIONS: A single administration of paclitaxel (10mg/kg) produced allodynia and hyperalgesia in mice, suggesting that it could be used as an animal model resembling the painful conditions observed in humans medicated with this drug. Using this model, Shakuyaku-kanzo-to was shown to relieve paclitaxel-induced painful peripheral neuropathy.

Anti-FGF23 neutralizing antibodies show the physiological role and structural features of FGF23.

Fibroblast growth factor (FGF)23 is proposed to play a physiological role in the regulation of phosphate and vitamin D metabolism; deranged circulatory levels of FGF23 cause several diseases with abnormal mineral metabolism. This paper presents a novel approach to analyze the mechanism of action of FGF23 using anti-FGF23 monoclonal antibodies that can neutralize FGF23 activities both in vitro and in vivo. We developed two antibodies (FN1 and FC1) that recognize the N- and C-terminal regions of FGF23, respectively. Both FN1 and FC1 inhibited FGF23 activity in a cell-based Klotho-dependent reporter assay. Their administration caused marked increases in serum phosphate and 1,25D levels in normal mice. These changes were accompanied by altered expression in the kidney of type IIa sodium-phosphate cotransporter, 25-hydroxyvitamin-D-1alpha-hydroxylase, and 24-hydroxylase. Thus, this study using neutralizing antibodies confirms that FGF23 is a physiological regulator of phosphate and vitamin D metabolism. We addressed the mechanism of action for these neutralizing antibodies. Structural analysis of the FGF23/FN1-Fab complex showed that FN1 masked putative FGF receptor-binding sites in the N-terminal domain of FGF23, whereas biochemical analyses showed that FC1 interfered with the association between FGF23 and Klotho by binding to the C-terminal domain of FGF23. Taken together, our results suggest that the N- and C-terminal domains of FGF23 are responsible for association with cognate FGF receptors and Klotho, respectively, and that these interactions are indispensable for FGF23 activity.