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Chronic kidney disease is characterized as impaired renal function along with the imbalance and dysregulation of mineral metabolism; recognized as chronic kidney disease-mineral and bone disorder. Hyperphosphatemia, characterized by altered phosphate homeostasis along with elevated fibroblast growth factor-23 and intact parathyroid hormone, is such an alteration of mineral metabolism. We discovered a novel inhibitor, EOS789, that interacts with several sodium-dependent phosphate transporters (NaPi-IIb, PiT-1, and PiT-2) known to contribute to intestinal phosphate absorption. This inhibitor dose-dependently increased the fecal phosphorus excretion rate and inversely decreased the urinary phosphorus excretion rate in normal rats, suggesting inhibition of intestinal phosphorus absorption. In rats with adenine-induced hyperphosphatemia, EOS789 markedly decreased the serum phosphate, fibroblast growth factor-23, and intact parathyroid hormone below values found in normal control rats. Notably, this pan-phosphate transporter inhibitor exhibited a more potent effect on serum phosphate than a NaPi-IIb-selective inhibitor in rats with hyperphosphatemia indicating that PiT-1 and PiT-2 play important roles in intestinal phosphate absorption. Moreover, in a long-term study, EOS789 sustained the suppression of serum phosphorus in parallel with fibroblast growth factor-23 and intact parathyroid hormone and ameliorated ectopic calcification of the thoracic aorta. Additionally, EOS789 treatment also ameliorated kidney deterioration in rats with progressive kidney injury, probably due to the strict phosphate control. Thus, EOS789 has potent efficacy against hyperphosphatemia and its complications and could provide a significant benefit to patients who are ineffectively treated with phosphate binders.

5a-Carba-β-D-glucopyranose derivatives were synthesized and identified as novel SGLT2-selective inhibitors. These inhibitors exhibited potent SGLT2 inhibition with high selectivity over SGLT1. Among the tested compounds, 6f indicated the most potent hSGLT2 inhibition and the highest selectivity over hSGLT1. Moreover, the pharmacokinetics data also showed that 6h, which had the same aglycon structure as sergliflozin-active (3-active), had a threefold longer half-life time (T(1/2)) than sergliflozin (3) with a high distribution volume in db/db mice. Subsequently, 6h lowered blood glucose levels as much as 3 and showed longer hypoglycemic action than 3 in db/db mice.

Werner syndrome (WS) is an autosomal recessive disorder associated with accelerated aging. It is well documented on systemic aging but it is unclear whether the brain with WS shows accelerated aging. A 55-year-old patient with WS was studied and it was found that a deletion mutation of exon 26 of the WRN gene was not associated with CNS pathology, such as amyloid plaques or NFT. Furthermore, additional genetic analysis showed an apolipoprotein E genotype of epsilon3/epsilon3 that did not play either an accelerating or inhibitory action on' amyloid deposition. Therefore, based on the genetic and neuropathological analysis, it was observed that the WS-associated aging seen in many organs did not extend to the CNS.

Both APP and PS-1 are causal genes for early-onset familial Alzheimer's disease (AD) and their mutation effects on cerebral Abeta deposition in the senile plaques were examined in human brains of 29 familial AD (23 PS-1, 6 APP) cases and 14 sporadic AD cases in terms of Abeta40 and Abeta42. Abeta isoform data were evaluated using repeated measures analysis of variance which adjusted for within-subject measurement variation and confounding effects of individual APP and PS-1 mutations, age at onset, duration of illness and APOE genotype. We observed that mutations in both APP and PS-1 were associated with a significant increase of Abeta42 in plaques as been documented previously. In comparison to sporadic AD cases, both APP717 and PS-1 mutation cases had an increased density (measured as the number of plaques/mm(2)) and area (%) of Abeta42 plaques. However, we found an unexpected differential effect of PS-1 but not APP717 mutation cases. At least some of PS-1 but not APP717 mutation cases had the significant increase of density and area of Abeta40-plaques as compared to sporadic AD independently of APOE genotype. Our results suggest that PS-1 mutations affect cerebral accumulation of Abeta burden in a different fashion from APP717 mutations in their familial AD brains.

Presenilin 1 (PS1) is a causative gene for chromosome 14-linked familial Alzheimer's disease. The gene product is known to be cleaved into N-terminal fragments (PS1-N) and C-terminal fragments (PS1-C). To understand the pathophysiological role of PS1, we conducted immunohistochemical studies using antibodies specific for PS1-N and PS1-C in sporadic Alzheimer's disease (AD). Both antibodies showed punctuate staining exclusively in neurons and their processes in both control and AD brains. PS1-N immunolabeling colocalized with neurofibrillary tangles (NFTs) in 36% of NFT-bearing neurons and with dystrophic neurites in 28% of senile plaques (SPs). PS1-C immunolabeling colocalized with dystrophic neurites in 70% of NFT-bearing SPs and with intraneuronal NFTs in 32% of NFT-bearing neurons. Both antibodies did not detect PHF-tau-positive neuropil threads and Abeta amyloid fibrils. The colocalization was also found in 33-38 % of NFT-bearing neurons in progressive supranuclear palsy. These results indicate that both PS1-N and PS1-C fragments are deposited in part of NFT-bearing neurons and dystrophic neurites in SPs; both are the pathologic hallmarks of AD.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes are associated with early-onset autosomal dominant familial Alzheimer's disease, and the gene products are endoproteolytically processed to yield N-terminal fragments (NTF) and C-terminal fragments (CTF). We have studied the cleavage site of the PS2 protein in stably transfected human neuroblastoma cells. The 23 kD PS2-CTF was isolated by a combination of anion exchange chromatography and affinity chromatography and directly sequenced. The N-terminus of the PS2-CTF started at residue 307, which indicated that the cleavage occurs between Lys306 and Leu307 in the proximal portion of the large hydrophilic loop. This site is close to the cleavage positions observed in the PS1 protein.

Inhibition of sodium glucose cotransporter 2 (SGLT2) has been proposed as a novel therapeutic approach to treat type 2 diabetes. In our efforts to discover novel inhibitors of SGLT2, we first generated a 3D pharmacophore model based on the superposition of known inhibitors. A search of the Cambridge Structural Database using a series of pharmacophore queries led to the discovery of an O-spiroketal C-arylglucoside scaffold. Subsequent chemical examination combined with computational modeling resulted in the identification of the clinical candidate 16d (CSG452, tofogliflozin), which is currently under phase III clinical trials.

The purpose of this study is to characterize the relationship between pharmacokinetics (PK) and pharmacodynamics (PD) of sodium–glucose cotransporter (SGLT) inhibitors. PK–PD studies of SGLT inhibitors (CH4941527 and T-1095), which have different half-life and selectivity to SGLT2, were performed using db/db mice. The time courses of compound concentration in plasma, blood glucose (BG), and renal glucose excretion were measured after a single oral administration of each SGLT inhibitor. An indirect-response PK–PD model was developed, in which it was assumed that an SGLT inhibitor enhances renal glucose excretion and the enhanced glucose excretion reduces BG. In the PK–PD study, both SGLT inhibitors increased renal glucose excretion and reduced BG in a dose-dependent manner. The present PK–PD model could suitably capture the effect of SGLT inhibitors and the effect shown suggested that the BG reduction could be explained by the enhanced renal glucose excretion. There were no great differences in the estimated PD parameters between the two inhibitors and they were comparable to the data from previously reported pharmacological studies. The present PK–PD model is helpful for understanding the plasma concentration-dependent effect of SGLT inhibitors on renal glucose excretion and BG. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:4347–4356, 2012

C-Aryl 5a-carba-β-d-glucopyranose derivatives were synthesized and evaluated for inhibition activity against hSGLT1 and hSGLT2. Modifications to the substituents on the two benzene rings resulted in enhanced hSGLT2 inhibition activity and extremely high hSGLT2 selectivity versus SGLT1. Using the created superimposed model, the reason for the high hSGLT2 selectivity was speculated to be that additional substituents occupied a new space, in a different way than known inhibitors. Among the tested compounds, the ethoxy compound 5h with high hSGLT2 selectivity exhibited more potent and longer hypoglycemic action in db/db mice than our O-carbasugar compound (1) and sergliflozin (2), which could be explained by its improved PK profiles relative to those of the two compounds. These results indicated that 5h might be a promising drug candidate for the treatment of type 2 diabetes.

Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential.

Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), is an autosomal dominant disorder caused by the Dutch mutation (E693Q) in the beta-amyloid precursor protein. This mutation produces an aberrant amyloid beta (Abeta) species (AbetaE22Q) and causes severe meningocortical vascular Abeta deposition. We analyzed the Abeta composition of the vascular amyloid in the brains of HCHWA-D patients. Immunohistochemistry demonstrated that the vascular amyloid contained both Abeta40 and Abeta42, with a high Abeta40/Abeta42 ratio. In Western blotting of cerebral microvessel fractions isolated from the brains, both wild-type and Dutch-type Abeta40 were observed as major species. Reverse-phase HPLC-mass spectrometric analysis of the fractions revealed both wild-type and Dutch-type Abeta38 as the other main components of the vascular amyloid. Moreover, we detected peaks corresponding to Dutch-type Abeta42 but not to wild-type Abeta42. These results suggest a pathogenic role for the mutant Abeta42 in addition to the mutant Abeta40 in the cerebral amyloid angiopathy of HCHWA-D.

Cerebrovascular deposition of the amyloid beta-protein (Abeta) is a common pathologic event in patients with Alzheimer's disease (AD) and certain related disorders. Such an Abeta vascular deposition occurs primarily in the medial layer of the cerebral vessel wall in an assembled fibrillar state. These deposits are associated with several pathological responses, including degeneration of the smooth muscle cells in the cerebral vessel wall. Severe cases of cerebrovascular Abeta deposition are also accompanied by loss of vessel wall integrity and hemorrhagic stroke. Although the reasons for this pathological consequence are unclear, altered proteolytic mechanisms within the cerebral vessel wall may be involved. We analyzed cerebral Abeta deposition in brains with AD and Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) on the basis of two amyloid species of Abeta(40) and Abeta(42/43) using specific monoclonal antibodies. Compared to Abeta deposition in senile plaques, the molecular composition of Abeta was distinguishable, indicating that the Abeta(40) species is the main component for vascular amyloid. Furthermore, we found Abeta(42/43) immunoreactivity was also much increased in amyloid angiopathy of all cases with HCHWA-D. Taken together, amyloid angiopathy in HCHWA-D may share an Abeta(42)-driven deposition mechanism with plaque amyloid, resulting in enhanced Abeta(40) deposition.

We describe a targeting technique that selects antigen-specific receptors on B lymphocytes using antigen driven selective production of monoclonal antibodies which are directed against functional peptide sequences within the presenilin 1 molecule that is believed to be related to the early-onset of familial Alzheimer's disease. Three different peptide sequences of presenilin 1 were constructed, one including the region around the amino acid position 300, where the putative cleavage site exists and the other two present in the N- and C-terminal regions of that site. The efficiency in production of the desired monoclonal antibodies was at least 5-40-fold that obtained with the poly(ethylene glycol) (PEG)-mediated method. In addition, monoclonal antibodies directed against each of the peptide sequence displayed a high specificity for the corresponding peptide, in contrast to the lack of success using the PEG method. Also, the selection of surface immunoglobulin receptors on B lymphocytes by the peptides of interest was confirmed by immunofluorescent analysis. Here we demonstrate that targeting B lymphocytes results in the successful and efficient production of highly specific monoclonal antibodies against the lower antigenic peptide sequences.

Background: Alterations in the metabolism of the amyloid precursor protein and the formation of b-amyloid (Ab) plaques are associated with neuronal death in Alzheimer disease (AD). The plaque subtype Abx-42 occurs as an early event, with Abx-40 plaques forming at a later stage. In dementia with Lewy bodies (DLB), an increase in the amount of cortical Ab occurs without severe cortical neuronal losses. Objective: To advance our understanding of the natural history of Ab in neurodegenerative diseases. Design: We evaluated the expression of Abx-40 and Abx-42 in DLB using monoclonal antibodies and immunohistochemical techniques in 5 brain regions. The data were compared with those elicited with normal aging and from patients with AD. Setting and Patients: A postmortem study involving 19 patients with DLB without concurrent neuritic degeneration, 10 patients with AD, and 17 aged persons without dementia for control subjects. Results: The Ab plaques were more numerous in patients with DLB than in controls in most brain regions, although the Abx-42 plaque subtype was predominant in both conditions. Overall, Abx-42 plaque density was similar in patients with DLB and those with AD, but Abx-40 plaques were more numerous in persons with AD than in those with DLB. The ratio of Abx-40 to Abx-42 plaques was significantly reduced in persons with DLB compared with patients with AD. Conclusions: The Ab plaques were more numerous in patients with DLB than persons with normal aging, but the plaque subtypes were similar. The relative proportion of the 2 Ab plaque subtypes in DLB is distinguishable from that in AD. Arch Neurol. 1999;56:1111-1118

To determine whether similar abnormalities of various soluble full-length and N-terminal truncated Abeta peptides occur in postmortem cerebral cortex of affected PS1 mutation carriers, we examined the amounts of two amyloid species ending at residue 40 or at residues 42(43) using sandwich ELISA systems. Our results indicate that PS1 mutations effect a dramatic accumulation in brain of the highly insoluble potentially neurotoxic long-tailed isoforms of the Abeta peptide such as Abeta1-42(43) and Abetax-42(43). This enhancing effect of PS1 mutation on Abetax-42(43) deposition was highly similar to that of a betaAPP mutation (Val717Ile) but the effects on Abetax-40 production were significantly different between these two causal genes. In contrast to previous studies of soluble Abeta in plasma and in supernatants from cultured fibroblasts of subjects with PS1 mutations, our studies also show that there is an increase in insoluble Abetax-40 peptides in brain of subjects with PS1 mutations.

In normal rats, consecutive administrations of AA-193 for 7 days maintained the dose-dependent uricosuric activity without significant changes of the plasma urate level. In clearance studies, AA-193 produced an increase in the fractional excretion of urate (FEua) namely an inhibition of the net urate reabsorption in the nephron, which was probably dependent on the plasma concentration of the agent. During in vitro studies, 1 mmol/L AA-193 had no effect on liver uricase activity and 0.2 mmol/L AA-193 did not inhibit xanthine dehydrogenase activity. Therefore, it is unlikely that AA-193 at physiologic doses has a significant effect on either the production or degradation of urate. To assess the hypouricemic effect of AA-193 derived from its uricosuric effect, we used uricase-inhibited rats produced by oxonate feeding. In the hyperuricemic rat model, consecutive administrations of AA-193 for 7 days increased urate excretion and decreased the plasma urate level. We conclude that AA-193 has a hypouricemic effect caused by increases in urate excretion in hyperuricemic rats.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of A beta1-42, indicating that PS-1 is involved in amyloidogenesis. However, PS-1 transgenic mice have failed to show amyloid plaques in their brains. Because PS-1 mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited A beta42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.