Kazuharu Ozawa

Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of Aβ1–42, 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 Aβ42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.

Discovery of Tofogliflozin, a Novel C-Arylglucoside with an O-Spiroketal Ring System, as a Highly Selective Sodium Glucose Cotransporter 2 (SGLT2) Inhibitor for the Treatment of Type 2 Diabetes

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.

Tofogliflozin, a potent and highly specific sodium/glucose cotransporter 2 inhibitor, improves glycemic control in diabetic rats and mice

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 Ki 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.

Both N-terminal and C-terminal fragments of Presenilin 1 colocalize with neurofibrillary tangles in neurons and dystrophic neurites of senile plaques in Alzheimer's disease

Presenilin 1 (PS1) is a causative gene for chromosome 14‐linked familial Alzheimers 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 Alzheimers 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 Aβ 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. J. Neurosci. Res. 53:99–106, 1998.

Amyloid-β-protein isoforms in brain of subjects with PS1-linked, βAPP-linked and sporadic alzheimer disease

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.

Distinguishable effects of presenilin-1 and APP717 mutations on amyloid plaque deposition.

Both APP and PS-1 are causal genes for early-onset familial Alzheimers 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.

Enhanced Aβ40 Deposition Was Associated with Increased Aβ42/43 in Cerebral Vasculature with Dutch‐Type Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA‐D)

Abstract: Cerebrovascular deposition of the amyloid beta‐protein (Aβ) is a common pathologic event in patients with Alzheimers disease (AD) and certain related disorders. Such an Aβ 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 Aβ 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 Aβ deposition in brains with AD and Dutch‐type hereditary cerebral hemorrhage with amyloidosis (HCHWA‐D) on the basis of two amyloid species of Aβ40 and Aβ42/43 using specific monoclonal antibodies. Compared to Aβ deposition in senile plaques, the molecular composition of Aβ was distinguishable, indicating that the Aβ40 species is the main component for vascular amyloid. Furthermore, we found Aβ42/43 immunoreactivity was also much increased in amyloid angiopathy of all cases with HCHWA‐D. Taken together, amyloid angiopathy in HCHWA‐D may share an Aβ42‐driven deposition mechanism with plaque amyloid, resulting in enhanced Aβ40 deposition.

Lack of amyloid plaque formation in the central nervous system of a patient with Werner syndrome

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 ɛ3/ɛ3 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.

Cerebral vascular accumulation of Dutch-type Aβ42, but not wild-type Aβ42, in hereditary cerebral hemorrhage with amyloidosis, Dutch type

Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA‐D), is an autosomal dominant disorder caused by the Dutch mutation (E693Q) in the β‐amyloid precursor protein. This mutation produces an aberrant amyloid β (Aβ) species (AβE22Q) and causes severe meningocortical vascular Aβ deposition. We analyzed the Aβ composition of the vascular amyloid in the brains of HCHWA‐D patients. Immunohistochemistry demonstrated that the vascular amyloid contained both Aβ40 and Aβ42, with a high Aβ40/Aβ42 ratio. In Western blotting of cerebral microvessel fractions isolated from the brains, both wild‐type and Dutch‐type Aβ40 were observed as major species. Reverse‐phase HPLC‐mass spectrometric analysis of the fractions revealed both wild‐type and Dutch‐type Aβ38 as the other main components of the vascular amyloid. Moreover, we detected peaks corresponding to Dutch‐type Aβ42 but not to wild‐type Aβ42. These results suggest a pathogenic role for the mutant Aβ42 in addition to the mutant Aβ40 in the cerebral amyloid angiopathy of HCHWA‐D.

Pharmacokinetic and Pharmacodynamic Modeling for the Effect of Sodium–Glucose Cotransporter Inhibitors on Blood Glucose Level and Renal Glucose Excretion in db/db Mice

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.