Justification of noninferiority margin: methodology considerations in an exposure-response analysis.

The pivotal clinical trial to support the indication of liver transplantation for everolimus was based on a noninferiority trial design. The unique trial design made it impossible to estimate the noninferiority margin at the design stage. Even though the trial was conducted based on a noninferiority margin of 12% for the primary efficacy endpoint, the lack of consensus on this margin made the efficacy results difficult to interpret. A novel pharmacometric approach was applied to derive a new margin. Even though it was smaller than 12%, the new margin was large enough so that the observed efficacy results became interpretable. This novel analysis was an important contributor to the "totality of evidence" approach that led to the approval of everolimus for the new indication. This approval represents the approval of a new drug in more than 10 years for the indication of liver transplantation.

Oxidation and rearrangements of flavanones by mammalian cytochrome P450.

To clarify the metabolic pathways of flavanones in mammals, the metabolism of (+/-)-flavanone and (+/-)-4'-methoxyflavanone by rat liver microsomes and recombinant human P450s in which structural changes are readily identifiable were examined. The beta-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation of flavone plus (+/-)-2,3-trans-flavanonol and of 4'-methoxyflavone plus (+/-)-2,3-trans-4'-methoxyflavanonol, respectively, by rat liver microsomes was observed. The same metabolites were generated by recombinant human P450s in addition to the formation of isoflavone from (+/-)-flavanone. The kinetic isotope effects in these reactions were examined using deuterated (+/-)-flavanone and (+/-)-4'-methoxyflavanone. There was a strong isotope effect in the production of flavanonols, but the isotope effect in the production of flavones was small. The results indicated that the P450-mediated conversion of (+/-)-flavanone and of (+/-)-4'-methoxyflavanone to the corresponding metabolites proceeded via abstraction of a hydrogen radical from the C-2- or C-3-position of the flavanone skeleton. The antioxidant properties of flavanone and its metabolites were examined by measuring superoxide-scavenging activity in a xanthine-xanthine oxidase-cytochrome c system. (+/-)-2,3-trans-Flavanonol had higher activity than that of other flavonoids. Flavanones are metabolized by mammalian P450s, providing important information relevant to the metabolism and pharmacological action of dietary flavanones.

Synthesis of cis- and trans-5,8-dihydroxy-5,6,7,8-tetrahydro-1,4-naphthoquinone.

Cis- and trans-5,8-dihydroxy-5,6,7,8-tetrahydro-1,4-naphthoquinone (1a, 1b) were for the first time synthesized from 5,8-dihydroxy-1,4-naphthoquinone (naphthazazine) (6) as a starting material and racemic triol (3) was first synthesized from 7. The configuration of 1a was determined by X-ray analysis.

Brain Abeta amyloidosis in APPsw mice induces accumulation of presenilin-1 and tau.

APPsw transgenic mice (Tg2576) overproducing mutant amyloid beta protein precursor (betaAPP) show substantial brain Abeta amyloidosis and behavioural abnormalities. To clarify the subsequent abnormalities, the disappearance of neurons and synapses and dystrophic neurite formation with accumulated proteins including hyperphosphorylated tau were examined. Tg2576 demonstrated substantial giant core plaques and diffuse plaques. The number of neurons was significantly decreased in the areas containing the amyloid cores compared with all other areas and corresponding areas in non-transgenic littermates in sections visualized by Nissl plus Congo red double staining (p<0.001). The presynaptic protein alpha-synuclein and postsynaptic protein drebrin were also absent in the amyloid cores. betaAPP and presenilin-1 were accumulated in dystrophic neurites in and around the core plaques. Tau phosphorylated at five independent sites was detected in the dystrophic neurites in the amyloid cores. Thus, the giant core plaques replaced normal brain tissues and were associated with subsequent pathological features such as dystrophic neurites and the appearance of hyperphosphorylated tau. These findings suggest a potential role for brain Abeta amyloidosis in the induction of secondary pathological steps leading to mental disturbance in Alzheimer's disease.

[Evaluation of the conjunctiva of leprosy patients using impression cytology].

OBJECTIVE: To evaluate ocular surface disorders (OSD) similar to cicatricial pemphigoid in leprosy patients. SUBJECT AND METHOD: Sixteen leprosy patients were examined. They were under treatment as inpatients at the Tama Zenshoen National Sanatorium, an institution for the treatment of leprosy. In addition to routine ophthalmological examinations, the patients conjunctival goblet cells were examined using impression cytology. OSD similar to ocular cicatricial pemphigoid were defined as the presence of at least two items of the following: symblepharon, cicatricial contraction of the conjunctival sac, corneal neovascularization, and palpebral entropion. RESULTS: OSD was present in 8 of the 16 patients (50%). Goblet cells were either decreased in number or absent in 7 cases(44%), and included 4 cases with OSD. Six of the 7 cases(86%) with reduced or absent goblet cells had been diagnosed as leprosy prior to 1944. CONCLUSION: Reduction or absence of goblet cells is a frequent feature in leprosy patients, particularly in longstanding ones of 56 years or more. Insufficient initial treatment appears to be a major cause of this finding.

Synthesis of phenanthridones using Diels-Alder reactions of 4-substituted 2(1H)-quinolones acting as dienophiles.

Diels-Alder reactions of 2(1H)-quinolones having an electron-withdrawing group at the 4-position with 1,3-butadiene derivatives were carried out to give the phenanthridones richly functionalized under the conditions of atmospheric and high pressure. Furthermore, the reactivities of 4-substituted 2(1H)-quinolones acting as a dienophile were examined using MO calculation.

The levels of cerebrospinal fluid Abeta40 and Abeta42(43) are regulated age-dependently.

Decreased levels of cerebrospinal fluid (CSF) Abeta42 is a diagnostic marker of Alzheimer's disease. To clarify the biological basis of this marker, the physiological alterations of CSF Abeta40 and Abeta42 by aging were studied. CSF samples from 92 normal subjects between 8 and 89 years old were measured using a specific ELISA for Abeta40 and Abeta42(43). High concentrations of Abeta40 and Abeta42(43) in the young group, under 29 years old, changed to be at low concentrations in the adult group between 30 and 59 years old. Subsequently, the levels increased again with age. Third order regression analysis showed a significant correlation between the levels of Abeta40 and age (Y = - 169 X(3) + 3.1X(2)- 0.02X + 4135; P < 0.034) and between the levels of Abeta42(43) and age (Y = - 46 X(3) + 0.9 X(2)- 0.005X + 992; P < 0.005). The levels of CSF Abeta40 and Abeta42(43) were physiologically regulated to show a U-shaped natural course in normal aging. These findings suggested that the physiological increase of Abeta42(43) over 59 years of age is selectively inhibited in Alzheimer's disease.

Abeta amyloidosis induces the initial stage of tau accumulation in APP(Sw) mice.

To clarify how Abeta deposits induce secondary tauopathy, the presence of phosphorylated tau, glycogen synthase kinase 3alpha (GSK3alpha), GSK3beta, cyclin-dependent kinase 5 (CDK5), mitogen-activated protein kinase (MAPK) and fyn were examined in the Tg2576 brain showing substantial brain Abeta amyloidosis and behavioral abnormalities. Phosphorylated tau at Ser199, Thr231/Ser235, Ser396 and Ser413 accumulated in the dystrophic neurites of senile plaques. The major kinase for tau phosphorylation was GSK3beta. Smaller contributions of GSK3alpha, CDK5 and MAPK were suggested. Thus, brain Abeta amyloidosis has a potential role in the induction of tauopathy leading to the mental disturbances of Alzheimer's disease.

Presynaptic inhibition of cerebellar GABAergic transmission by glutamate decarboxylase autoantibodies in progressive cerebellar ataxia.

Autoantibodies against glutamic acid decarboxylase (GAD) have been found in stiff-man syndrome, insulin dependent diabetes mellitus, and progressive cerebellar ataxia. A patient with progressive cerebellar ataxia is described who was positive for GAD autoantibodies, and had Sjögren's syndrome. Immunohistochemical studies using CSF and serum samples from the patient showed immunoreactivities in axon terminals of cerebellar GABAergic neurons. A whole cell patch clamp technique recording from rat cerebellar slices showed that the CSF, presumably through GAD autoantibodies, presynaptically inhibited GABAergic transmission. Intravenous administration of immunoglobulin failed to improve clinical symptoms and immunoreactivities examined after therapy. The findings suggest that GAD autoantibodies play a pathogenic part in reducing GABA release in in vitro slices.

Structure and stereochemistry of nardostachysin, a new terpenoid ester constituent of the rhizomes of Nardostachys jatamansi.

The structure and stereochemistry of a new terpenoid ester, nardostachysin (1), isolated from the rhizomes of Nardostachys jatamansi, were established as the 7',8'-dihydroxy-4'-methylene hexahydrocyclopenta[c]pyran-1'-one-8'-methyl ester of 7, 9-guaiadien-14-oic acid, by spectral and chemical studies.

Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer's disease brain.

The amyloid beta protein (Abeta) deposited in the Alzheimer's disease (AD) brain is heterogeneous at both its amino and carboxyl termini. Recent studies of the genetic forms of AD indicate that the aggregation and deposition of Abeta42 may be a common initiating event in all forms of AD. Here, we analyzed the amino termini of the Abeta species deposited in the AD brain, focusing specifically on species with amino-terminal pyroglutamate at position 3 (Abeta3(pE)). Immunocytochemical analysis of AD brains with an antibody specific for Abeta3(pE) confirmed that these species deposit in blood vessels and senile plaques. Using specific sandwich ELISAs, we determined the amounts of Abeta3(pE)-40 and Abeta3(pE)-42(43) in AD brain compared with other forms. This analysis showed that Abeta3(pE)-40 is closely correlated with the extent of Abeta deposition in blood vessels, whereas Abeta3(pE)-42(43) is not. In addition, Abeta3(pE)-42(43) is an important component of the Abeta deposited in senile plaques of the AD brain, constituting approximately 25% of the total Abeta42(43). In vitro comparison of Abeta1-42 and Abeta3(pE)-42 showed that Abeta3(pE)-42 is highly prone to oligomerization. These findings suggest that Abeta3(pE)-42 may be particularly important in AD pathogenesis.

Impaired neurotransmitter systems by Abeta amyloidosis in APPsw transgenic mice overexpressing amyloid beta protein precursor.

APPsw transgenic mice showing substantial features of brain Abeta amyloidosis such as senile plaques and behavioral abnormalities were examined by immunostaining to determine whether Abeta deposits could induce the subsequent disturbance of neurotransmitter systems including somatostatin, substance P and choline acetyltransferase (ChAT), which are prominent in the Alzheimer's disease brain. Somatostatin, substance P and ChAT disappeared in the areas of senile plaque and were accumulated in dystrophic neurites around the amyloid cores. These findings suggest a potential role of brain Abeta amyloidosis in disturbance of the neurotransmitter systems leading to memory disturbance of Alzheimer's disease.

Synthetic conversion of ACAT inhibitor to acetylcholinesterase inhibitor.

Natural product acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor pyripyropene A was synthetically converted to acetylcholinesterase (AChE) inhibitor via heterolitic cleavage of the 2-pyrone ring, followed by gamma-acylation/cyclization with several aroyl chlorides. The 4-pyridyl analogue selectively showed AChE inhibitory activity (IC50 7.9 microM) and no ACAT inhibitory activity IC50 = >1000 microM.

Accumulation of NACP/alpha-synuclein in lewy body disease and multiple system atrophy.

OBJECTIVES: NACP/alpha-synuclein is an aetiological gene product in familial Parkinson's disease. To clarify the pathological role of NACP/alpha-synuclein in sporadic Parkinson's disease and other related disorders including diffuse Lewy body disease (DLBD) and multiple system atrophy (MSA), paraffin sections were examined immunocytochemically using anti-NACP/alpha-synuclein antibodies. METHODS: A total of 58 necropsied brains, from seven patients with Parkinson's disease, five with DLBD, six with MSA, 12 with Alzheimer's disease, one with Down's syndrome, one with amyotrophic lateral sclerosis (ALS), three with ALS and dementia, one with Huntington's disease, two with progressive supranuclear palsy (PSP), one with Pick's disease, one with myotonic dystrophy, and three with late cerebellar cortical atrophy (LCCA), and 15 elderly normal controls were examined. RESULTS: In addition to immunoreactive Lewy bodies, widespread accumulation of NACP/alpha-synuclein was found in neurons and astrocytes from the brainstem and basal ganglia to the cerebral cortices in Parkinson's disease/DLBD. NACP/alpha-synuclein accumulates in oligodendrocytes from the spinal cord, the brain stem to the cerebellar white matter, and inferior olivary neurons in MSA. These widespread accumulations were not seen in other types of dementia or spinocerebellar ataxia. CONCLUSION: Completely different types of NACP/alpha-synuclein accumulation in Parkinson's disease/DLBD and MSA suggest that accumulation is a major step in the pathological cascade of both diseases and provides novel strategies for the development of therapies.

Age-related amyloid beta protein accumulation induces cellular death and macrophage activation in transgenic mice.

In view of the importance of amyloid beta protein accumulation in Alzheimer's disease, this paper examines age-related amyloid beta protein (Abeta) deposition and accompanying cellular changes in a mouse model in vivo. Transgenic mice were studied which expressed a gene encoding 18 residues of signal peptide and 99 residues of the carboxyl-terminal fragment (CTF) of the Abeta precursor, under the control of the cytomegalovirus enhancer/chicken beta-actin promoter. In the pancreas, Abeta accumulated in an age-dependent manner. Abeta deposits appeared as early as 3 weeks of age and increased in size and number from 4 to 16 months of age. The largest Abeta deposits were observed in the transgenic pancreas at 16 and 20 months of age. Haematoxylin and eosin staining, macrophage immunostaining, and electron microscopy showed that the Abeta fibril deposits closely correlated with degeneration of pancreatic acinar cells and macrophage activation. Abeta1-42 and Abetap3E-42 were predominant components of Abeta deposits among amino- and carboxyl-terminal modified Abeta species. These findings suggest that overproduction of Abeta causes age-related accumulation of Abeta fibrils, with accompanying cellular degeneration and macrophage activation in vivo.