Donanemab (LY3002813) Phase 1b Study in Alzheimer's Disease: Rapid and Sustained Reduction of Brain Amyloid Measured by Florbetapir F18 Imaging.

BACKGROUND: Donanemab (LY3002813) is an IgG1 antibody directed at an N­terminal pyroglutamate of amyloid beta epitope that is present only in brain amyloid plaques. OBJECTIVES: To assess effects of donanemab on brain amyloid plaque load after single and multiple intravenous doses, as well as pharmacokinetics, safety/tolerability, and immunogenicity. DESIGN: Phase 1b, investigator- and patient-blind, randomized, placebo-controlled study. SETTING: Patients recruited at clinical research sites in the United States and Japan. PARTICIPANTS: 61 amyloid plaque-positive patients with mild cognitive impairment due to Alzheimer's disease and mild-to-moderate Alzheimer's disease dementia. INTERVENTION: Six cohorts were dosed with donanemab: single dose 10-, 20- or 40- mg/kg (N = 18), multiple doses of 10-mg/kg every 2 weeks for 24 weeks (N = 10), and 10- or 20-mg/kg every 4 weeks for 72 weeks (N=18) or placebo (N = 15). MEASUREMENTS: Brain amyloid plaque load, using florbetapir positron emission tomography, was assessed up to 72 weeks. Safety was evaluated by occurrence of adverse events, magnetic resonance imaging, electrocardiogram, vital signs, laboratory testing, neurological monitoring, and immunogenicity. RESULTS: Treatment with donanemab resulted in rapid reduction of amyloid, even after a single dose. By 24 weeks, amyloid positron emission tomography mean changes from baseline for single donanemab doses in Centiloids were: -16.5 (standard error 11.22) 10-mg/kg intravenous; 40.0 (standard error 11.23) 20 mg/kg intravenous; and -49.6 (standard error 15.10) 40-mg/kg intravenous. Mean reduction of amyloid plaque in multiple dose cohorts by 24 weeks in Centiloids were: 55.8 (standard error 9.51) 10-mg/kg every 2 weeks; -50.2 (standard error 10.54) 10-mg/kg every 4 weeks; and -58.4 (standard error 9.66) 20-mg/kg every 4 weeks. Amyloid on average remained below baseline levels up to 72 weeks after a single dose of donanemab. Repeated dosing resulted in continued florbetapir positron emission tomography reductions over time compared to single dosing with 6 out of 28 patients attaining complete amyloid clearance within 24 weeks. Within these, 5 out of 10 patients in the 20 mg/kg every 4 weeks cohort attained complete amyloid clearance within 36 weeks. When dosing with donanemab was stopped after 24 weeks of repeat dosing in the 10 mg every 2 weeks cohort, florbetapir positron emission tomography reductions were sustained up to 72 weeks. For the single dose cohorts on day 1, dose proportional increases in donanemab pharmacokinetics were observed from 10 to 40 mg/kg. Dose proportional increases in pharmacokinetics were also observed at steady state with the multiple dose cohorts. Donanemab clearance was comparable across the dose levels. Mean donanemab elimination-half-life following 20 mg/kg single dose was 9.3 days with range of 5.6 to 16.2 days. Greater than 90% of patients had positive treatment-emergent antidrug antibodies with donanemab. However, overall, the treatment-emergent antidrug antibodies did not have a significant impact on pharmacokinetics. Donanemab was generally well tolerated. Amongst the 46 participants treated with donanemab, the following amyloid-related imaging abnormalities, common to the drug class, were observed: 12 vasogenic cerebral edema events (12 [19.7%] patients), 10 cerebral microhemorrhage events (6 [13.0%] patients), and 2 superficial siderosis events (2 [4.3%] patients). CONCLUSIONS: Single and multiple doses of donanemab demonstrated a rapid, robust, and sustained reduction up to 72 weeks in brain amyloid plaque despite treatment-emergent antidrug antibodies detected in most patients. Amyloid-related imaging abnormalities were the most common treatment-emergent event.

Transcriptional regulation of a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean tissue is linked to the phytochrome response.

The effects of white light, far-red light, and darkness on the transcription of a soybean ribulose-1,5-biphosphate carboxylase small subunit gene, SRS1, were investigated. RNA was labeled with [alpha-32P]UTP in nuclei isolated from plants grown under different conditions of light and darkness and used to probe Southern blots and dot blots. The levels of small subunit mRNA synthesis were normalized to ribosomal RNA synthesis. We demonstrate that the SRS1 gene is transcribed at a rate 16- to 32-fold higher in plants grown in the light than in those grown in darkness. Transcription of the small subunit increased dramatically when plants grown in darkness were given 30 min to 6 h of light and then leveled off after 24 to 48 h of exposure. When light-grown seedlings were exposed to greater than 2 h of darkness, a gradual decrease in transcription was detected. This decrease in transcription reached basal dark-grown levels after 48 h of exposure to darkness. The increase in transcription in etiolated seedlings treated with white light for 15 min could be reduced to basal levels if the treatment was followed by treatment with far-red light for 15 min. In addition, transcription in ligh-grown seedlings was reduced to basal levels when plants were exposed to far-red light for 15 min. The transcription of this ribulose-1,5-biphosphate carboxylase small subunit gene is strongly positively regulated by white light, is negatively regulated by far-red light, and exhibits a classic phytochrome-linked response.

GS32, a novel Golgi SNARE of 32 kDa, interacts preferentially with syntaxin 6.

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

Pyramidal neuron loss and "glycine-site therapy": a need for an animal model and study in late-life depression.

Localization and other studies of glutamatergic receptors are hampered, in part, as pyramidal neurons in the neocortex cannot yet be selectively destroyed in laboratory animals. Demonstration of glutamatergic neuron dysfunction in Alzheimer's disease may allow verification of a technique suitable for the study of these cells in disorders without histopathological hallmarks. This includes Major Depressive Disorder in the elderly which has a poor prognosis.

Morphology of intracellularly labeled interneurons in the dentate gyrus of the immature rat.

Although many aspects of the morphological development of interneurons in the dentate gyrus have been described, the full extent of their dendrites and local axon projections in immature rodents has not been examined. Here intracellular labeling was used to assess the branching patterns of interneurons in the dentate gyrus of rat pups between 7 and 9 days of age. Labeled neurons were located within or just below the granule cell layer, and most were classified as GABAergic basket neurons on the basis of their dendritic morphologies. All labeled interneurons exhibited immature characteristics. Spines were present on cell bodies and dendrites, and growth cones were visible on some dendrites and axons. In spite of these immature features, the dendrites and axon arbors of the labeled neurons were extensive. Many apical dendrites reached the top of the molecular layer, and a number of basal dendrites extended to the CA3 pyramidal cell layer of the hippocampus. Elaborate axon plexuses were present within the dentate gyrus itself, and axon collaterals of several neurons extended beyond the dentate gyrus to branch within regions CA3 and CA1 of the hippocampus. These results indicate that the dendrites and axon collaterals of dentate interneurons are extensive at a time when the principal neurons, the granule cells, are still proliferating. These data are consistent with the idea that GABAergic interneurons may influence granule cell development in the dentate gyrus, as well as pyramidal cell maturation in the hippocampus proper.

The use of Fas Ligand, TRAIL and Bax in gene therapy of prostate cancer.

Prostate cancer is the second leading cause of cancer death in the United States. Treatment options for confined disease are generally successful in prolonging life but long-term cures (10-15 years) are elusive for the majority of patients. The prognosis for advanced extra-capsular prostate cancer is grim. However, we are now entering the era of gene therapy options for treatment of prostate cancer. The human genome project coupled with genomics and protemics are providing information that will lead to selection of genes for treatment of prostate cancer. The problem is the science of delivery lags behind knowledge of gene function. Thus, it is important to develop therapies that do not require delivery to 100% of tumor cells but which nevertheless kills the entire cancer by virtue of the bystander effect or other means. This review covers the use, in gene therapy, of apoptotic inducing molecules such as Fas Ligand, and TRAIL which are believed to induce bystander killing activity and Bax which also may function in a similar way.

Ante mortem cerebral amino acid concentrations indicate selective degeneration of glutamate-enriched neurons in Alzheimer's disease.

There is little information about major constituents of the brain in Alzheimer's disease. In the case of amino acids most of the previous data are contradictory. These have been interpreted in an anatomic and neurotransmitter as well as a metabolic context. To help clarify this, the contents of 14 amino acids and ethanolamine were determined in samples of neocortex from diagnostic craniotomies of 15 demented patients (10 with Alzheimer's disease) and other neurosurgical procedures (57 patients, 18 with intractable depression). A comprehensive survey of the effects of possible complicating factors on the concentrations of amino acids showed that artefacts were few; this was in contrast to a post mortem series of brains (16 with Alzheimer's disease and 16 controls; six regions assayed). We have used the ante mortem data to provide the basis for an accurate comparison of amino acid values between Alzheimer and control samples. In Alzheimer's disease, the mean contents of many amino acids were slightly higher (sum of the increases of those significantly affected was 15 nmol/mg protein) whereas glutamate content alone was significantly reduced (by 16 nmol/mg protein). This was not a feature of depression or a group of patients with other dementias. Glutamate content of Alzheimer samples was related to pyramidal neuron density in cortical layer III. These alterations were detected relatively early during the course of Alzheimer's disease and are considered to be due to loss of corticocortical glutamatergic association pathways.

Terminal coma affects messenger RNA detection in post mortem human temporal cortex.

In situ hybridization histochemistry has been used to study the amount of M1 muscarinic receptor mRNA in temporal cortex from subjects with Alzheimer's disease and other neurodegenerative disorders, where the duration of terminal coma was known. Total polyadenylated mRNA and glutamate decarboxylase activity were also measured. Both muscarinic receptor mRNA and enzyme activity showed a significant decline with increasing duration of terminal coma, but were not related to diagnosis. Polyadenylated mRNA signal did not show an association with coma. These data indicate the need to consider the nature of the terminal illness in post mortem studies of mRNA as well as for neurochemical research.

Brain amino acid concentrations and Ca2+-dependent release in intractable depression assessed antemortem.

The concentrations of 3 putative neurotransmitters (glutamate, aspartate and gamma-aminobutyrate), 4 related amino acids and 5 non-transmitter-related amino acids have been measured in neurosurgical samples (frontal cortex) from patients with intractable depression and controls. In addition, the glutamate receptor agonist 2-amino-4-sulpho-butanoic acid (homocysteic acid) has been identified in human brain and measured in these samples. There were no changes in the concentrations of amino acids in depressed patients compared to control with the exception of aspartic and homocysteic acids which were elevated in a sub-group of patients with depression compared to control. The Ca2+-dependent release (K+-stimulated) of putative neurotransmitters has been demonstrated for the first time from brain tissue of depressed patients. Glutamate release was unaltered from the control value. Aspartate values showed unexplained variability but it's release and that of gamma-aminobutyrate were elevated in some depressed subjects. These results do not support the hypothesis of reduced amino acid function in depressive illness.

Somatostatin content and release measured in cerebral biopsies from demented patients.

Somatostatin-like immunoreactivity (SLIR) has been assayed in frontal and temporal cortex obtained at diagnostic craniotomy and post-mortem from patients with histologically verified Alzheimer's disease. SLIR content was not significantly different from controls in the frontal and temporal lobes, except in the temporal cortex post-mortem. The K+-stimulated release of endogenous SLIR from tissue prisms ('mini-slices') prepared from neocortex obtained at diagnostic craniotomy from Alzheimer patients was not below the control values. Indices of cholinergic varicosities in similar samples from the frontal and temporal lobes are reduced; accordingly, somatostatin does not seem to be as prominently involved in these regions. Patients with Alzheimer's disease underwent neuropsychological assessment shortly before sampling the temporal lobe. Scores for WAIS full scale and the verbal subscale and the Token Test (measure of language comprehension) significantly correlated with the SLIR content; mean values (fmole/mg protein) were 817, 1468 and 1363 for aphasic and non-aphasic Alzheimer patients and controls, respectively. Ventricular fluid obtained from Alzheimer patients during surgery, did not have a significantly different SLIR content compared to controls. SLIR contents of ventricular fluid and neocortex from demented patients, without any specific histological changes in the sample obtained at diagnostic craniotomy, were also not significantly different from controls. Previously, we have shown that these demented patients, as well as those with histologically verified Alzheimer's disease, have a reduced SLIR content of lumbar fluid so it seems that somatostatin neurones located outside the frontal and temporal lobes are affected relatively early in the disease process.

Topographical distribution of neurochemical changes in Alzheimer's disease.

Biochemical indices of cortical nerve cells affected in Alzheimer's disease have been proposed (excitatory dicarboxylic amino acid, EDAA, sodium-dependent carrier; phosphate-activated glutaminase activity; serotonin type 2 recognition site; somatostatin-like immunoreactivity). These and the content of EDAAs and two related amino acids, and choline acetyltransferase (ChAT) activity have been measured in up to 13 areas of cerebral cortex and the cerebellar cortex from 16 patients with Alzheimer's disease and 17 controls. Reduction of the index of the serotonin recognition site, somatostatin content and another biochemical index of interneurones coincide and indicate a rather unexpected focal loss of such neurones from the parietal lobe. No unequivocal measure of the integrity of pyramidal neurones could be established as the content of no amino acid was reduced, the index of the EDAA carrier showed evidence of change in few brain regions and glutaminase activity was subject to unexplained variability. ChAT activity alone closely paralleled a previous report of the distribution of morphological degeneration. The results are discussed in relation to therapy and positron emission tomography.

Influence of tetrahydro-9-aminoacridine on excitatory amino acid release.

Alzheimer's disease may be associated with an early and clinically relevant degeneration of some cortical excitatory amino acid-releasing neurons. Tetrahydro-9-aminoacridine (tacrine) might be an effective drug for the treatment of the disease. Various pharmacological paradigms (in rats) related to amino acid release are shown here to be modified both in vivo and in vitro by the drug. These effects are only observed with high concentrations, so it is unlikely that tacrine acts through amino acid release in humans.

The right to refuse treatment is not a right to be killed.

It is widely accepted now that a patient's right to refuse treatment extends to circumstances in which the exercise of that right may lead to the patient's death. However, it is also often effectively assumed, without argument, that this implies a patient's right to request another agent to intervene so as to bring about his or her death, in a way which would render that agent guilty of murder in the absence of such a request. But the right to refuse treatment can, logically, have no such implication, and the mistaken supposition that it does conflates a right to die with a right to be killed. Confusion over this issue is brought out by an examination of conflicting opinion concerning the permissible termination of ventilation for mentally competent patients. A wider lesson may be drawn regarding the need for the ethical assessment of new forms of life-sustaining medical technology.

The subunit structure of phosphoglucose isomerase from bakers' yeast.

Bakers' yeast phosphoglucose isomerase was studied by both chemical and physical methods to determine its submit structure. Gel filtration in 6 M guanidine HCl as well as acrylamide gel electrophoresis of sodium dodecyl sulfatedentured phosphoglucose isomerase showed two speices corresponding to one-half and one-fourth of the preparative molecular weight of 119,400 determined by equilibrium centrifugation. Further centrifugation studies showed that the enzyme could be completely dissociated to species of 30,000 molecular weight. Peptide maps of tryptic hydrolysates of denatured and chemically modified enzyme showed that the protein is composed of four identical or nearly identical sub-units. The results of amino acid analysis, except half-cystine content, were compatible with identical subunits. The appearent partial specific volume and extinction coefficient were also determined.

The mammalian ARF-like protein 1 (Arl1) is associated with the Golgi complex.

A rat cDNA clone was isolated which encodes a protein displaying characteristics of a ras-like small GTPase. The deduced amino acid sequence shows the highest amino acid identity (79%) with the Drosophila ARF-like protein 1 (dArl1) among all the known members of the ras-like small GTPase superfamily. The encoded protein was tentatively named rat Arl1 (rArl1). Northern blotting analysis revealed that the rArl1 gene is ubiquitously expressed in rat tissues. Recombinant rArl1 fused to glutathione-S-transferase (GST) to create GST-rArl1 binds GTP-gamma-S in a dose-dependent manner. Polyclonal antibodies raised against two unique rArl1 peptides recognized a 22 kDa protein in total NRK cell lysate. Immunofluorescence microscopy of NRK cells revealed discrete perinuclear labelling that could be competed out by GST-rArl1 but not GST. Examination of 8 additional cell lines revealed a similar labelling, suggesting that the antigen recognised by the antibodies is conserved and widely-expressed. Co-localization experiments in NRK cells with antibodies to mannosidase II and a newly identified cis-Golgi protein, p28, showed that rArl1 is localized to the Golgi complex. When cells were treated with nocodazole, the Golgi complex marked by mannosidase II and p28 was fragmented into punctate structures scattered throughout the cell, in which rArl1 was colocalized. Treatment with brefeldin A (BFA) resulted in the redistribution of rArl1 and mannosidase II into the cytoplasm and endoplasmic reticulum, respectively. The kinetics of the redistribution of rArl1 in response to BFA differ from those of ARF and beta-COP, two components of non-clathrin coated vesicles.

Lack of change in neurochemical markers during the postepileptic phase of intrahippocampal tetanus toxin syndrome in rats.

The chronic epileptic syndrome induced by injecting tetanus toxin into rat hippocampus causes functional changes that essentially are permanent, outlasting the period of active seizures by at least 1 year. These long-term changes have been characterized by an impaired performance on a range of behavioral tasks, which in turn have been associated with a physiologic depression of hippocampal evoked responses but not with any discernible histopathology. In the present study, we examined the hippocampi of rats in the postseizure phase of the tetanus toxin model and observed no significant changes in the concentration of neurochemical markers for six neurotransmitters. Therefore, the long-term reduction in hippocampal excitability cannot be attributed to any major loss of afferents or hippocampal neurons using aspartate, acetylcholine, gamma-aminobutyric acid (GABA), glutamate, norepinephrine (NE), or serotonin as their transmitters.

Interactions of glyceraldehyde-3-phosphate dehydrogenase with G- and F-actin predicted by Brownian dynamics.

Brownian dynamics (BD) was used to simulate the binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to G- and F-actin. High-resolution three-dimensional models (X-ray and homology built) of the proteins were used in the simulations. The electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations resulted in complexes of GAPDH with G- or F-actin involving positively charged surface patches on GAPDH (Lyses 24, 69, 110 and 114) and negatively charged residues of the N- and C-termini (Asps 1, 25 and 363 and Glus 2, 4, 224 and 364) of actin. The actin residues all belong to subdomain 1. Although the positively charged surface patches of GAPDH are not close enough to each other to enhance their electrostatic potential, occasionally two subunits of the GAPDH tetramer may simultaneously interact with two neighboring monomers of F-actin. These results are different from those of fructose-1,6-bisphosphate aldolase, where quaternary structure directly influenced binding by two subunits combining their electrostatic potentials (see previous study, Ouporov et al., 1999, Biophys. J. 76: 17-27). Instead, GAPDH uses its quaternary structure to span the distance between two different actin subunits so that it can interact with two different actin subunits simultaneously.

Extracellular concentrations of aspartate and glutamate in rat neostriatum following chemical stimulation of frontal cortex.

Extracellular concentrations of excitatory amino acids in the neostriatum of anaesthetized rats were studied by in vivo microdialysis and the influence of chemical stimulation of the neocortex assessed. Administration of gamma-aminobutyric acid (GABA) antagonists to the neocortex evoked an increase in the extracellular concentration of aspartate (to 315 and 210% of basal values for microinfusion and topical application, respectively), but not of extracellular glutamate. Such selectivity was also found in response to N-methyl-D-aspartate (NMDA, 348% increase in extracellular aspartate concentration). Yet co-administration of NMDA with GABA antagonists increased the extracellular concentration of glutamate (to 278% of basal values) as well as aspartate (to 611% of basal values). The results suggest that either the activated neurons are hypoglycemic or hypoxic, or that aspartate is the major neurotransmitter of corticostriatal neurons.

A SNARE involved in protein transport through the Golgi apparatus.

In eukaryotic cells, the Golgi apparatus receives newly synthesized proteins from the endoplasmic reticulum (ER) and delivers them after covalent modification to their destination in the cell. These proteins move from the inside (cis) face to the plasma-membrane side (trans) of the Golgi, through a stack of cisternae, towards the trans-Golgi network (TGN), but very little is known about how proteins are moved through the Golgi compartments. In a model known as the maturation model, no special transport process was considered necessary, with protein movement along the Golgi being achieved by maturation of the cisternae. Alternatively, proteins could be transported by vesicles or membrane tubules. Although little is known about membrane-tubule-mediated transport, the molecular mechanism for vesicle-mediated transport is quite well understood, occurring through docking of SNAREs on the vesicle with those on the target membrane. We have now identified a protein of relative molecular mass 27K which is associated with the Golgi apparatus. The cytoplasmic domain of this protein or antibodies raised against it quantitatively inhibit transport in vitro from the ER to the trans-Golgi/TGN, acting at a stage between the cis/medial- and the trans-Golgi/TGN. This protein, which behaves like a SNARE and has been named GS27 (for Golgi SNARE of 27K), is identical to membrin, a protein implicated earlier in ER-to-Golgi transport. Our results suggest that protein movement from medial- to the trans-Golgi/TGN depends on SNARE-mediated vesicular transport.