Buntanetap, a Novel Translational Inhibitor of Multiple Neurotoxic Proteins, Proves to Be Safe and Promising in Both Alzheimer's and Parkinson's Patients.

BACKGROUND: Previously we reported the clinical safety and pharmacological activity of buntanetap (known as Posiphen or ANVS401) in healthy volunteers and mild cognitive impaired (MCI) patients (21). The data supported continued clinical evaluation of buntanetap for treating Alzheimer's Disease (AD). Neurodegenerative diseases such as AD and Parkinson's disease (PD) share several pathological manifestations, including increased levels of multiple neurotoxic protein aggregates. Therefore, a treatment strategy that targets toxic species common to both disorders can potentially provide better clinical outcomes than attacking one neurotoxic protein alone. To test this hypothesis, we recently completed a clinical study in early AD and early PD participants and report the data here. OBJECTIVES: We evaluated safety, pharmacokinetics, biomarkers, and efficacy of buntanetap in treating early AD and PD patients. DESIGN: Double-blind, placebo-controlled, multi-center study. SETTING: 13 sites in the US participated in this clinical trial. The registration number is NCT04524351 at ClinicalTrials.gov. PARTICIPANTS: 14 early AD patients and 54 early PD patients. INTERVENTION: AD patients were given either 80mg buntanetap or placebo QD. PD patients were given 5mg, 10mg, 20mg, 40mg, 80mg buntanetap or placebo QD. MEASUREMENTS: Primary endpoint is safety and tolerability; secondary endpoint is pharmacokinetics of buntanetap in plasma; exploratory endpoints are 1) biomarkers in cerebrospinal fluid (CSF) in both AD and PD patients 2) psychometric tests specific for AD (ADAS-Cogs and WAIS coding test) or PD (MDS-UPDRS and WAIS coding test). RESULTS: Buntanetap was safe and well tolerated. Biomarker data indicated a trend in lowering levels of neurotoxic proteins and inflammatory factors and improving axonal integrity and synaptic function in both AD and PD cohorts. Psychometric tests showed statistically significant improvements in ADAS-Cog11 and WAIS coding in AD patients and MDS-UPDRS and WAIS coding in PD patients. CONCLUSIONS: Buntanetap is well tolerated and safe at doses up to 80mg QD in both AD and PD patients. Cmax and AUC increase with dose without evidence for a plateau up to 80mg QD. The drug shows promising evidence in exploratory biomarker and efficacy measures. Further evaluation of buntanetap in larger, longer-term clinical trials for the treatment of AD and PD are warranted.

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G.

The kainate receptor antagonist 2S,4R-4-methylglutamate attenuates mechanical allodynia and thermal hyperalgesia in a rat model of nerve injury.

Opioids and receptor antagonists of excitatory amino acids attenuate mechanical allodynia and thermal hyperalgesia in animal models of neuropathic pain. Recently, a kainate receptor antagonist, 2S,4R-4-methylglutamate, has been developed but has not been tested for antinociceptive effects in animal models of neuropathic pain. We evaluated whether 2S,4R-4-methylglutamate attenuated responses to mechanical and thermal stimuli in uninjured (control) rats and increased responsiveness in rats with chronic constriction injury. Rats were tested for a number of withdrawal responses using a calibrated von Frey filament (mechanical stimulus) and withdrawal latencies from a radiant heat source (thermal stimulus). In control rats, 2S,4R-4-methylglutamate produced a small but significant decrease in responses from the mechanical stimulus (25 mg/kg) and significantly increased withdrawal latencies from the thermal stimulus at the highest dose administered (100 mg/kg). In addition, 2S,4R-4-methylglutamate greatly attenuated increased responsiveness in rats with chronic constriction injury. At four to eight days following chronic constriction injury, animals that displayed increased responsiveness to mechanical and thermal stimuli were injected intraperitoneally with either dizocilpine maleate (0.1 mg/kg), morphine (4 mg/kg), vehicle as controls, or 2S,4R-4-methylglutamate (25, 50, 75 or 100 mg/kg). 2S,4R-4-Methylglutamate (25, 50, 75 and 100 mg/kg) significantly attenuated the frequency of responses to mechanical stimuli (Wilcoxon, P < 0.05) and the latency of responses to thermal stimuli (analysis of variance and Duncan's, P < 0.05). Dizocilpine maleate and morphine, as expected, also reduced these responses. These results suggest that, in addition to opioid and N-methyl-D-aspartate receptors, kainate receptors may play a role in the maintenance of mechanical allodynia and thermal hyperalgesia associated with peripheral nerve injury.

Allosteric modulators of the AMPA receptor: novel 6-substituted dihydrophthalazines.

Novel analogs of the allosteric AMPA receptor modulator SYM 2206 have been prepared. Structure/activity correlations of these novel analogs and other dihydrophthalazines (DHPs) reveal the important contribution of the heteroatom-based aryl substituents in this class of noncompetitive inhibitors. One of the analogs (6, SYM 2189) is equipotent with the early series, but with reduced sedation.

Differential effects of agents acting at various sites of the NMDA receptor complex in a place preference conditioning model.

A conditioned place preference paradigm was used to assess the potential rewarding properties of the uncompetitive NMDA receptor antagonist, MK-801 (dizolcipine), the two competitive NMDA receptor antagonists, CGP 37849 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentonoic acid) and its (R)-enantiomer CGP 40116, as well as the partial agonist at strychnine-insensitive glycine receptors, ACPC (1-aminocyclopropanecarboxylic acid). MK-801 (0.3 mg/kg), CGP 37849 (1.25-10 mg/kg) and CGP 40116 (1.25-10 mg/kg), administered in association with either the initially non-preferred or initially preferred side of the two-arm chamber, caused a significant increase in the time spent on that side in a post-conditioning test. In contrast, ACPC did not support the conditioned place preference. Thus, the time spent on the drug-associated side following conditioning with ACPC (50-400 mg/kg) did not significantly differ from that measured in the pre-conditioning test, irrespective of whether it was associated with the initially non-preferred black side or the initially preferred white side. These results are consistent with both clinical and pre-clinical data demonstrating differences in psychopharmacological properties among compounds acting at the multiple, allosteric regulatory sites on the NMDA receptor complex. Moreover, these results indicate that the abuse potential of ACPC, which acts as a functional NMDA receptor antagonist, may be lower than that of either uncompetitive or competitive NMDA receptor antagonists.

Synthetic analogues of conantokin-G: NMDA antagonists acting through a novel polyamine-coupled site.

Conantokin-G (con-G) is a 17-amino-acid polypeptide that acts as an N-methyl-D-aspartate (NMDA) antagonist. This action has been attributed to a specific but noncompetitive inhibition of the positive modulatory effects of polyamines at NMDA receptors. Con-G possesses several unusual structural features, including five gamma-carboxyglutamate (Gla) residues and a high degree of helicity in aqueous media. Previous structure-activity studies indicated that one or more Gla residues are necessary for NMDA antagonist activity. Con-G analogues were synthesized with alanine (Ala), serine (Ser), and phosphoserine substituted for Gla to assess the contribution of individual Gla residues to biological activity and secondary structure. Replacement of Gla in positions 3 and 4 resulted in polypeptides with markedly reduced and no NMDA antagonist actions, respectively. In contrast, Gla residues in positions 7, 10, and 14 are not required for NMDA antagonist actions because the potencies of con-G analogues containing Ser7, Ser10, Ala14, and Ser14 to inhibit spermine-stimulated [3H]MK-801 binding are similar to the parent peptide. Moreover, the Ala7 derivative of con-G was about fourfold more potent than the parent peptide both as an inhibitor of spermine-stimulated increases in [3H]MK-801 binding (IC50 of approximately 45 nM) and in reducing NMDA-stimulated increases in cyclic GMP levels (IC50 of approximately 77 nM) in cerebellar granule cell cultures. Although con-G and its analogues assumed mixtures of 3(10) and alpha-helices, no clearcut relationship was evinced between the NMDA antagonist properties of these peptides and the degree of helicity they assumed in aqueous solutions. Together with the inability of con-G to affect 5,7-dichloro[3H]kynurenic acid, [3H]CGP-39653, and [3H]ifenprodil binding, these data are consistent with the hypothesis that this polypeptide acts at a unique, polyamine-associated site on NMDA receptors.

Polyamine-like actions of peptides derived from conantokin-G, an N-methyl-D-aspartate (NMDA) antagonist.

Conantokins-T and -G are highly conserved polypeptides derived from Conus venoms. The N-methyl-D-aspartate (NMDA) antagonist properties of these compounds have been attributed to a potent noncompetitive inhibition of polyamine responses. Substitution of the highly conserved gamma-carboxyglutamate residues as well as modification of the N and C termini of conantokin-G abolished the inhibition of polyamine responses at the NMDA receptor complex. However, several of these modified polypeptides closely mimicked the neurochemical profile of polyamines at the NMDA receptor complex. One of these derivatives, Tyr0-conantokin-G, was found to be the most potent compound exhibiting polyamine-like actions at the NMDA receptor complex described to date, approximately 7-fold more potent than spermine. Circular dichroism studies demonstrate a significant alpha-helical content in conantokin-G (27% in aqueous medium). However, this alpha-helicity is not sufficient for the NMDA antagonist action of the parent peptide and is neither necessary nor sufficient for the polyamine-like behavior of several conantokin-G analogs. The modified conantokin-G derivatives described in this report should be useful probes for examining the role of both polyamines and the polyamine recognition site in the operation of the NMDA receptor complex.

Noncompetitive inhibition of N-methyl-D-aspartate by conantokin-G: evidence for an allosteric interaction at polyamine sites.

Conantokins T and G are polypeptide toxins present in snails of the genus Conus. These substances were recently reported to act as N-methyl-D-aspartate (NMDA) antagonists. In the present study, we examined the possible mechanisms producing this antagonism. Conantokin-G inhibited spermine- and spermidine-stimulated [3H]MK-801 binding to extensively washed rat forebrain membranes in a noncompetitive manner with IC50 values of approximately 507 and approximately 946 nM, respectively. In contrast, glutamate-enhanced [3H]MK-801 binding was unaffected by conantokin-G concentrations of less than or equal to 20 microM. At concentrations greater than or equal to 5 microM, conantokin-G effected a modest, noncompetitive inhibition of glycine-stimulated [3H]MK-801 binding and also produced a small enhancement of basal [3H]MK-801 binding. Conantokin-G reduced (IC50 approximately 1.08 microM) the NMDA-stimulated accumulation of cyclic GMP in cerebellar granule cell cultures to basal values, but did not affect kainate-mediated increases in cyclic GMP. These findings indicate that conantokin-G acts as a noncompetitive NMDA antagonist through an allosteric inhibition of polyamine responses. The neurochemical profile of this polypeptide is distinct from previously described noncompetitive NMDA antagonists.

Synthesis and characterization of a disulfide bond isomer of omega-conotoxin GVIA.

Solid phase peptide synthesis and air oxidation of omega-conotoxin GVIA yielded, in addition to the desired product, an isomeric peptide which could be completely separated from the native toxin by repeated HPLC. A chymotrypsin-trypsin digest of this peptide, when subjected to HPLC peptide mapping, provided peptides identical with synthetic disulfide containing peptides predicted for the omega-conotoxin isomer containing C1-C2, C3-C5, C4-C6 cystinyl pairings. The 'shaking' potency (ED50 = 1500 pmoles/kg, i.c.v.) of the isomeric peptide upon cannulated rats was 1.3% of the potency of native conotoxin (ED50 = 20 pmoles/kg). Considering that all three disulfide pairings in the isomer are different from the native toxin, its retention of biological activity is of interest.

Plasma somatotropin response to exogenous growth hormone releasing factor in lambs.

Two experiments were performed to examine the ability of human pancreatic growth hormone releasing factor (hGRF) administration to stimulate endogenous growth hormone (GH) secretion in lambs. Each study utilized eight Dorset wether lambs in replicated 4 X 4 Latin square experiments. Growth hormone response (integrated area under the curve for 150 min post-injection) for 0, 1, 5 and 10 micrograms hGRF/kg body weight averaged 13, 23, 92 and 134 units, respectively. While the 1-microgram hGRF dose was not different (P greater than .05) than the response to saline injection, there was an increased (P less than .01) GH response to 5 or 10 micrograms hGRF. Overall the GH response increased in a log dose-response fashion. There was distinct variation between lambs in their response to hGRF. Study II examined the optimal method to administer 40 micrograms hGRF/kg body weight to maximize GH concentration over 24 h. Continuous infusion (CI) was compared with eight (8X), four (4X), or two (2X) injections/d. Hourly blood samples were obtained from all lambs. Growth hormone response (area under the curve for 24 h) was 162, 305, 306 and 220 units for CI, 8X, 4X and 2X, respectively. Growth hormone response to CI was inferior to discrete injections, and the GH response to 4X or 8X was superior to 2X/d. Results demonstrate that, in spite of lamb-to-lamb variation, one can utilize exogenous hGRF to enhance GH secretion in lambs. Thus, the ability of exogenous hGRF to enhance growth performance merits further study.

Transport of proteins across the mitochondrial outer membrane. A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase.

Cytochrome c peroxidase, a cytoplasmically made enzyme located between the inner and outer membrane of yeast mitochondria, is synthesized as larger precursor in a reticulocyte cell-free lysate as well as in pulsed yeast spheroplasts. When the pulsed spheroplasts are chased, the precursor is converted to the mature apoprotein. When the in vitro synthesized precursor is incubated with isolated yeast mitochondria in the absence of protein synthesis, it is cleaved to the mature form; the mature form co-sediments with the mitochondria and is resistant to externally added proteases. These results, in conjunction with those reported earlier (Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347) suggest that the mechanism of protein transport into the mitochondrial intermembrane space is quite similar to that of protein transport into the matrix or the inner membrane.

Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast.

The mitochondrial F1-ATPase consists of five nonidentical subunits that are synthesized outside the mitochondria and imported across both mitochondrial membranes to the matrix side of the inner membrane. In order to study the mechanism of this import, we synthesized the F1-ATPase subunits of yeast either in vitro (in a reticulocyte lysate programmed with yeast RNA) or in vivo (in pulsed and pulsed-chased yeast spheroplasts). Both in vitro and in vivo, each of the three largest ATPase subunits was synthesized as a larger precursor. When the precursors that had been synthesized in vitro were incubated with isolated yeast mitochondria, they were converted to "mature" subunits that were no longer susceptible to externally added proteases. The uptake of the subunit into the mitochondria was thus accompanied by conversion of the precursor. Since uptake of precursors into mitochondria was independent of protein synthesis and since the precursors could also be detected in vivo, the transfer of proteins from the cytosol across both mitochondrial membranes does not occur by vectorial translation. Instead, the proteins destined for import are first made outside the mitochondria as precursors and only subsequently transported into the mitochondria. This step is accompanied by proteolytic conversion of the mature subunit.

Sugar-specific antibodies reactive towards cell-surface carbohydrates.

Antibodies generated against sugar-azoproteins have been purified by affinity chromatography using elution with monosaccharide. They have high specificity for monosaccharide and they react with terminal, non-reducing sugars on the surfaces of human erythrocytes. Some of them, either native or as Fab fragments, inhibit mitogenic stimulation of lymphocytes by concanavalin A.

Stimulation of lymphocytes by concanavalin A. Temperature-dependent effect of fatty acid replacements.

The membrane fatty acyl composition of lymphocytes was altered by growth in lipid-depleted serum containing fatty acid supplements, as well as avidin to block endogenous synthesis of fatty acids. Under these growth conditions over 50% of the total fatty acid in membrane phospholipid were derived from the added fatty acid. Enrichment of lymphocyte membranes with oleate (cis C18:1) or elaidate (trans C18:1) shifted the optimum temperature for mitogenic stimulation by concanavalin A as measured by [3H]thymidine incorporation. These results suggest that the fluidity of the membrane lipid phase plays a role in the process of lymphocyte stimulation by lectins.