SEN1500, a novel oral amyloid-ß aggregation inhibitor, attenuates brain pathology in a mouse model of Alzheimer's disease.

INTRODUCTION: Amyloid-ß (Aß) aggregation is thought to be a major pathogenic event underlying the neuropathology of Alzheimer's disease (AD). The development of new drugs inhibiting the Aß aggregation process is, therefore, important. SEN1500, an orally bioavailable and CNS-penetrant Aß aggregation inhibitor, has previously been shown to reduce spatial learning and memory deficits in an APP transgenic mouse model. To verify that the pharmacological properties of SEN1500 are not unique to this model, we investigated brain Aß pathology, neuroinflammation, as well as memory in a different mouse model of AD expressing the human amyloid precursor protein with Swedish and London mutations (APPSL). MATERIALS & METHODS: APPSL transgenic mice and non-transgenic littermates were treated with SEN1500 via food pellets from three months of age for four months. At the end of the treatment, animals were tested for memory deficits using the contextual fear conditioning test and brain tissue was analyzed for soluble and insoluble amyloid-ß1-38, -40, -42, ß-amyloid plaques, ß-sheet plaque cores, as well as for astrocytosis and activated microglia. RESULTS: SEN1500 treatment lowered insoluble Aß levels and ß-amyloid plaque load in the brain compared with control-treated APPSL mice. Activated microglia were significantly reduced in the cortex but not the hippocampus of SEN1500-treated APPSL mice. Memory deficits of APPSL mice could not be rescued by SEN1500. DISCUSSION: SEN1500 is not only able to reduce Aß pathology and activated microglia but also to improve learning and memory as previously shown, making SEN1500 a potential candidate for human AD treatment. This Aß aggregation inhibitor could be a promising therapeutic agent for the disease-modifying treatment of AD.

Aß oligomer toxicity inhibitor protects memory in models of synaptic toxicity.

BACKGROUND AND PURPOSE: Amyloid-ß (Aß) aggregation into synaptotoxic, prefibrillar oligomers is a major pathogenic event underlying the neuropathology of Alzheimer's disease (AD). The pharmacological and neuroprotective properties of a novel Aß aggregation inhibitor, SEN1269, were investigated on aggregation and cell viability and in test systems relevant to synaptic function and memory, using both synthetic Aß(1-42) and cell-derived Aß oligomers. EXPERIMENTAL APPROACH: Surface plasmon resonance studies measured binding of SEN1269 to Aß(1-42) . Thioflavin-T fluorescence and MTT assays were used to measure its ability to block Aß(1-42) -induced aggregation and reduction in cell viability. In vitro and in vivo long-term potentiation (LTP) experiments measured the effect of SEN1269 on deficits induced by synthetic Aß(1-42) and cell-derived Aß oligomers. Following i.c.v. administration of the latter, a complex (alternating-lever cyclic ratio) schedule of operant responding measured effects on memory in freely moving rats. KEY RESULTS: SEN1269 demonstrated direct binding to monomeric Aß(1-42) , produced a concentration-related blockade of Aß(1-42) aggregation and protected neuronal cell lines exposed to Aß(1-42) . In vitro, SEN1269 alleviated deficits in hippocampal LTP induced by Aß(1-42) and cell-derived Aß oligomers. In vivo, SEN1269 reduced the deficits in LTP and memory induced by i.c.v. administration of cell-derived Aß oligomers. CONCLUSIONS AND IMPLICATIONS: SEN1269 protected cells exposed to Aß(1-42) , displayed central activity with respect to reducing Aß-induced neurotoxicity and was neuroprotective in electrophysiological and behavioural models of memory relevant to Aß-induced neurodegeneration. It represents a promising lead for designing inhibitors of Aß-mediated synaptic toxicity as potential neuroprotective agents for treating AD.

Characterisation of the specific binding of the histamine H3 receptor antagonist radioligand [3H]GR168320.

We have examined the specific binding of the tritiated derivative of the potent histamine H3 receptor antagonist, [3,4-3H2]-cyclohex-yl-¿[4-(3H-imidazol-4-yl)-piperidin-l-yl] iminomethyl¿- amine ([3H]GR168320), to homogenates of rat cerebral cortex. Specific binding of [3H]GR168320 at 37 degrees C associated and dissociated rapidly. Binding was saturable (Bmax 412 +/- 89 fmol/mg protein) and of high affinity (Kd 0.12 +/- 0.11 nM). Saturation studies suggested the involvement of a single site. Histamine H3 receptor agonists and antagonists inhibited [3H]GR168320 binding with high affinity. Agonist and antagonist affinities correlated when compared with affinities obtained using the tritiated histamine H3 agonist radioligand N alpha-methylhistamine.

GR127935: a potent and selective 5-HT1D receptor antagonist.

GR127935 is the most potent 5-HT1D receptor antagonist yet described, possessing nanomolar affinity at human 5-HT1D receptors. Sumatriptan-induced contractions of the dog isolated basilar artery and saphenous vein are antagonised by GR127935 in an insurmountable manner indicative of its slow dissociation from the 5-HT1D receptor. 5-HT1D receptor-mediated hypothermia and rotational behaviour in guinea-pigs are antagonised potently, and with long duration, by GR127935, administered by a variety of routes. GR127935 also blocks central 5-HT1D autoreceptors in vitro and in vivo. GR127935 has much lower affinity at other 5-HT, and non-5-HT, receptors. In functional studies, GR127935 fails to affect 5-HT2 receptor-mediated 'wet dog shakes' in guinea-pigs and 5-HT1A receptor-mediated inhibition of 5-HT release in rat dorsal raphé nucleus. The compound has a good safety profile in all species tested. It is concluded that GR127935 is a useful pharmacological tool to characterise 5-HT1D receptor function.

Bromobenzofuran-based non-peptide antagonists of angiotensin II: GR138950, a potent antihypertensive agent with high oral bioavailability.

We have identified GR138950, a potent antagonist of the angiotensin II receptor with high oral bioavailability, as our second drug candidate to GR117289. Using GR117289, a compound with moderate bioavailability (20%) in man as a lead, we pursued a strategy aimed at enhancing bioavailability. The strategy was based on SAR established around the diacid GR117289, and from this, it was proposed that a monoacid, in particular a trifluoromethanesulfonamide, should be better absorbed after oral administration and have enhanced oral bioavailability. This led to the identification of GR138950, a potent antihypertensive agent in the renal hypertensive rat, causing sustained falls in blood pressure after oral administration. Oral bioavailability of GR138950 in rats and dogs is high, confirming that GR138950 is well absorbed after oral administration. Moreover, the low plasma clearance and long plasma half-life suggest that this compound will be suitable for once a day administration. Furthermore, the preliminary data indicate that the high bioavailability of GR138950 seen in rats and dogs translates to man. These results demonstrate clearly that GR138950 has the potential to be a clinically effective antihypertensive agent. Further studies are in progress to evaluate GR138950 in the treatment of hypertension.

4-[(Alkylamino)methyl]furo[3,2-c]pyridines: a new series of selective kappa-receptor agonists.

The synthesis of 5-(arylacetyl)-4-[(alkylamino)methyl]furo[3,2-c] pyridines (16-23, 26, 27) and their activities as kappa-opioid receptor agonists are described. kappa-Agonist potency was particularly sensitive to the nature of the basic moiety. In particular, in the rabbit vas deferens (kappa-specific tissue), the 3-pyrrolidinol analogue 17 (IC50 = 2.7 nM) was found to be approximately 5-fold more potent than the corresponding pyrrolidine analogue 16 (IC50 = 15 nM). In the rat and hamster vasa deferentia (mu-specific and delta-specific tissues, respectively), 17 showed only weak antagonist activity (pKB > 5.5), underlining its selectivity for the kappa-opioid receptor. The major activity for 17 is resident in the 4S,3'S-isomer 26 (rabbit vas deferens IC50 = 1.1 nM). Compound 26 displays excellent antinociceptive activity, as determined in the mouse acetylcholine-induced abdominal constriction test (ED50 = 0.001 mg/kg, sc).

A potent new class of kappa-receptor agonist: 4-substituted 1-(arylacetyl)-2-[(dialkylamino)methyl]piperazines.

The synthesis of 4-substituted 1-(arylacetyl)-2-[(alkylamino)methyl]piperazines (10-22, 26, 27, and 30-33) and their activities as kappa-opioid receptor agonists are described. This includes a range of 4-acyl and 4-carboalkoxy derivatives with the latter series showing the greatest kappa-agonist activity. In particular, methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl) methyl]-1-piperazinecarboxylate (18) displays exceptional potency and selectivity. It showed the following activities in functional in vitro assays: rabbit vas deferens (kappa-specific tissue) IC50 = 0.041 nM, rat vas deferens (mu-specific tissue) IC50 > 10,000 nM, and hamster vas deferens (delta-specific tissue) IC50 > 10,000 nM. Compound 18 is also a highly potent antinociceptive agent, as determined in the mouse acetylcholine-induced abdominal constriction test: ED50 = 0.000 52 mg/kg, sc. The activity of 18 resides solely in its 3(R)-enantiomer. The kappa-agonist activity in both the 4-acyl and the 4-carbamate series is sensitive to the size of the 4-substituent. In addition, it would appear that an appreciable negative electrostatic potential in this region of the molecule is an important requirement for optimal potency.

GR94839, a kappa-opioid agonist with limited access to the central nervous system, has antinociceptive activity.

1. The pharmacological profile of GR94839, a kappa-opioid agonist with limited access to the central nervous system, has been investigated. Its antinociceptive activity has been compared with that of GR103545, a centrally-penetrating kappa-agonist and ICI204448, the previously described peripherally-selective kappa-agonist. 2. GR94839 was a potent agonist in the rabbit vas deferens in vitro assay for kappa-opioid receptors (IC50: 1.4 +/- 0.3 nM; n = 6), but had limited activity at mu- or delta-opioid receptors. 3. In the mouse abdominal constriction test, GR94839 was 238 fold more potent when given i.c.v. (ED50: 0.008 (0.004-0.029) mg kg-1; n = 18) than when s.c. (ED50: 1.9 (0.7-3.1) mg kg-1; n = 30). In comparison, GR103545 was equipotent when given i.c.v. or s.c. 4. After intravenous administration, the maximum plasma to brain concentration-ratio attained by GR94839 was 18 compared with 2 for GR85571, a structurally-related kappa-agonist that is centrally-penetrating. 5. GR94839 inhibited the 2nd phase of the rat formalin response at doses 7 fold lower than those required to inhibit the 1st phase (ED50 vs 1st phase: 10.2 (6.7-17.1) mg kg-1, s.c.; ED50 vs 2nd phase: 1.4 (1.0-1.8) mg kg-1, s.c.; n = 18). GR103545 was equipotent against the two phases. 6. Intraplantar administration of the opioid antagonists, norbinaltorphimine (100 micrograms) or naltrexone (1 microgram), reversed the antinociceptive effect of systemic GR94839 (3 mg kg-1, s.c.) against the 2nd phase of the formalin response and intraplantar injection of GR94839 (30-100 micrograms) selectively inhibited the 2nd phase.7. GR94839 and IC1204448 reversed the hyperalgesia in the zymosan-inflamed rat paw at doses (ED50 GR94839: 2.0 (1.1-3.2) mg kg-', s.c.; ED50 IC1204448: 1.2 (0.8-1.7) mg kg-', s.c.), lower than those required to raise the noxious pressure threshold in the non-inflamed paw (EDSO GR94839: 16.4 (8.6-46.7) mg kg', s.c.; ED50 IC1204448: 68.0 (22.1-32000) mg kg', s.c.). GR103545 raised the noxious presure threshold in the inflamed and non-inflamed paws at the same doses.8. GR94839 was sedative in the rat rotarod test (ED50: 35 (12-245) mg kg-', s.c.) at doses higher than those required to inhibit the 2nd phase of the formalin response or reverse hyperalgesia in the zymosan-inflamed rat paw. The doses were comparable to those that inhibited the 1st phase of the formalin response and raised the noxious pressure threshold in the non-inflamed paw.9. The results suggest that GR94839 is a selective kappa-agonist which has antinociceptive activity against inflammatory pain at doses that produce limited central effects. These antinociceptive effects are probably mediated at peripheral opioid receptors.

New kappa-receptor agonists based upon a 2-[(alkylamino)methyl]piperidine nucleus.

The syntheses of some 1-[(3,4-dichlorophenyl)acetyl]-2- [(alkylamino)methyl]piperidines and their activities as kappa-opioid receptor agonists are described. Selected structural modifications are made to the basic moiety and at the 2-, 3-, 4-, 5-, and 6-positions on the piperidine nucleus to enable structure-activity relationships to be delineated. As a result, some highly potent and selective kappa-receptor agonists have been identified. In particular, this has been achieved by introduction of oxygen-containing functionality into the 4-position of the piperidine nucleus or the 3-position of the pyrrolidinylmethyl side chain. Thus, 1-[(3,4-dichlorophenyl)acetyl]- 2-[[1-(3-oxopyrrolidinyl)]methyl]piperidine (10) possesses high activity in the rabbit vas deferens (LVD, kappa-specific tissue) (IC50 = 0.20 nM) and is a potent antinociceptive agent, as determined by the mouse acetylcholine-induced abdominal constriction test (MAC) (ED50 = 0.06 mg/kg, sc). The spirocyclic analogue 8-[(3,4-dichlorophenyl)acetyl]-7-(1-pyrrolidinylmethyl)-1,4-dio xa-8- azaspirol4.5]decane (39) showed exceptionally potent activity: LVD, IC50 = 0.10 nM; MAC, ED50 = 0.001 mg/kg, sc. Both 10 and 39 displayed high selectivity for kappa-opioid receptors over both mu- and delta-opioid receptor subtypes.

Synthesis, antinociceptive activity, and opioid receptor profiles of substituted trans-3-(decahydro- and octahydro-4a-isoquinolinyl)phenols.

A series of trans-3-(6- and 7-substituted-decahydro-4a-isoquinolinyl)phenols and trans-3-(octahydro-4a-isoquinolinyl)phenols have been synthesized as potential opioid analgesics. Using a combination of in vitro and in vivo test systems, the receptor profiles of selected compounds have been assessed and in some instances distinguish between mu- and kappa-receptor agonists. In general, introduction of a 6-exocyclic methylene group into the trans-3-(decahydro-4a-isoquinolinyl)phenol system enhanced both antinociceptive activity and kappa-opioid receptor selectivity. For each series, analogues bearing an N-cyclopropylmethyl substituent exhibited greater kappa-receptor selectivity while N-methyl derivatives showed greater mu-receptor selectivity. The 7-substituted compounds (3b) were significantly less potent antinociceptive agents than their 6-substituted counterparts (3a), the octahydroisoquinoline analogues exhibiting intermediate activity. The axial 8-methyl-6-exocyclic methylene isoquinoline (20) is the most potent compound in the mouse abdominal constriction assay (ED50 = 0.05 mg/kg sc), whereas the equatorial 8-methyl isomer (16) was significantly less potent (ED50 = 3.3 mg/kg sc).

Neuroprotective actions of GR89696, a highly potent and selective kappa-opioid receptor agonist.

1. The effect of a novel, highly potent and selective kappa-opioid receptor agonist, GR89696, has been evaluated in two animal models of cerebral ischaemia: transient bilateral carotid artery occlusion in the Mongolian gerbil and permanent, unilateral middle cerebral artery occlusion in the mouse. 2. In the Mongolian gerbil model, administration of GR89696 (3 to 30 micrograms kg-1, s.c.), immediately before and at 4 h after insult, produced a dose-dependent reduction in the hippocampal CA1 neuronal cell loss resulting from a 7-min bilateral carotid occlusion. Similar effects were obtained with two other kappa-agonists, GR86014 (1 mgkg-1, s.c.) and GR91272 (1 mgkg-1, s.c.). The neuroprotective effect of GR89696 was completely blocked by prior administration of the opioid receptor antagonist, naltrexone, at 10 mgkg-1, s.c. Repeated post-treatment with GR89696 (100 micrograms kg-1, s.c.) or GR44821 (10 mgkg-1, s.c.) was also effective in protecting completely the hippocampal CA1 neurones from ischaemia-induced neurodegeneration. 3. In the permanent, unilateral middle cerebral artery occlusion model in the mouse, repeated administration of GR89696 at 300 micrograms kg-1, s.c. produced a 50% reduction in cerebrocortical infarct volume. In these experiments GR89696 was dosed 5 min, 4, 8, 12, 16, 20 and 24 h after occlusion on the first day and then three times daily for the next three days. GR89696 (300 micrograms kg-1) also produced a significant 35% reduction in infarct volume in this model when the initiation of dosing was delayed for 6 h after the insult. 4. The results indicate that the potent kappa-opioid receptor agonist, GR89696, is neuroprotective in both global and focal cerebral ischaemia models and suggest that, with this class of compound, there may be a considerable time window for pharmacological intervention.

A series of novel, highly potent and selective agonists for the kappa-opioid receptor.

1. This paper describes the opioid receptor pharmacology and in vivo activity of several novel benzene-acetamidopiperidine and benzeneacetamidopiperazine analogues. 2. These compounds all showed potent, naloxone-reversible, full agonist activity in the field-stimulated rabbit vas deferens, indicating that they are kappa-opioid agonists; but showed very little activity in the rat or hamster vas deferens, indicating good selectivity with regard to mu- and delta-opioid receptors. 3. They were all potent antinociceptive agents, the most potent compound, GR 103545, having an ED50 value in the mouse abdominal constriction test of 0.25 micrograms kg-1 s.c. The compounds also produced sedation and diuresis, but had little effect on respiration rate or gastrointestinal motility. 4. It is concluded that the seven novel compounds described are all potent and selective agonists for the kappa-opioid receptor.

Synthesis and antiviral properties of 5-(2-substituted vinyl)-6-aza-2'-deoxyuridines.

The following 5-(2-substituted vinyl)-6-aza-2'-deoxyuridines were synthesized: (E)-5-(2-bromovinyl) (2) (6-aza-BVDU), 5-(2-bromo-2-fluorovinyl) (a mixture of E and Z isomers) (3), (E)-5-(2-chlorovinyl) (4), (E)-5-[2-(methylthio)vinyl] (5), 5-(2,2-dibromovinyl) (6), and 5-(3-furyl) (7). The synthesis of 2-6 utilized Wittig-type reactions on 5-formyl-1-(2'-deoxy-3', 5'-di-O-p-toluoyl-beta-D-erythro-pentofuranosyl)-6-azauracil (16). 6-Aza-BVDU (and its alpha-anomer) was also synthesized from (E)-5-(2-bromovinyl)-6-azauracil (12) by using standard deoxyribosidation methodology. Compound 7 was prepared from 5-(3-furyl)-6-azauracil (33) via a ribosidation/deoxygenation sequence. An attempt to prepare the corresponding 5-(2,2-difluorovinyl) analogue afforded instead a mixture of the 5-[(2,2-difluoro-2-methoxy)ethyl] and 5-(2,2,2-trifluoroethyl) derivatives 29 and 30. Compounds 2-7, 29, and 30 were tested for in vitro activity against herpes simplex virus types 1 and 2 (HSV-1, HSV-2). 6-Aza-BVDU (2) exhibited ID50s of 8 micrograms/mL vs. HSV-1 and 190 micrograms/mL vs. HSV-2. BVDU (1) had ID50s of 0.015 and 1.6 micrograms/mL against HSV-1 and HSV-2, respectively. Compound 4 showed a similar profile of activity, but the other analogues were either weakly active or inactive.

Syntheses of all singly labeled [N]adenines: Mass spectral fragmentation of adenine.

Syntheses of all five of the singly labeled [(15)N]adenines are now provided. The presence or absence of two-bond (15)N-(1)H spin couplings in their (1)H NMR spectra confirm the location of the isotope in each case. The fragmentation patterns in their mass spectra are indicative of the sequential losses of HCN units and of CH(2)N(2) from adenine upon electron impact.

Effect of lin-benzoadenosine and lin-benzoadenosine 3':5'-monophosphate on cyclic AMP-dependent protein kinase activity in vitro.

We have shown that the fluorescent "stretched-out" analog of cyclic AMP, namely linear-benzo-cyclic AMP, maximally activates brain protein kinase and protein kinase from skeletal muscle. The corresponding linear-benzoadenosine inhibits kinase activity slightly less than does adenosine. Thus, the 2.4A-wider versions of cyclic AMP and of adenosine interact with protein kinase in a manner similar to that of the natural compounds.

Defined dimensional changes in enzyme cofactors: fluorescent "stretched-out" analogs of adenine nucleotides.

A concept is presented for testing the dimensional restrictions of enzyme-active sites by stretching the substrate or cofactor by known magnitude. These restrictions of enzyme-active sites specific for purine cofactors were tested by the synthesis and evaluation of lin-benzoadenosine 5'-triphosphate, 5'-diphosphate, and 3',5'-monophosphate with respect to enzyme binding and activity. These "stretchedout" (by 2.4 angstroms) versions of the adenine ribonucleotides bind strongly, slow the enzymatic rates, and have useful fluorescence properties.