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J A Kemp

Publications and source records attributed to J A Kemp.

At least 19 recordsLinked to original sources
PubMed2000

Controlling polymerization of beta-amyloid and prion-derived peptides with synthetic small molecule ligands.

The Alzheimer beta-amyloid peptide (Abeta) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting Abeta polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.

Amyloid beta-Peptides↗
PubMed1999

A randomized controlled trial of an enhanced patient compliance program for Helicobacter pylori therapy.

OBJECTIVES: To determine whether an enhanced compliance program (ECP) improves patient compliance with bismuth subsalicylate, metronidazole, and tetracycline hydrochloride (BMT) triple therapy for the treatment of Helicobacter pylori infection and to identify factors that affect compliance with therapy. DESIGN: A randomized controlled trial conducted in 4 staff-model health centers of a health maintenance organization in Massachusetts. PATIENTS AND METHODS: A total of 125 patients 18 years of age or older with peptic ulcer disease or dyspepsia whose clinicians prescribed BMT triple therapy for 14 days were randomized to a control group or to the ECP group. The ECP group received medication counseling (written and oral) from a pharmacist, along with a medication calendar and a minipillbox, as well as a follow-up telephone call after initiation of therapy. Compliance was assessed by a pill count, and factors affecting adherence to the regimen were identified by patients' reports. RESULTS: There was no statistically significant difference between the 2 groups in the number of patients taking more than 60% of the medications (89% of the control group vs 95% of the ECP group; P>.30). However, there was a statistically significant difference in the number of patients taking more than 90% of the medications (67% of the control group vs 89% of the ECP group; P<.01). An intention-to-treat analysis confirmed these results. The most frequently reported adverse effect was gastrointestinal intolerance. Other factors reported to affect compliance included the frequency of dosing and the number of pills. CONCLUSIONS: These findings suggest that although adverse effects were common, most patients were able to complete 60% or more of the 2-week regimen. An ECP further improved the percentage of medications taken.

Adult↗
PubMed1999

Screening for colorectal cancer with flexible sigmoidoscopy by nonphysician endoscopists.

PURPOSE: Screening with sigmoidoscopy reduces the risk of death from colorectal cancer. Only 30% of eligible patients have undergone sigmoidoscopy, in part because of a limited supply of endoscopists. We evaluated the performance and safety of screening sigmoidoscopic examinations by trained nonphysician endoscopists in comparison with board-certified gastroenterologists. SUBJECTS AND METHODS: Asymptomatic patients 50 years or older without evidence of fecal occult blood and no personal history or family history of a first-degree relative with colorectal cancer under age 55 years were offered sigmoidoscopy. All examinations were performed either by a gastroenterologist or a trained nonphysician endoscopist at a staff model health maintenance organization. Outcomes included the depth of examination, number and histology of polyps, and complications. RESULTS: Nonphysicians performed 2,323 sigmoidoscopic examinations, and physicians performed 1,378 examinations. The mean (+/-SD) depth of sigmoidoscopy examinations performed by nonphysicians was 52 +/- 10 cm compared with 55 +/- 9 cm (P <0.001) in physicians. Nonphysicians detected neoplastic polyps in a greater proportion of patients (7.8%) than physicians (5.8%), but this difference was not significant after adjusting for differences in the age, sex, and family history of the patients (P = 0.35). No major complications occurred. The cost per examination, including the nonphysician training cost, was lower for nonphysicians ($186 per examination) than for physicians ($283 per examination). CONCLUSIONS: Appropriately trained nonphysicians may be capable of performing safe and effective screening for colorectal cancer with flexible sigmoidoscopy. An increased use of nonphysicians to perform sigmoidoscopy may increase the availability and reduce the cost of the procedure.

Aged↗
PubMed1998

An allosteric interaction between the NMDA receptor polyamine and ifenprodil sites in rat cultured cortical neurones.

1. The atypical NR2B subunit-selective NMDA receptor antagonist ifenprodil was originally believed to act as a competitive antagonist at the polyamine binding site of the NMDA receptor. However, a number of studies have suggested that ifenprodil might bind to a distinct site. 2. Using whole-cell voltage clamp recordings, we have studied the interaction of spermine with both ifenprodil and the related NR2B selective antagonist Ro 8-4304 at the NMDA receptor in rat cultured cortical neurones in the presence of saturating concentrations of glycine. 3. Ifenprodil and Ro 8-4304 inhibited steady-state currents evoked by 100 microM NMDA in the absence of spermine with IC50 values of 0.3 and 0.6 microM, respectively. In the presence of 1 and 3 mM spermine, IC50 values for ifenprodil were 1.4 and 1.8 microM and for Ro 8-4304 they were 3. 0 and 7.5 microM, respectively. 4. In the presence of spermine, the on-time constant of receptor blockade by both antagonists was significantly slower than control and the off-time constant of recovery from receptor blockade following removal of Ro 8-4304 was significantly faster. 5. Fast application of spermine during an NMDA steady-state current in the continuous presence of a subsaturating concentration of either antagonist resulted in a biphasic increase in the current, consistent with a fast increase upon spermine binding and a slow increase resultant from dissociation of antagonist due to spermine binding-induced allosteric reduction in receptor antagonist affinity. In agreement with this, at higher, saturating concentrations of antagonist, the slow increase in current amplitude was markedly reduced or absent. 6. These observations are consistent with a non-competitive, allosteric interaction between spermine and the antagonists, such that spermine binding to the NMDA receptor results in a reduction in receptor affinity for the antagonists and vice versa. 7. The effects of Mg2+ on the NMDA-evoked currents and its interaction with ifenprodil were similar to those of spermine, supporting the suggestion that Mg2+ might be the physiological ligand acting at the spermine site mediating glycine-independent stimulation.

Allosteric Regulation↗
PubMed1998

Is colonoscopy indicated for small adenomas found by screening flexible sigmoidoscopy?

BACKGROUND: There is controversy over whether patients who have a small tubular adenoma on screening flexible sigmoidoscopy should undergo colonoscopic examination of the proximal colon. OBJECTIVE: To prospectively determine the prevalence of advanced polyps in the proximal colon among patients who have small adenomas on screening sigmoidoscopy. DESIGN: Prospective cohort study. SETTING: A health maintenance organization and a Veterans Affairs medical center. PATIENTS: Asymptomatic patients older than 50 years of age who had no risk factors for colon cancer and underwent sigmoidoscopy. INTERVENTION: At the time of sigmoidoscopy, all polyps were biopsied and characterized. All patients with distal adenomas were offered colonoscopy. MEASUREMENTS: The size and histology of polyps identified by sigmoidoscopy and colonoscopy were noted. Polyps were considered advanced if they were larger than 10 mm or were tubulovillous, villous, or malignant. The prevalence of advanced proximal polyps was determined, and patients were stratified by the size and number of distal polyps found by sigmoidoscopy. RESULTS: Among 4490 patients who underwent sigmoidoscopy, a neoplastic lesion was detected in 401 (8.9%) and colonoscopy was done in 301 (75%). Of 90 patients with a single tubular adenoma 1 to 5 mm in diameter in the distal colon, 0% (95% CI, 0.0% to 4.0%) had an advanced proximal polyp compared with 5.4% (CI, 2.4% to 10.4%) of those who had multiple distal polyps 1 to 5 mm or 6 to 10 mm in diameter and 7.9% (CI, 2.6% to 17.6%) of those who had advanced distal polyps (P = 0.013 for trend). The low-risk group with a single tubular adenoma 1 to 5 mm in diameter represented 44% of all patients with distal adenomas or cancers found at flexible sigmoidoscopy. CONCLUSIONS: Among patients undergoing screening sigmoidoscopy, those with single tubular adenomas of 5 mm or less had a low prevalence of advanced proximal polyps. These patients may not benefit from colonoscopy.

Adenoma↗
PubMed1998

Metabotropic glutamate group II receptors activate a G protein-coupled inwardly rectifying K+ current in neurones of the rat cerebellum.

1. The effects of the group II metabotropic glutamate receptor (mGluR) agonists DCG-IV and LY354740 were examined in neurones freshly dissociated from the rat cerebellum and olfactory bulb, using the whole-cell configuration of the patch-clamp technique. 2. Under experimental conditions in which K+ currents would be inward, rapid application of DCG-IV and LY354740 to interneurones expressing the group II mGluRs induced an inward current in a subpopulation of interneurones of the cerebellum, the unipolar brush cells. 3. The currents induced by DCG-IV and LY354740 had the major characteristics of a G protein-coupled inwardly rectifying K+ channel (GIRK) current; namely, rapid activation and deactivation upon agonist application and removal, G protein dependence, strong inward rectification, Cs+ and Ba2+ sensitivity, and K+ selectivity. 4. In Golgi cells of the cerebellum and interneurones of the accessory olfactory bulb, which also express group II mGluRs, LY354740 did not induce GIRK activation but inhibited voltage-gated Ca2+ channel currents. 5. These results demonstrate that, in unipolar brush cells, native group II mGluRs can functionally couple to activation of GIRKs. Thus, the absence of coupling in the majority of CNS neurones examined to date may be due to restricted cellular co-localization or co-expression of the appropriate proteins.

Animals↗
PubMed1998

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones.

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. L-glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), with EC50 values of 3.41+/-0. 39 and 5.69+/-1.64 microM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 microM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, L-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19+/-1.18 and 3.78+/-1.42 microM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S, 2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1998

Developmental changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal three distinct populations of NMDA receptors in individual rat cortical neurons.

Previous work with recombinant receptors has shown that the identity of the NMDA NR2 subunit influences receptor affinity for both glutamate and glycine. We have investigated the developmental change in NMDA receptor affinity for both glutamate and glycine in acutely dissociated parietal cortex neurons of the rat, together with the expression during ontogeny of NR2A and NR2B mRNA and protein. Whereas there is little change in NMDA receptor glutamate affinity with age, a population of NMDA receptors emerges in 14- and 28-d-old animals with a markedly reduced affinity for glycine (mKD = approximately 800 nM) and a reduced sensitivity to the NR2B subunit-selective NMDA antagonist ifenprodil. These changes are paralleled by a developmental increase in the expression of NR2A. Thus, in mature animals a population of NMDA receptors appears with a lower affinity for glycine that might not be saturated under normal physiological conditions. Ifenprodil (10 microM) inhibits virtually all of the NMDA receptor-evoked current in very young neurons that contain a single population of receptors exhibiting a high affinity for glycine (mKD = approximately 20 nM). In older neurons, which contain NMDA receptors with both high and low affinities for glycine, ifenprodil (10 microM) inhibits both the high-affinity population and a significant proportion of the low-affinity component, thus revealing three pharmacologically distinct populations of NMDA receptors in single neurons. Moreover, these observations suggest that ifenprodil might bind with high affinity to NMDA receptors containing both NR2A and NR2B subunits as well as those containing only NR2B.

Aging↗
PubMed1998

Multiple pathways for regulation of the KCl-induced [3H]-GABA release by metabotropic glutamate receptors, in primary rat cortical cultures.

In rat cortical primary cultures, group II- and III-metabotropic glutamate receptor-selective agonists concentration-dependently reduced KCl-induced [3H]GABA release, with IC50 values of 11 nM for LY354740, 80 nM for L(+)-2-amino-4-phosphonobutyric acid (L-AP4), 180 nM for DCG-IV, and 330 nM for L-SOP. The group II antagonists, LY341495 and EGLU, reversed the effect of LY354740, and the group III antagonist MTPG reversed the effect of L-AP4. In the presence of omega-conotoxin GVIA, LY354740 inhibited the remaining [3H]GABA release, whereas L-AP4 was inactive. In contrast, in the presence of nifedipine, L-AP4 inhibited the remaining [3H]GABA release, but LY354740 was no longer active. The PKA inhibitor, H89, blocked the effects of both L-AP4 and LY354740, whereas the PKC inhibitor Ro 31-8220 blocked only the effect of LY354740. Both Ro 31-8220 and H89 reduced the [3H]GABA release to 60% of control. In whole-cell, voltage-clamp experiments, LY354740 and L-AP4 inhibited voltage-gated calcium channel currents with IC50 values of 28 nM and 22 microM, respectively. The results suggest that, in these cells, KCl-induced [3H]GABA release is modulated by two different mechanisms, one involving group II receptors and a direct control of the Ca2+ channel activity, and the other mediated by group III receptors and possibly involving a regulation located downstream of the Ca2+ channel activation.

Animals↗
PubMed1998

State-dependent NMDA receptor antagonism by Ro 8-4304, a novel NR2B selective, non-competitive, voltage-independent antagonist.

1. Subunit-selective blockade of N-methyl-D-aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B-selective NMDA antagonist, 4-¿3-[4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxy-propoxy ¿-benzamide (Ro 8-4304), which exhibits >100 fold higher affinity for recombinant NR1(001)/NR2B than NR1(001)/NR2A receptors. 2. Ro 8-4304 is a voltage-independent, non-competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state-dependent mode of action similar to that described for ifenprodil. 3. The apparent affinity of Ro 8-4304 for the NMDA receptor increased in an NMDA concentration-dependent manner so that Ro 8-4304 inhibited 10 and 100 microM NMDA responses with IC50s of 2.3 and 0.36 microM, respectively. Currents elicited by 1 microM NMDA were slightly potentiated in the presence of 10 microM Ro 8-4304, and Ro 8-4304 binding slowed the rate of glutamate dissociation from NMDA receptors. 4. These results were predicted by a reaction scheme in which Ro 8-4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist-unbound resting state. Additionally, Ro 8-4304 binding resulted in a 3 4 fold increase in receptor affinity for glutamate site agonists. 5. Surprisingly, whilst exhibiting a similar affinity for NR2B-containing NMDA receptors as ifenprodil, Ro 8-4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B-selective compounds.

Animals↗
PubMed1998

Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes.

1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes. 2. [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg(-1) protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]-DCG IV binding. 3. Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nNM. 4. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPgamma35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-alpha-methylserine-O-phosphate (MSOP).

Animals↗
PubMed1998

L-687,414, a low efficacy NMDA receptor glycine site partial agonist in vitro, does not prevent hippocampal LTP in vivo at plasma levels known to be neuroprotective.

N-methyl-D-aspartic acid (NMDA) receptors are known to play a key role in the induction phase of long-term potentiation (LTP) at certain hippocampal synapses and to represent some component of spatial learning in animals. The ability of NMDA receptor antagonists (or gene knockout) to impair LTP has led to the suggestion that the therapeutic use of such antagonists may impair cognitive function in humans. The present study compares the effects on LTP of NMDA receptor ion channel block by MK-801 and glycine-site antagonism by 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414). In vitro experiments using rat cortical slices revealed L-687,414 to be approximately 3.6 fold more potent than its parent analogue, R(+)HA-966 at antagonizing NMDA-evoked population depolarizations (apparent Kbs: 15 microM and 55 microM, respectively). Whole-cell voltage-clamp experiments using rat cultured cortical neurones revealed L-687,414 to shift to the right the concentration-response relationship for NMDA-evoked inward current responses (pKb=6.2+/-0.12). L-687,414 affinity for the glycine site on the NMDA receptor complex was also determined from concentration-inhibition curves, pKi=6.1+/-0.09. In the latter experiments, L-687,414 and R(+)HA-966 were unable to completely abolish inward current responses suggesting each compound to be a low efficacy partial agonist (estimated intrinsic activity = approximately 10 and 20% of glycine, respectively). L-687,414 and MK-801 were compared for their effects on NMDA receptor-dependent LTP in the dentate gyrus of anaethestized rats following high frequency stimulation of the medial perforant path (mPP) afferents. Control rats, administered saline (0.4 ml kg(-1) followed by 0.0298 ml min(-1)), showed a robust augmentation of the population e.p.s.p. risetime (LTP) recorded in the dentate hilus following tetanic stimulation of the mPP. LTP was effectively abolished in a separate group of rats treated with an MK-801 dosing regimen (0.12 mg kg(-1) i.v. followed by 1.8 microg kg(-1) h(-1)) known to produce maximal neuroprotection in a rat stroke model but, by contrast, remained largely intact in a third group of animals given a similarly neuroprotective L-687,414 treatment (28 mg kg(-1) i.v. followed by 28 mg kg(-1) h(-1)). These experiments suggest that a low level of intrinsic activity at the glycine site may be sufficient to support NMDA receptor-dependent LTP but in circumstances where there is likely to be an excessive NMDA receptor activation the agonism associated with a low efficacy partial agonist, such as L-687,414, is dominated by the antagonist properties. Thus, an NMDA receptor partial agonist profile may offer a therapeutic advantage over neutral antagonists by permitting an acceptable level of 'normal' synaptic transmission whilst curtailing excessive receptor activation.

Animals↗
PubMed1998

In vitro binding properties in rat brain of [3H]Ro 25-6981, a potent and selective antagonist of NMDA receptors containing NR2B subunits.

The in vitro binding of a new subtype-selective NMDA receptor antagonist, [3H]Ro 25-6981, to rat brain membranes and sections was characterized. The compound bound to a single site on the membranes with a K(D) of 3 nM and a Bmax of 1.6 pmol/mg of protein. Specific binding, defined with a new NR2B-specific antagonist, Ro 04-5595 [1-(4-chlorophenyl)-2-methyl-6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoqu inoline], at 10 microM, was fully inhibited by several compounds with the following rank order of affinities--Ro 25-6981 > CP-101,606 > Ro 04-5595 = ifenprodil >> eliprodil > haloperidol > spermine > spermidine > MgCl2 > CaCl2--and partially inhibited by competitive glutamate recognition site antagonists. A high density of binding sites was detected, radioautographically, in several layers of the cerebral cortex, in the hippocampus, dentate gyrus, tuberculum olfactorium, caudate putamen, medium densities in the globus pallidus, thalamus, spinal cord dorsal horn, and motoneurons, whereas the cerebellum, pons, and medulla were, with a few exceptions, e.g., locus coeruleus, poorly labeled. Overall, the distribution of [3H]Ro 25-6981 binding sites correlated well with that of NR2B (but not NR2A) transcripts, revealed by in situ hybridization histochemistry. The high affinity of [3H]Ro 25-6981 for NR2B-containing receptors renders this compound the ligand of choice to study the regulation of NR2B-containing receptor expression.

Animals↗
PubMed1998

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain.

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.

Animals↗
PubMed1998

In vitro binding characteristics of a new selective group II metabotropic glutamate receptor radioligand, [3H]LY354740, in rat brain.

The in vitro binding of [3H]LY354740, the first high affinity group II-selective metabotropic glutamate (mGlu) receptor radioligand, was characterized in rat cortical, hippocampal, and thalamic membranes as well as in rat brain sections. [3H]LY354740 binding was saturable in all regions investigated. Nonspecific binding (in the presence of 10 microM DCG-IV) was approximately 8% of the total. Ionotropic glutamate receptor agonists, N-methyl-D-aspartate, (R,S)-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/kainate, a Na+-dependent glutamate uptake blocker as well as a group I-selective mGlu receptor agonist (all up to 1 mM) did not inhibit [3H]LY354740 binding to cortical membranes. However, several known metabotropic receptor ligands inhibited the binding with the following rank order of potency: LY354740 = LY341495 > (2S,2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine = (2S,1'S, 2'S)-2-(2-carboxycyclopropyl)glycine > glutamate = (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S, 2'S)-2-methyl-2-(2-carboxycyclopropyl)-glycine > quisqualate > ibotenate > L-2-amino-3-phosphonopropionic acid = (S)-alpha-methyl-4-carboxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-aspartyl-glutamate, (2S)-alpha-ethylglutamic acid, and (R, S)-alpha-methyl-4-phosphonophenylglycine inhibited [3H]LY354740 binding in a biphasic manner. Guanosine-5'-O-(3-thiotriphosphate concentration-dependently and almost completely inhibited the binding. Finally, in parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1-3 > 4-6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for presynaptic and postsynaptic mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to guanosine-5'-O-(3-thiotriphosphate show that [3H]LY354740 binding probably occurs to group II mGlu receptors in rat brain.

ATP-Binding Cassette Transporters↗
PubMed1997

Effect of plasma protein binding on in vivo activity and brain penetration of glycine/NMDA receptor antagonists.

A major issue in designing drugs as antagonists at the glycine site of the NMDA receptor has been to achieve good in vivo activity. A series of 4-hydroxyquinolone glycine antagonists was found to be active in the DBA/2 mouse anticonvulsant assay, but improvements in in vitro affinity were not mirrored by corresponding increases in anticonvulsant activity. Here we show that binding of the compounds to plasma protein limits their brain penetration. Relative binding to the major plasma protein, albumin, was measured in two different ways: by a radioligand binding experiment or using an HPLC assay, for a wide structural range of glycine/NMDA site ligands. These measures of plasma protein binding correlate well (r = 0.84), and the HPLC assay has been used extensively to quantify plasma protein binding. For the 4-hydroxyquinolone series, binding to plasma protein correlates (r = 0.92) with log P (octanol/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticonvulsant activity increases with in vitro affinity, but the slope of a plot of pED50 versus pIC50 is low (0.40); taking plasma protein binding into account in this plot increases the slope to 0.60. This shows that binding to albumin in plasma reduces the amount of compound free to diffuse across the blood-brain barrier. Further evidence comes from three other experiments: (a) Direct measurements of brain/blood ratios for three compounds (2, 16, 26) show the ratio decreases with increasing log R. (b) Warfarin, which competes for albumin binding sites dose-dependently, decreased the ED50 of 26 for protection against seizures induced by NMDLA. (c) Direct measurements of brain penetration using an in situ brain perfusion model in rat to measure the amount of drug crossing the blood-brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this is greatly reduced when the drug is delivered in plasma. In the 4-hydroxyquinolones glycine site binding affinity increases with lipophilicity of the 3-substituent up to a maximum at a log P around 3, then does not improve further. When combined with increasing protein binding, this gives a parabolic relationship between predicted in vivo activity and log P, with a maximum log P value of 2.39. Finally, the plasma protein binding studies have been extended to other series of glycine site antagonists, and its is shown that for a given log P these have similar protein binding to the 4-hydroxyquinolones, except for compounds that are not acidic. The results have implications for the design of novel glycine site antagonists, and it is suggested that it is necessary to either keep log P low or pKa high to obtain good central nervous system activity.

Animals↗