Three centuries on the science of coffee authenticity control.

Food authenticity relies on genuineness and reliability according to the information displayed on the package. Since the 18th century, when coffee became popularized in the West, adulteration began. Several methods have been developed to detect different kinds of frauds and they have evolved as demands increased and new technologies were introduced. The evolution of the science of coffee authenticity control in the past three centuries is reviewed, focusing on the discrimination between coffee and other foods or between coffee and its by-products. The earliest chemical and physical methods are presented followed by methods developed in the 20th and 21st centuries using microscopy, chromatography and spectroscopy associated with advanced statistical tools, and DNA-based methods. In addition to non-food material, before the 20th century, chicory was the most studied food-adulterant. From the 20th century on, corn, coffee by-products, and barley were the most studied, followed by chicory, rice and other food items. Matrix effects seem to be among the most challenging problems in these approaches, associated with variations in roast degree, particle size (particularly in spectroscopy-based methods), and lack of control over reference samples regarding species and purity. Limits of detection vary considerably within each category, with most limits being too high for commercial use. DNA-based methods appear to be promising to assess coffee authenticity, given that the limits of detection and quantification are low, and specificity is higher than in other methods. Nevertheless, as roast intensity increases, the sensitivity of the method decreases. So far, most reported methods have not been validated and only a few have been tested on commercial brands, except for those involving microscopy which has been highly used for monitoring coffee authenticity although not always efficiently enough.

Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4.

Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) is a positive allosteric modulator (PAM) of mGluR4 that has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency, and poor solubility. Via high-throughput screening, we discovered more than 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, (+/-)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle, and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD.

Drug evaluation: PRX-00023, a selective 5-HT1A receptor agonist for depression.

EPIX Pharmaceuticals Inc (formerly Predix Pharmaceuticals Inc) is developing PRX-00023, an oral aryl piperazine 5-HT1A agonist, for the potential treatment of depression. While initially in development for generalized anxiety disorder, EPIX is now only focusing on the development of PRX-00023 for depression. In November 2006, EPIX reported that it planned to initiate a phase II trial in patients with depression in the first half of 2007.

Drug evaluation: Vilazodone--a combined SSRI and 5-HT1A partial agonist for the treatment of depression.

Vilazodone is a combined selective serotonin reuptake inhibitor (SSRI) and a 5-HT(1A) receptor partial agonist that is being developed by Clinical Data Inc for the treatment of depression. In preclinical studies, vilazodone compared favorably to other antidepressants such as paroxetine and fluoxetine. Orally administered vilazodone inhibited ultrasonic vocalization in the rat after electrical foot shock (a model of anxiolytic activity). Yet, in the forced swimming test model of depression in rats, vilazodone administered intraperitoneally was active at 1 mg/kg but not at 3 or 10 mg/kg. During clinical trials, vilazodone completely abolished REM sleep for 8 h and demonstrated antidepressant efficacy that was equal to that of current antidepressant therapeutics. The author concludes that the success of vilazodone as an effective antidepressant agent will depend on whether the drug can produce a more rapid antidepressant effect than other SSRI agents, or if specific genetic markers of patients can be associated with clinical efficacy.

Substituent effects of N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides on positive allosteric modulation of the metabotropic glutamate-5 receptor in rat cortical astrocytes.

CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] was recently described as the first centrally active, positive allosteric modulator of rat and human metabotropic glutamate receptor (mGluR) mGluR5 subtype. We explored the structural requirements for potentiation of glutamate-induced calcium release in naturally expressed mGluR5 in cultured rat astrocytes and increasing affinity for the allosteric antagonist binding site by evaluating 50 analogues of CDPPB. In the fluorometric calcium assay, CDPPB exhibited an EC50 value of 77 +/- 15 nM in potentiating mGluR5-mediated responses in cortical astrocytes and a Ki value of 3760 +/- 430 nM in displacing [3H]methoxyPEPy binding in membranes of cultured HEK-293 cells expressing rat mGluR5. The structure-activity relationships showed that electronegative aromatic substituents in the para-position of the benzamide moiety of CDPPB increase potency. Both binding and functional activities were further increased with a halogen atom in the ortho-position of the 1-phenyl ring. These effects of substitution do not match those of either aromatic ring of MPEP [2-methyl-6-(phenylethynyl)pyridine] for the antagonist allosteric binding site. Combination of the optimal substituents and aromatic positions resulted in 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide (VU-1545) showing Ki = 156 +/- 29 nM and EC50 = 9.6 +/- 1.9 nM in the binding and functional assays, respectively.

Drug evaluation: Lymphoseek - Neoprobe's sentinel lymph node imaging agent for use in cancer patients.

Neoprobe, under license from the University of California San Diego Cancer Center, is developing 99mTc-DTPA-mannosyl-dextran (Lymphoseek) as a potential agent for imaging lymph nodes in patients undergoing treatment for breast cancer and melanoma. Phase II trials of Lymphoseek were initiated in mid 2006.

Chlorogenic acids and lactones in regular and water-decaffeinated arabica coffees.

The market for decaffeinated coffees has been increasingly expanding over the years. Caffeine extraction may result in losses of other compounds such as chlorogenic acids (CGA) and, consequently, their 1,5-gamma-quinolactones (CGL) in roasted coffee. These phenolic compounds are important for flavor formation as well as the health effects of coffee; therefore, losses due to decaffeination need to be investigated. The present study evaluates the impact of decaffeination processing on CGA and CGL levels of green and roasted arabica coffees. Decaffeination produced a 16% average increase in the levels of total CGA in green coffee (dry matter), along with a 237% increase in CGL direct precursors. Different degrees of roasting showed average increments of 5.5-18% in CGL levels of decaffeinated coffee, compared to regular, a change more consistent with observed levels of total CGA than with those of CGL direct precursors in green samples. On the other hand, CGA levels in roasted coffee were 3-9% lower in decaffeinated coffee compared to regular coffee. Although differences in CGA and CGL contents of regular and decaffeinated roasted coffees appear to be relatively small, they may be enough to affect flavor characteristics as well as the biopharmacological properties of the final beverage, suggesting the need for further study.

Distribution of Major Chlorogenic Acids and Related Compounds in Brazilian Green and Toasted Ilex paraguariensis (Maté) Leaves.

Ilex paraguariensis (maté) is one of the best sources of chlorogenic acids (CGA) in nature. When leaves are toasted, some isomers are partly transformed into 1,5-γ-quinolactones (CGL). Both CGA and CGL are important contributors to the brew's flavor and are thought to contribute to human health. In this study, we quantified 9 CGA, 2 CGL, and caffeic acid in 20 samples of dried green and toasted maté that are commercially available in Brazil. Total CGA content in green maté varied from 8.7 to 13.2 g/100 g, dry weight (dw). Caffeic acid content varied from 10.8 to 13.5 mg/100 g dw, respectively. Content in toasted maté varied from 1.5 to 4.6 g/100 g and from 1.5 to 7.2 mg/100 g dw, respectively. Overall, caffeoylquinic acid isomers (CQA) were the most abundant CGA in both green and toasted maté, followed by dicaffeoylquinic acids (diCQA) and feruloylquinic acids (FQA). These classes accounted for 58.5%, 40.0%, and 1.5% of CGA, respectively, in green maté and 76.3%, 20.7%, and 3.0%, respectively, in toasted maté. Average contents of 3-caffeoylquinolactone (3-CQL) and 4-caffeoylquinolactone (4-CQL) in commercial toasted samples were 101.5 mg/100 g and 61.8 mg/100 g dw, respectively. These results show that, despite overall losses during the toasting process, CGA concentrations are still substantial in toasted leaves, compared to other food sources of CGA and phenolic compounds in general. In addition to evaluating commercial samples, investigation of changes in CGA profile and formation of 1,5-γ-quinolactones was performed in experimental maté toasting.

Effect of roasting on the formation of chlorogenic acid lactones in coffee.

Of all plant constituents, coffee has one of the highest concentrations of chlorogenic acids. When roasting coffee, some of these are transformed into chlorogenic acid lactones (CGL). We have studied the formation of CGL during the roasting of coffee beans in Coffea arabica cv. Bourbon; C. arabicacv. Longberry; and C. canephora cv. Robusta. Individual CGL levels were determined by comparison of HPLC peaks with those of synthetic CGL standards. Seven CGL were identified: 3-caffeoylquinic-1,5-lactone (3-CQL), 4- caffeoylquinic-1,5-lactone (4-CQL), 3-coumaroylquinic-1,5-lactone (3-pCoQL), 4-coumaroylquinic-1,5-lactone (4-pCoQL), 3-feruloylquinic-1,5-lactone (3-FQL), 4-feruloylquinic-1,5-lactone (4-FQL), and 3,4-dicaffeoylquinic-1,5-lactone (3,4-diCQL). 3-CQL was the most abundant lactone in C. arabica and C. canephora, reaching peak values of 230 +/- 9 and 254 +/- 4 mg/100 g (dry weight), respectively, at light medium roast ( approximately 14% weight loss). 4-CQL was the second most abundant lactone (116 +/- 3 and 139 +/- 2 mg/100 g, respectively. The maximum amount of CGL represents approximately 30% of the available precursors. The relative levels of 3-CQL and 4-CQL in roasted coffee were reverse to those of their precursors in green coffee. This suggests that roasting causes isomerization of chlorogenic acids prior to the formation of lactones and that the levels of lactones in roasted coffee do not reflect the levels of precursors in green coffee.

The discovery of epidepride and its analogs as high-affinity radioligands for imaging extrastriatal dopamine D(2) receptors in human brain.

[(123)I]Epidepride, [(18)F]fallypride, and [(76)Br]isoremoxipride (FLB-457) and their corresponding [(11)C]labeled derivatives belong to a class of high-affinity radioligands for SPECT or PET imaging of dopamine D(2) receptors in the human brain. In contrast to previously used imaging agents, these ligands are capable of identifying extrastriatal dopamine D(2) receptors. The design of these substituted benzamides derive its origin from the atypical antipsychotic agent, remoxipride. Starting in the late 1970's, halogenated analogs of (S)-sulpiride were evaluated in binding assays and behavioral studies, leading to the discovery of remoxipride. Remoxipride was 10 times weaker than sulpiride in vitro but 50 times more potent in vivo. Search for a putative active metabolite of remoxipride led to the discovery of raclopride and eticlopride, the former becoming a useful radioligand as tritium or carbon-11 labeled form for receptor binding and PET studies, respectively. In the US, the mono-iodine analog of raclopride, [(123)I]iodobenzamide (IBZM), was found to have moderate putamen-to-cerebellum ratio in rat and human brain. Continued search for metabolites of remoxipride led to the discovery of its 3,6-dihydroxy derivative, NCQ-344, with an extremely potent in vivo activity in the rat. SAR studies of the metabolites of remoxipride led to the discovery of the 3-methoxy isomer, isoremoxipride (FLB-457) and its corresponding 6-hydroxy analog, FLB-463, both having affinities for the dopamine D(2) receptor in the 20-30 pM range. Later, the 5-[(123)I]iodo analog of FLB-463, [(123)I]ioxipride ([(123)I]NCQ-298), became a potential SPECT imaging agent. In the mean time, the deshydroxy analog of IBZM, [(125)I]iodopride, showed binding potential in the rat similar to [(125)I]IBZM. Epidepride was designed by combining the structure of isoremoxipride with that of iodopride. In 1988, epidepride was independently prepared and radiolabeled in three separate laboratories in Stockholm, Berkeley, and Nashville. Evaluation of seven [(125)I]iodine substituted analogs of raclopride, including IBZM, revealed the unusual high striatum-to-cerebellum ratio of 234 of [(125)I]epidepride in the rat. Subsequent SPECT images with [(123)I]epidepride demonstrated its ability to identify extrastriatal dopamine D(2) receptors in the human brain. Exploration of the structure of epidepride confirmed its exceptional properties, to be exceeded only by its N-allyl homolog, [(125)I]nalepride. The design by others of a series of potent 5-(3-[(18)F]fluoropropyl) substituted analogs of epidepride for PET imaging, lead to the discovery of [(18)F]fallypride. By elucidating the role of lipophilicity in the substituted benzamides, the excellent imaging characteristics of [(11)C]/[(123)I]epidepride, [(11)C]/[(76)Br]isoremoxipride and [(18)F]fallypride, could not only be explained but predicted with remarkable accuracy. By using the inverse product of the receptor affinity (K(D)), and the apparent partition constant of the radioligand (P((7.4))), estimates of maximal binding potential of any radioligand for imaging of any neurotransmitter receptor or transporter site seem possible.

ONO-2506. Ono.

ONO-2506 is an enantiomeric, three carbon atom homolog of valproic acid under development by ONO Pharmaceutical for the potential treatment of stroke, as well as Alzheimer's and Parkinson's disease. The injectable formulation (Proglia) is undergoing phase II trials in the US and Japan for acute-phase cerebral infarction, and the oral formulation (Cereact) is in phase I trials in the UK for Alzheimer's disease (AD) and Parkinson's disease (PD). Japanese and European phase I trials for AD, PD and amyotropic lateral sclerosis (ALS) had commenced by March 2002 and phase II trials for ALS are underway in Europe.

Perospirone (Sumitomo Pharmaceuticals).

Perospirone is a serotonin 5-HT2 antagonist and dopamine D2 antagonist developed by Sumitomo Pharmaceuticals for the potential treatment of schizophrenia and other psychoses [381769]. Its receptor binding profile and animal pharmacology resemble those of other atypical antipsychotic agents, in particular risperidone. In November 2000, CPAC's First Committee on Drugs recommended the approval of this product in Japan, [394007]; approval was granted in December 2000 [396121]. In January 2001, the NHI price was agreed by the Chuikyo [398222] and the drug was added to the NHI price list in February 2001 13982261. It was finally launched in Japan on February 8 2001 [399401]. In November 2000, Sumitomo and Welfide signed an agreement whereby Welfide's subsidiary Yoshitomi Yakuhin will copromote perospirone [394007]. Based on data from the Chuikyo, peak sales of perospirone are forecast to be yen9 billion in the sixth year following launch [398222], [398226]. In February 2001, Sumitomo predicted that perospirone would reach sales of yen10 billion within five to six years [399401].

Sumanirole Pharmacia.

Sumanirole is a dopamine D2 agonist under development by Pharmacia Corp (formerly Pharmacia & Upjohn) for the treatment of Parkinson's disease. The compound is currently undergoing phase II trials in Japan and phase III clinical trials in the US.

4-Caffeoyl-1,5-quinide in roasted coffee inhibits [3H]naloxone binding and reverses anti-nociceptive effects of morphine in mice.

RATIONALE: Cinnamoylquinides are formed from the corresponding chlorogenic acids during coffee roasting. Instant coffee has been shown to displace binding of the mu opioid receptor antagonist, [3H]naloxone, but the putative active agent, feruloylquinide, has not been characterized. OBJECTIVES: The goal was to identify the active agent(s) in coffee by measuring the binding affinity of individual cinnamoyl-1,5-quinides to the human mu opioid receptor, and determine the effects of these compounds on morphine-induced anti-nociceptive behavior in mice. METHODS: Cinnamoyl-1,5-quinides in extracts of decaffeinated instant coffee were quantified by reverse-phase HPLC comparisons with synthetic samples of 3-coumaroyl-1,5-quinide and 4-coumaroyl-1,5-quinide, 3-caffeoyl-1,5-quinide and 4-caffeoyl-1,5-quinide (4-CQL) 3-feruloyl-1,5-quinide and 4-feruloyl-1,5-quinides and 3,4-dicaffeoyl-1,5-quinide (DICAQ). Affinities of the cinnamoyl-1,5-quinides and decaffeinated instant coffee extract were determined by displacement of [3H]naloxone binding in cultured HEK-MOR cells. Inhibition of the anti-nociceptive activity of morphine (1 mg/kg IP) was determined in C57BL/6J mice using the hot plate test at 52 degrees C. RESULTS: Extract of decaffeinated instant coffee produced a displacement K(i) of 42+/-16 mg/l, while the K(i) of a synthetic sample of 4-CQL was 4.4+/-0.4 microM. Compounds with a cinnamoyl substituent in the 4-position of the quinide, i.e. 4-CQL, DICAQ, 3,4-diferuloyl-1,5-quinide, and 3,4-dicoumaroyl-1,5-quinide, had affinities for the mu opioid receptor in the low micromolar range. In the hot plate test, coffee extract, containing 0.78% of 4-CQL, reversed the anti-nociceptive effect of morphine at 10 mg/kg IP. Two cinnamoyl-1,5-quinides found in roasted coffee, DICAQ, and 4-CQL, were active at 1 and 0.1 mg/kg IP, respectively. CONCLUSIONS: These results suggest that the previously reported anti-opioid activity of instant coffee is caused primarily by the presence of 4-CQL, and to lesser extent by other cinnamoyl-1,5-quinides.

Dicinnamoylquinides in roasted coffee inhibit the human adenosine transporter.

Preliminary screening of a minor, non-xanthine constituent of roasted coffee, 3,4-diferuloyl-1,5-quinolactone (DIFEQ), showed inhibition of the adenosine transporter at low micromolar concentration. DIFEQ is a neutral derivative of the chlorogenic acids, i.e. isomeric mono- and di-substituted coumaroyl-, caffeoyl-, and feruloyl-esters of quinic acid, formed in the roasting process of coffee. Displacement of the adenosine transporter antagonist [(3)H](S)-(nitrobenzyl)-6-thioinosine binding by DIFEQ in cultured U-937 cell preparations, expressing the human adenosine transporter protein (hENT1), showed a K(i) of 0.96+/-0.13 microM. Extracts of regular and decaffeinated coffee showed binding activities equivalent to 30-40 mg DIFEQ per three cups of coffee. Acute administration of a high dose of DIFEQ (100 mg/kg i.p.) reduced open field locomotion in mice for 20 min in correlation with brain levels of DIFEQ. Both 3,4-dicaffeoyl-1,5-quinide and 3,4-dicoumaroyl-1,5-quinide, two close structural analogs of DIFEQ also present in roasted coffee, showed similar affinities for the adenosine transporter, while the corresponding 3- and 4-mono caffeoyl- and feruloyl-quinides were one to two orders of magnitudes less active. This suggests that 3,4-dicinnamoyl-1,5-quinides in coffee could have the potential to raise extra-cellular adenosine levels, thereby counteracting the stimulant effect of caffeine.

Antioxidant effects of dihydrocaffeic acid in human EA.hy926 endothelial cells.

Dihydrocaffeic acid (DHCA) is a metabolite of caffeic acid with potent antioxidant properties. Since DHCA has been detected in human plasma following coffee ingestion, we tested the hypothesis that DHCA protects the endothelium from oxidative stress in a model in human-derived EA.hy926 endothelial cells. During culture for 16-24 hours, the cells accumulated DHCA against a concentration gradient to low millimolar concentrations. In alpha-tocopherol-loaded cells, DHCA spared alpha-tocopherol during overnight culture in a dose-dependent manner. In response to oxidant stress induced by a water-soluble free radical initiator, both alpha-tocopherol and DHCA diminished oxidation of cis-parinaric acid that had been incorporated into the cells, although their antioxidant activities were not additive. DHCA also decreased intracellular oxidation of dihydrofluorescein due to redox cycling by menadione. This suggests that the protective effects of DHCA were caused by scavenging of intracellular reactive oxygen species. DHCA also increased nitric oxide synthase activity in a dose-dependent manner in cultured cells, which was associated with a comparable increase in endothelial nitric oxide synthase protein. Although the DHCA concentrations required for these effects are higher than those likely to be present in plasma or the interstitium, these results indicate that DHCA can function as an intracellular antioxidant.

Discrimination in 5-HT(3) receptor binding in murine brain and cultured cell preparations.

One-hundred ninety-one ligands were screened at 5-HT(3) receptors in membranes from rat brain and NCB20 cells for their ability to displace the selective, high-affinity 5-HT(3) receptor antagonist, [(125)I]DAIZAC ([(125)I]( S)-5-chloro-3-iodo-2-methoxy- N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide). Thirty-seven compounds having structures related to benzamide, dibenzepine, serotonin, phenylbiguanide, or arylpiperzine were selected for more extensive displacement studies in membranes from rat and mouse brains, from two cultured cell preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins (NCB20 and NG108-15 cell lines), and from recombinant Sf9 cells expressing homomeric 5-HT(3A) receptors. [(125)I]DAIZAC bound specifically to a single site in each of the five tissue preparations with high affinity ( K(D) 0.12-0.19 nM). The densities of [(125)I]DAIZAC-labeled 5-HT(3) receptors were 7.4-7.5 fmol/mg protein in membranes from murine brain, and 38, 99, and 1588 fmol/mg protein in membranes from cultured NCB20, NG108-15, and recombinant Sf9 cells, respectively. The affinity of substituted benzamides ( n=10) was similar in all five tissue preparations. The affinity of dibenzepines ( n=17) was significantly higher in membranes from cultured cells as compared to membranes from rat and mouse brain, but similar in the two brain membrane preparations, and in each of the cultured cell membrane preparations. Serotonin-, phenylbiguanide-, and quipazine-analogs ( n=10), which typically function as 5-HT (5-hydroxytryptamine) agonists, exhibited significantly higher apparent p K(i) values in membranes from rat brain and Sf9 recombinant cells than in membranes from the three preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins ( F=7.52, P<0.001). These findings confirm that there are both species and cell-type dependent differences in binding to 5-HT(3) receptors, and that care must be taken when comparing results between experimental paradigms that utilize different sources of 5-HT(3) receptors.

Interaction of novel positive allosteric modulators of metabotropic glutamate receptor 5 with the negative allosteric antagonist site is required for potentiation of receptor responses.

Exciting advances have been made in the discovery of selective positive allosteric modulators of the metabotropic glutamate receptor (mGluR) mGluR5. These compounds may provide a novel approach that could be useful in the treatment of certain central nervous system disorders. However, because of their low potencies, previously described mGluR5 potentiators are not useful for functional studies in native preparations. In addition, binding sites at which these compounds act have not been identified. It has been suggested that two allosteric potentiators, 3,3'-difluorobenzaldazine and 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), act by binding to the same allosteric site as the negative allosteric modulators of mGluR5 such as 2-methyl-6-(phenylethynyl)pyridine (MPEP). However, another mGluR5 potentiator, N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)m-ethyl]phenyl}-2-hydroxybenzamide, does not bind to this site, bringing this hypothesis into question. We have synthesized a series of CDPPB analogs and report that these compounds bind to the MPEP site with affinities that are closely related to their potencies as mGluR5 potentiators. Furthermore, allosteric potentiation is antagonized by a neutral ligand at the MPEP site and reduced by a mutation of mGluR5 that eliminates MPEP binding. Together, these data suggest that interaction with the MPEP site is important for allosteric potentiation of mGluR5 by CDPPB and related compounds. In addition, whole-cell patch-clamp studies in midbrain slices reveal that a highly potent analog of CDPPB, 4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (VU-29), selectively potentiates mGluR5 but not mGluR1-mediated responses in midbrain neurons, whereas a previously identified allosteric potentiator of mGluR1 has the opposite effect.

Biphenyl-indanone A, a positive allosteric modulator of the metabotropic glutamate receptor subtype 2, has antipsychotic- and anxiolytic-like effects in mice.

Previous studies indicate that agonists of the group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, may provide a novel approach for the treatment of anxiety disorders and schizophrenia. However, the relative contributions of the mGluR2 and mGluR3 subtypes to the effects of the group II mGluR agonists remain unclear. In the present study, we describe an alternate synthesis and further pharmacological characterization of a recently reported positive allosteric modulator of mGluR2 termed biphenyl-indanone A (BINA). In recombinant systems, BINA produced a robust and selective potentiation of the response of mGluR2 to glutamate with no effect on the glutamate response of other mGluR subtypes. In hippocampal brain slices, BINA (1 microM) significantly potentiated the mGluR2/3 agonist-induced inhibition of excitatory synaptic transmission at the medial perforant path-dentate gyrus synapse. BINA was also efficacious in several models predictive of antipsychotic- and anxiolytic-like activity in mice. The behavioral effects of BINA were blocked by the mGluR2/3 antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), suggesting that the in vivo effects of BINA are mediated by increased activation of mGluR2. Collectively, these results indicate that BINA is a selective mGluR2 positive allosteric modulator and provide further support for the growing evidence that selective allosteric potentiators of mGluR2 mimic many of the in vivo actions of mGluR2/3 agonists that may predict therapeutic utility of these compounds.

A novel class of positive allosteric modulators of metabotropic glutamate receptor subtype 1 interact with a site distinct from that of negative allosteric modulators.

We recently reported a novel class of compounds, represented by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CD-PPB), that act as positive allosteric modulators (potentiators) of metabotropic glutamate receptor (mGluR) subtype 5. Studies of CDPPB analogs revealed that some compounds in this series serve also as positive allosteric modulators of mGluR1. Although CDPPB is selective for mGluR5 relative to other mGluR subtypes, several CDPPB analogs also showed 2.5-fold potentiation of glutamate-induced calcium transients in cells expressing mGluR1 at 10 muM, with 4-nitro-N-(1,4-diphenyl-1H-pyrazol-5-yl)benzamide (VU-71) being selective for mGluR1. In previous studies, we found that two structural classes of mGluR5-selective allosteric potentiators, including CDPPB, share a common binding site with the allosteric mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine. Negative allosteric modulators of mGluR1, regardless of structural class, have been reported to bind to a common allosteric antagonist site on this receptor. However, neither the novel CDPPB analogs nor previously identified allosteric mGluR1 potentiators [e.g., (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)pyrrolidine (Ro 67-7476), ethyl diphenylacetylcarbamate (Ro 01-6128), and butyl (9H-xanthene-9-carbonyl)carbamate (Ro 67-4853)] displaced the binding of [(3)H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127), a high-affinity radioligand for the allosteric antagonist site on mGluR1 at concentrations several orders of magnitude higher than those required to induce allosteric potentiation of mGluR1 responses. These data suggest that allosteric potentiators of mGluR1 act at a site that is distinct from that of allosteric antagonists of mGluR1. Site-directed mutagenesis revealed that valine at position 757 in transmembrane V of mGluR1a is crucial for the activity of multiple classes of allosteric mGluR1 potentiators.