Dual A1 and A2A adenosine receptor antagonists, methoxy substituted 2-benzylidene-1-indanone, suppresses intestinal postprandial glucose and attenuates hyperglycaemia in fructose-streptozotocin diabetic rats.

BACKGROUND/AIM: Recent research suggests that adenosine receptors (ARs) influence many of the metabolic abnormalities associated with diabetes. A non-xanthine benzylidene indanone derivative 2-(3,4-dihydroxybenzylidene)-4-methoxy-2,3-dihydro-1 H-inden-1-one (2-BI), has shown to exhibit higher affinity at A1/A2A ARs compared to caffeine. Due to its structural similarity to caffeine, and the established antidiabetic effects of caffeine, the current study was initiated to explore the possible antidiabetic effect of 2-BI. METHODS: The study was designed to assess the antidiabetic effects of several A1 and/or A2A AR antagonists, via intestinal glucose absorption and glucose-lowering effects in fructose-streptozotocin (STZ) induced diabetic rats. Six-week-old male Sprague-Dawley rats were induced with diabetes via fructose and streptozotocin. Rats were treated for 4 weeks with AR antagonists, metformin and pioglitazone, respectively. Non-fasting blood glucose (NFBG) was determined weekly and the oral glucose tolerance test (OGTT) was conducted at the end of the intervention period. RESULTS: Dual A1/A2A AR antagonists (caffeine and 2-BI) decreased glucose absorption in the intestinal membrane significantly (p < 0.01), while the selective A2A AR antagonist (Istradefylline), showed the highest significant (p < 0.001) reduction in intestinal glucose absorption. The selective A1 antagonist (DPCPX) had the least significant (p < 0.05) reduction in glucose absorption. Similarly, dual A1/A2A AR antagonists and selective A2A AR antagonists significantly reduced non-fast blood glucose and improved glucose tolerance in diabetic rats from the first week of the treatment. Conversely, the selective A1 AR antagonist did not reduce non-fast blood glucose significantly until the 4th week of treatment. 2-BI, caffeine and istradefylline compared well with standard antidiabetic treatments, metformin and pioglitazone, and in some cases performed even better. CONCLUSION: 2-BI exhibited good antidiabetic activity by reducing intestinal postprandial glucose absorption and improving glucose tolerance in a diabetic animal model. The dual antagonism of A1/A2A ARs presents a positive synergism that could provide a new possibility for the treatment of diabetes.

Insight into the mechanism and toxicology of nitrofurantoin: a metabolomics approach.

Safety and effectiveness are the two ends of the balance in drug development that needs to be evaluated. The biotransformation of drugs within a living organism could potentiate biochemical insults in the tissue and compromise the safety of drugs. Nitrofurantoin (NFT) is a cheap clinical antibiotic with a wide array of activities against gram-positive and gram-negative organisms. The NFT scaffold has been utilized to develop other derivates or analogues in the quest to repurpose drugs against other infectious diseases. Several techniques were developed over the years to study the mechanism of NFT metabolism and toxicity, such as voltammetry, chromatographic analysis, protein precipitation, liquid-liquid extraction, etc. Due to limitations in these methods, the mechanism of NFT biotransformation in the cell is poorly understood. Metabolomics has been adopted in drug metabolism to understand the mechanism of drug toxicity and could provide a solution to overcome the limitations of current techniques to determine mechanisms of toxicity. Unfortunately, little or no information regarding the metabolomics approach in NFT metabolism and toxicity is available. Hence, this review highlights the metabolomic techniques that can be adopted in NFT metabolism and toxicological studies to encourage the research community to widely adopt and utilize metabolomics in understanding NFT's metabolism and toxicity.

Exploration of chalcones and related heterocycle compounds as ligands of adenosine receptors: therapeutics development.

Adenosine receptors (ARs) are ubiquitously distributed throughout the mammalian body where they are involved in an extensive list of physiological and pathological processes that scientists have only begun to decipher. Resultantly, AR agonists and antagonists have been the focus of multiple drug design and development programmes within the past few decades. Considered to be a privileged scaffold in medicinal chemistry, the chalcone framework has attracted a substantial amount of interest in this regard. Due to the potential liabilities associated with its structure, however, it has become necessary to explore other potentially promising compounds, such as heterocycles, which have successfully been obtained from chalcone precursors in the past. This review aims to summarise the emerging therapeutic importance of adenosine receptors and their ligands, especially in the central nervous system (CNS), while highlighting chalcone and heterocyclic derivatives as promising AR ligand lead compounds.

New fused pyrroles with rA1/A2A antagonistic activity as potential therapeutics for neurodegenerative disorders.

In a pilot study, eleven pyrrolopyridine and pyrrolopyrimidine derivatives (specifically, 7-azaindole and 7-deazapurine derivatives) were synthesised by Suzuki cross-coupling reactions and evaluated via radioligand binding assays as potential adenosine receptor (AR) antagonists in order to further investigate the structure-activity relationships of these compounds. 6-Chloro-4-phenyl-1H-pyrrolo[2,3-b]pyridine, with a 7-azaindole scaffold, was identified as a selective A1 AR antagonist with a rA1Ki value of 0.16 µM, and interestingly, the addition of a N-atom to the aforementioned fused heterocyclic ring system, creating corresponding 7-deazapurines, led to a dual A1/A2A AR ligand (2-chloro-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine: rA1Ki: 0.19 ± 0.02 µM; rA2AKi: 0.43 ± 0.01 µM). Introducing an additional N-atom into the heterocyclic ring system was tolerable for rA1 AR affinity and also led to rA2A AR affinity. This pilot study concluded that new 7-azaindole and 7-deazapurine derivatives represent interesting scaffolds for design of A1 and/or A2A AR antagonists.

Design, synthesis and evaluation of amino-3,5-dicyanopyridines and thieno[2,3-b]pyridines as ligands of adenosine A1 receptors for the potential treatment of epilepsy.

Due to the implication of adenosine in seizure suppression, adenosine-based therapies such as adenosine receptor (AR) agonists have been investigated. This study aimed at investigating thieno[2,3-b]pyridine derivatives as non-nucleoside A1 agonists that could be used in pharmaco-resistant epilepsy (PRE). Compound 7c (thieno[2,3-b]pyridine derivative), displayed good binding affinity to the rA1 AR (K i = 61.9 nM). This could be a breakthrough for further investigation of this heterocyclic scaffold as potential ligand. In silico evaluation of this compound raised bioavailability concerns but performed well on drug-likeness tests. The effect of intramolecular cyclisation that occurs during synthesis of thieno[2,3-b]pyridines from the lead compounds, amino-3,5-dicyanopyridine derivatives (6a-s) in relation to AR binding was also evaluated. A significant loss of activity against rA1/rA2A ARs with cyclisation was revealed. Amino-3,5-dicyanopyridines exhibited greater affinity towards rA1 ARs (K i < 10 nM) than rA2A. Compound 6c had the best rA1 affinity (K i = 0.076 nM). Novel compounds (6d, 6k, 6l, 6m, 6n, 6o, 6p) were highly selective towards rA1 AR (K i between 0.179 and 21.0 nM). Based on their high selectivity for A1 ARs, amino-3,5-dicyanopyridines may be investigated further as AR ligands in PRE with the right structural optimisations and formulations. A decrease in rA1 AR affinity is observed with intramolecular cyclisation that occurs during synthesis of thieno[2,3-b]pyridines (7a, 7d, 7c) from amino-3,5-dicyanopyridine derivatives (6a, 6f, 6g).

C3 amino-substituted chalcone derivative with selective adenosine rA1 receptor affinity in the micromolar range.

ABSTRACT: To identify novel adenosine receptor (AR) ligands based on the chalcone scaffold, herein the synthesis, characterization and in vitro and in silico evaluation of 33 chalcones (15-36 and 37-41) and structurally related compounds (42-47) are reported. These compounds were characterized by radioligand binding and GTP shift assays to determine the degree and type of binding affinity, respectively, against rat (r) A1 and A2A ARs. The chalcone derivatives 24, 29, 37 and 38 possessed selective A1 affinity below 10 µM, and thus, are the most active compounds of the present series; compound 38 was the most potent selective A1 AR antagonist (K i (r) = 1.6 µM). The structure-affinity relationships (SAR) revealed that the NH2-group at position C3 of ring A of the chalcone scaffold played a key role in affinity, and also, the Br-atom at position C3' on benzylidene ring B. Upon in vitro and in silico evaluation, the novel C3 amino-substituted chalcone derivative 38-that contains an α,ß-unsaturated carbonyl system and easily allows structural modification-may possibly be a synthon in future drug discovery. GRAPHIC ABSTRACT: C3 amino-substituted chalcone derivative (38) with C3' Br substitution on benzylidene ring B possesses selective adenosine rA1 receptor affinity in micromolar range.

C2-substituted quinazolinone derivatives exhibit A1 and/or A2A adenosine receptor affinities in the low micromolar range.

Antagonists of the adenosine receptors (A1 and A2A subtypes) are widely researched as potential drug candidates for their role in Parkinson's disease-related cognitive deficits (A1 subtype), motor dysfunction (A2A subtype) and to exhibit neuroprotective properties (A2A subtype). Previously the benzo-α-pyrone based derivative, 3-phenyl-1H-2-benzopyran-1-one, was found to display both A1 and A2A adenosine receptor affinity in the low micromolar range. Prompted by this, the α-pyrone core was structurally modified to explore related benzoxazinone and quinazolinone homologues previously unknown as adenosine receptor antagonists. Overall, the C2-substituted quinazolinone analogues displayed superior A1 and A2A adenosine receptor affinity over their C2-substituted benzoxazinone homologues. The benzoxazinones were devoid of A2A adenosine receptor binding, with only two compounds displaying A1 adenosine receptor affinity. In turn, the quinazolinones displayed varying degrees of affinity (low micromolar range) towards the A1 and A2A adenosine receptor subtypes. The highest A1 adenosine receptor affinity and selectivity were favoured by methyl para-substitution of phenyl ring B (A1Ki = 2.50 µM). On the other hand, 3,4-dimethoxy substitution of phenyl ring B afforded the best A2A adenosine receptor binding (A2AKi = 2.81 µM) among the quinazolinones investigated. In conclusion, the quinazolinones are ideal lead compounds for further structural optimization to gain improved adenosine receptor affinity, which may find therapeutic relevance in Parkinson's disease-associated cognitive deficits and motor dysfunctions as well as exerting neuroprotective properties.

Synthesis and evaluation of methoxy substituted 2-benzoyl-1-benzofuran derivatives as lead compounds for the development adenosine A1 and/or A2A receptor antagonists.

A series of fourteen methoxy substituted 2-benzoyl-1-benzofuran derivatives were synthesised and their affinities determined for adenosine A1 and A2A receptors via radioligand binding assays to establish the structure activity relationships pertinent for A1 and A2A affinity. Compound 3j (6,7-dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone exhibited A1 affinity (A1Ki (rat) = 6.880 µM) as well as A2A affinity (A2AKi (rat) = 0.5161 µM). Compounds 3a-b &3i-k exhibited selective affinity towards A1 with Ki values below 10 µM. The results indicate that C6,7-diOCH3 substitution on ring A in combination with meta (C3')-OCH3 substitution on ring B is beneficial for A1 and A2A affinity and activity. Compounds 3a-b &3j-k showed low cytotoxicity. Upon in vitro and in silico evaluation, compound 3j may be considered lead-like (i.e. a molecular entity suitable for optimization) and, thus, of value in the design of novel, potent and selective adenosine A1 and A2A receptor antagonists.

Benzopyrone represents a privilege scaffold to identify novel adenosine A1/A2A receptor antagonists.

Adenosine receptor antagonists are under investigation as potential drug candidates for the treatment of certain cancers, neurological disorders, depression and potentially improve tumour immunotherapy. The benzo-γ-pyrone scaffold is well-known in medicinal chemistry with diverse pharmacological activities attributed to them, however, their therapeutic potential as adenosine receptor antagonists have not been investigated in detail. To expand on the structure-activity relationships, the present study explored the adenosine A1 and A2A receptor binding affinities of a selected series of benzo-γ-pyrone analogues. In vitro evaluation led to the identification of 5-hydroxy-2-(3-hydroxyphenyl)-4H-1-benzopyran-4-one with the best adenosine A2A receptor affinity among the test compounds and was found to be non-selective (A1Ki = 0.956 µM; A2AKi = 1.44 µM). Hydroxy substitution on ring A and/or B play a key role in modulating the binding affinity at adenosine A1 and A2A receptors. Adenosine A1 receptor affinity was increased to the nanomolar range with hydroxy substitution on C6 (ring A), while meta-hydroxy substitution on ring B governed adenosine A2A receptor affinity. The double bond between C2 and C3 of ring C as well as C2 phenyl substitution was shown to be imperative for both adenosine A1 and A2A receptor affinity. Selected benzo-γ-pyrone derivatives behaved as adenosine A1 receptor antagonists in the performed GTP shift assays. It may be concluded that benzo-γ-pyrone based derivatives are suitable leads for designing and identifying adenosine receptor antagonists as treatment of various disorders.

Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders.

Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms have variable degrees of adenosine A1 receptor antagonistic activity. Prompted by this imidazo[1,2-α]pyridine analogues were synthesized and evaluated for their adenosine A1 and A2A receptor affinity via radioligand binding studies and subjected to a GTP shift assay to determine its adenosine A1 receptor agonistic or antagonistic functionality. Imidazo[1,2-α]pyridine, the parent scaffold, was found devoid of affinity for the adenosine A1 and A2A receptors. The influence of substitution on position C2 showed no improvement for either adenosine A1 or A2A receptor affinity. The addition of an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A1 or A2A receptor affinity. Surprisingly para-substitution on the phenyl ring at position C2 in combination with a cyclohexylamino group at position C3 led to adenosine A1 receptor affinity in the low micromolar range with compound 4d showing: (1) the highest affinity for the adenosine A1 receptor with a Ki value of 2.06µM and (2) adenosine A1 receptor antagonistic properties. This pilot study concludes that para-substituted 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridine analogues represent an interesting scaffold to investigate further structure-activity relationships in the design of novel imidazo[1,2-α]pyridine-based adenosine A1 receptor antagonists for the treatment of neurodegenerative disorders.

Discovery of 1,3-diethyl-7-methyl-8-(phenoxymethyl)-xanthine derivatives as novel adenosine A1 and A2A receptor antagonists.

Based on a previous report that a series of 8-(phenoxymethyl)-xanthines may be promising leads for the design of A1 adenosine receptor antagonists, selected novel and known 1,3-diethyl-7-methyl-8-(phenoxymethyl)-xanthine and 1,3,7-trimethyl-8-(phenoxymethyl)-xanthine analogs were synthesized and evaluated for their A1 and A2A adenosine receptor affinity. Generally, the study compounds exhibited affinity for both the A1 and A2A adenosine receptors. Replacement of the 1,3-dimethyl-substition with a 1,3-diethyl-substition pattern increased A1 and A2A binding affinity. Overall it was found that para-substitution on the phenoxymethyl side-chain of the 1,3-diethyl-xanthines decreased A1 affinity except for the 4-Br analog (4f) exhibiting the best A1 affinity in the submicromolar range. On the other hand A2A affinity for the 1,3-diethyl-xanthines were increased with para-substitution and the 4-OCH3 (4b) analog showed the best A2A affinity with a Ki value of 237nM. The 1,3-diethyl-substituted analogs (4a, and 4f) behaved as A1 adenosine receptor antagonists in GTP shift assays performed with rat whole brain membranes expressing A1 adenosine receptors. This study concludes that para-substituted 1,3-diethyl-7-methyl-8-(phenoxymethyl)-xanthine analogs represent novel A1 and A2A adenosine receptor antagonists that are appropriate for the design of therapies for neurodegenerative disorders such as Parkinson's and Alzheimer's disease.

Carbamate substituted 2-amino-4,6-diphenylpyrimidines as adenosine receptor antagonists.

A novel series of carbamate substituted 2-amino-4,6-diphenylpyrimidines was evaluated as potential dual adenosine A1 and A2A receptor antagonists. The majority of the synthesised compounds exhibited promising dual affinities, with A1Ki values ranging from 0.175 to 10.7 nM and A2AKi values ranging from 1.58 to 451 nM. The in vivo activity illustrated for 3-(2-amino-6-phenylpyrimidin-4-yl)phenyl morpholine-4-carboxylate (4c) is indicative of the potential of these compounds as therapeutic agents in the treatment of Parkinson's disease, although physicochemical properties may require optimisation.

1,3,7-Triethyl-substituted xanthines--possess nanomolar affinity for the adenosine A1 receptor.

Adenosine A1 receptors are attracting great interest as drug targets for their role in cognitive deficits. Antagonism of the adenosine A1 receptor may offer therapeutic benefits in complex neurological diseases, such as Alzheimer's and Parkinson's disease. The aim of this study was to discover potential selective adenosine A1 receptor antagonists. Several analogs of 8-(3-phenylpropyl)xanthines (3), 8-(2-phenylethyl)xanthines (4) and 8-(phenoxymethyl)xanthines (5) were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that the 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d), among each series, displayed the highest affinity for the adenosine A1 receptor with Ki values in the nanomolar range. This ethyl-substitution pattern was previously unknown to enhance adenosine A1 receptor binding affinity. The 1,3,7-triethyl-substituted analogs (3d, 4d, and 5d) behaved as adenosine A1 receptor antagonists in GTP shift assays performed with either rat cortical or whole brain membranes expressing adenosine A1 receptors. Further, in vivo evaluation of 3d showed reversal of adenosine A1 receptor agonist-induced hypolocomotion. In conclusion, the most potent evaluated compound, 8-(3-phenylpropyl)-1,3,7-triethylxanthine (3d), showed both in vitro and in vivo activity, and therefore represent a novel adenosine A1 receptor antagonist that may have potential as a drug candidate for dementia disorders.

The adenosine receptor affinities and monoamine oxidase B inhibitory properties of sulfanylphthalimide analogues.

Based on a report that sulfanylphthalimides are highly potent monoamine oxidase (MAO) B selective inhibitors, the present study examines the adenosine receptor affinities and MAO-B inhibitory properties of a series of 4- and 5-sulfanylphthalimide analogues. Since adenosine antagonists (A1 and A2A subtypes) and MAO-B inhibitors are considered agents for the therapy of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs that antagonize adenosine receptors and inhibit MAO-B may have enhanced therapeutic value. The results document that the sulfanylphthalimide analogues are selective for the adenosine A1 receptor over the A2A receptor subtype, with a number of compounds also possessing MAO-B inhibitory properties. Among the compounds evaluated, 5-[(4-methoxybenzyl)sulfanyl]phthalimide was found to possess the highest binding affinity to adenosine A1 receptors with a Ki value of 0.369 µM. This compound is reported to also inhibit MAO-B with an IC50 value of 0.020 µM. Such dual-target-directed compounds may act synergistic in the treatment of Parkinson's disease: antagonism of the A1 receptor may facilitate dopamine release, while MAO-B inhibition may reduce dopamine metabolism. Additionally, dual-target-directed compounds may find therapeutic value in Alzheimer's disease: antagonism of the A1 receptor may be beneficial in the treatment of cognitive dysfunction, while MAO-B inhibition may exhibit neuroprotective properties. In neurological diseases, such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs are expected to be advantageous over single-target treatments.

The adenosine A(2A) antagonistic properties of selected C8-substituted xanthines.

The adenosine A2A receptor is considered to be an important target for the development of new therapies for Parkinson's disease. Several antagonists of the A2A receptor have entered clinical trials for this purpose and many research groups have initiated programs to develop A2A receptor antagonists. Most A2A receptor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substituted heterocyclic compounds. In an attempt to discover high affinity A2A receptor antagonists and to further explore the structure-activity relationships (SARs) of A2A antagonism by the xanthine class of compounds, this study examines the A2A antagonistic properties of series of (E)-8-styrylxanthines, 8-(phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines. The results document that among these series, the (E)-8-styrylxanthines have the highest binding affinities with the most potent homologue, (E)-1,3-diethyl-7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a Ki value of 11.9 nM. This compound was also effective in reversing haloperidol-induced catalepsy in rats, providing evidence that it is in fact an A2A receptor antagonist. The importance of substitution at C8 with the styryl moiety was demonstrated by the finding that none of the 8-(phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines exhibited high binding affinities for the A2A receptor.

On the basis of sex: male vs. female rat adenosine A1/A2A receptor affinity.

OBJECTIVE: To ensure reproducibility in biomedical research, the biological variable sex must be reported; yet a reason for using male (instead of female) rodents is seldom given. In our search for novel adenosine receptor ligands, our research group routinely determines a test compound's binding affinities at male Sprague-Dawley rat (r) adenosine A1 and A2A receptors via in vitro radioligand binding studies. This pilot study compared the binding affinities of four adenosine receptor ligands (frequently used as reference standards) at male and female adenosine rA1 and rA2A receptors. RESULTS: The inhibition constant (Ki) values determined using female rats correspond well to the values obtained using male rats and no markable difference could be observed in affinity and selectivity of reference standards. For example, DPCPX the selective adenosine A1 receptor antagonist: male rA1Ki: 0.5 ± 0.1 nM versus female rA1Ki: 0.5 ± 0.03 nM; male rA2AKi: 149 ± 23 nM versus female rA2AKi: 135 ± 29 nM. From the limited data at hand, we conclude that even when using female rats for in vitro studies without regard for the oestrous cycle, the obtained data did not vary much from their male counterparts.

Novel sulfanylphthalimide analogues as highly potent inhibitors of monoamine oxidase B.

Monoamine oxidase (MAO) plays an essential role in the catabolism of neurotransmitter amines. The two isoforms of this enzyme, MAO-A and -B, are considered to be drug targets for the therapy of depression and neurodegenerative diseases, respectively. Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5-sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC(50) values in the nanomolar range. The most potent inhibitor, 5-(benzylsulfanyl)phthalimide, exhibits an IC(50) value of 0.0045 µM for the inhibition of MAO-B with a 427-fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy.

Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine oxidase B.

It has recently been reported that nitrile containing compounds frequently act as potent monoamine oxidase B (MAO-B) inhibitors. Modelling studies suggest that this high potency inhibition may rely, at least in part, on polar interactions between nitrile functional groups and polar moieties within the MAO-B substrate cavity. In an attempt to identify potent and selective inhibitors of MAO-B and to contribute to the known structure-activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC(50) values in the nanomolar range. In general, the sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding sulfanylbenzonitrile homologues. Among the compounds evaluated, 4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC(50)=0.025 µM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson's disease.

Paracetamol prevents hyperglycinemia in vervet monkeys treated with valproate.

Valproate administration increases the level of the inhibitory transmitter, glycine, in the urine and plasma of patients and experimental animals. Nonketotic hyperglycinemia (NKH), an autosomal recessive disorder of glycine metabolism, causes increased glycine concentrations in blood, urine, and cerebrospinal fluid (CSF), most likely due to a defect in the glycine cleavage enzyme or possibly deficits in glycine transport across cell membranes. We investigated the relationship between the hyperglycinemic effect of valproate and induced pyroglutamic aciduria via paracetamol in the vervet monkey. Firstly it was determined if valproate could induce hyperglycinemia in the monkey. The second aim was to increase glutamic acid (oxoproline) urine excretion using paracetamol as a pre-treatment and to assess whether valproate has an influence on the γ-glutamyl cycle. Hyperglycinemia was induced in healthy vervet monkeys when treated with a single oral dose of 50 mg/kg valproate. An acute dose of 50 mg/kg paracetamol increased oxoproline in the urine. Pre-treatment with paracetamol opposed the hyperglycinemic effect of valproate. However, the CSF:serum glycine ratio in a nonketotic monkey increased markedly after paracetamol treatment and remained high following valproate treatment. These results indicate that the γ-glutamyl cycle does indeed play a role in the hyperglycinemic effect of valproate treatment, and that paracetamol may have value in preventing and/or treating valproate-induced NKH.