Lars Jacob Stray Knutsen

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)‐N‐[4,4‐Bis(3‐methyl‐2‐thienyl)but‐3‐en‐l‐yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of γ‐[3H]aminobutyric acid ([3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50= 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5‐hydroxytryptaminelA receptors was inhibited by NO 328 at 5—30 μM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCI. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl‐dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake. It is concluded that NO 328 is a potent and selective inhibitor of neuronal and glial GABA uptake and that [3H]NO 328 is a useful radioligand for labeling the GABA uptake carrier in brain membranes. In the mouse brain in vivo, [3H]NO 328 likewise showed saturable and reversible binding that could be displaced by analogues of NO 328. Further studies are needed to demonstrate whether the uptake carrier is indeed labeled by [3H]NO 328 in vivo.

Characterization of tiagabine (NO-328), a new potent and selective GABA uptake inhibitor.

Tiagabine (NO-328) (R(-)-N-[4,4-bis(3-methylthien-2-yl)but-3-enyl]nipecotic acid, hydrochloride) is a new centrally acting GABA uptake inhibitor. The anticonvulsant activity of tiagabine was evaluated against seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), pentylenetetrazol, bicuculline, maximal electrostimulation (MES), or high intensity sound. The sedative actions of tiagabine were evaluated in tests for traction, rotarod performance and exploratory behavior. Finally, interoceptive properties of tiagabine were assessed using diazepam-, CGS 9896-, pentylenetetrazol-, or amphetamine-discriminating rats. Tiagabine was an effective anticonvulsant in doses which did not produce sedation or motor debilitation, although it was not potent against MES. In a manner similar to other anti-epileptic drugs, tiagabine potentiated dopaminergic function (methylphenidate-induced gnawing in mice) although it did not substitute for amphetamine in amphetamine-trained animals. Furthermore, although tiagabine antagonized DMCM-induced convulsions, it exhibited neither CGS 9896 or diazepam-like interoceptive effects, nor did it block (or potentiate) pentylenetetrazol-discrimination. Thus, GABA uptake inhibition represents a novel rationale for a valproate-like anticonvulsant drug therapy.

NNC-711, a novel potent and selective γ-aminobutyric acid uptake inhibitor: pharmacological characterization

NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride) is a novel, potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor. NNC-711 inhibited synaptosomal (IC50 = 47 nM), neuronal (IC50 = 1238 nM) and glial (IC50 = 636 nM) GABA uptake in vitro NNC-711 lacked affinity for other neurotransmitter receptor binding sites, uptake sites and ion channels examined in vitro. In vivo, NNC-711 was a potent anticonvulsant compound against rodent seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (ED50 (clonic) = 1.2 mg/kg i.p.), pentylenetetrazole (PTZ) (ED50 (tonic) = 0.72 mg/kg i.p., mouse; and ED50 (tonic) = 1.7 mg/kg, rat), or audiogenic (ED50 (clonic and tonic) = 0.23 mg/kg i.p.). At higher doses NNC-711 produced behavioral side effects characterized by inhibition of traction (ED50 = 23 mg/kg i.p.), rotarod (ED50 = 10 mg/kg i.p.) and exploratory locomotor activity (ED50 = 45 mg/kg i.p.) in the mouse. Following acute (3-h) in vivo pretreatment with NNC-711, behavioral tolerance developed to its motor impairing side effects (inhibition of traction, rotarod or exploratory locomotor activity) without corresponding tolerance to the anticonvulsant effects. These data suggest that NNC-711 will be useful for future in vitro and in vivo experiments to elucidate the role of the GABA uptake carrier in the central nervous system.

Discovery and Characterization of 6-{4-[3-(R)-2-Methylpyrrolidin-1-yl)propoxy]phenyl}-2H-pyridazin-3-one (CEP-26401, Irdabisant): A Potent, Selective Histamine H3 Receptor Inverse Agonist

Optimization of a novel series of pyridazin-3-one histamine H(3) receptor (H(3)R) antagonists/inverse agonists identified 6-{4-[3-(R)-2-methylpyrrolidin-1-yl)propoxy]phenyl}-2H-pyridazin-3-one (8a, CEP-26401; irdabisant) as a lead candidate for potential use in the treatment of attentional and cognitive disorders. 8a had high affinity for both human (K(i) = 2.0 nM) and rat (K(i) = 7.2 nM) H(3)Rs with greater than 1000-fold selectivity over the hH(1)R, hH(2)R, and hH(4)R histamine receptor subtypes and against an in vitro panel of 418 G-protein-coupled receptors, ion channels, transporters, and enzymes. 8a demonstrated ideal pharmaceutical properties for a CNS drug in regard to water solubility, permeability and lipophilicity and had low binding to human plasma proteins. It weakly inhibited recombinant cytochrome P450 isoforms and human ether-a-go-go-related gene. 8a metabolism was minimal in rat, mouse, dog, and human liver microsomes, and it had good interspecies pharmacokinetic properties. 8a dose-dependently inhibited H(3)R agonist-induced dipsogenia in the rat (ED(50) = 0.06 mg/kg po). On the basis of its pharmacological, pharmaceutical, and safety profiles, 8a was selected for preclinical development. The clinical portions of the single and multiple ascending dose studies assessing safety and pharmacokinetics have been completed allowing for the initiation of a phase IIa for proof of concept.

Unexpected neuroprotection observed with the adenosine A2A receptor agonist CGS 21680

The selective adenosine A2A receptor agonists 2‐[p‐(2‐carboxethyl)phenylethylaminol‐5′‐N‐ethylcarboxyamidoadenosine (CGS 21680), N‐[2‐(3,5‐dimethoxyphenyl)ethyl]adenosine (DPMA) and metrifudil were investigated for their ability to prevent the loss of pyramidal CA1 neurons in the hippocampus following 5 min of severe temporary forebrain ischemia in mongolian gerbils. CGS 21680, when administered at 18.7 μmol/kg 30 and 120 min following reperfusion, exhibited highly significant protection against neuronal loss, but was inactive at 5.6 μmol/kg. DPMA, a more potent agonist at A1 and A2A receptors, was inactive up to a dose of 19 μmol/kg. Metrifudil (equipotent with CGS 21680 at A2A >25 times more potent at A1) gave a modest degree of protection at 26 μmol/kg and was inactive at 7.8 μmol/kg. CGS 21680 showed an equal degree of hypothermia at 5.6 and 18.7 μmol/kg, suggesting that this was not the prime mode of action. While the effect of metrifudil may be the result of its higher A1 receptor affinity, the mode of action of CGS 21680 has not been established; the data, however, suggest that a non‐A1 non‐A2A receptor mechanism may possibly be involved. Drug Dev. Res. 39:108–114

Contrasting effects of adenosine A1 and A2 receptor ligands in different chemoconvulsive rodent models

The pro- and anticonvulsive properties of selective adenosine A1 and A2 receptor agonists and antagonists were investigated in mice using seizure models involving a specific blockade of adenosine A1 and A2 receptors, modulation of the gamma-aminobutyric acid/benzodiazepine receptor complex or activation with the excitatory amino acid glutamate. The selective adenosine A1 receptor agonists N-cyclopentyladenosine (CPA) and R-N-(phenylisopropyl) adenosine (R-PIA) in doses of 1 and 10 mg/kg i.p. potentiated seizures induced by the selective adenosine A1 receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]-phenyl]- 1,3-dipropylxanthine (XAC). Likewise, the selective adenosine A2 receptor agonists N-[(2-methylphenyl)methyl]adenosine (metrifudil) and N-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA), in doses of 30 and 100 mg/kg i.p., respectively, potentiated seizures induced by the selective adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). In contrast, the adenosine A1 and A2 receptor agonists both antagonized seizures induced by methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM--an inverse agonist at benzodiazepine receptors) and the adenosine A1 receptor agonists also protected against seizures induced by glutamate. Paradoxically, the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) antagonized DMCM- and pentylenetetrazole-induced seizures. Thus, it appears that adenosine A1 and A2 receptor agonists can be both pro- and anticonvulsive depending on the mechanism of action of the chemoconvulsant used in the seizure model. The findings with CPT suggest that other types of adenosine analogues than agonists may possess anticonvulsive properties.

The Chemistry of 2′-Deoxyribo-C-Nucleosides

Abstract The various synthetic approaches to 2′-deoxyribo-C-nucleosides are summarized. These approaches are divided into four groups. Emphasis is placed on the techniques used in determination of anomeric configuration in the products.

The synthesis and biochemical evaluation of new A1 selective adenosine receptor agonists containing 6-hydrazinopurine moieties

Abstract The synthesis and SAR of a series of novel derivatives of N-aminoadenosine is described, along with their in vitro effects in biochemical assays. The rat brain A1 adenosine receptor binding of these compounds is very dependent upon the purine 2-substituent. The novel agonist, 2-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine, exhibits a Li value for A1 receptor binding of

In vivo labeling of the central GABA uptake carrier with 3H-Tiagabine

The in vivo binding of 3H-Tiagabine to the central GABA uptake carrier in mouse brain was characterized. 3H-Tiagabine in vivo bound to a single class of binding sites with a Kd = 72.5 nM and a Bmax = 640 pmol/g tissue. 3H-Tiagabine binding in vivo was regionally distributed within the CNS, and showed a good correlation with 3H-Tiagabine binding in vitro. Pharmacological characterization of 3H-Tiagabine binding in vivo revealed a binding site exhibiting specificity for GABA uptake inhibitors. Experiments examining the in vivo receptor occupancy of the GABA uptake carrier for a series of GABA uptake inhibitors revealed that 20-30% of the GABA uptake sites were occupied at the ED50 for inhibiting DMCM-induced clonic convulsions, while a 50-62% receptor occupancy in vivo was needed to inhibit rotarod performance. These data suggest that 3H-Tiagabine in vivo binding may be a useful method for assessing GABA uptake inhibitor penetration into the CNS, and may be a useful tool for studying the physiological regulation of the GABA uptake carrier.

Anticonvulsant Actions of Novel and Reference Adenosine Agonists

A host of potential mechanisms exist by which manipulation of neurotransmission in the central nervous system (CNS) can lead to inhibition of seizures in mammals. This reflects the complex nature of the diverse disease states that result in the group of symptoms collectively known as epilepsy. The research group at Novo Nordisk has previously discovered agents with clinical anticonvulsant activity, such as Tiagabine (structure 1, Fig. 51–1), an inhibitor of the synaptosomal uptake of gamma-aminobutyric acid [1] and Abecarnil (2, Fig. 51–1), a βcarboline benzodiazepine partial agonist [2], both of which facilitate inhibitory neurotransmission. Antagonists at the excitatory amino acid (EAA) receptor subtypes, such as the AMPA receptor, the metabotropic glutamate receptor [3], and glycine receptors [4] (3, 4 and 5, Fig. 51–1), have also been shown to exhibit anticonvulsant properties in rodent seizure models.