Gary A. Koppel

Isolation and measurement of the endogenous cannabinoid receptor agonist, anandamide, in brain and peripheral tissues of human and rat

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.

Isolation of three separate anaphylatoxins from complement-activated human serum

Recent methodologies used in preparing anaphylatoxins from complement-activated serum are described. Activation of the alternative pathway generates C3a and C5a; however, activation of the classical pathway is required to generate the anaphylatoxin from C4. This article describes an activation scheme that simultaneously generates all three of the anaphylatoxins (e.g., C3a, C4a and C5a) in human serum and outlines a procedure for isolating each as homogeneous products. Purification of intact anaphylatoxins directly from complement-activated serum takes place only if an exopeptidase in serum, known as carboxypeptidase N (SCPN), is properly inhibited. A new series of mercapto derivatives of arginine analogs are introduced as potent and effective inhibitors of SCPN. These inhibitors permit normal complement activation but prevent degradation of the released activation fragments C3a, C4a or C5a. The SCPN inhibitor previously used was 6-aminohexanoic acid (EACA), but it required a 1 M concentration for effective inhibition, the substituted mercapto-guanido compounds prove to be effective in the mM range.

Orally active vasopressin V1a receptor antagonist, SRX251, selectively blocks aggressive behavior

Arginine vasopressin functions as a neurochemical signal in the brain to affect social behavior. There is an expanding literature from animal and human studies showing that vasopressin, through the vasopressin 1A receptor (V1A), can stimulate aggressive behavior. Using a novel monocylic beta lactam platform, a series of orally active vasopressin V1a antagonists was developed with high affinity for the human receptor. SRX251 was chosen from this series of V1a antagonists to screen for effects on serenic activity in a resident-intruder model of offensive aggression. Resident, male Syrian golden hamsters were given oral doses of SRX251 or intraperitoneal Manning compound, a selective V1a receptor antagonist with reduced brain penetrance, at doses of 0.2 microg, 20 microg, 2 mg/kg or vehicle. When tested 90-120 min later, SRX251, but not Manning compound, caused a significant dose-dependent reduction in offensive aggression toward intruders as measured by latency to bite and number of bites. The reduction in aggression persisted for over 6 h and was no longer present 12 h post treatment. SRX251 did not alter the amount of time the resident investigated the intruder, olfactory communication, general motor activity, or sexual motivation. These data corroborate previous studies showing a role for vasopressin neurotransmission in aggression and suggest that V1a receptor antagonists may be used to treat interpersonal violence co-occurring with such illness as ADHD, autism, bipolar disorder, and substance abuse.

Synthesis and structure-activity relationships of benzophenones as inhibitors of cathepsin D

Abstract Non peptide inhibitors of cathepsin D, an aspartyl protease that has been implicated in many disease states including Alzheimers disease, were prepared and evaluated. The most potent inhibitor of cathepsin D in this series was found to be (Z)-5-[[4-(4-benzoyl-3-hydroxy-2-propylphenoxy) methylphenyl]methylene]-2-thioxo-4-thiazolidinone ( 3f , IC 50 = 210 nM).

Vasopressin antagonists as anxiolytics and antidepressants: recent developments.

A compelling case for the potential utility of vasopressin (AVP) antagonists as a novel therapeutic class for the treatment of stress-related affective illness has emerged based on observations in depressed individuals, findings in animal models of anxiety and depression, and an understanding of changes in hypothalamic-pituitary-adrenal (HPA) axis regulation under chronic stress. The scientific bases for vasopressin antagonists as a pharmacotherapy for anxiety and depression include: 1) the neuroadaptation and dysregulation of HPA function that accompanies chronic stress in affected humans and in animal models of anxiety and depression, 2) recognition that AVP, not corticotrophin releasing factor (CRF), drives HPA function associated with chronic psychological stress, 3) the CNS localization of vasopressin V1a and V1b receptors in limbic system regions involved in HPA regulation and control of social behaviors, and 4) preclinical data showing efficacy in animal models employed as screens for anxiolytic and antidepressant activity. The public health need for new pharmaceutical treatments for stress-related affective illness is well documented. In the United States alone, anxiety and depression affect some 40 million people each year and carry a conservatively estimated annual total economic burden of at least

Clavulanic acid: A competitive inhibitor of beta-lactamases with novel anxiolytic-like activity and minimal side effects

125 billion. Existing pharmacotherapies for both indications are not uniformly effective and frequently have undesirable side effects. These limitations demonstrate that a new treatment approach through vasopressin receptor antagonism in the CNS may offer significant opportunities for improved outcomes. In this review, the development of compounds in this class since 2005 is considered. The most advanced clinical candidates and newer compounds described in recent patents are presented.

Rapid parallel synthesis applied to the optimization of a series of potent nonpeptide neuropeptide Y-1 receptor antagonists

Clavulanic acid is a member of the beta lactam family of antibiotics with little or no intrinsic antibacterial activity of its own; instead, it is used to enhance the activity of antibiotics by blocking bacterial beta-lactamases. Because clavulanic acid by itself is very safe, orally active and shows good brain penetrance, we sought to determine if it had any potential as a psychotherapeutic. Clavulanic acid was a tested across three mammalian species, hamsters, rats and cotton-top tamarin monkeys in a series of behavioral assays designed to screen for anxiolytic activity. In addition, several studies were done in rodents to compare the behavioral profile of clavulanic acid to the commonly prescribed benzodiazepines, particularly with respect to their unwanted side effects of motor depression, amnesia and neuroendocrine dysregulation. Our findings show that clavulanic acid is a highly potent anxiolytic in rodents without altering motor activity in the open field test, normal learning and memory in the Morris water maze, or normal stress hormone release. Orally administered clavulanic acid significantly reduces measures of anxiety in male/female pairs of cotton-top tamarins. In addition, male tamarins showed a highly significant increase in sexual arousal as measured by the number of penile erections. The fact clavulanic acid has anxiolytic activity in the tamarin holds the promise that this drug may be an effective therapeutic for the treatment of anxiety disorders in humans.

A novel class of 5-HT2a receptor antagonists: Aryl aminoguanidines

Abstract This study describes the integrated application of parallel synthesis and computational chemistry to the design of potent nonpeptide antagonists for the neuropeptide Y-1 (NPY1) receptor. A lead molecule was modeled in the active site of the NPY1 receptor, and a potentially fruitful region for analog construction was identified. Synthesis of suitable scaffolds followed by solution phase generation of a small library of analogs produced a compound with 5-fold improvement in binding over the already potent lead. This new compound was shown to be an unanticipated side product of the parallel synthesis reaction.

Use of peptide combinatorial libraries in drug design: the identification of a potent serotonin reuptake inhibitor derived from a tripeptide cassette library

Local delivery of serotonin (5-HT) produces a rapid edematous response in soft tissues via increased fluid extravasation which is prevented by 5-HT2 antagonists such as ketanserin or mianserin. Here we report the effects of a new class of aminoguanidine 5-HT2 antagonists, with relative selectivity for 5-HT2A receptors which are potent inhibitors of 5-HT-induced paw edema in the rat. Radioligand binding studies with 125I DOI on human 5-HT2A and 5-HT2C receptors and with 3H-5-HT on human 5-HT2B receptors demonstrated that, LY314228, and LY320954 displayed some selectivity for the 5-HT2A receptor. When compared to binding at other 5-HT2 receptor subtypes, LY314228 had an 18.6-fold greater affinity for the 5-HT2A site over the 5-HT2B site, and 2.6 fold greater at the 5-HT2C site. LY320954 displayed similar preference for 5-HT2A sites. Both compounds also inhibited 5-HT-induced paw swelling in rats, with ED50s of 6.4 and 4.8 mg/kg (for LY314228 and LY320954, respectively). These studies offer evidence for a novel class of pharmacophores for the 5-HT2 receptor family which show greater relative affinities for the 5-HT2A receptor subclass.

Schultz-Dale reaction in mouse trachea.

BACKGROUND Medicinal chemistry traditionally requires the identification of biologically active molecules by synthesizing and screening each purified substrate. Further progress in drug discovery then requires definition of the structure-activity relationship of the lead compound. More recently, combinatorial chemistry has emerged as a way to examine structure-activity relationships by screening a large mixture of compounds synthesized in a predictably random manner, without the labor-intensive costs of molecular isolation and purification. We set out to use this approach to examine the structural requirements for peptide binding to serotonin and dopamine transporters. RESULTS We screened a tripeptide cassette library for serotonin and dopamine reuptake inhibition using cloned transporter assay systems. The method has afforded a number of tripeptide pharmacophores with inhibitory IC50 values ranging from 10 microM to < 1 microM in the dopamine and serotonin reuptake systems. The conformation of one of these tripeptides, N-acetyl-D-Trp-L-Phe-D-Lys-CONH2 (which inhibits serotonin uptake with an IC50 of 10 microM) was compared to that of the serotonin uptake inhibitor s-fluoxetine, and was shown to be more similar in conformation to fluoxetine than was an analogous tripeptide containing L-Lys (IC50 > 50 microM). CONCLUSIONS We have identified five tripeptides with inhibitory IC50 values of < 10 microM in the serotonin reuptake system. One tripeptide was predicted to have pharmacophore features similar to that of fluoxetine, a selective and potent non-peptide serotonin reuptake inhibitor. Our results suggest that tripeptides derived from combinatorial libraries will help to define the important structural elements of pharmacophores.