Nullifying drug-induced sensitization: behavioral and electrophysiological evaluations of dopaminergic and serotonergic ligands in methamphetamine-sensitized rats.

Repeated exposure to methamphetamine produces a persistent enhancement of the acute motor effects of the drug, commonly referred to as behavioral sensitization. Behavioral sensitization involves monoaminergic projections to several forebrain nuclei. We recently revealed that the ventral pallidum (VP) may also be involved. In this study, we sought to establish if treatments with antagonists or partial agonists to monoaminergic receptors could "reverse" methamphetamine-induced behavioral and VP neuronal sensitization. Behavioral sensitization was obtained in rats with five once-daily s.c. injections of 2.5mg/kg methamphetamine, an effect that persisted for at least 60 days. After the development of sensitization, 15 once-daily treatments of mirtazapine (a 5-HT(2/3), alpha(2) and H(1) antagonist), SKF38393 (D(1) partial agonist) or SCH23390 (dopamine D(1) antagonist) nullified indices of motor sensitization as assessed by measuring the motoric response to an acute methamphetamine challenge 30 days after the fifth repeated methamphetamine treatment. VP neurons recorded in vivo from methamphetamine-sensitized rats at the 30-day withdrawal time also showed a robust downward shift in the excitatory responses observed to an acute i.v. methamphetamine challenge in non-sensitized rats. This decreased excitatory effect was reversed by mirtazapine, but not by other antagonists that were tested. These data suggest a potential therapeutic benefit for mirtazapine in the treatment of methamphetamine addiction, and point to a possible role for the VP in the sensitization process to methamphetamine.

Therapeutic potential of histamine H3 receptor agonists and antagonists.

The histamine H3 receptor was discovered 15 years ago, and many potent and selective H3 receptor agonists and antagonists have since been developed. Currently, much attention is being focused on the therapeutic potential of H3 receptor ligands. In this review, Rob Leurs, Patrizio Blandina, Clark Tedford and Henk Timmerman describe the available H3 receptor agonists and antagonists and their effects in a variety of pharmacological models in vitro and in vivo. The possible therapeutic applications of the various compounds are discussed.

Design, synthesis, and structure-activity relationships of acetylene-based histamine H3 receptor antagonists.

New, potent, and selective histamine H3 receptor antagonists have been synthesized by employing the use of (1) an appropriately positioned nonpolar acetylene spacer group, (2) either a two-carbon straight chain linker or a conformationally restricting trans-cyclopropane ring between the C-4 position of an imidazole headgroup and the acetylene spacer, and (3) a Topliss operational scheme for side-chain substitution for optimizing the hydrophobic domain. Compounds 9-18 are examples synthesized with the two-carbon straight chain linker, whereas 26-31 are analogues prepared by incorporation of the trans-(+/-)-cyclopropane at the C-4 position of an imidazole headgroup. Synthesis of both the (1R,2R)- and (1S, 2S)-cyclopropyl enantiomers of the most potent racemic compound 31 (Ki = 0.33 +/- 0.13 nM) demonstrated a stereopreference in H3 receptor binding affinity for the (1R,2R) enantiomer 32 (Ki = 0.18 +/- 0.04 nM) versus the (1S,2S) enantiomer 33 (Ki = 5.3 +/- 0.5 nM). (1R,2R)-4-(2-(5,5-Dimethylhex-1-ynyl)cyclopropyl)imidazole (32) is one of the most potent histamine H3 receptor antagonists reported to date.

Effects of selected histamine H3 receptor antagonists on tele-methylhistamine levels in rat cerebral cortex.

The H3 antagonist thioperamide is thought to act on brain H3 autoreceptors to increase both the release and metabolism of neuronal histamine (HA). Our studies investigated the effects of several new brain-penetrating H3 antagonists on rat cerebral cortical levels of the HA metabolite tele-methylhistamine (t-MH). Animals were pretreated with H3 antagonists (0.3 to 30 mg/kg; 1-4 hr; i.p.) in the presence or absence of the monoamine oxidase inhibitor pargyline to prevent metabolism of t-MH. Cortical t-MH levels were measured by both radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS). Pargyline (60 mg/kg; 1 hr; i.p.) produced an approximately 2-fold increase in t-MH levels as measured by either GC-MS or RIA. Thioperamide (+/- pargyline) increased t-MH levels as measured by both GC-MS and RIA. In contrast, neither 5-cyclohexyl-1-(4-imidazol-4-ylpiperidyl)pentan-1-one (GT-2016) (+/- pargyline), 4-(6-cyclohexylhex-cis-3-enyl)imidazole (GT-2227) (+/- pargyline), nor clobenpropit (minus pargyline) increased t-MH levels as measured by GC-MS. A good agreement was found between t-MH levels as determined by either RIA or GC-MS except after treatment with GT-2016, which increased apparent t-MH brain levels according to the former but not the latter method. Subsequent studies suggest the in vivo formation of a GT-2016 metabolite, which can cross-react in the t-MH RIA. Although all H3 receptor antagonists studied to date seem capable of enhancing brain HA release, only thioperamide presently was found to enhance cortical t-MH levels. Thus, H3 receptor antagonists may differentially affect HA release and turnover, and brain t-MH levels may not be reliable predictors of H3 agonist, partial agonist, or antagonist in vivo activity.

Determination of plasma and brain concentrations of SCH 39166 and their correlation to conditioned avoidance behavior in rats.

Plasma and brain concentrations of the dopamine D1 receptor antagonist, SCH 39166, were measured and compared to behavioral activity in the conditioned avoidance response paradigm (CAR). SCH 39166 was administered at two behaviorally active doses (1 mg/kg, SC and 10 mg/kg, PO) and the time course for CAR activity was compared with the plasma and brain concentrations of unconjugated SCH 39166. Conjugation and N-demethylation of SCH 39166 after oral administration were also determined and first pass metabolism examined. Results from these studies demonstrated a similar time-dependent disappearance of unconjugated SCH 39166 from both the plasma and brain, independent of route of administration. Brain concentrations of SCH 39166 were approximately 5-fold higher than corresponding plasma concentrations, regardless of route. However, plasma and brain concentrations of unconjugated SCH 39166 were higher after SC administration of 1.0 mg/kg, than after PO administration of 10 mg/kg, suggesting a substantial first pass metabolism of SCH 39166. In addition, total (conjugated and unconjugated) plasma concentrations of SCH 39166 were at least 10-fold higher than unconjugated concentrations of SCH 39166 after PO administration of 10 mg/kg, demonstrating that a high proportion of drug was conjugated. Metabolism to the N-desmethyl analog, SCH 40853, was observed after PO administration of 10 mg/kg SCH 39166 and a high proportion of conjugation of the desmethyl analog was also seen. Finally, plasma concentrations of unconjugated SCH 39166 exhibited a high positive correlation (r = 0.934, P < 0.001) with brain concentrations of unconjugated SCH 39166.(ABSTRACT TRUNCATED AT 250 WORDS)

Highlights of D1 dopamine receptor antagonist research.

SCH 39166 is now undergoing clinical trials in schizophrenics as a selective D1 dopamine receptor antagonist. It differs from SCH 23390, the prototype D1 receptor antagonist, by having reduced affinity for serotonin receptors and a longer duration of action in primates, as measured in the squirrel monkey conditioned avoidance paradigm. Further studies on this difference in primates indicates that it may be attributable to reduced affinity of SCH 39166 compared to SCH 23390 for the hepatic glucuronosyltransferase system. Their affinities are similar in rats, a species in which both have similar duration of action. These and other studies presented are consistent with the novel profile of SCH 39166.

Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A).

Ethylcholine aziridinium (AF64A) has been proposed as a specific cholinergic neurotoxin. In earlier studies, using AF64A, we reported that slow infusion of 1-2 nmol of this compound into each lateral ventricle of Sprague-Dawley rats resulted in small, and transient decreases in noradrenaline (NA) and serotonin (5-HT) levels in the hippocampus, while inducing a permanent and significant cholinergic hypofunction in the same brain region. The experiments described in this paper were designed to test the hypothesis that such noradrenergic and serotonergic changes after small doses of AF64A are secondary to the changes observed in cholinergic neurons. Levels of NA, and of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were measured concurrently with levels of acetylcholine (ACh), in various brain regions of rats in which the effect of AF64A was attenuated, and in respective control animals. The effect of AF64A was diminished by inhibiting the interaction of AF64A with the high affinity transport site for choline (HAChT). This was achieved using hemicholinium-3 (HC-3), which does not cross the blood-brain barrier, and A-4 (a bis 4-methylpiperidine analog of HC-3), which is centrally active following its peripheral administration. A-4 (20 or 40 mg/kg i.p.) or HC-3 (10 micrograms/ventricle) had no effect on ACh, NA, 5-HT or 5-HIAA levels in saline-treated rats. However, all treatments significantly attenuated the decrease in ACh content produced by AF64A pretreatment. Transient decreases in NA, 5-HT and 5-HIAA contents after AF64A treatment were prevented or reduced by prior treatment with A-4 or HC-3. These results indicate that changes in noradrenergic and serotonergic neurons following AF64A administration are not due to non-specific toxicity of AF64A, but may be the result of adaptation of these neurons to withdrawal of cholinergic input, which would normally inhibit the release of NA and 5-HT. These results also indicate that AF64A can be used to produce specific lesions of hippocampal cholinergic nerve terminals.

Effect of SCH 39166, a novel dopamine D1 receptor antagonist, on [3H]acetylcholine release in rat striatal slices.

SCH 39166 is a novel and selective dopamine D1 receptor antagonist. It has been reported to have potential antipsychotic properties and reduced extrapyramidal side-effect liabilities (EPS). The current studies investigated the pharmacological effects of SCH 39166 on striatal cholinergic function in order to further characterize its dopamine D1 receptor selectivity and to address its EPS liability. Electrically stimulated [3H]acetylcholine (ACh) release from rat striatal slices was measured and comparisons were made between SCH 39166, SCH 23390, (-)-sulpiride, haloperidol or apomorphine on their effect on [3H]ACh release. Results indicated that apomorphine inhibited [3H]ACh release from striatal slices (IC50 = 0.31 microM). (-)-Sulpiride and haloperidol completely reversed the inhibition of [3H]ACh release seen with apomorphine. In contrast, SCH 39166, as well as, SCH 23390 did not reverse the inhibition of [3H]ACh release induced by apomorphine. These findings indicate that dopamine D2 receptors are primarily involved in modulation of [3H]ACh release. Furthermore, selective dopamine D1 receptor antagonists, such as SCH 39166, are ineffective in modulating striatal [3H]ACh release, suggesting that striatal cholinergic hyperactivity and possibly EPS will not be a consequence of dopamine D1 receptor blockade.

Evaluation of 4-methylpiperidine analogs of hemicholinium-3.

A series of substituted piperidine analogs of hemicholinium-3 was evaluated for their ability to inhibit neuromuscular transmission, to decrease acetylcholine content of caudate slices, to inhibit choline acetyltransferase activity, and to produce toxicity. Quaternary and tertiary amine derivatives of 4-methyl- and 4-hydroxyl-substituted piperidine analogs containing beta-carbonyl or beta-hydroxyl substitutions in the phenylethyl spacing moiety were tested. 4-Methyl piperidine derivatives maintained potent hemicholinium-3 like activity. Reduction of activity was seen with the 4-hydroxyl piperidine analogs. Compounds with beta-hydroxyl substitution were more potent than those with beta-carbonyl substitution. The tertiary amine, 4-methyl piperidine derivative with a hydroxyl group on the beta-carbon of the ethyl side chain also possessed hemicholinium-3 like activity. However, tertiary amine analogs were substantially less potent than hemicholinium-3 or their quaternary amine analogs.

High antagonist potency of GT-2227 and GT-2331, new histamine H3 receptor antagonists, in two functional models.

GT-2227 (4-(6-cyclohexylhex-cis-3-enyl)imidazole) and GT-2331 ((1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole) were developed as new potent histamine H3 receptor antagonists. The functional activity of these ligands on the histamine H3 receptor-mediated inhibition of neurogenic contraction of the guinea-pig jejunum and histamine H3 receptor-mediated inhibition of norepinephrine release from guinea-pig heart synaptosomes were investigated. GT-2227 and GT-2331 both antagonized the inhibitory effects of (R)-alpha-methylhistamine on the contraction induced by electrical field stimulation in the guinea-pig jejunum with pA2 values of 7.9+/-0.1 and 8.5+/-0.03, respectively. In addition, GT-2227 and GT-2331 antagonized the inhibition of norepinephrine release in cardiac synaptosomes by GT-2203 ((1R,2R)-trans-2-(1H-imidazol-4-yl)cyclopropylamine), a histamine H3 receptor agonist. The current results demonstrate the antagonist activity for both GT-2227 and GT-2331 in two functional assays for histamine H3 receptors.

The binding of SCH 39166 and SCH 23390 to 5-HT1C receptors in porcine choroid plexus.

SCH 39166 is a novel benzonaphthazepine, which has been characterized as a potent and selective D1 antagonist. Recently, its D1 selective benzazepine predecessor, SCH 23390, has been shown to bind to 5-HT1C binding sites in the choroid plexus. Therefore, the present studies were undertaken to determine if SCH 39166 has any measurable affinity for 5-HT1C binding sites. Our results indicate that SCH 39166 exhibited poor affinity for the 5-HT1C receptor, with a Ki of 1327 nM. In contrast, SCH 23390 inhibited [3H]-mesulergine binding to 5-HT1C receptors with a Ki of 30 nM. The non-selective 5-HT antagonist, methysergide, inhibited binding with a Ki of 2.4 nM. Finally, studies with the stereoisomers of SCH 39166 and SCH 23390 demonstrated that stereoselectivity at the 5-HT1C site is significantly less than for the D1 site.

Characterization of a rat liver glucuronosyltransferase that glucuronidates the selective D1 antagonist, SCH 23390, and other benzazepines.

SCH 23390 is a novel benzazepine that selectively blocks dopamine receptors of the D1 subtype. Glucuronidation of this selective D1 antagonist was studied in vitro using rat liver microsomes. Methods to separate SCH 23390 glucuronide from SCH 23390 were developed which utilized either HPLC techniques or solvent extraction of SCH 23390 with 3-heptanone. Formation of a SCH 23390 glucuronide was confirmed upon incubation of SCH 23390 and UDPGA with naive rat liver microsomes. Liver enzyme activity for SCH 23390 glucuronidation was also enhanced after addition of the detergents, Lubrol or Triton X-100, to the naive liver microsomes. Kinetic analyses indicated an apparent Vmax and Km for UDPGA as 120.9 pmol/mg protein/min and 0.63 mM, and an apparent Vmax and Km for SCH 23390 as 282.4 pmol/mg protein/min and 0.41 microM. Further characterization of the liver enzyme responsible for the glucuronidation of SCH 23390 revealed a stereoselective substrate preference similar to that seen with the D1 dopamine receptor. Substrate inhibition studies indicated that SCH 23390, haloperidol, apomorphine, and alpha-naphthol demonstrated the highest affinity for the glucuronosyltransferase enzyme. However, (-)-sulpiride, raclopride, and endogenous substrates such as dopamine, serotonin, epinephrine, and norepinephrine demonstrated low affinity for the liver enzyme. These studies describe a rat liver glucuronosyltransferase with a unique substrate specificity toward selected dopaminergic agents. Finally, induction profiles revealed that neither phenobarbital (100 mg/kg, ip, for 3 days), beta-naphthoflavone (100 mg/kg, ip, for 4 days), nor 3-methylcholanthrene (80 mg/kg, ip, for 4 days) enhanced liver glucuronosyltransferase activity for SCH 23390 glucuronidation.

Methanol toxicity in the monkey: effects of nitrous oxide and methionine.

Methanol poisoning in monkeys and humans is characterized by the development of formic acidemia, metabolic acidosis and ocular toxicity. Formate, the metabolite associated with the toxicity of methanol, is oxidized to carbon dioxide by a tetrahydrofolate-dependent pathway. Nitrous oxide treatment was used to inhibit the tetrahydrofolate-generating enzyme, 5-methyltetrahydrofolate homocysteine methyltransferase (methionine synthetase, E.C. 2.1.1.13.), to delineate the role of this enzyme in regulating formate oxidation in the monkey. The importance of methionine in the regulation of formate oxidation in the monkey also was evaluated. Nitrous oxide inhibited the oxidation of formate generated from the metabolism of methanol (1 g/kg i.p.) in the monkey, resulting in the development of severe metabolic acidosis and high blood formate levels in these animals compared with air-breathing monkeys administered the same dose of methanol. Treatment of nitrous oxide-exposed monkeys with repetitive doses of methionine (100 mg/kg 10, 12 and 14 hr after methanol) reversed the effects of nitrous oxide on formate oxidation, resulting in a marked decrease in blood formate levels and an increase in the rate of [14C]O2 formation from methanol. Methionine treatment also reversed the development of metabolic acidosis and bicarbonate depletion observed in nitrous oxide-exposed monkeys. These results indicate that hepatic methionine synthetase is important in the regulation of tetrahydrofolate-dependent metabolism in the monkey and that the generation of tetrahydrofolate by this enzyme is a major factor in determining the sensitivity of a species to methanol poisoning.

New acetylene based histamine H3 receptor antagonists derived from the marine natural product verongamine.

New histamine H3 receptor antagonists were developed using an acetylene moiety as a replacement for the amide-oxime functionality of verongamine 5. Optimization of receptor binding was performed by following aliphatic Topliss tree guidelines. These new H3 ligands demonstrate excellent blood-brain barrier penetration.

Hybridization between sika deer (Cervus nippon) and axis deer (Axis axis).

We report an incidence of hybridization from natural mating between sika deer (Cervus nippon) and axis deer (Axis axis). A female exhibiting physical characteristics intermediate between the two species was born on a Tennessee deer farm sometime in 1995. Gel electrophoresis of three blood proteins (TF, HBB, and SOD) from the putative hybrid, the putative sika deer sire and three axis deer hinds from the herd (not necessarily including the dam) initially verified that hybridization had occurred. Q-banded karyotypes further identified the offspring as a hybrid (2n = 67) between sika deer (2n = 68) and axis deer (2n = 66). Fertility of the hybrid remains to be assessed, although it is now of reproductive age.

Catalytic antibodies: a new window on protein chemistry.

Catalytic antibodies have created a new dimension in protein chemistry. In these studies it is particularly valuable to investigate systems for which natural enzymic catalysts are unknown. At Strathclyde we have examined several ways of preparing homochiral building blocks for organic synthesis. Antibodies that catalyse the Diels-Alder reaction have been characterized. The target reaction was the addition of acetoxybutadiene to N-substituted maleimides, a reaction that should give a pentafunctional homochiral building block. Catalytic antibodies can give insight into the mechanism of catalysis by proteins. We have investigated an adventitious hydrolytic antibody that cleaves activated esters. We have also shown that an antibody raised to ampicillin for analytical purposes catalyses hydrolysis of the beta-lactam ring.

Optical isomers of some piperidine-derived hemicholinium congeners containing secondary alcohol groups: preparation and biological activities.

Previous publications on the title compounds, 1 and 2 ('A-4' and 'A-5'), reported pharmacological data on what were probably mixtures of optical isomers of unknown composition. The compounds can exist as an enantiomeric pair as well as a diastereomeric meso isomer. In the present work, all possible stereoisomers (meso and dextrorotatory and levorotatory isomers) of the piperidine derivatives 'A-4' and 'A-5' have been isolated, identified, and characterized, and pharmacological data have been obtained on all products. In all of the bioassays conducted, optical isomers of each structure exhibited similar qualitative and quantitative effects. We conclude that the stereochemical nature of the chiral centers bearing secondary alcohol groups in these molecules does not play a critical role in interactions with in vivo receptors, and therefore that the alcohol groups in 'A-4' and 'A-5' are not prime sites of interaction(s) with receptor(s).

Alterations in acetylcholine metabolism in rat striatal slices by a 4-methyl piperidine analog of hemicholinium-3.

The in vitro effects of a tertiary amine, 4-methyl piperidine analog of hemicholinium-3 (A-4), were investigated on acetylcholine (ACh) metabolism in rat striatal slices. Rat striatal slices were incubated in the presence of 1.0 microM [3H]-choline in the presence or absence of 0.1 mM A-4. High pressure liquid chromatography with electrochemical detection was utilized to separate and measure total and [3H]-labeled ACh and choline. The effects of A-4 on [3H]-choline uptake, ACh and choline content, ACh release, and specific activity of ACh and choline tissue pools were investigated. Results indicated that A-4 inhibited the uptake of [3H]-choline into the striatal slices. Addition of 0.1 mM A-4 also produced a significant reduction in ACh content and ACh release and reduced the specific activity of the tissue choline and ACh pools. The effects of A-4 were more prominent upon immediate incubation than after a 60-min preincubation. These studies demonstrate that the pharmacological effects exhibited by A-4 are consistent with inhibition of choline uptake, with subsequent reduction in ACh synthesis and release.

Stereoisomers of quaternary and tertiary 4-methyl piperidine analogs of hemicholinium-3.

Previous studies have shown that quaternary and tertiary 4-methyl piperidine derivatives of hemicholinium-3 (A-5 and A-4, respectively) are potent inhibitors of choline uptake. The d-, l-, and mesostereoisomers of A-5 and A-4 were separated and the potency and reversibility were compared. Isomeric forms of each compound were found to be approximately equipotent inhibitors in the following preparations: inhibition of rabbit neuromuscular transmission using the sciatic nerve-gastrocnemius muscle preparation, reductions in acetylcholine content in rat caudate tissue slices and inhibition of choline uptake in neuroblastoma cells, line NB41A3. Because these results show no difference in potency or reversibility for the stereoisomers of A-5 or A-4, these studies indicate that hydroxyl substitutions in these agents do not play a role in their biologic activity. Perhaps only 2-point attachment is required for inhibition of choline transport by hemicholinium-like compounds.

A-4, a bis tertiary amine derivative of hemicholinium-3 produces in vivo reduction of acetylcholine in rat brain regions.

The pharmacological effects of A-4, a bis 4-methylpiperidine tertiary amine derivative of hemicholinium-3, were investigated. Systemic administration (i.p.) of this compound produced a dose-dependent reduction in acetylcholine content of several brain regions. A dose of 40 mg/kg of A-4 produced a 40% reduction in acetylcholine content in the corpus striatum and this reduction was maintained for over 4 hr. Increased choline content was found concurrent with the reduction in acetylcholine content. Reversal of the A-4-induced reductions in acetylcholine content was seen with eserine and oxotremorine but not with choline chloride. Acute treatment with A-4 appeared selective for cholinergic neurons as no significant changes were seen in norepinephrine or serotonin parameters of any of the brain regions assayed. Dopamine turnover was increased in the striatum. The neurochemical changes produced by A-4 were not seen with the bis 4-methylpiperidine quaternary amine, A-5, or hemicholinium-3 after systemic administration of doses which produced pronounced behavioral and toxic effects. Intraventricular administration of A-5 or hemicholinium-3 produced a selective reduction in acetylcholine content. No changes were seen in dopamine, norepinephrine or serotonin parameters. Thus, A-4 represents a novel hemicholinium-3 analog in its ability to act centrally after systemic administration and may be potentially useful as a pharmacological tool in understanding cholinergic mechanisms in the central nervous system.