Synthesis and Myorelaxant Activity of 1,3-Bis(5-ammoniopentyl)-6-methyluracil Dihalides

A series of 1,3-bis(5-ammoniopentyl)-6-methyluracil dihalides with the anticholinesterase type of activity have been synthesized and characterized with respect to toxicity and myorelaxant activity. The obtained compounds exhibit high toxicity with respect to mice and belong to the class of low/practically zero toxicity with respect to daphnia. Under conditions of functional loading (treadmill running test) in mice, the compounds with small-volume electron-acceptor substitutes (F, NO₂, CN, CH₂ OCH₃, C(O)OCH₃ etc.) at the quaternary nitrogen atoms in the benzyl radical are more effective (upon intraperitoneal injection) and safer than the reference drugs (proserine and BW284c51) and induce the development of a clearly pronounced myorelaxant effect with a duration of no less than 1 day with ED₅₀ = 0.04 – 0.09 μ-M/kg and LD₅₀/ED₅₀ up to 56.25. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 4, pp. 13 – 15, April, 2005.     

Synthesis and Myorelaxant Activity of 1,3-Bis-(5-ammoniopentyl)-6-methyluracil Dibromides

Some representatives of 1,3-bis-(5-ammoniopentyl)uracil dibromides with the anticholinesterase type of activity are classified as highly/moderately toxic in mice and as slightly toxic/practically nontoxic in daphnia. Under the conditions of functional testing (treadmill running test in mice, i.p.), compounds with less bulky substituents (H, F, Br, CH₃, CN, NO₂, and CH₃O groups) at positions 5 and 6 of the uracil cycle are more effective and safer than proserine and BW284c51: (a) they induce the development of a clearly pronounced myorelaxant effect lasting over not less than one day (ED₅₀ = 0.03 – 0.11 μM/kg), (b) the ratio LD₅₀/ED₅₀ is below 76.67. An increase in length of the aliphatic radical in position 5 of the uracil cycle leads to a gradual increase in the toxicity with respect to daphnia, while the toxicity with respect to mice exhibits a decrease. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 6, pp. 12 – 14, June, 2005.     

Comparison of the effects of calmidazolium, morphine and bupivacaine on N-methyl-d-aspartate- and septide-induced nociceptive behaviour

We have recently shown that spinal calmodulin inhibitors (W-7 and calmidazolium) dose-dependently inhibit the nociceptive reaction (biting, scratching, licking, BSL) evoked by intrathecal N-methyl-d-aspartate (NMDA) and septide, an agonist of the neurokinin (NK) NK₁ receptor. To compare this effect with that induced by standard analgesics, we now report a study of the effects of calmidazolium (14–420 nmol), bupivacaine (29–582 nmol) and morphine (26–260 nmol) when coadministered intrathecally with either NMDA (4 μg) or septide (0.5 μg). Calmidazolium had the highest potency for inhibiting septide-induced nociceptive behaviour, acting over a dose range of 34–130 nmol (dose eliciting a half-maximal response, ED₅₀, 67 nmol) lower than that of bupivacaine [ED₅₀ 234 (115–475) nmol]. Only the highest dose of morphine (260 nmol) inhibited septide-evoked BSL [ED₅₀=133 (69–255) nmol]. Higher doses of morphine could not be tested due to the appearance of an excitatory aversive reaction. Both calmidazolium [ED₅₀=232 (138–388) nmol] and bupivacaine [ED₅₀=123 (59–256) nmol] dose-dependently reduced NMDA-induced BSL reaching an almost maximal inhibition at the highest doses assayed (420 and 291 nmol, respectively). In contrast, morphine had less effect on NMDA-induced behaviour, inducing only a partial reduction of BSL even with the highest dose assayed (260 nmol). Overall, it can be concluded that the calmodulin inhibitor calmidazolium inhibits septide- and NMDA-evoked nociceptive behaviour with a potency and efficacy at least as high as those of morphine and bupivacaine. Received: 1 April 1998 / Accepted: 4 September 1998     

Synthesis and analgesic and antiinflammatory properties of new benzodiazepine derivatives

Several new N-(2,3-dihydro-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-2-carboxamides have been synthesized and characterized with respect to acute toxicity (LD₅₀), analgesic and antiinflammatory properties (formalin-and carrageenan-induced foot edema models), and interaction with morphine-induced antinociception in mice. The new compounds were prepared by acyl coupling of 2-aminobenzophenones with α-(benzotriazol-1-yl)-N-acylglycines followed by displacement of the benzotriazole ring with ammonia and cyclization of the resulting monoacyl aminals. The LD₅₀ of the synthesized compounds exceeds 1000 mg/kg. Three compounds produced significant analgesic action in doses 100–150 μg/kg in the early (painful) phase of the formalin test and potentiated the morphine-induced antinociception in this test. The synthesized drugs neither showed antinociception in the second (inflammatory) phase of the formalin test nor decreased the carrageenan-induced foot edema growth. Thus, the synthesized compounds produce analgesic action but do not possess antiinflammatory properties. The analgesic activity is probably due to the interaction with μ-and δ-opioid receptors. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 12, pp. 21–23, December, 2005.     

Endothelium-derived hyperpolarizing factor, but not nitric oxide or prostacyclin release, is resistant to menadione-induced oxidative stress in the bovine coronary artery

The interaction of superoxide anions (O₂ ^–) generated by menadione with the synthesis and/or action of nitric oxide (NO), prostacyclin (PGI₂) and endothelium-derived hyperpolarizing factor (EDHF) was investigated in segments of the left anterior descending coronary artery (LAD) isolated from bovine hearts. EDHF and NO release were monitored by superfusion bioassay in segments pre-constricted with the thromboxane mimetic, U46619, PGI₂ release in addition by enzyme immunoassay for 6-keto-prostaglandin F_1α, and generation of O₂ ^– by lucigenin-enhanced chemiluminescence. Bradykinin (1–1,000 pmol) elicited a prominent, endothelium-dependent, relaxant response, 50–60% of which was insensitive to combined blockade of cyclooxygenase with diclofenac (1 μM) and NO synthase with N ^G-nitro-l-arginine (50 μM). This diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response was completely abrogated in the presence of tetrabutylammonium (3 mM), a non-selective inhibitor of Ca²⁺-dependent K⁺ channels, or when the segments were pre-constricted with potassium chloride (60 mM) instead of U46619, and thus most likely mediated by EDHF. Despite causing a two- to fourfold increase in the concentration of O₂ ^– in or at the vessel wall, menadione (30 μM) did not affect the diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response to bradykinin. When administered in the absence of N ^G-nitro-l-arginine, however, menadione significantly inhibited the relaxant response to bradykinin, presumably by attenuating the NO-mediated relaxation. Menadione also abolished the bradykinin-stimulated release of PGI₂ from luminally perfused segments of the LAD. This effect was more pronounced in the absence of N ^G-nitro-l-arginine, indicating that PGI₂ release in this preparation may be more sensitive to inhibition by peroxynitrite, the reaction product of NO and O₂ ^–, than to O₂ ^– alone. These findings suggest that, in contrast to NO and PGI₂, the release, and presumably also the mechanism of action, of EDHF in the bovine LAD is resistant to an increase in the local concentration of O₂ ^– or peroxynitrite which is thought to play an important role in coronary heart disease. Received: 27 August 1998 / Accepted: 18 November 1998     

Eukaliuric natriuresis and diuresis in response to disprocynium24: studies on the tubular site of action

We previously described that 1,1’-diisopropyl-2,4’-cyanine (disprocynium24, DP24) exerts an eukaliuric diuresis and natriuresis in the anesthetized rat. The purpose of the present study was to localize the tubular site of action of DP24. Employing micropuncture experiments in anesthetized rats, we first tested the effect of systemic application of DP24 (300 μg/kg+300 μg/kg h, i.v.) on whole kidney excretion rates as well as on fluid, sodium and potassium ion delivery to the early distal tubule (V_ED, Na⁺ _ED, K⁺ _ED). It was found that the eukaliuric diuresis and natriuresis in response to DP24 was accompanied by a substantial increase in V_ED and Na⁺ _ED, suggesting a predominant tubular site of action upstream to the early distal tubule, most likely in the proximal tubule. DP24 caused a comparable fractional, although minor absolute increase in K⁺ _ED as compared to Na⁺ _ED . Second, application of DP24 into the first surface loop of the proximal tubule significantly increased V_ED and Na⁺ _ED at a concentration of about 10^–7 M, indicating that DP24 may act from the intratubular site. Third, microperfusion of tubular segments revealed that effects of DP24 on the proximal convoluted tubule and the loop of Henle accounted for about 70 and 30%, respectively, of its diuretic and natriuretic action upstream to the early distal tubule. With regard to the loop of Henle, the quantitative effect of DP24 on fluid and Na⁺ reabsorption proposed a predominant effect on the straight part of the proximal tubule rather than the thick ascending limb. Intratubular DP24 did not affect reabsorption in the distal tubule. In summary, the present findings indicate that: (1) the diuretic and natriuretic effect of DP24 resides predominantly in the proximal tubule, and (2) DP24 may act from the intratubular site. Since DP24 increased V_ED and Na⁺ _ED without apparently affecting sodium or potassium ion transport in the distal tubule, the mechanism of the eukaliuric response remains unclear. Received: 13 February 1998 / Accepted: 11 May 1998     

Radiorecovery and prophylactic-treatment efficacies of manganese(III)2(II)(μ3-O)(μ-3,5-diisopropylsalicylate)6 in γ-irradiated mice

Survival and changes in body mass of whole-body irradiated mice were determined to examine the radiorecovery and prophylactic-treatment efficacies of manganese(lIl)₂(ll)(μ₃-O)(μ-3,5-diisopropylsali-cylate)₆(Mn₃(O)(3,5-DIPS)₆). To determine the radiorecovery efficacy, four groups of 25 C57BL/6 female mice were LD_50/30 γ-irradiated and treated with 0, 30,40 or 50 umol Mn₃(O)(3,5-DIPS)₆/kg of body mass 3 h after irradiation. Survival increased from 28% in the vehicle-treated control group to 84% (p ≤0.00007), 92% (p ≤ 0.00005), or 88% (p ≤ 0.00002) in the 30, 40 or 50 μmol/kg-treated groups, respectively. Postirradiation treatment with Mn₃(O)(3,5-DIPS)₆ enabled rapid recovery of radiation-induced loss of body mass and an overall elevated body mass when compared with the vehicle-treated control mice. To determine the combined prophylactic/therapeutic activity of this complex, four groups of 25 C57BL/6 female mice were treated with 50 umol Mn₃(O)(3,5-DIPS)₆/kg of body mass 3 h prior to LD100/30 γ-irradiation and then treated with 0,10,20 or 40 umol Mn₃(O)(3,5-DIPS)₆/kg on days 2, 4 and 6 after irradiation. Survival rates were increased to 28% (p ≤ 0.006), 12% or 16% in the 10, 20 or 40 μmol/kg postirradiation-treated mice compared with no survival in control animals. Body mass determinations revealed that mice treated with the 10 μmol/kg dose recovered from the radiation-induced loss of body mass at a more rapid pace than all other mice. These results support the hypothesis that Mn₃(O)(3,5-DIPS)₆ is an effective radiorecovery/radioprotective agent and that it is effective when given in a combined prophylactic/therapeutic regimen to LD_100/30 whole-body-irradiated mice.     

Dopamine transporter binding without cocaine-like behavioral effects: synthesis and evaluation of benztropine analogs alone and in combination with cocaine in rodents

Rationale: Previous SAR studies demonstrated that small halogen substitutions on the diphenylether system of benztropine (BZT), such as a para-Cl group, retained high affinity at the cocaine binding site on the dopamine transporter. Despite this high affinity, the compounds generally had behavioral effects different from those of cocaine. However, compounds with meta-Cl substitutions had effects more similar to those of cocaine. Objectives: A series of phenyl-ring analogs of benztropine (BZT) substituted with 3′-, 4′-, 3′,4′′- and 4′,4′′-position Cl-groups were synthesized and their pharmacology was evaluated in order to assess more fully the contributions to pharmacological activity of substituents in these positions. Methods: Compounds were synthesized and their pharmacological activity was assessed by examining radioligand binding and behavioral techniques. Results: All of the compounds displaced [³H]WIN 35,428 binding with affinities ranging from 20 to 32.5 nM. Affinities at norepinephrine ([³H]nisoxetine) and serotonin ([³H]citalopram) transporters, respectively, ranged from 259 to 5120 and 451 to 2980 nM. Each of the compounds also inhibited [³H]pirenzepine binding to muscarinic M₁ receptors, with affinities ranging from 0.98 to 47.9 nM. Cocaine and the BZT analogs produced dose-related increases in locomotor activity in mice. However, maximal effects of the BZT analogs were uniformly less than those produced by cocaine, and were obtained 2–3 h after injection compared to the relatively rapid onset (within 30 min) of cocaine effects. In rats trained to discriminate IP saline from 29 μmol/kg cocaine (10 mg/kg), cocaine produced a dose-related increase in responding on the cocaine lever, reaching 100% at the training dose; however, none of the BZT analogs fully substituted for cocaine, with maximum cocaine responding from 20 to 69%. Despite their reduced efficacy compared to cocaine in cocaine discrimination, none of the analogs antagonized the effects of cocaine. As has been reported previously for 4′-Cl-BZT, the cocaine discriminative-stimulus effects were shifted leftward by co-administration of the present BZT analogs. Conclusions: The present results indicate that although the BZT analogs bind with relatively high affinity and selectivity at the dopamine transporter, their behavioral profile is distinct from that of cocaine. The present results suggest that analogs of BZT may be useful as treatments for cocaine abuse in situations in which an agonist treatment is indicated. These compounds possess features such as reduced efficacy compared to cocaine and a long duration of action that may render them particularly useful leads for the development of therapeutics for cocaine abusers. Electronic Publication     

Locomotor response to novelty predicts a rat's propensity to self-administer nicotine

Rationale: Zolmitriptan is an anti-migraine agent with action at 5-HT_1B/D receptors. It penetrates into the central nervous system and, like other 5-HT_1B/D agonists, its pharmacotherapeutic profile may include significant anti-aggressive effects. Objectives: To examine whether zolmitriptan has potential anti-aggressive effects by studying two kinds of aggressive behavior in mice – species-typical and aggression under the influence of alcohol. A second objective was to study whether pre- or post-synaptic receptors mediate these anti-aggressive effects. Methods: Initially, the anti-aggressive effects of zolmitriptan were studied in male CFW mice during 5-min resident–intruder confrontations. To confirm the 5-HT_1B receptor as a critical site of action for the anti-aggressive effects, the zolmitriptan dose–effect determinations were repeated after pretreatment with GR 127935 (10 mg/kg, i.p.). In further experiments, mice were treated concurrently with alcohol (1.0 g/kg, p.o.) and zolmitriptan (1–30 mg/kg, i.p.) in order to compare the effects of this agonist on species-typical and alcohol-heightened aggression. Finally, mice were infused with the neurotoxin 5,7-DHT (10 μg) into the raphé area to eliminate somatodendritic and presynaptic autoreceptors. The anti-aggressive effects of zolmitriptan (17 mg/kg, i.p.) or CP-94,253 (10 mg/kg, i.p.) were assessed 10 days after the lesion, and levels of 5-HT and 5-HIAA were measured in the hippocampus and prefrontal cortex. Results: Zolmitriptan exerted behaviorally specific anti-aggressive effects. The reduction in aggression was antagonized by GR 127935, indicated by a rightward shift in the dose–effect curves of zolmitriptan, showing the specificity for the 5-HT_1B receptors. Zolmitriptan also decreased alcohol-heightened aggression with equal efficacy. The anti-aggressive effects of CP-94,253 and zolmitriptan remained unaltered by 5,7-DHT lesions that depleted cortical and hippocampal 5-HT by 60–80%. Conclusions: Zolmitriptan proved to be an effective and behaviorally specific anti-aggressive agent in situations that engender moderate and alcohol-heightened levels of aggression. These effects are potentially due to activation of post-synaptic 5-HT_1B/D receptors. Electronic Publication     

Modulation of the hyperpolarization-activated inward current (If) by antiarrhythmic agents in isolated human atrial myocytes

The hyperpolarization-activated inward current (I _f) has been discussed to contribute to arrhythmias in human atrial myocardium. I _f was found to be increased by β-adrenergic stimulation. In the present study, we evaluate the modulation of I _f by carbachol, adenosine and by class Ic, III and IV antiarrhythmic drugs in isolated human atrial myocytes. The whole-cell patch-clamp technique was used to record I _f in isolated myocytes from 18 human right atrial appendages. A typical time- and voltage-dependent hyperpolarization-activated inward current could be recorded in all cells investigated (n=56). Mean current density recorded at –130 mV was –2.8±1.2 pApF^–1. Both adenosine and carbachol were found to directly inhibit I _f in human atrial myocytes by shifting the activation curves to more negative potentials. Adenosine 10^–5 mol/l shifted the potential of half-maximal activation by –5.9±0.4 mV from –99.4±0.6 mV to –105.3±0.4 mV (n=8; P<0.05), and carbachol 10^–5 mol/l by –5.7±0.5 mV from –99.2±0.5 mV to –104.9±0.6 mV (n=6; P<0.05). The concentration-response curve of adenosine calculated by a Hill function yielded a half-maximal effect of adenosine (EC₅₀) at a concentration of 3.6±0.5 μmol/l, a maximal shift of –6.5±0.3 mV, and a Hill coefficient (h) of 2.40. We did not observe any effect of flecainide (10^–5 mol/l; n=8), sotalol (10^–5 mol/l; n=6), amiodarone (10^–5 mol/l; n=6) or verapamil (10^–5 mol/l; n=5) on I _f in human atrial myocytes. However, propafenone (10^–5 mol/l) was found to reversibly reduce I _f current size (9/13 cells) by shifting the activation curve by –5.2±0.4 mV (P<0.05). In human atria adenosine- and muscarinic receptor stimulation might function as endogenous protective mechanisms inhibiting the initiation of ectopic tachycardia by reducing I _f current size. Propafenone may be more effective in some patients with atrial tachycardias that do not respond to other class Ic, III and IV antiarrhythmic drugs. However, it has yet to be defined whether these agents suppress atrial tachycardias via an inhibition of I _f in vivo. Received: 27 May 1998 / Accepted: 15 September 1998     

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels

In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II–III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95% O₂–5% CO₂ with 95% N₂–5% CO₂) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K_ATP) channels, diazoxide (3–300 μM). The effect of 30 but not 300 μM diazoxide was reversed by washout. Tolbutamide (300 μM), an antagonist of K_ATP channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 μM) on the HD. Neither diazoxide (3–300 μM) nor tolbutamide (300 μM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of –90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 μM) or tolbutamide (300 μM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between –130 and –50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 μM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri- to diphosphate ratios progressively declined for ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxia was increased from 5 to 30 min. Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassium current failed to indicate an allosteric regulation of a plasmalemmal K_ATP channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in combination with a hypoxic stimulus; it is suggested that both plasmalemmal and mitochondrial K_ATP channels are involved under these conditions. The hypoxic depolarization may be due to blockade of K⁺,Na⁺-ATPase by limitation of energy supplying substrate. Received: 3 June 1998 / Accepted: 20 July 1998     

Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site

Effects of the intravenous anaesthetic ketamine on the desipramine-sensitive noradrenaline transporter (NAT) were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NAT (bNAT). Incubation (1–3 h) of adrenal medullary cells with ketamine (10–300 μM) caused an increase in appearance of catecholamines in culture medium. Ketamine (10–1000 μM) inhibited desipramine-sensitive uptake of [³H] _{noradrenaline} (NA) (IC₅₀=97 μM). Saturation analysis showed that ketamine reduced V _max of [³H]NA uptake without changing K _m, indicating a non-competitive inhibition. Other inhibitors of NAT, namely cocaine and desipramine, showed a competitive inhibition of [³H]NA uptake while a derivative of ketamine, phencyclidine, showed a mixed type of inhibition. Ketamine (10–1000 μM) also inhibited the specific binding of [³H]desipramine to plasma membranes isolated from bovine adrenal medulla. Scatchard analysis of [³H]desipramine binding revealed that ketamine increased K _d without altering B _max, indicating a competitive inhibition. In transfected Xenopus oocytes expressing the bNAT, ketamine attenuated [³H]NA uptake with a kinetic characteristic similar to that of cultured adrenal medullary cells. These findings are compatible with the idea that ketamine non-competitively inhibits the transport of NA by interacting with a site which partly overlaps the desipramine binding site on the NAT. Received: 18 December 1997 / Accepted: 17 June 1998     

Celiprolol: agonist and antagonist effects at cardiac β1- and vascular β2-adrenoceptors determined under in vivo conditions in the rat

Celiprolol is a β-adrenoceptor antagonist which has desirable ancillary properties since it is relatively cardioselective and can exert direct vasodilator and bronchodilator effects. Here agonist and antagonist effects of celiprolol at cardiac β₁- and vascular β₂-adrenoceptors were determined under in vivo conditions in the rat. All experiments were carried out in catecholamine-depleted, pentobarbital anesthetized and vagotomized rats, placed under artificial respiration. I.v. administrations were madevia the femoral vein. Blood pressure was measured from the cannulated right carotid artery and heart rate was recorded with a cardiotachometer. Celiprolol (10 μg/kg to 1 mg/kg i.v.) produced dose-related increases in heart rate and decreases in mean carotid artery blood pressure which were of longer duration than those mediated by standard agonists of β₁-(isoprenaline) or β₂-(salbutamol) adrenoceptors respectively. Although the maximal increase in heart rate by celiprolol (110±4 beats/min, n=7) was approximately half that of isoprenaline (198±1 beats/min, n=5), isoprenaline acted at doses 200-fold lower than celiprolol. Betaxolol (0.03-0.3 mg/kg i.v.), a β₁-adrenoceptor antagonist, inhibited strongly and with similar potency the tachycardic effects of celiprolol (DR₁₀ = 45 μg/kg i.v.) as well as isoprenaline (DR₁₀ = 45 μg/kg i.v.). On the other hand, the hypotensive effects of celiprolol and salbutamol were antagonized markedly and with similar potency by ICI118,551, a relatively selective β₂-adrenoceptor antagonist (DR₁₀ = 15 and 25 μg/kg i.v. respectively). In rats pretreated with celiprolol (0.03 to 0.3 mg/kg i.v.), the heart rate dose-response curves to isoprenaline were shifted to the right of those determined in matched groups of vehicle-pretreated animals. In this respect, celiprolol was half as potent as betaxolol in blocking cardiac β₁-adrenoceptors. Furthermore, celiprolol also antagonized the hypotensive effects of salbutamol, but, in this respect, celiprolol was 90-fold less potent than ICI 118,551. In conclusion, these results clearly indicate that celiprolol has the ability of stimulating and blocking not only cardiac β₁- but also vascular β₂-adrenoceptors. The effects on cardiac β₁-adrenoceptors as well as the agonism of β₂-adrenoceptors are produced by similar doses of celiprolol. These doses are notably lower than those necessary to block β₂-adrenoceptors. Thus, this pharmacological profile, which has also been demonstrated in humans, indicates that celiprolol is a modulator of cardiac β₁-adrenoceptors with vascular β₂-adrenoceptor agonist properties. Received: 26 June 1996 / Accepted: 3 March 1997     

Histaprodifen, methylhistaprodifen, and dimethylhistaprodifen are potent H1-receptor agonists in the pithed and in the anaesthetized rat

Selective H₂- and H₃-receptor agonists, exhibiting an at least tenfold higher potency than histamine itself at the respective receptors, have been known for several years. Selective H₁-receptor agonists with a potency exceeding that of histamine have become available only recently; the most potent are methylhistaprodifen and dimethylhistaprodifen [N ^α-methyl- and N ^α,N ^α-dimethyl-2-(3,3-diphenylpropyl)histamine, respectively] with 3.4- and 2.4-fold higher potencies than histamine in vitro (in the guinea-pig ileum). The aim of the present study was to examine whether these compounds and the parent compound histaprodifen are potent H₁-receptor agonists in the pithed and in the anaesthetized rat. In pithed, vagotomized rats diastolic blood pressure was decreased by 2-(2-thiazolyl)ethanamine i.v. (which was used as a reference H₁-receptor agonist) and by histaprodifen, methylhistaprodifen, and dimethylhistaprodifen; the maximum decrease was about 45 mmHg for each compound, and the potencies, expressed as pED₅₀, the negative logarithm of the dose (in mole per kilogram body weight) eliciting a half-maximal response, were 7.23, 7.55, 8.43 and 8.12, respectively. The dose/response curves of the four compounds were shifted to the right to about the same extent by the H₁-receptor antagonist dimetindene (1 μmol/kg i.v.). The vasodepressor response was not affected by combined i.v. administration of the H₂- and H₃-receptor antagonists ranitidine and thioperamide, by combined i.v. administration of the α₁- and α₂-adrenoceptor antagonists prazosin and rauwolscine, and by the β-adrenoceptor antagonist propranolol i.v. but was attenuated by the inhibitor of NO synthase, N ^ω-nitro-l-arginine methyl ester i.v. In anaesthetized rats 2-(2-thiazolyl)ethanamine, histaprodifen, methylhistaprodifen and dimethylhistaprodifen i.v. also decreased diastolic blood pressure in a manner sensitive to dimetindene i.v. Our data show that histaprodifen and, in particular, methyl- and dimethylhistaprodifen are highly potent H₁-receptor agonists in vivo. Received: 3 September 1998 / Accepted: 23 October 1998     

Effects of serotonin-3 receptor antagonists on the intracranial self-administration of ethanol within the ventral tegmental area of Wistar rats

Abstract Rationale. Previous work from our laboratory indicated that Wistar rats will self-administer ethanol (EtOH) directly into the posterior ventral tegmental area (VTA) and that 5-HT₃ antagonists will inhibit EtOH-stimulated somatodendritic release of dopamine within the VTA. Objectives. The objective of this study was to use the intracranial self-administration procedure to determine the involvement of 5-HT₃ receptors in mediating the reinforcing effects of EtOH within the VTA, and to increase our understanding of central nervous system mechanisms involved in the rewarding effects of EtOH. Methods. Adult female Wistar rats were stereotaxically implanted with guide cannulae aimed at the posterior VTA. After 1 week, rats were placed into standard two-lever experimental chambers for a total of seven sessions (4-h sessions separated by 48 h) and allowed to self-administer vehicle alone, a 5-HT₃ antagonist alone, 200 mg% EtOH alone, or combinations of 200 mg% EtOH with different concentrations of a 5-HT₃ antagonist (n=6–9 per group). Results. Throughout all seven sessions, Wistar rats self-infused more 200 mg% ETOH (25±5 infusions) than vehicle (5±4 infusions) or 5-HT₃ antagonist (6±4 infusions) (P<0.05), and responded significantly more (P<0.05) on the active than inactive lever (e.g., 50±12 vs 12±8 responses in session 1). Co-administration of 50 μM or 100 μM ICS 205,930 with 200 mg% EtOH completely prevented the acquisition and maintenance of EtOH self-infusion into the posterior VTA. Similarly, co-administration of either 25–100 μM LY-278–584 or 10–100 μM zacopride with 200 mg% EtOH completely blocked EtOH-maintained intracranial self-administration behavior. Conclusions. The results of this study suggest that the reinforcing effects of EtOH within the posterior VTA of Wistar rats require activation of local 5-HT₃ receptors. Electronic Publication     

Heterogeneous characteristics of imidazoline-induced insulin secretion

Imidazolines are regarded as a pharmacological group of insulin secretagogues with one uniform mechanism of action, namely closure of ATP-dependent K⁺ channels (K_ATP channels) and, in consequence, depolarization of the plasma membrane, Ca²⁺ influx and stimulation of secretion. This assumption was investigated by measuring insulin secretion from perifused pancreatic islets in response to three imidazoline compounds and comparing the characteristics of secretion with changes in membrane potential and cytosolic Ca²⁺ concentration [Ca²⁺]_i of single β-cells. Phentolamine (32 μM) stimulated insulin secretion from perifused mouse islets in the presence of stimulatory (10 mM and 30 mM) and substimulatory (5 mM) glucose concentrations and even in the absence of glucose. Idazoxan in concentrations up to 500 μM was virtually ineffective in the presence of 5 mM glucose. At 10 mM glucose, there was a moderate but significant increase of secretion by idazoxan, 20 μM being nearly as effective as 100 μM. The effect of phentolamine was of slow onset and irreversible in the time frame of the experiments, while the effect of idazoxan was of fast onset and reversible. Alinidine also stimulated secretion in the presence of 10 mM glucose with fast and reversible kinetics, but in contrast to idazoxan, 100 μM was clearly more effective than 20 μM. These heterogeneous characteristics of secretion were reflected by changes of [Ca²⁺]_i: the increase of [Ca²⁺]_i by phentolamine was slow and only partially reversible, whereas idazoxan led to a smaller, but faster and reversible response. The increase of [Ca²⁺]_i by phentolamine and idazoxan was abolished by the Ca²⁺ channel blocker D 600. Surprisingly, all three compounds depolarized the β-cell plasma membrane from a resting potential of –71 mV to about –36 mV. Again, the effect of phentolamine was slow and that of idazoxan and alinidine fast. Thus, the characteristics of phentolamine-induced secretion appear to be attributable to the consequences of K_ATP channel closure. It is unclear, however, why all three test compounds achieved the same degree of depolarization in spite of their known different efficiency to close K_ATP channels. Apparently, there are additional mechanisms involved in the action of idazoxan and alinidine, which may contribute to the obvious differences in the characteristics of secretion. Received: 2 October 1998 / Accepted: 21 December 1998     

Naltrexone and β-funaltrexamine antagonism of the antinociceptive and response rate-decreasing effects of morphine, dezocine, and d-propoxyphene

  Rationale: Patterns of competitive and insurmountable antagonism provide important data to guide the classification and characterization of different types of opioid agonists as well as infer the mechanism of action for agonists. Objective: Experiments with the competitive antagonist, naltrexone, and the insurmountable antagonist, β-funaltrexamine (β-FNA), were conducted to determine whether the antinociceptive and rate-decreasing effects of the opioid agonists dezocine and d-propoxyphene are 1) mediated through μ opioid receptors in rats, and 2) differ from morphine with respect to relative efficacy. Methods: The rat tail-withdrawal assay was used to measure antinociception and a fixed ratio 20 (FR20) schedule of food delivery was used to measure rate suppression. Results: Naltrexone (0.01–1.0 mg/kg) was approximately equipotent as an antagonist of the antinociceptive and rate-decreasing effects of both morphine and dezocine and as an antagonist of the antinociceptive effects of d-propoxyphene. Naltrexone failed to block the rate-decreasing effects of d-propoxyphene. β-FNA (5 and 10 mg/kg) also antagonized the antinociceptive and rate-decreasing effects of morphine and dezocine as well as the antinociceptive effects of d-propoxyphene. β-FNA failed to produce a dose-dependent antagonism of the rate-decreasing effects of d-propoxyphene. Conclusions: These data suggest that the antinociceptive effects of morphine, dezocine, and d-propoxyphene and the rate-decreasing effects of morphine and dezocine are mediated through μ opioid receptors. Overall, high doses of β-FNA produced a greater degree of antagonism of the behavioral effects of dezocine than morphine or d-propoxyphene, confirming other reports that dezocine is a lower efficacy agonist than morphine. Additionally, the degree of antagonism produced by β-FNA was greater for the antinociceptive effects of all three compounds than for the rate-decreasing effects. Received: 14 August 1998 / Final version: 4 December 1998     

Endomorphin-1 and endomorphin-2 activate µ-opioid receptors in myenteric neurons of the guinea-pig small intestine

The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central μ-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM–1 μM) inhibited (–log EC₅₀ values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective μ-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), caused a concentration-dependent (30 nM–1 μM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA ₂ values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective δ-receptor antagonist) or 30 nM nor-binaltorphimine (a selective κ-receptor antagonist). These results demonstrate that endomorphins selectively activate μ-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists. Received: 6 August 1998 / Accepted: 5 October 1998     

Comparison of the discriminative and neuroendocrine effects of centrally penetrating kappa-opioid agonists in rhesus monkeys

Abstract Rationale. The discriminative effects of κ-agonists may be mediated centrally, whereas their effects in a neuroendocrine biomarker assay (prolactin release) may be mediated by κ-receptors in hypothalamic areas outside the blood-brain barrier. Prolactin may thus be a useful biomarker, due to its potential to provide quantitative pharmacodynamic data for κ-opioid ligands in vivo. The potency of centrally penetrating κ-agonists could be similar in these two assays, due to their ability to occupy κ-receptor pools inside and outside the blood-brain barrier, following SC administration. Objective. To compare the potency of centrally penetrating κ-agonists in producing U69,593-like discriminative stimulus effects (U69,593 is considered a selective κ-agonist), and in producing prolactin release in rhesus monkeys. Methods. Cumulative dose-effect curves of κ-agonists (R84760, bremazocine, spiradoline and U50,488) were investigated in a food-reinforced U69,593 discrimination (n=3), and compared to those for the μ-opioids fentanyl and nalbuphine and the δ-agonist SNC80. Selected κ-opioids (R84760 and spiradoline) were compared to fentanyl, nalbuphine and SNC80 in the neuroendocrine biomarker assay, in intact female rhesus monkeys (n=4). Results. All the selective κ-agonists caused dose-dependent generalization (i.e. at least 90% drug-appropriate responding) in the U69,593 discriminating subjects, and caused robust, dose-dependent prolactin release in female rhesus monkeys. By contrast, fentanyl, nalbuphine and SNC80 did not cause generalization in these subjects. Fentanyl and nalbuphine also caused prolactin release; quantitative antagonism (apparent pK_B) experiments following nalmefene (0.01, 0.1 mg/kg) differentiated the effects of a selective κ-agonist (spiradoline) from those of a selective μ-agonist (fentanyl). A positive correlation (r=0.99) was noted between the mean log ED₅₀ of κ-agonists in the discrimination and neuroendocrine assays, from these and previous determinations. Conclusions. The potency of centrally penetrating κ-agonists in causing their neuroendocrine effects is similar to their potency in causing discriminative effects. Furthermore, apparent pK_B experiments with nalmefene differentiated the receptor mediation (i.e. κ or μ) of these compounds in the neuroendocrine biomarker assay. Electronic Publication     

Synthesis and anticholinesterase activity of 2-(dimethylamino)ethyl and choline esters of n-substituted α, β-dehydroamino acids

A new method for the synthesis of tertiary and quaternary aminoesters of N-substituted α,β-dehydroamino acids is described. Aseries of 16 dehydroamino acids esterified to choline or to its tertiary analog have been synthesized by the proposed method with a yield of 84–93%, and their interactions with human erythrocyte acetylcholinesterase (ACE) and human plasma butyrylcholinesterase (BCE) has been studied. The half-inhibiting concentrations IC₅₀ of the synthesized compounds (determined with respect to cholinesterase hydrolysis of a model substrate, 0.1 mM ATC) vary within a broad range (0.16–1840 μM). The values of traditional parameters of the wave functions of ligands estimated using the Hartree-Fock method do not explain the observed pattern of the anticholinesterase activity. The specific properties of the molecules, especially in their quaternary ammonium salt forms, are probably related to their structural features, in particular, to the ability of the inhibitors to form cyclic conformations (so-called crown structures). Such structures are probably stabilized as a result of the formation of intramolecuar hydrogen bonds between protons of the choline residue and oxygen of the terminal peptide group. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 3, pp. 18–23, March, 2006.