In vitro and in vivo effects of SCA40 on guinea pig airways

SCA40 (6-bromo-8-methylaminoimidazo[1,2-a]- pyrazine-2-carbonitrile), a compound which had been described as an opener of Ca²⁺-dependent large conductance potassium channels (BK_Ca channels), was investigated in comparison with salbutamol for in vitro and in vivo bronchospasmolytic effects and for the ability to reverse airways hyperreactivity in guinea pigs. SCA40 reduced the spontaneous tone of isolated guinea pig tracheal rings with a biphasic concentration-response curve (first phase: pD₂ = 8.0, E_Max = 29.7% of maximal effect; second phase: pD₂ = 6.4, E_Max = 72.6%). The salbutamol curve was monophasic (pD₂ = 8.0, E_Max = 100%). Total lung resistance (R_L) was determined in anaesthetized, ventilated guinea pigs. Bronchoconstriction, measured as an increase in R_L, was elicited in normoreactive animals by i.v. infusion of bombesin (100 ng/kg/min) or by i.v. injection of histamine (1.8–5.6 μg/kg). Airways hyperreactivity was induced by acute i.v. administration of pre-formed immune complexes. Intravenous bolus injections of histamine (2.4 μg/kg) were used to define the sensitivity of the airways prior to and after the exposure to immune complex. Following intratracheal (i.t.) administration, SCA40 reversed bombesin-induced bronchoconstriction with an ED₅₀ of 43 μg/kg (E_Max = 57%). The ED₅₀ for salbutamol was 0.8 μg/kg i.t. (E_Max = 78%). Histamine-induced bronchoconstriction in hyperreactive guinea pigs was inhibited by SCA40 with an ED₅₀ of 13 μg/kg i.t. (E_Max = 82%). Salbutamol completely inhibited histamine-induced bronchospasm with an ED₅₀ of 9 ng/kg i.t. In normoreactive guinea pigs, SCA40 prevented histamine-induced bronchoconstriction with an ED₅₀ of 100 μg/kg i.t.; for salbutamol the ED₅₀ in this test was 0.48 μg/kg i.t. Thus, for both SCA40 and salbutamol, the effects obtained at low doses in hyperreactive guinea pigs represent a true reversal of airways hyperreactivity, whereas at higher doses, anti-hyperreactive and bronchospasmolytic properties may account for the observed effects. In conclusion, SCA40 relaxes guinea pig airways smooth muscle in vitro and in vivo, and it partly reverses airways hyperreactivity. With respect to both potency and efficacy, SCA40 is markedly less active than the β-adrenoceptor agonist salbutamol. Received: 23 August 1996 / Accepted: 11 October 1996     

The role of vasopressin on the effect of U-50,488 to block the development of morphine tolerance and physical dependence

U-50,488, a selective κ-opioid receptor agonist, has been reported to inhibit the development of antinociceptive tolerance to morphine in mice, rats and guinea pigs, but the mechanism involved in this action remains unknown. Since U-50,488 has been reported to supress the plasma vasopressin level, we investigated the role of vasopressin with U-50,488 in the male Sprague Dawley rat in this study. Animals (230–270 g) were chronically treated with morphine (10 mg/kg, i.p.) twice a day for 6 days in order to induce tolerance to antinociceptive effect measured by tail-flick test. Withdrawl symptoms were precipitated by naloxone (10 mg/kg, i.p.) on day 7. U-50,488 (i.p.) or AVP (i.p. or i.c.v.) or U-50,488 and AVP was (were) coadministered with chronic morphine to investigate their effects on morphine tolerance and dependence. We found that coadministration of 8 mg/kg U-50,488 (i.p.) with morphine almost completely block morphine tolerance and partially block withdrawal symptoms. In contrast, coadministration of AVP (0.3 μg/kg, i.p., or 0.01 μg, i.c.v.) with morphine and U-50,488, the effects of U-50,488 to block morphine tolerance and dependence were reversed. In addition, treatment of AVP antagonist (dPTyr(Me)AVP, 0.5 μg/kg, i.p. or 0.5 μg, i.c.v.) has the similar effect as U-50,488 to block morphine tolerance. In summary, the effect of U-50,488 to block morphine tolerance and dependence may relate to its inhibitory effect on AVP release. Received: 20 February 1996 / Accepted: 14 October 1996     

Effects of WEB 2086, a novel antagonist of platelet activating factor, in active and passive anaphylaxis

WEB 2086, a novel specific platelet activating factor (PAF) antagonist derived from triazolodiazepines, inhibited in a dose-related manner the PAF-dependent component of anaphylaxis as well as PAF-induced effects in mice and guinea pigs. In mice a lethal anaphylactic shock and a PAF-induced (100 micrograms/kg i.v.) death was inhibited by i.v. WEB 2086. The ED50 values were 13.6 and 0.37 mg/kg i.v., respectively. In actively sensitized guinea pigs, the anaphylactic lung reaction (bronchoconstriction), but not the corresponding hypotension, was prevented by oral (0.05-0.5 mg/kg) doses of WEB 2086. In contrast, in passively sensitized animals a dose-dependent inhibition of the pulmonary (bronchoconstriction) and blood pressure (hypotension) reaction due to anaphylaxis was achieved by i.v. WEB 2086. Similarly, oral (0.05-0.5 mg/kg) and i.v. (0.005-0.05 mg/kg) WEB 2086 inhibited PAF-induced reduction in respiratory flow (bronchoconstriction) and hypotension in guinea pigs. The ED50 values were 0.070 and 0.066 mg/kg p.o., and 0.017 and 0.015 mg/kg i.v., respectively. In conclusion, PAF seems to play a more major role in passive than in active anaphylaxis in guinea pigs. These results provide further evidence for an important role of PAF in anaphylaxis and support the hypothesis that PAF is involved in asthma and other allergic diseases.     

Triazolodiazepines are potent antagonists of platelet activating factor (PAF) in vitro and in vivo

Summary The triazolodiazepines brotizolam, triazolam and alprazolam inhibited PAF-induced human platelet aggregation in vitro (IC₅₀ = 0.54, 7.6 and 13.7 M, respectively) but showed only a weak or no effect against other aggregating agents (ADP, adrenaline, collagen, serotonin, arachidonic acid). In comparison, flunitrazepam and diazepam, two diazepines without the triazole ring, showed IC₅₀-values of 42 and 260 M, respectively. Flunitrazepam does not possess the specificity shown by the other compounds. Brotizolam and triazolam also inhibited PAF-induced human neutrophil aggregation in vitro, with IC₅₀-values 0.21 and 6.6 M, respectively.In anaesthetized guinea pigs, brotizolam (2.5 to 10 mg/kg p.o. or 0.1 to 0.5 mg/kg i.v.) or triazolam (20 to 100 mg/kg p.o.) inhibited dose-dependently the intrathoracic accumulation and aggregation of ¹¹¹Indium labelled platelets induced by an i. v. infusion of PAF (30 ng/kg × min).Brotizolam at doses of 1 to 10 mg/kg p. o. and 0.1 to 0.5 mg/kg i. v. inhibited dose-dependently the reduction in tidal volume (bronchoconstriction), the systemic hypotension and the lethal effect due to i. v. PAF in guinea pigs. Triazolam inhibited these effects of PAF at doses of 50 to 200 mg/kg p.o.PAF-induced systemic hypotension in rats can be reversed by cumulative i. v. doses (0.05 to 1.0 mg/kg) of brotizolam.In conclusion, these results show that triazolodiazepines, like brotizolam and triazolam, are potent inhibitors of PAF-induced effects in vitro and in vivo. Send offprint requests to J. Casals-Stenzel at the above address     

The effect of the selective PAF antagonist CIS-19 on PAF- and antigen-induced bronchoconstriction, microvascular leakage and bronchial hyperreactivity in guinea-pigs

We investigated the effects of a novel platelet-activating factor (PAF) receptor antagonist, CIS-19 [cis-2-(3, 4-dimethoxyphenyl)-6-isopropoxy-7-methoxy-1-(N-methylformamido)-1, 2, 3, 4-tetrahydronaphthalene], on PAF-, histamine-, substance P- and antigen-induced bronchoconstriction and microvascular leakage, as well as PAF- and antigen-induced bronchial hyperreactivity to methacholine in urethane-anesthetized guinea-pigs. Administration of CIS-19 (0.5–5 mg/kg, i.v.) inhibited the increase in lung resistance induced by PAF (30 ng/kg, i.v.) in a dose-dependent manner, but failed to inhibit the increase induced by histamine (30 μg/kg, i.v.) or substance P (6.5 μg/kg, i.v.). CIS-19 (5 mg/kg, i.v.) did not inhibit the increase in lung resistance induced by ovalbumin (2 mg/kg, i.v.) in actively sensitized guinea-pigs. PAF (30 ng/kg, i.v.)-induced microvascular leakage, measured by the extravasation of Evans blue dye, was dose-dependently inhibited by CIS-19 (0.5–5 mg/kg, i.v.) in the trachea, main bronchi and intrapulmonary airways, but it did not affect histamine (30 μg/kg, i.v.)- or substance P (6.5 μg/kg, i.v.)-induced microvascular leakage at all airway levels. CIS-19 (2.5 and 5 mg/kg) did not affect ovalbumin (2 mg/kg, i.v.)-induced microvascular leakage in all airway levels in actively sensitized guinea-pigs. CIS-19 (2.5 and 5 mg/kg, i.v.) significantly inhibited PAF-induced enhancement of the bronchial response to methacholine, but had no effect on ovalbumin (0.05 mg/kg, i.v.)-induced bronchial hyperreactivity in actively sensitized guinea-pigs. It is concluded that CIS-19 is a potent PAF receptor antagonist which inhibits PAF- but not antigen-induced bronchoconstriction, microvascular leakage and bronchial hyperreactivity. These results suggest that PAF plays little or no role in early airway responses following antigen challenge. Received: 29 April 1996 / Accepted: 10 October 1996     

Pharmacological studies of loperamide, an anti-diarrheal agent. II. Effects on peristalsis of the small intestine and colon in guinea pigs (author's transl)

Effects of loperamide on peristalsis in the guinea pig intestines were investigated in comparison with those of morphine and atropine. The following results were obtained. The ejection of intraluminal fluid produced by the peristaltic contraction of the isolated ileum was suppressed by loperamide at a concentration of 10(-8) or 2 X 10(-8) g/ml. Peristalsis in the intestinal loop of anesthetized guinea pigs was inhibited by i.v. administration of loperamide at a dose of 0.03 mg/kg. Morphine (0.03 mg/kg i.v.) and atropine (0.05 mg/kg i.v.) also inhibited the peristaltic contraction. The effect of loperamide continued longer than that of morphine. Peristalsis in the colonic loop of anesthetized guinea pigs was inhibited by i.v. administration of loperamide at a dose of 0.01 or 0.03 mg/kg. Morphine (0.1 mg/kg i.v.) and atropine (0.03 mg/kg i.v.) also inhibited the peristaltic contraction of the colonic loop. Loperamide (0.01 or 0.03 mg/kg i.v.) and morphine (0.1 mg/kg i.v.) caused a slight and temporary increase of resting level of intraluminal pressure with inhibition of peristalsis in the colonic loop. These results suggest that loperamide suppresses the peristaltic contraction caused by distension of the intestinal lumen.     

Effects of flutropium bromide, a new anti-asthma drug, alone or in combination with salbutamol, aminophylline and disodium cromoglycate on the acetylcholine induced bronchoconstriction

Effects of flutropium bromide, a new bronchodilator with an anticholinergic action, alone or in combination with other antiasthma drugs were investigated in guinea pigs by using an index of inhibition of the acetylcholine (ACh)-induced bronchoconstriction. Single inhalation of flutropium bromide (0.0003%) into the airways of guinea pigs inhibited the ACh (i.v.)-induced bronchoconstriction without changing the fall in blood pressure induced by ACh. When salbutamol (3 micrograms/kg, i.v.), aminophylline (5 mg/kg, i.v.) or disodium cromoglycate (10 mg/kg, i.v.) was administered in combination with flutropium bromide (0.0003%), bronchodilation was enhanced as compared with single administration of the respective antiasthma drugs. From the above results, it is indicated that inhalation of flutropium bromide provides a more efficient bronchodilation in combination with other antiasthma drugs that possess different mechanisms of antiasthma effects.     

Effects of SR141716 and WIN 55,212-2 on tolerance to ethanol in rats using the acute and rapid procedures

Rationale Our previous findings have shown rapid cross-tolerance between ethanol and Δ⁹-tetrahydrocannabinol and that intraperitoneal (i.p.) injection of cannabinoid receptor type 1 (CB1R) antagonist SR141716 (SR) does not interfere with tolerance to either of these drugs in mice. Objectives This study investigates the effects of SR, alone or in combination with the CB receptor agonist WIN 55,212-2 (WIN), on the development of acute and rapid tolerance to the incoordinating effect of ethanol in rats. Materials and methods Male Wistar rats received SR, through i.p. (0.5–2.0 mg/kg) or intracerebroventricular (i.c.v.) injections (0.5–4.0 μg), alone or together with WIN (1.0 μg, i.c.v.), in combination with ethanol (2.7 g/kg, i.p.). Another group received WIN (1.0 μg, i.c.v.) in combination with ethanol (2.3 g/kg), and the rats were tested for motor coordination. Rapid tolerance was assessed 24 h later by administering ethanol to all animals and retesting them under the same dose regimen. Acute tolerance was evaluated for 75 min after ethanol (3.0 g/kg, i.p.) in animals treated with SR or WIN (i.c.v.). Results The reduced motor impairment on day 2 (i.e., rapid tolerance) was blocked by SR (i.p. and i.c.v.). WIN (1.0 μg, i.c.v.) facilitated rapid tolerance and also prevented the blockade of rapid tolerance by SR (1.0 μg, i.c.v.). In the acute tolerance procedure, SR did not affect the motor incoordination induced by ethanol. Conclusions The results suggest that the endocannabinoid system may contribute to the development of rapid tolerance to ethanol.     

Comparative study of the absorption,elimination and acute hepatotoxic action of ethanol in guinea pigs and rats

In fed guinea pigs, an oral dose of 6.4 g/kg of ethanol given as a 40% solution (v/v) produced a maximal blood alcohol level of 6.8±0.3 mg/ml, whereas in fed rats, blood alcohol levels after the same dose did not exceed 2.1±0.2 mg/ml. Maximal blood alcohol levels in fasted animals after an oral load of 4.8 g/kg of ethanol were 6.3±0.2 mg/ml in guinea pigs and 3.7±0.3 mg/ml for rats. However, i.v. injected ethanol (1 g/kg) was eliminated at the same rate in both species (275 mg per kg · h), and ADH activity of the liver related to body weight was by 20% greater in guinea pigs than in rats.Therefore, absorption of ethanol occurs at a much slower rate in rats than in guinea pigs. This is possibly due to the fact that high ethanol concentrations strongly delay emptying of the rat stomach. Lowering the ethanol concentration accelerates absorption rate in the rat. However, even after gavage of a 10% solution peak levels of blood alcohol were still lower by 36% in rats than in guinea pigs.In guinea pigs, increased serum activities of GOT, GPT, and GLDH occurred after an oral dose of 4.8 g/kg or 6.4 g/kg of ethanol, respectively. SGOT already increased after 1.6 g/kg of ethanol p.o. After 6.4 g/kg of ethanol given to rats serum transaminase levels increased only slightly, and GLDH activity not at all. Vacuolar degeneration was the morphological substrate of ethanol-induced liver damage in guinea pigs and rats. In guinea-pigs, it occurred already after 1.6 g/kg of ethanol, whereas in rats only after 6.4 g/kg.In conclusion, the guinea pig seems to be better suited for research on alcohol toxicity than the rat.     

Studies on antiplatelet effect of OP-41483, a prostaglandin I2 analog, in experimental animals. I. Effect on platelet function and thrombosis

Antiplatelet and antithrombotic effects of OP-41483, a PGl2 analog, were studied in experimental animals, and the following results were obtained: 1) With 10 min-intravenous infusion to guinea pigs, OP-41483 inhibited platelet adhesiveness and platelet aggregation at 300-1000 ng/kg/min and 1000 ng/kg/min, respectively. In these effects, OP-41483 was 1-3 times more potent than carbacyclin and 3 times less potent than PGl2. 2) With oral administration to guinea pigs, OP-41483 given as its alpha-cyclodextrin clathrate (OP-41483 alpha-CD) inhibited platelet adhesiveness at doses higher than 1.0 mg/kg (expressed in terms of OP-41483), whereas PGl2 and carbacyclin did not at 10 mg/kg. OP-41483 alpha-CD also inhibited platelet aggregation after a single dose of 3 mg/kg and repeated doses of 3 mg/kg/day for 7 days. 3) In the electrically induced thrombosis model of guinea pig mesenteric artery, OP-41483 (300-1000 ng/kg/min, i.v.-infusion) and OP-41483 alpha-CD (1.0-3.0 mg/kg, p.o.) inhibited thrombus formation, but heparin (1.0-10 U/kg/min, i.v.-infusion) did not. 4) In the rabbit extracorporeal circulation thrombosis model, OP-41483 (100 and 300 ng/kg/min, i.v.-infusion) inhibited thrombus formation in the extracorporeal shunt and prevented the decrease in platelet count, hematocrit and fibrinogen level in circulating blood. Heparin (1.0-3.0 U/kg/min, i.v.-infusion) also inhibited the thrombus formation and the decrease in fibrinogen level, but did not inhibit the decrease in hematocrit and platelet count.     

Pharmacological evidence for the presence of functional β3-adrenoceptors in rat retinal blood vessels

The aim of this study was to examine whether stimulation of β₃-adrenoceptors dilates rat retinal blood vessels and how diabetes affects the vasodilator responses. Images of ocular fundus were captured with an original high-resolution digital fundus camera in vivo. The retinal vascular responses were evaluated by measuring diameter of retinal blood vessels contained in the digital images. Both systemic blood pressure and heart rate (HR) were continuously recorded. The β₃-adrenoceptor agonist CL316243 (0.3–10 μg/kg/min, i.v.) increased diameter of retinal arterioles (at 10 μg/kg/min, a 31% increase) and decreased mean blood pressure (at 10 μg/kg/min, a 21% decrease) in a dose-dependent manner. CL316243 produced a small but significant increase in HR (at 10 μg/kg/min, a 9% increase). Both SR59230A (1 mg/kg, i.v.) and L-748337 (50 μg/kg, i.v.), β₃-adrenoceptor antagonists, significantly prevented CL316243-induced retinal vasodilator responses. Similar observations were made with another β₃-adrenoceptor agonist, BRL37344. The β₂-adrenoceptor agonist salbutamol also increased diameter of retinal arterioles (at 10 μg/kg/min, a 43% increase), whereas the drug produced greater decrease in blood pressure (at 10 μg/kg/min, a 46% decrease) and increase in HR (at 10 μg/kg/min, a 16% increase), compared with β₃-adrenoceptor agonists. The retinal vasodilator responses to CL316243 and BRL37344 observed under blockade of β₁/β₂-adrenoceptors with propranolol (2 mg/kg, i.v. bolus followed by 100 μg/kg/min infusion) were unaffected 2 weeks after induction of diabetes by the combination of streptozotocin treatment and d-glucose feeding. On the other hand, the vasodilator responses to salbutamol of retinal arterioles were significantly reduced in diabetic rats. These results suggest that stimulation of β₃-adrenoceptors causes the vasodilation of retinal arterioles in vivo and the vasodilator responses are unaffected at the early stage of diabetes.     

Prolonged treatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) differently affects the serotonergic modulation of cortical acetylcholine release in male and female guinea pigs

Acetylcholine (ACh) release from the cerebral cortex was studied in freely moving guinea pigs, implanted with epidural cups. A single dose of either 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT, 0.1 mg/kg s.c.) or 5-hydroxytryptamine (5-HT, 250 μg i.c.v.) increased cortical ACh release, both in male and in female guinea pigs. In female guinea pigs chronically treated with 8-OHDPAT (0.1 mg/kg daily s.c., for 14 days) the facilitatory effect of 8-OHDPAT and 5-HT was maintained. In chronically 8-OHDPAT-treated male guinea pigs, 8-OHDPAT no longer modified ACh release, while 5-HT inhibited it. The inhibition was prevented by the 5-HT₃ antagonist MDL 72222, 1 mg/kg s.c. These results indicate that differences exist between male and female guinea pigs in the adaptive responses to prolonged treatment with the selective 5-HT_1A agonist 8-OHDPAT.     

Prevention of reocclusion following tissue type plasminogen activator-induced thrombolysis by the RGD-containing peptide, echistatin, in a canine model of coronary thrombosis

We evaluated the effect of the RGD-containing peptide, echistatin, on thrombolysis time and acute reocclusion in a canine model of coronary thrombosis/thrombolysis. Occlusive thrombus formation was induced by electrical injury, via a stimulating electrode, to the endothelial surface of the circumflex coronary artery in the open-chest, anesthetized dog in the presence of a critical stenosis. Fifteen minutes after occlusive thrombus formation, dogs received either an intravenous infusion of vehicle (saline at 0.1 ml/min) or echistatin (15 micrograms/kg/min i.v.). Heparin was given as an initial bolus (100 U/kg i.v.) 15 min after thrombus formation and repeated at hourly intervals (50 U/kg). This dose of heparin increased activated partial thromboplastin time to 1.5- to 2.5- fold over control. Thrombolysis was induced with recombinant tissue-type plasminogen activator (tPA) at a total dose of 1 mg/kg, intravenously administered over 90 min with 10% given as an initial bolus. The vehicle-treated animals reperfused at 48 +/- 9 min with a reperfusion incidence of 60% (3/5). The echistatin-treated animals reperfused at 46 +/- 5 min with a reperfusion incidence of 100% (5/5). After stopping the tPA infusion, acute reocclusion occurred in 100% (3/3) of the vehicle-treated dogs and in only 20% (1/5) of the echistatin-treated dogs. Echistatin caused a greater than 5-fold increase in buccal mucosa bleeding time and almost completely inhibited ex vivo platelet aggregation to ADP, collagen, and U-46619. Residual thrombus wet weight, determined at the end of the experiment, was significantly lower for the echistatin group (2.1 +/- 0.2 mg) compared to the vehicle group (5.8 +/- 0.7 mg).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism of the renal vasodilator activity of dopamine by metoclopramide

Summary The interaction of metoclopramide with renal dopamine receptors has been characterized in anesthetized dogs surgically prepared with arterial blood pressure catheters and renal artery blood flowprobes.In normal dogs, i. v. dopamine (3 g/kg) produced consistent and selective decrements in renal vascular resistance (RVR) and increments in renal blood flow over a 220 min test period; mean arterial blood pressure and cardiac rate were minimally affected. Pretreatment with metoclopramide, 1 and 10 mg/kg i. v., resulted in dose-related inhibition (maximum inhibition 44% and 94%, respectively) of the renal vasodilator activity of dopamine without altering baseline parameters. The duration of antagonism produced by 1 mg/kg of metoclopramide was approximately 30 min, while 10 mg/kg resulted in significant attenuation for the entire test period. Decreases in RVR produced by prostaglandin A₁ (0.03 and 0.3 g/kg, i. v.) and bradykinin (3 and 15 g/kg, i. v.) that were comparable to those of dopamine were unaltered by metoclopramide. Furthermore, metoclopramide did not affect the diastolic blood pressure responses to noradrenaline (0.1–3 g/kg, i. v.) or isoproterenol (0.03–0.3 g/kg, i. v.), nor did it alter dopamine-induced vasoconstriction of the iliac vasculature.In phenoxybenzamine (3 mg/kg, i. v.) treated dogs, dopamine (0.3–30 g/kg, i. v.) produced dose-related reductions in RVR. Administration of metoclopramide (10 mg/kg, i. v.) resulted in a 10-fold parallel displacement, to the right, of the RVR dose-response curve to dopamine.These findings demonstrate that metoclopramide is an effective antagonist of renal dopamine receptors following systemic administration in the dog. The results are not consistent with the classification of metoclopramide as a selective antagonist of D-2 receptors.     

The effects of bisantrene on human platelets

Summary Bisantrene, (9,10-anthracenedicarboxaldehyde bis [(4,5-dihydro-lH-imidazol-2yl)hydrazone] dihydrochloride) is one of a series of anthracene dicarboxaldehyde compounds in Phase II trials. Preliminary studies suggested that bisantrene has anti-platelet activity. Therefore, in vitro studies of its effects on platelets were undertaken. Bisantrene in clinically attainable concentrations of 0.625 – 10 M, caused a 95 ± 1% decrease in maximal platelet aggregation to collagen (1 g/ml), and epinephrine (5–40 M) and 90 ± 10% inhibition of arachidonic acid (50 g/ml) induced aggregation. Collagen induced platelet shape change was not affected. Aggregation to calcium ionophore A23187 was inhibited by 10–30%. No effect on ADP induced aggregation was seen at clinically relevant bisantrene concentrations. This inhibition was time dependent, reaching a maximum when platelets were preincubated with bisantrene for 10 minutes before exposure to agonist. Inhibition persisted after bisantrene was removed by washing. To determine the mechanism of platelet inhibition, platelet prostaglandin metabolism, oxygen consumption and release reaction were measured. In the presence of drug: 1. normal cyclooxygenase activity was demonstrated by O₂ consumption studies and normal secondary wave ADP (3.2 M) aggregation; 2. lipoxygenase activity, by O₂ consumption was also normal. 3. There was 30–50% inhibition of thromboxane A2 synthesis induced by arachidonic acid or collagen; 4. ADP, collagen and AA induced release of serotonin was decreased by 30–60% but was never abolished; 5. No effect on basal platelet cAMP levels nor additive effect on PGE₁ induced elevation of platelet cAMP was detectable. These data demonstrate that bisantrene has potent antiplatelet activity probably mediated by several different mechanisms. The inhibition may have important clinical and theoretical consequences.Abbreviations PGl ₂ prostaglandin I₂ - PGEi prostaglandin E₁ - PRP platelet rich plasma - PPP platelet poor plasma - ACD acid citrate dextrose - TBX A₂ thromboxane Az₂ - TBX B₂ thromboxane B₂ - PAF platelet activating factor-acetyl glyceryl ether phosphoryl choline     

L8027 and 1-nonyl-imidazole as non-selective inhibitors of thromboxane synthesis

The inhibition of prostaglandin-related actions on the platelets, respiration and hemodynamics of guinea pigs by 1-(isopropyl-2-indolyl)-3-pyridyl-3-ketone (compound L8027) and by 1-nonyl-imidazole (NI) was studied. L8027 (1–10 μg/kg, i.v.) inhibited arachidonic acid (AA)-initiated bronchoconstriction, thrombocytopenia and hypotension in a dose-dependent and reversible manner. NI, however, in doses as high as 8 mg/kg did not inhibit these actions. AA-induced platelet aggregation was antagonized by L8027 (50–500 nM) and NI (25–100 μM), and this action was dose-dependent and competitive. Thromboxane A2 (TxA2) synthesis in blood and platelets, as measured by the rabbit aorta bioassay, was suppressed in a concentration-related manner by L8027 at anti-aggregatory doses. NI did not inhibit TxA2 synthesis in guinea-pig platelet-rich plasma (PRP) at concentrations which blocked platelet aggregation. However, it did block TxA2 synthesis in rabbit and human PRP and in washed guinea-pig platelets, indicating a species or plasma specificity. Both compounds inhibited ATP release from platelets as they inhibited aggregation. The radioimmunoassay revealed that L8027 decreased formation of both TxB2 and PGE2, unlike imidazole which only blocked formation of TxB2 and resulted in an increase of PGE2. These results show that although L8027 does block TxA2 formation, it is not a selective antagonist and it inhibits cyclo-oxygenase as well. NI appears to have the ability to act via mechanisms other than prostaglandin metabolism.     

Differential alterations in levels of hepatic microsomal cytochrome P450 isozymes following intracerebroventricular injection of bacterial lipopolysaccharide in rats

To investigate the effect of central inflammation due to bacterial infection, such as meningitis, on the activities of hepatic cytochromes P450 (CYPs), rats were injected intracerebroventricularly (i.c.v.) with 0.1 μ g of bacterial lipopolysaccharide (LPS). The LPS i.c.v. injection significantly decreased the total P450 contents (by 30% of the levels of control rats treated with saline i.c.v.), the contents of CYP1A (48%), 2B (54%), 2C11 (37%) and 3A (40%) and related drug metabolizing activities, 7-ethoxycoumarin O-deethylation (36%), imipramine N-demethylation (41%) and erythromycin N-demethylation (33%) in liver microsomes 24 h after the treatment. In contrast, intraperitoneal (i.p.) injection of LPS at the same dose as i.c.v. (0.1 μ g) did not significantly affect the hepatic microsomal contents of total P450 or the content of each individual CYP isozyme and its activity. CYP2D1 protein and the activity of imipramine 2-hydroxylase were not significantly decreased by LPS injection regardless of the route of administration. The inhibitory effects of 0.1 μg i.c.v. LPS on the activities of these CYPs were almost equal to those of 10 μg i.p. LPS, and 0.01 μg of i.c.v. LPS significantly decreased the activity of imipramine N-demethylase only. Therefore, the LPS i.c.v. injection resulted in CYP isozyme-selective inhibition at an ineffective dose when injected i.p.. It is suggested that a central inflammation, such as meningitis, differentially decreases the levels of hepatic CYP isozymes. A possible involvement is discussed of the central nervous system in this down-regulation. Received: 2 September 1997 / Accepted: 5 May 1998     

Toxicokinetics of soman in cerebrospinal fluid and blood of anaesthetized pigs

The toxicokinetics of the four stereoisomers of the nerve agent C(±)P(±)-soman was analysed in cerebrospinal fluid (CSF) and blood in anaesthetized, spontaneously breathing pigs during a 90-min period after injection of soman. The pigs were challenged with different intravenous (i.v.) doses of C(±)P(±)-soman corresponding to 0.75–3.0 LD₅₀ (4.5, 9.0 and 18 μg/kg in a bolus injection and 0.45 μg/kg per min as a slow infusion). Artificial ventilatory assistance was given if, after soman intoxication, the respiratory rate decreased below 19 breaths/min. Blood samples were taken from a femoral artery and CSF samples from an intrathecal catheter. The concentrations of the soman isomers were determined by gas chromatography coupled with high resolution mass spectrometry. All four isomers of soman were detected in both blood and CSF samples. The relatively non-toxic C(±)P(+) isomers disappeared from the blood stream and CSF within the first minute, whereas the levels of the highly toxic C(±)P(−) isomers could be followed for longer, depending on the dose. Concurrently with the soman analyses in blood and CSF, cholinesterase (ChE) activity and cardiopulmonary parameters were measured. C(±)P(−) isomers showed approx. 100% bioavailability in CSF when C(±)P(±)-soman was given i.v. as a bolus injection. In contrast, C(±)P(−) isomers displayed only 30% bioavailability in CSF after slow i.v. infusion of soman. The ChE activity in blood decreased below 20% of baseline in all groups of pigs irrespective of the soman dose. The effect of soman intoxication on the respiratory rate, however, seems to be dose-dependent and the reason for ventilatory failure and death. Artificial ventilation resulted in survival of the pigs for the time-period studied. Received: 3 March 1998 / Accepted: 5 May 1998     

14-methoxymetopon,a highly potent μ opioid agonist,biphasically affects ethanol intake in Sardinian alcohol-preferring rats

Rationale Increased opioidergic activity is thought to increase the propensity to consume ethanol. However, the dose monotonicity and receptor subtype for this effect remain uncertain. 14-methoxymetopon is a centrally acting, selective μ opioid receptor agonist with greater systemic antinociceptive potency than morphine and a putatively improved therapeutic index. Objective To determine whether 14-methoxymetopon influenced voluntary ethanol intake in Sardinian alcohol-preferring (sP) rats. Methods Male sP rats with continuous 2-bottle choice access to ethanol (10% v/v) or water were subjects. The effects of systemic 14-methoxymetopon administration (2, 5, 12.25, 30 μg/kg, s.c.) on 4-h ethanol intake were determined. The ability of naltrexone (50 μg/kg, s.c.), an opioid antagonist, to block actions of 14-methoxymetopon (12.25, 30 μg/kg, s.c.) was examined as were the effects of 14-methoxymetopon (12.25 μg/kg, s.c.) on self-administered blood alcohol levels (BALs) and clearance of a passive ethanol bolus (1 g/kg). Finally, the effects of central 14-methoxymetopon administration (0.0003–100 ng, i.c.v.) on 4-h ethanol intake were evaluated. Results Systemic 14-methoxymetopon very potently and dose-dependently suppressed ethanol and food intake for 30 min, followed by a greater, longer-lasting, and behaviorally specific increase in ethanol intake. The increased ethanol intake led to threefold higher BALs, was naltrexone-reversible, and not due to altered ethanol clearance. Intracerebroventricular 14-methoxymetopon administration rapidly altered ethanol intake per an inverted U-shaped dose-response function, increasing it at a 10 pg dose, while suppressing it at a 10,000-fold higher dose. Conclusions The novel μ analgesic increases ethanol intake, a potential therapeutic liability, and results suggest a non-monotonic influence of brain μ opioid receptor stimulation on ethanol intake. Valentina Sabino and Pietro Cottone contributed equally to this work.     

Cardiovascular effects of intracerebroventricular GABA,glycine and muscimol in the rat

Summary Intracerebroventricular (i.c.v.) injections of gammaaminobutyric acid (GABA, 125–2,000 g), glycine (250–2,000 g) and muscimol (0.5–4 g) caused dose-dependent reductions of blood pressure and heart rate in conscious rats. Low doses of muscimol (<0.12 g) were hypertensive. The cardiovascular depression induced by GABA was antagonized by bicuculline (3.5 mg/kg i.p.), while that induced by glycine was counteracted more effectively by strychnine (0.7 mg/kg i.v.) than by bicuculline. The cardiovascular response to GABA, but not to glycine, was potentiated by pretreatment with pentobarbitone (50 mg/kg i.p.). It was prolonged by diazepam (5 mg/kg i.p.) and aminooxyacetic acid (AOAA, 25 mg/kg i.p., 4h) but not by AOAA (1 h) or nipecotic acid (1–100 g i.c.v.). Following treatment with d-amphetamine (5 mg/kg i.p.) or reserpine (10 mg/kg i.p., 6h) the cardiovascular depression induced by GABA was attenuated. Propranolol (2.5+2.5 mg/kg i.v. + s.c.) or atropine (2 mg/kg i.v.) alone partially counteracted and in combination completely prevented the negative chronotropic response to GABA. Neither central noradrenaline depletion by means of -MMT (after carbidopa) and -MT nor pretreatment with phenoxybenzamine (20 g i.c.v.) influenced the response to GABA. The arterial hypotension induced by GABA was prolonged after a high dose of atropine (10 mg/kg i.v.) but appeared attenuated by a low dose (2 mg/kg i.v.) or by physostigmine (0.1 mg/kg i.v.). Central serotonergic activation by means of 5-hydroxytryptophan after benzerazid antagonized the cardiovascular actions of GABA. Spiroperidol (0.05 mg/kg i.v.) and apomorphine (1 mg/kg i.v.) did not affect the blood pressure reduction in response to GABA.It is concluded that i.c.v. GABA causes cardiovascular depression by activation of GABA receptors and that the response is mediated by the sympathetic and vagal systems. There were no indications for an involvement of central noradrenergic of dopaminergic mechanisms while a decreased central cholinergic and an increased serotonergic activity possibly contribute to the cardiovascular effects of GABA.