Antagonism of serotonin3 (5-HT3) receptors within the blood-brain barrier prevents cisplatin-induced emesis in dogs.

Recently discovered serotonin3 (5-HT3) receptor antagonists are potent antiemetics in cytotoxic drug-induced vomiting. The specific site where 5-HT3 receptor antagonists act to abolish emesis is controversial. The major objective of this study was to determine whether the antiemetic effect of 5-HT3 receptor antagonists is exerted in the brain areas that reside inside or outside of the blood-brain barrier. Tropisetron, zatosetron (LY277359 maleate) and its quaternary analog zatosetron-QUAT were used in this study. Zatosetron and zatosetron-QUAT showed high affinity and selectivity for 5-HT3 receptors in radioligand binding studies. Both compounds antagonized 5-HT-induced bradycardia in rats with an approximate ID50 of 0.7 and 0.2 microgram/kg i.v., respectively. Zatosetron and tropisetron significantly inhibited cisplatin-evoked emesis in dogs (estimated ID50 values of 34.4 +/- 2.3 micrograms/kg and 108.3 +/- 4.8 micrograms/kg i.v., respectively). Zatosetron-QUAT (0.01-1.0 mg/kg i.v.) had no effect. [14C]-zatosetron-QUAT (100 micrograms/kg) was not detected in the brain after i.v. administration to rats, consistent with the inability of charged compounds to achieve significant brain concentrations. However, i.c.v. administration (100 ng/kg) of zatosetron-QUAT reduced emetic episodes significantly (11.6 +/- 1.6 vs. 2.8 +/- 1.2). These studies suggest that, in dogs, antagonism of 5-HT3 receptors located within the blood-brain barrier is important to block cisplatin-induced emesis.

In vitro metabolism of zatosetron. Interspecies comparison and role of CYP 3A.

The major in vivo human metabolite of zatosetron is 8-alpha-methyl,8-beta-oxo zatosetron [N-O (1) zatosetron]. N-Desmethyl zatosetron (NdM zatosetron) and 3-hydroxy-zatosetron (3-OH-zatosetron) are minor human metabolites. In the rat, the primary in vivo metabolite is 3-OH-zatosetron. The enzyme kinetics of zatosetron metabolism were determined using human, rat, and monkey hepatic microsomal incubations. In the rat, the intrinsic clearance (IC; IC = Vmax/KM = microliter/min/mg of protein) of 3-OH-zatosetron (IC = 5.2) was favored over N-O (1) zatosetron (IC = 3.0) and NdM zatosetron (IC = 1.4). In the monkey, N-O (1) zatosetron exhibited the highest IC (IC = 7.0) relative to that for 3-OH-zatosetron (IC = 4.4) and NdM zatosetron (IC = 2.9). Monkey microsomes also formed 8-beta-methyl,8-alpha-oxo zatosetron (IC = 2.2). In two human samples (identified as HL-E and HL-J), the metabolic clearance of zatosetron favored the formation of N-O (1) zatosetron (HL-E, IC = 6.9; HL-J, IC = 2.9) over NdM zatosetron (HL-E, IC = 0.6; HL-J, IC = 0.3) and 3-OH-zatosetron (HL-E and HL-J, IC = 0.1). These results indicate that the monkey is better than the rat as a model for the exposure of humans to zatosetron and its metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

Subchronic toxicity, metabolism, and pharmacokinetics of the aminobenzamide anticonvulsant ameltolide (LY201116) in rhesus monkeys.

Studies were undertaken to define the subchronic toxicologic profile of ameltolide, an aminobenzamide anticonvulsant, in young adult rhesus monkeys. Daily doses of ameltolide, dissolved in 10% aqueous acacia, were administered orally via nasogastric intubation at dosages of 5, 10, 20, 45, and 100 mg/kg. Deaths occurred in two monkeys, one each at 45 and 100 mg/kg, which were directly attributable to the effects of the compound. The exact cause of death in these monkeys was not readily apparent. A third monkey (100 mg/kg) was killed moribund on Day 82 of the study due to conditions not directly related to treatment. Clinical signs in monkeys treated with 100 mg/kg included convulsions, diarrhea, weakness, inappetance, vomition, and ataxia. Plasma concentrations of the N-acetyl metabolite of ameltolide were greater than parent drug concentrations by one to two orders of magnitude. Mean area under the plasma-time curve (AUC) values for ameltolide were larger than expected at doses of 20 mg/kg or greater, while AUC values for the metabolite were less than expected at 45 and 100 mg/kg. These findings suggest a saturation of metabolism and/or excretion at the two higher doses. Similar nonlinearity was seen with mean peak concentrations for both parent and metabolite. No specific target organ toxicity was found on histological evaluation of tissue sections. Methemoglobin concentration was increased in monkeys given 45 or 100 mg ameltolide/kg. This change was not considered to be toxicologically important as there were no corroborative clinical, gross, or histopathological findings. Ameltolide administered by nasogastric intubation at doses up to 20 mg/kg/day for 3 months did not cause any toxicologically important alterations in rhesus monkeys.

Relation of plasma and brain concentrations of the anticonvulsant ameltolide to its pharmacologic effects.

Ameltolide, a newly described anticonvulsant, was studied to determine the relation between dose administered, plasma and brain concentrations, and pharmacologic effects. The relation of the N-acetyl metabolite and the OH-N-acetyl metabolite to the dose administered and to the pharmacologic effects was also determined. Ameltolide plasma concentrations in both mice and rats were linearly related to dose administered over the entire dose range from low doses, at which the anticonvulsant effects were noted, to high doses, at which neurologic impairment occurred. The plasma concentrations of the metabolites were not as consistently linearly dose-related in the two species. In rats, the brain concentrations of ameltolide were highly correlated with plasma concentrations and the doses administered. Ameltolide was shown to have a phenytoin (PHT)-like anticonvulsant profile in vitro in the cortical slice preparation. These data confirm the potent anticonvulsant profile of ameltolide and the lack of significant activity of the metabolites.

Evaluation of LY203647 on cardiovascular leukotriene D4 receptors and myocardial reperfusion injury.

In vitro studies have shown LY203647 to be a selective antagonist of contractile responses to leukotriene (LT) D4 and LTE4 in guinea pig ileum, trachea and lung parenchyma. In pithed rat, i.v. injection of LTD4 produced pressor responses that were selectively antagonized by LY203647 in a dose-dependent manner [ED50 7.5 (6.0-9.5) mg/kg, i.v.]. In normal anesthetized rats and dogs, LTD4 reduced aortic blood flow and stroke volume in association with systemic vasoconstriction, variable blood pressure responses and no change in cardiac rate. LTD4 did not alter myocardial contractility corrected for alterations in afterload. Pretreatment of rats and dogs with LY203647 (1-10 mg/kg, i.v.) produced dose-related inhibition of the myocardial and systemic hemodynamic effects of LTD4, whereas coadministration of LY203647 reversed established myocardial depression and systemic and pulmonary vasoconstriction during continuous infusion of LTD4 in dogs. LY203647 (10 mg/kg over 90 min, i.v.) infusion in normal dogs abolished or greatly antagonized hemodynamic responses to LTD4 for 6 hr. In subsequent experiments, myocardial infarct size was measured after 1 hr of occlusion of the circumflex coronary artery and 5 hr of reperfusion. LY203647 (10 mg/kg over 90 min, i.v.) treatment did not alter cardiovascular parameters when compared to time-related alterations observed in control dogs. ST segment deviation and the intensity and duration of cardiac arrhythmias associated with coronary artery occlusion and reperfusion also were similar between groups. Resultant infarct sizes were 46 +/- 1 and 45 +/- 1% of the left ventricular mass placed at risk in control and LY203647-treated dogs, respectively. Present data illustrate the prominent cardiac and systemic hemodynamic effects of LTD4 and indicate that LY203647 produces selective and sustained antagonism of cardiovascular LTD4 receptors. Lack of containment of infarction by LY203647 suggests that endogenous cysteinyl-LT do not contribute to reperfusion injury of ischemic myocardium.

Preclinical studies on LY237733, a potent and selective serotonergic antagonist.

8B-N-cyclohexyl-6-methyl-1(1-methylethyl)ergoline-8-carboxamide (LY237733) is an ergoline with potent and highly selective 5-hydroxytryptamine (5-HT) antagonist activity. The in vitro radioligand displacement studies showed that LY237733 has a preferential affinity for 5-HT1c and 5-HT2 receptors compared to other monoaminergic receptors. This characteristic is shared with other previously described ergoline 5-HT antagonists, such as LY53857 and sergolexole. In parallel ligand displacement assays, LY237733 had a similar potency to sergolexole. LY237733 was equipotent to sergolexole, but slightly less potent than LY53857 in the antagonism of 5-HT-induced elevation in blood pressure and quipazine-induced elevation in corticosterone levels, which are considered to be measures of 5-HT2 and possibly 5-HT1c antagonist activity. LY237733 failed to antagonize pergolide or 8-hydroxy-2-(di-n-propylamino)tetralin-induced elevations in serum corticosterone levels, indicating selectivity for the 5-HT1c/2 receptor, relative to 5-HT1a and D2 dopaminergic receptors. The only in vivo response that could be detected after administration of LY237733 alone in doses less than 1 mg/kg was the amplification of male rat sexual behavior. LY237733 was 10 to 100 times more potent than LY53857 or sergolexole in augmenting sexual responses of male rats with different levels of sexual response capacity. LY237733 has a much longer serum half-life than sergolexole. These studies have provided the pre-clinical rationale to evaluate the effects of this compound in the treatment of sexual disorders such as psychogenic erectile dysfunction, and other therapeutic indications for a 5-HT2 antagonist, including depression, anxiety, schizophrenia and migraine.

Synthesis and pharmacological evaluation of a major metabolite of ameltolide, a potent anticonvulsant.

The 4-aminobenzamides have provided several anticonvulsants that have been extensively investigated. Ameltolide, 4-amino-N-(2,6-dimethylphenyl)benzamide (compound 2,LY201116), is the most potent analogue studied to date. This drug is inactivated in vivo by metabolic N-acetylation and addition of a hydroxy moiety to one of the methyl substituents, resulting in compound 7,N-[4-[[[2-(hydroxymethyl)-6- methylphenyl] amino] carbonyl] phenyl] acetamide. This metabolite was prepared in five steps from a readily available starting material. Compound 7 and its nonacetylated analogue 6 were compared to ameltolide as anticonvulsants. After oral administration to mice, the MES ED50 values of ameltolide, 6, and 7 were 1.4, 10.9, and greater than 100 mg/kg, respectively, demonstrating that hydroxylation and acetylation dramatically decrease the anticonvulsant potency of ameltolide. This rank order of MES anticonvulsant potency was also seen after iv administration to mice, suggesting that these data reflect intrinsic pharmacological activities. After oral administration of 2.0 mg/kg of ameltolide to mice, parent drug, N-acetyl metabolite 3, and the hydroxy metabolite 7 were detected in plasma; the Cmax values were 572, 387, and 73 ng/mL, respectively. Compound 7 was the primary metabolite excreted in urine. These data indicate that 7 is a major metabolite of ameltolide, but does not contribute significantly to the pharmacological effects seen after administration of ameltolide to mice.

Antagonism of leukotriene B4 receptors does not limit canine myocardial infarct size.

Injection of leukotriene (LT)B4 (0.1-3 micrograms/kg i.v.) in normal anesthetized dogs produced dose-related leukopenia that was accompanied by arterial hypotension and tachycardia at higher tested doses. LTD4 (0.1-3 micrograms/kg i.v.), in contrast, increased arterial blood pressure, lowered cardiac rate and produced little change in arterial blood leukocyte count. Continuous infusion of LY255283 [(1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)- heptyloxy)phenyl)ethanone] (0.33 mg/kg/min i.v.), a selective LTB4 receptor antagonist, resulted in near complete inhibition of leukopenic, hypotensive and tachycardic responses to LTB4 (3 micrograms/kg i.v.) challenge over a 6-hr test period. Persistent antagonism of canine LTB4 receptors was associated with high circulating levels of LY255283 that were bound extensively to plasma proteins. In subsequent experiments, myocardial infarct size was measured following 1 hr of occlusion of the circumflex coronary artery and 5 hr of reperfusion in control dogs infused with vehicle, and in dogs receiving LY255283 (0.33 mg/kg/min i.v.). Drug and vehicle were infused continuously beginning 15 min before coronary artery occlusion. LY255283 treatment essentially did not alter base-line cardiovascular parameters or myocardial oxygen demand when alterations were compared to time-related changes observed in control dogs. LY255283 infusion also did not alter the degree of myocardial ischemia or the intensity and duration of cardiac arrhythmias associated with coronary artery occlusion and reperfusion. Resultant infarct sizes were 43 +/- 5% of the left ventricle placed at risk in control dogs and 32 +/- 5% in dogs given LY255283; this difference was not statistically significant. The extent of left ventricle placed at risk was similar between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptamine2 receptor antagonist activity of the acid metabolite (1-isopropyl dihydrolysergic acid) of the ergoline ester, sergolexole (LY281067).

The ergoline esters, LY53857 [6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid 2-hydroxy-1-methylpropylester (Z)-2-butenedioate] and sergolexole (LY281067) ([trans-(8 beta)]6-methyl-1-[1-methylethyl]ergoline-8-carboxylic acid, 4-methoxycyclohexyl ester (maleate salt] are potent 5-hydroxytryptamine2 (5-HT2) receptor antagonists in vivo and in vitro. Ester hydrolysis of either compound results in the formation of 1-isopropyl dihydrolysergic acid which in rats is a major metabolite of these ergoline esters. The present study details the pharmacological activity of 1-isopropyl dihydrolysergic acid and examines its relative contribution to the 5-HT2 receptor antagonism seen after sergolexole in rats. In vitro, 1-isopropyl dihydrolysergic acid was a competitive antagonist of 5-HT2 receptors in the rat jugular vein, with a dissociation constant approximating 10(-7) M. After i.v. administration to pithed rats, 1-isopropyl dihydrolysergic acid also antagonized the pressor response produced by serotonin, an in vivo estimate of vascular 5-HT2 receptor blockade. In fact, after i.v. administration, 1-isopropyl dihydrolysergic acid was nearly one-third as potent as sergolexole. After i.p. administration, 1-isopropyl dihydrolysergic acid was approximately one-tenth to one-thirtieth as potent as sergolexole. Likewise, 1-isopropyl dihydrolysergic acid antagonized central 5-HT receptors as measured by blockade of quipazine-induced increases in serum corticosterone concentration in rats and was approximately one-twentieth as potent as sergolexole.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism, disposition, and pharmacokinetics of a potent anticonvulsant, 4-amino-N-(2,6-dimethylphenyl)benzamide (LY201116), in rats.

The metabolism, disposition, and pharmacokinetics of 4-amino-N-(2,6-dimethylphenyl)benzamide (LY201116) have been studied in rats. 14C-LY201116 was well absorbed (approximately 94%) from the gastrointestinal tract following oral administration. Of the dose administered, 64.5% was excreted in the urine and 29% in the bile; with the majority being excreted during the first 24 hr. Peak plasma levels of LY201116 were observed at 0.75 hr, whereas peak plasma concentrations of radioactivity were seen at 2 hr after dosing. Maximum levels of radioactivity were observed at 2 hr in all of the tissues studied. The elimination of radioactivity from the tissues was monophasic with a mean half-life of 3.4 hr. Biotransformation of LY201116 in rats was investigated by quantitating and isolating metabolites from urine and plasma. The major route of metabolism was N-acetylation to form 4-(acetylamino)-N-(2,6-dimethylphenyl)benzamide (ADMP), and subsequent hydroxylation to form 4-(acetylamino)-N-(2-hydroxymethyl-6-methylphenyl)benzamide (HADMP). Two hr after oral dosing with 14C-LY201116, ADMP and HADMP comprised 92% of the total radioactivity in the plasma. The major urinary metabolite, accounting for 63% of the radioactivity in the urine, was HADMP. The elimination of LY201116 from the systemic circulation following iv administration was monophasic, with a terminal t1/2 of 9.4 min. The volume of distribution was 911 ml/kg and the plasma clearance was 66.9 ml/min/kg.

Metabolism of the prodrug DEGA (N-(2,6-dimethylphenyl)-4-[[(diethylamino)acetyl]amino]benzamide) to the potent anticonvulsant LY201116 in mice. Effect of bis-(p-nitrophenyl)phosphate.

In mice, the diethylglycineamide analogue of LY201116, DEGA (N-(2,6-dimethylphenyl)-4-[[(diethylamino)acetyl]amino]benzamide), is metabolized by consecutive N-deethylations for form MEGA and GA; the monoethylglycineamide and glycineamide analogues of LY201116, respectively. All of these compounds are in turn hydrolyzed to form LY201116 [4-amino-N-(2,6-dimethylphenyl)benzamide]. LY201116 is N-acetylated to form the N-acetyl metabolite, NAC. NAC is also deacetylated to reform LY201116. All of the above compounds inhibit maximal electroshock-induced seizures (MES) in mice. After oral administration, the potencies of these compounds were similar at their time of peak anticonvulsant effect. However, the MES ED50 values for the above compounds 5 min after iv dosing were 43, 13, 2, and 0.5 mg/kg for DEGA, MEGA, GA, and LY201116, respectively. Similar plasma levels of LY201116 were produced in mice 5 min after iv dosing with the respective ED50 values of the above compounds, which suggested that all of the compounds produced their anticonvulsant effects via LY201116. The in vivo metabolism of DEGA and MEGA but not GA to LY201116 was inhibited by the acylamidase inhibitor bis-(p-nitrophenyl) phosphate (BNPP). Mice predosed with BNPP were not protected by DEGA and MEGA from MES-induced seizures and the plasma samples contained little or no LY201116. The metabolism of GA to LY201116 was not inhibited by BNPP, and GA was an active anticonvulsant in BNPP-pretreated mice. The apparent iv potency of DEGA increased dramatically with time after dosing, again suggesting time-dependent, metabolically mediated liberation of the more potent anticonvulsant LY201116.

Electrophysiologic and antiarrhythmic activities of 4-amino-N-[2-(diethylamino)ethyl]-3,5-dimethylbenzamide, a sterically hindered procainamide analogue.

Procainamide is a widely used antiarrhythmic that is fraught with therapeutic limitations such as a short half-life, production of autoimmune antibodies and a lupus-like syndrome, and complex pharmacokinetics. We synthesized the congeners of procainamide possessing one or two methyl substituents ortho to the 4-amino moiety (compounds 4 and 5, respectively), in order to sterically encumber the 4-amino substituent and prevent or diminish the rate of metabolic N-acetylation. Moreover, we anticipated that this structural alteration might eliminate the autoimmune toxicities associated with procainamide. Like procainamide, the two methylated analogues significantly reduced the rate of rise and amplitude of the action potential when studied in isolated canine Purkinje fibers. Whereas procainamide caused no significant change in action potential duration (APD), both methylated congeners significantly reduced APD at 70% and 95% repolarization. Moreover, the dimethylated congener was significantly more efficacious than procainamide in reducing ERP (effective refractory period) and increasing the ERP/APD70. The ability of these compounds to block ouabain-induced arrhythmias was studied in anesthetized dogs. Addition of two methyl groups ortho to the amine produced an increase in potency: The conversion doses for procainamide and the monomethyl and dimethyl congeners were 19.0, 18.3, and 14.3 mg/kg, respectively, following iv administration. After iv administration to rats, procainamide was extensively metabolized to N-acetylprocainamide and displayed a half-life of 0.4 h. In contrast, dimethylprocainamide was not metabolized by N-acetylation, had a half-life of 1.4 h, and provided greater peak plasma concentrations. Thus, addition of methyl substituents ortho to the 4-amino group of procainamide alters the electrophysiological characteristics of the compound, increases its potency against ouabain-induced arrhythmias in vivo, increases its plasma half-life, and prevents N-acetylation.

Preclinical pharmacology of a new serotonergic receptor antagonist, LY281067.

The preclinical pharmacologic activity of LY281067 shows it to be a potent and highly selective serotonergic (5-HT2) receptor antagonist. Based upon binding studies with 5-HT2 receptors in brain cortical membranes and block of 5-HT-induced contractions in the rat jugular vein, LY281067 showed high affinity at 5-HT2 receptors with a dissociation constant of approximately 1 nM. Furthermore, LY281067 was a highly selective 5-HT2 receptor antagonist without appreciably binding to 5-HT1, D1 or D2 receptors or interacting with histamine (H1), cholinergic, beta adrenergic or alpha-1 adrenergic receptors in smooth muscle. LY281067 had modest affinity at alpha-2 receptors with a dissociation constant of approximately 100 nM. Oral bioavailability of LY281067 in spontaneously hypertensive rats was excellent with an oral to i.v. dose ratio approximating 4, based upon blockade of pressor responses to 5-HT as an in vivo estimate of 5-HT2 receptor antagonist activity. Furthermore, LY281067 blocked quipazine-induced increase in serum corticosterone concentration, an increase thought to be mediated by activation of central 5-HT receptors. After oral administration to rats, LY281067 antagonized vascular 5-HT2 receptors with a relatively long duration of action (greater than 6 hr), an observation likely to be related to plasma concentrations of both the parent and an active metabolite. Lastly, LY281067 was relatively nontoxic, possessing a therapeutic index of approximately 1000 after oral administration to rats. In summary, LY281067 is a potent and highly selective, orally active 5-HT2 receptor antagonist with a relatively long duration and wide margin of therapeutic safety.

Discovery and anticonvulsant activity of the potent metabolic inhibitor 4-amino-N-(2,6-dimethylphenyl)-3,5-dimethylbenzamide.

Compound 2 [4-amino-N-(2,6-dimethylphenyl)benzamide] is an effective anticonvulsant in several animal models. For example, following oral administration to mice, it antagonized maximal electroshock (MES) induced seizures with an ED50 of 1.7 mg/kg. During drug disposition studies with 2, we found that it was rapidly metabolized by N-acetylation. Thirty minutes after oral administration of 1.7 mg/kg of 2 to mice, plasma concentrations of parent drug and the N-acetyl metabolite 5 were 1.09 and 0.41 microgram/mL, respectively. Six hours postadministration the concentrations were 0.23 and 0.22 microgram/mL, respectively. In order to sterically preclude or diminish the rate of metabolic N-acetylation, we synthesized analogues of 2 possessing either one (3) or two (4) methyl groups ortho to the 4-amino substituent. Both compounds antagonized MES-induced seizures after administration to mice; oral ED50 values for 3 and 4 were 3.5 and 5.6 mg/kg, respectively. Compound 3 was rapidly metabolized by N-acetylation. However, 4 provided exceptionally high and long-lived plasma concentrations of parent drug; no N-acetyl metabolite could be detected. While 2 and 3 had no pharmacologically relevant effects on hexobarbital-induced sleeping time in mice, 4 was a potent, dose-dependent potentiator of sleeping time. Oral administration of 375 micrograms/kg led to a 61% increase in sleeping time relative to control values. Thus, 4 represents one of the most potent potentiators of hexobarbital-induced sleeping time described to date.

Single-dose and steady-state pharmacokinetics of tomoxetine in normal subjects.

A pharmacokinetic profile of tomoxetine, a selective norepinephrine uptake inhibitor, was developed in human volunteers following single and multiple oral administrations. Following the administration of a single 90-mg oral dose of tomoxetine to four normal volunteers, the plasma half-life was 4.3 +/- 0.5 hours. Mean plasma clearance was 0.60 +/- 0.14 L/Kg/hr, and the mean volume of distribution was 3.7 +/- 0.9 L/kg. Multiple doses of tomoxetine (20 mg bid and 40 mg bid) for seven days were administered to an additional seven subjects. The data appeared to have a bimodal distribution. The mean plasma half-life determined following the last dose was 4.6 +/- 0.5 hours in five subjects. The other two subjects, one at each dose level, demonstrated accumulation of tomoxetine occurring from the first to last dose where tomoxetine disappeared from plasma with a mean half-life of 19 hours.

Inhibition of drug metabolism by fluoxetine.

Fluoxetine hydrochloride (Lilly 110140: 3-p-trifluoromethylphenoxy-3-phenyl-N-methyl-propylamine hydrochloride) inhibited the metabolism of hexobarbital and ethinamate in rodents and prolonged the hypnotic effects of these drugs.