Species differences in the metabolism of ritobegron in vitro and assessment of potential interactions with transporters and cytochrome P450 enzymes.

Ritobegron, a selective ß3-adrenoceptor agonist, is the prodrug of the active compound, KUC-7322. We investigated species differences in its metabolism in vitro and the potential for drug-drug interactions with ritobegron. In rat, dog, monkey, and human liver microsomes, ritobegron was not metabolized by cytochrome P450 enzymes (CYPs). KUC-7322 was the only metabolite observed. Hydrolysis of ritobegron to KUC-7322 was likely catalyzed by carboxylesterases in human liver microsomes. The maximum velocity of the reaction (V(max))/Michaelis-Menten constant (K(m)) for hydrolysis of ritobegron to KUC-7322 was much higher in rat serum than those in other species. There were also species differences in the conjugation of KUC-7322. Sulfate conjugates of ritobegron were detected in all species, whereas glucuronide and glutathione conjugates of KUC-7322 were only observed in rat liver subcellular fractions. Ritobegron and KUC-7322 did not affect the CYP-mediated metabolism of probe substrates in human liver microsomes and organic anion transporter 1 (OAT1)-, OAT2-, OAT3-, organic cation transporter 2 (OCT-2)-, OCT3-, or organic cation/carnitine transporter 1 (OCTN1)-mediated uptake of probe substrates in S2 cells. Ritobegron, but not KUC-7322, inhibited P-glycoprotein-mediated digoxin transport in Caco-2 cells. Significant uptake of KUC-7322 was observed in OAT3-expressing S2 cells. Therefore, CYP-mediated drug-drug interactions are not likely when ritobegron is administered with CYP substrates or inhibitors. Ritobegron may increase the plasma concentrations of P-glycoprotein substrates, such as digoxin, and the plasma concentration of KUC-7322 may increase when it is administered in combination with OAT inhibitors such as probenecid.

Effects of ritobegron (KUC-7483), a novel selective ß3-adrenoceptor agonist, on bladder function in cynomolgus monkey.

We evaluated the pharmacological profile of ritobegron [KUC-7483; (-)-ethyl 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)-2,5-dimethylphenyloxy]acetate monohydrochloride] and its effects on the bladder in cynomolgus monkeys by in vitro and in vivo experiments. In vitro, ritobegron decreased the resting tension of the isolated bladder in a concentration-dependent manner (EC(50) 8.2 ± 2.3 × 10(-7) M; maximal relaxation 88.7 ± 3.7%). The ß(3)-adrenoceptor (AR) antagonist 3-(2-allylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol hydrochloride (SR58894A) produced a rightward shift of this concentration-response curve without altering the maximal response (pK(B) value 6.56 ± 0.35). In isolated atria, ritobegron increased the atrial rate only at high concentrations (EC(50) 6.5 ± 1.2 × 10(-5) M). Ritobegron had no effect on tracheal contraction at concentrations from 10(-9) to 10(-4) M, and even at the highest concentration tested, 10(-3) M, the maximal relaxation it induced was only 26.7 ± 8.1%. Tests of the selectivity of ritobegron for the bladder gave values of 79.3- and 1200-fold higher versus atria and trachea, respectively. In the in vivo study ritobegron significantly decreased intravesical pressure (ED(50) 1.44 mg/kg) without affecting either mean blood pressure or heart rate. In conclusion, ritobegron displayed potent and selective ß(3)-AR agonistic activity and relaxed the monkey isolated bladder, and in vivo it decreased intravesical pressure without affecting cardiovascular parameters. These results suggest that ritobegron may be a promising potential agent for the treatment of overactive bladder.

Multiple Direct Effects of the Dietary Protoalkaloid N-Methyltyramine in Human Adipocytes.

Dietary amines have been the subject of a novel interest in nutrition since the discovery of trace amine-associated receptors (TAARs), especially TAAR-1, which recognizes tyramine, phenethylamine, tryptamine, octopamine, N-methyltyramine (NMT), synephrine, amphetamine and related derivatives. Alongside the psychostimulant properties of TAAR-1 ligands, it is their ephedrine-like action on weight loss that drives their current consumption via dietary supplements advertised for 'fat-burning' properties. Among these trace amines, tyramine has recently been described, at high doses, to exhibit an antilipolytic action and activation of glucose transport in human adipocytes, i.e., effects that are facilitating lipid storage rather than mobilization. Because of its close structural similarity to tyramine, NMT actions on human adipocytes therefore must to be reevaluated. To this aim, we studied the lipolytic and antilipolytic properties of NMT together with its interplay with insulin stimulation of glucose transport along with amine oxidase activities in adipose cells obtained from women undergoing abdominal surgery. NMT activated 2-deoxyglucose uptake when incubated with freshly isolated adipocytes at 0.01-1 mM, reaching one-third of the maximal stimulation by insulin. However, when combined with insulin, NMT limited by half the action of the lipogenic hormone on glucose transport. The NMT-induced stimulation of hexose uptake was sensitive to inhibitors of monoamine oxidases (MAO) and of semicarbazide-sensitive amine oxidase (SSAO), as was the case for tyramine and benzylamine. All three amines inhibited isoprenaline-induced lipolysis to a greater extent than insulin, while they were poorly lipolytic on their own. All three amines-but not isoprenaline-interacted with MAO or SSAO. Due to these multiple effects on human adipocytes, NMT cannot be considered as a direct lipolytic agent, potentially able to improve lipid mobilization and fat oxidation in consumers of NMT-containing dietary supplements.

Inhibition of indoleamine 2,3-dioxygenase 1/2 prevented cognitive impairment and energetic metabolism changes in the hippocampus of adult rats subjected to polymicrobial sepsis.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection that may affect the brain. We investigated the role of indoleamine 2,3-dioxygenase (IDO-1/2) inhibition on long-term memory and energetic metabolism after experimental sepsis by caecal ligation and perforation (CLP). Experimental sepsis increased the activity of complexes I, II-III and IV at 24h after CLP, and IDO-1/2 inhibition normalized the activity of these complexes in the hippocampus. Wistar rats presented impairment of habituation and aversive memories 10days after CLP. Adjuvant treatment with the IDO inhibitor prevented long-term cognitive impairment triggered by sepsis.

Mechanism of D-amphetamine inhibition of protein synthesis.

At 1 h after intraperitoneal administration of D-amphetamine sulphate (15 mg/kg), rat brain polyribosomes show disaggregation accompanied by reduced capacity for in vitro peptide chain elongation. The direct action of amphetamine on cell-fine protein-synthesizing systems was therefore explored. When brain or liver polyribosomes from untreated rats were incubated with pH 5 enzyme, peptide chain elongation was not inhibited by the addition 4 mM amphetamine to the medium. On the other hand, an initiation-dependent system consisting of rat liver of brain mRNA and wheat germ S-30 fraction showed inhibition of [3H]leucine incorporation by 50% when 4 mM amphetamine were added. The metabolites of amphetamine, p-hydroxyamphetamine and p-hydroxynorephedrine, had no inhibitory action in either system, but the potent neurotoxin p-chloroamphetamine was a more powerful inhibitor of initiation than amphetamine. By using [3H]amphetamine, it was shown that amphetamine binds to the 80-S ribosomes of the wheat germ system. This binding depended on the presence in the system of natural liver or brain mRNA or several synthetic mRNAs, but was not promoted by polyuridylic acid as the messenger. Significantly, polyuridylic acid-dependent polyphenylalanine synthesis by the wheat germ system was not inhibited by amphetamine or p-chloroamphetamine. Therefore, it was concluded that amphetamine inhibits protein synthesis by interfering with initiation through a step related to formation of the mRNA ribosome complex.

Involvement of alpha-adrenoceptors in para-hydroxyamphetamine-induced head-twitch response.

The effect of some drugs with a higher selectivity for either the alpha 1- or alpha 2-adrenoceptors on the head-twitches induced by intracerebroventricular administration of p-hydroxyamphetamine (p-OHA) in mice, have been studied. Pretreatment with yohimbine increased the number of head-twitches induced by p-OHA, whereas pretreatment with clonidine or prazosin reduced the number of responses. The decrease in head-twitches produced by clonidine was completely antagonized by pretreatment with yohimbine. Pretreatment with 6-hydroxydopamine prior to the combined treatment with clonidine and p-OHA, resulted in recovery of the reduced level head-twitches to the level induced by p-OHA alone. Pretreatment with 6-hydroxydopamine alone resulted in a marked increase in the number of p-OHA-induced head-twitches. These results clearly indicate that a noradrenaline system in the brain may, at least in part, be involved in the p-OHA-induced head-twitches in mouse, most probably by modulating a serotonergic system which is responsible for the head-twitch response.

Sympathetic pupillary activity in infants.

In an attempt to learn the contribution of the sympathetic system to smaller pupillary size in infants, pupillary responses were tested to agents acting on this system in 12 ihfants and 23 young adults. Phenylephrine dilated the pupils of both groups by the same ratio, with infants' pupils reaching a lesser diameter. The responses to cocaine and hydroxyamphetamine were lower in children. It is concluded that in the first months of life the postganglionic sympathetic nerve releases less norepinephrine. This may be due to a lower number of sympathetic neurons. The post-synaptic apparatus may not be fully developed in infants.

Superior cervical ganglionectomy in monkeys: surgical technique.

Seven cynomolgus monkeys underwent a histologically confirmed left superior cervical ganglionectomy (SCGx). Unilateral ocular sympathetic denervation persisting for at least 2 yr was confirmed by ipsilateral ptosis, miosis, supersensitivity of pupillary dilation to topical phenylephrine, and profound pupillary hyporesponsiveness to topical hydroxyamphetamine. Intraocular pressure 8-9 and 23-27 months postoperatively were identical in the denervated and contralateral control eyes. This model should facilitate studies of aqueous humor physiology and pharmacology.

Hydroxyamphetamine increases intraocular pressure in rabbits.

OBJECTIVE: To determine the effect of norepinephrine (NE) released from endogenous ocular stores on intraocular pressure (IOP) and aqueous flow in rabbits. METHODS: The IOP was measured with a pneumatonometer, the aqueous flow with a scanning fluorophotometer, and the aqueous NE by methylation with catechol-O-methyltransferase in the presence of S-adenosyl-L-[methyl-(3)H]methionine. RESULTS: Hydroxyamphetamine increased IOP in a dose-dependent fashion. Surgical removal of the superior cervical sympathetic ganglion eliminated the increase in IOP and pupil diameter; preganglionic section of the cervical sympathetic trunk did not. Hydroxyamphetamine increased the concentration of NE in the aqueous. Increased IOP was not accompanied by increased aqueous flow and was eliminated by blockade of alpha(1)-adrenergic receptors but not beta- or alpha(2)-adrenergic receptors. CONCLUSIONS: Increased IOP after hydroxyamphetamine application is consistent with earlier suggestions that the nocturnal circadian increase in IOP in rabbits is mediated in part by NE released from ocular sympathetic nerves. However, failure of hydroxyamphetamine to increase aqueous flow and of beta-adrenergic blockade to blunt the increase in IOP does not support our suggestion that the nocturnal increase in IOP results in part from NE stimulation of ciliary process beta-adrenergic receptors and increased aqueous flow. CLINICAL RELEVANCE: In addition to increasing pupil diameter, hydroxyamphetamine increases IOP.

Effects of 4-hydroxyamphetamine on in vivo brown adipose tissue thermogenesis and feeding behavior in the rat.

The influence of 4-hydroxyamphetamine (4-OHAM) on food and water intake and in vivo brown adipose thermogenesis was examined in two experiments. In Experiment 1, female rats were treated with 0.00, 0.25, 0.50, 1.00, or 2.00 mg/kg 4-OHAM (ip) prior to assessment of interscapular brown adipose tissue (IBAT) thermogenesis. The 4-OHAM treatment induced dose-dependent activation of IBAT thermogenesis consistent with the enhanced serum levels of norepinephrine and epinephrine observed in 4-OHAM-treated rats immediately after temperature measurement. In Experiment 2, the influence of 4-OHAM on food and water intake was assessed during 120-min test intervals in female rats fed food and water ad lib. Although there was a trend for 4-OHAM to increase water intake, there was no significant effect of 4-OHAM (0.40, 0.80, 1.00, 2.00, and 4.00 mg/kg) on either food or water intake. These data suggest that brown adipose thermogenesis does not play a role in the anorexia induced by amphetamine or in the regulation of feeding.

4-OH amphetamine enhances retention of an active avoidance response in rats and decreases regional brain concentrations of norepinephrine and dopamine.

An .82 mg/kg dose of dl-4-OH amphetamine administered ip immediately following training in a one-way active avoidance task enhanced retention performance measured 24 hr later. In contrast, 4-OH amphetamine in a dose range of .41--1.65 mg/kg ip did not affect retention of a swim escape task. The behaviorally active dose of .82 mg/kg 4-OH amphetamine decreased dopamine concentrations in the amygdala and hippocampus. A dose of 8.2 mg/kg 4-OH amphetamine administered ip to naive untrained rats decreased concentrations of norepinephrine measured in the amygdala, cortex, hippocampus, hypothalamus, and midbrain; decreased concentrations of dopamine in the amygdala, cortex, hippocampus, and striatum; and significantly reduced concentrations of norepinephrine and epinephrine in the adrenal medulla. In addition, because the integrity of the adrenal medulla is necessary for the enhancing action of 4-OH amphetamine and because 4-OH amphetamine reduces concentrations of catecholamines in the brain and adrenal medulla, it is possible that 4-OH amphetamine affects retention performance by a dual action on the brain and the adrenal medulla.

Role of training conditions in discrimination of central nervous system stimulants by rats.

Amphetamine and some relate compounds were compared in rats trained to discriminate (+)-amphetamine (0.4, 1.0 or 1.6 mg/kg) or cocaine (10.0 mg/kg) from the non drug condition in a standard, two-bar procedure with food reinforcement (n=5-6). Amphetamine and cocaine were generalized completely with each other, in most cases at dose levels which did not greatly reduce the overall numbers of responses. The ED50 values for amphetamine and cocaine varied with the drug and dose used for training, and it was concluded that the stimuli produced by the two drugs were similar but may not be identical. There was an excellent correlation between ED50 values derived from indices of bar selection and percentage-responding on the drug-appropriate bar. Apomorphine was generalized with amphetamine only in the rats trained with the higher doses of amphetamine, and only when administered in doses which greatly reduced the overall number of responses. Para-hydroxyamphetamine increased responding on the drug-appropriate bar only when administered in high doses to the rats trained with the lowest dose of amphetamine (0.4 mg/kg). The results strengthen the evidence that the particular drug and dose level used for training can significantly affect the outcome of generalization tests, and challenge the notion that the discriminability of drugs is an immutable property that is amenable to absolute measurement.

Paradoxical effect of amphetamine in an endogenous model of the hyperkinetic syndrome in a hybrid dog: correlation with amphetamine and p-hydroxyamphetamine blood levels.

A telomian-beagle hybrid has been studied as a possible model for the hyperkinetic syndrome in children. Behavior tests showed that hybrids, like children, exhibit hyperactivity, impulsiveness, and impaired learning. Two groups of hybrid could be differentiated; the behaviour of one improved after amphetamine (responders) while that of the other did not (nonresponders). Moreover hybrids were less responsive than beagles to other effects of amphetamine such as stereotyped behaviour and hyperthermia. Measurement of blood levels of amphetamine and its active metabolite p-hydroxyamphetamine (pOA) showed that hybrids form less pOA. We propose that the lesser response of hybrids to toxic effects of amphetamine is due to this difference in amphetamine metabolism. Responders showed higher peak blood levels of amphetamine than nonresponders and their improvement on amphetamine correlated with blood levels of amphetamine. Therefore high levels of amphetamine appear to be necessary for its 'paradoxical' effect in this model. This suggests that amphetamine acts by activating both noradrenergic and dopaminergic neuronal systems in the CNS.

The contribution of the metabolite p-hydroxyamphetamine to the central actions of p-methoxyamphetamine.

RATIONALE: Para-methoxyamphetamine (PMA) is a substituted amphetamine that has been responsible for a number of fatalities in Australia and North America. Previous investigators have shown that p-hydroxyamphetamine (PHA), the primary metabolite of PMA, has effects on central neurotransmitter kinetics in vitro that are similar to those of the parent compound. In order to understand the role of PHA, it is necessary to determine both the in vivo actions and the concentrations achieved relative to those of PMA. OBJECTIVES: The effects of PHA and PMA on 5-hydroxytryptamine (5HT) and dopamine kinetics in brain were determined and the concentrations of each compound measured in blood and brain. METHODS: Animals were housed at 20-22C on a standard 12/12-h light/dark cycle. High speed chronoamperometry was used to compare the ability of PMA and PHA to alter 5HT and dopamine kinetics in the rat striatum in vivo. Concentrations of PHA and PMA in blood, whole brain and striatum were determined following a dose of PMA (10 mg/kg, IP.) using HPLC with fluorescence detection. RESULTS: PHA was more effective than PMA at evoking neurotransmitter release and inhibiting the uptake of dopamine. However, both compounds were approximately equipotent 5HT uptake inhibitors. PMA and PHA concentrations in whole brain and striatum peaked within 30 min of the administered dose, whereas blood concentrations of both compounds peaked 1 h after the dose. PHA concentrations in both blood and brain were consistently much lower than PMA concentrations. CONCLUSIONS: These data indicate that although PHA is more effective than PMA at altering 5HT and dopamine kinetics in vivo, it is unlikely to achieve sufficient brain concentrations to contribute to the central effects of PMA.

Systemic and nigral application of amphetamine both cause an increase in extracellular concentration of ascorbate in the caudate nucleus of the rat.

Unmodified microvoltammetric electrodes and electrochemically modified cylindrical electrodes, both constructed from carbon fibers, were used to determine changes in ascorbate concentration in the caudate nucleus of the anesthetized rat following administration of amphetamine. The increased ascorbate levels with systemic amphetamine originate from a CNS source, since the response is not eliminated in adrenalectomized animals, and does not occur when p-hydroxyamphetamine, an agent with peripheral actions similar to amphetamine, is used as a stimulus. Local application of amphetamine in the caudate nucleus results in an increase in catechols. However, this procedure results in a decrease of the extracellular concentration of ascorbate. Unilateral nigral infusion of dopamine or amphetamine leads to a significant increase in the concentrations of ascorbate detected bilaterally in the caudate nuclei with in vivo electrochemistry. In contrast, the paradigm causes a decrease in extracellular catechols on the ipsilateral side and an increase on the contralateral side. These experiments demonstrate that the extracellular level of ascorbate is not correlated with dopamine secretion. Furthermore, the data show that the action of amphetamine on ascorbate concentration in the caudate nucleus is in part mediated by the nigral actions of amphetamine.

The effects of peripheral D-amphetamine, 4-OH amphetamine, and epinephrine on maintained discharge in the locus coeruleus with reference to the modulation of learning and memory by these substances.

D-Amphetamine, 4-OH amphetamine, and epinephrine have been shown in many behavioral studies to facilitate memory when given post-training. The effect of these drugs on the maintained discharge of cells in the locus coeruleus (LC) was investigated using a route of administration (intraperitoneal) and a log-dose range of these drugs comparable to those used in the behavioral experiments. D-Amphetamine profoundly suppressed maintained discharge: an inhibitory effect was observed at every dose (0.1, 1.0, 10.0 mg/kg). In contrast, only the highest dose of 4-OH amphetamine (8.2 mg/kg) inhibited activity in the LC, and this effect was a modest one. Unlike the amphetamines, epinephrine (500 micrograms/kg) elevated maintained discharge. These results are discussed in the context of the hypothesized involvement of the LC in the enhancement of memory by these drugs.

Attenuation of amphetamine-induced enhancement of learning by adrenal demedullation.

These experiments investigated the effect of immediate posttrial administration of peripherally acting DL-4-hydroxyamphetamine on retention of a one-trial inhibitory avoidance response in intact, adrenal medullectomized, sympathectomized, and medullectomized and sympathectomized rats. In intact rats, 0.82 mg/kg of DL-4-OH-amphetamine enhanced retention performance. In rats sympathectomized by peripheral 6-hydroxydopamine, 24 h prior to training, a lower dose of 4-OH-amphetamine (0.21 mg/kg) was most effective in enhancing retention. Adrenal demedullation abolished the memory enhancing effects of DL-4-OH-amphetamine and also D-amphetamine. These findings suggest that the memory enhancing effects of DL-4-OH-amphetamine and D-amphetamine involve adrenal medullary catecholamines.

Aqueous humor dynamics in a series of patients with third neuron Horner's syndrome.

We studied aqueous humor dynamics in a group of 21 human subjects, each of whom had third neuron Horner's syndrome in one eye and a normal fellow eye. The diagnosis had been made at least four months before this study began. We estimated the degree of involvement with Lowenstein-Loewenfeld pupillography with and without hydroxyamphetamine. Surprisingly, aqueous humor dynamics were normal in the eyes with Horner's syndrome. The mean intraocular pressure was 1 mm Hg less than that of the normal eyes. Aqueous humor flow and tonographic facility of outflow were normal, as was the response to the beta-blocker timolol. However, the response of the eyes with Horner's syndrome to epinephrine was abnormal. The normal eyes showed an increase in aqueous humor flow after epinephrine administration and the eyes with Horner's syndrome showed a decrease.

Hydroxyamphetamine mydriasis in normal subjects.

Hydroxyamphetamine eyedrops are used to help localize the lesion in Horner's syndrome. Because normal variability in the response to the eyedrops may influence the interpretation of test results in patients with Horner's syndrome, we studied both the interocular variability of the drug's mydriatic effect within each normal subject and the variation between individuals. We used photographs to document the variability among 26 normal subjects. Hydroxyamphetamine hydrobromide 1% eyedrops (Paredrine) were placed in both eyes of normal subjects in the same way that patients with Horner's syndrome are tested. The drug produced a mean increase in pupil size of 1.96 mm (+/- 0.61 S.D.) in the 52 eyes tested. In normal subjects, the mydriatic effect of hydroxyamphetamine was symmetric in each pair of eyes. The mean interocular asymmetry of mydriasis as measured by the difference in dilation (right eye dilation minus left eye dilation) was -0.087 mm (+/- 0.29 S.D.). Thus, the variability of hydroxyamphetamine mydriasis from one eye to the other in a single subject was much lower than the variability between subjects.

Hydroxyamphetamine mydriasis in Horner's syndrome.

We studied hydroxyamphetamine hydrobromide 1% (Paredrine) mydriasis in 54 patients with Horner's syndrome to determine its effectiveness in distinguishing preganglionic lesions from postganglionic lesions. The difference in pupillary dilation between the unaffected and affected sides was used as a measure of the hydroxyamphetamine effect. We found that patients who had clinical evidence of damage to the postganglionic neuron of the oculosympathetic pathway had less pupillary dilation on the affected side. In contrast, almost all patients judged to have clinical evidence of preganglionic lesions dilated more on the affected side. We determined the probability that a given difference in pupillary dilation between the involved and uninvolved side is the result of a postganglionic lesion.