Search

Pravadoline is a new chemical entity with analgesic activity in humans. This report describes the pharmacology of pravadoline and compares the activity of pravadoline with that of two major classes of analgesics, the opioids and the nonsteroidal anti-inflammatory drugs (NSAIDs). Like the NSAIDs, pravadoline inhibited the synthesis of prostaglandins (PGs) in mouse brain both in vitro (IC50, 4.9 microM) and ex vivo (ED50, 20 mg/kg p.o.) and displayed antinociceptive activity in rodents subjected to a variety of chemical, thermal and mechanical nociceptive stimuli. Administration of pravadoline prevented the writhing response induced by i.p. administration of acetylcholine (ED50, 41 mg/kg p.o.) or PGE2 (ED50, 24 mg/kg p.o.) and prolonged the response latency induced by tail immersion in hot water at a temperature of 55 degrees C (minimum effective dose, 100 mg/kg s.c.). In the rat, treatment with pravadoline prevented acetic acid-induced writhing (ED50, 15 mg/kg p.o.), brewer's yeast-induced hyperalgesia (Randall-Selitto test) (minimum effective dose, 1 mg/kg p.o.), the nociceptive response induced by paw flexion in the adjuvant-arthritic rat (ED50, 41 mg/kg p.o.) and bradykinin-induced head and forepaw flexion (ED50, 78 mg/kg, p.o.). The antinociceptive activity of pravadoline cannot be explained by an opioid mechanism, because pravadoline-induced antinociception was not antagonized by naloxone (1 mg/kg s.c.) and pravadoline did not bind to opioid receptors at concentrations up to 10 microM. However, like the opioid analgesics, pravadoline diminished the electrically induced twitch response of mouse vas deferens preparations, but, in contrast to opioids, this action of pravadoline was not attenuated by naloxone. The possibility is discussed that this effect of pravadoline upon isolated tissues may contribute to its antinociceptive activity. In contrast to NSAIDs, pravadoline was more potent ex vivo as an inhibitor of the formation of PGs in brain vs. stomach. In addition, pravadoline failed to produce gastrointestinal lesions when administered p.o. to rats or mice, and did not possess significant anti-inflammatory activity at antinociceptive doses. Also unlike NSAIDs, pravadoline inhibited rat gastrointestinal transit when administered at doses similar to those which were antinociceptive. The overall pharmacologic profile of pravadoline suggests that the compound may be capable of managing more diverse or more severe pain than is achieved by anti-inflammatory analgesics, without producing side effects commonly associated with either the opioid or the nonopioid analgesics.

Napamezole is an alpha-2 adrenergic receptor antagonist and a selective inhibitor of 5-hydroxytryptamine re-uptake in vitro. In the present study, napamezole was evaluated in vivo for its ability to antagonize alpha-2 adrenergic receptors and to inhibit 5-hydroxytryptamine re-uptake. The alpha-2 blocking activity of napamezole was demonstrated by its ability to: 1) antagonize clonidine-induced antinociception in mice (ED50 value, 36 mg/kg p.o.; 3 mg/kg s.c.); 2) enhance norepinephrine turnover in rat brain (minimum effective dose, 30 mg/kg p.o.); and 3) enhance locus coeruleus neuronal firing (active at doses greater than or equal to 1 mg kg i.v.) and to reverse clonidine-induced suppression of locus coeruleus firing in rats. The rank order of potencies of napamezole and reference alpha-2 antagonists to inhibit clonidine-induced antinociception (based upon s.c. ED50 values) were: idazoxan greater than yohimbine greater than rauwolscine greater than or equal to napamezole greater than tolazoline greater than or equal to piperoxan greater than RS21361. The relative potencies of compounds to enhance alpha-methyltyrosine-induced depletion of forebrain norepinephrine following p.o. administration were: idazoxan = yohimbine greater than mianserin greater than napamezole greater than RS21361. The ability of each of these compounds to enhance alpha-methyltyrosine-induced depletion of rat-forebrain norepinephrine was reversed by the administration of clonidine. These results indicate that napamezole blocks alpha-2 adrenergic receptors in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Napamezole (2-[3,4-dihydro-2-naphthalenyl)methyl]-4,5-dihydro-1H- imidazole-monohydrochloride) is a selective alpha-2 adrenergic receptor antagonist and a monoamine re-uptake inhibitor in vitro. The alpha adrenergic antagonist activity of napamezole was determined in vitro in rat brain receptor binding assay using [3H]clonidine and [3H]prazosin for alpha-2 and alpha-1 receptors, respectively. The Ki values for napamezole were 28 nM (alpha-2) and 93 nM (alpha-1). The relative potencies for inhibiting [3H]clonidine binding were: phentolamine greater than idazoxan greater than napamezole greater than mianserin greater than yohimbine greater than piperoxan greater than rauwolscine greater than tolazoline much greater than prazosin; and for inhibition [3H]prazosin binding they were: prazosin greater than phentolamine greater than mianserin greater than napamezole greater than yohimbine greater than idazoxan greater than tolazoline. Alpha adrenoceptor antagonism was also assessed in the isolated rat vas deferens. Napamezole reversed clonidine-induced decreased in twitch height in the electrically stimulated rat vas deferens (alpha-2 antagonism with a Kb of 17 nM). The rank order of potency as an alpha-2 antagonist relative to other compounds was phentolamine greater than idazoxan greater than yohimbine greater than piperoxan = napamezole greater than mianserin much greater than prazosin. Napamezole also antagonized methoxamine-induced contractions (alpha-1) of the rat vas deferens with a Kb of 135 nM. The rank order of potency of these compounds as alpha-1 antagonists was prazosin greater than phentolamine greater than mianserin greater than yohimbine greater than napamezole greater than idazoxan.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of the present study was to develop a biochemical marker of inhibition of cyclooxygenase in the central nervous system following oral administration of cyclooxygenase inhibitors. An ex vivo method was developed wherein the brain was removed, incubated at room temperature for 2 min permitting prostaglandins to be synthesized from spontaneously released arachidonic acid. Indomethacin, zomepirac Na, naproxen Na, ibuprofen, aspirin and acetaminophen inhibited the ex vivo production of prostaglandin E2 in a dose-related manner at doses that correlated well with both their potency to inhibit mouse brain cyclooxygenase in vitro, and their antinociceptive potency in a mouse abdominal constriction test. When the brains were frozen immediately after the mice were killed, the above drugs did not reduce the endogenous prostaglandin E2 level. Thus while cyclooxygenase inhibitors did not reduce normal prostaglandin E2 levels in brain, they did attenuate post-mortem increases in prostaglandin E2. This novel assay permits an estimate of cyclooxygenase inhibitory effects in vivo. The data further support the suggestion that many of these drugs may have a central as well as a peripheral effect.

An enzymatic-radioisotopic assay was used to measure free choline in unextracted tissue. The lowest concentration of free choline in any tissue studied was present in human cerebrospinal fluid (mean, 5.7 microM; range, 1.8 + 31.2 microM). A postmortem increase in concentration of free choline occurred in blood (0.2 nmol/min.ml), kidney (13 nmol/min.g) and liver (22 nmol/min.g) of mice. The concentration of free choline in these tissues was estimated by extrapolation to be 5, 77, and 29 nmol/g (or ml), respectively. Several treatments were found to increase the concentration of free choline. For example, intraperitoneal administration of choline or 2-amino-2-methyl-propanol (a choline oxidase inhibitor) induced an increase in the level of choline i blood, kidneys, liver, and brain of mice, and administration of 2-dimethylaminoethanol (deanol) caused an increase in kidney and liver choline. The level of choline in blood was increased when rats were treated orally with either antibiotics or esters of choline such as phosphorylcholine, glycerylphosphorylcholine, laroylcholine, or propionylcholine. The results show that the concentration of free choline may be regulated by intestinal metabolism, availability of esterified precursors, and activity of enzymes that metabolize choline.

The rank order of potency to activate central dopamine autoreceptors of seven compounds known to possess central nervous system dopamine agonist activity were assessed with the following techniques: (1) inhibition of dopaminergic neuronal firing in anesthetized rats, (2) inhibition of dopamine synthesis in rats pretreated with gamma-butyrolactone, and (3) inhibition of mouse locomotor activity. The compounds were also examined for their ability to induce stereotypic behaviors in rats as an index of postsynaptic dopamine receptor activation. The compounds under investigation were apomorphine, N-n-propyl-norapomorphine, lergotrile, bromocriptine, RU 24926 and 6-ethyl-9-oxaergoline (EOE). There was a high degree of correlation between the rank order of potency of the compounds in all three of the presumptive autoreceptor tests and with minor variations the following rank order of potency was found: N-n-propylnorapomorphine greater than or equal to EOE greater than apomorphine greater than lergotrile greater than or equal to RU 24926 greater than bromocriptine. However, in the induction of stereotypies, the rank order of potency was considerably different: N-n-propylnorapomorphine greater than apomorphine greater than EOE greater than RU 24926 greater than lergotrile greater than bromocriptine. There was a poor and statistically significant degree of correlation between the rank order of potency of the test compounds to induce stereotyped behaviors and any of the other three test procedures. Altogether, these data confirm and extend the suspected dopaminergic agonist properties of the compounds under investigation and additionally lend credence to the assumption that the three putative autoreceptor assays employed do in fact reflect dopaminergic autoreceptor activation.

The putative dopamine (DA) autoreceptor agonists, N-n-propyl-3-(3-hydroxyphenyl)-piperidine (3-PPP) and 6, 7-dihydroxy-2-dimethylaminotetralin (TL-99) were compared with apomorphine in a series of tests indicative of DA receptor activation. All three agents displaced [3H] apomorphine and [3H] spiroperidol from DA recognition sites in rat brain and caused contralateral turning in the 6-hydroxydopamine lesioned rat. Apomorphine and TL-99 were generally more potent than 3-PPP. All three agents were also active at the DA autoreceptor that controls the synthesis of dopamine as indicated in vivo using the gamma-butyrolactone (GBL) procedure and in vitro using a synaptosomal preparation. In addition, all agents produced emesis in beagles. clear differences in the drugs' actions were observed in other test procedures. In the rat, apomorphine was the only compound which caused stereotypy or rotation following a reversible KCI-induced lesion of the striatum. Conversely, TL-99 and 3-PPP lacked activity in these procedures. Presumably, activity in these two tests indicates postsynaptic DA receptor activation. Each of the putative autoreceptor agonists produced a monotonic dose-related decrease in the mouse locomotor activity as opposed to the biphasic effect exerted by apomorphine. This action on the mouse locomotor activity, coupled with the results for the GBL test, provides an index of autoreceptor activation. In contrast to 3-PPP, both apomorphine and TL-99 increased locomotor activity in animals pretreated with reserpine and alpha-methyl-p-tyrosine and caused rotation in unilaterally caudectomized mice. In these test procedures thought to reflect activity at the postsynaptic DA receptor, TL-99 differed in its action from 3-PPP in a manner which suggests 3-PPP may be a more selective DA autoreceptor agonist.

– The concentration of free choline in peripheral tissues (duodenum, heart, kidney, liver, stomach and plasma) of rats was found to be related to the amount of free choline in the diet. Under steady-state conditions, the concentration of free choline in plasma varied from a minimum of approx 6 nmol/ml (in rats fed a choline-deficient diet) to a maximum value not exceeding 21 nmol/ml. The concentration of plasma choline was elevated above 21 nmol/ml for a short time after parenteral administration of choline chloride or one of its precursors (CDP choline or phosphorylcholine), but was not affected by stress, endocrine manipulations, drug treatments or the time of day when rats were killed. The metabolism of intravenously administered [methyl-3H] choline was accelerated in peripheral tissues (except plasma) of choline-deficient rats, indicating that free choline is not preserved during choline deficiency by a reduction in its rate of turnover. Furthermore, the decrease in concentration of plasma choline that occurred in rats fed a choline-deficient diet was prevented by addition of deanol (dimethylaminoethanol) to the diet. These results indicate that free choline in peripheral tissues of rats is derived from both free choline in the diet, and from precursors of choline present within the diet. In contrast to the effects in peripheral tissues, the concentration of free choline in brain was not reduced by dietary deprivation of free choline; however, the increase in free choline that occurred when rats were decapitated was reduced in brains by deficiency of choline, suggesting a decrease in the concentration of esterified forms of cerebral choline. The concentration of acetylcholine was not reduced in the brain, duodenum, heart, kidney or stomach of 21-week old rats raised from birth on a choline-deficient diet, in the duodenum of rats given a choline-deficient diet for 1, 5 or 11 days, or in brains of rats deprived of free choline for 1 or 11 days. However, the rate of in vivo synthesis of ACh from [methyl-3H]choline was accelerated in cholinergic tissues that were depleted of free choline (i.e. duodenum, heart and stomach).

The biosynthesis of acetylcholine and the fate of intravenously administered choline [methyl- 3-H] were studied in guinea pigs anesthetized with pentobarbital. Choline and acetylcholine were isolated by paper electrophoresis and estimated by use of a specific enzymatic (choline kinase) - radioisotopic assay. The concentration of acetylcholine ranged from 25.5 to 1.1 nmol/g in the following tissues (in order of decreasing concentration): duodenum, corpus striatum, stomach, cerebral cortex, spinal cord, abdominal fat, submaxillary gland, kidney, adrenal gland, spleen, liver, lung, heart and diaphragm. Choline [methyl- 3-H] was converted in the tissues to acetylcholine within 3 minutes after intravenous administration of the precursor. Virtually all the radioactivity in plasma at that time was present as free choline, suggesting that free choline from plasma is the immediate precursor for acetylcholine synthesized in the tissues cited. The concentration of free choline in tissues ranged from 344 nmol/g in adrenals to 40 nmol/g in heart, while that in plasma was 15 nmol/g. The initial half-life of choline in plasma, estimated from the rate of disappearance of choline after intravenous administration of either a tracer dose of choline [methyl- 3-H] (0.031 mumol/kg) or a high dose of choline chloride (200 mumol/kg), was less than 1 minute. This rapid removal of choline from plasma resulted from uptake (or binding) by tissues, with kidney and liver removing about 50% of the administered dose of choline [methyl- 3-H] within 3 minutes after its administration. Uptake of choline occurred in all tissues cited above, but there was a 20-fold difference in the uptake by the most active tissues (kidney and adrenals), as compared to that of the least active (central nervous system). Within 60 minutes after administration of choline [methyl- 3-H], most of the radioactive choline taken up by tissues had been converted to organic-soluble metabolites and to water-soluble metabolites that behaved like either phosphorylcholine or betaine during paper electrophoresis and chromatography. Betaine was the principal metabolite of choline in plasma. Radioactivity was excreted slowly into urine, which contained primarily free choline, betaine and a large amount of an unidentified metabolite. These findings indicate that the principal mechanism for the rapid removal of choline from plasma is uptake into tissues followed by metabolism.

Kojic amine (KA; 2-aminomethyl-5-hydroxy-4H-pyran-4-one), a compound which shares some structural features with gamma-aminobutyric acid (GABA) and muscimol, has been examined in a variety of test systems for GABAmimetic activity. In several in vitro central nervous system receptor binding assays employing rat brain membrane preparations, KA exhibited selective activity to displace 3H-muscimol but with a relatively high IC50 of 4.4 muM. KA did not alter the binding of 3H-diazepam. Iontophoretically applied KA exerted a pronounced (comparable to GABA on the basis of ejection currents)i inhibition of the firing of cerebellar Purkinje cells and spontaneously active or glutamate-activated neurons in the cerebral cortex. The inhibitory effects of KA, which were longer lasting than those of GABA, were antagonized by bicuculline and enhanced in the presence of 2,4-diaminobutyric acid. On the isolated amphibian (Bufo marinus) spinal cord, KA was less than 1/3 as potent as GABA in depolarizing primary afferent terminals. In this preparation KA caused a marked decrease in the dorsal and ventral root potentials evoked by electrical stimulation of an adjacent or corresponding dorsal root. KA is a poor substrate for GABA uptake systems into rat brain synaptosomes, has no effect on GABA release in vitro, and does not inhibit GABA transaminase activity. Altogether, these data suggest that KA does have some GABAmimetic actions (which are perhaps restricted to hyperpolarizing post-synaptic GABA receptors) but also exerts other pharmacological effects as well.

Administration of 2-dimethylaminoethanol (deanol) to mice induced an increase in both the concentration and the rate of turnover of free choline in blood. Treatment with deanol also caused an increase in the concentration of choline in kidneys, and markedly inhibited the rates of oxidation and phosphorylation of intravenously administered [3H-methyl]choline. In the liver, deanol inhibited the rate of phosphorylation of [3H-methyl]choline, but did not inhibit its rate of oxidation or cause an increase in the level of free choline. These findings suggest that deanol increases the choline concentration in blood by inhibition of its metabolism in tissues. Deanol may ultimately produce its central cholinergic effects by inhibition of choline metabolism in peripheral tissues, causing free choline choline to accumulate in blood, enter the brain, and stimulate cholinergic receptors.

Most of the work on transmitter replacement therapy by means of precursor loading has been directed toward manipulation of neurotransmitters synthesized from aromatic amino acids (47). However, beginning in the mid 1970’s, clinical investigators became interested in the use of choline (Ch) to treat disorders in which too little acetylcholine (ACh) was formed in the brain, or in which the formation of an excess of the neurotransmitter was believed to be desirable. This interest in Ch as a therapeutic agent was triggered by basic research showing that the administration of Ch to laboratory animals could cause an increase in the level of ACh in the brain, a phenomenon first described in 1972 by Kuntscherova (32). Although the beneficial effects of Ch in some disorders such as tardive dyskinesia (see 2) may reside in other properties of the molecule (e.g. a direct postsynaptic muscarinic action of Ch has been demonstrated (9,20,31)), there is evidence that part of the effect of Ch is elicited through stimulation of ACh synthesis and release. This evidence is reviewed in the first part of this report.

[3H]Substance P was used to label putative substance P receptors in rat brain membranes. [3H]Substance P binding was saturable, reversible, and displaceable by non-radioactive substance P. Association, dissociation and saturation experiments suggest a single site interaction of [3H]substance P to its binding site (k-1/k+1 = 0.56 nM; KD = 0.9 nM, Bmax 86.5 fmol/mg protein.

6-Ethyl-9-oxaergoline (EOE) and its enantiomers were compared with apomorphine in a number of tests designed to measure dopamine (DA) agonist activity within the central nervous system. In rats, the tests were: interaction with DA receptors labeled with 3H-apomorphine or 3H-spiroperidol; the effects on DA synthesis as assessed by the -butyrolactone procedure; turning in 6-OHDA lesioned animals; stereotypy; and, slowing of DA cell firing rates. In the mouse, locomotor activity, hypothermia and postural asymmetry in Caudectomized animals were studied. Emesis in the beagle was also examined. The (-)-enantiomer of EOE was more potent than either the (+)-enantiomer or the racemate in all tests. With the exception of inducing stereotypy and the displacement of 3H-apomorphine from rat striatal membranes, (-)-EOE was equi- or more potent than apomorphine in all test procedures. (-)-EOE was effective following oral administration and exhibited a longer duration of action than apomorphine. The results indicate EOE is a potent DA agonist.

In order to assess the involvement of alpha-2 adrenergic receptors in nociception, the in vitro potencies of seven alpha-2 adrenergic agonists (clonidine, guanabenz, guanfacine, BHT-920, ICI 106270, xylazine and lofexidine) were compared with their ability to prevent the writhing response elicited by i.p. administration of phenyl-p-guinone. Administration of each compound elicited antinociception, and this effect was attenuated by pretreatment with the alpha-2 adrenergic antagonists, yohimbine. The potency of these compounds to cause antinociception was correlated with their potency to displace [3H]clonidine from its binding site on brain membranes and with their ability to inhibit the twitch of the electrically stimulated vas deferens, suggesting an alpha-2 involvement in the antinociceptive action. In addition to causing antinociception, administration of these agonists also impaired rotorod performance in mice. These agonists were 2.5 to 72 times more potent in inhibiting writhing than in impairing rotorod performance, and, except for ICI 106270, there was a correlation between antinociceptive and ataxic potency. ICI 106270 was a notable exception to this correlation, however, producing only minimal ataxia, which unlike the other agonists was not reversed by yohimbine. These results indicate that alpha-2 adrenergic agonists can produce antinociception and further suggest that this may be dissociable from the ataxia.

Serologic, biochemical, and pathogenic characteristics of 11 porcine isolants of Erysipelothrix rhusiopathiae that could not be placed in any of 16 established serotypes, were examined. On the basis of double-diffusion percipitin reactions, isolants were divided into 4 serologic variants, given serotype designations 17, 18, 19, and 20. Biochemical activity of the isolants was typical of E rhusiopathiae. One or more isolants of each serologic variant were pathogenic for both mice and swine.

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min−1 mg−l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS04, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm) to phosphorylcholine [32P] in the presence of ATP [y32P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two Km values for choline at pH 9.5; 32 μm and 0.31 mm. At pH 7.4, the higher Km was not observed and enzyme activity was maximal with 0.1 mm choline. The Km for ATP was 1.1 mm. Enzyme activity was inhibited by ATP (20 mm), AMP, ADP, cytidine diphosphocholine (1 or 10 mm), and activated by choline esters (1.0 mm), NaCl or KCl(200 mm).

SEVERAL lines of evidence indicate that release of dopamine (DA) within the corpus striatum mediates turning behaviour in the rat1,2. The turning occurs in a direction away from the striatum with the greatest activation of DA neurones. Contraversive turning also occurs after unilateral microinjection of agonists of γ-aminobutyric acid (GABA) into the substantia nigra (SN)3–6, the site of origin of the dopaminergic nigrostriatal pathway. This finding was unexpected, as GABA inhibits neurones7,8 thought to be DA-containing cells9,10 in the SN. Hence, a GABA agonist would be expected to cause a decrease in the rate of release of striatal DA on the microinjected side and result in ipsiversive turning. Therefore, it is unclear whether a GABA agonist applied to the SN alters DA release in the ipsilateral striatum to evoke turning, or whether the turning results from a non-dopaminergic mechanism5. The dopamine antagonist, haloperidol, has been reported either to reduce significantly3,6 or exert no action on4,5 the contraversive turning response evoked by intranigral injection of a GABA-like compound. To clarify the action of GABA on the nigrostriatal dopaminergic system, we examined the effect of muscimol, a potent GABA agonist11, on striatal DA metabolism and release. We report here that intraperitoneally (i.p.) administered muscimol increases the concentration of striatal homovanillic acid (HVA) and that intranigral perfusion of muscimol simultaneously evokes both contraversive turning and DA release from the ipsilateral striatum.