The use of total quality principles to achieve regulatory compliance in research laboratories.

Since the late 1980s, the U.S. Food and Drug Administration (FDA) has increased its focus on regulatory compliance. This is evidenced by an increase in warning letters, product seizures, injunctions, prosecutions, and recalls. This increased level of FDA enforcement activity is evidenced across all of the compliance regulations. Thus, the ability to comply with the Good Laboratory Practices (GLPs). Good Clinical Practices (GCPs) and Good Manufacturing Practices regulations could result in FDA enforcement activities and delays in the approval process for new products. Therefore, the ability to achieve compliance with the GLPs, GCPs, and GMPs in research laboratories is a key to business success in the 1990s. Applying the principles of total quality is one way to address these important business issues. The principles of total quality (customer focus, improvement, involvement, leadership, and measurement) provide a methodology for ensuring success in a rapidly changing regulatory environment. The FDA needs to be treated as a customer by research organizations. Valid regulatory requirements need to be negotiated with the agency. Once these customer requirements have been identified, the quality unit needs to play a leadership role in translating these requirements into action plans. Quality units, no matter how skillful or good, cannot achieve success without involvement of the scientific staff and management group of their organizations. Without their involvement there will be no organizational commitment to compliance programs undertaken to meet the valid requirements of the FDA. Meeting these valid customer requirements will require improvement efforts. The many tools of quality improvement in achieving the desired improvements.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical studies on quinelorane, a potent and highly selective D2-dopaminergic agonist.

Quinelorane (LY163502) has the endocrine, neurochemical and behavioral profile of a potent and highly selective D2-dopaminergic agonist. The administration of quinelorane produced dose-related decreases in serum prolactin concentration of reserpinized, male rats and increases in serum corticosterone concentration of male rats. The minimum effective doses (MED) for these effects were 10 and 30 micrograms/kg i.p., respectively. Quinelorane induced increases in 3-methoxy-4-hydroxyphenylglycol-sulfate levels in the brain stem (MED, 30 micrograms/kg i.p.) and decreases in hypothalamic epinephrine levels (MED, 100 micrograms/kg i.p.) in male rats as determined by high-pressure liquid chromatography with electrochemical detection methods. Quinelorane induced increases in extracellular ascorbic acid as determined by in vivo voltammetry in the nucleus accumbens and striatum of male rats. Quinelorane produced concentration-dependent suppression of K+-evoked release of acetylcholine from superfused caudate slices, with an IC50 of approximately 10(-8)M. Quinelorane administration produced dose-related increases in compulsive, contralateral turning in male rats with unilateral nigrostriatal lesions and increases in locomotor activity and stereotypic behavior in male rats. In dogs, quinelorane administration produced dose-related increases in emetic response with an ED50 of 7 micrograms/kg i.v. Quinelorane administration also produced dose-related decreases in the striatal concentrations of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic (MED, 1 microgram/kg i.p. for both metabolites) as determined by high-pressure liquid chromatography with electrochemical detection methods and decreases in extracellular concentrations of homovanillic acid in the nucleus accumbens and striatum as determined by in vivo voltammetry., Quinelorane produced concentration-dependent decreases in K+-evoked dopamine release from superfused striatal slices (IC50 = 3 X 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of beta-funaltrexamine to determine mu opioid receptor involvement in the analgesic activity of various opioid ligands.

Systemic administration of beta-funaltrexamine (beta-FNA) 24 hr before analgesic testing produced approximately a 10-fold parallel shift in the dose-response curves of the prototypic mu agonists morphine, I-methadone, fentanyl and etorphine in the mouse abdominal constriction test. In contrast, prior administration of beta-FNA produced no appreciable shift in the analgesic dose-response curve of the selective kappa agonist, U-50, 488H. These results suggest that beta-FNA is selective for mu over kappa receptors under the conditions used in this study. The dose-response curves for ethylketazocine and proxorphan were affected only to a small extent by beta-FNA pretreatment, suggesting that these compounds have analgesic actions mediated primarily through nonmu, probably kappa receptors. The dose-response curves for cyclazocine, buprenorphine, butorphanol, nalorphine and nalbuphine were shifted markedly to the right and frequently not in a parallel fashion by the prior administration of beta-FNA. These results seem to indicate a major role for the mu receptor in the analgesic actions of these compounds.

Differential effects of selected dopaminergic agents on locomotor activity in normotensive and spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) exhibit a significantly higher level of spontaneous locomotor activity than age-matched normotensive controls (WKY). The direct-acting dopamine agonists, apomorphine and pergolide, produced a biphasic effect on locomotor activity levels in normotensive controls. Low doses of these agonists decreased activity levels, while higher doses of these agonists dramatically stimulated activity. In marked contrast to these results was the effect observed in the SHR, in which these agonists at all doses tested decreased activity. Amphetamine, a dopamine releaser, stimulated activity levels in both the WKY and SHR; however, the magnitude of the increase was somewhat attenuated in the SHR.

Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors.

The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with 3H-naloxone or 3H-D-Ala2-D-Leu5-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties.

Picenadol.

Picenadol is a unique opioid mixed agonist-antagonist analgesic currently under clinical evaluation. Structurally, picenadol is a 4-phenylpiperidine derivative and a racemic mixture whose mixed agonist-antagonist properties are a consequence of the d-isomer being a potent opiate agonist, whereas the l-isomer is an opioid antagonist. In the mouse writhing and rat tail heat tests, the analgesic potency of picenadol is estimated to be 1/3 that of morphine. Picenadol itself has weak antagonist activity, whereas the antagonist potency of the l-isomer is approx. 1/10 that of nalorphine. Evaluation of picenadol's affinity for opioid receptors reveals that picenadol, unlike other mixed agonist-antagonists has high affinity for both the mu and delta receptors but a markedly lower affinity for the kappa receptor. Extensive pharmacological investigations show picenadol to have a low potential to produce opiate-like side effects, including a low liability for abuse and physical dependence.

Nitrous oxide generalizes to a discriminative stimulus produced by ethylketocyclazocine but not morphine.

The discriminative stimulus properties of nitrous oxide, an analgesic and anesthetic gas, were evaluated in rats trained to discriminate the effects of morphine or ethylketocyclazocine. Administration of nitrous oxide in concentrations as high as 80 percent did not produce generalization to the discriminative cue produced by morphine. Nitrous oxide did, however, generalize in a concentration-dependent manner in rats trained to discriminate ethylketocyclazocine, a psychotomimetic opioid. Naltrexone, a potent narcotic antagonist, did not block the generalization of nitrous oxide to ethylketocyclazocine. These results suggest that the subjective effects of nitrous oxide are similar to those produced by psychotomimetic drugs rather than those produced by morphine. These findings are in close agreement with those generated in man. Thus, nitrous oxide exhibits some pharmacological properties similar to those of morphine, for example, naloxone reversible analgesia. Yet, it has other properties such as subjective effects that are dissimilar from morphine.

Characterization of the alpha adrenoceptor-mediated effects and antihypertensive activity of ICI 106270: comparison with clonidine.

Clonidine and ICI 106270 are centrally acting antihypertensive agents which act through stimulation of medullary alpha-2 adrenoceptors. Both compounds are equipotent agonists at alpha-2 adrenoceptors as assessed in functional studies in isolated organs and in radioligand binding studies. In addition, clonidine and ICI 106270 possess the same degree of selectivity for alpha-2 adrenoceptors over alpha-1 adrenoceptors. Clonidine and ICI 106270 are equipotent antihypertensive agents after intracisternal administration to spontaneously hypertensive rats, consistent with the observations made in vitro that both compounds are equipotent alpha-2 adrenoceptor agonists. Both compounds were less potent in lowering blood pressure after i.v. administration than after intracisternal administration, thus confirming a central mechanism of action. Interestingly, clonidine was a more potent antihypertensive agent than ICI 106270 after i.v. administration, which, in view of the equal potencies observed after intracisternal administration, suggests that diffusion of ICI 106270 into the central nervous system is selectively retarded, relative to clonidine, by the blood-brain barrier. The differences observed in the rate of penetration of the blood-brain barrier are consistent with the higher pka and corresponding higher extent of ionization (at physiological pH) and lower lipophilicity of ICI 106270 relative to clonidine. A relatively large difference between the i.v. and p.o. antihypertensive potencies was observed for ICI 106270 which indicates poor p.o. absorption of ICI 106270 relative to clonidine, again likely resulting from the greater proportion of ICI 106270 existing in the ionized species.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of alpha-adrenoceptor agonist antinociceptive activity by nisoxetine: a selective inhibitor of noradrenergic uptake.

The alpha-adrenoceptor agonists, clonidine and xylazine, produced a dose-dependent antinociceptive effect in the mouse writhing assay as does morphine. Fluoxetine, a highly-specific inhibitor of serotonin uptake, enhanced the antinociceptive effect of morphine in this test but not that of clonidine or xylazine. In contrast, nisoxetine, a selective inhibitor of noradrenergic uptake, significantly potentiated the antinociceptive activity of morphine, clonidine, and xylazine. These findings strengthen the evidence for an involvement of a noradrenergic mechanism in the antinociceptive effects of alpha-adrenoceptor agonists.

Stimulation of presynaptic dopamine autoreceptors by 4-(2-di-n-propylaminoethyl) indole (DPAI).

The dopaminergic activity of 4-(2-di-n-propylaminoethyl)indole (DPAI) was investigated. In animal models for postsynaptic dopaminergic activity DPAI showed only very weak or no effects. In rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal tract, very weak contralateral turning behavior was induced in 4/6 rats. DPAI did not induce stereotyped behavior but caused a pronounced reduction in locomotor activity. In male rats pretreated with reserpine, DPAI lowered serum prolactin levels. Levels of homovanillic acid (HVA) were monitored in the striatum of the chloral hydrate anesthetized rat by means of in vivo voltammetry. DPAI administration reduced the faradaic peak corresponding to HVA. In rats pretreated with the decarboxylase inhibitor, NSD-1015, DPAI blocked the accumulation of dopa in response to gamma-butyrolactone. The results of this study indicate that DPAI possesses a high degree of selectivity for presynaptic dopamine autoreceptors, and little or no effect on postsynaptic dopamine receptors.

Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand, D-Ala2-D-Leu5 enkephalin in mouse brain.

The binding characteristics of the delta opioid receptor ligand, 3H-D-Ala2-D-Leu5 enkephalin, were markedly altered in brains obtained from mice fed an ethanol-containing diet for five days. Control mice exhibited both a high and low affinity site for 3H-D-Ala2-D-Leu5 enkephalin, whereas those consuming the ethanol diet were found to possess only one binding site. This singular site has an intermediate KD value with an increase in receptor number when compared to the high and low affinity sites observed in control mice. The in vitro addition of ethanol to a brain membrane preparation obtained from untreated mice, at a concentration equivalent to that found in the blood of the ethanol-treated mice, did not markedly affect D-Ala2-D-Leu5 enkephalin binding characteristics. No alteration in the binding characteristics of 3H-naloxone, a mu receptor ligand, was noted following five days of ethanol consumption. Mice maintained on the ethanol-containing diet were tolerant to the activity-stimulating effects of acute ethanol administration. These results suggest that mice consuming an ethanol diet in sufficient quantities to render them tolerant exhibit a specific loss of a 3H-D-Ala2-D-Leu5 enkephalin binding site, while the binding of 3H-naloxone was unchanged.

Antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, a novel analog of clonidine: role of opioid receptors and alpha-adrenoceptors.

N-(4-hydroxyphenacetyl)-4-aminoclonidine, a derivative of the alpha-adrenoceptor agonist p-aminoclonidine, was found to exhibit dose-dependent antinociceptive activity in the mouse writhing assay. In this measure of antinociceptive activity it was less potent than clonidine or xylazine. Naloxone, an opioid receptor antagonist, at a dose sufficient to abolish the antinociceptive activity of morphine, did not affect the antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, clonidine or xylazine. In contrast, yohimbine, a alpha-adrenoceptor antagonist, reduced the antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, clonidine and xylazine, but not morphine. The affinity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, clonidine and xylazine for alpha-adrenoceptors in rat aorta was correlated highly with the relative potency for writhing inhibition. These results suggest that the antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine is mediated by alpha-adrenoceptors.

Altered behavioral response to a D2 agonist, LY141865, in spontaneously hypertensive rats exhibiting biochemical and endocrine responses similar to those in normotensive rats.

LY141865, a dopamine agonist selective for D2 dopamine receptors, caused hypomotility at low doses (0.01 and 0.1 mg/kg) and hypermotility at high doses (1 and 10 mg/kg) after i.p. injection into normotensive rats (WKY). In age-matched hypertensive rats (SHR), LY141865 caused hypomotility (not hypermotility) at all of these doses. The basal locomotor activity was higher in SHR than in WKY, but striatal concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and of serotonin and its metabolite, 5-hydroxyindoleacetic acid, were not different between the two groups of rats. The specific binding of a dopamine receptor radioligand, tritiated pergolide, in striatum and mesolimbic regions, did not differ in SHR compared with WKY. In contrast to the lack of locomotor stimulation in SHR, other dopaminergic responses to LY141865 occurred in SHR as well as WKY. For instance, LY141865 decreased striatal and mesolimbic concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid, increased striatal and mesolimbic concentrations of acetylcholine, decreased hypothalamic concentrations of epinephrine, increased serum corticosterone concentration and decreased serum prolactin concentration in SHR as in WKY. Because radioligand-labeled dopamine receptors and several LY141865 responses mediated by dopamine receptors did not differ appreciably in SHR compared with WKY, the lack of behavioral hypermotility in response to LY141865 in SHR may be due to abnormalities in some synaptic mechanisms beyond dopamine receptor activation that are involved in the expression of increased locomotion in response to the dopamine agonist.

Resolution and absolute configuration of an ergoline-related dopamine agonist, trans-4,4a,5,6,7,8,8a,9-Octahydro-5-propyl-1H(or 2H)-pyrazolo[3,4-g]quinoline.

The title compound (+/-)-5 (R = Pro) (LY141865) has been resolved into a (-) isomer and a (+) isomer as the D- and L-tartrate salts, respectively. Biological studies have shown that dopamine agonist activity is a property of only the (-) isomer. Crystallographic analysis has proven that the absolute configuration of the active (-) isomer is the same as that of the natural ergolines.

Catecholamine mechanisms in the stimulation of mouse locomotor activity by nitrous oxide and morphine.

Nitrous oxide, like morphine, produces a significant increase in mouse locomotor activity. This stimulation of locomotor activity is antagonized by the narcotic antagonist naloxone (10 mg/kg). The catecholamine synthesis inhibitor alpha-methyl-p-tyrosine (300 mg/kg) also significantly reduced the nitrous oxide stimulation of mouse locomotor activity. Administration of naloxone (10 mg/kg) to mice previously treated with alpha-methyl-p-tyrosine resulted in a further decrease in nitrous oxide stimulated activity levels. Haloperidol (0.08-0.64 mg/kg), a potent dopamine receptor antagonist, dose dependently reduced the activity stimulation resulting from exposure to nitrous oxide. Administration of haloperidol (0.08 mg/kg) in conjunction with naloxone (10 mg/kg) further reduced the nitrous oxide effect. In view of the fact that morphine's activity stimulating effects are reduced by alpha-methyl-p-tyrosine and haloperidol, the results reported here suggest additional similarities in the pharmacology of morphine and nitrous oxide. The stimulation of mouse locomotor activity produced by nitrous oxide appears to be mediated by both endogenous opioid and catecholamines.

Opioid antagonists and drinking: evidence of kappa-receptor involvement.

Diprenorphine, naloxone, MR-2266-BS and WIN-44,441-3 were compared for their ability to antagonize morphine analgesia and to decrease deprivation-induced drinking in rats. Diprenorphine and naloxone were markedly more potent (32x) than MR-2266-BS and WIN-44,441-3 in antagonizing the analgesic effects of morphine. In contrast, diprenorphine, naloxone and MR-2266-BS decreased deprivation-induced drinking over a similar dose range. The doses required to reduce fluid consumption were higher than those necessary to antagonize morphine. WIN-44,441-3 was ineffective in decreasing drinking. The relatively similar potencies of diprenorphine, naloxone and MR-2266-BS for decreasing deprivation-induced drinking suggest that the effect on drinking involves antagonist activity at a kappa-opioid receptor.

Degree of selectivity of pergolide as an agonist at presynaptic versus postsynaptic dopamine receptors: implications for prevention or treatment of tardive dyskinesia.

Pergolide mesylate is a potent dopamine agonist that is being evaluated clinically in Parkinson disease, hyperprolactinemia, and other diseases. Pergolide activates both presynaptic and postsynaptic dopamine receptors, with some apparent selectivity for the presynaptic dopamine autoreceptors. In rats, low doses of pergolide (0.01 mg/kg or less, intraperitoneally) decreased dopamine turnover in brain, decreased serum prolactin concentration, and reduced blood pressure in spontaneously hypertensive rats. At somewhat higher doses (0.05 mg/kg or more, intraperitoneally), pergolide caused contralateral turning in nigrostriatal-lesioned rats, elevation of serum corticosterone, and hypermotility with stereotyped behavior. All of these actions are thought to be due to stimulation of dopamine receptors at various sites, but the data suggest that pergolide may have preferential affinity for presynaptic dopamine receptors. If low doses of pergolide can reduce dopaminergic transmission by activating presynaptic receptors that control dopamine release, then this action might be therapeutically useful in treating schizophrenia without causing tardive dyskinesia or in the treatment of tardive dyskinesia. The long duration of action of pergolide seen in animal and human studies could be an important advantage over some other dopamine agonists such as apomorphine.

Cross-tolerance studies distinguish morphine- and metkephamid-induced analgesia.

In vitro data demonstrate that metkephamid (LY127623), an analog of methionine enkephalin, has a high affinity for the delta opioid receptor, as well as the mu-receptor. Data generated utilizing two in vivo measures of receptor selectivity, furthermore, indicate that metkephamid's analgesic activity is in part mediated by delta opioid receptors. The analgesic activity of metkephamid was investigated in the mouse writhing assay following chronic treatment with morphine, the prototypic mu agonist. Mice were treated chronically with increasing doses of morphine or saline and the inhibition of writhing measured in response to an acute injection of morphine or metkephamid. The dose response curve for morphine was shifted to the right 3- to 4-fold following chronic administration of morphine. In contrast, no such shift in the dose response curve for metkephamid was observed in these morphine-tolerant mice. In a further series of tests, a 50 mg/kg dose of naloxazone 20 hr prior to the assessment of morphine or metkephamid analgesia in the mouse hot plate test substantially shifted the dose-response curve for morphine to the right, while leaving the dose-response curve for metkephamid unchanged. These results suggest that delta-receptor activation contributes to the analgesia produced by metkephamid.