Inhibition of cyclic flow variations and reocclusion after thrombolysis in dogs by a novel antagonist of platelet-activating factor.

To determine whether platelet-activating factor is a specific mediator of cyclic flow variations in damaged stenotic arteries and whether it contributes to reocclusion after thrombolysis, femoral arteries in anesthetized dogs were subjected to mural injury and high grade stenosis to induce cyclic flow variations (28 +/- 4/h) or methods selected to elicit platelet-rich and fibrin-rich thrombosis. Oral administration of a novel triazolobenzodiazepine (U46,195 [10 mg/kg]) that selectively inhibits platelet-activating factor abolished cyclic flow variations within 120 min and for greater than or equal to 2 h thereafter compared with persistent flow variations in dogs given saline solution. Platelet aggregation induced ex vivo with platelet-activating factor was inhibited in parallel with in vivo inhibition of cyclic flow variations after administration of U46,195. However, buccal mucosa bleeding time was not affected. After thrombosis, administration of U46,195 before thrombolysis was induced with human recombinant tissue-type plasminogen activator (1.7 mg/kg intravenously over 60 min) prevented reocclusion within 120 min in six of eight and six of seven arteries by platelet-rich and fibrin-rich thrombosis, respectively. In contrast, in dogs given saline solution, reocclusion occurred in eight of eight (p = 0.007 compared with U46,195) and five of eight arteries by platelet-rich and fibrin-rich thrombosis, respectively. Thus, both cyclic flow variations and reocclusion after thrombolysis appear to be mediated in part by platelet-activating factor. The results suggest that inhibition of platelet-activating factor with specific antagonists may be useful in reducing platelet-mediated occlusion of coronary arteries without eliciting bleeding.

Effects of lazaroids and a peroxynitrite scavenger in a cell model of peroxynitrite toxicity.

We developed a cerebellar granule cell model of peroxynitrite toxicity and showed that certain sulfhydryl-containing compounds (e.g., penicillamine) present as concurrent treatments could inhibit this toxicity. In the present study, 21-aminosteroid and pyrrolopyrimidine lazaroids were tested for cytoprotection in this peroxynitrite toxicity model. In addition, we tested for added protection using a peroxynitrite scavenger concurrent treatment combined with a lazaroid post-treatment. The toxicity assay utilized cells that were previously exposed to 100 microM L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of gamma-glutamyl-cysteine synthetase, for 24 h. This sublethal concentration of BSO shifted the peroxynitrite (1-1000 microM) toxicity curve to the left by more than one-half of a log unit. The half-maximal toxicity concentration (TC50) of peroxynitrite in cells treated with BSO was 50 microM. The 21-aminosteroids, U-74006F and U-74500A, and the pyrrolopyrimidines, U-91736B and U-101033E, were tested as post-treatments. U-74006F and U-74500A had EC50 values of approximately 100 microM (concentrations which blocked 50% of the toxicity). U-91736B and U-101033E had EC50 values of 1 microM and showed 100% protection at 3-10 microM. Treatment with either 100 microM U-74006F or 1 microM U-101033E resulted in a right-hand shift (protection) in the peroxynitrite toxicity curve. Further, combination treatment of lazaroids with 1 mM penicillamine resulted in additive protection compared to either treatment alone.

Azulenyl nitrones: colorimetric detection of oxyradical end products and neuroprotection in the gerbil transient forebrain ischemia/reperfusion model.

We present analytical and neuroprotective data on a unique spin trapping agent derived from a novel chemical class known as an azulenyl nitrone (AZN). Based on Colorimetric properties, AZN was used to assess the formation of free radicals in a bilateral carotid occlusion (BCO) model in gerbils by monitoring the conversion of the nitrone to the aldehyde in affected tissue. In addition, AZN was tested as a neuroprotectant in this model regarding the preservation of CA1 pyramidal cells of the hippocampus following transient ischemia/reperfusion. AZN was electrochemically oxidized to give the aldehyde using an HPLC system with on line electrochemical oxidation. The oxidation potential associated with a 50% loss of AZN occurred at about 600 mV (half-wave potential versus palladium electrode). The major product detected as AZN oxidation occurred in an aqueous methanolic medium was the corresponding azulenyl aldehyde. Oxidation of AZN was inversely related to the formation of the aldehyde. Based on this test, we considered the in vivo conversion of AZN to aldehyde to be a measurement of oxidative stress in tissue. Results show that 0.3% of hippocampal AZN was converted to aldehyde in animals treated as shams. However, in gerbils subjected to a 7-min ischemic insult plus 7-min reperfusion, the conversion rate was about 3 times higher at 1.0%. In this model, surviving CA1 hippocampal neurons were counted from gerbils that were subjected to 7 mins of BCO followed by 5 days of reperfusion. In sham animals, about 89 cells were counted in a selected field of CA1 neurons. With injury, only 27 cells on average survived (70% loss) and were counted from this selected field. Under similar conditions and AZN treatment, 57 cells survived (36% loss). We conclude, therefore, that the demonstrated neuroprotection occurs because AZN neutralizes radicals which contribute to neuronal damage following ischemia/reperfusion.

4-Amino-4-arylcyclohexanones and their derivatives, a novel class of analgesics. 1. Modification of the aryl ring.

Investigation of central nervous system activity of phenylcyclohexylamines was continued by preparation of "reversed" analogues. Following the unexpected finding of analgesic activity with 1-(dimethylamino)-1-phenylcyclohexylamine, the SAR of the series was investigated. Synthesis starts by double Michael reaction of acrylate on arylacetonitriles. Following cyclization, decarboxylation, ketalization, and saponification, the geminally substituted acid is rearranged to the isocyanate by means of (C6H5O)2PON3. Isocyanates were then converted to the title compounds. Analgesic activity is very sensitive to the nature and position of the substitutent on the aromatic ring. The most potent compounds in this series (p-CH3, p-Br) showed 50% the potency of morphine. Deletion of the ring oxygen abolishes activity.

4-amino-4-arylcyclohexanones and their derivatives: a novel class of analgesics. 2. Modification of the carbonyl function.

The effect on potency of modification of the carbonyl function of analgesics derived from 4-(dimethylamino)-4-arylcyclohexan-1-one was studied by reduction and by addition of nucleophiles. The resulting amino alcohols were separated and assigned structures on the basis of X-ray crystallography, NMR, and TLC mobility. The trans (OH and N) isomers were invariably more potent than the cis. Inclusion of flat lipophilic moieties (phenyl, cyclohexenyl) at a distance of at least two carbon atoms from the carbon bearing hydroxyl led to increases in potency by orders of magnitude. The possible significance of this on receptor interaction is discussed.

1-(Aminoalkyl)-6-aryl-4-H-s-triazolo[4,3-a][1,4]benzodiazepines with antianxiety and antidepressant activity.

A series of 1-(aminoalkyl)-6-aryl-4H-s-triazolo[4,3-a][1,4]benzodiazepines has been prepared and evaluated for central nervous system activity. We have found that members of this series have activity in pharmacological test systems designed to detect both anxiolytic and antidepressant activity. Each type of activity could be varied independently by appropriate substituent selections.

Centrally acting serotonergic agents. Synthesis and structure-activity relationships of C-1- or C-3-substituted derivatives of 8-hydroxy-2-(di-n-propylamino)tetralin.

The synthesis and structure-activity relationships (SAR) of C-1- or C-3-substituted derivatives of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) are described. These analogs were synthesized via alkylation of the tetralone derivatives followed by reductive amination. All of the analogs were inactive at the dopamine D2 receptor. Among the 8-OMe or 8-OH C-1,N-disubstituted analogs synthesized, the cis analogs were more potent in the 5-HT1A binding assay than the corresponding trans analogs. However, in the case of 1-(cyclopropylmethyl)-N-n-propyl analogs, the trans isomer has a slightly higher 5-HT1A affinity than its cis counterpart. The order of binding potency for C-1 substitution was found to be allyl > hydroxymethyl > n-propyl > cyclopropylmethyl >> carbomethoxy. Interestingly, the 5-OMe analogs were found to be inactive in both the 5-HT1A and dopamine D2 binding assays. In the C-3 allyl-substituted analogs, 5-HT1A agonist activity was found to be considerably lower. In these examples, the trans analogs showed weak 5-HT1A binding activity whereas the cis analogs were inactive. Analogs with C-1,N,N-trisubstitution also showed a marked decrease in 5-HT1A binding affinity. Overall, the SAR study showed that cis C-1 substitution maintains the 5-HT1A agonist activity of 8-OH-DPAT whereas trans C-1 substitution displays somewhat diminished activity. On the other hand, the trans C-3 substitution shows modest agonist activity whereas cis C-3 substitution removes the activity completely.

High-affinity partial agonist imidazo[1,5-a]quinoxaline amides, carbamates, and ureas at the gamma-aminobutyric acid A/benzodiazepine receptor complex.

A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.

3-Phenyl-substituted imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas that have high affinity at the GABAA/benzodiazepine receptor complex.

A series of imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas containing substituted phenyl groups at the 3-position was developed. Compounds within the imidazo[1,5-alpha]quinoxaline urea series had high affinity for the GABAA/benzodiazepine receptor complex with varying in vitro efficacy, although most analogs were partial agonists as indicated by [35S]TBPS and Cl- current ratios. Interestingly, a subseries of piperazine ureas was identified which had biphasic efficacy, becoming more antagonistic with increasing concentration. Analogs within the imidazo[1,5-alpha]quinoxalin-4-one series had substantially decreased binding affinity as compared to the quinoxaline urea series. These compounds ranged from antagonists to full agonists by in vitro analysis, with several derivatives having roughly 4-fold greater intrinsic activity than diazepam as indicated by Cl- current measurement. Numerous compounds from both series were effective in antagonizing metrazole-induced seizures, consistent with anti-convulsant properties and possible anxiolytic activity. Most of the quinoxaline ureas and quinoxalin-4-ones were active in an acute electroshock physical dependence side effect assay in mice precluding further development.

Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality.

A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.

Thyroid hormonal modulation of the binding and activity of the GABAA receptor complex of brain.

Thyroid hormones, which are known to act by genomic mechanisms in peripheral tissues, were found to influence the binding and function of the GABAA receptor complex in brain membranes. Submicromolar concentrations of triiodothyronine and thyroxine stereospecifically stimulated the binding of [35S]t-butylbicyclophosphorothionate (a convulsant ligand for the GABAA receptor complex) to highly washed rat brain membranes, while higher concentrations of the hormones inhibited radioligand binding. GABA-stimulated 36Cl-flux in isolated brain membrane sacs was inhibited by L-triiodothyronine with a half-maximally inhibitory concentration (IC50) of 10(-7) M. Patch-clamp analysis of recombinant GABAA receptor subunits expressed in human embryonic kidney-293 cells showed an inhibition of chloride currents by thyroid hormones. This effect required only the alpha 1 beta 2 subunits, and was not blocked by the benzodiazepine antagonist flumazenil. Since thyroid hormones are known to be concentrated in nerve terminal preparations and subsequently released, the hormones may have non-genomic mechanisms of action as putative neurotransmitters or neuromodulators in brain and act through GABAA receptors.

Comparison of pyrrolidinyl and piperidinyl benzamides for their anticonvulsant activity and inhibitory action on sodium channel.

1. A pair of benzamide analogues containing a pyrrolidinyl or piperidinyl group was examined for their anticonvulsant activity against the electroshock-induced seizures in mice and the ability to block the voltage-gated Na channel in N1E-115 cells, in comparison with the prototype compound, U-54494A, (+/-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-ben zam ide , a potent anticonvulsant and a Na channel blocker. 2. The pyrrolidinyl benzamide (U-49524E) was found to be effective against the electroshock-induced seizures (ED50 = 35 mg kg-1, i.p.) whereas the benzamide with a piperidinyl moiety (U-49132E) was inactive (ED50 greater than 100 mg kg-1). 3. Using whole-cell patch clamp techniques we found that U-49132E was several times less potent, with an IC50 of 396 microM as compared to 118 microM for U-49524E at the holding potential of -80 mV, and was much slower in blocking Na channels with a half-time of 10.7 +/- 1.1 min vs. 2.2 +/- 0.4 min for its counterpart. 4. Qualitatively, their general modes of interaction with Na channels were similar to each other and to that of U-54494A in that they interacted with the resting and slowly-inactivated states of the channels and exhibited a use-dependent inhibition because of a slow recovery from the inactivated state in the presence of the drugs. 5. Comparison of their physicochemical properties, shows the less potent and slowly acting U-49132E is more hydrophobic and bulkier than U-49524E, but has the same pKa. This suggests that the drugs approach the Na channel through a narrow and hydrophilic pathway.6. Overall, this study underscores the importance of inhibiting the Na channel to the anticonvulsant activity of the benzamide compounds.

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABA(A) receptors but with the opposite functionality.

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA(A) receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence. 2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response. 3. These functional characteristics were monitored with Cl- currents measurements in cloned rat alphaxbeta2gamma2 subtypes of GABA(A) receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA(A) receptors with binding of [35S]-TBPS, which is a high affinity ligand specific for GABA(A) receptors with exquisite sensitivity to allosteric modulations. 4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

PNU-107484A with alpha isoform-dependent functional changes in alpha(x)beta2gamma2 subtypes of rat recombinant GABA(A) receptors.

1. We discovered a novel gamma-aminobutyric acidA (GABA(A)) receptor ligand displaying seemingly opposite functionalities, depending on the alpha isoform of the alpha(x)beta2gamma2 subtypes. PNU-107484A enhanced GABA-induced Cl- currents in the alpha1beta2gamma2 subtype, but inhibited the currents in the alpha3beta2gamma2 and alpha6beta2gamma2 subtypes, and its half-maximal concentrations in the subtypes were 3.1 +/- 0.5, 4.2 +/- 1, and 3.5 +/- 0.2 microM, respectively, without showing much dependency on alpha isoforms. 2. In the alpha1beta2 subtype, the drug at concentrations up to 40 microM showed no effect on GABA-induced Cl- currents, suggesting the requirement of the gamma subunit for its action. 3. PNU-107484A behaved like a positive allosteric modulator of the alpha1beta2gamma2 subtype with its binding site distinct from those for benzodiazepines, barbiturates and neurosteroids. With the alpha3beta2gamma2 subtype, the drug behaved like a non-competitive inhibitor of GABA, thus blocking Cl- currents by GABA alone or in the presence of pentobarbitone and neurosteroids. 4. It appears that PNU-107484A is a unique GABA(A) receptor ligand with alpha isoform-dependent functionalities, which may provide a basis for development of alpha isoform-selective ligands, and it could be useful as a probe to investigate the physiological roles of the various alpha isoform subtypes.

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes of cloned GABAA receptors.

1. In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the alpha 1 beta 2 gamma 2 and the alpha 6 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2. The prototype compounds, U-85575 (12-chloro-5-(5-cyclopropyl-1',2',4'- oxadiazol-3'-yl)-2,3-dihydro-diimidazo [1,5-a;1,2-c]quinazoline), and U-92330 (5-acetyl-3-(5'-cyclopropyl-1',2',4'-oxadiazole-3'-yl)-7-chloro-4,5-d ihy dro [1,5-a]quinoxaline), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA-mediated Cl- currents in both subtypes were sensitive to Ro 15-1788, a classical benzodiazepine antagonist. 3. Minor changes in the ring substituents of the drugs reduced their affinity to the alpha 6 beta 2 gamma 2 subtype more pronouncedly than to the alpha 1 beta 2 gamma 2 subtype. The diimidazoquinazoline containing a 2-methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the alpha 6 beta 2 gamma 2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5-benzoyl group showed a lower affinity to the alpha 6 beta 2 gamma 2 subtype than the 5-acetyl counterpart. In particular, the 5-benzoyl analogue containing a 6-fluoro group showed no interaction with the alpha 6 beta 2 gamma 2 subtype even at the concentration of 10 microM, probably due to stabilization of the benzoyl group in the out-of-plane region by the steric and electrostatic effects of the 6-fluoro group.4. We propose that the benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype shares overlapping regions with that of the alpha 1 beta 2 gamma 2 subtype, but has a sterically restricted out-of-plane region, which may be also incompatible with the 5-phenyl group of classical benzodiazepines.

Triazolobenzodiazepines: a new class of stimulators of tissue-type plasminogen activator synthesis in human endothelial cells.

In our search for compounds that can stimulate endogenous fibrinolysis, we have found that certain triazolobenzodiazepines enhance the production of tissue-type plasminogen activator (t-PA) by vascular endothelial cells maintained in vitro, with no or even a lowering effect on plasminogen activator inhibitor type-1 (PAI-1) production. The most active compounds tested, U-34599, U-46195 and U-51477, were studied in more detail and showed a time- and dose-dependent increase in the production of t-PA by human umbilical vein endothelial cells. At optimal stimulatory concentrations (about 10 microM), the three compounds stimulated t-PA expression about 2-fold after 24 hr and maximally about 4-fold after 48 hr of incubation; this maximal increase in t-PA synthesis was sustained at prolonged incubations of 72 or 96 hr. The triazolobenzodiazepine effects on t-PA production were accompanied by parallel increases in t-PA mRNA levels, without marked changes in PAI-1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA concentrations. Numerous analogues of the three lead compounds were then tested to determine the relationship between benzodiazepine structure and the ability to stimulate t-PA production. No positive correlation was found between the ability of the various triazolobenzodiazepines to stimulate t-PA production and their affinity for the benzodiazepine receptor. In agreement with this, no specific binding of [3H]flunitrazepam, a specific ligand for benzodiazepine receptors, to endothelial cell membrane preparations was observed. Thus, it is unlikely that the triazolobenzodiazepines act through central-type benzodiazepine receptors to stimulate t-PA production. Similarly, no evidence was found for the presence of peripheral-type benzodiazepine receptors on endothelial cell membranes. The ability of the benzodiazepines to stimulate t-PA production, however, appeared to be related to their platelet-activating factor (PAF) antagonist activity. Despite this finding, several non-benzodiazepine PAF antagonists did not stimulate t-PA production. While the precise mechanism of action is not yet clear, selected benzodiazepine analogues possessing PAF antagonist activity stimulate the production of t-PA by endothelial cells in vitro.

Prostaglandin E2, cyclic adenosine monophosphate and morphine analgesia.

Intravenous administration of prostaglandin E2 (PGE2) to mice results in a significant distribution of PGE2 into the brain and an elevation of cyclic adenosine monophosphate (cAMP) in the midhindbrain and corpus striatum. Neither acute morphine administration nor morphine tolerance alters this response. Morphine alone also elevates cAMP levels. Although these elevations are blocked by the narcotic antagonist, naloxone, they only occur at supra-analgesic doses. Therefore morphine analgesia does not correlate with either elevation of cAMP levels of antagonism of PGE2-induced elevations of cAMP.

Neuroprotective effects of the dopamine D2/D3 agonist pramipexole against postischemic or methamphetamine-induced degeneration of nigrostriatal neurons.

We have examined the neuroprotective efficacy of the selective dopamine (DA) D2/D3 receptor agonist pramipexole in two models of nigrostriatal (NS) degeneration. The first involves the delayed (28-day) postischemic retrograde NS degeneration that takes place in gerbils following a 10-min episode of bilateral carotid arterial occlusion-induced forebrain ischemia. In vehicle (40% hydroxypropyl cyclodextrin)-treated male gerbils, there was a 40-45% loss of NS cell bodies in the pars compacta and pars reticulata (TH immunohistochemistry and Cresyl violet histochemistry) by 28 days after ischemia/reperfusion. Daily postischemic oral dosing (1 mg/kg p.o., b.i.d., beginning at 1 h after insult) decreased the 28-day postischemic loss of NS DA neurons by 36% (P < 0.01 vs. vehicle-treated). The effect was specific for dopamine neurons since no significant salvage of hippocampal CA1 neurons was observed. In a second model, pramipexole's effects were examined on methamphetamine-induced (10 mg/kg, i.p. X 4, each 2 h apart) NS degeneration in male Swiss-Webster mice. In vehicle-treated mice, there was a 40% loss of NS neurons by day 5. In contrast, pramipexole dosing (1 mg/kg, p.o., 1 h after the last methamphetamine dose, plus daily) attenuated the NS degeneration from 40% to only 8% (P < 0.00001 vs. vehicle). We postulated that pramipexole acts in both of these models to reduce the elevated DA turnover and the associated elevation in hydroxyl radical production secondary to increased MAO activity that could be responsible for oxidative damage to the NS neurons. Indeed, in the gerbil ischemia model, we documented by HPLC-ECD a 135% postreperfusion increase in DA turnover (DOPAC + HVA/DA) at 5 min after reperfusion. Pramipexole at the 1 mg/kg, p.o., dose level was able to significantly reduce the increased DA turnover, but by only 16%. Thus, it is conceivable that other mechanisms may also contribute to pramipexole's dopaminergic neuroprotection. Based on a preliminary examination of pramipexole's oxidation potential, it appears that the compound may possess significant intrinsic antioxidant properties that might contribute to its neuroprotective effects.

Involvement of biogenic amines with the mechanisms of novel analgesics.

The analgesic activity of the kappa opioid agonist, U-50,488H, was markedly antagonized by pretreatment with reserpine, p-chlorophenylalanine, and ketanserin. Likewise, analgesic doses of U-50,488H enhance serotonin metabolism. These results suggest that kappa analgesia requires serotonin acting through 5-HT2 receptors. The non-opioid analgesic, nefopam HCl, though a blocker of biogenic amine uptake, displays an analgesic spectrum of action more similar to that of amphetamine than that of the tricyclic antidepressants or serotonin uptake blockers. Likewise p-chlorophenylalanine and ketanserin do not block nefopam analgesia nor do naloxone, atropine, yohimbine, propranolol or haloperidol. However, as reserpine does block nefopam analgesia, biogenic amines acting at other receptors may be involved. The observation that m-tyrosine causes behavioral effects similar to high doses of nefopam suggested that they might be acting through similar mechanisms. However, although m-tyrosine causes analgesia, it is blocked by yohimbine. This suggests that alpha2-adrenoreceptors are involved in m-tyrosine analgesia and that it differs in mechanism from nefopam analgesia.

Properties of a selective kappa agonist, U-50,488H.

U-50,488H has been shown to be a naloxone antagonizable analgesic in rodents. However, the dose of naloxone needed for antagonism is higher than it is for morphine. U-50,488H does not produce physical dependence; however it does produce tolerance upon chronic administration. U-50,488H is cross tolerant with bremazocine but not with morphine. Monkeys trained to discriminate ethylketocyclazocine (EKC) from saline show a complete generalization to U-50,488H but not to morphine. The evaluation of U-50,488H in 3H-EKC site-selective binding indicated that U-50,488H has a high affinity for the kappa receptor (Ki = 114 nM) and a low affinity for the mu receptor (Ki = 6100 nM). The ratio of Ku/Kk was 0.08 for morphine, 0.4 for dynorphin, and 53.5 for U-50,488H. The data suggest that U-50,488H is a selective agonist at the opioid kappa receptor.