Interactions of intrathecally administered ziconotide, a selective blocker of neuronal N-type voltage-sensitive calcium channels, with morphine on nociception in rats.

Ziconotide is a selective, potent and reversible blocker of neuronal N-type voltage-sensitive calcium channels (VSCCs). Morphine is an agonist of mu-opioid receptors and inhibits N-type VSCC channels via a G-protein coupling mechanism. Both agents are antinociceptive when they are administered intrathecally (spinally). The present study investigated the acute and chronic (7-day) interactions of intrathecally administered ziconotide and morphine on nociception in several animal models of pain. In the acute study, intrathecal bolus injections of morphine and ziconotide alone produced dose-dependent inhibition of formalin-induced tonic flinch responses and withdrawal responses to paw pressure. The combination of ziconotide and morphine produced an additive inhibition of formalin-induced tonic flinch responses and a significant leftward shift of the morphine dose-response curve in the paw pressure test. After chronic (7-day) intrathecal infusion, ziconotide enhanced morphine analgesia in the formalin test. In contrast, chronic intrathecal morphine infusion produced tolerance to analgesia, but did not affect ziconotide antinociception. Antinociception produced by ziconotide alone was the same as that observed when the compound was co-administered with morphine to morphine-tolerant rats. In the hot-plate and tail immersion tests, chronic intrathecal infusion of morphine lead to rapid tolerance whereas ziconotide produced sustained analgesia with no loss of potency throughout the infusion period. Although ziconotide in combination with morphine produced an apparent synergistic analgesic effects during the initial phase of continuous infusion, it did not prevent morphine tolerance to analgesia. These results demonstrate that (1) acute intrathecal administrations of ziconotide and morphine produce additive or synergistic analgesic effects; (2) chronic intrathecal morphine infusion results in tolerance to analgesia but does not produce cross-tolerance to ziconotide; (3) chronic intrathecal ziconotide administration produces neither tolerance nor cross-tolerance to morphine analgesia; (4) intrathecal ziconotide does not prevent or reverse morphine tolerance.

Antinociceptive properties of fenfluramine, a serotonin reuptake inhibitor, in a rat model of neuropathy.

Fenfluramine is an indirect agonist of 5-hydroxytryptamine (5-HT) receptors that acts by evoking 5-HT release and blocking 5-HT reuptake in neuronal cells. The current study compared the antinociceptive properties of fenfluramine with those of the tricyclic antidepressants amitriptyline and desipramine in rat models of acute, persistent, and neuropathic pain. In a rat model of neuropathic pain produced by tight ligation of the L(5)/L(6) spinal nerves, i.v. bolus injection of fenfluramine resulted in a dose-dependent and long-lasting (>4 h) blockade of mechanical allodynia (ED(50) = 3.5 mg/kg; 95% confidence interval, 2.2-5.4 mg/kg) and cold allodynia (ED(50) = 2.4 mg/kg; 95% confidence range, 1.2-4.6 mg/kg). Fenfluramine also prevented tonic pain evoked by the s.c. injection of dilute (5%) formaldehyde solution (formalin), into the dorsal hindpaw. The i.v. administration of amitriptyline (4.7 mg/kg) or desipramine (13.5 mg/kg) at maximum tolerated doses did not block either allodynia in rats with spinal nerve ligation-induced painful neuropathy or tonic pain in the formalin test. Fenfluramine had differential effects on acute behavioral responses to noxious thermal (heat), chemical (5% formaldehyde solution), and mechanical stimuli; it completely inhibited nociceptive behavior in the acute phase of the formaldehyde solution test and partially inhibited licking and jumping responses in the hot-plate test but did not alter nociceptive thresholds in either the paw pressure test or the tail immersion test. Intracerebroventricular bolus injection of 240 microg of fenfluramine significantly increased mechanical allodynia thresholds; however, the same dose administered spinally by intrathecal bolus injection was ineffective. The inhibitory effects of fenfluramine on mechanical allodynia (and tonic pain behavior in the formaldehyde solution test) were prevented by pretreatment with 10 mg/kg metergoline, a selective antagonist of 5-HT receptors, but not with the mu-opioid receptor antagonist naloxone. These results suggest that fenfluramine produces analgesia in the formaldehyde solution test and the spinal nerve ligation model of neuropathic pain by potentiating, at least in part, supraspinal 5-HT mediated processes.

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

The neuroprotective effects of intrathecal administration of the selective N-type calcium channel blocker ziconotide in a rat model of spinal ischemia.

PURPOSE: Spinal cord ischemia and resulting paraplegia represent a major complication associated with surgical repair of the thoracoabdominal aorta. Although the mechanism of spinal neuronal degeneration during ischemia is unclear, it may involve excessive calcium influx via N-type voltage-sensitive calcium channels (VSCCs). The neuroprotective capacity of intrathecal (IT) administration of the selective N-type VSCC blocker ziconotide, previously shown to be potently analgesic, was studied. METHODS: In a rat aortic occlusion model, spinal cord ischemia was induced for 8, 9, or 10 minutes by occluding the descending thoracic aorta. Ziconotide was administered IT as (1) a continuous infusion of 300 or 600 ng/kg/h initiated 24 hours before ischemia and continuing an additional 24 hours or (2) a 0.3 microgram bolus injected 45 minutes before the induction of ischemia. Animals were allowed to live for 24 hours, and recovery of motor function was evaluated during this period. Spinal cords were processed using a silver impregnation technique and microtubule-associated protein type II (MAP2) immunohistochemistry. RESULTS: Continuous IT infusion of ziconotide provided significant protection against 8- and 9-minute occlusions, but not 10-minute occlusions, as indicated by recovery of motor function, degree of spinal neuronal degeneration, and loss of MAP2 immunoreactivity. Acute IT pretreatment with ziconotide provided transient protection during the initial 4 hours of reperfusion; however, this protective effect was no longer present at 24 hours. CONCLUSION: These data implicate N-type VSCC activation in spinal neuronal degeneration caused by transient spinal ischemia, because selective blockade of this channel by continuous IT infusion of ziconotide was protective against injurious intervals of spinal ischemia. Based on these findings, ziconotide may provide both neuroprotection and preemptive analgesia for aortic aneurysm surgery.

Peripheral versus central potencies of N-type voltage-sensitive calcium channel blockers.

The ability of a series of synthetic analogues of omega-conopeptides MVIIA (SNX-111) and TVIA (SNX-185) to prevent electrically-evoked norepinephrine release from rat tail artery and hippocampal slice preparations was determined in an effort to identify voltage-sensitive calcium channel (VSCC) blockers that selectively target N-type VSCCs in central nervous system tissue. Electrical field stimulation (3 Hz, 1 ms in duration. 80 V for 1 min) caused a high and consistent tritium outflow from rat tail artery and hippocampal slice preparations preloaded with [3H]-norepinephrine. All conopeptides, chosen for their selective affinities for high-affinity SNX-111 binding sites (i.e., N-type VSCCs) over high-affinity omega-conopeptides MVIIC (SNX-230) binding sites (i.e., P/Q-type VSCCs), produced a concentration-dependent inhibition of calcium dependent electrically-evoked tritium outflow from both tail arteries and hippocampal slices: IC50s ranged from 1.2 nM to 1.2 microM. Blocking potencies (IC50s) in the tail artery assay were significantly correlated with those measured in the hippocampal slice preparation (r = 0.91, P = 0.00000012). There was a significant correlation between IC50s for blockade of hippocampal norepinephrine release and the inhibition of high-affinity [125I]-SNX-I11 binding in rat brain synaptosomes (r = 0.76, P = 0.00028). Blockade of hippocampal norepinephrine release was not significantly correlated with the inhibition of high-affinity SNX-230 binding (r = 0.46, P = 0.056). Maximum inhibition of tritium outflow in the tail artery assay was 22+/-1.4% of control, approximating the value (20.9+/-16.0% of control) obtained in the absence of extracellular Ca2+. In contrast, the maximum inhibition of tritium release from hippocampal slices was 36.8+/-2.5% of control (P < 0.05, compared to that of the tail artery assay). These results suggest that (1) N-type VSCCs alone mediate low frequency electrical stimulation-evoked neurotransmitter release from peripheral sympathetic efferents (tail artery) while both N-type and non-N type(s) mediate neurotransmitter release from CNS neurons (hippocampus); and (2) analogues of omega-conopeptides MVIIA and TVIA do not differentiate between N-type VSCCs mediating norepinephrine release from central and peripheral neural tissues.

Use of intrathecal SNX-111, a novel, N-type, voltage-sensitive, calcium channel blocker, in the management of intractable brachial plexus avulsion pain.

OBJECTIVE: The objective was to assess the analgesic, antihyperesthesic, and anti-allodynic properties of SNX-111 in neuropathic pain. DESIGN: We describe a patient with refractory, severe deafferentation pain successfully treated with SNX-111 in an open-label, baseline-controlled Phase I/II trial. SETTING: The patient was hospitalized for treatment and observation. PATIENT: The patient was a 43-year-old man with intractable deafferentation pain of 23 years' duration secondary to brachial plexus avulsion. INTERVENTION: SNX-111, the first neuron-specific, N-type, voltage-sensitive calcium channel blocker developed for clinical use, was administered by continuous, constant-rate, intrathecal infusion via an indwelling cervical catheter. OUTCOME MEASURES: The primary outcome measures were the Visual Analog Scales of Pain Intensity (VASPI) and Pain Relief (VASPR). RESULTS: The patient experienced complete pain relief (VASPI = 0.0 cm and VASPR = 10.0 cm) with elimination of hyperesthesia and allodynia. CONCLUSIONS: SNX-111, administered intrathecally by continuous, constant-rate infusion, produced dose-dependent pain relief in a 43-year-old male patient with a 23-year history of intractable deafferentation and phantom limb pain secondary to brachial plexus avulsion and subsequent amputation. Dizziness, blurred vision, and lateral-gaze nystagmus were dose-dependent side effects that resolved with decreasing dose levels. Complete pain relief was achieved in this patient without side effects after dose adjustment. We conclude that SNX-111 is a potent analgesic, antihyperesthesic, and antiallodynic agent. Controlled studies of SNX-111 in patients with malignant and nonmalignant pain syndromes are warranted and are under way.

Selective blockade of N-type voltage-sensitive calcium channels protects against brain injury after transient focal cerebral ischemia in rats.

The neuroprotective efficacy of the selective N-type voltage-sensitive calcium channel blocker, SNX-111, was evaluated in spontaneously hypertensive rats subjected to 60 min of focal cerebral ischemia by permanent ligation of the right common carotid artery and temporary occlusion of the right middle cerebral artery. Intravenous infusion of 167 microg/kg per min SNX-111 for 30 min (5 mg/kg), initiated immediately after reperfusion, significantly reduced cortical infarct volumes measured 24 h after the ischemic insult.

Selective N-type neuronal voltage-sensitive calcium channel blocker, SNX-111, produces spinal antinociception in rat models of acute, persistent and neuropathic pain.

Male Sprague-Dawley rats were used to evaluate the antinociceptive properties of the selective N-type voltage-sensitive calcium channel (VSCC) blocker, SNX-111, when the compound is administered spinally by either bolus injection or continuous, constant-rate infusion into the subarachnoid space. SNX-111 produced significant, dose-dependent antinociceptive effects by suppressing both the acute (phase 1: ED50, 14 ng/hr) and tonic (phase 2: ED50, 0.82 ng/hr) phases of the formalin test when it was infused for 72 hr immediately before testing. Phase 2 nociceptive responses were suppressed by bolus injections of 100 ng SNX-111. SNX-111 was approximately 1000-fold more potent than morphine in blocking phase 2 responses when the compounds were administered by intrathecal bolus injection. In rats with an experimentally induced painful peripheral neuropathy, intrathecal bolus injections of 30 to 300 ng SNX-111 blocked mechanical allodynia in a dose-dependent manner. Subacute administration of SNX-111 (1, 10 and 100 ng/hr) by continuous intrathecal infusion produced a reversible blockade of mechanical allodynia without apparent development of tolerance. These results show that: 1) selective N-type VSCC blockers are potent and efficacious antinociceptive agents when they are administered by the spinal route; 2) selective N-type VSCC blockers are effective in rat models of acute, persistent and neuropathic pain; and 3) N-type VSCCs play a significant role in the spinal processing of noxious somatosensory input.

Differential blockade of voltage-sensitive calcium channels at the mouse neuromuscular junction by novel omega-conopeptides and omega-agatoxin-IVA.

This investigation assessed the ability of a variety of calcium channel blocking peptides to block synaptic transmission in the isolated mouse phrenic nerve-hemidiaphragm. The synthetic version of the naturally occurring N-type voltage-sensitive calcium channel (VSCC) blocker omega-conopeptide MVIIA (SNX-111) had no effect on nerve-evoked muscle contractions. The non-N-, non-L-type VSCC blocker, omega-conopeptide MVIIC (SNX-230), blocked neuromuscular transmission completely, as did the selective P-type VSCC blocker, omega-Aga-IVA. Subsequent evaluation of other synthetic omega-conopeptides and analogs disclosed a significant positive correlation between the test compounds' affinities for high-affinity SNX-230 brain binding sites and their neuromuscular blocking potencies. Quantal analysis of transmitter release showed that SNX-230 abolished evoked endplate potentials completely, but had little effect on the amplitude and frequency of spontaneous miniature endplate potentials. Perineural focal recordings of presynaptic currents showed that SNX-230 did not block the neuronal action potential. These and other findings indicated that SNX-230 prevents transmitter release at the mouse neuromuscular junction by blocking calcium channels at presynaptic nerve endings. These calcium channels correspond pharmacologically to VSCCs associated with high-affinity binding sites in rat brain and are most probably either of the P- or Q-type.

Cardiovascular effects of omega-conopeptides in conscious rats: mechanisms of action.

We examined the effects of omega-conopeptides, a novel class of neuronal voltage-gated calcium channel antagonists, on hemodynamic responses in rats. Intravenous (i.v.) injections of SNX-111 (omega-conopeptide MVIIA) dose-dependently decreased arterial blood pressure (BP) in conscious rats. Intracerebroventricular (i.c.v.) SNX-111 injections (580 pmol) tended to increase BP and, after an initial decrease, to increase heart rate (HR). The dose-response curve for SNX-111 administered i.v. in conscious rats was biphasic. Beginning at subdepressor doses, SNX-111 caused a long-lasting blockade of pressor responses elicited by sympathetic nerve stimulation in pithed animals but did not prevent increases in BP evoked by exogenously administered norepinephrine (NE). Pretreatment of rats with histamine antagonists partially blocked the hypotensive effects of the higher (870 and 2,900 nmol/kg) doses of SNX-111. Substitution of alanine for arginine at position 10 ([Ala10]-MVIIA) markedly attenuated the histamine-mediated component of the vasodepressor response. Together, these findings indicate that SNX-111 administered i.v. decreases systemic BP by a combination of blockade of sympathetic neurotransmission and mast cell degranulation; the latter function appears to be dependent on the arginine residue in position 10 of the amino acid sequence of SNX-111.

CNS monoamines and their metabolites in canine narcolepsy: a replication study.

In two separate studies a significantly greater concentration of DA (dopamine) and its metabolite, DOPAC (3,4-dihydroxyphenylacetic acid), was observed in the amygdala of narcoleptic canines. DOPAC was also significantly elevated in the reticularis parvicellularis, whereas NE (norepinephrine) was significantly elevated in the reticularis oralis, but depressed in the preoptic hypothalamus. No changes were observed in concentrations of serotonin or its metabolite, 5-HIAA (5-hydroxyindoleauric acid) in any region in the narcoleptic canine brain. Results of the two studies were similar, except that previously observed differences between narcoleptic and control canines in DOPAC levels in the caudate and reticularis oralis failed to replicate. Thus, steady state measures of neurotransmitter/metabolite tissue concentrations suggest region-specific alterations in DA and NE metabolism, rather than a global deficit in catecholamine neurotransmission in canine narcolepsy.

Evidence for multiple [3H]prazosin binding sites in canine brain membranes.

Two classes of alpha 1 adrenoceptors were identified in canine brain and liver using conventional radioligand binding methods. Scatchard plots of specific [3H]prazosin binding to brain and liver membranes prepared from 100-150-day-old Doberman pinscher dogs were consistently curvilinear and best fit a two-site binding model (frontal cortex, Kd1 = 57.7 +/- 10.0 pM, Bmax1 = 64.6 +/- 17.1 fmol/mg protein, Kd2 = 1.5 +/- 0.5 nM, Bmax2 = 159 +/- 37.6 fmol/mg protein; liver, Kd1 = 82.6 +/- 36 pM, Bmax1 = 7.0 +/- 5.1 fmol/mg protein, Kd2 = 0.8 +/- 0.2 nM, Bmax2 = 62.1 +/- 8.7 fmol/mg protein). Kinetically derived affinity constants from association and dissociation experiments agreed with those obtained by Scatchard analyses of equilibrium binding data. Binding sites were saturable, heat labile, bound ligand reversibly, and appeared to be appropriately distributed in relation to endogenous catecholamine. [3H]Prazosin also bound with high affinity to two classes of binding site in porcine and bovine brain membrane but [3H]prazosin binding in monkey and rat brain was best described by a single-site binding model. Affinities obtained were in between values obtained for high and low affinity Kds in the other species. Competitions for [3H]prazosin binding sites in canine frontal cortex were conducted with the following antagonists: WB-4101, corynanthine, phentolamine, benoxathian, phenoxybenzamine, chlorethylclonidine, thymoxamine, prazosin, yohimbine and agonists: methoxamine, (-)-norepinephrine, and clonidine. All ligands but prazosin, norepinephrine and clonidine competed for specific [3H]prazosin binding in a statistically significant biphasic manner. Benoxathian and WB-4101 displayed the highest affinities (benoxathian: Ki1 = 0.26 nM, WB-4101: Ki1 = 0.20 nM) and selectivity (high affinity/low affinity: benoxathian = 1640, WB-4101 = 13204) for the high affinity [3H]prazosin binding site; chlorethylclonidine had highest affinity (Ki2 = 91 nM) and selectivity (low affinity/high affinity = 405) for the lower affinity [3H]prazosin binding site. As defined, the two sites were similar to the alpha 1a and alpha 1b recently described in the rat and rabbit. A noticeable difference was that the subtypes described in dog brain had a 30-fold difference in affinity for prazosin.

The influence of ambient temperature on sleep characteristics in the aged cat.

Sleep characteristics were compared in young adult and aged cats over a range of environmental temperatures from 5 to 35 degrees C. Although both groups exhibited sleep disruptions as ambient temperatures decreased, transient arousals were increased at temperature extremes in the aged group compared to young adults. Declining efficiency of thermoregulatory control and increased sensitivity to environmental temperature in the elderly may contribute to the changes in sleep quality which occur during the aging process.

Influence of freezer storage time on cerebral biogenic amine and metabolite concentrations and receptor ligand binding characteristics.

Brains from breed and age-matched canines stored at -80 degrees C for between 3 and 44 months showed a time-dependent decline in the concentration of norepinephrine in the amygdala and dopamine and norepinephrine in the caudate. No changes were seen in the density or ligand affinities of prazosin or spiperone binding sites in the same areas nor were there changes in quinuclidinyl benzylate binding sites in the frontal cortex. The changes in dopamine concentrations in the caudate were not accompanied by changes in the concentrations of dopamine metabolites. The chromatograms from which the dopamine and norepinephrine concentrations were estimated contained several unidentified, amperometrically detectable, extraneous peaks which increased in size in the older tissue samples. These results suggest that the decline in dopamine and norepinephrine concentrations was not the result of enzymatic breakdown, but probably the result of chemical decomposition. These findings have significance for the measurement of dopamine and norepinephrine concentrations in autopsied brains kept frozen in storage.

Modulation of activity of the striatal dopaminergic system during the hibernation cycle.

To evaluate how the activity of a well-established neurotransmitter pathway is modulated by a behavioral state, 3H-spiperone binding sites and dopamine (DA) and DA metabolite concentrations were measured in the striata of ground squirrels in 5 phases of the hibernation cycle. Whereas levels of striatal DA and its deaminated metabolite DOPAC did not change significantly, the concentrations of the O-methylated-deaminated metabolite, homovanillic acid (HVA), decreased in all phases of hibernation relative to euthermia. Striatal 3H-spiperone binding sites declined across the hibernation cycle in parallel with the reduction of HVA concentration; receptor binding affinity was unchanged by arousal state. In conjunction with previously reported findings, these results are consistent with the hypothesis that hibernation is associated with a down-regulation of the postsynaptic D2 receptors secondary to increased extracellular DA concentration and reduced DA degradation.

Brain dopamine receptor levels elevated in canine narcolepsy.

Concentrations of dopamine D2 receptors in discrete brain areas differed significantly between dogs with the genetically transmitted form of narcolepsy, and age- and breed-matched controls. D2 receptors were assayed and quantified with Scatchard analysis using [3H]spiperone. Receptor densities in the nucleus accumbens, rostral caudate, and amygdala were consistently higher in narcoleptic animals. In amygdala, dopamine receptor abnormalities were associated with elevated dopamine and 3,4-dihydroxyphenylacetic acid concentrations, but no change in 3-methoxytyramine or homovanillic acid concentrations. These data indicate mesolimbic system involvement in canine narcolepsy and point to impaired dopamine release as a possible etiologic factor.

Brain benzodiazepine receptor characteristics in canine narcolepsy.

A regional analysis of brain benzodiazepine (BDZ) receptors was conducted in narcoleptic and normal dogs to determine whether these sites are altered in narcolepsy. It was postulated that BDZ receptors play a role in the excessive sleepiness of narcolepsy because activation of these sites in freely behaving normal animals is hypnogenic. [3H]Flunitrazepam binding sites were assessed in 11 discrete areas of the forebrain and brainstem. No consistent or statistically significant group differences in either receptor densities (Bmax) or binding affinities (Kd) were found. These findings do not support the assertion that BDZ receptors are involved in the pathogenesis of canine narcolepsy.

Muscarinic cholinergic receptors and the canine model of narcolepsy.

The role of the muscarinic cholinergic receptor in narcolepsy was examined using radioligand binding to various brain regions of normal and genetically narcoleptic Doberman pinschers. In this multi-litter study, a previous report of a proliferation of muscarinic cholinergic receptors in the brainstem was confirmed, and the concentration of the M2 receptor subtype, in particular, was elevated. This up-regulation of brainstem cholinergic receptors suggests a problem with release of acetylcholine, which, together with previous reports of an impairment of dopamine release, may be indicative of a fundamental membrane problem in narcolepsy.