Effect of inhibition of neuropeptidases on the pain threshold of mice and rats.

The effect of the inhibition of aminopeptidase and enkephalinase A on the pain threshold of mice and rats was investigated, using bestatin and thiorphan as selective peptidase inhibitors. The results indicate that both enzymes are relevant to the catabolism of enkephalins in vivo; however, their simultaneous activation requires particular conditions. These conclusions are based on the following observations: (1) Only concomitant intracerebral treatment with both inhibitors led to an increase in the threshold of animal pain, whereas, in the presence of exogenous peptides, the concomitant injection of both inhibitors in mice elicited an analgesic response greater than the sum of the effects of each single inhibitor. (2) This response could be seen only after acute trauma; in fact, when the drugs were injected through a plastic cannula, only enkephalinase A inhibition was effective in increasing analgesia induced by exogenous peptides.

Combined analgesic/neuroleptic activity in N-butyrophenone prodine-like compounds.

Some 4-phenyl-4-piperidinols, corresponding esters, and related compounds with a p-fluorobutyrophenone chain on nitrogen were synthesized and evaluated in in vitro and in vivo tests in order to examine their ability to interact contemporaneously with opioid and dopamine receptors. The propionyloxy derivatives showed a good combination of analgesic and neuroleptic activity. With a 3-methyl substituent on the piperidine ring, the beta-configuration was the more active form not only for analgesic activity, as expected from previous results on prodines, but also for neuroleptic activity. Haloperidol and its propionate were also tested as reference compounds.

N-(4-Isoxazolylthiazol-2-yl)oxamic acid derivatives as potent orally active antianaphylactic agents.

A series of N-(4-isoxazolylthiazol-2-yl)oxamic acid derivatives was synthesized and tested on the passive cutaneous anaphylaxis (PCA) model in rats to verify its potential antianaphylactic activity. These compounds were prepared by reaction of an appropriate bromoacetylisoxazole with thiourea to give the corresponding aminothiazole and subsequent condensation with an oxalic acid monoester chloride to yield, following the usual process, the oxamic acid derivatives. Most of the new compounds exhibited, by intraperitoneal route in rats, a very potent antianaphylactic activity on PCA response, higher than that of the reference compound disodium cromoglycate (DSCG). The new derivatives, in contrast with DSCG, were effective on PCA even by oral route. The most interesting derivative of the new series was N-[4-(3-methyl-5-isoxazolyl)-2-thiazolyl]oxamic acid 2-ethoxyethyl ester (49), which was also active and more potent than DSCG in experimental models involving either IgE- or IgG-mediated anaphylactic responses at bronchopulmonary level.

Common stereospecificity of opioid and dopamine systems for N-butyrophenone prodine-like compounds.

The two optical isomers of 1-[3-(p-fluorobenzoyl) propyl]-3-methyl-4-phenyl-4-propionoxypiperidine (FPP) were obtained by resolution of (+/-)-r-3-methyl-4-phenyl-c-4-piperidinol followed by N-alkylation and O-propionylation. These, as well as the racemate, were evaluated for their antinociceptive, opioid, and neuroleptic properties using in vivo and in vitro test systems. The results are remarkable in two respects, namely, the dextrorotatory isomer is consistently the most potent on all tests, and it acts on both opioid (mu) and neuroleptic (D2) receptors.

Neurotolerability of contrast agents in rats with brain ischemia induced by transient middle cerebral artery occlusion: EEG evaluation.

RATIONALE AND OBJECTIVES: Because contrast agent (CA) formulations are injected intravenously to patients who may have a disrupted blood-brain barrier, their neurotolerability should be tested by using appropriate animal models. In the present study, a model of rat brain ischemia evaluated in terms of the electroencephalogram (EEG) was validated and then used to compare the neurotolerability of gadobenate dimeglumine to that of gadodiamide, a well-documented CA for brain MRI. METHODS: Rats were prepared for EEG recording about 15 days before ischemia induction. Ischemia was induced in the right hemisphere by 2-hour middle cerebral artery (MCA) occlusion and 3-day reperfusion. Model validation in terms of EEG, on day 3 after MCA occlusion, was performed by using iopromide, a poorly neurotolerated iodinated CA in rats, intravenously injected at 7 g iodine/kg. The EEG recording was analyzed for pathological tracings and for changes in spectral content in terms of the frequency index (FI) at 1, 2, and 3 hours after test compound injection. The comparative study between gadobenate dimeglumine and gadodiamide was performed at 2.0 mmol/kg. D-Mannitol was used as a control compound. The presence of CA in the rat brain was verified by measuring the total gadolinium content by using inductively coupled plasma-atomic emission spectrometry analysis. Given the absence of metabolism for both CAs, the values of gadolinium content can be interpreted as representing unmetabolized CA. RESULTS: On days 1, 2, and 3 after transient MCA occlusion, the lesioned hemisphere of rats presented a decreased FI value with respect to the basal value. The unlesioned hemisphere, after a slight, nonsignificant decrease in the FI value on the first 2 days, presented a normal FI value on day 3. Thus, ischemic rats on day 3 after transient MCA occlusion were chosen for these neurotolerability studies. Iopromide injected intravenously into ischemic rats at a dose 10 times higher than the maximum clinical dose caused bilateral spikes on the EEG and increases in FI values for the unlesioned hemisphere without affecting the lesioned hemisphere. Gadobenate dimeglumine, like gadodiamide when injected into ischemic rats, did not cause spikes or further changes in the FI value of the lesioned hemisphere and did not modify the normal FI value of the unlesioned hemisphere. Furthermore, no significant differences between gadobenate dimeglumine, gadodiamide, and D-mannitol were found when postinjection FI values were compared. Finally, higher levels of gadolinium were found in the lesioned hemisphere with respect to the unlesioned hemisphere after both gadobenate dimeglumine and gadodiamide administration. CONCLUSIONS: We can therefore conclude that (1) on the EEG, ischemia induced by transient MCA occlusion is an appropriate model for evaluating CA neurotolerability because ischemic and CA effects can be clearly differentiated; (2) the higher level of CA in the lesioned hemisphere compared with the unlesioned one (two to three times), even 3 hours after injection, demonstrates that the CA effectively penetrated the brain; if it were neurotoxic, any negative effects would have been detected; and (3) gadobenate dimeglumine, like gadodiamide, injected intravenously at a dose 20 times higher than the intended clinical dose for brain MRI is well tolerated and, also like gadodiamide, is suitable for use in neurological diseases for which contrast-enhanced MRI is indicated.

Comparative study between gadobenate dimeglumine and gadobutrol in rats with brain ischemia: evaluation of somatosensory evoked potentials.

RATIONALE AND OBJECTIVES: The present study had two main objectives: to validate a rat model of brain ischemia in terms of somatosensory evoked potentials (SEPs) and to compare, using the validated model, the potential activity on the somatosensory function of the new, approved contrast agent gadobenate dimeglumine with that of gadobutrol, a specific contrast agent, for magnetic resonance imaging of the brain. METHODS: Rats were prepared for SEP recording at least 5 days before ischemia induction. Ischemia was induced by 30-minute right middle cerebral artery occlusion and 3-day reperfusion. The SEP responses, evoked contralaterally to the stimulated upper limb, were recorded from the primary somatosensory cortical area. Model validation, on day 3 after occlusion, was performed using iopromide, an iodinated contrast agent poorly neurotolerated in rats, intravenously injected at 7 g I/kg. The comparative study between gadobenate dimeglumine and gadobutrol was performed at an intravenously injected dose of 2 mmol/kg. Somatosensory evoked potential responses were measured as peak latencies (P2 and N2) and peak-to-peak amplitude (P2N2). The brain concentration of iopromide was measured by high-performance liquid chromatography, whereas that of the gadolinium contrast agent was measured by inductively coupled plasma-atomic emission spectrometry analysis; given the absence of metabolism for both contrast agents, the gadolinium content values can be interpreted as representing unmetabolized contrast agent. RESULTS: In the ischemic rats, the SEP responses of the lesioned hemisphere showed significant increases in P2, N2, and interpeak N2-P2 latencies and a significant reduction in peak-to-peak (P2N2) amplitude. In the validation experiments, iopromide dramatically increased the P2N2 amplitude of the SEP responses recorded from both hemispheres of ischemic rats without affecting the P2, N2, and interpeak N2-P2 latencies. The iopromide effect was coupled with high concentrations of the contrast agent in the brain. Iopromide had no effect on healthy rats. In the comparative study, gadobenate dimeglumine did not induce any alteration in SEP components of either the lesioned or unlesioned hemisphere of ischemic rats. In fact, no significant difference was found between responses obtained before and after gadobenate dimeglumine injection. Gadobutrol, administered at the same dose, on the whole showed the same behavior as gadobenate dimeglumine, although a slight but significant decrease in the P2 latency, a sign of excitatory activity, was observed 2 hours after injection. Analytic data indicated higher levels of contrast agent in the lesioned hemisphere versus the unlesioned hemisphere 2 hours after injection. CONCLUSIONS: Based on these results, three conclusions can be drawn: (1) the evaluation of SEPs in ischemic rats is a useful tool for assessing the potential neurological effects of a new contrast agent because ischemic and contrast agent effects can be clearly differentiated; (2) the experimental conditions used allow the contrast agents to penetrate into the brain, where their activity can be manifested and evaluated; and (3) the complete absence of neurological activity of gadobenate dimeglumine shows its safety profile and confirms its suitability for use in neurological diseases for which contrast-enhanced magnetic resonance imaging is indicated.

Neurotolerability of gadobenate dimeglumine. Evaluation of brain dopamine concentration in freely moving rats by microdialysis.

RATIONALE AND OBJECTIVES: Magnetic resonance imaging with contrast agents (CAs) is a procedure currently used for the diagnosis of neurologic pathologies. These pathologies are often characterized by blood-brain barrier disruptions, which determines a direct contact between CA and brain tissue. For this reason, an accurate assessment of neurotolerability is useful for the development of new CAs. The present study was designed to evaluate the neurotolerability of a new CA for MRI, gadobenate dimeglumine, employing a neurochemical method. The effect of gadobenate dimeglumine on the striatal levels of neurotransmitters was determined. In particular, the brain concentrations of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic and homovanillic acid, were measured using microdialysis, after the direct application of gadobenate dimeglumine into the rat corpus striatum. Gadopentetate dimeglumine and gadoteridol were employed as reference compounds. METHODS: A microdialysis probe for brain extracellular fluid sampling and a stainless-steel cannula for CA application were chronically inserted into the right corpus striatum of rats. All CAs were administered at a dose of 5.4 nmol/rat. Dopamine and metabolite concentrations were analyzed using high-performance liquid chromatography. RESULTS: Gadobenate dimeglumine did not induce any significant changes in the extracellular levels of dopamine or dopamine metabolites up to 2 hours after administration. Gadoteridol produced similar results. Gadopentetate dimeglumine caused a moderate but not significant increase in dopamine levels throughout the duration of the experiments. CONCLUSIONS: Gadobenate dimeglumine directly administered into the corpus striatum of freely moving rats did not affect the dopaminergic system. This result demonstrates the safety of gadobenate dimeglumine under the experimental conditions used, thus confirming previous behavioral and electrophysiologic findings.

Neurotolerability of gadobenate dimeglumine in a rat model of focal brain ischemia: EEG evaluation.

RATIONALE AND OBJECTIVES: The neurologic pathologies for which contrast-enhanced MRI is indicated are often accompanied by a disruption of the blood-brain barrier (BBB), which allows the contrast agent to come into contact with the nervous tissue. Thus, assessment of the neurologic safety for a new contrast agent is of crucial importance. The objective of this study was to assess the neurotolerability of the new MRI contrast agent gadobenate dimeglumine using EEG in the presence of focal lesions of the BBB. METHODS: Lesions of the BBB were obtained inducing a photochemical ischemia in rats. Gadobenate dimeglumine was intravenously administered at 4.0 mmol/kg. An EEG was recorded during sleep/awake behavior and was analyzed for pathologic tracing and for changes in spectral content in terms of total power and frequency index. The presence of the BBB lesions was verified using high-performance liquid chromatography measurement of the gadobenate ion content in the brain. RESULTS: Gadobenate dimeglumine did not have any epileptogenic effect in ischemic rats. However, it caused a transitory shift of the EEG power spectrum toward the 0.5 to 9 Hz frequency bands of the lesioned hemisphere during quiet wake. In the lesioned cortex, higher levels of gadobenate ion were found until 3 hours after administration. CONCLUSIONS: In experimental conditions of focal brain ischemia associated with BBB lesions, gadobenate dimeglumine was well tolerated up to doses even 10 times higher than the maximum clinical dose (0.3 mmol/kg) intended for brain imaging procedures.

Involvement of monoaminergic and peptidergic components in cathinone-induced analgesia.

Evidence has been obtained suggesting that cathinone-induced analgesia depends upon stimulation of alpha-adrenoceptors, followed by release of opioid peptides and by activation of serotonergic pathways. This hypothesis is supported by the following. (1) Cathinone potentiated morphine analgesia and the whole effect was antagonized by naloxone whereas onto the cathinone potentiation was counteracted by phenoxybenzamine. (2) Bestatin potentiated cathinone-induced analgesia and this effect was sensitive to both naloxone and phenoxybenzamine blockade. (3) The analgesic effect of cathinone + bestatin was further potentiated by the serotonin uptake inhibitor citalopram.

Effect of bestatin and thiorphan on [Met5]enkephalin-Arg6-Phe7-induced analgesia.

We investigated the effect of bestatin, a specific inhibitor of aminopeptidase and thiorphan, a specific inhibitor of enkephalinase A, on the analgesic effect induced by the intracerebral injection of heptapeptide [Met5]enkephalin-Arg6-Phe7 (MEAP) in cannulated rats. In contrast with the results obtained when [Met5]enkephalin (ME) was used, bestatin clearly potentiated the analgesic effect of MEAP, but thiorphan was totally ineffective. These observations indicate that the predominant inactivating mechanism for MEAP is the action of an aminopeptidase whereas this enzyme seems to be little involved in the catabolism of ME. The existence of two different catabolic pathways for MEAP and ME suggests that MEAP may act not only as a precursor of ME but also as an independent neuromodulator.

Bivalent ligand type beta-adrenolytics related to practolol.

The synthesis and pharmacological evaluation of a number of symmetrical bivalent ligand type beta-adrenolytics related to practolol are reported. The best results have been observed with N,N'-bis[3-[2-hydroxy-3-[1-methylethyl)amino]propoxy]phenyl] ethanediamide.

Bis-arenoxypropanolamines as potential beta-adrenolytics: a reinvestigation.

A number of symmetrical as well as asymmetrical bis-arenoxypropanolamines have been synthesized and evaluated for their beta-adrenolytic properties. The pharmacological characterization of these compounds was performed in vitro by direct studies (3H-dihydroalprenolol was used as the specific ligand for beta-receptors), and by determining the beta-blocking capacity on isoproterenol (ISO)-induced increased heart rate (isolated guinea-pig atrium); and in vivo by evaluating the antagonism on ISO-induced hypotension and tachycardia, as well as the intrinsic sympathomimetic activity (ISA) on reserpinized and vagotomized rats. The best result was observed with the simplest derivative, i.e. the bis-phenoxypropanolamine compound, which shows in vitro and in vivo potencies almost comparable to propranolol and, moreover, significant bronchodilating activity.

Gadobenate dimeglumine and cerebral glucose metabolism. Continuous monitoring of striatal lactate levels in freely moving rats.

PURPOSE: Brain contrast-enhanced MR imaging reflects the leakage of contrast material into the brain tissue due to blood-brain barrier (BBB) disruption. The contact between brain tissue and contrast material requires a high level of neurotolerability of the contrast agent (CA). In the present study, we investigated the neurotolerability of the paramagnetic CA gadobenate dimeglumine, locally applied into the corpus striatum of freely moving rats, by evaluating its potential effects on cerebral glucose metabolism based on lactate levels. MATERIAL AND METHODS: Lactate levels were monitored using a microdialysis technique coupled with an enzyme reaction. A microdialysis probe for extracellular fluid sampling, together with a stainless steel cannula for CA administration, were inserted into the right corpus striatum of rats. Lactate levels were monitored for 2 h after gadobenate dimeglumine administration at 120 nmol/rat, at fixed volume of 1.2 microl. The same volume of artificial cerebrospinal fluid (aCSF) was administered to control rats. RESULTS: Gadobenate dimeglumine did not induce any significant changes in the lactate striatal levels over the 30-min period after administration. Small, but significant, reductions in lactate concentration were found from the 45-min control point after gadobenate dimeglumine administration. Lactate response showed the same pattern in rats given aCSF. CONCLUSION: Gadobenate dimeglumine, intracerebrally administered, did not affect cerebral glucose metabolism in rats as it showed the same behaviour as aCSF on cerebral glucose utilization. The gradual attenuation in the endogenous lactate release observed 45 min after test compound administration is possibly due to a slight reduction in the probe recovery. The present findings confirm the neurotolerability of gadobenate dimeglumine previously shown in behavioural and electrophysiological studies.