The formalin test does not probe inflammatory pain but excitotoxicity in rodent skin.

The most widely used formalin test to screen antinociceptive drug candidates is still apostrophized as targeting inflammatory pain, in spite of strong opposing evidence published. In our rat skin-nerve preparation ex vivo, recording from all classes of sensory single-fibers (n = 32), 30 units were transiently excited by formaldehyde concentrations 1-100 mM applied to receptive fields (RFs) for 3 min, C and Aδ-fibers being more sensitive (1-30 mM) than Aß-fibers. From 30 mM on, ~1% of the concentration usually injected in vivo, all RFs were defunctionalized and conduction in an isolated sciatic nerve preparation was irreversibly blocked. Thus, formaldehyde, generated a state of 'anesthesia dolorosa' in the RFs in so far as after a quiescent interphase all fibers with unmyelinated terminals developed a second phase of vigorous discharge activity which correlated well in time course and magnitude with published pain-related behaviors. Sural nerve filament recordings in vivo confirmed that higher formalin concentrations (> 42 mM) have to be injected to the skin to induce this second phase of discharge. Patch-clamp and calcium-imaging confirmed TRPA1 as the primary transducer of formaldehyde (10 mM) effects on mouse sensory neurons. However, stimulated CGRP release from isolated skin of TRPA1+/+ and TRPA1-/- mice showed a convergence of the saturating concentration-response curves at 100 mM formaldehyde, which did not occur with nerve and trachea preparations. Finally, skin-nerve recordings from C and Aδ-fibers of TRPA1-/- mice revealed a massive reduction in formaldehyde (30 mM)-evoked discharge. However, the remaining activity was still biphasic, thus confirming additional unspecific excitotoxic actions of the fixative that diffuses along still excitable axons as previously published. The multiplicity of formaldehyde's actions requires extensive discussion and literature review, leading to a fundamental reevaluation of the formalin test.

Early-stage Mycosis Fungoides: Epidemiology and Prognosis.

Most patients with mycosis fungoides are diagnosed with early-stage disease. However, prevalence of early-stage disease is unknown, and evidence of its burden is scarce. The aim of this study is to estimate the prevalence of early-stage mycosis fungoides, how long patients live with early-stage disease and to characterise these patients. Data were obtained from 4 key publications and from US cancer registries (Surveillance, Epidemiology and End Results Program; SEER). The derived incidence of early-stage mycosis fungoides was 0.26/100,000 (UK), 0.29/100,000 (US) and 0.38/100,000 (US-SEER) and the prevalence was 4.8/100,000 (UK), 5.2/100,000 (US) and 6.6/100,000 (US-SEER). Early-stage disease may last for 18 years. From SEER registries, 3,132 were diagnosed at early stage (mostly stage IA). Median age at diagnosis was 58 years. Compared with stage IA, the relative risk of death was 1.3 for stage IB and 3.5 for stage IIA. We confirm the rarity of early-stage mycosis fungoides, a differential prognosis and the potential for elevated burden of disease.

The metabotropic glutamate receptor 5 radioligand [11C]AZD9272 identifies unique binding sites in primate brain.

The metabotropic glutamate receptor 5 (mGluR5) is a target for drug development and for imaging studies of the glutamate system in neurological and psychiatric disorders. [11C]AZD9272 is a selective mGluR5 PET radioligand that is structurally different from hitherto applied mGluR5 radioligands. In the present investigation we compared the binding patterns of radiolabeled AZD9272 and other mGluR5 radioligands in the non-human primate (NHP) brain. PET studies were undertaken using [11C]AZD9272 and the commonly applied mGluR5 radioligand [11C]ABP688. Autoradiography studies were performed in vitro using [3H]AZD9272 and the standard mGluR5 radioligands [3H]M-MTEP and [3H]ABP688 in NHP tissue. Competition binding studies were undertaken in vivo and in vitro using different mGluR5 selective compounds as inhibitors. In comparison to other mGluR5 radioligands radiolabeled AZD9272 displayed a distinct regional distribution pattern with high binding in ventral striatum, midbrain, thalamus and cerebellum. While the binding of [11C]AZD9272 was almost completely inhibited by the structurally unique mGluR5 compound fenobam (2.0 mg/kg; 98% occupancy), it was only partially inhibited (46% and 20%, respectively) by the mGluR5 selective compounds ABP688 and MTEP, at a dose (2.0 mg/kg) expected to saturate the mGluR5. Autoradiography studies using [3H]AZD9272 confirmed a distinct pharmacologic profile characterized by preferential sensitivity to fenobam. The distinctive binding in ventral striato-pallido-thalamic circuits and shared pharmacologic profile with the pro-psychotic compound fenobam warrants further examination of [11C]AZD9272 for potential application in psychiatric neuroimaging studies.

AZD9272 and AZD2066: selective and highly central nervous system penetrant mGluR5 antagonists characterized by their discriminative effects.

The metabotropic glutamate receptor 5 (mGluR5) antagonists fenobam, MPEP (2-methyl-6-(phenylethynyl)pyridine), and MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine) were previously shown to not cause N-methyl-D-aspartate antagonist-like psychoactive effects in phencyclidine (PCP) drug discrimination studies, but to cause MTEP-like discrimination in rats, suggesting that the psychoactive and psychotomimetic effects reported with fenobam in humans were likely mediated by mGluR5 antagonist mechanisms. The present study was designed to characterize AZD9272 (3-fluoro-5-(3-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol5-yl)benzonitrile) and AZD2066 [4-(5-{(1R)-1-[5-(3-chlorophenyl)isoxazol-3-yl]ethoxy}-4-methyl-4H-1,2,4-triazol-3-yl)pyridine], two mGluR5 antagonists taken to clinical development for analgesia. AZD9272 was evaluated in several groups of rats trained to discriminate cocaine, PCP, chlordiazepoxide, (-)-Δ(9)-tetrahydrocannabinol [(-)-Δ(9)-THC], or MTEP from no drug. AZD9272 shared discriminative properties with MTEP only. The discriminative half-life was 3.23 hours for MTEP and 21.93 hours for AZD9272 in rats trained to discriminate MTEP from no drug. Other rats were successfully trained to discriminate AZD9272 from no drug. Due to the long duration of action of AZD9272, discrimination training was conducted every other day. AZD9272 caused a dose-dependent increase in AZD9272-appropriate responding. PCP did not cause AZD9272-appropriate responding, whereas MTEP, fenobam, and the mGluR5 antagonist AZD2066 did. The discriminative half-life of AZD9272 was 24.3 hours in rats trained to discriminate AZD9272 from no drug. It is concluded that the discriminative effects of AZD9272 and AZD2066 are similar to those of previously investigated mGluR5 antagonists and dissimilar to those of cocaine, PCP, chlordiazepoxide, and (-)-Δ(9)-THC. The discriminative half-life of AZD9272 is approximately 7-fold longer than for MTEP. These data support and extend previous findings suggesting that mGluR5 antagonism causes psychoactive effects selectively mediated by mGluR5 mechanisms.

mGluR5 antagonist-induced psychoactive properties: MTEP drug discrimination, a pharmacologically selective non-NMDA effect with apparent lack of reinforcing properties.

Fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea], a potent metabotropic glutamate mGluR5 receptor antagonist, reported to have analgesic effects in animals and anxiolytic effects in humans, also caused adverse events, including psychostimulant-type effects and "derealization phenomena." Recent electrophysiologic, pharmacologic, and anatomic data show that the mGluR5 antagonists 2-methyl-6-(phenylethynyl)pyridine (MPEP) and (E)-2-methyl-6-styryl-pyridine (SIB-1893) can inhibit NMDA receptor-mediated activity and that mGluR5 receptors are highly expressed in limbic and forebrain regions. The present studies first evaluated the potential of mGluR5 receptor antagonists to cause PCP-like psychoactive effects in a rat drug discrimination procedure and, second, explored and characterized the selective mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) as a discriminative stimulus and compared MTEP with other drugs known to be psychoactive in humans. Additionally, the reinforcing potential of MPEP and MTEP was compared with phencyclidine (PCP) in a rat intravenous self-administration procedure. Dizocilpine [(+)-MK-801] and ketamine caused full PCP-appropriate responding. Memantine and the mGluR5 antagonists caused no or weak partial PCP-appropriate responding. In MTEP-trained rats, MTEP, MPEP, and fenobam caused full and equipotent MTEP-appropriate responding. (+)-MK-801 and memantine caused MTEP-appropriate responding below 70%, whereas PCP, chlordiazepoxide and LSD caused MTEP-appropriate responding below 50%. Δ(9)-Tetrahydrocannabinol, yohimbine, arecoline, and pentylenetetrazole all caused MTEP-appropriate responding below 20%. Rats self-administered PCP but not MPEP or MTEP, indicating a lack of reinforcing effects of the mGluR5 antagonists. These data suggest that the mGluR5 antagonists appear not to have reinforcing properties, that the discriminative effects of mGluR5 antagonists and PCP are dissimilar, and that mGluR5 antagonists may produce psychoactive effects different from NMDA-antagonists and other drugs with known psychotomimetic properties.

The interphase of the formalin test.

The formalin test still surprises with its biphasic pain-related behavior resulting from a quiescent interphase that does not occur with other algogenic compounds and remains unexplained. The first phase has been attributed to TRPA1-mediated excitation of nociceptors, the second phase to their inflammatory and/or spinal sensitization. We show that the second and interphase require higher formaldehyde concentrations to emerge, and that from 12 mM on calcium influx is induced in TRPA1-deficient sensory neurons as well as in native HEK293T cells. After a short depolarizing and excitatory period in a subset, all wild-type neurons showed a concentration-dependent hyperpolarization, a reduction of voltage-activated sodium currents, and a progressive increase of the input resistance, which, after about 10 min restored the transiently lost excitability, enabling smaller and wider action potentials to be evoked than before formaldehyde (30 mM). The hyperpolarizing effect was absent if extracellular sodium was replaced, and largely prevented by a high but not low concentration of tetrodotoxin. In rat skin in vivo, the spatiotemporal redistribution of injected formalin and the plasma extravasation were studied using Evans blue. The parameters gained were entered into a computational model to predict the activation pattern of primary afferents. The model supports a peripherally generated biphasic response, the time course matching the behavioral results. In conclusion, the interphase is a result of hyperpolarization and transient inactivation by formaldehyde of the surviving neurons; their recovery and the centrifugal spread of formalin in the skin induce a second phase of nociceptive activity before the formalin concentration falls below threshold.

A positron emission tomography study in healthy volunteers to estimate mGluR5 receptor occupancy of AZD2066 - estimating occupancy in the absence of a reference region.

AZD2066 is a new chemical entity pharmacologically characterized as a selective, negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). Antagonism of mGluR5 has been implicated in relation to various diseases such as anxiety, depression, and pain disorders. To support translation from preclinical results and previous experiences with this target in man, a positron emission tomography study was performed to estimate the relationship between AZD2066 plasma concentrations and receptor occupancy in the human brain, using the mGluR5 radioligand [(11)C]-ABP688. The study involved PET scans on 4 occasions in 6 healthy volunteers. The radioligand was given as a tracer dose alone and following oral treatment with different doses of AZD2066. The analysis was based on the total volume of distribution derived from each PET-assessment. A non-linear mixed effects model was developed where ten delineated brain regions of interest from all PET scans were included in one simultaneous fit. For comparison the analysis was also performed according to a method described previously by Lassen et al. (1995). The results of the analysis showed that the total volume of distribution decreased with increasing drug concentrations in all regions with an estimated Kipl of 1170 nM. Variability between individuals and occasions in non-displaceable volume of distribution could explain most of the variability in the total volume of distribution. The Lassen approach provided a similar estimate for Kipl, but the variability was exaggerated and difficult to interpret.

Palladium mediated ¹¹C-cyanation and characterization in the non-human primate brain of the novel mGluR5 radioligand [¹¹C]AZD9272.

INTRODUCTION: The aims of the present positron emission tomography (PET) study were to set up a system for (11)C-cyanation labeling of the selective mGluR5-antagonist [(11)C]AZD9272 and to perform the first in vivo characterization of [(11)C]AZD9272 binding in cynomolgus monkeys. METHODS: [(11)C]AZD9272 was labeled using palladium mediated (11)C-cyanation. Altogether seven PET measurements were performed in three cynomolgus monkeys including baseline and co-injection experiments with unlabelled AZD9272 (0.04 and 0.4 mg/kg). Radiometabolites in plasma were measured using HPLC. RESULTS: [(11)C]AZD9272 was prepared in over 50% incorporation yield from hydrogen [(11)C]cyanide in a total synthesis time of 45-50 min. The radiochemical purity of the radioligand in its final formulation was high (>99%) and the mean specific radioactivity was 47 GBq/ µmol (1278 Ci/mmol, n=7) calculated at end of bombardment (EOB). In the baseline measurements 10% of the total injected radioactivity was present in monkey brain at five minutes after i.v. injection. The radioactivity concentration was high in the caudate, cingulate gyrus and thalamus whereas it was moderate in the temporal cortex and lower for the cerebellum. After co-injection with cold AZD9272 the binding of [(11)C]AZD9272 was reduced in a dose-dependent fashion. Analysis of radiometabolites showed relatively slow metabolism and resulted only in hydrophilic radiometabolites. CONCLUSION: A fast and efficient method was developed to label AZD9272 with (11)C. PET-examination in Cynomolgus monkeys showed that [(11)C]AZD9272 entered the brain to a high extent, that binding was saturable and that the regional radioactivity pattern was in accordance with the known distribution of mGluR5. The results support further examination of [(11)C]AZD9272 binding in human subjects.

In vitro pharmacological characterization of a novel TRPA1 antagonist and proof of mechanism in a human dental pulp model.

AZ465 is a novel selective transient receptor potential cation channel, member A1 (TRPA1) antagonist identified during a focused drug discovery effort. In vitro, AZ465 fully inhibits activation by zinc, O-chlorobenzylidene malononitrile (CS), or cinnamaldehyde of the human TRPA1 channel heterologously expressed in human embryonic kidney cells. Our data using patch-clamp recordings and mouse/human TRPA1 chimeras suggest that AZ465 binds reversibly in the pore region of the human TRPA1 channel. Finally, in an ex vivo model measuring TRPA1 agonist-stimulated release of neuropeptides from human dental pulp biopsies, AZD465 was able to block 50%-60% of CS-induced calcitonin gene-related peptide release, confirming that AZ465 inhibits the native human TRPA1 channel in neuronal tissue.

Discovery and characterization of AZD9272 and AZD6538-Two novel mGluR5 negative allosteric modulators selected for clinical development.

AZD9272 and AZD6538 are two novel mGluR5 negative allosteric modulators selected for further clinical development. An initial high-throughput screening revealed leads with promising profiles, which were further optimized by minor, yet indispensable, structural modifications to bring forth these drug candidates. Advantageously, both compounds may be synthesized in as little as one step. Both are highly potent and selective for the human as well as the rat mGluR5 where they interact at the same binding site than MPEP. They are orally available, allow for long interval administration due to a high metabolic stability and long half-lives in rats and permeate the blood brain barrier to a high extent. AZD9272 has progressed into phase I clinical studies.

Non-linear mixed effects modelling of positron emission tomography data for simultaneous estimation of radioligand kinetics and occupancy in healthy volunteers.

The aim of this work was to develop a model simultaneously estimating (11)C-AZD9272 radioligand kinetics and the relationship between plasma concentration of AZD9272 and receptor occupancy in the human brain. AZD9272 is a new chemical entity pharmacologically characterised as a noncompetitive antagonist at the metabotropic glutamate receptor subtype 5 (mGluR5). Positron emission tomography (PET) was used to measure the time course of ((11)C-AZD9272) in the brain. The study included PET measurements in six healthy volunteers where the radioligand was given as a tracer dose alone as well as post oral treatment with different doses of unlabelled AZD9272. Estimation of radioligand kinetics, including saturation of receptor binding was performed by use of non-linear mixed effects modelling. Data from the regions with the highest (ventral striatum) and lowest (cerebellum) radioligand concentrations were included in the analysis. It was assumed that the extent of non-displaceable brain uptake was the same in both regions while the rate of CNS uptake and the receptor density differed. The results of the analysis showed that AZD9272 binding at the receptor is saturable with an estimated plasma concentration corresponding to 50% occupancy of approximately 200 nM. The density of the receptor binding sites was estimated to 800 nM and 200 nM in ventral striatum and cerebellum respectively. By simultaneously analysing data from several PET measurements and different brain regions in a non-linear mixed effects framework it was possible to estimate parameters of interest that would otherwise be difficult to quantify.

Organization of projections from the spinal trigeminal subnucleus oralis to the spinal cord in the rat: a neuroanatomical substrate for reciprocal orofacial-cervical interactions.

The organization of efferent projections from the spinal trigeminal nucleus oralis (Sp5O) to the spinal cord in the rat was studied using the anterograde tracer Phaseolus vulgaris leucoagglutinin. Sp5O projections to the spinal cord are restricted to the cervical cord. No labeled terminal can be detected in the thoracic and lumbar cord. The organization of these projections happens to critically depend on the dorso-ventral location of the injection site. On the one hand, the dorsal part of the Sp5O projects to the medial part of the dorsal horn (laminae III-V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the ipsilateral one. Ipsilateral labeled terminals are distributed throughout laminae VII to IX but tend to cluster around the dorso-medial motor nuclei, especially at C3-C5 levels. Within the contralateral ventral horn, label terminals are found particularly in the region of the ventro-medial motor nucleus. This projection extends as far caudally as C3 or C4 level. On the other hand, the ventral part of the Sp5O projects to the lateral part of the dorsal horn (laminae III-V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the contralateral one. Contralateral labeled terminals are distributed within the region of the dorso- and ventro-medial motor nuclei at C1-C4 levels whereas they are restricted to the dorso-medial motor nucleus at C5-C8 levels. These findings suggest that Sp5O is involved in the coordination of neck movements and in the modulation of incoming sensory information at the cervical spinal cord.

Phenyl ureas of creatinine as mGluR5 antagonists. A structure-activity relationship study of fenobam analogues.

Fenobam (1) was developed by McNeil Laboratories as an anxiolytic agent with an unknown molecular target in the late 1970s. In a recent publication, it was revealed that fenobam is a non-competitive mGluR5 antagonist. Herein, we present the structure-activity relationship of fenobam and its analogues and similarities between the SAR of mGluR5 antagonism and the SAR of CNS properties originally reported by McNeil are discussed.

Recent advances in non-competitive mGlu5 receptor antagonists and their potential therapeutic applications.

Extensive research into the functions of glutamate and glutamate receptors in the central nervous system (CNS) has shown an essential role of metabotropic glutamate (mGlu) receptors in normal brain functions, but also in neurological and psychiatric disorders. The precise functions of these receptors remain undefined, and progress toward understanding their functions has been hampered by the lack of selective ligands with appropriate pharmacokinetic properties. The Group I mGlu receptor, mGlu5, is well positioned to regulate and fine-tune neuronal excitability and synaptic transmission through its modulation of various signal transduction pathways and interactions with other transmitter systems. Therefore, the mGlu5 receptor may be an important therapeutic target for the treatment of disorders of the central nervous system. The discovery of MPEP 3, a non-competitive mGlu5 receptor antagonist, provided a potent, selective, systemically active tool compound for proof of concept studies in animal models of various disease states. These studies have led to greater understanding of possible therapeutic applications of mGlu5 receptor antagonists in recent years, suggesting their use in a number of disease states, including chronic pain, various psychiatric and neurological disorders, substance abuse and withdrawal, obesity and gastroesophageal reflux disease (GERD). Together, these findings have intensified efforts to find other non-competitive mGlu5 receptor antagonists and have led to the discovery of several second-generation compounds, a few of which are in preclinical evaluations. There have been several recent reviews on mGlu receptor. This article highlights recent efforts on the design, synthesis and development of novel, non-competitive mGlu5 receptor antagonists and studies to understand their in vitro mechanisms of action and in vivo pharmacological profiles. Emphasis is also given to recent advances in the potential therapeutic applications of non-competitive mGlu5 receptor antagonists.

The orofacial formalin test.

The subcutaneous injection of formalin into the rat upper lip generates behavioral responses that last several minutes. The time course of the response is similar to what is observed following formalin injection into the paw, i.e. biphasic, with an early and short-lasting first phase followed, after a quiescent period by a second, prolonged (tonic) phase. The applied chemical stimulus (formalin) can be qualified as noxious since it produces tissue injury, activates Adelta and C nociceptors as well as trigeminal and spinal nociceptive neurons and is felt as painful in man. In addition, increasing the concentration of formalin causes a parallel aggravation of histological signs of tissue inflammation and injury. The measured behavioral response (face rubbing) is a relevant end-point: prolonged face rubbing is evoked by formalin but not saline injection and a positive relationship between the amplitude of the response and the formalin concentration is observed, at least up to 2.5%. At higher formalin concentrations, the use of other or additional end-points should be considered. Finally, the behavioral response in the orofacial formalin test is sensitive to various opioid and non-opioid analgesics. The orofacial formalin test can then be considered as a reliable way of producing and quantifying nociception in the trigeminal region of the rat.

Organization of parabrachial projections from the spinal trigeminal nucleus oralis: an anterograde tracing study in the rat.

In recent years, we have accumulated data showing that the spinal trigeminal nucleus oralis (Sp5O) contributes to the processing of somatosensory inputs from the orofacial region. Although the parabrachial area (PB) represents the main brainstem relay for autonomic, nociceptive, and gustatory afferents, few data are available regarding the topographical distribution of the efferent projections from the Sp5O to the PB. We have addressed this question with the rat, by using the anterograde tracer Phaseolus vulgaris leucoagglutinin. A dense trigeminoparabrachial pathway from the Sp5O toward, predominantly, the ipsilateral PB was revealed. Projections come mainly from the dorsal part of the Sp5O that was found to innervate densely the medial, external medial, and ventral lateral subnuclei. In contrast, the ventral part of the Sp5O projected almost exclusively to an as yet not formally described region, located dorsally and laterally to the lateral tip of the brachium conjunctivum, close to the Kölliker-Fuse nucleus. These results suggest that distinct regions within the Sp5O may be involved in the processing of gustatory and nociceptive information.

The orofacial formalin test in rats: effects of different formalin concentrations.

In this study of the orofacial formalin test in rats, the effects of different formalin concentrations (0.2%, 0.5%, 1.5%, 2.5%, 5% and 10%) on the behavioural nociceptive response (face rubbing) was investigated. The histological responses of the skin were also evaluated. Increasing the concentration of formalin caused a parallel aggravation of histological signs of tissue inflammation and injury. All concentrations provoked an early phase of nociceptive response, but its intensity was not concentration-dependent. The 2nd phase of response to formalin only occurred for concentrations of 1.5% and higher. A positive relationship between the formalin concentration and the amplitude of the rubbing activity measured between 12 and 45 min after injection could be observed until 2.5% but with the highest concentrations (5 and 10%), the amplitude of the response decreased. Our findings indicate that the orofacial formalin test should be carried out using concentration between 0.5 and 2.5%. This is essential to assess increase as well as decrease in pain intensity. Moreover, this will have the effect of minimizing the suffering of the experimental animal.

Evidence for a peripheral origin of the tonic nociceptive response to subcutaneous formalin.

The orofacial formalin test in the rat is a valid and reliable model of nociception and is sensitive to various classes of analgesic drugs. The noxious stimulus consists in an injection of diluted formalin (2.5% in saline) into the upper lip. The behavioural nociceptive response is measured in terms of the amount of time the animal spends rubbing the injected area. Two distinct periods of intense rubbing activity can be identified, a first phase occurring in the first 3 min and a second phase lasting from 12 to 39 min after formalin injection. The present study verified the peripheral origin of the first phase of the formalin response and examined whether the second phase is produced by peripheral activation of afferent fibres and/or by a phenomenon of central facilitation induced by the neural activity that occurs during the first phase. This was determined by assessing the effect of a local anaesthetic agent (lidocaine) administered into the formalin injection site, before or after the first phase of the formalin response. Local injection of 50 microliters of lidocaine prior to formalin completely abolished the first phase of the formalin response but this blockade did not significantly influence the appearance and development of the second phase. Thus, the primary afferent activity that normally occurs during the first phase of the formalin response is not a prerequisite for the expression of the second phase. A higher dose of lidocaine (150 microliters) induced, in addition, inhibition of the first part of the second phase.(ABSTRACT TRUNCATED AT 250 WORDS)