Novel Antischizophrenia Treatments: 213 (Handbook of Experimental Pharmacology)

Summary This volume tries to put current therapy - achievements, shortcomings, remaining medical needs - and emerging new targets into the context of increasing knowledge regarding the genetic and neurodevelopmental contributions to the pathophysiology of schizophrenia. Some of the chapters also deal with respective experimental and clinical methodology, biomarkers, and translational aspects of drug development. The volume concentrates on reviewing the ongoing research attempting to identify novel treatments for the cognitive deficits and negative symptoms of schizophrenia, which are not treated adequately by current antipsychotic medications.

Notes Includes bibliographical references and index. Keefe and Philip D. Young, Nurith Amitai and Mark A. McKinzie and Frank P. Bymaster -- Glutamatergic synaptic dysregulation in schizophrenia: You are browsing titles by their Library of Congress call number classification. Library Staff Details Staff view. Access Note UW-Madison does not have a subscription for electronic access to this title, which may only be available online to patrons at other UW System campuses.

The mixture was stirred overnight allowing the cooling bath to expire. When the ice melted, the aqueous phase was extracted with EtOAc and the organic phase was dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure. The crude title compound 1. To a solution of 3-hydroxymethylphenyl -acetonitrile 1. The suspension was refluxed for 24 h and the solvent was evaporated under reduced pressure.

The residue was dissolved in CH 2 Cl 2 and washed with water and brine. The organic layer was dried over Na 2 SO 4 , filtered and the solvent evaporated under reduced pressure. The crude was purified by flash chromatography petroleum ether: To a solution of 3-butoxymethylphenyl acetonitrile 0. The reaction was quenched by addition of diethylentriamine 0. The solvent was removed under reduced pressure and the residue re-dissolved in EtOAc, washed with water, dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure.

The crude was purified by flash chromatography silica, petroleum ether: The title compound was prepared from [2- 3-butoxymethylphenyl -ethyl]-carbamic acid tert-butyl ester according to the standard procedures described in Steps 3 and 4 of Example 1. Obtained as white solid. Analogously, starting from 2,6-difluoromethoxyphenyl -acetonitrile the following compound was prepared:.

To a solution of 3-methoxyphenyl -acetonitrile 8. The reaction was stirred for 30 min, and MeI 3. The reaction was stirred 1 h at room temperature. The reaction was stirred at room temperature overnight. DMF was evaporated and the crude diluted with brine and extracted with Et 2 O. The organic phase was washed with water, dried over Na 2 SO 4 , the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography petroleum ether: The title compound was prepared from 2- 3-methoxyphenyl methylpropionitrile according to the procedure described in Example 5.

To a solution of 3-iodophenyl -acetonitrile 2. The reaction mixture was diluted with AcOEt and filtered over a celite pad. The organic phase was washed with water and brine, dried over Na 2 SO 4 and the solvent was evaporated under reduced pressure. The title compound was prepared from 3-butylthiophenyl -acetonitrile according to the procedure described in Example 5.

To a solution of 2-[2- 3-butylthiophenyl - tert-butoxycarbonyl ethylamino]-N,N-dimethylacetamide hydrochloride 0. The mixture was stirred at room temperature for 2 h. The mixture was partitioned between water and CH 2 Cl 2 , the organic was washed with water and brine, dried over Na 2 SO 4 and the solvent was evaporated under reduced pressure.

Pharmacology of Itch

The title compound was prepared from 2-[2- 3-butylsulfonylphenyl - tert-butoxycarbonyl ethylamino]-N,N-dimethylacetamide according to the standard procedure described in Step 4 of Example 1. To a solution of 2-[2- 3-butoxyphenyl - tert-butoxycarbonyl ethylamino]-N,N-dimethylacetamide 0. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography petroleum ether: The title compound was prepared from 2-[2- 3-butoxyphenyl - tert-butoxycarbonyl ethylamino]-N,N-dimethylthioacetamide according to the standard procedure described in Step 4 of Example 1.

To a solution of 2- 3-butoxyphenyl -ethanol 3. The organic layer was separated, dried over Na 2 SO 4 , filtered and evaporated under reduced pressure. The residue containing the title compound 2. To a suspension of 2- 3-butoxyphenyl -acetaldehyde 2. The reaction was left at room temperature overnight and then extracted with diethylether. The organic layer was dried over Na 2 SO 4 , filtered and evaporated under reduced pressure.

The residue containing the desired oxime intermediate 2. The organic phase was dried over Na 2 SO 4 , filtered and concentrated to dryness under vacuum. The crude residue was purified by column chromatography, petroleum ether: These cells lack of TTXr sodium channels as shown by the absence of their respective transcripts. The Membrane Potential Kit Assay Molecular Devices , based on a negatively charged fluorescent dye able to monitor changes in membrane potential caused by the sodium influx due to the channel opening, was used for the assay.

Then, nM of the toxin Anemonia sulcata used as enhancer of the channel opener response alone or in the presence of TTX as reference standard or test compound were added for further 15 minutes. The inhibition curves were calculated from 5 concentrations, each in triplicate, and the IC 50 determined using linear regression analysis. The compounds of the present invention inhibit TTXs sodium channels with pharmacologically significant IC 50 values.

The results, obtained with some compounds which are representative of the entire class of compounds of the invention, compared with the standards ralfinamide and safinamide, are reported in Table 1. The results, obtained with some compounds which are representative of the entire class of compounds of the invention, compared with the standards ralfinamide and safinamide, are reported in Table 2.

Procedures involving animals and their care were conducted in conformity with institutional guidelines in compliance with national D. National Research Council, Cortical neurons were prepared from embryonic Wistar rats EE A female rat at date of pregnancy was anesthetized and sacrificed. The uterus and placenta were removed, the fetuses were decapitated and the heads were placed in ice-cold Hank's solution. The skin of the head was removed using a pincer, the scalp was opened cutting laterally from the back till the eyes, and the brain was taken out using a curved pincer.

The brain was cut in two halves, the outer connective tissue membrane was removed with a pincer, and, keeping the brain upside down, the cerebellum, the brainstem and the diencephalon was removed using a curved pincer trying to clean as much as possible the inside of the cortex. Each cortex was cut in smaller parts with a scissors, the pieces were transferred to a 15 ml centrifuge tube using a 5 ml pipette and washed twice with Hank's solution. The solution was removed except ml and the tissue was first dissociated with a 5 ml pipette then with two fire-polished Pasteur pipettes medium and small opening, respectively.

Supernatant was removed and 5 ml of complete Neurobasal medium was added NB medium Life tech. Cortical neurons were used from day 6 th till day 11 th after plating, and once a week Neurobasal medium was changed. Experiments on cortical neurons were carried out using standard whole cell patch clamp methods Hamill et al. Protocol playing and data acquisition were controlled online with Axon pClamp8 software. Measuring and reference electrodes were AgCl—Ag electrodes. Cells were continuously superfused with extracellular solutions, using a solution changer Biologic RSC Drug concentration-inhibition curves were obtained by plotting tonic blocks in the resting and depolarized condition, versus drug concentrations.

Dose-response curves were fitted to the tonic block data, according to the logistic equation: Control bath solution contained mM: Internal pipette solution consisted of mM: They were diluted to the final concentrations in the external solution. The results obtained with 2-[2- 3-butoxyphenyl -ethylamino]-N,N-dimethylacetamide hydrochloride NW , a representative compound of the chemical class of this invention, compared with our standards safinamide and ralfinamide, are reported in Table 3.

The molecular identity of the channels responsible for the TTX-s conductance is unknown but it is presumed that TTX-s conductance arises from the activity of a mixed population of sodium channels. The results obtained with 2-[2- 3-pentyloxyphenyl -ethylamino]-N,N-dimethylacetamide hydrochloride NW and 2-[2- 3-butoxyfluorophenyl -ethylamino]-N,N-diethylacetamide, representative compounds of the chemical class of this invention, compared with our standard raffinamide, are reported in Table 4. Data expressed as IC 50 value at p. Male Wistar rats Harlan, Italy weighing g were sacrificed under light anaesthesia and brains were rapidly removed and homogenized in 8 volumes of ice-cold 0.

The crude homogenate was centrifuged at rpm for 10 minutes and the supernatant recovered.

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The pellet was homogenized and centrifuged again. The reaction was stopped by adding 0. As a matter of fact a significative MAO-B inhibition is considered when the IC 50 values are in the sub-micromolar range such as our standards safinamide and ralfinamide. According to a modified protocol from Rosland et al. Pain behaviour was quantified by counting the cumulative licking time s of the injected paw. Measurements were taken during the early phase min and late phase min after formalin injection Tjolsen A.

The test compound was administered p. Control group was treated with vehicle. The orally and subcutaneous administered compounds of the invention can be found active in this experimental model. The results expressed as ED 50 value, obtained with one compound administered at 0. Adult male Wistar rats weighing g were used. After measurement of a basal allodynic threshold, test compounds dissolved in distilled water was administered orally at the doses of 0. Control rats were treated with vehicle. Neuropathy was produced according to a modified method described by Kim S.

Rats were allowed to recover after surgery for about days before testing. Mechanical allodynia thresholds were determined according to the method of Chaplan et al. The paw withdrawal thresholds of the hind paws of the rats were determined in response to probing with 8 calibrated von Frey filaments Stoelting, Wood Dale, Ill. Each filament was applied perpendicularly to the plantar surface of the ligated paw of rats. A maximal cut-off of 15 g was used.

The mechanical allodynia thresholds both in the sham and operated animals was measured before pre-drug and at 15, 30, 60, 90, , , , , and min after p. A 24 h threshold was also measured in both treatment schedules. The test was carried out between 9 a. The observers were blind to the experimental and treatment conditions.

Thermal hyperalgesia was assessed using the plantar test Ugo Basile, Varese, Italy. The rats were placed in Plexiglas enclosures on a clear glass plate. With the rat standing relatively still, a radiant heat source beneath the glass floor was aimed at the plantar surface of the hind paw, and the withdrawal latency was measured. Orally administered representative compounds of this invention were found active in this experimental model. Effect of 2-[2- 3-butoxyphenyl -ethylamino]-N,N-dimethylacetamide hydrochloride NW orally administered in thermal hyperalgesia in SNL rats.

A group of naive rats were used as control. The CFA injection produced an area of localized oedema and inflammation starting from few h after injection, with a progressive reduction in the mechanical withdrawal threshold. Each animal was allowed to develop the arthritis over a period of days before testing. Visceral pain is still one of the most common forms of pain, which seeks medical care. Despite the conventional belief that visceral pain is a variant of somatic pain, it differs in neurological mechanisms and transmission pathways.

Visceral pain is characterized by referral hyperalgesia and also it is not always linked to tissue injury. The acetic acid-induced visceral pain model is widely used in experimental research to produce abdominal contractions Korster R et al. Male CD1 mice weighing g were used. Each treated group was allowed 30 min to habituate to laboratory surroundings in individual polypropylene transparent boxes. The visceral pain was scored by counting the number of writhes for 10 min after i.

The number of writhes after acetic acid administration were evaluated. Both complete body stretching complete writhe or partial stretching with a clear contracting of the abdomen partial writhe were counted. Data are expressed as the mean number of writhes during the 10 min observation period. Orally administered representative compounds of this invention were found active in this experimental model reducing the number of writhes induced by acetic acid.

The maximal electroshock test MES is used commonly in the screening of anti-epileptic drugs in rodent models. Male CD1 mice weighing 25 g were used. The procedure described by White et al. Antiepileptic Drugs 4th ed: The stimulus was delivered intra-aurally through clip electrodes in mice 0.

The acute effect of compounds administered intraperitoneally i. Ten mice were studied per group. Complete suppression of the hindlimb tonic extensor component of seizures was taken as evidence of anticonvulsant activity.

Cognitive impairment in schizophrenia.

The compounds of the invention were administered p. The results, expressed as ED50 values obtained with some compounds representative of the entire chemical class of the invention, are reported in Table 7 and in Table 8, demonstrate that these compounds are active as anticonvulsant drugs. N,N-dimethylacetamide 2-[2- 3-Butoxyphenyl -ethylamino]-N,N- 60 6. In this model, mice are treated with a mixture of d-amphetamine plus an anxiolytic dose of the benzodiazepine, chlordiazepoxide Rushton R, Steinberg H.

Combined effects of chlordiazepoxide and d-amphetamine on activity of rats in an unfamiliar environment. Evaluation of the effects of lamotrigine, valproate and carbamazepine in a rodent model of mania Behavioural Brain Research, The model has been claimed to mimic some aspects of mania in bipolar disorder. Importantly, the hyperactivity induced by the mixture of d-amphetamine and chlordiazepoxide could be prevented by prior administration of the established mood stabilizer, lithium, as well as other mood stabilizers drugs e. Therefore, this model has face and predictive validity as a model of bipolar disorder and represents a valuable tool to determine, if a test compound could be a potential mood stabilizer drug candidate.

The locomotor activity was recorded using Opto-M3 System Columbus Instruments which is multi-channel activity monitor. Opto-M3 system has 10 infrared emitters and respective amount of receivers 0. Thus the system differentiates forward locomotion ambulation from stereotyped like movement total counts. Mice were pretreated with the test compound 0. After successive 30 min. The locomotor activity ambulation and total activity count was evaluated for 30 min.

Each group consisted of mice. Results show that amphetamine and amphetamine-chlordiazepoxide CDZ administration induced a significant increase in locomotor activity. Orally administered representative compounds of this invention were found active in this experimental model decreasing amphetamine and amphetamine-chlordiazepoxide induced hyperactivity. The method which detects antiamnesic activity, follows that described by Morris Learn.

The water is made opaque by addition of a non-toxic coloring agent e. The animals are given 4 training sessions over 4 consecutive days Day 1 to Day 4. Each training session consists of 4 trials in the Morris Maze, each separated by 1 minute. For each trial the animal is placed in the maze at one of four starting points equally distributed around the maze and allowed to find the escape platform.

The animal is left on the escape platform for 60 seconds before being returned to its home cage. If the animal does not find the platform within seconds the experimenter removes it from the water and places it on the platform for 60 seconds before replacing it in its home cage. During the 4 trials the animals start the maze once from each starting point in a randomly determined order per animal. The trials are video-recorded and the behaviour of animals is analyzed using a video-tracking system Panlab: The principal measure taken is the escape latency at each trial.

Additional measures are the swim speed and distance traveled. A probe test will be performed on Day 5. The platform is removed from the maze and the animal allowed to swim freely in the maze for 60 seconds. The principal measure taken is the time spent in the target quadrant i. Twelve scopolamine-treated rats are studied per group.

The experiment also includes a normal control group receiving saline instead of scopolamine. The test substance is evaluated at 3 doses, administered p. The experiment will therefore include 5 groups. Data are analyzed by comparing treated groups with scopolamine control using unpaired Student's t tests. The object recognition test consists of a sample trial and a choice trial separated by an inter-trial interval ITI. On the sample trial, two identical objects are presented. On the choice trial, one of the objects presented in sample trial termed as familiar objects is replaced by a new object.

Therefore, the object recognition task with a 1 h ITI allows to detect the amnesic effect of a drug and whether this amnesic effect is reduced by another drug. The object recognition test with a 24 h ITI allows to detect a drug-induced enhancement of memory. Numerous studies have been conducted in order to assess memory effects of drugs. For example, past studies demonstrated that nicotine improves memory in the h ITI condition and reduces scopolamine-induced amnesia in the 1-h ITI condition. The apparatus of the object recognition task is an open box made of grey opaque Plexiglas 40 cm L, 40 cm W, 40 cm H.

The objects to be discriminated 3. They are a white door button round shape and a grey door button star shape. Apparently, they have no natural significance for rats and they had never been associated with reinforcement. In order to rule out the possibility of scent traces left on the objects and therefore the dependency of the recognition capacity of rats on the olfactory cue, the objects and the ground of the box are washed with clear water and dried between each trial.

A video camera is fixed to the ceiling above the box to monitor the animals' activity. Experiments take place over 2 days scopolamine-induced amnesia or 3 days natural forgetting. The test consists of a min habituation trial day 1 , a 3-min sample trial day 2 and a 3-min choice trial day 2 or day 3. On Day 1 The rat is allowed to explore the apparatus for 15 min. On day 2 the rat is placed in the apparatus with two identical objects presented in two corners of the box for 3 min sample trial. After 1 hr the rat is placed in the apparatus with two objects; one of the objects presented in sample trial termed as familiar objects is replaced by a new object.

Twenty four hours later day 3 the rat is placed in the apparatus with two objects; one of the objects presented in sample trial termed as familiar objects is replaced by a new object.

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The basic measurement is the time spent by the rats in exploring the objects during the sample trial and during the choice trial. Object recognition task indices include the following parameters:. Orally administered representative compounds of this invention were found active in this experimental model reducing scopolamine-induced amnesia in the object recognition task with a 1-h inter trial interval 1 hr ITI and improving memory in a natural forgetting situation, i.

They are based on a common phenomenon: The reason of agitation is searching, it is highly energy consuming, while the purpose of immobility is energy conservation. Animals after antidepressant treatment struggle more even in desperate situation, and they spend less time with immobility. Some aspects of neurotic depression can be studied with the aid of these models.

The present method detects antidepressant and anxiolytic activity and follows that described by Stem et al Psychopharmacology, 85, , Rodents, suspended by the tail, rapidly become immobile. Antidepressants decrease the duration of immobility, whereas tranquillizing agents increase the duration of immobility. The behaviour of the animal is recorded during 6 minutes automatically using a computerized device Itematic-TST developed by Stem et al Prog. Psychiatry, 11, , Two parameters are recorded:.

The test is not performed blind but the randomization schedule generated by the Itematic-TST ensures a homogeneous distribution of the treatments both in time and in the position of each animal in the apparatus. The experiment therefore includes 6 groups. Data are analyzed by comparing treated groups with vehicle control using unpaired Student's t tests. Cognitive impairment is often associated with schizophrenia and it has come to be recognized as a core element of the disorder, bearing on patient's recovery and re-integration into society.

Particular interest has recently attracted a pharmacological model of cognitive dysfunctions in schizophrenia, which is based on the effects of glutamate NMDA receptor antagonists such as phencyclidine PCP and ketamine Javitt et al. Food Rieper, Italy was available ad libitum. The animals had two hours of access to water at the end of each day's testing. The test apparatus consisted of four USA , as previously described Greco et al. The running program for the 5-CSRT task was custom-written.

Mice were handled for one week and their body weight recorded. They were then water-deprived by allowing them 2-h access to water in the early evening until their body weight had stabilised 8 days. On the following two days mice were habituated to the operant boxes.

First, mice had to learn that every 5 sec the liquid reward was available in a small cup in the receptacle hole.

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During this period head entries were recorded. During the next period, mice were trained to poke their noses into the illuminated holes. Immediately after a poke in the water receptacle a LED at the rear of one of the holes was turned on. A nose-poke in the lighted hole extinguished the light stimulus and the liquid dipper provided a 0. Any response in one of the other four holes had no consequence and was not recorded. The light stimulus was presented in all five holes in random order. A mouse was switched to the 5-CSRT task after it had completed at least 50 rewarded nose-poke trials in one min session.

The start of the session was signalled by illumination of the house-light and the delivery of a 0. Nose poking in the receptacle hole began the first trial. After a fixed delay the inter-trial interval, ITI , the LED at the rear of one of the holes came on for a short period. The LED stimulus was presented the same number of times in each hole during a complete session, with the order of presentation randomised by the computer.

While the light was on, and for a short period afterwards the limited hold , responses in the hole that was illuminated correct response resulted in the liquid reward. Responses in the holes that had not been illuminated incorrect responses or failure to respond within the limited hold omissions caused the house-lights to be turned off for a short period time out.

Responses in the holes while the house-light was off restarted the time out. After the delivery of the liquid reward, or at the end of time out, the mouse started the next trial by poking its nose into the receptacle hole. Responses made in the holes after a correct response perseverative responses , or after the end of time out before nose-poking into the receptacle hole, resulted in a period of time out. Responses in the holes during the ITI anticipatory responses also resulted in a period of time out.

After anticipatory responses a nose-poke into the receptacle hole restarted the current trial. Each daily session consisted of trials or 30 min of testing, whichever was completed sooner, after which all lights were turned off and further responses had no effect. In the first session of the test schedule, the stimulus and limited hold each lasted 1 min and, depending on individual performance, they were progressively reduced to 1 sec.

The stimulus duration was reduced in the following sequence: The ITI and time out both lasted 2 sec during the first session and the ITI was raised to 5 sec in subsequent sessions; time out was not changed. Throughout the whole period of training and experiments each mouse had one session per day on a 5-CSRT task. The test compound is dissolved in water and is administered intraperitoneally i.

Five minutes after the treatment mice were injected with vehicle saline or PCP 1. In each experiment the various combination of the test compound with vehicle or PCP are administered according to a Latin-square design. At least 48 h are left between the drug testing days. During these intervening days the mice are tested on the 5-CSRT task to re-establish baseline performance and to check for any residual effects of drugs. The main dependent variables selected for analysis are: Subsequently the treatment group means are compared using a post-hoc Tukey-Kramer test.

Prepulse inhibition PPI is a cross-species phenomenon ie, it is present in mammals ranging from mice to humans , yet it is relatively absent among schizophrenic patients. The reduced ability to filter out among irrelevant auditory stimulation is a characteristic thought to contribute to certain manifestations of these conditions including inattention, distractibility, and cognitive deficits. In the acoustic startle model of sensorimotor gating a weak acoustic stimulus prepulse decrease the reflexive flinching response startle produced by a second, more intense, stimulus the pulse.

Drugs like dizocilpine MK or amphetamine disrupt PPI and represent an animal model of schizophrenia. Antipsychotic drugs are able to prevent PPI deficit. The test is quite useful to screen potential antipsychotic drugs. The startle apparatus San Diego Instruments, CA consisted of 12 plastic transparent cages, placed individually in a sound-proof cabinets, equipped with a movable platform floor attached to a sensor recording vertical movements of the platform.

Startle reaction was evoked by acoustic stimuli delivered by a loudspeaker suspended above the cages and connected to an acoustic generator. The transient force resulting from the movements of the platform evoked by the startle reaction was recorded with a PC computer during a recording window of ms measured from the onset of the acoustic stimulus, digitalized and stored in the computer for further evaluation. The amplitude of the startle response was measured during the whole recording window ms and an average value of amplitude was taken for further evaluation.

The control and treated rats were placed in the testing cages individually. After 5 min of habituation background white noise, 65 dB , two types of acoustic stimuli were used in random order: During each experimental session 20 trials of each type were presented with interstimulus interval of 20 s. The amplitudes were averaged for each individual animal, separately for both types of trials stimulus alone or stimulus preceded by the prepulse.

The percent PPI was calculated with the following formula: Deficits of sensorimotor gating was induced by MK 0. Orally administered representative compounds of this invention were found active in this experimental model, reversing PPI deficit induced by MK FIG. Detection of acoustic startle response and PPI was performed as described in Bortolato et al Psychopharmacology, , October; 3: In each experiment, mice were assigned to receive either compounds of the invention or vehicle and were tested in the PPI session using a between-subjects design.

The magnitude of the acoustic startle response was calculated as the average response to all of the pulse-alone trials, excluding the first and last blocks of five pulse-alone trials presented. The psychopathological and neurobiological relationships between sleep and psychotic phenomena have been evidenced by a number of classical clinical observations and psychological reports.