Psychopharmacology (2003) 170:287–293
`DOI 10.1007/s00213-003-1545-4
`ORIGINAL INVESTIGATION
`Leandro F. Vendruscolo · Reinaldo N. Takahashi ·
`Gustavo R.
`Br/C252ske · Andr/C216 Ramos
`Evaluation of the anxiolytic-like effect of NKP608,
`a NK1-receptor antagonist, in two rat strains that differ
`in anxiety-related behaviors
`Received: 12 December 2002 / Accepted: 20 May 2003 / Published online: 12 August 2003
`/C23 Springer-Verlag
`2003
`Abstract Rationale: NKP608, a selective NK1 receptor
`antagonist, has been shown to produce anxiolytic-like
`effects in rodents tested in different anxiety models.
`However, most of these findings are based on social
`behaviors and, to our knowledge, there is no report
`concerning the effects of NKP608 in the elevated plus-
`maze (EPM) and the open field (OF), two classical
`models of anxiety/emotionality. Moreover, this com-
`pound has never been tested in rodent strains that display
`contrasting levels of anxiety-related behaviors. Objec-
`tives: To investigate the anxiolytic-like effects of NKP608
`in Lewis and SHR inbred rats, proposed as a genetic
`model of anxiety for showing high and low indices of
`anxiety, respectively. Methods: Lewis and SHR rats of
`both sexes were tested in the EPM and OF tests following
`acute administration of NKP608 (0.003, 0.03 or 0.3 mg/
`kg) or chlordiazepoxide (CDZ, 5 mg/kg). Measures of
`approach/avoidance towards the open arms of the EPM
`and the central area of the OF were used as indices of
`anxiety. Results: All doses of NKP608 produced anxio-
`lytic-like effects, similar to those of CDZ, in SHR males
`tested in the OF but not in the EPM. Conversely, this
`compound had a partial anxiolytic effect in LEW males
`(and, to a lower degree, in SHR females) in the EPM, but
`not in the OF. LEW females were unaffected following all
`pharmacological treatments. Conclusions: These findings
`indicate that the anxiety-related effects of NKP608 are
`strain-, sex- and test-dependent. Moreover, the present
`data confirm and extend the therapeutic potential of NK1
`receptor antagonists for the treatment of anxiety.
`Keywords NK-1 receptor antagonist · Spontaneously
`hypertensive rats · Lewis · Anxiety · Open field · Elevated
`plus-maze
`Introduction
`Substance P (SP), a neuropeptide belonging to the
`tachykinin family, is widely distributed in the mammalian
`central nervous system, along with its preferring receptor
`NK1, including particularly those areas involved in the
`control of fear and anxiety such as the amygdala,
`hypothalamus and periaqueductal gray (Otsuka and
`Yoshioka 1993; Ribeiro-da-Silva and H/C246kfelt 2000).
`Studies on different animal species indicate that the
`injection of SP into these limbic areas, which are classical
`targets for anxiolytic and antidepressant drugs, provoke
`either anxiogenic- or anxiolytic-like effects, depending on
`the doses used and on the way of administration (Aguiar
`and Brand¼o 1996; Hasen/C246hrl et al. 1998; Gavioli et al.
`1999).
`Stimulated by the aforementioned evidence, several
`NK1 receptor antagonists have been synthesized and
`tested in a variety of animal models of anxiety and
`depression where they have been shown to display
`anxiolytic- and/or antidepressant-like effects (Kramer et
`al. 1998; Papp et al. 2000; Boyce et al. 2001; Cheeta et al.
`2001; Varty et al. 2002). Moreover, as revealed by studies
`using knockout mice, the genetic deletion of either SP or
`its receptor NK1 caused a decrease in anxiety-related
`behaviors (Rupniak et al. 2000; Santarelli et al. 2001;
`Bilkei-Gorzo et al. 2002). In humans, the NK1 antagonist
`MK-869 has been reported to reduce indices of depression
`and anxiety in depressed patients (Kramer et al. 1998).
`Taken together, these findings suggest that the blockade
`of NK1 receptors is potentially useful in the treatment of
`anxiety- and depression-related disorders.
`G. R. Br/C252ske · A. Ramos ())
`Laborat/C243rio de
`Gen/C216tica do Comportamento,
`Departamento de Biologia Celular, Embriologia e Gen/C216tica,
`Universidade Federal de Santa Catarina,
`88.040-900 Florian/C243polis SC, Brazil
`e-mail: andre@ccb.ufsc.br
`Tel.: +55-48-3315153
`Fax: +55-48-3315148
`L. F. Vendruscolo · R. N. Takahashi
`Departamento de Farmacologia,
`Universidade Federal de Santa Catarina,
`88.040-900 Florian/C243polis SC, Brazil
`HELSINN EXHIBIT 2055
`Azurity Pharmaceuticals, Inc. v. Helsinn Healthcare S.A.
`IPR2025-00945
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`NKP608 is a selective, potent, orally active and CNS-
`penetrant
`NK1 receptor antagonist both in vivo and in
`vitro, with a quite comparable high affinity at the rat, the
`gerbil and the human NK1 receptor (Vassout et al. 2000).
`Anxiolytic-like effects, similar to those of benzodi-
`azepines, have been described for this compound in the
`social interaction and the social exploration tests in rats
`(Vassout et al. 2000; File 2000) and the social investi-
`gation test in gerbils (Gentsch et al. 2002). However, this
`compound showed only weak, partial anxiolytic-like
`effects in the stress-induced hyperthermia test in mice
`(Spooren et al. 2002), a paradigm that does not involve a
`social component. Taken together, these results suggest
`that the anxiolytic-like effects of NKP608 may depend on
`the context (social/non-social) of the test. Animal tests
`involving conflict (e.g. the elevated plus-maze) are often
`seen as models of generalized anxiety disorder, whereas
`social interaction tests have been suggested as models of
`social phobia and panic (Gyerty/C181n 1992). Therefore, an
`evaluation of NKP608 in further classical models of
`anxiety/emotionality, such as the elevated plus-maze and
`the open field tests, might be helpful to provide a better
`evaluation of the therapeutic potential of this molecule.
`The influence of genetic factors on sub-clinical forms
`of anxiety as well as on anxiety-related psychopathologies
`has been repeatedly demonstrated (van de Wetering et al.
`1999; Lesch 2001). Therefore, the use of genetic models
`(i.e. inbred strains of rodents that differ for anxiety-related
`traits) has proven to be a useful tool for the study of the
`biological and molecular mechanisms underlying anxiety
`(Gray et al. 1999). As pointed out by Spooren et al.
`(2002), the anxiolytic effects of NKP608 were so far seen
`only in "normal" animals, whose behavioral reactions
`could be considered as adequate from the adaptive point
`of view. Hence, it remains to be determined whether
`NKP608 and other NK1 receptor antagonists will be
`equally effective in animal strains with contrasting
`baseline levels of anxiety, which might represent a better
`model of psychopathological states in humans.
`It has been recently proposed that the inbred rat strains
`Lewis (LEW) and spontaneously hypertensive rats (SHR)
`constitute a useful genetic model for the study of anxiety
`(Ramos et al. 1997, 1998, 2002). These two strains
`display contrasting behavioral responses when submitted
`to a variety of tests of anxiety/emotionality such as the
`elevated plus-maze, open field, black/white box and
`elevated T-maze. LEW rats of both sexes display higher
`levels of anxiety-related behaviors when compared with
`SHR rats, without differing in relation to several measures
`of general locomotion in either novel or familiar
`environments (Ramos et al. 1997, 1998, 2002; Ramos
`and Morm/C155de 1998).
`An F2 intercross between the LEW and SHR strains
`has been successfully used to identify and map, for the
`first time in rats, QTLs (quantitative trait loci) affecting
`emotionality-related behaviors (Ramos et al. 1999). This
`study revealed a locus on chromosome 4 which strongly
`affected the locomotion of rats in the central area of the
`open field test (a putative measure of anxiety). This
`genomic region was found to also harbor the gene Tac1r,
`which codes for the NK1 receptor (Ramos et al. 1999).
`This finding points to a possible implication of the NK1
`receptor in the genetic differences in emotionality
`observed between LEW and SHR rats. If this hypothesis
`holds true, one could expect to find a different reactivity
`between these two strains to an antagonist of NK1
`receptors.
`The aim of the present study was thus to evaluate the
`effects of NKP608 in LEW and SHR rats of both sexes in
`two behavioral tests widely used to measure anxiety/
`emotionality, namely the elevated plus-maze and the open
`field tests, two paradigms which do not involve social
`stimuli.
`Materials and methods
`Animals
`Colonies of the two inbred rat strains Lewis (LEW) and sponta-
`neously hypertensive rats (SHR), were maintained in our laboratory
`under a system of brother-sister mating. In total, 200 rats (50 per
`sex and strain) were used in this study. The animals were weaned
`and separated by sex at 4 weeks of age and, thereafter, were kept in
`collective plastic cages (40/C14832/C14816.5 cm) with five rats/cage having
`food and water available ad libitum under a L12:D12 cycle (lights
`on at 0700 hours) at 23€2/C30C. At 9 weeks of age the average
`(€SEM) weight of the animals was 232€3 g and 160€1 g for male
`and female SHR and 234€4 g and 172€2 g for male and female
`LEW rats, respectively. These rats were submitted to the elevated
`plus-maze and, immediately after, to the open field test with five
`different pharmacological treatments being applied prior to test.
`Males and females were tested in alternate days. On each day, both
`LEW and SHR rats treated with any of the five treatments were
`tested in alternation, with the five treatments being randomly
`assigned. All tests were carried out between 1300 and 1800 hours.
`The present studies were in accordance with the local regulations
`for the ethical use of animals in research (CEUA/UFSC) and
`covered by the valid permission No. 23080.002412/2001-26.
`Drugs
`NKP608 (quinoline-4-carboxilic acid [ trans-(2R, 4S)-1-(3,5-bis-
`trifluoromethyl-benzoyl)-2-(4-chloro-benzyl)-piperidin-4-yl]-amide)
`was synthesized by Novartis Pharma AG (Basel, Switzerland). The
`drug was suspended in 0.5% methylcellulose (Methocel) and
`administered by oral gavage (2 ml/kg) at doses of 0.003, 0.03 or
`0.3 mg/kg; note that the doses were selected based on the findings
`of Vassout et al. (2000). The control group received a similar
`volume of 0.5% Methocel. An additional group treated with 5 mg/
`kg chlordiazepoxide-HCl (Profarmaco S.R.L., Italy) served as
`positive standard. All treatments were given 90 min prior to the first
`test. Ten rats were used per treatment per strain and per sex.
`Elevated plus-maze (EPM)
`The apparatus was made of wood covered with a layer of black
`Formica and had four elevated arms (52 cm above the floor) 50 cm
`long and 10 cm wide. The arms were arranged in a cross-like
`disposition, with two opposite arms being enclosed (by 40 cm high
`walls) and two being open, having at their intersection a central
`platform (10/C14813.5 cm) that gave access to any of the four arms. The
`open arms were surrounded by a raised ledge (1 mm thick and
`5 mm high) to avoid rats falling off the arms. The central platform
`was under 70 lux of illumination. Each rat was placed onto the
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`central platform facing an open arm and the following behaviors
`were
`registered for 5 min: the number of entries and the time spent
`(with all four paws) inside each type of arm and the percentage of
`open-arm entries in relation to the total number of arm entries. The
`behavior of each animal was recorded via a video camera
`positioned above the maze and monitored in another room via a
`closed circuit TV camera. The floor of the apparatus was cleaned
`with a wet sponge and then dried with a paper towel between rats.
`Open field (OF)
`The apparatus, made of wood covered with impermeable Formica,
`had a white floor of 100/C148100 cm (divided by black lines into 25
`squares of 20/C14820 cm) and 40 cm high white walls. Illumination
`inside the open field was 330 lux. Each rat was placed in the center
`of the open field and the following variables were registered for
`5 min: number of peripheral squares (adjacent to the walls) crossed
`(peripheral locomotion), number of central squares (away from the
`walls) crossed (central locomotion), time in the central area (central
`time) and total number of faecal boli (defecation). Also the floor of
`the apparatus was cleaned with a wet sponge and then dried with a
`paper towel between rats.
`Statistics
`Statistical analysis was performed separately for each sex using a
`two-way ANOVA for strain and treatment effects. Duncan’s
`multiple range test was used for post-hoc comparisons of means
`whenever a significant treatment or treatment/strain effect was
`detected by the ANOVA. Specific comparisons between vehicle-
`treated LEW and SHR rats were performed for each sex by using a
`Student t-test. For one specific variable (time spent in the open
`arms of the EPM), for which the two-way ANOVA detected an
`almost significant effect of treatment, we also performed a one-way
`ANOVA for treatment effect in male LEW rats only.
`Results
`Elevated plus-maze
`For males, the results grouped by strain and treatment are
`presented in Table 1 and Fig. 1. The two-way ANOVA
`revealed a significant effect of the treatments on the time
`spent in the open arms ( F=3.73, P<0.01), the number of
`open-arm entries ( F=12.43, P<0.01), the percentage of
`open-arm entries ( F=6.09, P<0.01) and the time spent in
`the closed arms ( F=3.46, P<0.05). Post-hoc comparisons
`indicated that LEW rats treated with CDZ (5 mg/kg) spent
`more time in the aversive open arms ( P<0.05) and made a
`higher percentage of open-arm entries ( P<0.01) than their
`vehicle controls. In addition, LEW rats treated with CDZ
`or NKP608 (0.3 mg/kg) made more open-arm entries
`(P<0.01 for CDZ and P<0.05 for NKP608) and spent less
`time in the closed arms ( P<0.01 for CDZ and P<0.05 for
`NKP608) than their control group. A post-hoc comparison
`following a one-way ANOVA for treatment effects on
`LEW males only, revealed that both CDZ and NKP608
`(0.3 mg/kg) had significantly increased the time spent in
`the open arms in this specific sub-group. SHR rats treated
`with CDZ made a higher percentage ( P<0.05) and number
`(P<0.01) of entries into the open arms than their vehicle
`controls. The ANOVA also revealed overall strain effects
`for the time spent ( F=25.75, P<0.01) and percentage of
`entries ( F=6.47, P<0.05) in the open arms (SHR>LEW)
`and time spent ( F=57.34, P<0.01) and number of entries
`(F=8.03, P<0.01) in the closed arms (LEW>SHR). The
`interstrain comparisons indicated that SHR rats treated
`with vehicle spent more time in the open arms ( t=/C1593.04,
`P<0.01) and less time in the closed arms ( t=4.21, P<0.01)
`when compared with their LEW counterparts.
`Table 1 Elevated plus-maze measures (mean€SEM) of 9-week-old LEW and SHR rats of both sexes
`Treatment Dose
`(mg/kg,
`GO)
`Males Females
`Open-arm
`entries Open-arm entries (%) Open-arm entries Open-arm entries (%)
`LEW SHR LEW SHR LEW SHR LEW SHR
`Vehicle 0 2.0€0.7 3.5€0.7 20.5€6.0 34.4€6.3 6.4€1.0 6.6€0.9 40.2€4.2 43.2€3.0
`CDZ 5 8.1€1.3** 8.8€0.7** 49.0€4.2** 55.6€1.2* 8.7€1.0 14.7€0.6** 47.7€3.0 68.4€2.6**
`NKP608 0.003 3.6€1.1 3.1€0.6 25.6€6.9 32.5€5.0 6.0€1.2 7.5€0.9 40.8€5.7 50.0€4.0
`0.03 3.3€0.7 4.7€0.9 31.9€6.4 49.3€7.3 4.9€0.9 8.3€0.9 33.9€5.4 52.2€3.5
`0.3 5.1€1.0* 3.7€0.8 33.4€5.0 35.2€6.7 7.1€0.7 6.7€0.7 44.1€2.4 44.3€2.6
`For each strain, significant treatment differences in relation to the vehicle control group are represented by * and ** ( P<0.05 and P<0.01)
`Fig. 1 Time spent in the open arms in the elevated plus-maze of
`LEW and SHR male rats treated with CDZ, methocel (0.5%) or
`NKP608 at three different doses. Bars and vertical lines represent
`the means and SEM of animals grouped by strain and treatment
`(n=10). For each strain, significant treatment differences in relation
`to the vehicle control group are represented by * ( P<0.05).
`Significant interstrain differences for vehicle-treated rats are
`represented by ## (P<0.01, Student t-test). DSignificant treatment
`difference in relation to the vehicle control group when one-way
`ANOVA was applied for LEW rats only ( P<0.05)
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`For females, the results grouped by strain and treat-
`ment
`are presented in Table 1 and Fig. 2. The two-way
`ANOVA revealed a significant overall treatment effect
`for the time spent in the closed arms ( F=5.45, P<0.01),
`only. A significant treatment/strain interaction was found
`for the time spent in the open arms ( F=2.49, P<0.05), the
`number of open-arm entries ( F=4.27, P<0.01) and the
`percentage of open-arm entries ( F=2.81, P<0.05). Post-
`hoc analyses showed that SHR females treated with CDZ
`made more entries into the open arms, displayed a higher
`percentage of open-arm entries and spent less time in the
`closed arms ( P<0.01) than their vehicle control group.
`Yet, SHR females treated with both CDZ and NKP608
`(0.03 mg/kg) spent more time in the open arms ( P<0.01
`for CDZ and P<0.05 for NKP608) than their controls.
`The ANOVA also revealed an overall strain effect
`(LEW>SHR) for the number of entries ( F=4.90,
`P<0.05) and the time spent ( F=67.23, P<0.01) in the
`closed arms. The interstrain comparison of vehicle-treated
`rats also indicated that SHR females spent less time in the
`closed arms ( t=2.93, P<0.01) than their LEW counter-
`parts.
`Open field
`For males, the results grouped by strain and treatment are
`presented in Fig. 3. The two-way ANOVA revealed a
`significant treatment/strain interaction for central loco-
`motion ( F=3.33, P<0.05) and central time ( F=2.89,
`P<0.05). Post-hoc analyses showed that males from the
`SHR strain treated with CDZ or with any of the NKP608
`doses crossed more squares and spent more time in the
`aversive central area of the open field than the vehicle-
`treated control rats. No treatment-effect was observed in
`LEW males. The ANOVA also revealed an overall strain
`effect for peripheral locomotion (LEW>SHR). The inter-
`strain comparison of vehicle-treated rats revealed that
`SHRs crossed more squares ( t=/C1594.79,P<0.01) and spent
`more time ( t=-2.41, P<0.05) in the central area than
`LEWs.
`Fig. 2 Time spent in the open arms in the elevated plus-maze of
`LEW and SHR female rats treated with CDZ, methocel (0.5%) or
`NKP608 at three different doses. Bars and vertical lines represent
`the means and SEM of animals grouped by strain and treatment
`(n=10). For each strain, significant treatment differences in relation
`to the vehicle control group are represented by * and ** ( P<0.05
`and P<0.01)
`Fig. 3 Time spent in the central area and locomotion in the central
`and total areas of the open field of LEW and SHR male rats treated
`with either CDZ, methocel (0.5%) or NKP608 at three different
`doses. Bars and vertical lines represent the means and SEM of
`animals grouped by strain and treatment ( n=9). For each strain,
`significant treatment differences in relation to the vehicle control
`group are represented by ** ( P<0.01). Significant interstrain
`differences for vehicle-treated rats are represented by # and ##
`(P<0.05 and P<0.01, Student t-test)
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`For females, the results grouped by strain and treat-
`ment
`are presented in Fig. 4. The two-way ANOVA
`revealed no effect of the pharmacological treatments but
`showed an overall strain effect (SHR>LEW) on central
`locomotion ( F=172.21, P<0.01), central time ( F=123.76,
`P<0.01) and total locomotion ( F=21.19, P<0.01). For
`vehicle-treated animals, SHR females displayed higher
`locomotion ( t=/C1595.46,P<0.01) and spent more time
`(t=/C1593.76,P<0.01) in the central area of the open field
`than LEW females.
`Discussion
`The potent and selective NK1 receptor antagonist
`NKP608 has been recently shown to produce anxiolytic-
`like effects both in rats and gerbils (File 2000; Vassout et
`al. 2000; Gentsch et al. 2002). These previous findings, as
`well as those of the present study, reinforce the emerging
`notion that NK1 receptor antagonists have an anxiolytic
`profile and they corroborate the hypothesis that tachyki-
`nins are involved in anxiety- and/or depression-related
`disorders (Kramer et al. 1998; File 2000; Papp et al. 2000;
`Stout et al. 2001; Gentsch et al. 2002; Spooren et al. 2002;
`Varty et al. 2002). The present results also suggest,
`however, that the psychopharmacological effects of
`NKP608 may depend on the genotype and gender of the
`animals tested and on the psychological context of the
`behavioral test.
`In the EPM, the first test the rats were exposed to in the
`present study, the highest dose of NKP608 (0.3 mg/kg)
`showed an anxiolytic-like effect (though less pronounced
`than that caused by CDZ) in male LEW rats, by
`increasing their number of entries and time spent in the
`open arms and decreasing their time spent in the closed
`arms, without affecting their main measure of general
`locomotion (entries in the closed arms). For SHR females,
`the intermediate dose of NKP608 increased only one
`index of anxiety, the time spent in the open arms, again
`without altering general locomotion. These findings point
`to a weak and genotype-dependant anxiolytic effect of
`this NK1 antagonist in the EPM. In this same test, the
`classical benzodiazepine CDZ, at the dose of 5 mg/kg,
`showed a more robust anxiolytic effect in males of both
`strains and in SHR females, without causing any stimu-
`lant or sedative effect (as judged by the number of closed-
`arm entries) in any of the groups tested. No anxiolytic
`effect of CDZ was observed in LEW females, who may
`be less sensitive than the other groups to benzodiazepines.
`In the OF test, all doses of NKP608 and CDZ (5 mg/
`kg) were similarly effective in SHR males, increasing
`their level of approach towards the aversive central area
`while causing no change in either total or peripheral
`locomotion, which is consistent with the anxiolytic profile
`of the two drugs. However, the same pharmacological
`treatments had no effect in any of the other groups of rats
`(LEW males; LEW and SHR females) in this test. As far
`as SHR females are concerned, it is possible that a ceiling
`effect had already been attained by the control group. For
`LEW rats of both sexes, on the other hand, the lack of
`anxiolytic-like effect suggests that the high-anxiety
`profile of these rats may be resistant to both CDZ and
`NKP608 in this specific test. It should be noticed,
`however, that when rats were submitted to this OF test
`they were no longer test naive, since they had been
`exposed immediately beforehand to an EPM apparatus.
`Taken together, the present results indicate that NK1
`Fig. 4 Time spent in the central area and locomotion in the central
`and total areas of the open field of LEW and SHR female rats
`treated with either CDZ, methocel (0,5%) or NKP608 at three
`different doses. Bars and vertical lines represent the means and
`SEM of animals grouped by strain and treatment ( n=10). Signif-
`icant interstrain differences for vehicle-treated rats are represented
`by ## (P<0.01, Student t-test)
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`receptors may modulate fearfulness in the OF test and that
`such
`a modulation depends on the gender and genotype of
`the rats used. Both these findings would be in agreement
`with our aforementioned hypothesis that the gene for the
`NK1 receptor (which we have mapped, through the use of
`a LEW/SHR intercross, near a locus influencing central
`locomotion in the OF) may be involved in the control of
`emotionality in LEW and SHR rats (Ramos et al. 1999).
`The EPM is a model of anxiety that is based on the
`natural tendency of rodents to avoid open alleys (Mont-
`gomery 1955; Pellow et al. 1985; Treit et al. 1993). The
`approach towards its open arms can be increased by
`benzodiazepines and decreased by anxiogenic substances
`(Handley and McBlane 1993), whereas serotonin-related
`compounds produce variable results (Pellow et al. 1985;
`Handley and McBlane 1993; Treit et al. 1993; Hogg
`1996). The OF, on the other hand, was originally
`developed as a test of emotionality (Hall 1934, 1936).
`In this novel large arena, rodents tend to avoid the center
`and concentrate their ambulation in the peripheral area,
`where they can physically touch the walls (Treit et al.
`1993; Ramos and Morm/C155de 1998). The effects of
`anxiolytic drugs have not been universally verified in
`this test (Fisher and Hughes 1996; Angrini et al. 1998)
`but, as suggested by Prut and Belzung (2003), the OF
`seems to be a good model to test classical benzodi-
`azepines and 5-HT 1A agonists.
`Although the doses of NKP608 employed herein have
`not yet been demonstrated to cause blockade of NK1
`receptors in the CNS of rats, the present findings are in
`general agreement with the demonstration that various
`NK1-receptor antagonists (including NKP608) produce
`anxiolytic-like effects in rodents (File 2000; Vassout et al.
`2000; Gentsch et al. 2002). The NK1 receptor antagonists
`MK-869, L-742,694, L-733.060, CP-99,994 and CP-
`122,721 produced anxiolytic-like effects in the gerbil
`EPM (Varty et al. 2002). On the other hand, FK888 had
`anxiolytic effects in the EPM in mice (Teixeira et al.
`1996) but not in rats (De Lima and Ribeiro 1996),
`whereas RP67580 produced anxiolytic effects in mice
`tested in the EPM and the ultrasonic vocalization test
`(Santarelli et al. 2002). Moreover, CGP49823 and L-
`760735 increased social interaction in rats (File 1997) and
`gerbils (Cheeta et al. 2001).
`In agreement with our previous findings (Ramos et al.
`1997; 1998, 2002), the present results suggested that SHR
`rats were less fearful than their LEW counterparts in both
`the EPM and OF tests. Nevertheless, there is increasing
`evidence for the fact that different tests may assess
`distinct types of emotionality in rodents (Ramos and
`Morm/C155de 1998). Factorial studies have shown, for
`example, that the central locomotion in the OF may not
`correlate with measures of anxiety from the EPM (Ramos
`et al. 1997, 1998). Therefore, the effects of NKP608 that
`were shown to depend, in the present study, on the strain
`and behavioral test used, are likely to result from the
`different types of stress that the EPM and the OF tests
`may evoke in either LEW or SHR rats.
`Previous studies had shown that NKP608 caused
`anxiolytic-like effects in rats and gerbils tested in a
`variety of models involving a social context (File 2000;
`Vassout et al. 2000; Gentsch et al. 2002). However, in the
`test of stress-induced hyperthermia, a non-social para-
`digm, Spooren et al. (2002) have reported only a partial
`anxiolytic effect of NKP608 in mice. To the best of our
`knowledge, this is the first study to investigate the effects
`of NKP608 in two widely recognized, non-social models
`of emotionality/anxiety, the EPM and the OF. In the
`latter, this NK1 antagonist has shown a strong anxiolytic-
`like effect in SHR males, whereas in the former, it
`produced a mild anxiolytic-like effect in males of the
`LEW strain. Since only a limited dose range of NKP608
`has been tested, the present results should be interpreted
`with some caution. Moreover, similar studies with other
`NK1 antagonists will be necessary before one can infer
`that the effects (or lack of effects) reported herein are
`compound or class-of-compound specific.
`In the present study, females were less sensitive to the
`pharmacological treatments than males, especially in the
`LEW strain, which may be related with the known
`influence that sex hormones have on mood and on the
`effects of psychoactive drugs (Fink et al. 1996; Godfroid
`1999). Therefore, further studies aiming to investigate sex
`and sex/genotype differences in drug-induced emotional
`responses are needed and, ideally, they should also
`control for the stage of the females’ estrous cycle.
`In summary, the present study showed that an oral,
`acute administration of NKP608 produced clear-cut
`anxiolytic-like effects in SHR males when tested in the
`OF, but not in the EPM. Conversely, this compound had a
`partial anxiolytic effect in LEW males (and, to a lower
`degree, in SHR females) in the EPM, but not in the OF.
`LEW females were unaffected following all pharmaco-
`logical treatments. The present results demonstrate that
`the complexity of emotional behaviors requires compre-
`hensive studies and that ignoring sex, strain and test
`differences in pre-clinical research may lead to oversim-
`plified explanations of drug action. The present data
`confirm and extend the therapeutic potential of NKP608
`for the treatment of anxiety and provide further evidence
`for the involvement of tachykinins in the control of fear-
`related behaviors.
`Acknowledgements We wish to thank Dr. Conrad Gentsch from
`Pharma Novartis in Basel for providing to us NKP608. A. Ramos
`and R.N. Takahashi had fellowships from CNPq. L.F. Vendruscolo
`had a scholarship from CAPES. G.R. Br/C252ske had a scholarship
`from PIBIC-CNPq.
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