.European Journal of Pharmacology 366 1999 243±252
`Inhibition of emesis by tachykinin NK receptor antagonists inSuncus1
`/murinus house musk shrew
`John A. Rudd), Man P. Ngan, Man K. Wai
`Department of Pharmacology, Faculty of Medicine, The Chinese Uni˝ersity of Hong Kong, Shatin, N.T., Hong Kong, China
`Received 16 September 1998; revised 25 November 1998; accepted 4 December 1998
`Abstract
` . .  . .The anti-emetic potential of CP-122,721q -2S,3S -3- 2-methoxy-5-trifluoromethoxybenzyl amino-2-phenylpiperidine , CP-99,994
`  . .  . .   . .  .q -2 S,3S -3- 2-methoxybenzylamino -2-phenylpiperidine , CP-100,263y -2 R ,3R -3- 2-methoxybenzylamino -2-phenylpiperi-
`.  . w . x .dine , RP 67580 3 R ,7 a R -7, 7-diphenyl-2- 1-imino-2- 2-methoxyphenyl ethyl po-hydroisoindol-4-one , FK 888
` 2 w.  . x . .N -4 R -4-hydroxy-1- 1-methyl-1H-in-dole-3-yl carbonyl-L-propyl -N- methyl-N-phenylmethyl-l-3- 2-naphthyl -alaninamide and GR
`w 94 10x . .  .82334 D -Pro spiro-g-lactam Leu -physalaemin- 1±11 was investigated to inhibit nicotine 5 mgrkg, s.c. -, copper sulphate
`.  .pentahydrate 120 mgrkg, intragastric - and motion 4 cm horizontal displacement at 1 Hz for 5 min -induced emesis inSuncus murinus.
`.A 30 min intraperitoneal pre-treatment with CP-122,721, CP-99,994, RP 67580 and FK 888 significantlyP -0.05 antagonized
`nicotine-induced emesis with ID values of 2.1, 2.3, 13.5 and 19.2 mgrkg, respectively CP-100,263, the less active enantiomer of50
`CP-99,994, was inactive at doses up to 10 mgrkg. Infusion of GR 82334, CP-122,721, CP-99,994 and FK 888 into the dorsal vagal
`complex of the hindbrain also antagonized nicotine-induced emesis yielding ID values of 1.1, 3.0, 3.3 and 58.0mgrdorsal vagal50
`complex, respectively RP 67580 and CP-100,263 were inactive. RP 67580 and FK 888 failed to antagonize copper sulphate-induced
`emesis but CP-122,721 and CP-99,994 were active yielding ID values of 2.2 and 3.0 mgrkg, i.p., respectively. CP-99,994 also50
`.completely prevented motion-induced emesis at 10 mgrkg, i.p. P -0.05 and RP 67580 produced a significant reduction of
`.motion-induced emesis at 10 mgrkg, i.p.P -0.05 . These studies provide evidence of a central site of action of tachykinin NK1
`receptor antagonists to inhibit nicotine-induced emesis inS. murinusand confirm the broad profile of inhibitory action. The rank order of
`potency of the antagonists following the intra-dorsal vagal complex administration suggests that theS. murinustachykinin NK receptor1
`has a unique pharmacological profile.q 1999 Elsevier Science B.V. All rights reserved.
`()Keywords: Tachykinin NK receptor; Emesis;Suncus murinus1
`1. Introduction
`Evidence has accumulated to indicate the importance of
`substance P in the emetic reflex. For example, tachykinin
` .NK receptor antagonists, such as CP-99,994 2S,3S -3-1
`.2-methoxybenzylamino -2-phenylpiperidine; McLean et
`.  .al., 1993 and GR 203040 2S,3 S -2-methoxy-5-tetrazol-
`. .l-yl-benzyl - 2-phenyl-piperidin-3-yl -amine; Beattie et al.,
`.1995 , are highly effective in reducing the emesis induced
`by a wide variety of challenges including centrally- e.g.,
`.apomorphine, morphine, and nicotine and peripherally-
`) Corresponding author. Tel.:q852-2609-6789; Fax:q852-2603-
`5139; E-mail: jar@cuhk.edu.hk
`.acting emetics e.g., intragastric copper sulphate and
`provocative motion Bountra et al., 1993; Gardner et al.,
`1995a, 1996; Lucot et al., 1997; Tattersall et al., 1993,
`.1994, 1995 . The pre-clinical studies have yielded informa-
`tion relevant to the clinical utilization and mechanism of
`action of the compounds. Only tachykinin NK receptor
`1
`antagonists that penetrate the blood brain barrier are active
`to antagonise emesis and a site of anti-emetic action within
`the vicinity of the nucleus tractus solitarius has been
`.proposed Tattersall et al., 1996 . The nucleus tractus
`solitarius is a logical site for a `broad spectrum' anti-emetic
`agent to act since it is a site at which vagal afferents from
`the gastrointestinal tract converge with inputs from the
`area postrema the classical `chemoreceptor trigger zone'
`.for emesis and other brain areas associated with emesis
`.control Leslie and Reynolds, 1992; Tattersall et al., 1996 .
`0014-2999r99r$ - see front matterq 1999 Elsevier Science B.V. All rights reserved.
`.PII: S 0 0 1 4 - 2 9 9 9 9 8 0 0 9 2 0 - 0
`HELSINN EXHIBIT 2060
`Azurity Pharmaceuticals, Inc. v. Helsinn Healthcare S.A.
`IPR2025-00945
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`()J.A. Rudd et al.rEuropean Journal of Pharmacology 366 1999 243±252244
`Most of the pre-clinical investigations into the role of
`tachykinin NK receptor antagonists to inhibit drug-in-1
`duced emesis have been conducted in the ferret, a species
`that has a human-like tachykinin NK receptor Tattersall1
`.et al., 1996 . However, a few studies have usedSuncus
`.murinus the house musk shrew , a species of insectivore,
`since this animal can be easily utilized to assess the
`anti-emetic potential of drugs to inhibit motion-induced
`.emesis Ueno et al., 1988; Gardner et al., 1995a,b . There
`are however, known species differences in the pharmacol-
`ogy of the tachykinin NK receptor, particularly between
`1
`human and rodents Gitter et al., 1991; Fardin and Garret,
`.1991; Beresford et al., 1992 . The species differences are
` . wseen with RP 67580 3 R ,7 aR -7,7-diphenyl-2- 1-imino-
`. x .2- 2-methoxyphenyl ethyl po-hydroisoindol-4-one , a
`compound that displays approximately 20 times higher
`.affinity for rodent-likeK s3 nM than for human-like
`i
`tachykinin NK receptorsK s56 nM; see Tattersall et1 i
`.al., 1996 . InS. murinus, it is interesting that RP 67580 is
`only active to reduce emesis when used at relatively high
`doses and radioligand binding studies indicate a very low
`.affinityK )1 mM for S. murinus tachykinin NK
`i 1
`.receptors Tattersall et al., 1995 . Taken together, these
`results strongly indicate a further species difference for the
`tachykinin NK receptor and suggest thatS. murinusmay
`1
`not be ideally suitable to screen for active tachykinin NK1
`receptor antagonists to prevent emesis in man.
`In the present studies, we decided to investigate further
`the pharmacology and role of the tachykinin NK receptor1
`in S. murinusby using a range of potent and selective
`tachykinin NK receptor antagonists. The tachykinin NK11
`receptor antagonists used were CP-99,994 McLean et al.,
`.  .  .1993 , CP-100,263 2 R ,3R -3- 2-methoxybenzylamino -
`2-phenylpiperidine; the less active enantiomer of CP-
`.  . 99,994 , CP-122,721 2 S,3S -3- 2-methoxy-5-trifluoro-
`.methoxybenzyl amino-2-phenylpiperidine; McLean et al.,
`.  2 w. 1996 , FK 888 N -4 R -4-hydroxy-1- 1-methyl-1H-in-
`. xdole-3-yl carbonyl-L-propyl -N-methyl-N- phenylmethyl-L
`. .-3- 2-naphthyl -alaninamide; Fujii et al., 1992 , RP 67580
`.  w 9Garret et al., 1991 and GR 82334 D -Pro spiro-g-
`4 10 x . .lactam Leu -physalaemin- 1±11 ; Hagan et al., 1991 and
`their rank order of anti-emetic potency was determined
`against nicotine-, copper sulphate- and provocative mo-
`tion-induced emesis. Some of the investigations utilized an
`intracerebral administration of the antagonists into the
`dorsal vagal complex of the brainstem to inhibit emesis.
`The dorsal vagal complex essentially comprises the area
`postrema, nucleus tractus solitarius and dorsal motor nu-
`.cleus of the vagus nerve Leslie and Reynolds, 1992 . The
`studies may be considered important to characterize the
`tachykinin NK receptors in the emesis models since the
`1
`direct delivery of compounds into the brainstem avoids the
`logistical problems associated with the use of drugs that
`have poor central nervous system penetration. The studies
`should help to clarify if theS. murinustachykinin NK
`1
`receptor is `human-like', `rat-like' or completely novel.
`2. Materials and methods
`2.1. Animals
`The experiments were performed on adult male or
`.femaleS. murinus30±85 g , bred at the Chinese Univer-
`sity of Hong Kong. The breeding animals were originally
`obtained from the Central Institute for Experimental Ani-
`.mals Kanagawa, Japan in 1993. Prior to the experiments,
`they were housed in a temperature controlled room at
`24"18C under artificial lighting, with lights on between
`0700 h and 1730 h. They were allowed free access to
`
`wwater and pelleted cat chow Feline Diet 5003, PMI
`.Feeds, USA . All experiments were conducted in accor-
`dance with the Animal Research Ethics Committee, The
`Chinese University of Hong Kong.
`2.2. Measurement of emesis
`On the day of the experiment, the animals were trans-
`ferred to clear perspex observation chambers 21=14=13
`3.cm for the assessment of emetic behaviour. Animal
`behaviour was recorded by a trained observer. Episodes of
`emesis were characterized by rhythmic abdominal contrac-
`tions which are either associated with the oral expulsion of
`solid or liquid material from the gastrointestinal tract i.e.,
`.vomiting or not associated with the passage of material
`.i.e., retching movements . Episodes of retching andror
`.vomiting bouts were considered separate when an animal
`changed its location in the observation chamber, or when
`the interval between retches andror vomits exceeded 2 s.
`2.3. Stereotaxic surgery
`The stereotaxic surgery performed inS. murinuswas
`similar to that previously carried out in the ferret see
`.Tattersall et al., 1996 . Animals were anaesthetized with
`.sodium pentobarbitone 40 mgrkg, i.p. and placed into a
`stereotaxic frame equipped with custom made ear-bars and
`.mouth pieces David Kopf Instruments, Tujunga, USA .
`An incision was made in the skin from just behind the
`nose to the back of the head and the temporalis muscles on
`either side of the sagittal crest were displaced. The skull
`areas in the immediate vicinity of the crest were then
`cleared of connective tissue. A burr hole was made midline
`0.3 mm from the posterior edge of the surface of the skull
`.and a guide cannula 23 gauge was lowered into the brain
`to a 2 mm depth below the surface of the dura i.e., 2 mm
`.above the dorsal vagal complex and fixed with dental
`cement to a brass anchor screw that was secured to the
`skull. Once the cement had dried, the operative area was
`closed with a number of interrupted stitches around the
`guide cannula. Animals were allowed 72 h to recover from
`the operative procedure prior to the commencement of the
`emesis studies.
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`()J.A. Rudd et al.rEuropean Journal of Pharmacology 366 1999 243±252 245
`2.4. Drug-induced emesis studies
`.The effect of nicotine 0±10 mgrkg, s.c. or intragas-
`.tric copper sulphate pentahydrate solution 0±200 mgrkg
`was investigated over a 30 min observation period to
`ascertain the optimum doses of the emetogens to use in the
`anti-emetic studies.
`To assess the anti-emetic potential of an intra-dorsal
`vagal complex administration of tachykinin NK receptor
`1
`antagonists, the animals were first injected subcutaneously
`.with nicotine and then an injection needle 30 gauge was
`immediately inserted into the dorsal vagal complex via the
`guide cannula. After 3 min, the animals received a 4 min
`intra-dorsal vagal complex infusion of drug or vehicle 1
`.mlrmin and were monitored for the development of
`retching andror vomiting episodes. At the end of the 30
`min observation period, the animals were anaesthetized
`.with pentobarbitone 60 mgrkg, i.p. and 2ml of Evans
`Blue dye was infused into the site of injection. We injected
`2 ml of dye since this volume enabled a more accurate
`assessment of the centre of the site of drug infusion than
`the 4 ml volume used for the actual drug administration
`studies. The brains were then fixed in situ by cardiac
`perfusion with phosphate buffered paraformaldehyde and
`dissected for histological examination using a binocular
`.light microscope=10 magnification to confirm the site
`of injection.
`Selected tachykinin NK receptor antagonists were also
`1
`administered intraperitoneally as a 30 min pre-treatment to
`assess the effect of a peripheral administration of the drugs
`on the emesis induced by either nicotine or copper sul-
`phate.
`2.4.1. Motion-induced emesis studies
`In these experiments, the animals were initially screened
`for their susceptibility to motion-induced emesis 2 weeks
`prior to receiving the antagonists on the cross-over. On the
`first occasion, the animals received an intraperitoneal in-
`jection of the respective tachykinin NK receptor antago-
`1
`nist vehicle followed by a 5 min motion-induced emesis
`test. To conduct the test, the animals were placed on a
`linear reciprocating desktop shaker Heidolph Promax
`.2020, Labplant, England that was set to produce a 4 cm
`horizontal displacement, delivered at 1 Hz, as previously
`.described Gardner et al., 1995a,b; Ueno et al., 1988 .S.
`murinus that failed to retch or vomit during the 5 min test
`were excluded from the studies. On the cross-over, the
``motion-sensitive' animals were randomized to receive
`either an intraperitoneal administration of tachykinin NK
`1
`receptor antagonist or respective vehicle 30 min prior to
`the motion test.
`2.5. Statistical analysis
`The total number of episodes was recorded in each
`animal following the administration of the respective eme-
`togens or the start of the motion-induced emesis test. For
`the drug-induced emesis experiments, the significance of
`difference between treatments was assessed by One-way
`.analysis of variance ANOVA followed by a Fisher's
`.Protected Least Significant Difference PLSD test
`
`w .Statsview , Abacus Concepts, USA . To assess an effect
`on motion-induced emesis the data was subject to analysis
`by a repeated measures two-factor ANOVA with compar-
`isons of specified means by Planned Contrasts Super-
`w .ANOVA , Abacus Concepts, USA . The latter procedure
`is very efficient for comparing a limited subset of possible
`contrasts. This is useful for testing hypotheses about data
`that are more specific than the hypothesis automatically
`tested for by each term in the ANOVA model Gagnon et
`.al., 1989 . Differences were considered significant when
`P -0.05. ID values were determined on the mean data
`50
`by non-linear regressional analysis Kailidagraphe, Syn-
`.ergy Software, USA .
`2.6. Drug formulation
`. Both y -nicotine di-D -tartrate Research Biochemicals
`.International and copper sulphate pentahydrate British
`.Drug Houses were dissolved in distilled water and admin-
`.istered in a volume of 2 mlrkg. CP-99,994 Pfizer ,
`. .CP-100,263 Pfizer , CP-122-721 Pfizer and GR 82334
`.Research Biochemicals International were dissolved in
`.  .saline 0.9% wrv . RP 67580 Rhone-Poulenc Rorer wasÃ
`first dissolved with a few drops of 0.1 M hydrochloric acid
`and was made up in NaCl 0.9%wrv; the final pH was
`.adjusted to 6 with 0.1 M NaOH . FK 888 Fujisawa
`.Pharmaceutical was formulated in polyethylene glycol
`200 for the peripheral studies. For the central administra-
`tion studies, FK 888 was formulated in a solution of 2:1
`Table 1
`.The emetic action of nicotine 1.25±10 mgrkg s.c. and copper sulphate
`.pentahydrate solution 40±200 mgrkg intragastric inS. murinusduring
`a 30 min observation period
`.Latency min Episodes RV rT
`()Nicotine mgrkg
`0.0 ± 0.0"0.0 0r3
`1.25 ± 0.0"0.0 0r3
`2.5 11.3 "3 10.7"5.0 3r3
`a5.0 4.4"2.0 20.0"1.2 3r3
`10.0 5.1"0.7 13.3"3.2 3r3
`()CuSO P5H O mg rkg42
`0.0 ± 0.0"0.0 0r4
`40.0 2.1"0.4 8.5"0.7 4r4
`80.0 2.7"0.6 11.0"4.5 4r4
`a120.0 1.5 "0.2 20.0"2.4 4r4
`a200.0 2.1 "0.3 17.0"4.3 4r4
`Data represent the mean"S.E.M. of 3±4 determinations.
`.RVr T indicates the number of animals either retching or vomiting RV
`.out of the number of animals testedT .
`Significant differences between the respective vehicle treatments 0.0
`.mgrkg and nicotine or copper sulphate pentahydrate treated animals are
`a indicated asP -0.05 One-way ANOVA followed by Fisher's PLSD
`.test .
`Page 3 of 10
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`()J.A. Rudd et al.rEuropean Journal of Pharmacology 366 1999 243±252246
`.  . Fig. 1. The emetic profile of nicotine 5 mgrkg, s.c. , copper sulphate pentahydrate 120 mgrkg, intragastric and motion 4 cm horizontal displacement, 1
`.Hz for 5 min inS. murinus. Values are the means"S.E.M. of 5±14 determinations.
`.dimethyl sulfoxide:saline 0.9% wrv . For the peripheral
`administration studies, CP-99,994, CP-100,263, CP-122-
`721 and FK 888 were administered in a volume of 2
`mlrkg; RP 67580 was administered in a volume of 4
`mlrkg. Doses are expressed as the free base weight unless
`otherwise indicated.
`3. Results
`3.1. Drug-induced emesis
`The subcutaneous administration of nicotine produced
`emesis at doses as low as 2.5 mgrkg and was maximally
`effective at 5 mgrkg; higher doses were associated with a
`.reduction of emesis Table 1 . A dose of nicotine 5
`mgrkg, s.c., was selected for the anti-emetic studies. This
`dose reliably induced emesis within 4.4"2.0 min of
`injection and comprised 20.0"1.2 episodes; the duration
`.of emesis from the first to the last episodes was 6.0"1.8
`.min Fig. 1 .
`The intragastric administration of copper sulphate pen-
`tahydrate produced emesis at doses as low as 40 mgrkg
`within approximately 2 min of administration. Higher doses
`.up to 200 mgrkg were also associated with emesis
`.Table 1 . A dose of copper sulphate pentahydrate 120
`mgrkg was selected for the anti-emetic studies and pro-
`duced emesis within 1.5"0.2 min of administration and
`.  .Fig. 2. The effect of an intra-dorsal vagal complex DVC administration of tachykinin NK receptor antagonists on nicotine 5 mgrkg, s.c. -induced1
`emesis inS. murinus. Values are the means"S.E.M. of 3±8 determinations. The number of animals retching andror vomiting out of the number of
`animals tested is indicated as a `fraction' for each treatment group. Significant differences from vehicle treatment during the 3±30 min observation period
`a .are indicated asP -0.05 One-way ANOVA followed by a post-hoc Fisher's PLSD test .
`Page 4 of 10
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`()J.A. Rudd et al.rEuropean Journal of Pharmacology 366 1999 243±252 247
`comprised 20.0"2.4 episodes; the duration of action was
`.12.2"2.6 min Fig. 1 .
`3.2. Antagonism of nicotine-induced emesis
`3.2.1. Central administration of antagonists
`The site of injection of the antagonist or vehicle was
`confirmed by the infusion of Evans Blue dye at the end of
`the experiment. Only data from animals showing bilateral
`staining of the primary target site restricted to the dorsal
`vagal complex were analyzed see Won et al., 1998, for a
`.description of brainstem anatomy inS. murinus; data
`from brains showing extensive staining of the central canal
`were rejected. In a preliminary set of experiments, the
`.intra-dorsal vagal complex infusion of saline 1mlrmin
`.or 2:1 dimethyl sulfoxide:saline 1mlrmin , for 4 min,
`was associated with a reduction of exploratory behaviour.
`It is possible that the intra-dorsal vagal complex infusion
`technique andror surgery may have interfered with the
`emetic potential of nicotine since only approximately 7±15
`episodes of retching and or vomiting could be observed
`.Fig. 2 compared to the 17±24 episodes observed in
`.normal non-operated animals Fig. 3 . While it is possible
`that the physical infusion of vehicle into the brain may
`have affected the emetic reflex, it is also possible that the
`handling of the animals to insert and remove the infusion
`needle could have also interfered with the retching and
`vomiting response. Nevertheless, infusion of CP-99,994
`inhibited nicotine-induced emesis at doses as low as 10
`.mgrdorsal vagal complexP -0.05 but its less active
`enantiomer, CP-100,263, was without significant effect
`.P )0.05 over a similar dose range and CP-122,721
`produced a significant 88% reduction of emesis at 30
`.mgrdorsal vagal complex Fig. 2;P -0.05 . There was a
`non-significant trend for FK 888 to reduce dose-depen-
`dently nicotine-induced emesis the reduction at a dose of
`.100 mgrdorsal vagal complex was 65%,P )0.05 but RP
`67580 was essentially inactive at 30mgrdorsal vagal
`.complex Fig. 2; P )0.05 . GR 82334 was active to
`antagonise emesis at doses as low as 3mgrdorsal vagal
`.complex Fig. 2;P -0.05 but could only produce a 64%
`maximum reduction of emesis when tested at higher doses
`.up to 30mgrdorsal vagal complex,P -0.05 .
`Based on ID values, the rank order of potency of the
`50
`antagonists to reduce emesis was: GR 82334 IDs1.1250
`.mgrdorsal vagal complex ) CP-122,721 ID s 3.050
`.mgrdorsal vagal complex ) CP-99,994 ID s 3.350
`.mgrdorsal vagal complex)FK 888 ID s58 mgrdor-50
`.sal vagal complex . The ID values for CP-100,263 and50
`RP 67580 could not be calculated but RP 67580 appeared
`less potent than FK 888 to inhibit emesis.
`3.2.2. Peripheral administration of antagonists
`CP-99,994, CP-122,721 and RP 67580 dose depen-
`.dently reduced nicotine-induced emesis Fig. 3;P -0.05 .
`The anti-emetic effects of CP122,721 and CP-99,994 were
`evident at 1 to 3 mgrkg, i.p. but with RP 67580 only
`significantly inhibiting emesis at 30 mgrkg, i.p. Fig. 3;
`.P -0.05 . Similarly, FK 888 was only active to antagonise
`nicotine-induced emesis when used at 30 mgrkg, i.p. and
`.Fig. 3. The effect of an intraperitoneal administration of tachykinin NK receptor antagonists on nicotine 5 mgrkg, s.c. -induced emesis inS. murinus.1
`Values are the means"S.E.M. of 4±5 determinations. The number of animals retching andror vomiting out of the number of animals tested is indicated
`a as a `fraction' for each treatment group. Significant differences from vehicle treatment are indicated asP -0.05 One-way ANOVA followed by a
`.post-hoc Fisher's PLSD test .
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`()J.A. Rudd et al.rEuropean Journal of Pharmacology 366 1999 243±252248
`Fig. 4. The effect of an intraperitoneal administration of tachykinin NK receptor antagonists on copper sulphate pentahydrate 120 mgrkg,1
`.intragastric -induced emesis inS. murinus. Values are the means"S.E.M. of 3±9 determinations. The number of animals retching andror vomiting out of
`the number of animals tested is indicated as a `fraction' for each treatment group. Significant differences from vehicle treatment are indicated asaP -0.05
`.One-way ANOVA followed by a post-hoc Fisher's PLSD test .
`produced a 65% reduction in the number of emetic episodes
`.Fig. 3;P -0.05 . The anti-emetic effects of CP-99,994
`and CP-122,721 were comparable at 10 mgrkg, i.p. with
`. .89 P -0.05 and 87% P -0.05 reductions, respec-
`tively of the number of emetic episodes but CP-100,263
`only produced a 39% reduction that was not statistically
`.significant Fig. 3;P )0.05 . Based on ID values, the50
`rank order of potency of the antagonists to reduce emesis
`. was: CP-122,721 ID s2.1 mgrkg GCP-99,994 ID50 50
`. .s2.3 mgrkg )RP 67580 ID s13.5 mgrkg )FK50
`.888 ID s19.2 mgrkg . The ID value of CP-100,26350 50
`could not be determined.
`3.3. Antagonism of copper sulphate-induced emesis
`Only CP-99,994 and CP-122,721 were capable of sig-
`nificantly reducing copper sulphate-induced emesisP -
`. .0.05 and CP-100,263 and FK 888 were inactiveP )0.05
`.Fig. 5. The effect of tachykinin NK receptor antagonists on motion 4 cm horizontal displacement at 1 Hz for 5 min -induced emesis inS. murinus.1
`Values are the means"S.E.M. of 3±7 determinations. The number of animals retching andror vomiting out of the number of animals tested is indicated
`.as a `fraction' for each treatment group. Significant differences between data generated in the pre-test open histograms and test phase of the experiment
`. a  .hatched histograms are indicated asP -0.05 repeated measures two-factor ANOVA with comparisons of means by Planned Contrasts .
`Page 6 of 10
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`.at the doses tested Fig. 4 . RP 67580 displayed a trend to
`antagonise dose dependently copper sulphate-induced eme-
`sis but this was not statistically significant Fig. 4;P )
`.0.05 . At the doses tested, none of the antagonists were
`capable of completely preventing the emesis induced by
`copper sulphate. The maximum reduction observed was
`79.5% by CP-122,721 at 10 mgrkg, i.p.
`Based on ID values, the rank order of potency of the
`50
`antagonists to reduce emesis was: CP-122,721 IDs2.250
`. .mgrkg GCP-99,994 ID s3.0 mgrkg )RP 6758050
`.ID s28.6 mgrkg . The ID values for CP-100,263 and50 50
`FK 888 could not be calculated but both antagonists
`appeared less potent than RP 67580 to inhibit emesis.
`3.4. Antagonism of motion-induced emesis
`Analysis of the control data generated from the first arm
`of the motion experiments revealed a latency to onset of
`emesis of 2.2"0.3 min comprising 6.4"1.5 episodes
`during the 5 min test interval see Fig. 1 for a profile of the
`.emetic response . After randomization of the animals to
`receive vehicle or drug administration, the control animals
`exhibited emesis following a latency of 2.0"0.3 min and
`comprised 5.2"1.2 episodes on the second motion test
`.Fig. 5 .
`We used a repeated measures two-factor ANOVA with
`comparisons of specified means by Planned Contrasts to
`avoid misinterpreting changes caused by simple desensiti-
`zation of the animals to the motion stimulus as a drug
`effect. Using this approach, analysis of the data showed
`that CP-99,994 completely prevented emesis in four out of
`four animals at the doses of 1 this was not statistically
`. .significant;P )0.05 and 10 mgrkg, i.p. P -0.05 .
`However, CP-100,263 only inhibited emesis in two out of
`five animals at the high dose of 30 mgrkg, i.p. this was
`.not statistically significant,P )0.05; Fig. 5 . RP 67580
`produced a dose-related antagonism of motion-induced
`emesis that was statistically significant at 10 mgrkg, i.p.
`causing a 64% reduction in the number of emetic episodes
`four out of six animals were protected from retching
`.andror vomiting,P -0.05; Fig. 5 .
`4. Discussion
`The present studies have used a number of selective and
`potent tachykinin NK receptor antagonists to characterize
`1
`the tachykinin NK receptors in the emetic reflex ofS.1
`murinus. Using an intra-dorsal vagal complex administra-
`tion, we demonstrated a central site of anti-emetic action
`of tachykinin NK receptor antagonists that is consistent
`1
`with the mechanism previously described for inhibition of
`vomiting in the ferret Gardner et al., 1994; Tattersall et
`.al., 1996 . In our studies, GR 82334, CP-122,721 and
`CP-99,994 were potent inhibitors of nicotine-induced eme-
`sis following injection into the dorsal vagal complex and
`FK 888 was less active. The ability of CP-99,994 to
`antagonize the emesis induced by three emetic challenges
`with purported different mechanisms of action confirm the
`broad anti-emetic potential of the tachykinin NK receptor
`1
`antagonists. Importantly, these studies revealed that RP
`67580 was inactive following central administration, con-
`firming the suspicion that theS. murinustachykinin NK
`1
`receptor has a unique pharmacological profile see Tatter-
`.sall et al., 1995 .
`However, while RP 67580 was inactive to inhibit nico-
`tine-induced emesis following an intra-dorsal vagal com-
`plex injection, it was capable of antagonizing emesis fol-
`lowing peripheral administration. A significant 78% reduc-
`tion of nicotine-induced emesis was observed at 30 mgrkg,
`i.p. which is similar to the level of inhibition reported by
`.Tattersall et al. 1995 . We have, therefore, established that
`RP 67580 is not likely to be acting at the dorsal vagal
`complex to suppress emesis and given the poor penetration
`of this compound into the central nervous system V.
`.Fardin, Personal communication , a peripheral mechanism
`of action may be involved. While the precise mechanism
`of action of RP 67580 to suppress nicotine-induced emesis
`in S. murinusis unknown, it has been hypothesized to
`2q involve a block of Ca channels Rupniak et al., 1993;
`.Tattersall et al., 1995 . Such a mechanism is possible and
`further studies using selective calcium channel blockers
`could be designed to help resolve the mechanism. Cer-
`tainly, the studies may be interesting given the trend by RP
`67580 to also reduce copper sulphate-induced emesis and
`to antagonise motion-induced emesis. Of further impor-
`tance to the classification of tachykinin NK receptors in
`1
`S. murinuswas the similar inhibition of emesis by CP-
`122,721 and CP-99,994. There was an equipotent reduc-
`tion of nicotine-induced emesis either by intra-dorsal vagal
`complex ID values ranged from 3.0±3.3 mgrdorsal
`50
`.vagal complex or intraperitoneal ID values ranged from50
`.2.1±2.3 mgrkg administration to suggest that these com-
`pounds have similar penetration into the brain and similar
`affinity forS. murinus tachykinin NK receptors. Both
`1
`compounds were also as effective, in terms of ID values50
`.ranges2.2±3.0 mgrkg, i.p. to antagonise copper sul-
`phate-induced emesis. Though not directly comparable, the
`data generated from the nicotine and copper sulphate
`studies suggest that CP-99,994 is approximately 10 times
`less active in theS. murinusthan in the ferret to prevent
`.emesis Bountra et al., 1993; Watson et al., 1995 . Consis-
`tent with the difference in anti-emetic potency, radioligand
`binding studies have revealed that CP-99,994 has six times
`less affinity for theS. murinustachykinin NK receptor
`1
`.IC s12 nM; Tattersall et al., 1995 compared to the50
`affinity for the ferret tachykinin NK receptor ICs1.9715 0
`.nM; Watson et al., 1995 . No data are available to indicate
`the affinity of CP-122,721 for either ferret orS. murinus
`receptors but this compound is a non-competitive antago-
`nist at human tachykinin NK receptors expressed in IM-9
`1
`.cells approximate IC values0.2 nM and has a similar50
`Page 7 of 10
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`()J.A. Rudd et al.rEuropean Journal of Pharmacology 366 1999 243±252250
`.affinity to CP-99,994 McLean et al., 1996 . Like CP-
`99,994, CP-122,721 was also 2.5±10 times less active to
`inhibit emesis inS. murinusthan in the ferret following a
`peripheral administration.
`An antagonism of motion-induced emesis was also
`produced by CP-99,994 which is consistent with the use of
`the racemic compound in S. murinus Gardner et al.,
`.1995b . The anti-emetic action was clearly evident at 10
`mgrkg, i.p. but a complete inhibition of emesis was also
`.seen at 1 mgrkg P )0.05 which is much lower than
`required to prevent nicotine- or copper sulphate-induced
`emesis. CP-99,994 appears more potent in this test possi-
`bly because provocative motion is the weakest emetic
`stimulus used in the studies. However, CP-100,263, the
`enantiomer of CP-99,994, was less active against motion-
`induced emesis even when used at the high dose of 30
`mgrkg, i.p. The data provide evidence that tachykinin
`NK receptor antagonism is probably the mechanism of
`1
`anti-emetic action of CP-99,994; such stereoselective ef-
`.fects are also reported in the cat Lucot et al., 1997 .
`CP-100,263 was also inactive to prevent nicotine-induced
`emesis to confirm the stereoselective action present stud-
`.ies; Tattersall et al., 1995 and we also observed no effect
`with this compound against copper sulphate-induced eme-
`sis.
`Some of our studies used FK 888, a peptide based
`antagonist that has high affinity for human-like tachykinin
`.NK receptors K s1.2±3.6 nM; Goso et al., 1994 . FK
`1 i
`888 failed to antagonise copper sulphate-induced emesis
`but did show a trend to reduce nicotine-induced emesis.
`The antagonism of the emesis was evident following either
`.a central IDs58 mgrdorsal vagal complex or periph-
`50
`.eral administration IDs19.2 mgrkg . The peripheral50
`activity of the compound was initially unexpected given
`the reports of a failure of intravenously administered FK
`.888 1 mgrkg to prevent cisplatin-induced emesis in the
`.ferret Rupniak et al., 1997 . However, following an intra-
`venous administration, FK 888 is able to antagonise ID
`50
`.values3.7 mgrkg the foot-tapping induced by the cen-
`tral administration of the tachykinin NK receptor agonist,1
` . w 91 0 x GR 73632 q -Ava L-Pro , Me±Leu substance P- 7±
`..11 ; Rupniak et al., 1997 to suggest that this compound
`does cross the blood brain barrier. In our studies, the
`intra-dorsal vagal complex administration of FK 888 was
`approximately 13 times less active than CP-99,994 to
`reduce nicotine-induced emesis but was only eight times
`less active following the peripheral administration. FK 888
`is known to have approximately four times lower affinity
`for human tachykinin NK receptorsK s1.2±3.6 nM;
`1 i
`.Goso et al., 1994 than CP-99,994K s0.3 nM; Tatter-i
`.sall et al., 1996 but since CP-99,994 is also slightly less
`active atS. murinustachykinin NK receptors, the small
`1
`difference in potency of FK 888 may be expected. We can
`only speculate that the lack of activity of FK 888 in the
`ferret model may relate to the low dose used andror a
`metabolism of the compound during the 4 h assessment
`period; the present studies assessed anti-emetic activity
`over a shorter 30 min observation time.
`As we have reported, some of our studies used copper
`sulphate to induce emesis to assess the