® 1995 Stoddon Press AD rights r11served 0007-U88j95 $9.00 ~
`Pharmacological characterization of RS 25259-197 a novel
`and selective 5;.. HT 3 receptor antagonist, in vivo
`'
`'R.M. Eg1en, C.-H. Lee, W.L. Smith. L.O. Johnson, *R. Oark, R.L. Whiting &
`S.S. Hegde
`
`Institute oC Phanuacology, •Institute of Organic Chemistry, SyDtex Discovery Rescan:b, 3401 1iiJJmw Avenue, Palo
`Alto, CA-94304, U.S.A.
`
`~==logical effects in YI'VQ, of RS 25259-197, a selective S-liT3 receptor antagonist, have
`In anaesthetized rats, RS 25259-197, &dmfulstercd by tho intravenous intraduodeDal or tmnscbmal
`2.
`route, doso-dc~Uy inhibited
`the von Bezold-Jarlsc.b
`refleX
`lnctuced by 2-mcthyl S-HT
`(ID.!!":"0.04p~kg-•, a.v., 3.2JCgkg-1, i.d .. aod 32.8f11 Pet c:bambcr, ~ely). In this regan~. when
`admiDistcrtd mtiadoodenaJly, RS 2S2S9-l97 was more: potent and exhibited a longer duration of fCdon
`than either oudansetrou or gtanisetron.
`3 -In c:oDBclous ferrets, RS 25259-197, administered intravenously or orally, dose-dependently inhibited
`emesis ln~uced by cisplafin. The IDso estimates of RS 25259-197 wero l.lllSk&-1, i.v •. and 3.2f'gkg-•,
`p.o. In this n:spect, RS 25259-197 waa more potent than ondansetron aud cquipcrteot with zranisetrou.
`In conscious dogs, R8 25259-197, administcrt:d Intravenously or orally, doae.dcpcmdeotly Inhibited
`4
`emesis induced by dspJatiD (ID,-1.9f'Skg-1, i.v. and B.Sf.llkg-1, p.o.), dacarbazine (ID.so•4.1 pg
`kg-•. i.v. amS 9.7pgkg-1, p.o.), actincxnyc:ln D (ID,.,.,.4.9J1gkg-•, i.v. and Z.Spgkg·t, p.o.) and
`mechlo.tetba.uUne (IDso-4:4JCgkg-1, i.v. and 3.0J&gq-1, p.o.). Against each of tho lmlCtogenic agents
`RS 2Sl59·197 was very mucll more potent than olldausctron. When tested at equi-eft"ectiw intravono~
`doses agai.Dst ciaplatiu-indllcod emesis m dogs, R.S 25259-197 had a longer duration oC BJrti.cmetic
`adivity (7h) than ondansetron (4h). At doses up to and indndiug lOOOpgkg-•, p.o,, neither RS
`2S259-197 nor onda&metton was capable of inhibiting apomorphine-induced emesis.
`5 At doses up to 1000 I'B q-1, i.v., RS 25259--197 produced no mcaJdnaf\d haemodynamio c;:hanges In
`anaesthetized dop.
`lD ~. RS 25259·197 is a novel, bigbbr potent and o.raJly active S-HT3 l'Cil8J)tor antagoDist tn
`6
`vivo. W'rth respec;t to its anti-eMetic BDtivity, R8 25259-197 appears to be a significant improvement over
`oudansetcou in ·terms or potency aud dutation of ~Wtiou.
`Keywords: 5-HT, antagoDist; RS 252.59-197; RS 2S2S9; ondansetron; emesis; cisptatin; cancer chemothena.py; anti-emetic; von
`Bezold-Jarlsc:h reflex
`.
`·
`
`It is DOW weD established that the sido-cft'ects of nausea and
`emesis assoclatecl with antk:aDOer tbt:rapy are duo to activa-
`tion of s..m, rcc:eptom at a peripheral and/or central IOCU$
`(Xilpatrick et al., 1990; Tyers, 1991). The vast majority of
`evidence favoun a peripheral mechanism involving activation
`of S..m', receptors, localized on vagal aft'mmts wpplying the
`upper gastrointA:Siinal tm:t. by 5-HT released from entero-
`dttomaJBn c:eUs as a consequence or upGSunl to cytotoxic
`drngs or X-ray imldiation (Costall & Naylor, 1992). The
`advent of selectivo S-HT1 m:eptor aatagonista has shown
`that, in cancer patients, these drugs &~e hf&hl7 effective in
`pnmntiDg chcmothefapy..mduced emesis and therefore im-
`proving the quality of life.
`Numerous 5-BT3 n:ceptor utagonists ha-ve been IIJ)l·
`the8bed to dato. The most widely c:bancterized compounds
`im:lude ODdanscb:on, granlsctron, zacopriclo and t.ropisctron
`(Ox.l'ord a llL. 1992). Emerging evidence indicates that
`significant difl'cnmc:es in potency. efficacy and duration of
`action exist between tb11 compounds which JDay haw an
`Impact on their cllD,«:al uscfulnasa (Marr et al., 1991; And-
`rewa et al.. 1992). For example, the anti-emetic effects haw
`been reported to be dose-dependent for granisetron but not
`for ondansotron (Andrews et a/., 1992). Also, in tha ferret.
`
`1 Author for conespoDC!eucc.
`
`gnmisetron was reported to be more potent as an llJ1ti-cluetic
`. whon admiuistcrecJ by the oxal route than the intravenous
`route, whcteas the revenc was true for ondansetron (Frt2pat·
`rick et al., 1990). Ondansetron, the most cxteDsively studied
`s-HT, rceeptor antagonist, is poorly effi:ctive in controlling
`mild and delayed emesis (Krls Ill a/,, 1992; Lcvf!i el al.,
`1993). Furthermore, none of the CUJ:rently awilable S-HT,
`receptor antagonists is capable of completely blocking the
`inoidencle of nausea and emesis· in cancer patients. Rqtorts or
`sido-effccts, such as chest pala and cmapynunidal symptoms
`with ondaDsetron (Ballard et al., 1992; Halpern & Murphy,
`199.2), and anhna1 toxicity &Uch as granisctron-induced
`bcpatio can:inoma (Joss & Dott, 1993) may also limit the
`dinica1 utility of these compounds. HBIW8, there is clearly
`a n=d for the clevdopment of sdeotlvc 5-Hr, receptor anta-
`gonjsta which IU1I mOM safv. potellt and ellkadous. RS 2S259-
`197((3aS)·2-{(S)·I-azabicyoJo(2.2.lJoct-3-yl)2.3,3a,4,S,6-hexa-
`hydro-1-oxo-1-lB-beoz[cle] isoquinolinc hydrochloride) is a
`novel selective S-HT3 rea:ptor antagouist (Woog et ar •• 1995)
`(Figura 1). Homogenate radiollpnd binding and ftmctioual
`pharmacological studiell have established that RS 252.59·197
`exhibits a high affinity at 5-HT3 recc:ptoJS (pKj"" 10.4 at
`S-HT, rcceptom in rat cortex and pA2'""8.8 at S.HT1 recep-
`tors in guinea-pig ileum). In the ptaiCilt paper, we present
`our findings on the fn vivo pharmacological etrccts of this
`compound using ondansetron and/or granisctron as reti:n::nce
`compounds.
`
`Dr. Reddy’s Laboratories, Ltd., et al.
`v.
`Helsinn Healthcare S.A., et al.
`U.S. Patent No. 9,(cid:20)(cid:26)(cid:22),(cid:28)(cid:23)(cid:21)
`Reddy Exhibit 1025
`
`Exh. 1025
`
`

`
`R.M. Eglen eta/
`
`Chiral
`
`RS-25269·197
`Flpre 1 Chemical llnlcturc of RS 25259-197 ((3aS)-2-[(S)-I-aza-
`bh;ydo[2. 2. 2]oct-3-y1)·2,3, 3a,4,S, 6-hcxabydro-1-oxo-1-1 H·benz[dc]
`isoquinoline hydroc:hlorid.e).
`
`Methods
`
`Studies on the 110n Bezold-Jarlsch rtiflex in rata
`Intravenously admblistc:red S-Hr or 2-methyl 5-HT elldta an
`Immediate aad ~ vagaiiy mediated reflex brady..
`cardia (refemd to as the Von Bezold-Jadsch reftax) which la
`spc:rifically mediated by S..HT3 receptors and is antagonized
`by selective S..fiT, receptor antagonists (Fozant. 1984). RS
`25259-197 was evaluated for its ability to inhibit this n:~Ocx,
`using ondansetron and granisctron as reCcn:nce compounds.
`Male Sprague Dawley rats (Cbarlee River, 250-380 g)
`were given food and water ad libllum, em:pt those used for
`intraduodenal drug admhlistratloo; tbcsc rats wen: deprived
`of food overnight. Rats wmo anaesthetized with uretbaDo
`(1.5 gkg-1, i.p.) and placed on an aquatic K. module heating
`pad to maintain body temperature at 3TC. Tho left j!Jgular
`vein or duodenum, trachea and left femoral vein were can-
`nulated for drug administration (i.v. or i.d.), facilitation of
`respiration and injection of 2-mc.thyl S.HT, respectively.
`Heart rate was derived from a limb lead n ECG moniton:d
`via subdermal platinum elc:drodes and was recorded with
`BCO/Blotach amplifiers connected to a Gould recorder (RS
`) 3800). A dose-response ourve to 2-methyl 5-HT (S-100 1'8
`Jtg-1, i.v.) was c:onstructcd in eacll rat to establish a sulunax-
`imal dose (usually 10 or 20 f'S kg-1, Lv.) which would elicit a
`reproducibJc bradycardic response. Each rat then received a
`single dose of RS 2S2S9-197, ondansetron or granisetcon and
`was then c:ballengecl with 2-inethyl .5-HT at S, 15, 30, 60, 120,
`180, 240. 300, 420 and 480mln post dosing. A separate
`group of rats .receiving \'Cbialc (saline for i. v., deionized water
`for i.d.) was aimUariy tested in c:ach study. Preliminary
`experiments showed that then: was no cviclence of tachy-
`phylaxis in the responses to 2-mcthyl S..HT. Duration of
`action of the compounds was uscssed by dcteaninlog the
`period of time for which the inhibitory efl'ects remained
`significantly dift'cront from wbicle controls.
`In a separate serlea of experiments, RS 2.5259-197, wlu:D
`applied topically on the abdominal skin of rats. was
`evaluated for its ability to inhibit the reflex. An aqueous
`solution or the drug was absorbed onto a Hill Top chamber
`(2S mm in diameter) in a volume of 0.4 mL A submaximal
`dose or .z..mcthyl 5-HT that could elicl~ a reproducible
`bradycardlo response was lint dctenniued in each rat. R8
`25259-197 (0.01-lOOOJlg per chamber) was them applied
`topicaJJy to the depilitated abdominal area and the 11lt was
`challenged with 2-methyl S..HT at S, 15, 30, 60, 120, 180, 240
`and 300 min po~~t dose.
`
`861
`
`RS 25259-197 11 potllllt 5-111'11 antagonist In vlw
`Anti-emetic studies in ferrets and.tloga
`The fem:t (Fiorczyk et al., 1982) and the dog (Gylys el al.,
`1979) are well established animal models of emesis wbich
`respond to cancer chemotherapeutic agents in a manner
`similar to that observed in man.
`Adult male ferrets (l-1.41cg, MarahaU farms) were ran-
`domly assigned to different tn:abncnt groups. Each animal
`was anaesthetized with mctofane inhalant. A jugular vein was
`cannulated and exteriorized from the outside of the neck.
`Following recovery from anaesthesia, each animal was dosed
`with either P.S 25259-197 (1-lOOJAgkg- 1, i.v. or 0.3-IOOJlg
`tg-1, p.o.), ondansetron (30-1000 1'8 ~cg-•, p.o.) or vehicle
`(lmllqC 1, i.v. or f•o.) 30min prior to administration or
`cisplatin (lOmg kg-. i.v.). The compouads woro giveo orally
`ill hard gelatin capsules. Bach animal was counted for the
`number of emetic episodes for S h following cisplatin
`administration.
`Adult male dogs (8-20 kg) (Hazleton Research Products)
`were randomly assigm:d to different treatment groups. Eadi
`animal was dosed with either RS 25259-197 (O.J-300pg
`q- 1, l.v. or p.o.), vehicle c:ontrol (0.1 m1 kg-1, i.v. or p.o.),
`ondansetron (1-1000 pg lcg-1, L v. or p.o.) or baloperidol
`(Smg.kg-1, p.o.) at JOmin prior to the administration of
`apomorphine (0.1 msklC 1• s.c.), or 120min prior to the
`administration of either dacarbazine (30 mg tg-1, i.v.) or
`mecblorethamino (OAmgkg·•, i.v.) and actinomycin D
`(0.1.5 mg ~cg-•, i.v.) or at 60 min after tho administration of
`dsplatin (3mske'" 1, i.v.). Tho c:ompounds wcra given intra-
`venously via the c:epbalic vein or orally in bard gelatin c:ap-
`sules. Each anima) was counted for the number of 11metic
`episode~ for S h followiDg the administration of cadi emetic.
`An emetic episode was defined as the BUCalsSful evaeuation of
`stomach contents. In separate experiments designed to
`c:xamine the duration of antHmetic activity, dogs were
`pretreated with vehicle (0.) m1 kg-•, i.v.), RS 25259-197
`(30J.&gkg- 1, i.v.) or ondansetron (3001'3 kg-1, i.v.) at 24, 12,
`8, 7, 6, .5, 4, 3, 2 or 1 h prlot to the administration or
`cispladn, after which each animal was observed for an addi-
`tional S h.
`
`H~modynomit: studies in arulUthetized dogs
`Mongn:l dogs (4 male. 4 female, 11-16 kg, Hazelton Labor-
`atories) were anaesthetized with puntobarbitonc sod1um
`(36m.gtg-1, i.v.). The dogs were then a.rtUiciaDy ventilated
`with Harvanl RIS,Pirators. A mtheter inserted into a carotid
`artery was used for measurement or arterial pressure and for
`blood sampling (pH/blood gas analysis with a Coming
`Model Hi8 analyser). The ipsilateral femoral vein was can-
`.nulated for the administratio11 of teat compound or vehicle
`and for tho admiuiatration of a maintenance infusion of
`pentobarbitone sodium (3-Smgkg- 1 b- 1). A Millar 7F
`MiJcro... np Catheter pressure transducer was inserted into the
`left vcnlridc for the measurement of left ventricular systolic
`pressure (LVP) alld l.dl ventricular end-diastolic pressuR
`(LVBDP). and dpftll- was obtained by clmromc
`differentfatlon of tho L VP signal The ript cxtemal jugular
`vein was isolated and a SF Swan-Gaaz catheter (Spectramed
`SPSIOSH) piKed in the pulmonary artery to permit deter-
`mination of ~ac output (CO) by thermodllutfon using a
`Gould cardiac Index Computer (Modd 435). Heart rate
`(HR) was measured with a eardiotachomcter triggered by the
`R.-wavc of a limb lead n BOO n:corded from needle elect-
`rodes inserted subcutaneously. Mean arterial pressure (MAP)
`was cak:uJatcd as 1/3 (systolic arterial pressure-diastolic
`arterial pressure)+ dJastoHc a.rtc$1 pressuR. Di1'cctly
`monitored parameters as well as electronically derived dpf
`dt..,.. and HR were n:GOrded on a polygraph (Gould RS
`3800).
`FoUowlng completion of instrumentation, each dog was
`allowed to equiUbrate for 30-60 min befpre beginning an
`experiment. Each dog recdvm vehicle (0.02 m1 q-1, i.v.) or
`
`Exh. 1025
`
`

`
`R.M. Eglen eta/
`a single dose of RS 25259-197 (10, 100 and lOOOf&gJcs-1,
`i.v.). Recorded panunetem were measuml immediately prior
`to the first treatment dose and at 2, S, IS, 30, 4S and 60 min
`afb:r each dose.
`.
`
`Statistical analysis
`Statistical analysis of the data was performed by a repeated
`measure analysis of variance {ANOV A) and, in some cases,
`was followed by paitwise·compari110ns agaiDst control at each
`time period using F'tsbcr's LSD multiple comparison test.
`Statistical significanc:o wns defined as P<O.OS. In emetic
`studies, an m,. (dose requin:d to produce: SO% of the max-
`imal inhibition) was calculated wbcrevcr appropriate. 1030
`was calculated with NONLlN84 software, a nonlinear
`modelling programme.
`
`Materials
`RS 25259·197, graniaetron and ondansetron were synt]iesizlld
`at the Institute of Organic Chemistry, Syntex Rcseatch. S..
`HT, 2-mcthyl 5-HT, actinomycin D, ~platin, dacaxbazlne,
`mechlorethamine and apomorphine were obtamed from
`Sigma Chemical Co (St Louis, MO, U.S.A.).
`
`RS 25259-197 a pcatant 5-HT.t antagonist lit vwo
`methyl s-HT induced bradycardic response, yielding ID,.,
`estimates (9S% confidence interval) of 0.04(0.02-0.06), 2.2
`(O.S-3.96) and 0.1(0.01-0.19)pgkg-1, respectively (Figure
`2a). When administered intraduodenaOy, R.S 252.59-197,
`ondaosetron and granisctron prodlllled doso-dcpendent in-
`hibition of the bradyc;atdic response (F"agUrc 2b). By the i.d,
`route, RS 25259-197 was much more potent (1030 (9S%.
`coufidenco interval)): [3.2(2.7-3.7Jlgkg-1) than oudansctron
`[ 144(53.2-234.2) 11£ q-1) and granisetron [49.1(21.2-76.9)
`pg kg'"~. Tho duration of inhibitory effects was dose-
`dependent for aU three compounds (YJgDrc 3a-c). At equi-
`eft'ective doses (lowest dose required to produce 100%
`Inhibition)
`the duration of the Inhibitory effects for
`RS 25259-197 (IO!'gkg-1, Ld., 420min} was greater than
`ld., 300min) and
`that of ondansctron (lOOOpgkg-1,
`granisetron (300 p.g kg- 1, i.d., 300 min).
`Transdermal administration of R8 25259--197 (0.01-1000
`H/cbambcr} produced a dosrrdcpendcnt Inhibition of the
`bradycardia indUQCd by 2-methyl S.Hf (Figure 4a) yielding
`an ID,. (95% confidence interval) of 32..8 (12..8-52.. 7) p.g per
`c:hamber. The onset of the inhibitozy cft'ccts was inversely
`proportional to the concentration of the drug in the chamber
`(Figure 4b). At doses of 1-lOOpg per chamber,
`the
`inbi"bitory effects of the drug lasted for greater tban 300 min.
`
`Studies on the von Bezold-Jarisch reflex
`When admiDistered intravenously, RS 25259--197 (O.Ol-10pg
`kg-1), ondansctron (1-300~&~kg'" 1) and granisetroo (0.03-
`300f£Skg-l) produced dose-dependent iulu'bition of tho 2-
`
`120a
`
`100
`
`110
`
`c: E 60
`~
`.5
`~
`
`lZOb
`
`100
`
`80
`
`}
`
`120 8
`too
`c:::
`80
`0
`~ 60
`:c
`.E
`40
`"d!.
`.w
`0
`-20
`
`0
`1.W b
`too
`~ 80
`0
`~ 60
`;Q
`.s
`J;;
`40
`1ft.
`20
`0
`-211
`
`0
`
`100
`c
`80
`0
`~ 60
`:.::
`40
`.5
`"d!.
`20
`0
`
`100
`
`200
`
`300
`
`coo
`
`500
`
`100
`
`200
`
`300
`
`400
`
`600
`
`-20 0.1
`
`10
`Dose (Jig kg-1,1.d.l
`lllgur$ ~ J'llhibltol)' effi!Cts of RS 2S2S9-197 (0), ondaasctron <•>
`and gl'll1\isetro1l (A). admlnistmed intnwonomly {a) and intra-
`clllodenally (b), on tim 2.-metbyl s-HT -Induced btadycudia. Bach
`point repre:ICUIS the mean± a.c.mean, II"' 8-10.
`
`1000
`
`100
`
`400
`
`300
`200
`1ime(mln)
`JlJgate 3 DumtioD of the 1nhfbitory dfecta of R8 25259-197 (a).
`ouda.usctrou (b) and gran)setroD (c) OD the 2.-mcthyl-S..HT-induced
`bnutyc:arctia. {a) RB ZS:zs!J-197, <•> 3psts-•. Ld., <•> tOpgJta-•,
`Ld., (4) 30!1Rkg-1, l.d., (b) Ondaaaetrou, <•> 300p.gkg- 1, i.d., (A)
`tOOOpgJcg-1,-l.d., (O) 3000flsJtx-', i.d. (o) Oran~tron, <•>
`IOOf',kg-1, Ld., (A) 300Jlgkg-1, l.d., (0) l000ji8kg-1, i.d. Each
`point n:pi'C:RIItS the mean± a.cmcan, n- 8-JO.
`
`Exh. 1025
`
`

`
`R.M. E'glen eta/
`
`Anti-emetic activity In ferrets and dogs
`Compared with vehicle control, RS 2m9-197 (l-30f1gkg-•,
`i.v. and l-30f'g.kg~ 1, p.o.), ondansetron (30-IOOOf'g.tcg-•,
`p.o.) and gnmisetron (3-300J&gkg- 1, p.o.) given 30min
`prior to clsplatia producocl aigiJ.ificant and dose-dependent
`rcd1ICtioa in tho number or emetic episodes in fcm:ts (Figure
`S, Table 1). When admiDfstcrc:d orally, RS 25259-197 was 2
`and 13 fold more poamt than granisetron and ondansetron,
`respectively.
`As shown in F'JgUtC 6 and Table 2, RS 25259-197
`(0.3-lOOpgkg-1, i.v. and l-l00f1glcg-1, p.o.) and ondau-
`
`•
`
`120
`100
`80
`
`c
`
`I 80
`
`.c
`,5 40
`~
`
`20
`
`10
`0.1
`RS 25259-197 (JAg/chamber)
`
`*
`
`*
`
`*
`
`• ..
`
`0
`-20
`0.001
`
`100
`c 80
`.2
`:}i 60
`:c .5 40
`
`~
`
`20
`
`0
`
`I'IS 25259-197 a potent ~HT, antagonist In vlro
`863
`satron (3-300pgkg- 1, i.v. and 10-lOOpgkg-t, p.o.) pw-
`dua:d dose-dependent inhibition of the emesis indumi by
`cisplatin, actinomycin D, dacarbazlne and mechlorethamine
`in dogs, IDlO estimates are shown in Table 3. When
`admini.tcred orally, RS 25259-197 was about 30 fold more
`potent than ondallsetron as an anti-emetic against each of
`tha ometogenic ageats. In studie. designed to munine the
`duration of aoti-cmedc IWtivity, RS 2S2S9-197 (30J&Bfc&-',
`i.v.) was effective for 7 h in inhibiting cispJatiD-induced
`eDICils. whereas the anti-emetic activity or ondansctron
`(300 Ji& kg-1, i.v.) lasted for 4'h (F'J.gUrC 7).
`Neither RS 25259-197 (1-1000 Jlgkg-1, p.o.) nor ondan·
`setron . (1000 118 kg-', p.o.) wexv effective
`In inhibiting
`apomorphine induced emesis in dogs (data not shown). In
`contrast, haloperidol (Smgkg- 1, p.o.) produced significant
`blhibitlon of apomorphine induced emesis (data not shown).
`
`Haemody1UIItric effects in anaesthetized dogs
`Baseline values for the measured parameters did not cllifer
`signiftcantly between the vehicle and RS 25259-197 treatment
`groups. Administration of vehicle or RS 25259-197 had no
`significant dl'ects on MAP, CO, dP/dT and SVR (data not
`
`Tabla I
`lDso estimates of RS 252S9·l97, OlldaDsctron and
`gJ'IUiiretroD ap.inst c:ispladn-iftduced emeaiB in fem:ts
`IDso (las tg-1 (9S% c:onlidem:e Intervals))
`p.o.
`Lv.
`1.1 (1.0-1.2)
`3.2 (1.6-4.8)
`ND
`43 (18.0-100)
`. 6.9 (1.0-40)
`ND
`
`ND • not detcnninecl.
`
`~~4---r--,---r--,---r--.--;r--,
`-60
`.,
`110
`100 150 200
`250 300
`350
`lime(mln)
`Ff&are 4 IDhibitory cft'ectt or RS 25259-197 (admluisten:d tranader-
`mally) on the 2-mctbyl ~BT -lDcllwed bradyaudia. (a) DOIC-rc:spomcl
`cune toRS 252.W-197. (b) DuratioD of the inbi'bitOJY ctrec:u of RS
`) 2$259-197, (•) I PI per dumlber, (.) 10 fl.g per chamber, (b.)
`100 1'1 per chamber. Eaeb point rcpn:senlll tho mean± II.C.me&n.
`n .. 10, •Siguilica&\tly difll:rcnt (P<O.~ from 'VCbldc control
`
`0
`
`I E :I z
`
`Veh
`
`0.3
`
`30
`10
`3
`1
`RS 25259-197 (pg kg-1, l.v.)
`
`,00
`
`:8 -a
`
`0 ·; .. ~ a> e ...
`
`'ti
`jj
`E :I z
`
`1
`
`•
`•
`*
`30
`10
`3
`RS 26269-197 (lagkg-', l.v.)
`~ 5 Inhibitory c&cls of ini1'8VCSloilsly admiulatmed RS 25259-
`197 011 dsplatJD4nduted emesis in fmcts.. Jbwh column rcpreseab
`tbo mcan:ts.c.mean. """''· •Sigoiflcaudy diflC:Rnt (P<O.OS) from
`vdJiclc eoutrol (Veh).
`
`10
`OndansBlron {llg kg-1, l.v.)
`lnhlbltory ell'ecls of intravenously administcml RS 2.5259-
`l'fpue 6
`197 (a) and ontfaDsetroD (b) on dsplatin-induccd emesis ha dogs.
`Ead:l polllt reprullltl tho lllCilll ± s.o.mcau, n • 6. •Significantly
`diffenmt (P<O.OS) from vabielc control (Veh).
`
`Exh. 1025
`
`

`
`shown) • .RS 25259-197 produced a siguificant decnase in HR
`at 1000p.gkg-1,1.v, but only at 4Smin post-dtug (Figun: 8).
`
`R.M. £glen st aJ
`
`n~seww~o.
`
`The data obtained in the present study show that RS 25259-
`197, a novel S..HT, receptor antagonist, dlaplays several
`pharmacodynamic and pbarmacoklnetio diffen:IICilll from the
`S-HTJ m:eptor antagonists, ondansetron and graoisetron.
`
`TUie l ADti-cmatio activity of RS 2SlS9-197 iu dogs
`
`RS 25259-197 a potent S.HT1 antag0111st In vlrll
`Studies on the Von BezoltJ..Jarisch reflex
`Inhibition of the 2-metbyl S-HT ind1wcd bradycardia (Von
`Bezold-Jariscb reftwt) is a useful test for the evaluation of
`S.HT, receptor antagonists In Ylvo. In this assay, R.S 2S2S9-
`197 administered either Intravenously, intracfuodeaally or
`transdennally, was effective- in inhibiting the responses to
`2-methyl S-HT. By the intravenous route, RS 25259-197 was
`approximately 3 and SS fold more potent than granisetron
`and ondansetron, respectively. The affinity of RS 25259-197
`
`Dacarbazlne
`(30mgkg-1, l.v.)
`
`Number oft!IMtk episJJtlu
`Actinomycin D
`MdloretluBnble
`(O.lS~~~gtg- 1, lv.)
`(0.4mgk,-1, i.v.)
`9.5 ± l.!J
`NT
`4.5 ± 1.4
`2.8± 1.~
`0.3±'0.3•
`o±~
`Nf
`5.7±1.0
`B.S± 1.3
`2.8± t.s•
`o±o•
`
`14.5±2.3
`NT
`5.8 ±0.6"
`6.7± u•
`3.3± 1.94'
`1.0±0.,.
`Nf
`9.8±3.1
`10.3± 1.9
`4.2±0.8•
`1.5±0.6•
`
`Clqlatilt
`(3 IJI8kg"1, LV.)
`
`14.2± 3.3
`12.8 t 1.9
`16.2±2.)
`4.0± 1.6•
`2.7±0.,.
`o±o•
`12.7 ± 1.2
`18.3±3.4
`IO.S ± 1.3
`3.2 ±o_s.
`2.0±0,.
`
`D~~~g
`
`Vehlde (mlkj-1, p.o.)
`RS 25259-197
`(llgq-•, p.o.)
`
`Oudansetroo
`{JlJJcg-•, p.o.)
`
`Dou
`0.1
`14.2± 1.6
`Nf
`I
`12.8 ± 2.7
`3.2
`10
`6.8±0.2•
`G.l± D.l•
`31.6
`o±o•
`100
`10
`Nf
`10.8± 1.1
`31.6
`6.2± 2.2"'
`100
`3.0± 1.2•
`316
`1000
`0.1±0~
`AD values aro expnssed as mean± s.c.mcan (r1"" 6 per JPOUP).
`Nf- not teltcd.
`"Statiatlcally algniflamt from vchidc control (P<O.OS).
`
`TaNe 3 JDSil (ltgkg- 1) csthnab:s of RS 25259-197 and ondausetroo against vadoua emetoganic agents In dogs
`Emetogenlc agent
`
`D~lne
`
`4.1
`(2.1-6.1)
`9.7
`(7.3-12.0)
`80.0'
`83.0'
`
`200.0
`(47.0-900)
`160.0'
`
`A.ctiilomyctn D M~
`4.9
`(0.45-54)
`2.SS
`
`4.4
`(2.1-9.1)
`3.0
`(0.8$-10.0)
`36.0'
`280.0'
`
`Druz
`RS 2S2S9-1517
`
`Qudansdron
`
`Rout11 of
`admfni8tratlon
`
`i.v.
`p.o.
`
`Lv.
`
`p.o.
`
`Clsp/alln
`1.9
`(0.28-13.0)
`8.5
`(3.6-20)
`46.0
`. (17.0-74.0)
`160.0
`(4B.D-270)
`1Meaoblgi\ll contideDI:a intervals could not bo obtained bccaiUC of Jack: of dosc-clcpendency.
`
`.~ D.
`
`.. -8
`Ql i E Q)
`J E ::J z
`
`'5
`
`•
`2
`
`0
`0
`
`l'1pJe 7 Dumtlon of tho inhibitory elfec:UI or vehlde (0.1 m1 tg- 1)
`<•>. RS 25259-197
`(30flgkg- 1,
`i.v.) (e) and ondansetron
`(300flg q-1, i.V.) (0) OD clspJatin-badiJced emesia iD dogs, Dog1
`were ~tseaCI:d wilh the dmp at 24, 12, 8, 7, 6, S. 4, 3, 2 and I h
`prior to the admiDistradoo of alsplali11. Each point repmcp.ts the
`me1111 ± s.e.mean, n • 6 •Sigoific:antly different (P<O.OS) lium vdli-
`do control.
`
`200
`
`~ 175
`e e
`a 150
`~
`t:
`Ill
`II> 1Z5
`:J:
`
`~ t
`.I
`
`*
`
`100~--~--4---~--~~~~~-T--~
`co . 50
`-10
`0
`10
`30
`60
`70
`2l)
`limu <miru
`lliglme 8 Eft'ccra of Intravenously adminisllm!d RS 25259-197 011
`heart rate (HR? in anacsthctit.ed clop. (0) Vllhlale, <•> R8 2S2S9-
`l97 (101'8lcr, l.v.), (b) RS 2SlS9-I!n (IOpgkg- 1, l.v.), (.0) RS
`252S9-197 (1000pglcg"1, Lv.). Each point reprcscotB tho mean±
`s.e.mean, n-6. ·~tty ditrcrcnt (P<O.OS) from vehicle con-
`trol.
`.
`
`Exh. 1025
`
`

`
`RS 25259-197 a potent S.KT1 antasonlst In vivo
`865
`Besides cisplatin. other cancer chemotherapeutic agents
`sm:h as dacarbazine. mechlorethamine and actinomycin D
`also CllU8C emesis although of a Jesser severity. Thes~ agents
`aJSQ induced emesis in dog& wbich was dose-dependently
`inhibited by RS 2S2S9-197 Indicating that the anti-emetic
`eft'ecta of this compound are not reatricted to cisplatln alone,
`In tho caso of daalrbazine and mecbiiX"Cthamine, complete
`Inhibition of the emetiQ episodes was achieved with
`IOOJ&gkg-•, p.o. of RS ZS2S9-197. Ondansetron was also
`cft'ecdve against the above cnwtDgenio agents although it was
`about 100 fold l.ess potent than RS 2SlS9-197. Also, at the
`doses tested, ondansetron could produce compktc blockad~
`of the emetic episodes only against mechlorethamine bnt not
`agaimlt cisplatio., dacarbazinc and actinomycin D. RS 25259-
`197 was incft'ective against apomorphine-induced emesis sug-
`gesting that tho anti-emetic effects are not a result of
`dopamine n:ccptor antagonism. Such an interpretation is
`consistent with Ugand binding studies which ba~ shown that
`RS 2Sl59-197 does not interact with D1 and ~ receptors
`(Wong e.t aL, 1995).
`
`Haemodynarnic studies
`1'hG cffeots of RS 2S259-197 on bacmodynamics in dogs were
`studied to determine whether the compound evoked car-
`diovascular effects at doses that showed anti-emetic activity.
`This study was undertaken in view of the reported clini<:al
`catdiovascular adverse effects reported with ondan~tl'On
`(Ballard et al., 1992). RS 25259-197 produced a modest but
`significant dccretUIC in HR at lOOOf'gkg-1• However, In
`dogs, anti-emetic dl"ects of RS 2S2S9-l97 were observed at
`doses as low as If'S kg- 1• Thus. R8 2S2S9-197 was essen-
`tially devoid of any adverse cardiovasculBr aide-effects at
`doses that produced anti-emetic effects.
`In summary, RS 25259-197 appears to be a novel, orally
`active 5-HT, receptor antagonist that is motc potent and has
`a longer duration oC action than either ond~tron or
`gnmisetron. lu discussed In the Introduction. wmmtly
`available 5-HT, receptor antagonists such as ondansetron
`S1lft'er from drawbaclcs most notable of wblch is thuir poor
`effectiveness in controlliDg mild and delayed emesis. It is
`unclear whether this is due to the intrinsic property of these
`drugs or is a commonality for all S-BT3 receptor antagODists.
`The present study bas shown that, at least in animal models,
`RS 25259-197 repn:sents a significant improvement over
`ondansctron as an anti-elb.Ctic agent with respect to potency
`and duration of action. It should be noted that pnisetron
`bas also been reported to possess the same advantages o.-
`ondansetron (Andrews et al., 1992}. As the present study
`lacks ex.tcnsivo anli-cmetic efficacy data with granisetron, jt is
`unclCIU' as to how RS 25259-197 would compare with graui-
`setroa. Future studies should, therefore. be aimed at a direct
`comparison of the two druga. Studies aro also in progress to
`evaliJat.C the dinical eft'cx:tiveness of RS 2$259-197.
`
`R.M. Eg!en 6t aJ
`for displacing rH]-quipazinc in the rat cerebral cortex is lOA
`(Wong et al., 1994) whereas the reported p~ values for
`ondansetron and gmnisetron in rat tissues range from
`8.3-9.0 (for ondansetron) and from 9.2-9.8 (for granisetron)
`(Kilpatdc:k et al., 1990). Thus the order of potency in
`inhibiting the Von BezDid-Jarisch reflex para1h:ls the relative
`affinities of the thm1 compounds at .5-HT1 receptors. By the
`illtraduodcmal route, R8 2S2S9-J.91 was about 15 and 45 fold
`more potent than ondansctron and granisetron. The i.d./l.v.
`potency ratio for RS 25259-197, ondansetz:on aDd gnmlsetton
`was calculated to be 80, 65 and 490, respectively. Thesu data
`BUggeSt that RS 25259-197 and ondansctron possess better
`bioavailabllity obaracteristics than granlsctron. Comequently,
`by the intraduodenal route. the better bioavaiJability and
`higher affinity of RS 25259-197 for 5-BT, receptors malre it
`the mORt potent or the three compounds. Besides being :mon::
`potent, RS 25259-197 also appears to have a longer duration
`of action; its lnhlbitory effects persisted longer than those of
`granisetron and ondaDsetron wheo all were tested at equi-
`effcctive doses.
`Oral administration of drugs to cancer patimta undergoing
`chemotherapy is problmnatic owinf. to the hi&h incidence of
`emesis in these patients. Thus. availability of a S-HTJ recep-
`tor antagonist in the fonn of a tmnsdennal formulation
`would greatly facilitate the management of emesis in these
`l plllicnts. The present study has shown that RS 2S2.59-197,
`when adminis~ered by the lransdennal route, causes potent
`and
`inhibition of the 2-methyl-5-HT-induced
`IUStained
`bradycardia. These data suggest that transdermal foJlJI.lJla-
`tions of RS 25259-197 may prove to be oonvenient drug-
`delivery systems In patients experiencing emesis due to cancer
`chemotherapy.
`Anti-emetic studiu
`The emctDgenic dfccts of anti-c:8.1U:er drugs is most pro-
`nounced with platinum dmgs such as cisplatin. Cisplatin,
`when administered to dogs and ferrets, produces an emetic
`aod behavioural profilo which most closely resembles the
`clinical symptoms (Gylys et -al., 1919; Florczyk et al., 19&2).
`The present study has shown that, RS 25259-197, admini-
`stered intravenously or orally, potently inhibits cisplatin
`induced emesis in both the fcnet and dog. Comparison of the
`oral anti-emetic potency in the ferret showed that RS 25259-
`197 was approximately 2 and 13 fold mOte potent than
`gramsetron and ondansetron, respectively. In the dog, RS
`25259-197 was about 30 fold more potent than ondanselron
`.~. . in inhibiting c:isplalin-induced enu:sls. The !ower potency of
`l ondansetron relative toRS 25259--197 is agam couslstcnt with
`·
`the relative aflinity of these compounds for 5-HT, receptors.
`When tested at equi-dfcctivo doses in dogs, RS 252S9-J97
`appeared to have a loaget" duration of anti-emetio adion
`(7 h) than oudaml:tron (4 h).
`RS 2S2S9-l97 produced complete inhibition of emesis
`lnduc:cd by dsplatin in ferrets (Lv. but not p.o. route) and
`dogs (p.o. but not i.v. route). This is a pertinent ~
`became, ln the clinical setting, it is critical to produce com-
`plete block rather than parlial attenuation in the number of
`CDletlc episodes, .s GYeD mild nausea and emesis can severely
`comproblise the suc:ccss or chemotherapy. The maximal
`inhibitory effeots of RS 25259-197 appear to depend on the
`route of administmtion. The precise reason for this paradox-
`ical finding Is unclear at present.
`Rer.,rem:es
`ANDJtEWS, P.L.ll., llRANDARI, P., DAR.VEV, P.T., BINOHAM, S.,
`MAJtR., U.B. A BLOWER. P.R. (1992). Ara aD S.HT, receptor
`antagonists the aarnd! Eur. J. Cancer, 28A (Suppl. 1}, S2-S6.
`BALLARD, U.S.. BOTl'INO, 0. & BO'ITJNO, I. (l992). Ondansc:troa.
`and West pain. Lancet. :WO. 1107,
`COSTALL. B.&: NAYLOR, R.I. (1992). NCIII"ophannacology ofauu:sls
`lu rcllltioo to dinlcal responso. Br. J. er.-r, 19 (Suppl),
`SlZ-SJ3.
`
`· ·
`
`We wish 10 thauk Luplta Jacob&on. Susan Young, Craig Coombs,
`Willy Ladmit, Mary Fulks, Larry Wood. George Faurot, MaN
`Pmy, Arnold Wong, Jill Oiaucttoni, Paul Ku aad Merrie Va.n.Syoc
`for their el'J'Crl tcchnlcal aaaista"IICO,
`
`FriZPATIUCIC. L.R., LAMDBR.T, R..M., PHNDLEY, C.S., MAKT1N.
`G.B., BOSTWICK, J,S., OElSNER, O.W., AIRBY, J.B., YQUS-
`SEfYI!II. R.D., PBNOBLTON, R.G. & DECKTOR, D.L. (1990). RG
`11915: a potent 5-hydroxytrypmmlne-3 anta&ooist that is an
`orally ctrcc:tlvc inhibitor of cytotoxlo drug-induced emesis lu tile
`fenct llDCI dog. J. .PIIonll6col. Exp. Tfler., 254. 4S0-4SS.
`
`Exh. 1025
`
`

`
`866
`
`R.M. Eaten et al
`
`FLORCZYK,. AJ'., SCHUIUG. J.B. &: Blt,ADNER, W.T. (1982), Cis-
`plallu-indliCCd cmesl• in tho 1Crret: a new animal model c-
`Thrat. kp .. li6, 187-189.
`FOZAllD, J.ll. (1984), MDL 7222.2: a potun1 lllld hlghly sclci;IWo
`B.Dtagonfst at mmonal 5-hydroxytcyptamiue reocpton. NCIUII)In·
`Sclrmled. Arch. Plulnrua:ol,. 321, 36-44.
`OYLYS, I.A., DORAN, K.M. &: BUYNISltl, J.P. (1979). Antqoniam of
`c:iaplatin-lnduccd Cl1lesb In the dog. CollllftiDI. CMm. Patltol.
`Phamracol., 23, 61-68.
`'ffALPBRN. J.ll. a: Ml,JRPHY, II. (1992). Bxlrap)'CSD~idal n:amioo to
`ondametron. Omur, 69. 1275.
`JOSS. R.A. & DD'lT, C.S. (1993). Ctiolcal atuclics with Granlsetron, a
`new S·RT3 receptor antagoaist for the treatment of ~
`chemotherapy..lnducc:d aiDillia. .&r. J. Cam:er, 29A, sn-829.
`IOLPATIUCK. GJ., BUNCE, K. T. & TYBllS, M.D. (1990}. S.Hf, RICe(l-
`tors. Med. Ra. . .R.n•., 10, 441-475.
`KRJS, M.G., TYSON, L.B., CLARK, R.A. & GRAU.A, R.J. (1992). Oral
`ondall5dron for the 4;0ntrol of dclayr.d emt.sls afrer dsplalln.
`c-. 10 (Supp1 4). to12-1o16.
`UM1T, M,. WAIUt, D,. YELLE, L., RAYBR, H.L., LOFTERS, W.S.,
`PBRAULT, D.l., WILSON, K.S., IATREILLB, J., POTVJN, M.,
`WAIUilBR, B., l'lUTCHAIID, IU., PALMBR, M., LBII. B. & PA111R,
`J.L. (1993). On~ c;ompated with daamethasonc and
`111Ctodopramidc as aotic:metks in the chamotherapy of bmut
`CIUHlCI' with cyclophosphamide, methotruate and llaorouradl N.
`&gt. J. Mm., 3211, tosJ-1084.
`
`RS 25259-197 • potant S.HT1 111tqanfsl/n vlro
`MARK. II.B., DAllVBY, P.T. & BARTLBIT, AJ, (1991), Bmarging
`dlft'el'CDI:C8 bctwccn S.Hf3 receptor antaaonists. Anllr:GIIt!el'
`Drup. 2, 513-518.
`OXFORD. A.W., BBU.., J.A., ICILPATIUCJt, G.l,. UWLAND, SJ, A
`'J'YBRS, M.D. (1!192). Ondausctron and related 5-HTJ rceeptor
`antqonislll: rcamt advaiiCIIJ. Prog. Med. Chem., 29, 239-270 ..
`T