Pharmacological Characterization of the Novel ACh Releaser α‐tropanyl 2‐(4‐bromophenyl ...

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PHARMACOLOGICAL CHARACTERIZATION OF THE NOVEL ACH
RELEASER A-TROPANYL 2-(4-BROMOPHENYL)PROPIONATE (PG-9).
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PHARMACOLOGICAL CHARACTERIZATION OF THE NOVEL ACH RELEASER A-TROPANYL 2-(4-
BROMOPHENYL)PROPIONATE (PG-9) / C. GHELARDINI; N. GALEOTTI; M. ROMANELLI; F. GUALTIERI; A.
BARTOLINI. - In: CNS DRUG REVIEWS. - ISSN 1080-563X. - STAMPA. - 6:(2000), pp. 63-78.
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18 February 2023
CNSDrugReviews
Vol.6,No.1,pp.63–78
©2000NevaPress,Branford,Connecticut
Pharmacological Characterization
of the Novel ACh Releaser (cid:3)-tropanyl
2-(4-bromophenyl)propionate (PG-9)
C.Ghelardini,N.Galeotti,M.N.Romanelli,*
F.Gualtieri,*andA.Bartolini
DepartmentofPharmacologyand*DepartmentofPharmaceuticalSciences,
UniversityofFlorence,Florence,Italy
Key words: Acetylcholine release—Analgesia—Antinociception—Cholinergic system—
Learning—Memory—Pain—PG-9.
INTRODUCTION
Ghelardinietal.(27)reportedthatatropineatverylowdosesinducescentralantinoci-
ceptioninrodentsthroughanenhancementofcholinergictransmission.Soonafter,itwas
discoveredthattheR-(+)-enantiomerofatropine,R-(+)-hyoscyamine,wasresponsiblefor
the antinociceptive activity of the racemate, while the S-(–)-enantiomer, S-(–)-hyoscy-
amine, was devoid of any antinociceptive action (29). R-(+)-hyoscyamine, in the same
range of analgesic doses, was also able to prevent amnesia induced by antimuscarinic
drugs (35). It is interesting to note that this antinociceptive activity, different from that
produced by direct muscarinic agonists and cholinesterase inhibitors, was not accom-
panied by typical cholinergic symptoms (e.g., tremors, sialorrhea, diarrhea, rhinorrhea,
lacrimation). An investigation of the antinociceptive and antiamnesic effect of atropine
hasdemonstrated,usingmicrodialysistechniques,thatR-(+)-hyoscyamine,atcholinomi-
metic doses, produced an increase in acetylcholine (ACh) release from the rat cerebral
cortexinvivo,indicatingthatitactsviaapresynapticmechanism(35).
On this basis, a synthetic program to modify the chemical structure of atropine was
started,whichaimedtodevelopcholinergicamplifiersendowedwithmoreintensiveanti-
nociceptiveandantiamnesicactivitiesthanatropinebut,likeatropine,lackingcholinergic
sideeffects.Thesecompoundswould,therefore,bepotentiallyusefulasanalgesicsand(cid:1)or
in pathological conditions characterized by cholinergic deficits (e.g., Alzheimer’s
Address correspondence and reprint requests to Dr. Carla Ghelardini, Department of Pharmacology, viale
G.Pieraccini,6,I-50139Florence,Italy.
Tel.:[phone].Fax:[phone].E-mail:[email protected]
63
64 C.GHELARDINIETAL.
Fig.1. ChemicalstructureandsynthesisofPG-9.
disease). Of the many compounds synthesized and studied, (cid:3)-tropanyl 2-(4-bromophe-
nyl)propionatelabeledPG-9(45)(Fig.1)showedaninterestingpharmacologicalprofile.
CHEMISTRY
The chemical structure of the lead compound, R-(+)-hyoscyamine, was extensively
manipulatedinordertoovercomethestabilityproblemsassociatedwithtropicacidderiv-
ativesandtoobtainmoreefficaciousantinociceptivecompounds.Theremovalofthehy-
droxylgrouptoform(cid:3)-tropanyl2-phenylpropionate(ET-103)(43)gaveacompound70-
timeslesspotentbutwithsimilarefficacyastheleadcompound(45).Betterresultswere
obtainedwiththeseriesofsubstituted2-phenylpropionicacidesters;infact,withtheap-
propriate substituent on the phenyl ring (4-Br, 4-F, 4-OMe, 2-, 3-, or 4-Cl) compounds
whichshowedlowerpotencythanET-103butamuchhigheranalgesicefficacywereob-
tained (45). In this series, as well as in that of the 2-phenoxypropionic acid derivatives,
antinociceptiveactivitywaslimitedtothe(cid:3)-tropanylester(44).Amongallcompoundsof
the series, (cid:3)-tropanyl 2-(4-bromophenylpropionate (PG-9) showed the best combination
ofpotencyandefficacyandwasselectedforfurtherstudies.Thesyntheticpathwayused
for its synthesis is shown in Fig.1. Single enantiomers were synthesized and studied be-
cause PG-9 possesses a stereogenic center (68,39). They showed enantioselectivity in
pharmacological activity, although, unlike atropine enantiomers (35), both stereoisomers
of PG-9 had analgesic and nootropic properties. In any case, the most potent and effica-
cious stereoisomer (S-(+)-PG-9) possesses the same spatial arrangement at the chiral
centerastheleadcompound,R-(+)-hyoscyamine(68).
INVIVO STUDIES
AntinociceptiveProperties
PG-9 induced antinociception in mice and rats. Antinociception was elicited, re-
gardless of the noxious stimulus used: thermal (hot-plate and tail flick tests), chemical
CNSDrugReviews,Vol.6,No.1,2000
PG-9 65
Fig.2. Dose-responsecurvesofPG-9i.p.(a)andi.c.v.(c)inthemousehot-platetest.ThePG-9timecourseof
40mg(cid:1)kgi.p.isreportedinband20(cid:6)gpermousei.c.v.indfromthesametest.Verticallinesgive (cid:10) S.E.M..
Eachpointisthemeanofatleast10mice.^P<0.05;*P<0.01vs.controls.InaandcPG-9wasadministered
15minbeforethetest.
(abdominal constriction test) or mechanical (paw pressure test). The methods used were
thoseofO’CallaghanandHoltzman(62),D’AmourandSmith(16),Kosteretal.(53),and
Leightonetal.(56),respectively.
In the mice hot-plate test PG-9, by systemic administration (i.e., i.p., p.o., i.v.), pro-
duced a dose-dependent increase in the pain threshold (Fig.2; ref.37). A similar effect
wasobtainedwiththeabdominalconstrictiontestinmice(Fig.3,panelA).Themaximal
CNSDrugReviews,Vol.6,No.1,2000
66 C.GHELARDINIETAL.
antinociceptiveeffectofPG-9wasreachedat15minafteritsadministration.Thereafter,
theeffectslowlydiminished(Fig.2,panelsBandD).PG-9alsoproducedanincreasein
Fig.3. AntinociceptiveeffectofPG-9andantagonismbyhemicholinium-3(HC-3)(1(cid:6)gpermousei.c.v.),at-
ropine(5mg(cid:1)kgi.p.),anddicyclomine(10mgpermousei.p.)ontheenhancementofpainthresholdinducedby
PG-9(20mg(cid:1)kgs.c.)inthemouseabdominalconstrictiontestinducedby0.6%aceticacid(a)andintherat
paw-pressuretests(b).HC-3,atropineordicyclominewereinjected,respectively,5h,15minand10minbefore
testing.Intheabdominalconstrictiontestthenociceptiveresponseswererecorded15minafterPG-9adminis-
tration.VerticallinesshowS.E.M.^P<0.05;*P<0.01vs.salinecontrols.°P<0.01vs.PG-9(20mg(cid:1)kgs.c.).
Numbersinsidethecolumnsindicatethenumberofmiceorrats.
CNSDrugReviews,Vol.6,No.1,2000
PG-9 67
thepainthresholdinrats(pawpressure:Fig.3,panelB;tailflick:ref.37)withapharma-
cological profile similar to that in mice. Both PG-9 enantiomers dose-dependently in-
creased the pain threshold in mouse hot-plate and abdominal constriction test, although
R-(+)-PG-9wasslightlymoreeffectivethanS-(–)-PG-9(37,68).
PG-9isendowedwithcentralantinociceptiveactivity.Itwas,infact,possibletoreach
the same intensity of analgesia by injecting PG-9 directly into the cerebral ventricles by
methodspreviouslydescribed(46)at10–30(cid:6)gpermouse(Fig.2,panelC;ref.37).Itsac-
tivityatsuchlowdosesruledoutthepossibilitythattheantinociceptioncouldhavebeen
duetoretrodiffusionofthedrugfromthecerebralventriclestotheperiphery.
PG-9 showed good antinociceptive efficacy in comparison to R-(+)-hyoscyamine or
wellknownanalgesicdrugs,suchasmorphine,diphenhydramine,orclomipramine.Asa
matteroffact,acomparisonoftheareasundertheactivitycurvesoftheabove-mentioned
compoundsatthehighestdosesthatdonotimpairnormalbehaviorofmicerevealedthat
PG-9wasaseffectiveasmorphineandmoreeffectivethanR-(+)-hyoscyamine,diphenhy-
dramine,orclomipramine(37).
Atdoseslowerthan1mg(cid:1)kg,PG-9reducedthenumberofabdominalconstrictionsin-
ducedbyi.p.injectionof0.3%aceticacidandreversedhyperalgesiainducedbymorphine
withdrawal.
PG-9antinociceptionisnotduetoanantiinflammatoryaction.Atconcentrationsupto
10–4mol(cid:1)L PG-9 did not inhibit inducible COX activity, while indomethacin (IC )
50
27(cid:1)10–6mol(cid:1)Lwaseffective.Furthermore,PG-9failedtosuppresscarrageenan-induced
pawedemaatanalgesicdoses(Table1).
AntiamnesicActivity
PG-9 ameliorates impaired cognitive processes in mice. In the passive avoidance test
this compound prevented amnesia induced by scopolamine, dicyclomine (Fig.4), or
(–)-ET-126 (36). Complete prevention of amnesia was obtained with PG-9 at 10mg(cid:1)kg
i.p. (Fig.4) or 20(cid:6)g i.c.v. (36). At these doses PG-9 had only weak analgesic activity in
the hot-plate test. To prevent amnesia, PG-9 was administered at 20min before the
training session, since the time-course of the antiamnesic activity of PG-9 indicates that
thecompoundreachesitsmaximaleffectat15–30minafterinjection.
TABLE1.EffectofPG-9oncarrageenan-inducedpawedemainrats
Pretreatment Treatment Dose(mg(cid:1)kgi.p.) Pawvolume(ml(cid:10)S.E.M.)
Saline Saline 1.25(cid:10)0.08
Carrageenan Saline 2.31(cid:10)0.06
Carrageenan PG-9 20 2.37(cid:10)0.08
Carrageenan PG-9 40 2.39(cid:10)0.10
Carrageenan Indomethacin 1 1.45(cid:10)0.07*
Indomethacin was used as positive control; n=5 rats per group. *P<0.05 in comparison with
carrageenan-salinecontrols.PG-9wasinjected15minbeforethetest.
CNSDrugReviews,Vol.6,No.1,2000
68 C.GHELARDINIETAL.
Fig.4. Effect of PG-9 (i.p.), aniracetam (p.o.), and physostigmine (i.p.) on dicyclomine-induced amnesia in
mousepassiveavoidancetestand,underthesameexperimentalconditions,effectofPG-9onscopolamineam-
nesia.Punishmentconsistsofafallintocoldwater(10°C).PG-9,aniracetam,andphysostigminewereinjected
20minbeforethetrainingsession.Scopolamineanddicyclominewereinjectedimmediatelyafterthetraining
session.*P<0.01vs.salinecontrols.Eachcolumnrepresentsthemeanofatleast25mice.
Inthepassiveavoidancetest,animprovementincognitionofanimalswithnomemory
impairment is difficult to demonstrate. As a matter of fact, PG-9 as well as well-known
nootropicdrugs,suchaspiracetam,aniracetam,androlipramorcholinomimetics,suchas
physostigmineandoxotremorine,donotshowanymemoryfacilitationinnormalanimals
(15,41).TheprocognitiveactivityofPG-9wasunmaskedbyusingasociallearningtest,
whichwasperformedaccordingtomethodsdescribedbyMondadorietal.(61).PG-9ex-
erted beneficial effects on cognitive performance by prolonging the time normally re-
quiredbyratstodeletemnemonicinformation(datanotshown).
EffectofPG-9onAnimalBehavior
PG-9produceditsmaximalantinociceptiveeffectat40mg(cid:1)kgs.c.withoutanyvisible
modification in gross behavior of either mice or rats. Moreover, mice treated with the
same dose of PG-9 retained motor coordination in the rotating rod test (method of Kuri-
bara et al., 54) (Table2). Under these experimental conditions, the effects of PG-9 were
comparedtothoseofpilocarpineorphysostigmineatequi-effectivedoses(Table2).Both
the muscarinic agonist and the inhibitor of cholinesterase produced a statistically signif-
icant reduction in endurance time on the rotating rod. The spontaneous motility of mice,
CNSDrugReviews,Vol.6,No.1,2000
PG-9 69
TABLE2.EffectofPG-9incomparisonwithpilocarpineandphysostigmineintherotarodtest
Endurancetimeonrotarod(sec)
Aftertreatment
Before
Treatments.c. treatment 15min 30min 45min
Saline 104.3(cid:10)6.2(18) 106.7(cid:10)6.0(18) 107.8(cid:10)5.9(18) 103.5(cid:10)6.2(18)
PG-9(40mg(cid:1)kg) 101.2(cid:10)5.9(10) 96.7(cid:10)8.2(10) 95.6(cid:10)9.4(10) 106.2(cid:10)8.4(10)
Pilocarpine(10mg(cid:1)kg) 108.2(cid:10)8.4(11) 66.5(cid:10)7.1*(11) 61.3(cid:10)9.6*(11) 74.4(cid:10)8.7*(11)
Physostigmine(200(cid:6)g(cid:1)kg) 93.4(cid:10)5.7(9) 61.4(cid:10)6.8*(9) 54.4(cid:10)8.1*(9) 52.3(cid:10)8.8*(9)
*P<0.05vs.salinecontrols.Thenumberofmiceisshowninparentheses.
evaluated by the Animex apparatus, as well as the exploratory behavior, studied by the
hole-board test (36), were normal after the administration of PG-9 at 40mg(cid:1)kg s.c. or
30(cid:6)g per mouse i.c.v. Impaired motor coordination and spontaneous motility were ob-
served in mice after PG-9 was administered alone at doses starting at 100mg(cid:1)kg s.c.,
whereasitsLD was400mg(cid:1)kgs.c.
50
EffectonIntestinalMotility
PG-9didnotmodifyintestinaltransittimeinmiceatanalgesicandantiamnesicdoses
as determined by the technique of Reynell and Spray (67) (data not shown). In contrast,
otheranalgesicdrugs,suchasmorphine,significantlyretardedgastrointestinalpropulsion,
whereas the cholinesterase inhibitor, neostigmine, accelerated net propulsion (72). The
lack of effect of PG-9 on intestinal motility indicates that this compound has the same
analgesic activity and may be superior to opioid analgesics, which produce constipation,
ortoclassicalcholinomimetics,whichcausediarrhea.
INVITRO STUDIES
EffectonEndogenousNerveGrowthFactor
InvitroadministrationofPG-9increasedthesecretionofNerveGrowthFactor(NGF)
byastrocytesinadose-dependentmanner(36).AfteradditionofPG-9themaximalNGF
levelswere17.6timegreaterthatthecontrolvalue.Inculturedastrocytes,effectivecon-
centrationsofPG-9producednomorphologicalchanges.However,slightcelltoxicityor
morphological changes were observed with PG-9 at 1mg(cid:1)mL. (36). Cultured quiescent
astrocytescanbeusedtostudytheeffectsofdrugsonNGFsynthesissinceNGFsynthesis
isregulatedinagrowth-dependentmannerinculturedastrocytes;mostoftheastrocytesin
the brain are in the quiescent phase and do not express the NGF gene with in vivo
administration.
CNSDrugReviews,Vol.6,No.1,2000
70 C.GHELARDINIETAL.
Fig.5. Dose-response curves of PG-9 on nicotine (4(cid:6)M) ((cid:1)) and electrically (0.1Hz; 0.5msec; double
thresholdvoltage)evokedcontractions((cid:2))ofguinea-pigileummyentericplexuslongitudinalmusclestripex-
pressedaspercentagevariationofcontractions.Eachpointrepresentsthemeanofatleast6experimentsandver-
ticallinesgiveS.E.M.*P<0.05calculatedintherangebetween0.1pM–0.1nM.
EffectonIsolatedGuineaPigIleum
Whenaddedtotheorganbathatconcentrationsrangingfrom1pmol(cid:1)Lto0.1nmol(cid:1)L,
PG-9 potentiated contractions evoked by either nicotine (4(cid:6)mol(cid:1)L) or electrical stimu-
lation [0.1Hz, 0.5 msec, voltage double threshold, method of Paton and Vizi (64)]
(Fig.5).Asmeasuredbytheareaunderthecurve,PG-9potentiatedcontractionsinduced
by nicotine to a greater extent than those induced by electrical stimulation. The
potentiation was no longer observed when the concentration of PG-9 in the medium
reached 0.1nM. At 10nmol(cid:1)L PG-9 began to inhibit both types of evoked contractions.
Nicotine-evokedcontractionswereaboutfourtimeslargerthanthoseevokedelectrically
(Fig.5). The difference in the magnitude of contraction is probably due to the fact that
duringelectrically-evokedcontractionsbothintramuralcholinergicandsympatheticfibers
are activated, whereas only cholinergic fibers are activated during nicotine-evoked con-
tractions.Norepinephrinereleasedduringelectricalstimulationislikelytolimittheeffect
of ACh released after the administration of low concentrations of PG-9. The greater
potentiation of the nicotine-evoked contractions compared to those elicited by electrical
stimulationcanbeexplainedbytheinhibitoryeffectofnorepinephrine,whichisreleased
onlyduringelectricalstimulation(27).
CNSDrugReviews,Vol.6,No.1,2000
PG-9 71
Fig.6. Dose-response curves of PG-9: (a) on electrically evoked phrenic nerve-hemidiaphragm contractions
(0.2Hz, 0.5msec, twice threshold voltage); (b) on tritium efflux from the same preparation preloaded with
[3H]choline.Eachpointrepresentsthemeanof6experiments.Verticallinesgive (cid:10)S.E.M.*P< 0.05calculated
intherangebetween10–9–10–12M.
RatPhrenicNerve-HemidiaphragmPreparation
PG-9(1pmol(cid:1)L–1nmol(cid:1)L;>1(cid:6)mol(cid:1)L)potentiatedcontractionsofhemidiaphragm
evoked by electrical stimulation of the left phrenic nerve (Fig.6, panel A). ACh release
(Fig.6,panelB),studiedbythemethodofBülbring(9)asmodifiedbyWesslerandKil-
binger (75), was also enhanced by PG-9, while contractions evoked by the direct stimu-
lation of the diaphragm were not modified by the drug. At concentration greater than
1(cid:6)mol(cid:1)L, numerous muscarinic antagonists, such as atropine, pirenzepine, dicyclomine,
orglycopyrrolateareknowntoenhancehemidiaphragmcontractionsbyblockingmusca-
CNSDrugReviews,Vol.6,No.1,2000