5S
`
`ISSN 1i21.13 tY'
`
`'f 1 .
`Acta Phannacol Sin
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`i #; i
`-Au Jul ;
`har: wwu . chinaphar . cum
`
`: 5:41- 541
`
`1
`
`Original Research
`Effects of calcitonin gene - related peptide and BIBN4O96BS on
`myocardial ischemia in anesthetized rats
`
`Dong-fvlei Vv1Jt , Pieter A van 2WLETEN2, Henri N LXX)DS'
`( Cardiusclsc-ulur Research , Bcrehrirtl;er h1gelhrirn
`Pharmu KG , Birkenciot;ifèr Str 65 . D-88.397 Biberach . Gernurrrv ; 2Deparrnrerrt of Phurrrracotheru¡ry. . Academic Medi-
`cal Centre , University of Amsterdam .Meibergdrecf 15 , 1105 AZ Arn,sterdum , The Netherlands)
`
`KEY WORDS
`calcitonin
`gene -related peptide;
`B[BN4096BS; myocardial infarction; reperfusion injury
`
`CGRP is a potent myocardial protective substance.
`
`ABSTRACT
`
`INTRODUCTION
`
`l
`
`AIM The cardioproiective effect of calcitonin gene -
`related peptide (CGRP) was investigated in an ischemia
`rat model . METHODS :
`ischemia-reperfusion
`injury
`was provoked by 60 min left main coronary artery occlu-
`sion followed by 60 min of reperfusion in anesthetized
`rats. The transverse slices of ventricles were stained by
`2,3, 5- triphenyltetrazolium chloride to determine the in-
`fant area.
`Plasma creatine phosphokinase levels were
`determined by means of a creatine phosphokinase ( CPK )
`A radioirnmunoassay was used to determine plasma
`kit .
`levels. RESULTS:
`CGRP
`Intravenous
`infusion of
`CGRP (
`I nmol kg -
`' ) 10 min before occlusion until
`h
`the end of reperfusion reduced
`infarct size by 89 % ±
`5 %
`The reduction in infarct size was accompanied by
`a decrease in circulating levels of creatine phosphokinase.
`Infusion of the same dose of CGRP commencing from the
`start of reperfusiort until its end induced a 40 % ± 3 %
`reduction of the infarct size. The cardioprotective effects
`of CGRP were blocked by the novel CGRP antagonist
`BIBN4OEXXBS (20 nmol kg- t h- 1) . Although cardiac
`ischemia resulted in an almost 54) % increase in plasma
`CGRP levels in blood sampled from right cardiac ventri-
`intravenous
`cle ,
`infusion of
`the CGRP antagonist
`BIBN4 96BS before occlusion until the end of reperfusion
`had no statistically significant effect on the infarct size.
`CONCLUSION; The present study demonstrates
`that
`
`Now in Division ev`. Neonatology, Mount Sinai ,Medical Center,
`Miami Beach. Florida, :131411, USA
`Correspondence to Henri N D00DS. Ph D.
`Fax '19-7351-512171 .
`Plrn 49-7351 -1424)20 .
`E -mail henni . doedsC be . boehrin
`r- ingelheitn. corn
`Received 2000 -09-21
`Accepted 2001 -03 -23
`
`t I
`
`,
`
`f
`
`Calcìtonin gene - related peptide
`( CGRP)
`is a 37
`amino acid peptide that is predominantly synthesized and
`stored in sensory neurons.
`It can be released from both
`the central and peripheral axons of these neutims. i
`CGRP containing nerve
`fibers have been
`identified
`throughout the cardiovascular system, in association with
`blood vessels .
`in particular
`the coronary arteries . and
`around
`the sinoatrial and atdoventdeular nodes'" i l
`CGRP is a potent vasodilator peptide and it exerts positive
`isotropic effects
`chronotrrl'pic and
`rats and hu-
`in
`mans(' -'1.
`It has been shown to exert extremely potent
`vasodilator activity in isolated bovine. porcine. and hu-
`man coronary arteries' " - r "' .
`In addition. CGRP appears
`to be a more potent vasodilator in
`the small- diameter
`coronaries when compared with the large- diameter coro-
`naries t
`In patients with acute myocardial infarction.
`an almost two- fold increase of plasma CGRP level was
`observed within 24 h after hospital admission . This rise
`may result from the reflex release of CGRP in response to
`the reduction in myocardial perfusion' 12.1I" . CGRP has
`been tested in patients with chronic stable angina caused
`by coronary artery disease.
`It was shown to dilate coro-
`nary arteries at the site of atheromatous sienoses and to
`delay the onset of myocardial ischemia during exercise
`testing- 14
`We previously reponed on the first small molecule
`selective CGRP antagonist: BiBN- 104X)BS,
`; R -( R.
`- ail- 2 -[ [ 3- amino- I -
`( R `
`1 4 !. 1- pyridinyl 1- 1 -
`. S '
`)
`piperazinyl ]carbonyl , pentyl amino 1-1-1 t 3 ..)- dibromo -4-
`hydrox }phenyl) methyl ] -2- oxoethyl J --i- (
`. 4- dihydro- 3-
`( 2H )- quinazolinyl )- ,
`0x0-3
`l- piperidinecarboxamide
`which possesses high affinity and selectivity for human
`CGRP -receptors' l') . For the putative CGRP- J (cg, car-
`
`.
`
`.
`
`I
`
`Lilly Exhibit 1284
`Eli Lilly & Co. v. Teva
`Pharms. Int'I GMBH
`
`

`

`ISSN ()LLxi -97.5% Arta Phannawl Sin
`E -mail ups
`. ;H:. en
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`'l' 14 tai =f
`2IN11 Jul; 22 (7:,
``1Ei
`Phn:'Fat 86-21-6.174-'21)29
`
`diac tissue) and CGRP -2 receptors in rat tissue the affinity
`(p Kb) of BIEN 4096BS amounts to 8.5 and 7.1. re-
`In the present study . we investigated the
`spectihelyt Ir'1.
`effects CGRP and BEBN4096BS on myocardial ischemia
`provoked by left main coronary artery occlusion followed
`by reperfusion in anaesthetised rats.
`
`MATERI_AIS AND METHODS
`Male Wistar rats ( Chbb: Thom) , weighing 35() -
`380 g were used in this study. All experiments were
`performed according to institutional guidelines.
`Measurement of myocardial infact size One
`day fasted rats were anaesthetised with sodium pentobarbi-
`induction with 60 mg kg- I
`ip and then main -
`tone:
`tainence with an infusion of 30 mg kg -1 h -1 sc in the
`abdominal skin through a 23 G needle using a solution of
`I . The trachea was cannulated and the animals
`10 g L
`were artificially ventilated (80 strokes min -') with room
`The body temperature was
`air supplemented by oxygen .
`maintained at 37 t with a heating pad.
`The right
`femoral artery and left jugular vein were cannulated for
`the continuous measurement of arterial blood pressure and
`intravenous infusion of test agents ( or vehicle: saline) .
`from the blood
`Heart rate was derived
`respectively.
`pressure signal.
`A left sided thoracotomy was performed at the level
`A 5 -0 silk suture was
`of the fifth intercostal space.
`placed around the left anterior descending coronary artery
`approximately. 1 -2 min from its origin .
`Four- pieces of
`ligated along with
`the
`number 16 sewing cotton were
`The coronary
`coronary artery to facilitate reperfusion
`artery was occluded for a period of 60 min followed by
`60 min of reperfusion. Reperfusion was instituted by re-
`moving the ligature. Blood pressure and heart raie were
`measured continuously throughout the experiment. At
`the end of the reperfusion period, the heart was removed.
`Both atria and the roots of the great vessels were re-
`The entire ventricle was cut from the apex to
`moved .
`base into four transverse slices and incubated in 2,3 .5-
`triphenyltetrazolium chloride (I0 mg mL' I
`in phosphate
`
`buffer) for a period of 10 min at 37 t to visualize the
`
`ur.J
`
`. Each section was scanned by a colour
`infarct area` 17'
`image scanner ( Hewlett Packard ScanJet Iicx , Germany )
`and infarct size was determined using the Photoshop 4.0
`program.
`saline, CGRP
`agents
`treatment: Test
`Drug
`(1 nmol kg -' . h -1) , BEBN4096BS (20 nmol kg-1
`
`.
`
`I
`
`) .
`
`h -') + BlBN40968S
`h- ) , and CGRP (1 nmol kg- I
`(20 nmol - kg -' h -')
`were infused into the jugular
`- t
`vein at a rate of 3.3 mL kg- I - h
`In the first series
`the infusion of each com-
`of experiments ( protocol
`pound commenced 10 min before occlusion and was
`In the second
`maintained until the end of reperfusion.
`series of experiments ( protocol II ) , the infusion of each
`compound commenced from the start of reperf ision and
`was maintained until the end of reperfiision .
`The doses
`were selected on the basis of prior testing: infusion of
`CGRP ( 1 nmol kg- I h- I . for 2 h ) ,
`induced a 25 Cí.
`decrease in blood pressure from 10 min after starting of
`in anesthetized rats.
`infusion until the end of infusion
`h - I ) and BIBN4090BS (20
`When CGRP (1 nmol kg-
`h -' ) were infused together . B1BN4096BS
`nmol kg -
`reduced the effect of CGRP on blood pressure by 50 % .
`Measurement of creatine phosphokinase ac-
`In protocol l groups, femoral artery blood samples
`tivity
`were removed before infusion of the test agent, at min af-
`ter occlusion, and 50 min after reperfusion. The blood
`
`I
`
`samples were promptly centrifuged at 14 (MX) x g , 4 t .
`
`for 15 min, and the plasma thus obtained wa.N stored at
`-80 `C until assay. Creatine phosphokinase (CPK) lev-
`els were determined by means of a CPK kit.
`Measurement of CGRP levels Animals were di-
`ht group 1, blood samples were
`vided into three groups .
`taken from the right ventricle of the heart after anaesthesia.
`III, animals were subjected to 4) min
`In groups 11 and
`tyt) min Of
`by
`followed
`artery occlusion
`coronary
`infusions of Saline and CGRP
`and
`the
`reperfusion ,
`I) were commenced 10 min before (Y.:-
`(I limo! kg" I h
`elusion and were maintained until the end of reperfusion,
`Femoral artery blood samples were removed
`respectively .
`before occlusion and 50 min after reperfusion from group v
`animals. Blood samples from the right ventricle of the
`heart were removed 50 min after reperfusion frotn both
`group II and group m animals. The blood samples were
`promptly centrifuged at 14 000 x g ,
`
`1. t, far 15 min, and
`the plasma was stored at -80 t until assay.
`Peptide extraction : Plasma samples were mixed with
`a double volume of 0.1 % trifluoroacetic acid (TFA) (v/
`V) and centrifuged at 14 000x g , 4 ` , for 15 min . The
`
`supernatants were pooled and applied on preprimed C18
`Sep -Pak cartridges ( Waters Corporation, Massachusetts,
`USA) . The Sep -Pak cartridges were washed with 20 mL
`, and then din-
`0.1 % TEA at a flow rate of 3 mL min - r
`ed with 3 mL of 60 % acetonitril (v /v) containing 0. I %
`TFA (v /v) at a flow rate of 2 mL min -I. The eluates
`were freeze dried, and stored at
`
`-810 t until radioim-
`
`

`

`590
`
`ISSN rY':>:i-<t?Sr, Acta Pliann:to:l Sin
`E-mail lip; @, rnail dx-ne .<x'. CIr
`
`21NII Jul: 22 (
`1'14
`Ptut 'fax ft,-21 4, I7 1-:'J.i.x9
`
`and then adjusted to pH o..) - 7 M by NaOH
`l moi. L.
`Solutions were diluted to final concentrations with saline.
`
`RESULTS
`
`munoassay was performed. Recovery was determined by
`addition of I2' I -CCGRP to plasma prior to extraction, and
`was (75.4±1.1) % (n =8).
`Radioimmunoassay; A competiuve radioimmunoas-
`say was used to specifically measure rat ( r) CGRP con-
`centrations in plasma extracts. The procedure was ap-
`plied according to the instructions of the rCGRP RIA kit.
`Plasma extracts were assayed in duplicate using an anti-
`label ('= "' 1 -rCGRP )
`The
`body raised against rCGRP.
`was added after the samples had been incubated with the
`antibody ( rabbit anti- tCGRP) for 1 d.
`Subsequent to
`another I d incubation, the antibody bound 12''1 -rCGRP
`was separated using goat anti- rabbit antibody, and the ra-
`dioactivity counted on a gamma counter
`( Canberra -
`Rat CGRP was used as
`Packard GmbH , Germany ) .
`standard.
`Statistical analysis Myocardial infarct size was
`expressed as a percentage of the surface of the transection
`of the ventricles. All results are expressed as x ± .c. ,
`n = 5 - O. Comparisons were carried out by means of
`analysis of variance ( ANOVA )
`followed by Dunnett' s
`P < 0.05 were considered to be significant.
`test .
`Drugs used The following drags and analytical
`tools were used; h -aCGRP was purchased from Polypep-
`tide, Wolfenbüttel, Germany;
`the CCGRP RIA kit was
`purchased from DRG Instruments GmbH , Marburg. Ger-
`many; the CPK kit was purchased from Sigma, Stein -
`synthesized by
`heim, Germany ; BIBN4096BS was
`Boehringer Ingelheint Pharma KG, Biberach an der Riss.
`Germany. BIBN40%BS was dissolved in a small vol-
`) HCl 1 inol!L, further diluted with saline.
`ume (20
`
`changes during
`coronary
`Hemodynamic
`artery occlusion and reperfusion in control and in
`drug treated rats As shown in Tab 1, Lxclusitm of
`in a reduction in
`the main left coronary artery resulted
`mean blood pressure and heart rate. ami remained un-
`changed throughout the experiment .
`Intravenous infusion
`1 nmol kg-l h-
`) before occlusion resulted
`of CGRP (
`significant decrease
`blood pressure
`in moan
`in
`a
`Infusion of
`to 82 ± T.
`from 110 ± 8
`( mini -1g ) ,
`h -I) and CGRP (I nmol
`BEBN40(X)BS (20 nmol kg- I
`h `' ) did
`) + B1BN4096BS (20 nmol kg- I
`kg-
`- h
`not induce significant changes in mean blood pressure.
`there was no significant difference between
`However,
`treated groups and control group after coronary artery
`occlusion and reperfusion.
`Immunoreactive CGRP levels We observed a
`small but significant increase in plasma CORP leve! in the
`blood sampled from the right ventricle of the heart SO min
`, compared to the basal level
`after reperfusion in group II
`The levels were ( 1;3;3 ± I -1 )
`of control animals (Fig 1) .
`ng L-
`t. 8,1 ± 12 ) rig
`in ischemic hearts compared to
`in the controls ( P < 0.0S). We found no increase
`L- I
`In CGRP
`in the systemic blood ( data not shown
`the CGRP level was ( 798 ± 103)
`treated animals ( III ) ,
`ng L
`in the blood sampled from the right ventricle of
`the heart 50 min after reperfusion.
`
`)
`
`.
`
`I
`
`Tab 1.
`
`Ilemodyrkunics in control and drug treatment groups.
`
`n =6. x t sx .
`
`by <0.05 vs the control group.
`
`MBP (mmHg)
`Control
`CGRP (Pre -Isch)
`BIBN41)% ( Pre -Isch )
`CGRP+ BIBN4O% (Pre - ls h)
`CGRP ( Pre -Rep)
`BIBh1O% Pre -Rep)
`
`HR (bpm)
`Control
`CGRP ( Pre-Inch)
`131 B l 4íO9(
`( Pre - I sch )
`CGRP + BIBMO96 (Pre- Inch)
`CGRP ( Pre-Rep)
`BBN4(7 IO ( Pre-Rep)
`
`Basel i ne
`
`117±9
`11U±ä
`116 ±R
`1'22 ±9
`112±8
`Ill ±8
`
`427 ± 22
`418 ±23
`413 ±24
`425 ±2,b
`422 1: 20
`447 ± 21
`
`After .5 min
`drug treatment
`
`Occlusion
`3(I min
`
`llfi±9
`ti2t7h
`117±9
`11(ftR
`112±R
`112±R
`
`42f,f22
`:t;36±2:4
`41(t±25
`129 t 2't
`4'2.2±21
`418 ± 23
`
`(,.5±5
`(li ± r)
`07
`
`± 4
`l£3 ± (
`(f3 ± i
`
`:;1'?f I)3
`35.5 ± 17
`313 ±'}tt
`.328 # 22
`341 ±
`321 ± 19
`
`Eleperl usi<,n
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`1h
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`tr1±
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`tYt ± :>
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`:318 ± 22
`307 ± 18
`:338 ±211
`:3111± 20
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`2h
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`(t: s ri
`7:í
`t>ii±
`r,1±:,
`;1I±.i
`t>t1 ±.i
`
`31 ± toi
`;i.î(I± 17
`320 ± 211
`312 ± loi
`344 _± )0
`:il I ± 19
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`

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`Fig I . CGRP levels in the blood samples taken from
`the right ventricle of the rat heart. The influence of is.
`chemin- nperftrsioa and CGRP treatment of the animals
`n z 6. x r s r .
`< 0.05 vs saline control .
`is shown.
`
`The release of creatine phosphokinase Tab 2
`illustrates the difference in CPK activity in each group.
`Plasma c teatime phosphokinase levels w,cte significantly
`elevated 50 min after occlusion of the coronary :rite.
`This increase was further enhanced 50 min after the start
`I mild kg' I
`of reperfusiom. nealmcnt witá CGRP (
`) reduced the increase during the reperfusion period
`h-
`( P < 0.051. Treatment with B18=190S ( 20 nmol
`) did not influence plasma creatine pho phoki-
`kg -111-
`t» activity compandl to saline
`treatment controls.
`However, RIR141096BS significantly attenuated the effect
`of CGRP on CPK activity ( P <0.03) .
`Myocardial infarct size Oucdusinn of the tell
`anterior tksctcnding coronary artery for a period or ti) min
`folkaml by ti) min of teperfwm rc.u1Isd in Salistanaial
`In a group of con. onirt rats oc
`injury to the myn ardium .
`elusion and reperfucion produced vn infarct amounting to
`(33± 4 I 'tr of the area cf ventricles (fig 2) .
`Fig 3 stows the infiuvi sire expressed as a percent.
`
`Control
`
`CGRP 0
`
`Fig 2. Scanning graphs of the infarct area ; wufuce of
`t'och slice) .
`
`a
`
`( I 111111
`
`I
`
`Cf,RP
`
`s. 409611S,
`
`Fist 3. The effects of CGRP (1 nnal kg"' h' ` ),
`BIBN4496BS (20 nmof. kg -' h 11, and CGRP f 1 om&i
`kg"' h'') 81BN4096BS 120 rrmoi kg- ' h'') on in
`farte sire, expressed as a perventae of the surface of
`both ventricles. n = 6. x t s,.
`' P < 0.03 ac with
`saline cannot. "P<0.05 ras the group receiving CGRP
`alone.
`
`age of both ventricles after treatment with saline and let
`agents, respectively. The infusion was started 10 min
`before ligation of (he cxmotlauy artery and continued until
`
`13131ü114O9611S (20 mnol kg- I h'') , and CGRP (I nmol kg'' h "') #
`'Me effects of CGRP (1 nmol kg'' h '
`Tlrh 2.
`1318N40968S (20 nnml kg - ' h-1 I on creatine phosphokinase levels (U nÍL "' ) in the blood of anaesthetized mis. The
`left descending coronary urtery was occluded for a 60-min period fallmved by 60 min of mperfuskin and femoral artery
`blood samples were taken S min before compound infus locus, SO min after occlusion. and 50 min after reprfusion, re
`spectively. n @ S - 6. x t sx . kr < 0.05 Vs saline control group. "P <0 .05 as the CGRP treatment group. SP <0 .05
`us the base level ( before oecirsion) .
`
`flrfir;
`occlusion
`at min AM +xt Iuµn
`30 mn after rrprrfasion
`
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`
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`

`- 59?
`
`ISSN (Y2.:- I.075t, Acta l'ha moot Sin
`E -mail apsCd mail. shcnc
`. en
`
`1'14
`Phn Fax
`
`sk
`20(11 Jul : 22 (
`214,174-2r,29
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`,
`
`'
`
`the end of reperfusion . Treatment with CGRP (1 nmol
`infarct size by 89 % ± 5 9
`kg -
`h 1) reduced the
`The reduction
`infarct size by CGRP was coun-
`in
`teracted by BIBN4 %BS
`However,
`treatment with
`- 1
`BIBN4O%BS alone (20 nmol kg-1 h
`) had no statisti-
`cally significant effect on myocardial infarct size.
`Fig 4 shows
`the infarct size afler treatment with
`saline, CGRP ( l nmol kg -1 h- I) , and BIBN409613S
`(20 nmol kg- I
`h- 1
`respectively , when the com-
`) ,
`pounds were infused at the start of the reperfusion and
`maintained until the end of reperfusion. CGRP reduced
`the infarct size by (44) ± 3 )
`'o . BIBN4O%BS showed
`no influence on
`infarct size compared
`the
`to control
`group.
`
`40
`
`35 "
`30 '
`
`25
`
`20
`
`15
`
`10'
`
`5"
`
`Con froi
`
`CGRP
`
`i3It3N40968S
`
`Fig 4. The effects of infusion of CGRP (1 nrnol kg -1
`) and BIBN4096BS (20 snot kg -1 - h -1 ) from the
`h- r
`start of reperfusion and maintained until the end of
`reperfusion on infarct size, expressed as a percentage
`of the surface of both ventricles. n =6. x t sx .
`°p <
`0.05 vs with saline control.
`
`DISCUSSION
`
`Early reperfusion of an occluded coronary artery is a
`well known and effective means of reducing ischemia-
`induced myocardial damage( 1`' -22.1. However, reperfu-
`sion has been shown to cause significant canliac injury by
`itself. 2:1.' 4 .
`In the present study, occlusion of the left
`anterior descending coronary artery for a period of 60 min
`followed by 60 min of reperfusion resulted in substantial
`In a group of control rats,
`injury to the myocardium.
`occlusion and reperfusion produced an infarct of (33
`4) % of
`In accordance with
`the ventricle.
`litera-
`Lure ''`' , a progressive rise in the CPK release during
`reperfusion. resulting from myocardial reperfusion injury,
`was observed.
`In order to investigate the cardioprntective effect of
`
`)
`
`CGRP in anaesthetized rats subjected to coronary artery
`occlusion followed by reperfusion,
`two different experi-
`mental protocols were used. When the
`infusion was
`started 10 min before ligation of the coronary artery until
`the end of reperfusion. CGRP (1 nmol kg -
`h -1 ) re-
`duced the infarct size by (89 ± S) % compared to con-
`trols. The sane dose of CGRP reduced the infarct size
`by (40 ± :3) % while the infusion commenced from the
`start of reperfusion until the end of reperfusion. CGRP
`did not alter CPK levels during ischemia but there was a
`marked decrease
`release during
`reperfusion,
`in CPK
`These findings indicate that posi- ischemia tissue damage
`induced by reperfusion can be markedly attenuated by
`CGRP. CGRP appears to maximize the myocardial sal-
`vage achieved by reperfusion and may hence preserve
`ventricular function.
`In both protocols,
`infusion of
`I h- I
`, a novel CGRP an-
`BIBN4O96BS (20 nmol kg -
`tagonist, did not signigicantly influence myocardial infarct
`size and CPK activity by itself. However, BIBN40%BS
`h-
`(20 nmol kg- t
`significantly antagonized the
`effects of CGRP on cardioprotection as well as on CPK
`activity
`La patients with congestive heart failure or myocar-
`dial infarction , an almost two told increase of endogenous
`levels of CGRP level has been observedd 12'13' .
`it seems
`likely that circulating CGRP acts primarily as a vasodila-
`tor and it may thus play a role in the regulation of sys-
`flow.
`temic blood pressure and regional organ blood
`Since CGRP is stored in the peripheral terminal of sensory
`nerves, it can he speculated that when the tissue is sub-
`ischemia or during the excitation of sensory
`jected to
`nerves, CGRP might be released by lei axon reflex mech-
`anism,
`increasing
`local blood
`flow.
`CGRP -
`thus
`immunoreactive nerves are abundantly present around the
`coronary artery, and CGRP exerts extremely potent coro-
`the regulation of
`nary vasodilator activity. Therefore,
`coronary blood flow by endogenous CGRP can be imag-
`ined. We observed an almost 50 % increase in plasma
`in blood from the right ventricle of the
`CGRP levels
`hearts 50 min after reperfusion compared to the basal lev-
`in systemic
`el . However. no increase was observed
`blood samples. BIBN4O%BS did not significantly in-
`crease in infarct size which is somewhat in contrast to a
`, showing that capsaicin pre-
`study by Kallner et (21 [37
`This could
`treatment augmented myocardial infarction .
`that receptor antagonism
`be explained by the fact
`is a
`more specific way to block CGRP mediated effects than
`depleting neumpeptides from sensory nerves through cap -
`saicin treatment. Alternatively, it can be imagined that
`
`

`

`ISSN 02M-1)756 Acta Phannard Sin
`E-mail aps@ Inaul. sl)cnr. ac. en
`
`2(101 Jul ; 22 (; t
`42 A
`Phni Fax M-21 -f474-2(;24
`
`iy.,
`
`-
`
`ischemia preconditioning ' .
`
`)
`
`,
`
`the release of endogenous CGRP in the experimental set -
`ing used is insufficient to produce effective tandioprotec-
`tion. Only high plasma levels CGRP may cause. cardio-
`treated with CGRP
`protection.
`that were
`the rats
`In
`the CGRP levels were ahout 10
`( i nrnol kg -
`h -
`fold higher than the basal level of the control group when
`blood was sampled from the right ventricle.
`that CGRP
`it has recently been shown
`Although
`the
`involved
`might be
`in
`exact mechanism and role for CGRP release under patho-
`physiological condition is not yet fully understood.
`It
`has been proposed that CORP is not released during the
`acute phase of myocardial infarction or anginal pain
`in
`human but could he considered as an inflammatory reac-
`tion rather than a response to ischemiat2 ") .
`the present investigation demonstrates
`Nevertheless,
`a cardioprotective effect of CGRP in anaesthetized rats
`which were subjected to coronary artery occlusion fol-
`lowed by reperfusion . Accordingly, these results support
`the hypothesis that CGRP is a very potent myocardial pro-
`tective substance. However. the endogenous release of
`CGRP under experimental ischemia conditions used in this
`study was not sufficient to produce effective cardioprotec-
`lion.
`
`We
`I3ritty
`ACKNOWLEDGEMENTS
`thank Mrs
`MISSLER for technical help in conducting the radioim-
`mu noassay .
`
`REFERENCES
`
`1
`
`Brain SD. Cambridge H. Calcitonin gene- related peptide;
`vasoaetive effects and potential therapeutic mle. Gen Phanna-
`col 1996: 27: rß07 - 11.
`2 Muldeny PK, Ghatei MA. Radrigo J, Allen JM. Rosenfeld
`c,r c1f. Calcitonin gene -related peptide in
`MG. Polak TM,
`1 t:
`o I' the rat. Neuroscience
`l0B5:
`cardiovascular tissues
`41.? -M.
`3 Saito A. Kimura S, Gott) K. Calcitonin gene related peptide
`as potential neurotransmitter in guinea pig right atrium. Am J
`Physiol lcllth; 150: H693 - H698.
`4 Brain SD. Williruns TJ. Tippins RJ, Morris HR, Maclntyre I.
`Calcitonin gene -related peptide is a potent vasodilator. Nature
`19/15; 31:1 ::41 - 6.
`Calciionin gene - related peptide and
`h-4nc(-Cen ceda A.
`human eñicardial coronary arteries: presence,
`release and
`vasodilator effects.. Br J Pharmaeol 1991; 102: 5Ot - 111.
`fi Ohmura T. Nislnio M. Kigoshi S, Muraniatsu 1.
`Electro-
`physiological and mechanical -effects of ca3citonisn gene -related
`peptide on guinea -pig atria. Br J Pharmacol 1990; 1(X); 27 -
`
`5
`
`3t1.
`
`9
`
`{1
`
`1
`
`l
`
`7 Gennari C. Fischer JA. Cardiovascular action of calcitonin
`gene - related peptide in humans. Calcif Tissue Im 1935; :17:
`581 -4.
`8 Greenberg B. Rhoden K, Barnes P. Calcitonin gene -related
`peptide ( CORP) k a potent non-endothelium-dependent
`in-
`hibitor of coronary va.otmotor tone. Br J Phamrtcol 1987;
`92: 789-'M.
`ishihara H. Ishikawa T, Saito A. Gate K . Va-
`Shoji T,
`sodilatoxy effects of human and rat calcitonin gene -related pep-
`tides in isolated Ravine coronary artery. Naunyn Schntícde-
`bergs Arch P7ulnnaccrl 108.7; 33if: 438-44.
`Potent elects of neu
`Franco-Cereceda A, Lundberg A1.
`rupeptide Y and calcitonin gene-related peptide on human c(iro-
`in vitro. . Ac to Physiol Scand I987 ;
`nary vascular tone
`I :31
`159 -6Cl.
`Foulkes R. Shaw N. Bose C. Hughes B.
`Differential
`vasodilator profile of calcikmin gene - related peptide in porcine
`large and small diameter coronary artery rings. Eur J l'h;urna-
`col 1991; 2(11; 14.3 -o).
`12 Mair J. Lechleimer P. Laengle T. Wiedermann C. I)ienstl F,
`infarction.
`in acute myocardial
`Plasma CGRP
`Saria A.
`Lancet 1990; 335; I08.
`13 Dzau VJ. Contribution of neuroerxlccrine and [neat atmm rive
`puractine mechanisms to the pathopttysiolagy and pharmacology
`of congestive heart failure. Am .1 Cardiol 1988; (>8: 76L -
`81E.
`14 Uren AG. Seydoux C. Davies GJ. Effect of intravenous cal -
`cilonin gene - related peptide on ischaetnia threshold and coro-
`nary stetnxsis severity in humans. Cardiovasc Res 1992; 27:
`1477- 81 .
`15 Doods H, Hallermayer G. Wu D. Entzeroth M. Rudolf K.
`Pharmacological profile of BiBN4(96BS.
`Engel W. et crl.
`the first selective small molecule CGRP antagonist. Br
`J
`42(1 -3.
`Pharmacol2(Xxl; 12
`l6 Wu D. Eberlein W, Rudolf Klaus. Engel W. Hallertnayer U,
`Dcxxls H. Characterisation of cakitonin gene - related peptide
`receptors in rat atrium and vas deferenst evidence for at
`( Cys
`(Er 2.7 )hCGRP-prcfcrring receptor. Eur J Ph:trmacol 20110;
`400..:ít3 -n.
`17 Petty MA. Dow J. Grisar Al. Jong WDE. Effect of a car -
`dioselective a- tocopheral analogue on repertusion Injury in rats
`J Phannacol 1901;
`induced by myocardial ischaemia. Eur
`102; 383 - 8.
`18 Hayashi N. Fujimurd Y, Yamauncrto S. Kornctani M. Nakao
`Pharmacological profile of vaLsartan, a non - peptide art -
`K.
`giotensin II type 1 rewpt(tr antagonist. Arzneimittellimchung
`1997: 47; (i'2.î -9.
`` wavefnrnt phe nornenon * of
`lo Reimer KA. Jennings RB. The
`iI. T- ansmural progression
`myocardial ischaemic cell death.
`of necrosis within the framework of ischacrnic tied size (trty-
`Lab Invest 1979: 11):
`oc;miium at risk ) and collateral flow.
`(1i :3
`20 Reimer KA, Lowe JE. Rasmussen MM, Jennings RB. The
`wavetront phenonrtenon of ischaemic cell death,
`I. Myocar-
`dial infarct size vs duration of coronary occlusion in dogs.
`Circulation 1977; 56: 786-44.
`
`

`

`544
`
`ISSN (7Z33-475g Acta Phanmwaol Sin
`E -mail aps@ m,il. shnc, ar c rn
`
`'' #k
`'l(X)I Jul; 22 ,. ?1
``I' Gil Pi
`Phn/Faa tV) 21 4,474-2029
`
`29 WVahrborg P. Ehasson T. Edvardsson N, Erman R.
`Mannheimer C, Nedner T. Acute ischetnic chest pain is not
`associated with increased calciton gene -related peptide (CGRP)
`the coronary circulation.
`levels in peripheral plasma nor
`in
`Scand Cardiovasc 1 I999; :i3: 295 -9.
`
`fitf %.*Nffi 411X tk#1 BIBN4096BS v-gm*pg,rl.tAli
`fikIIII ll iVf)
`
`Dong-Mei V1rì.lt , Pieter A van Z1+4'lETEN2 , Henri N
`
`DOODS{
`( Cardiovascular Research . Brehrrnger 1n,s;elheirn Phar-
`ma KG , Birkendorfer Str b5 , D-88397 Biberach, Ger-
`"'Department of Phannacrtherapy , Academic
`mcrrty;
`Medical Centre , University of Amsterdam , Aleiber,Kdrtcef
`1:5 , 1105 AZ Amsterdam , The Netherlands)
`
`9titi4 4 t i A 4
`
`; ßIBN4096BS;
`
`(*1-1YA 44
`
`g
`
`)
`
`97
`
`28
`
`22
`
`24
`
`25
`
`21 Kloner RA, Ellis SG. Lange R, I3raunwald F.
`Studies of
`experimental coronary artery reperfusion. Effects on infarct
`size, myocardial function. biochemistry, ultrastructure and mi-
`crovascular damage. Circulation 1983; 68 (Suppl I. 8-
`15.
`Dewood MA. Spores J, Hensley GR . Simpson CS . Eugster
`GS, Sutherland KJ , es al . Coronary arteriographic findings
`in acute transmural myocardical infarction. Circulation 1483;
`683; 39-49.
`Li Brawiwald E, Kloner RA. Myocardial rrperfusion: A dcw-
`J Clin Invest 198.5; 76: 1713 -9.
`Ne- edged sword.
`Flaherty JT, Weisfèldt ML. Reperfitsion injury.
`Free Rad
`Biol Med 1488; 3: 409-19.
`Shell WE Kjekshus JK, Sobel BE. Quantitative assessment
`of the extent of myocardial infarction in the conscious dog by
`means of analysis of serial changes in serum marine phospho-
`J Clin Invest 1971; 50: 2(]4 - 3.
`kinase activity.
`2(, Blumenthal NM, Wang HH. Liu iMP. Experimental coro-
`Effects on enzymes, elec-
`nary artery occlusion and release.
`trocardiograms, myocardial contractility and reactive hyper-
`emia. Am J Car-diol 1975; 36; 225-3.3.
`Kallner G. Gown A. Franco- Cereceda A. Calcitonin gene -
`related peptide in myocardial ischemia and reperfusion in the
`pig. Cardiovasc Res 1998; 38: 4931 -9.
`Evidence for calcitonin gene -
`Lu R, Li YJ , Deng FEW.
`related peptide - mediated ischemia- preconditioning
`the
`rat
`in
`Regul Pept 199); kV: 53 - 7.
`heart .
`
`