ELSEVIER
`
`Regulatory Peptides 79 (1999) 153-158
`
`REGULATORY
`
`PEPTIDES
`
`CGRP 27-37 analogues with high affinity to the CGRP1 receptor show
`antagonistic properties in a rat blood flow assay
`
`Beate Rista, J. Silvain Lacroixb, Michael Entzeroth\ Henry N. Doodsc,
`Annette G. Beck-Sickingera·*
`
`'Department of Pharmacy, ETH Zurich, Willterthurer Str. 190, CH 8057 Zurich, Switzerland
`•Department of Otorhinolaryngology, Hopital Cantonal Universitaire de Geneve, Geneva, Switzerland
`cDepartment of Biochemical Research, Dr. Karl Thomae GmbH, Biberach, Germany
`
`Received 5 May 1998; received in revised form 6 November 1998; a_ccepted 6 November 1998
`
`Abstract
`
`CGRP Y0 -28-37 is known as a selective CGRP1 receptor antagonist. We succeeded in optimising the CGRP1 receptor affinity of this
`fragment by multiple amino acid replacement. The analogues [P34
`, F 3 5)CGRP 27-37 and [D 31
`, P 34
`, F 35)CGRP 27-37 exhibit a 100-fold
`increased affinity compared to the unmodified segment. Receptor binding studies were performed with human neuroblastoma cells
`SK-N-MC, which selectively express the hCGRP, receptor. Blood flow, which is increased by exogenous CGRP, was measured in the
`right femoral artery. Preincubation of the rats with [P34
`, F 35]CGRP 27-37 and [D 31
`, P 3 4
`, F 35]CGRP 27-37 led to a significant decrease in
`CGRP induced increase in vascular conductance indicating the antagonistic properties of these compounds. Interestingly, an exchange of
`the amino acid Asn 31 to Asp 31 in [P3 4
`, F 35]CGRP 27- 37 shortened the period of the antagonistic effect significantly, suggestive of a
`different rate of metabolism for the two ligands. Secondary structure investigations obtained by circular dichroism measurements revealed
`that an increase in ordered structure correlates with high binding affinity. © 1999 Elsevier Science B:V. All rights reserved.
`
`Keywords: CGRP, receptor antagonist; Neuroblastoma cells; Vascular conductance; Circular dichroism; Synthetic peptides; Peptide
`mime tics
`
`1. Introduction
`
`Calcitonin gene-related peptide (CGRP) is a 37-amino
`acid peptide generated by alternative tissue-specific splic(cid:173)
`ing of the primary transcript of the calcitonin [1,2]. In
`contrast to calcitonin which is predominantly expressed in
`the C-cells of the thyroid, two forms (a. and (3) of CGRP
`are produced in a variety of human tissues which are
`mostly of neuronal origin [3,4]. In fact, CGRP is co(cid:173)
`localised with substance P in sensory nerves, with acetyl(cid:173)
`choline in motorneurones, and with various other transmit(cid:173)
`ters in the brain (for review see Ref. [5]).
`A variety of biological effects have been reported for
`
`*corresponding author. Tel.: + 41-1-635-6063; fax: + 41-1-635-6884;
`e-mail: beck-sickinger@pharma.ethz.ch
`
`CGRP such as peripheral blood vessel dilatation, an
`increase in heart rate, regulation of calcium metabolism
`and insulin secretion, reduction of gastric acid secretion,
`increase in body temperature, and decrease in food uptake
`[5]. These effects have been suggested to be mediated by
`at least two receptor subtypes (CGRP1 and CGRP2 ) [6,7]
`which are both G-protein coupled. The hCGRP1 receptor
`has been cloned recently [8].
`Structure-activity studies of reduced size CGRP ana(cid:173)
`logues showed that the C- and N-terrninal region of the
`hormone interact independently with their receptors. Since
`we were interested in antagonistic ligands at the hCGRP1
`receptor, we focused on the optimisation of the structurally
`poorly defined C-termina'l part of the hormone. The
`segment CGRP Y0-28- 37 which is reported as a selective
`CGRP1 receptor antagonist [9,10], however with low
`
`0167-0115/99/$- see front matter © 1999 Elsevier Science B:V. All rights reserved.
`PII : SOI67-0115(98)00159-l
`
`1
`
`EX2008
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01427
`
`

`

`154
`
`B. Rist et al. I Regulatory Peptides 79 (1999) 153-158
`
`affinity. No activity has been found up to the micromolar
`concentration [11] which is in agreement with our results.
`In order to improve binding affinity, multiple amino acid
`replacement within this segment has been performed [12]
`and resulted in a series of high affinity ligands, including
`, F 35]CGRP 27-37 and [D 31
`, P 34
`, F 35]CGRP 27-37.
`[P 34
`Within the present study we could confirm the suggested
`antagonistic properties of C-terminal analogues of CGRP
`Y0-28-37 using a rat in vivo assay. Furthermore, we were
`able to identify a portion of the peptide sequence which
`appears to be sensitive to proteolytic cleavage but does not
`affect receptor binding affinity.
`Circular dichroism studies have been performed in order
`to elucidate the difference in secondary structure between
`peptidic ligands exhibiting poor and high receptor binding
`affinities, respectively.
`
`2. Methods
`
`2.1. Peptide synthesis
`
`The linear peptides were synthesised by automated
`multiple solid phase peptide synthesis using a robot system
`(Syro, MultiSynTech, Bochum). In order to obtain a
`peptide amide, 4-(2' ,4' -dimethoxyphenyl-Fmoc-amino(cid:173)
`methyl)-phenoxy resin was used. The polymer matrix was
`polystyrene-! %-divinylbenzene (30 mg; 15 1-1mol). The
`side chain protection was chosen as follows: Tyr(tert(cid:173)
`butyl), Glu(tert-butyl), His(trityl), Gln(trityl), Asn(trityl),
`Thr(tert-butyl),
`and Lys(tert-butyloxy-carbonyl), Ser(cid:173)
`(trityl). Double coupling procedures were performed with
`diisopropylcarbodiimide /1-hydroxy-benzotriazole activa(cid:173)
`tion, sevenfold excess and a coupling time of 40 min.
`The linear peptide amides were cleaved with trifiuoro(cid:173)
`acetic acid/thioanisole/thiocresol within 3 h and precipi(cid:173)
`tated from cold diethyl ether. The products were collected
`by centrifugation and resuspended twice in diethyl ether.
`Finally they were lyophilised from water/tert-butyl alcohol
`(1:2).
`The peptides were purified to homogeneity higher than
`94% by reversed-phase HPLC on a nucleosil C-18 column,
`6 j.Lm, 25 X 300 mm (Waters), using acetonitrile/water
`mixed with 0.1% trifiuoroacetic. acid as eluent. !socratic
`conditions between 20-30% acetonitrile were applied,
`depending on the polarity of the peptide. Analysis of the
`peptides was performed on a nucleosil C-18 column, 5
`j.Lm, 3 X 125 mm (Merck-Hitachi) by a gradient of 15-
`60% acetonitrile over 30 min.
`Correct mass was identified by ion-spray mass spec(cid:173)
`trometry (API III, Sciex, Toronto).
`
`2.2. Circular dichroism
`
`Circular dichroism spectra were measured over 250-190
`nm on a Jasco 1720 spectrapolarimeter at room tempera-
`
`ture in nitrogen atmosphere. The peptides were dissolved
`in 2,2,2-trifiuoroethanol/phosphate buffer (pH= 7, 10
`mM) in the ratio 317, at concentrations of 0.20-0.22 mM.
`All measurements were performed three times using a
`sample cell with path length of 0.2 mm. Response time
`was set to 2 s at a scan speed of 20 nm/min, a sensitivity
`range of 10 mdeg, a band width of 2.0 nm and a step
`resolution of 0.1 nm. High-frequency noise was reduced by
`means of a low-path Fourier-transform filter. The circular
`dichroism spectra of pure solvents were subtracted from
`the peptide solution to eliminate interference from cell,
`solvent and optical equipment. The circular dichroism data
`are given as mean residue ellipticities [ e] m.
`
`2.3. Membrane preparation
`
`SK-N-MC cells were cultivated in Dulbecco's modified
`Eagle medium. The medium of confluent culture was
`removed and the cells· were washed twice with PBS-buffer.
`The cells were detached by the addition PBS-buffer,
`supplemented with 0.02% EDTA. Resuspended in 20 ml
`Balanced Salts Solution (BSS (in mM): NaCI 120, KCl
`5.4, NaHC0 3 16.2, MgS0 4 0.8, NaH2 P0 4 1.0, CaC1 2 1.8,
`o-glucose 5.5, HEPES 30, pH 7.40) the cells were cen(cid:173)
`trifuged twice at 100 X g and resuspended in BSS. After
`determination of their number, the cells are homogenised
`using an Ultra-Turrax and centrifuged for 10 min at
`3000 X g (4°C). The supernatant was discarded and the
`pellet was resuspended in Tris buffer (10 mM Tris, 50 mM
`NaCI, 5 mM MgCl 2 , 1 mM EDTA, pH 7.40) sup(cid:173)
`plemented with 1% bovine serum albumin and 0.1%
`bacitracin, recentrifuged and resuspended (1 ml/1 000 000
`cells). The homogenate was frozen at - 80°C. The mem(cid:173)
`brane preparations are stable for more than 6 weeks.
`
`2.4. Receptor binding
`
`After thawing the homogenate was diluted 1:10 with
`assay buffer (50 mM Tris, 150 mM NaCI, 5 mM MgC1 2 , 1
`mM EDTA, pH 7.40) and treated for 30 s with an Ultra(cid:173)
`Turrax. Then, 230 1-11 of the homogenate were incubated
`for 180 min at
`room
`temperature with 50 pM
`125I]iodotyrosyl-calcitonin gene-related peptide and in(cid:173)
`[
`creasing concentrations of the test compound in a total
`volume of 250 1-11. The incubation is terminated by either
`centrifugation for 10 min at 2900 X g or filq-ation through
`polyethylene imine (0.1% )-treated GF /B glass fibre filters
`using a cell harvester. The protein-bound radioactivity is
`determined in a gamma-counter. The non-specific binding
`is defined as radioactivity bound in the presence of 1 1-1M
`human CGRP1 during the incubation period. Half maximal
`inhibition of the specific binding of [125l]iodohistidyl(cid:173)
`calcitonin gene-related peptide of three separate experi(cid:173)
`ments each performed in triplicate is given as the K1 value
`(Table 1) ±S.E.M. (Fig. 1). Data analysis was performed
`with GraphPad.
`
`2
`
`

`

`B. Rist et al. I Regulatory Peptides 79 (1999) 153-158
`
`155
`
`2.5. Blood flow
`
`Experiments were carried out in adult female Wistar rats
`weighing between 250 and 280 g anaesthetized with
`sodium pentobarbitone (Nembutal, Boehringer-Ingelheim;
`60 mg/kg, intraperitoneal). Intravenous supplements of
`pentobarbitone were given to maintain anaesthesia. The
`trachea was cannulated and connected to a positive pres(cid:173)
`sure rodent ventilator (Ugo Basile 6025). The left femoral
`artery was cannulated for continuous recording of arterial
`blood pressure via a Gould-Statham physiological pres(cid:173)
`sure transducer (P23XL) which was connected to one
`channel of a pen recorder (Graphtec WR7500). Tempera(cid:173)
`ture was continuously monitored via a rectal probe (Di(cid:173)
`gitron Model 1808) and kept in the range 36± 1 °C. The
`electrocardiogram was
`recorded
`through subcutaneous
`needle electrodes and displayed on a storage oscilloscope.
`The electrocardiogram was used to obtain beat-by-beat
`pulse interval (PI) after processing with Neurolog modules
`(Digitimer, UK, NL200,304,600). Triggering was checked
`with a counter. PI was preferred to heart rate because of
`the linear relation between PI and frequency of vagal
`stimulation. Both vagus nerves were cut high in the neck.
`This was done to eliminate vagally-mediated reflex effects
`on the heart which occur when blood pressure is decreased
`by CGRP. The left femoral vein was cannulated for
`administration of CGRP and analogues of CGRP, as well
`as further doses of anaesthetic. The right femoral artery
`was exposed and separated from the right femoral vein and
`saphenous nerve, and the perivascular portion of the flow
`probe was placed around the femoral artery. Blood flow
`was measured in the right femoral artery using a perivascu(cid:173)
`lar flow probe (Model 0.5VB 152 Transonic systems). The
`cavity created for the probe was filled with K-Y lubricating
`jelly (Johnson & Johnson) to provide acoustic coupling to
`the probe. The flowprobe was connected to an ultrasonic
`volume flowmeter (Model T106 Transonic Systems) which
`in turn was connected to a polygraph (Grass Model 79) for
`data analysis.
`Doses of CGRP (0.5 nmol!kg) were administered
`intravenously to obtain a control response. This dose was
`selected because it decreased the resting blood pressure by
`an average 40 mmHg. When controls were obtained a
`bolus injection of the CGRP analogue, 0.17 ~J.mol/kg or
`0.068 f.LmOl!kg was administered followed by a dose of
`CGRP within 10 min. Additional doses were given if the
`response to CGRP was attenuated. Experiments were
`performed in quadruplicate and data are shown±S.E.M.
`(Fig. 3). Data were analyzed using Student's t-test and
`P < 0.05 was found for significant values.
`
`2.6. Materials
`
`Fmoc-protected amino acids were obtained from Alexis
`(Uiufelfingen, Switzerland), 4-(2' ,4'-dimethoxyphenyl(cid:173)
`Fmoc-aminomethyl)-phenoxy resin from NovaBiochem
`
`(Uiufelfingen, Switzerland), diisopropylcarbodiilnide and
`thiocresol from Aldrich, 1-hydroxybenzotriazole, diiso(cid:173)
`propylethylamine, 2,2,2-trifl.uoroethanol,
`trifl.uoroacetic
`acid, thioanisole and piperidine from Fluka and dimethyl(cid:173)
`formamide (p.a. grade), diethyl ether, acetonitrile and tert(cid:173)
`butyl alcohol from Merck. HEPES, Tris, bovine serum
`albumin, bacitracin were purchased from Sigma, poly(cid:173)
`ethylenimine from Serva, Dulbecco's modified Eagle
`medium and PBS-buffer (041-04190 M) from Gibco.
`EDT A and all salts for buffer preparation were either from
`
`Fluka or from Merck. e25I]Iodohistidyl-calcitonin gene
`
`related peptide was ordered from Amersham.
`
`3. Results
`
`3.1. Receptor binding studies
`
`The peptides that are listed in Table 1 have been
`obtained by multiple automatic solid phase peptide syn(cid:173)
`thesis. They have been characterised by analytical HPLC
`and electrospray mass spectrometry. CGRP1 receptor af(cid:173)
`finity was investigated at human neuroblastoma cells SK(cid:173)
`N-MC (Fig. 1). The most potent peptidic compounds are
`derived from CGRP 27-37 and [D 31 ]CGRP 27-37 which
`themselves only have a minor potency (Ki 728 and 2390
`nM, respectively). The introduction of a proline in position
`34 (no. 2, 5) leads in both segments to a 6- to 10-fold
`increased affinity. Within these modified segments, the
`replacement of Glu in position 35 by Phe (no. 3, 6)
`furthermore improves binding by one order of magnitude,
`thus leading to analogues with Ki values of 19 and 14 nM.
`
`3.2. Circular dichroism investigations
`
`In Fig. 2a the circular dichroism spectra of the ligands
`with low affinity (no. 1, 4) are compared with those in
`which proline in position 34 was introduced (no. 2, 5). The
`secondary structure in solution does not seem to be
`signi:ficantly affected by the exchange of S 34 to P 34
`,
`because the circular dichroism spectra of CGRP 27-37 and
`[D 3 1]CORP 27-37 are very similar to
`the ones of
`[P3'lCGRP 27-37 and [D 3
`\ P 34]CGRP 27-37, respective-
`
`Table I
`Sequences of CORP 27-37 analogs and their affinity to the CORP,
`receptor, investigated on human neuroblastoma SK-N-MC cells
`
`No.
`
`Peptide
`
`Sequence
`
`hCORP,
`K; (nM)
`
`CGRP 27-37
`[P34]CGRP 27-37
`[P 34
`, F" ]CGRP 27-37
`[D"]CGRP 27-37
`, P34]CGRP 27- 37
`[D 31
`[D", P34
`, F"]CGRP 27-37
`
`FVPTNVGSEAF
`FVPTNVGPEAF
`FVPTNVGPFAF
`FVPTDVGSEAF
`FVPTDVGPEAF
`FVPTDVGPFAF
`
`728
`123
`19
`2390
`226
`14
`
`2
`3
`4
`5
`6
`
`3
`
`

`

`156
`
`B. Rist et al. I Regulatory Peptides 79 (1999) 153-158
`
`a
`
`120
`
`'0
`§ 100
`.8
`ll. 80
`a:
`~
`0
`N ...
`ID-
`"ifl.
`
`0
`-11
`
`-10
`
`b
`
`120
`
`'0
`§ 100
`.8 eo
`ll. a:
`~
`0
`N ...
`ID-
`40
`"ifl. 20
`
`60
`
`-6
`-5
`-7
`-8
`-9
`Competitor log [M]
`
`-4
`
`-3
`
`0
`-12
`
`-11
`
`.g
`-6
`-10
`-7
`-8
`Competitor log [M]
`
`-5
`
`-4
`
`Fig. 1. Inhibition of specific [ 125I]CGRP binding to SK-N-MC cell
`membranes by peptide no. I <•), no. 2 (_.), no. 3 (T) (a) and by peptide
`no. 4 (+), no. 5 (0) and no. 6 (D) (b) as described in Section 2. Data
`represent tbe mean±S.E.M. of three determinations.
`
`---- FVPTDVGSEAF
`-----· ~GSEAF
`FVPTDVGPEAF
`- FVPTNVGPEAF
`
`-
`
`-
`
`Wavelength[ run]
`
`250
`
`---- FVPTDVGPFAF
`----- · FVPTNVGPF AF
`-
`FVPTDVGPEAF
`- - - ~GPEAF
`
`15000
`
`a
`
`Mol. Ellip.
`
`-15000
`190
`
`15000
`
`b
`
`Mol. Ellip.
`
`-15000L------1----~----....1...----~--___JL..._ __ ---l
`250
`190
`Wavelengtb[nm)
`
`Fig. 2. Comparative circular dich~oism spectra of the CGRP 27-37
`analogs.
`
`·ly. Therefore we conclude that the proline in position 34
`does not induce an unusual three dimensional arrangement,
`moreover it serves to stabilise a tum which is already
`present in the unmodified segment.
`Fig. 2b shows the effect on secondary structure of the
`[P 34
`] peptides, when Glu in position 35 is replaced by Phe,
`additionally. In this case, a significant effect on the
`secondary structure could be observed, which is indicated
`by a change-of the symmetry of the obtained curves. The
`characteristic wavelength at which the molar ellipticity of
`zero is detected, has shifted to a higher wavelength in the
`[P 34
`, F 35
`] peptides compared to [P 34
`] analogues. Further- ·
`more, a shift of the maximum and minimum of the circular
`dichroism spectra has been detected. The intensity of the
`positive Cotton effect in the range of 192-196 nm is
`, F35
`[P34
`[D 3
`p 34
`increased considerably.
`and
`]-
`,
`\
`F 35]CGRP 27-37 exhibit the most ordered structures
`within the whole set of peptides. The increase in binding
`affinity which is gained by the introduction of Phe in
`
`position 35 correlates with a particular change in sec(cid:173)
`ondary structure.
`
`3.3. Blood flow measurements
`
`In order to investigate the functional role of the CGRP
`segments, the changes in vascular conductance in the
`femoral artery (Vcfa) of rat have been measured. A dose of
`CGRP which decreased the resting blood pressure by 40
`mmHg was applied. CGRP led to an increase in Vcfa of 85
`to 90% compared to control animals without CGRP
`administration (Fig. 3a and b, grey bars). The effect of
`CGRP is reduced to less than 20% increase in Vcfa after 8
`min. For testing the CGRP analogues, the peptides were
`pre-injected to the application of CGRP. The increase of
`Vcfa was significantly reduced compared to the effect of
`CGRP without pre-injection of the peptides (65% in the
`case of 170 nmollkg [D 3
`\ P 34
`, F 35]CGRP 27-37, 58% in
`
`4
`
`

`

`B. Rist et al. I Regulatory Peptides 79 (1999) 153-158
`
`157
`
`100
`
`1 a
`
`80
`
`FVPTDVGPFAF
`
`•CGRP
`• analog+ CGRP
`D 30 min+ CGRP
`
`~60
`
`~-.. .. ..
`
`~ 40
`.5
`~
`> 20
`
`Omin
`
`1 min
`
`2 min
`
`4 min
`
`8 min
`
`FVPTNVGPFAF
`
`DCGRP
`• analog + CGRP
`030 min+ CGRP
`D 60 min + CGRP
`
`0
`
`-20
`
`b
`
`100
`
`80
`
`~ 60 c .. ..
`"' ~ 40
`.5
`.!!!
`0
`> 20
`
`0
`
`Omin
`
`1 min
`
`2 min
`
`4min
`
`8 min
`
`Fig. 3. Effects of i.v. injection of endogenous CORP (0.5 nmol/kg) on
`the vascular conductance in the femoral artery (Vcfa) of the rat in vivo
`, F 35]CGRP 27-37
`before and after i.v. pretreatment with (a) [D", P34
`(170 nmollkg) and (b) [P 34
`, F 35]CGRP 27-37 (170 nmol/kg).
`
`, F 35]CGRP 27-37, com(cid:173)
`the case of 170 nmollkg [P 34
`pared to 90% and 85%, respectively). At a dose of 68
`nmollkg the effect was less pronounced but still signifi(cid:173)
`cant. Even after 8 min, there was still a difference between
`the pre-injection of CGRP segments and without peptides
`(Fig. 3, dark bars). After 30 min CGRP was injected again.
`, F35JCGRP 27-37 no significant
`In the case of [D 31
`, P34
`difference neither in immediate Vcfa increase nor after 8
`min was found compared to the application of CGRP
`without pre-injection of the CGRP segment (Fig. 3a, white
`bars). However, in the case of [P 34
`, F 35]CGRP 27-37, the
`blocking effect of the CGRP segment was further in(cid:173)
`creased after 30 min to an increase of Vcfa to only 33%
`(Fig. 3b white bars) (compared to 85% ). Only a third
`application of CGRP after 60 min showed the same Vcfa
`increase as before the injections of the CGRP segment
`(Fig. 3b light grey bars). Injection of the CGRP segments
`
`, F35]CGRP 27-37 and [P34
`, P34
`[D 31
`showed no change in Vcfa per se.
`
`, F 35]CGRP 27-37
`
`4. Discussion and conclusion
`
`,
`
`[P 34
`analogues
`corresponding
`the
`Comparing
`,
`F 35]CGRP 27-37 and [D 31
`, P 34
`, F 35]CGRP 27-37, which
`only differ in position 31, it became evident that Asp 31
`instead of Asn 31 has no significant effect on both receptor
`recognition and secondary structure. In contrast, however,
`significant differences were found in the rat blood pressure
`in vivo assays. Both analogues show no agonistic effect
`per se, but inhibit the effect of exogenous CGRP. Thus,
`both peptides act as antagonists. However, although the
`two peptides do not distinguish in their primarily antago(cid:173)
`nistic effect at the tested concentrations (estimated pA2
`values were 6.60 and 6.64, respectively), they show
`significant differences in their half life.
`p 34
`[D 3
`Whereas
`the
`inhlbition of CGRP by
`\
`F 35]CGRP 27-37
`[P 34
`lasts
`for maximal 30 min,
`,
`35 F ]CGRP 27-37 shows a strong effect up to an hour.
`Further investigations revealed that the extent as well as
`the duration of the effect which is caused by [D 3
`\ P 34
`,
`F 35]CGRP 27~37 is very similar to that of the known
`[P 34
`antagonist CGRP 8-37
`[13-15], whereas
`for
`,
`F 35]CGRP 27-37 the duration is at least twofold in(cid:173)
`creased.
`Differences in the noncompetitive, irreversible receptor
`binding of the two analogues could be speculated, however
`no differences in the background of the binding assays
`were found.
`Since both ligands exhibit receptor binding affinities in
`the same range (Ki = 19114 nM), we suggested that the
`analogue with Asp 31 is faster metabolised, perhaps because
`of an increased susceptibility for proteases. This effect
`could be confirmed by preliminary results with other
`analogues (no. 2, 5) containing either Asp or Asn in
`position 31. In this case as well, the effect of the Asp(cid:173)
`containing peptides was significantly increased.
`Thus we conclude that circular dichroism measurements
`allow discrimination between the analogues which exhibit
`high receptor affinity (no. 3, 6) and those with low binding
`affinity (no. 1, 4). We could how that binding of the
`analogues CGRP 27-37 and [D 3 1]CGRP 27-37 is very
`sensitive to changes in position 34 35. A p 31 instead of
`Asn 31 does not effect affinity, but has major effect upon
`the half life of the analogues in vivo. Asn 31 seems to be
`necessary to prevent rapid degradation of [P34
`, F 35]CGRP
`27-37 and may therefore result in longer lasting antago(cid:173)
`nistic effects.
`
`Acknowledgements
`
`The present study was supported by grants from the
`
`5
`
`

`

`158
`
`B. Rist eta/. I Regulatory Peptides 79 (1999) 153-158
`
`Swiss National Fund for Scientific Research No. 32-
`37872.93. We thank Margaret Smith-White for expert
`technical assistance.
`
`References
`
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`native RNA processing in calcitonin gene expression generates
`rnRNAs
`encoding different polypeptides products. Nature
`1982;298:240-4.
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