Pharmacological characterization of new cholecystokinin analogues

EJP 5’7.35

Pharmacological

characterization

of new chollecystokinin ana

Received Y April IYYL revised MS receiwd 7 August 1902. acceptedI I August 1092

New analogucs of ch~~i~cyst[~kinin-7 (CCK-7) modified at aminu acid residues 5 and 7 wcx assayed tor their cffccl on pall bladder, pancreatic sccrction. food intake (anorcctic activity). amount of roaring (scdativc activity) and analecsia. ;1swell as their ability lo inhibit “‘I-CCK-X binding to pancreatic ccl1 membrane rcccptors and brain membrane receptors. The results wcrc compared it, the activities of standard compounds, CCK-8. ccrulcin, BOC-CCK-7 (BOC = tcrtbu~y[~xy~drb[)nyi) and BOC[Nlc’,Nlc’]CCK-7. All analogucs tlxhibitcd agonistic cffccls. Their anorcctic activity was significantly prolonged.

CCK-7 anabgucs: Roccptor binding; Amylasc sccrction: Gall bladder contractions; Anorexia; Analgesia: Scdatinn

1. Introduction The C-terminal octapeptide of cholecystokinin (CCK-8; H-Asp-Tyr(SOH ,)-Mct-Gly-Trp-Met-AspPhe-NH,) possesses the tuli biological profiie of choiccystokinin, a linear polypeptide hormone composed of 33 amino acid residues (for reviews, see Martinez, 1989; Silver and Morley, 1991; Woodruf and Hughes, 1991; Zetler, 1985) and plays a major role both in the gastrointestinal tract and the central nervous system. This pcptidc interacts with at least two pharmacologically distinct receptors that have been described in various central and peripheral tissues (Dourish et al., 1989). CCK binding sites have been classified as peripheral (CCK,) or brain-type (CCK,,) CCK receptors. The CCK, receptors have high affinity for CCK-8 and low affinity for CCK-4 and desuifated CCK-8. CCK, receptors show high affinity for these different fragments of CCK-8. In contrast to the biological activities of CCK assigned either to CCK, receptors (gall bladder contractions, pancreatic enzyme secretion, gut motility) or to CCK I+ receptors (sedation, analgesia, thermoregulation), the regulation of food intake by CCK is more complicated. CCK appears to play a physiological role in the termination of food intake and controls the Correspondence to: L. MaletinskB. Institute of Organic Chemistry and Biochemistry. Czechoslwvak Academy of Sciences, Flemingovo I nam. 2, lhhl0 Prague 6. Cz~~h(l~ll~v~ki~.Tel. 42 (2) 331 23’11, RX 42 (2)

31 I 07x4.

decrease of food intake both centrally and peripherally (Silver and Morley, 1991). However, the degree of potency after central or peripheral administration depends on the anima1 species (Morley, 1989; Morley et al., 1985; Zetlcr and Morsdorf, 1984; Delia-Fera et al., 1981). At present, several classes of sclectivc Iigands for CCK, or CCK, receptors have been described. Several analogues of CCK-7 were synthesized and partially biologically characterized in our Prague laboratory. Alterations in the central part of the molecule caused complete suppression of the biological activity (Hiav;llEek et al., 1991a). However, analogues modified at the C-terminus of CCK-8 by methyiation of the Phe aromatic side-chain or by modification of the Met side-chain kept the biological activity (Hlavicek et al.. 1991b,c; in press). We now describe the pharmacological characterization of several CCK analogues. These anaiogues were modified at amino acid residues in positions 5 and 7 (for chemical structures see table 1). Methionine in position 5 was replaced by neopent~Igiyc~ne and/or Phe in position 7 by its derivative with

(Neo)

methyl groups in positions 2,4 and 6 of the aromatic side-chain. Syntheses of these anaiogues have been described elscwhere (Hiav$Eek et al., 1991b,c; in press). These analogues were assayed in the following biological tests in vivo and in vitro: amyiase release from and binding to rat pancreatic acini, binding to guinea pig brain membranes, guinea pig gall bladder contractions, anorectic, sedative and analgesic test on mice.

logical Associates (Bethesda. MD), bovine plasma albumin (fraction V) from Miles 1 aboratories inc. (Elkhart, IN). Phadebas amylase test from Pharmacia Diagnostics (Piscataway. NY), and 13-‘1-labeled N-succinimidyl-3-(4-hydroxyphenyl)propionyl-CCK-8 from Amersham Corp. (Buckinghamshire, UK). BOC[Nle’.NleS]CCK-7 was synthesized in the laboratory of J. Martinez (Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France). Cerulein was a gift from Farmitalia-Carlo Erba (Milan, Italy), cholecystokinin octapeptide was purchased from Cambridge Biochemicals (UK). The new analogues and BOCCCK-7 were synthesized in the Institute of Organic Chemistry and Biochemistry, Prague.

2.3 Tissue preparatiotu The activity of these compounds was compared that of CCK-S literature.

and potent analogues described

tn addition

to

with in the

CCK-8. we used as standard

~rn~?~nd cerulein. the decapeptide related to CCK-S (H-Pyr-Gfu-Asp-Tyr(SO;H)-Thr-Gly-Trp-Met-Asp-

PheSH 21 which shows a similar spectrum of biological activities to CCK-S. but greater potenq (Zetler. 19855). We also tested the amino-protected

carboxy-terminal

heptapeptide oqcarbonyl)

BOC = tertbutyl-

of CCK

(BOC-CCK-7:

for which a spectrum of biologica! activities similar to that of CCK-S was described (Jensen et al., IYS2; Pan et al.. IYSI). Analogues synthesized in our laboratory were compared with the potent CCK malogue.

JMV

236

(BOC-[Nle’.Nle5]CCK-7,

TyrCSO;H)-Nle-Gly-Trp-Nle-Asp-PheNH

BOC?).

2.4. Atnylase secretion

BOC-

has been reported to exhibit the same activity as CCK-8 in pharmacological tests (Rodriguez et al.. 1987; Ruiz-Gayo et al., IYSS). !NIe’.Nle’lCCK-7

2. Materials

Dispersed acini from rat pancreas were prepared according to the method of Jensen et al. (1982). Guinea pig brain membranes were prepared following the procedures of Pelaprat et al. (1985). The standard incubation solutions contained (mM): 24.5 HEPES (pH 7.4!, 98 NaCI, 6 KCI, 2.5 NaH,PO,, 5 sodium pyruvate, 5 sodium fumarate, 5 sodium glutamate, 2 glutamine, I I.5 glucose, 0.5 CaC12, 1 MgCIZ, 0.5 mg/ml bacitracin, 0.2% (w/v) albumin, 0.03% (w/v) soybean trypsin inhibitor, 1% (w/v) essential amino acid mixture, and 1% (w/v) esseniial vitamin mixture.

and methods

Male Wistar rats (180-200 g) were obtained from Effa-Credo (Saint Get-main I’Arbresle. France), male guinea pigs (280-300 g) from le Centre d’Elevage d’Animau.. de Laboratoire (Ardenay, Francei and from Velaz (Prague. Czechoslovakia). Male NMRI-mice weighing 25-30 g were obtained from the Institute of Pharmacy and Biochemistry (KonArovice. Czechoslovakia).

HEPES was purchased from Boehringer-Mannheim, collagenase from Serva (Garden City Park, NY), soybean trypsin inhibitor from Sigma (St. Louis, MO), Eagle’s basal amino acid medium from Gibco (Grand Island. NY). essential vitamin mixture from Microbio-

Amylase release was measured using a previously described procedure (Jensen et al., 1982; Rodriguez et al., 1990). Acini were resuspended in the standard incubation solution supplemented with 1% bovine serum albumin containing about 1 mg of protein/ml and samples (0.5 ml) were incubated at 37°C for 30 min. Amylase activity was determined by the method of Ceska and Birath (1969) using the Phadebas reagent. Amylase release was determined as the difference in amylase activity in the samples with and without a peptide to be tested. It was expressed as the percentage of maximum stimulation obtained with BOC[Nle’,Nle’]CCK-7 as a standard. 2.5. Binding of labelled CM-8 to ral pancreatic acini The method was described previously (Jensen et al., 1982; Rodriguez et al.,1990). Samples (0.5 ml containing about 1 mg/ml of protein) were incubated with the appropriate peptide concentrations for 30 min at 37°C in the presence of 10 pM “‘I-CCK-8. After dilution and centrifugation at 10 000 x g for 10 min, the radioactivity associated with the acinar pellets was measured. The specXc activity of “‘I-CCK-8 was 2 000 Ci/mmol. The total binding in the absence of unla-

helled CCK pcptidc was about 10% of the total radi~i~ctivity in the sample. The non-specific binding found in the presence of 1 PM BOC-[Nle’,NlcSICCK-7 was less than 15% of the total binding. 2.6. ~i~idi~lgof labe~~edCCK-8 brartes

i5

guima pig brain mm-

The test was performed according to a method described previously (Fclaprat et al., 1985; Rodriguez et al., 1990). The buffer contained 50 mM Tris/HCI. 5 mM MgClz and 0.1 mg/ml bacitracin, pH 7.4. Experiments were performed by incubation of brain membranes (approximately 0.5 mg of protein/ml) in the presence of 10 pM ‘““I-CCK-8, plus various concentrations of peptides in a total volume of 0.5 ml for 60 min at 25°C. Non-specific binding was determined in the presence of I PM BOC-[Nle’,Nle5]CCK-7 and was less than 25% of the total binding. The total binding was about 15% of the total radioactivity in the sample. 2.7. Guinea pig gall bladder contractions The test was performed according to Lonovics et al. 119801. The guinea pig was decapitated, its gall bladder was removed and cut longitudinally to form a strip which was placed into a bath (37°C) containing a solution composed of (mM): 137 NaCI, 2.68 KCI, 1.8 CaCl?, 0.49 M&O,, 0.42 NaH,PO,, 11.9 NaHCO, and 5.5 glucose and bubbled with O&O,. The strip was fixed to a magnetoelectric recorder (tension of 0.5 g was applied constantly) and its contractions were measured. The compounds were tested at a concentration range from I.lO- “’ to 3.10Wh mol/l. The activity was compared with that of standard solutions of CCK-8 or cerulein. The contractile response-concentration curve for each peptide was based on the percentage of the maximum contraction caused by this peptide. The concentration producing 50% of the maximal response was the EC&,.

TABLE

2.8. &da titxl

effkts

Anti-rearing activity (Zetler, 1983a.b) was evaluated in a hmall open-field test. The mouse was put into ;i glass cylinder 10 min after the i-p. injection (tt.2 mf of sample per mouse) and its exploratory activity was observed. The number of rearings in a 5-min observation period was determined. The number of rearings in the control group (group with injected saline) - tcIo% of activity - was compared with the number of rearings of mouse groups treated with various doses of analogues (each group consisted of five mice, each dose was tested in at least three separate experiments). The dose of analogue causing a 50% decrease of the number of rearing was taken as the EDS,,. 2.9. AFtaigesic acti~i~ This was determined with mice and the hot-plate test (53°C) after the sedative test (the same mice after i.p. injection of 0.2 ml of the sample, i.e. 15 min after administration of the peptide) and the latency of jumping was measured. The prolongation of the jumping time was expressed as the percentage of the maximal latency, which was calculated as % of the difference between the time of jumping after administration of 40 pg/kg of cerulein and time of jumping in saline-treated mice. 2.10. Anorectic activity Food intake was followed as previously described (HlavaEek et al., 1991a). Mice had free access to food over 5 days from 8:00 to 16:00 h and were fasted overnight. They had free access to water during the whole 24-h period. On the sixth day, these mice. having fasted for 16 h, were injected i.p. (0.2 ml of sample Per mouse) with the compound to be tested and were placed in separated cages ifive mice per group, one group in one cage). After 10 min following the injec-

2

lnhibition of “‘I-CCK-8

binding hy CCK analogues.

Means~ S.E.M. of at least three separate experimenk

Rat pancreatic nrini __....

CCK-ti BOC-[Nle~,Nies~CK-7 BOC-[Nec?]CCK-7 BOC-[Phe(2,4.h-triMe)‘]CCK-7 BOC-INeo’,Phe(2.4.6-triMe)‘]CCK-7 BOC-[Phe(2,4,6-triM&]CCK-7 i* Values taken from Antblard et al. (1991).

2.100+ 0.52 ;’ l.lSOf 0.43 3.167k 0.29 4.500 * 0.87 IO.h7Ort 3.75 193.3 + 32.15

Guinea pig brain membranes ‘.110+_ tt.317+ 0.353 *

0.1x iI O.IO 0.03

1.550+ 0.13 2.667 k 0.23 975 &318

2

125

a c

2 3ummarizes

Tabk

IC+,

~~~~~~~~~~~ds in the two diffcrcnt

values ohtaincd

for

all

1 illus-

tissues. Figure

trztcs the ~~~i~~ti~~n cun’es of the analogucs and of BOC-~NIc-‘.?JiEc~]CCK-7 used as a standard. In rat pancrcatic

acini

rchtive

affinities

CONCENTRATION

of the tested peptidcs

decreased as foHows: BOC-[Nlc’.NleS]CCK-7 > BOCEke‘ CK-7 = BOC-[Phe( Z.-l&triMc )‘]CCK-7 > BOC&Neoi.Phc(‘.J.h-triMc)‘]CCK-7 EPhi~l.l.ti-triMc)‘ICCK-7

(tip.

B

125

BOC-

Uvl)

1

I. top). The interaction

~sith cxntr& CCK rcccptars (fig. I. hottom) was similar. CCK anstogues were able to inhibit the binding of “il-CCK-S to guinea pig brain membranes. The anatogue BW-[NedlCCK-7 ws as potent as BOC[NIe’.Nle’KCK-7. On the contrary the binding affinity of the analogue BOC-[Phc~1.3.h-triMe)i]CCK-7 tmi receptors was significantly decreased.

to cen-

‘p

50

z $ s!

25 0

Ertch of the peptides was ahlc to stimulate amylase secretion (see table 3 and fig_ 3, top) and with good Fig.

of ai lea>t three separate

<‘cqWund~

(TK-s

Cmuhn -C’C‘K-7

cxprrimenls.

ED,,,.

10”

of compounds, (01,

BOC-[Phe(2.4.6-triMe)‘]CCK-7

binding to rat pancreatic

and guinea pig brain membranes

(bottom).

as 7: of the value obtained with labelled CCK-X alone.

required

to elicit half-maximal

contraction.

(nM) Guinea pig gall

rat pancreatic

hladder contraction

acini

0.0.3 -_+ 0 .00.5 iI

7.57*

NT

2.23 * (I.67

2.23

10.10~3.x5 11.40+1.51

BOC_(NcoijCCK-7

0. I47 f 0.04

12.05 + h.OO

BOC_IPhrf2_l.~triMe)‘K‘CK-7

0. IS3 f 0.07

10.7x+4.31

BOC~Nro’.Phet~l.h-triMe)‘]CCK-7

0.433 f 0.08

53.15k4.31

6.500 * 2. I2

acini (top)

The values are expressed

Amylase release from

et al. (IYY I): NT = not tested.

BOCBOC-

( A ). BOC-[Phe(2.4.6-lriMek’]CCK-7

of ‘%CCK-8

NT

taken from Amblard

(0). (+ ),

(01 on inhibition

0.039 + 0.01

_LVa!ur

10-6

[M1

BOC-]Nle’,NleS]CCK-7

BOC-]Nle’.Nlei]CCK-?

BOC-]Phrf’.~.~triMe)~]CCK-7

10-T

[Neo’.Phe(2,4.6-triMe)‘]CCK-7

dose of peptidr

ED,,,

1. Effects

[Nro’]CCK-7

dose-reswnse cun;e for stimulated amylase secretion (amylasc release grows to a ma?timum and then dec~asfs at supram~~imal concentrations of the agonists).

t S.E.M.

10-s

CONCENTRATION

correlation with the binding data for pancreatic acini. The analogues are typical CCK agonists with a biphasic

Mean\

10-l”

10-l’

> IO .I

3.3. Guineu pig gui/ Ihddcr

contractions

The ability of the anaiogues to contract gall bladder smooth muscle was compared with that of CCK-8 and cerulein. The results are given in table 3 and are illustrated in fig. 2, bottom. The activity of BOC[Nle’,Nle-SICCK-7 was similar to that of tested compounds. The ECz,, value of the analogue BOC[Ph&,4,-6-triMe)‘]CCK-7 was very low (out of the range in which the contractions were measured). 3.4. Food intake Figure 3 and table 4 show the effects of the compounds tested on the food intake of fasted mice. All the peptides inhibited feeding in a dose-dependent manner (fig. 3, top), with relative affinities being

DOSE IFS/kg,

0

lo-”

I’ 0

10-10

1~-9

10-8

lo-7

I

40

I

I

70100130

,

I

190

IPI

I

250

I

310

I

370

lo-8

TIME [MINI CONCENTRATION

[Ml

Fig. 3. Anorexic effect of compounds CCK-X
125 1

$ z 3

50 25 0

I lo-‘*

,

&’

I

”

10-s

I

10”

I

1

10-T

10’”

Fig. 2. Effects of compounds. CCK-X (Cl 1.cerulein fA ),BOC-CCK-7 (0). DOC-[Ne&CK-7 fob, BOC[Phe(2.4,6-triMef]CCK-7 (* 1, BOC-[Neo5,Phe(2AB-triMe)‘JCCK-7 (A ) in tests in vitro: (top) on amylase release from rat pancreatic acini (in % of BOC-[Nle~.Nles~CK-7 maximal response); (~~tt~nl~ on the contraction of guinea pig gall bladder strips (Q of maximal response of each peptide).

f Ei1, B~C-lNle~.Nlc~~CK-7

cerulein > BOC-CCK-7 > BOC-[Nle~,Nle~~CCK-7 = BOC-[Neo5]CCK-7 = CCK-8 > BOC-[Phe(2.4.6-triMe)‘]CCK-7 = BOC-[Neo~,Phe~2,4,6-triMc~‘]CCK-7 zz=.BOC-[Phe(2,4,6-triMe)“JCCK-7. N-BOC protected analogues of heptapcptide of CCK-8 showed a prolonged inhibition of feeding (fig. 3, bottom). When we compared the effects of the same dose of CCK-8 or cerulein with those of BOGCCK-7 analogues, BOCCCK-7 aqd BOC-[Nle”,Nle”]CCK-7 exhibited a verv prolonged inhibition of feeding in mice. BOC[Nle’,Nle”]CCK-7 at a dose of 1 CL!:per mouse I40 pg/kg) could completely inhibit food intake for about 2 h, the same dose of cerulein doing so for less than 1 h.

~~~~~~~ itL?)r ;kaptongt&c most potent of the peptides test&_ ~~~~~c~crm \va1; measured.

Four a~~~~og~e~of BOC-CCK-7 wtrc tested in in xi\-o and in \-itro tests in order to find their biological activity in the central nen~us system and in the pe~p~e~~ s>%em. AI! the analogues exhibited agonistic effects. however with different potencies. Their structure was rn~i~~~d in positions 5 and 7. The results were compared with the values for standard CCK-8, ceruEein and BOC-CCK-7. A potent analogue, BOCN’fc’,Nfe’lCCK-7 L?MV 36) described in detail in the itcrature (Rodriguez et al.. 1987). was retested under r-*-r rulnrl;tinnc .,.&. C...lb_. . . . .. . t,, . . . h?.... ..us b 2” kmrl-ahnyg@-w . . .._..... _” ‘2 c-nmn.ar&w C”...@d.. L.... >*.. The first snalogue had a non-coded amino acid. ~e~?~~~~g~~c~n~. in position 5. This amino acid has the same characteristics as norleucine. which is often used to replace methionine without loss of activity. However it is more compact at~d bulligv and somewhat restricts the ~~~fo~atio~~~ freedom of the C-terminal end, As cdn he seen from the results. this change preserves the ogicai activities of CCK-8, however. it decreases ut d-fold the affinity and activity at the CCK,

E%ct> on CmFrd

rcccptor. The affinity for the CCK, receptor remains unchanged and the central effects are stronger. This is in agrecmcnt with similar analogues having Nle in position 5 (Rodriguez et al,, 1987). The second analogue had been modified in the C-terminal end - the phenylalanine in position 7 was made bulky by substituting the aromatic ring by three methyl groups. This change results in a decrease in the aromatic character of the residue. Changes in position 7 have interesting results. Suppression of the C-terminal amide function results in analogues able to cause maxima1 amylasc secretion with no decrease in the response at supramaximal concentration (Galas et al., 19X8). These analogues showed the unique property of being agonists at the high-affini~ CCK binding site and antagonists at the low-affinity CCK binding site. Our results showed that the analogue in which phenylalanine in position 7 had been replaced had decreased affini~ and activity in pancreas and gall bladder. Interestingly. while the affinity to the CCK, receptor was not decreased the central effects were slightly decreased ( 1S-2 times). Combination of the hvo structural modifications (third analogue, BOC-[Neo5,Phe(2,4,6-triMe)‘]CCK-’7) caused a further decrease of the affinity and activity with pancreas and gall bladder (CCK, receptor). The affini~ for brain receptors was also decreased. A decrease of the central activities - sedation and analgesia ccrrrzpondcd Kiih thk ikling. Tilt: oniy activity which was decreased only slightly was anorexia, The analogue was selective by about IO-fold to cause a decrease in food intake as compared to the other analogues. It has dissociated anorectic, sedative and analgesic activity in favor of the anorectic activity. The last analogue having trimethylated phenylalanine in position 5 had a more than 200-fold decreased affinity at both receptors, in accordance with their bjological activities. Replacement of methionine hy a

mice after i.p. administration. Meens+ S.E.M. of at least three wparate experiments. ED,,, values. calculated in 96 of activity of in the case of analgesia. the difference hrtween the effect saline and cerulein (40 @s/k& wa:s taken for IOf!~i.

group (saline-ireaied):

Anorexia 40 min after injection Y.Ih*4.0 2. i 2 * 0.3h 5.4X + 3.56 7.47* 1.35 8.20~5.04

I232 * 0.44 14.96 & 5.56 > 640

Sedative effecl to.w*

Analgesia (hot-plate lest) 4.16

2.36+ 0.M 3.4X* 8.4%* X.32+ 15.92 + 1n1.20+ > h40

0.92 3.08 6.12 4.20 16.40

20.04 + 6.68 0.95 * 0.08 6.73+_0.51 13.72&0.1 I 9.20+ 2.94 > 360 > 1fiO > Iii0

2.ZY

125

1

= 100 5 5

75-

c!J g

50-

ii! n 25-

00 0.1

1

10

100

1000

DOSE [jig/kg.IPI

1

125 ti s 2 100

BOC-[Nle’.NlcS]CCK-7, was previously tested for the influence on food intake in non-fasting rats (6ourch et al., 1990) and it was found that the anorectic effect was prolonged (for more than 4 h). Our results using a different model (HlaviEek et al., 1991a), i.e. using fasting mice, confirm this finding. The EDs,, value at 40 min level was comparable with that of CCK-8, however its effect was prolonged (the duration was significantly longer than in the case of other analogues). On the other hand the anorectic effect of the analogue. BOC[Nec’]CCK-7, was found to be slightly less than we previously reported (HlaviEek et al., 1991~). However this difference might be explained by the different strain of mice used for testing. While the first experiments were done using the DBA/Z strain of mice from our breeding colony; commercial NMRI strain of mice was used for the present work. The prolonged action of all BOC-CCK-7 analogues is probably due partly to the presence of the protecting group in position 1. The presence of this group could decrease the degradation of the peptide, resulting in an effect prolonged in comparison to that of CCK-8 and cerulein.

Acknowledgement This

ae

0 0.1

1

Fig. 4. Dose-response and

cerulein

analgesic

( A 1.BOC-CCK-7

[Neo5]CCK-7

(0).

Number

compounds

(It ), BOC-[Nle-?,NleS]CCK-7 ( A)

IO min before

on mice.

measurement

Doses

effect

CCK-8

(0).

(0).

BOC-

(* 1,

BOC-

(fig/kg

of rearing

i.p.)

activity.

of rearings was followed for 5 min and expressed as percent-

age of the corresponding Analgesic

of

BOC-!Phe(2,4.6-triMe)‘]CCK-7

[NeoS,Phe(2,4,h-triMe)‘]CCK-7 were administered

for sedative (anti-rearing)

(hottom)

test (hot-plate

number of rearings of saline-treated test, 53°C)

was performed

supported

hy

Grant

No.

75542

of

the

of Sciences.

References

[pg/kg, IPI

relationship effect

was

Academv

1000

100

10 DOSE

(top)

research

C7rrhn+vak

after

group. sedative

test; latency of jumping was measured.

Amhlard,

M.. M. Rodriguez,

and J. Martinez.

M.C.

Galas.

1992. Biolofical

M.F.

Lignon, N. Bernad

activities

of cionin and some

synthetic analopues. in: Peptides. Proceedings of the 12th American Peptide (ESCOM,

Symposium

Leiden!

Ceska, M. and K Birath. determination

1991. eds. J.A.

Smith and J.E. Rivier

p. 474. IYhY. A new rapid method for the clinical

of a-amylase

activities in human serum and urine.

Clin. Chim. Acta 26, 437. Della-Fera,

M.A..

C.A.

Baile. B.S. Schneider

and J. Grintrr.

19X1.

CCK antibody injected in cerebral ventricles stimulates feeding in sheep. Science 2l?,hX7. Dourish.

bulky aromatic amino acid seems to be incompatible with an active conformation of the molecule. Of the activities tested, anorexia yielded the most interesting results. Using trained mice fasted before the experiment, the anorectic effect of a compound could be followed very precisely in a short-term experiment (up to 6 h). All the analogues tested were more active than CCK-8, not only 40 min after i.p. injection, but throughout the period followed (fig. 3, bottom). All the analogues displayed a significantly prolonged effect, i.e. the quantity of food consumed returned to the reference level after a much longer time than in the case of CCK-8 and even of cerulein. The analogue,

C.T.. W. Rycroft

satiety by blockade

and SD.

Iversen.

1989, Postponement

of brain cholecystokinin

(CCK-8)

of

receptor.

Science 245. 1509. Galas. M.C..

M.F.

Lignon. M. Rodriguez,

Laur and J Martinez. on cholecystokinin:

C. Mendre.

198X. Structure-activity

P. Fulcrand. J.

relationship

studies

analogues with partial agonist activity. Am. J.

Physiol. 254. Gl76. Gourch, A., M. Orosco, M. Rodriguez. Jacquot.

1990, Effects

J. Martinez.

Y. Cohen and C.

of a new cholecystolinin

236) on food intake and brain monoamines

analopue (JMV

in the rat. Neuropep-

tides 15. 37. Hlavii-ek.

J.. V. i’eiovsk$.

Slaninovci, l99la. ified

in central

Cflmmun. IBaviEek.

J. Pirkovi.

Analogues part

of the molecule.

L. Maletinski octapeptide

Collect.

and J. mod-

Czech.

Chem.

J. Slaninrwi

and L.

56. 1963.

J., J. Pirkov;i. P. Majcr.

Maletinski.

P. Majer.

of cholecystokinin

M. i?ertov&

lYY I b. Cholecystokinin

analogues with alkyla~cd car-

Pclapr;at.

D..

S;IU~I

J.M.

and

nab k~hrllcd pancrew. Rodrigutx,

Zajac.

B.P.


Roqucs.

lisand for C’CK receptors: X1.F. Lignon.

A. Aumelas.

M.C.

tapcptide

of cholecystokinin.

Honda. J. Med.

Chcm.

M.. M.F. fw

B

P. Fulcrand.

C. Mendre.

analogue~

of the C-terminal

hep-

of the peptide

30. 136h.

Lipnon.

M.C.

rat and guinea

Mol. Pharmxol. Ruir-Ciayo.

A. It.

lYS7. Synthesis und hiologi-

On the importance Galas.

IYYO. Cyclic cholecystokinin

Itxtive

Morgat.

“]C’CK2,

binding on brain and on

G&IS.

J. Laur and J. Martinez.

cal activities of pscudoprptide

tinez.

J.L.

Life Sci. 37. 2-W. M..

Rodriguez.

Duricux.

[ ‘I1]BOC[Nle’”

IYSS.

M. Amblard

analogucs

pig central

and J. Mar-

that are highly se-

chnlecystokinin

receptors.

3X. 323.

M.. V. Dauge.

I. hlcnxnt.

D. Brgue.

G. Gacrl

and B.P.

Roqucs. IYS5. Synthe>i> and biological activity of BOC[Nle’“. y,c 31]C‘CK J7 II. highly potent CCK, analopi;c. Peptides 6. -II<. Silvrr. .\.i:

and J:E. Morley.

IYYI. Role of CCK

intake. Frog. Neurohiol. Wwdruf.

G.N.

and J. Hughes.

Ann. Rev. Pharmacol. Zetler.

iYY1. Cholerystokinin

and ceruletidc effects

interactions

date and picrotoxin.

Eur. J. Pharmacol.

G.. IYS.‘;. Cacruloin

properties. Zetler.

G.

Prptides

and

cholecystokinin

K.H.

effects

from those of mor-

92. 151.

G.. lYS3h. Nrurolrptic-like

tokinin nctapeptide: Zetler.

antagonists.

separates the antinociceptive

octapeptide

phinc. Eur. J. Pharmacnl. Zetler.

of food

Toxicol. 31. -WY.

G.. lYS3a. Apnmorphine

of cholrc~~tokinin

in regulation

26. 23.

oicrruletidc

with spomcwphine.

and cholecysmrthylpheni-

Y1. %I.

and its ;nxtlogucs: neuropharmacolofiical

6. 33. Morsdorf.

octapcptide

deh. Arch. Pharmacol.

33.

IYXJ. on eating

319.

Effects

of cerulrtide

and

in mice. Naunyn-Schmie-