Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 98/27113 PCT/NL97/00703
MELANOCORTINS
The present invention relates to t:he field of melanocortin
peptides.
Melanocortins (which used to be called melanotropines also)
are peptides originally derived from a larger precursor
protein named pro-opiomelanocortin. The natural melanocortins
share the heptapeptide core sequence Met-Glu-His-Phe-Arg-Trp-
Gly. These melanocortins include cK-MSH (cx-melanocyte
stimulating hormone), (3-MSH, y-MSH, y-LPH (y-lipotropin
hormone) and ACTH (adrenocorticotrope hormone).
Melanocortins have a wide range of: biological activities. They
have been known for a long time to stimulate pigmentation and
corticosteroidgenesis, but they hive also been shown to induce
excessive grooming behaviour in the rat, to stimulate
conditioned active avoidance response, to increase blood
pressure and heart rate, to accelerate nerve regeneration and
to modulate immune responses. Quite recently five neuropeptide
receptors for melanocortins have r>een identified and cloned.
These receptors have different di~;tribution patterns (in
presence as well as in abundance) over the different tissue
types. They belong to the family of so-called G-protein
coupled receptors. Melanocortin rs~ceptor 1 (MCR-1) is
expressed in melanocytes, whereas MCR-2 is the ACTH receptor
expressed in for instance the adrs~nal gland. Melanocortin
receptors 3, 4 and 5 have been found to be expressed in the
central nervous system. The cognate ligands of these receptors
have profound neuropharmalogical effects of, such as
facilitated arousal, motivation, attention, memory and
learning. The ligands have also been implicated in food-
motivated behaviour. Further a relation with antipyretic
activity has been disclosed.
Many different (synthetic) analogs of melanocortins have been
. prepared and suggested to be of use in activating or blocking
one or more of the MC-receptors. Z~his agonistic or
antagonistic action has then been suggested to be useful in
applications relating to pigmentation, nerve regeneration,
etc.
. . _.,.
r.
~;,~,~~~,, .
Y' x ~.iWr
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However, the melanocortins developed so far lack n .
specificity (selectivity) for the receptor* expr0 sed in the
nsxvous system, and/or they lack ,i.n suffici~nt b ding
.~ affinity or capability to,induce the receptor med at~d
respoase or to block said response. Typically it ld ba
desired. that a drug targeting the MC-raoeptors ba highly
potent, orally adma.nistexabla, r~~asonalily resista~t to
breakdaarn in the body (have a ~uf~Eicient half-lif~) cad able
to cross the blood-brain barrier.
..
l0 The present invention provides peptides or peptid -like
structures that meat the structural requirements o be useful
as essentially 1tC-receptor specif;i.c drugs.
Thus th~ invention provides a pap~tida having spsc~.fic binding
affinity for a melanocortino rec~ptor, preferabiylthe mc3, mc4
or mc5 recaptox conprisiag th~ sego~nve ~ .
X-Y--His-(D-2-Thienyl-Ale)-ar9-(Z)
or
x-x-His- ( 3-.pyridyi-D-Al~a ) -Arq- ( ~ )
whereby X and Y a~ amino acid residues and $ is Ian aromatic
amino acid residue. ,
According to the prest~nt inv~entioxi the above
po ides era
aqonists for MsH aotivity ~rhfch as.~ highly potan. A vest'
25~ isportant contributioa to the high potency attributsd
can
to the 7-position (oountvd gut in t:h,~ oriqinsl -moiaculsr,)
A
which should bs D-2--thianyl~Ala o~~ 3-pyridyi-D- a and for
which only wty limited and very similar residue may be
substituted without losi~xg the inc~raaso in aqonit potency.
AnothQr important contribut3.on to M88 agonistict3.Wity i*
the omission of residu~s 1-3 and/a~r the o~n3ssiono~ rasidusa
11-13. A hiqh.contribution to activity is also ovidad by
p the
pretence of an aromtic amino acid. residua at po ition~9.
positions 4 and 5 should normally not bs omitted these
residuts should ba pras~nt though it is tar critical
lass
wdich aaa~no acid residues era preson~t at said itions. rt
is
clear that at least consarrvad substitutions era ilow*d for
these positions, but also lass conserwd substitu tions grill
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3 ~ . .
histidine residue at position 6 .and the arginine esidve at
position 8 ar~ quite important for activity and ~ ould only be
replaced by very consorvatiw suJbstitutions, if a ~a11.
5~~ Especially the more important residues in general should not
be. all replaced by substitutes i~z on~ and the sam~ molecule.
The preferred residue at position is Naftyi-alaai#ie, bo it. in
the D-or is the L-configuration.
The presenc~ of this residue 1~ads to a fuxth~r i~ncreae~e i.r~
potency.
An a~n.W o acid residue at position 10 is not esse~ial. for the
activity of the molecul~, but it does seem to have some
effect. If a residue fs present j.t is pref~rred t,~at this
residue is Qlycine or lysine, whe~rlby the latterihas the
additional. advantage that it provides a reactive ;moiety whicb
can be used to couple the peptids~ tv other molecules or to
make the peptide cyclic. In the e~wnt that a cyc is paptida is
to be produoed, which~is preferzed, since the ha f~lit~ of
such a cyclic peptide is improved over the half-~ifo of the
lin~ar fo~n, then a reactiv~s moiety at the other~end shouxd
also be provided. Another advantage of having a ~yalic paptida
is that these peptides tend to have a higher sal~axivity for
MC-receptors, in particular an disulphide bridge'inareasee the
selectivity for the MC-4 receptor. Cyclic peptid~s oan ho~nsyer
also be produced by providing rea~"tivs moieties tside the
essential sore that enable closury! of the loo ~uch ax
p~ ~ '
reactive moieties leading to a lacaam.
The preferred residues it positions X and x, (nr~~ninq 4 and 5
in the original ~CTB-core) are mle for x and G1y! or lisp for 7r,
whereby the presence of Asp loads to a further alivantage in
having a raaatiw moiety for making a lactam.
The peptides.accordinq to the invention are gen~aliy more
potent than irt~a itself. The prefQx~red peptides h~w poteneias
of up to 100 timQS the potency of leg. Less pots t
are within the scope of the present inwntioa, s ncelpoteency'
~ is not the only criterion which is required for ~a successful
peptide-based drug. As already mentioned, half-l~~ife arid
selectivity are also important par,~ters.
g~nerally not lead to'a significant loss of poten~y. The
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9, dU~d. 1999 15; Ol ~'ER OCTR~JOIBUREAUX (3i ~ 334227319 . N0, 4345 P. 20
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. The tests used for~detes~aining the potency of thelpeptides are .
the regular in itro t~ate as well as an i vivo ~est i,n rats
whereby the growing behaviour i;s measured: The.r~eults in a -
groos~ng assay are good indications that the.pept~.des will be
5' able to activate the receptor and thereby the G-protein
cascad~ coupled to said receptor and thus the pepltides can be
used as agonists for ~sC-receptors . Targeting the ~.-receptor
in particular of the MC..4 rec~pt«r fox which a t~cularly
selective peptide has been provided by the pres invention
in an agonistic manner is useful in the treatment of Ci~s-
disorders, neurophat6ies, obesity, and in particuilar for
diabetes related neuropathy, as 'sell as neuropat~y ~ a result
of cytotoaic treatments (chemotherapy aird the if.ke ) .
Th~retore tho pres~nt invention also provides ph~rmscantical
co~ositic~ns capable of treating the above conditions. Dosages
for suoh treatments will usually be given once n~day in doses
of about 1 erg to about 100 mg p~z~ dosage unit, preferably
10 erg - 10 mq, more preferably 5b~ uq - 1 mg. The~dosage should
ZO zmsult is a concentration in the body of between!0,1 nay at~d
1 um. preferably 1 n~1 - 100 n~i, aaost preferably ~0 - 50 nl~l.
The compositions msy comprise the usual additivs~ for usual
dosage formats for peptide drugs or for peptide ~leriv~sd drugs,
The format is preferably an oral formulation :ua~ as s tablet,
Z5 a granulate, a powder or a~liquid formulation, a~though
enteral and parerrteral administrations may find ~ppiication as
wail. particularly preferred are ~~o~positions~ wherein a
peptide according to the xrrventio~a is combined w~.th a drug
aiuiing to prevent or that leads t~~ neuropathy, s~ch as~insulin
3 0 arrd cytotoxio agents . '
The invention w~.ll be explained in spore detail the
following eape~r3,m~enta3, part .
I
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9. JU?u', 1999 13. ~2 VER OCTRO~aISLTREAUK (31; 3342273:9 NG, 4343 P, 2I
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BXPBRII~~
Material sad ~tstbods
' l~le,Ianocortin Ifgand receptor activity
5 v guman embryonal kidney (8810 293) cells mere stabi~r transfected
with the human MC3(Gantz et al. ~tBC 1993. 268a824~6-8250),
human MC4 (Gaeta et al. 1993. JHC; 268a15174015179~) or human
MC5 receptor constructs using the calcium ph4spha~t
precipitation method. A6 a reporter plasmid 10 ~ ~ of the pCRE-
LaGz vector (Chon tt al. 1995. Anal.Biochem. ZZ6~349-354), in
'vrhich a c~ responsiv! element drives ezprsssio~ of the Lacx
gene, was transfected at ttu begj.aning of each ~~rioent. The
day after pCRE~LacB transfection, cells were split in 96-wells
plates.. After 48 hours cells were stimulated with var5ring
concentrations of the MC recepto=~ ligands in ass~y miodium
Dl~t t 0 :1 mg/ml HBA, 0 . i mi~~ ZBMx ) for 6 house . ' Cells were
lysed in lysir buffer_by a freQae-thaw round, suk~stratQ buffer
(60~! sodium phosphate, i mt~t MqClZ) 5 m!t ø-merea~tosthanol,
200 NQ/ml ONPG) was added and col.ls were incubat~rd at 37~C for
1 hour. The activation of the cAll~ signal~transd~ction pathway
upon receptor activation was detected in a micro;plat~ reader
(8forad le'del 3500) a 405nm using a colorimetri~ assay as
dQecribed by Chen et a1.)
Animals, 3aqpiantatson o! carrnctlar~, fntracerebrov$ritricti3ar
injection .
dale ~Pistar rats woigt~ing 120-130 g were implant~d with
cannulas into the foramen intrave~ntriculars uade~r hypno~o
anaesthesia (8rakkee at al., Life 3cieace'vol. ih 19'79, ).
hats were allomsd to recover for .3 days and usodj for
experiments during the neat 10 da;rs. In case that rats were
used for more than one grooming e;~perimrat they ere allomsd
to recover for at least 3 days between subsequo~itwxpe~riments,
peptides (15 nq)~dissolvod in 3 N~~ saline (154 m~ NaCl) were
injected i.c.v. by means of a Ha~~lton syring~s. 't3ruom,ing taste
' were performed according to (Gispc~n~ et al, Lab. ~llnim. Sci. 29
1975). Rats were placed into the obsorvatioa boos ima~ediately
after the infection. obsezvaticn started 15 miniafter the
i
i
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u. JUN, !9~'~ _~ 5: Q? UER UC'fROnBUREAUX i3!' 3~~4~~'l3' ~ 1VU, 4~?4'~
1.
fi , r .
injection and continued for 50 min. Grooming was cored each
15 sec over 50 min, thus the msxa.mal grooming scope for a rat
is 200. ~ j _
5~ syntbl,ia of peptides
Pur'~f i ca -i on o ~~ ~ '
Preparative BpLC was carried out using a eaters P~sp 4000 .
liquid chromato9raph, eguipped wj.th a Waters aCM ~modui~ with
two prepBak cartridges plus c~uardl cartridge ( 25:2~.0 mm) filled
with Delta-Pak C18 material. Peptides tm~re detec~ad at 230 nm
using a Waters 486 spectrophoton~a~ter with a prap~rativrr cell.
purifications wexe performrad in g~radfonts a*in9 v~ater with
0.11 trifiuoroacetic acid (TFA) and aaetonitrile.with O.lt
TPA.
.1S i
l~tettiods for synthesis and cyclisation of a-r~iSH p~ptides a
~ltinl~es~.id~ Svnthnw~t*
We .used a eanilton 1r11crolab 2200 to synthesise u~ to 40
peptides sinaltaneously at 30 ~1 scan. Thi 8a~iiton
Microlab 2200 xaa programesd to delivor washing ~olveats and
roagents to a rack with 40 individual 4 ml columsi* with
filter, containing Rf.dk ( 4- ( 2 ~ , 4,' .-dinsthosypheny~-I s'nrx-
aminos~hyl)-pheno:y) resin for peptide *ynthssf.~. The columns
wets drained automatically after ~eaoh atop by vacuum. The
2S coupling Gyclo.rras based on Fmoc/»BTV (2-(1H-ben~otria~sol-1-
yi)-1,1,3.,3-tetraa~thyZuronium hoaafluorophospha~e) chemistry
[Fields ~t al.~ Peptide Research ~~, 95-101] usin~ double
coupling steps of 40 minutes. Pep~tidos ire depr~tec~ted and
cl~avod in 2 hre using 1.5 ml of as mixture o! tr~fiuoroacvtic
acid/phenol/thioanisolelwater/ethanedithioi j
10/0.7510.5/0.5/0.25 and then precipitated twicelby adding
hr~ane/disthy3.ether 111. The precipitate was dri~d and
lyophi7.ised from water/acetoaitri7:e. ,
C~~~,n_ of n nt; ~. .
PLC purified' or crnd~e peptides wore used for cyriization. via
a disulfide bridge with cysteiaes or via a lactajs br3,.dQo w3.th
the side ehaias of aspartio acid and lysiaes~ !
CA 02275442 1999-06-16
WO 98/27113 PCT/NL97/00703
7
A. disulfide bridge: crude MBJ06 (40 mg) was dissolved in 40
ml of a 0.5~ ammoniumbicarbonate~ solution at pH 8 and stirred
overnight. After 24 hours no free sulphydryl groups were
detected using Ellman's reagent and the product was
lyophilized after addition of 0.5 ml of acetic acid. The
y peptide was dissolved in 3 ml of 40~ acetic acid and purified
by preparative HPLC in a gradient of 14$ to 21$ acetonitrile
in water (containing 0.1~ TFA) i.n 30 min. Yield after
purification: 22.8 mg.
B. Lactam bridge: a mixture of 20 ml of DMF (peptide grade),
26 mg of Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP, 0.05 mmol) and 0.017 ml of DIEA
(0.1 mmol) was added to crude MH~J07 (20 mg, 0.012 mmol). The
cyclization reaction was followed by analytical HPLC. After 2
. hours the mixture was acidified to pH 4 using 0.1 M HC1. The
product was purified by preparative HPLC after dilution with
30 ml of water in a gradient of 21~ to 30~ acetonitrile in
water, containing 0.1~ TFA, in 30 min. Yield 15.4 mg.
Results
Screening of modified MSH peptides generated by PEPSCAN~
revealed several amino acids that increased MSH potency. D-2-
thienyl-Ala and 3-pyridyl-D-Ala at position 7 of the MSH
peptide was the most potent contributors of increased MSH
potency. Naftyl-Ala at position 9 also increased MSH potency
(figure 1b). Deletion of positions 1-3 and 11-13 further
increased MSH activity. Figure 1 shows the dose-response
curves for the following peptides:
MBU 23 *2GH6R7G# linear
MBU 24 *2GH6R8G# linear
MBU 27 *CGH6R8C# cyclic disulphate
MBU 28 *2DH6R8K# cyclic lactam
MBU 29 *2DH6R7K cyclic lactam
* acetyl # carboxamid 2= Norleucine, 6= D-Thienyl,
7= 2-naftyl-L-alanine, 8= 2-naftyl-D-alanine
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9. 1999 1;03 VER OCTROOIBUREAUX ('j1)
JUN.3~4~~'~31~
'
Nu, 434
r
.
. 8 ( .
.
. ,
, (figures
Positions 4~and 5 may not be critical 3 sand
4) -
At position 9 an aromatic acid (1~,H,Y)is highly
~reterred
figure 2 ) . Figures 3 to 9 show ithe
~ffect' of the' different -
amina acid substitutions'.at positions4 to 10
of ale-I~cSS,
5' xespeatively.
. I
. induction
The activity of these peptides can ref 9roodi.ng
the
behaviour ~ following intracer~hro~rentricular $tions is.
in jec
shown in figure 6. 1500 nQ is thr~
lowest dose of iMBH that
induces excessive grooming ~behav~Lour.The five
cQmpouads
tested hers are 100x pore potent than Ms8 in
viva.
Table 1. Calculated 8C50 values f:or
the three di~ferQnt cell
lures . '
hHC3 hMC:4 hl~lC~
l~tJ 23 5.510-9 ~.~i,~0-9 a.dr
i
. 4.80-$
i~u 24 3.010-9
1.?v10-8
,
i
I~1 Z7 I.3I0-~ 6.G~10-g 2~.5a a-s
MeU 28 6.310-7 1.710 8 4.5~0~8
i '
I~1 24 . 1.410-9 ~ 1.510-8 2.3~,Q~8
i
a-MSH 1.410-a 2.610-8 .3.0x0'"$
i
Legends '
3o Pi~urs 1. Dose response curves ~:or the differsr~t ldSB peptides
on eBlC 293 cells aabiy e=pressing husr~n MC3, KC~4 sad MC5
receptors. M8U 27 shows specificity for the HC4 ;receptor. 1~U
23 is a potent li9aad on all three receptor .
~ignre 1b. effect of the different aainoacids ~.t position 7
and 9 togeth~e= with MHtI 23 and IKB~J 24. All peptides wero
tested at 100 ruK oa a8x 293 cells stabiy expreseinq the three
different 1~C receptorsf MC3, MC4 ~~~d MC5 rtoieptc~rs. values
i
CA 02275442 1999-06-16
I
9. JUA, 19u ~ '~ ~, ; f?? E'R'R OCTR~JG i BURE~~n (3', i 334??"3~ ~ N0. 434 P,
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i
represent the percenteg~ activit~~ as cao~pared to the maximal
acti~rity of 100 n1~1 8n1?-MBH.
' Eiguro ~ . 8ffect of sini~le aminoacid Bubstitutiqns at
5~ aminoacid position number.9 of t~P-MS8. On the x-p~cis the
aminoacids replacing~the endogenous aminoacid (Try) are
depicted. values represent~th~a percentage activity as cospared
to thQ maximal activity of 10 nM at-MISH. All pept'~des were
tested at 10 nlK on H81C 293 cells stably ~apress~g the. three
different 1KC receptorsfi~C3,~ MC4 s.nd L~t05 receptors. at this
position on an NDp-M8g background. an aromatic amino acid ie
higtily preferred.
i~iqure 3. affect of single ami,noacid substitnti~tss at
aminoacid position numbtr 4 of Nle-MSg. On the Xrtazis the
aminoacid~t replacing the endogenous aminoacid (lit) err
depicted. Values represent the pert~ntag~ activity as comparod
to' the masimal activity , of 100 nl~!', a-M88. A11 peptides were
tested at 100 aM oa 8810 293 cells etably eapress~ng the. three
different NC receptors= i~C3, MC4 and' 1~C,5 receptors.
FiQnre ~. 8ffect of single aminoacid substitutilons at
aaiinoacid position numtior 5 of l~Ie~1i88. On tho x~-asis the
aminoacids replacing the endogenous aa~inoaaid (G~.u) are
depicted. Values represent the percentage activity as cvosparetl
to ttLe ma~cfaval activity of 100 nl! a-1~3H. All pep~ti.des were .
tasted at 100 n1~ on HBx 293 cells stably a:pressiing t1~ three
diffQrent 1KC recaptorsi MC3, KC4 and MC5 rec~pto~rs.
Figure 5. 8tfect of singlo aminoacid substitutions at
aminoaeid position number d of Nl~e-MBH. 0:1 the 7~-eels the
as~inoacids replacing the endogenous aminoacid (bis) are
depicted. valves represent the perc~ntagQ activ~:ty as compared
to the ma:imsl activity of 100 nM a--MS8. rrll peptides mere
tested at 100 nip op B8R 293 calls stably a=pressing the three
different 1~C reoeptors=MC3, MC4 and- ICS receptors.
i
CA 02275442 1999-06-16
q. JUIV, 1996 i~~:~3 VER ~.CTR;'~CrBURE"L''r 31; 3 ?""
r s 3~~ c r 319 ~ NC-, 435 F. 26
. 10 . '
Fignre '6. Effect of ~ single aminoacid substitutions at
aminoacid poeitiou number 7 of N7.e-MSH. On the X-!axis the
aminoacids replacing the endogenous aminoacid (Pte) are
depicted. Values represent the perc~atage activity as coa~ared
to th~ maximai'activity of 100 nr!t a-l~H. A11 pep#id~s were
tested at l00 nli on eSE 293 call~i stably ezpress~lag the three
different MC receptor:; MC3) MC4 and HCS racapto~s. ~ -
Pfgure ~. Effect of single aminoacid substitution: at
amfnoacid position n~ober 8 of Nl,e-1188. Ori the Z+axis the
~ami.noacids replacing the endogenous aminoacid (A~g) airs
depiotrd. values represent tho percentage activity as comparod
to the maximal activity of 100 n~!t a-M88. 7111 peptides arere
tosted ~ at 100 ate on B~ 293 cell~~ stably expressing tlu~ three
different MC receptors;MC3, MC4 and 1~C~ receptors. Only fiv~
aminoacid~substitutioria mere reefed.
Figure o. Hffect of single amincxcid substitutibris at
aainoacid position nunber 9 of Nl.e~MSB. oa tho 3~aais the
aminoacids replacing tho tr~dog~nous ~0.i.noacid (Tlrp) are
dQpiotod. Valuos~repreetat the pa~rcantage activity as compared
to the ma~zimal activity of 100 nl~t~ a-M88. 1111 ptpitides were
tested at 100 nH on 881 293 cells aably eupross~iaQ tho tdrQe
d~,ftorerit iIC-rlceptorap M~C3, KC4 and MC5 receptors.
, -
Figure !, affect of single amincxoid substitutions act
aoinoacid position numba,t 10 of 1~'1Q-MBH. 0n the~X-a:is t~
aminoacids replaci~aq the endogenous anninoacid ( (~ly ) are
depictod. values represent the percentage activity as co~mparod
to thc~ mazimel activity of 100 nil a-~tSH. 1111 ps~tides were
ttst~d at 100 ~u~t ~ on 293 collr stably e:pres~ting the three
different MC' receptors ~ ~IC3, Mc4 and xC5 receptors .
rigur~ lo. .
Peptide ~faductd groaioiag ~ .
' 3 ;ri saline, ~or 3 ;rl saline with ~eithor 15 ng 1~Q peptides or
190 ng a-l~lBH or 1500 ng a-~18s wau in jeoted i.c.v. in rats and
grooming behaviour was snored (mean t s.d.).
CA 02275442 1999-06-16
