Peptide-based compounds as new inhibitors of metallo-
ectopeptidases, compositions comprising said compounds and their
pharmaceutical and cosmetic uses
The present invention relates to new peptide-based compounds as
new inhibitors of metailo-ectopeptidases and compositions comprising said
compounds. The present invention also relates to their pharmaceutical and
cosmetic uses.
Mammalian zinc ectopeptidases, located at the surface of cells in
nervous and systemic tissues, play important roles in turning off neural and
hormonal peptide signals at the cell surface, notably those processing sensory
information. Among the neuronal peptide-signals are enkephalins, which are
potently and rapidly inactivated in vivo by two ecto-enzymes, NEP (Neutral
EndoPeptidase; EC 3.4.24.11) and AP-N (AminoPeptidase-N; EC 3.4.11.2).
Enkephalins are involved in regulating pain and nociception in the body.
Enkephalins play a crucial role in the dynamic control of neurotransmission
pathways of pain and in the modulation of cerebral structure activity governing,
among others, motivation and the adaptive equilibrium of emotional states.
Their action is specifically transmitted, like that of morphine (an alkaloid opiate),
via µ - and 3- opioid membrane receptors. The identification of the mechanisms
which control the upstream regulation of enkephalin signals is of fundamental
significance to physiological and therapeutic studies because of the importance
of the biological constants regulated by the endogenous opoid system.
An inhibitor of enkephalinin activating zinc ectopeptidases in
humans, named opiorphin, has been recently discovered (Wisner et al., 2006).
It is a QRFSR peptide that inhibits the human neutral ecto-endopeptidase,
hNEP, and the human ecto-aminopeptidase, hAP-N. Opiorphin displays potent
analgesic activity in chemical and mechanical pain models by activating
endogenous opioid-dependent transmission. The function of opiorphin is
closely related to the bovine spinorphin peptide (Nishimura et at., 1993 ;
Yamamoto et al., 2002) and to the rat sialorphin peptide (Rougeot et al., 2003).
European application EP 1 577 320 describes the QRFSR peptide
and its use in the prevention and treatment of pain. This application also
describes a YQRFSR peptide and a Glp-RFSR peptide, in which Glp is
pyroglutamate.
Inventors have now identified new peptide-based compounds,
derived from the QRFSR peptide. These new compounds exhibit inhibitory
properties against metallo-ectopeptidases, in particular hNEP and/or hAP-N.
Advantagously, the peptide-based compounds of the invention
enhance the bioavailability of the compounds, because of their structure. They
may also advantageously have other biological properties, such as anti-
inflammatory, anti-oxidant or antiseptic properties.
Description of the invention
The present invention relates to a compound that inhibits a metallo-
ectopeptidase, preferably the Neutral EndoPeptidase (EC 3.4.24.11) and/or the
AminoPeptidase-N (EC 3.4.11.2), said compound being selected in the group
consisting of x-aa2-aa1-z, x-aa3-aa2-aa1-z, x-aa4-aa3-aa2-aa1-z and x-aa5-
aa4-aa3-aa2-aa1-z, wherein:
aa1 and aa4, independently from each other, represent an arginine amino
acid, an histidine amino acid, an asparagine amino acid or a lysine amino acid,
aa2 represents a serine amino acid, a glycine amino acid, a threonine
amino acid or an alanine amino acid,
aa3 represents a phenylalanine amino acid, a leucine amino acid, an
isoleucine amino acid, a tyrosine amino acid or a tryptophan amino acid,
aa5 represents a glutamine amino acid, a glutamic acid amino acid, an
asparagines amino-acid or a lysine amino acid,
and wherein x and z, independently from each other, represent:
-H;
- OH, SH ;
- a halogen atom,
- an amino group,
- an alkyl, haloalkyl or heteroalkyl group containing from 1 to 30 carbon
atoms, linear or branched,
- an alkenyl or alkynyl group containing from 2 to 30 carbon atoms, linear
or branched,
- an acyl group containing from 1 to 30 carbon atoms, saturated or
unsaturated, linear or branched,
- a cycloalkyl, cycloakenyl, cycloalkynyl or an heterocyclic group
containing from 3 to 30 carbon atoms, linear or branched,
- one or more aryl or heteroaryl groups containing from 3 to 10 carbon
atoms per cycle,
- a alkoxy, thioalkyl, sulf onylalkyl, aminoalkyl containing from 1 to 30
carbon atoms, linear or branched,
- one or more heterocyclic group, containing from 5 to 10 carbon atoms
per cycle,
said groups being optionally substituted by one or more halogen atoms, alkyl
groups, hydroxy groups, alkoxy groups, aryloxy groups, acyloxy groups,
carbamoyloxy groups, carboxy groups, mercapto groups, alkylthio groups,
acylthio groups, arylthio groups, aryl groups, heterocyclic groups, heteroaryl
groups or amino groups.
The peptide is preferably selected from the group consisting of
x-SR-z, x-FSR-z, x-RFSR-z and x-QRFSR-z.
More preferably, z is H or NH2 and x is a saturated or unsaturated,
linear or branched fatty acid containing from 2 to 24 carbon atoms and possibly
substituted by one or more heteroatoms.
The invention also relates to a composition comprising the above-
mentioned compound, in association with an acceptable carrier. Said
composition may be a cosmetic or pharmaceutical topical composition.
Another object of the present invention is the use of the above-
defined compound for the preparation of a medicament for treating of
preventing pain, or in a topical cosmetic formulation.
Finally, the invention relates to a process of cosmetically treating
human skin or a mucous membrane comprising applying an effective amount of
the topical composition to said skin or mucous membrane.
Peptide-based compounds
The compounds of the invention are selected in the group
consisting of
x-aa2-aa1-z (I)
x-aa3-aa2-aa1-z (II)
x-aa4-aa3-aa2-aa1-z (III)
x-aa5-aa4-aa3-aa2-aa1-z (IV)
wherein:
aa1 and aa4, independently from each other, represent an arginine amino
acid, an histidine amino acid or a lysine amino acid,
aa2 represents a serine amino acid, a glycine amino acid, a threonine
amino acid or an alanine amino acid,
aa3 represents a phenylalanine amino acid, a leucine amino acid, an
isoleucine amino acid, a tyrosine amino acid or a tryptophan amino acid,
aa5 represents a glutamine amino acid, a glutamic acid amino acid, an
asparagines amino-acid or a lysine amino acid.
The compounds of the invention have at least a peptide moiety. "Peptide"
refers to a polymer in which the monomers are alpha amino acids joined
together through amide bonds. According to the invention, the peptide moiety is
two to five amino acid monomers long. Preferably, the peptide moiety is three
to five amino acid monomers long.
Amino acid residues in peptides are abbreviated as follows:
Ala or A is alanine
Arg or R is arginine
Asn or N is asparagine
Asp or D is aspartic acid
Cys or C is cysteine
Glu or E is glutamic acid
Gln or Q is glutamine
Gly or G is glycine
His or H is histidine
lie or I is isoleucine
Leu or L is leucine
Lys or K is lysine
Met or M is methionine
Phe or F is phenylalanine
Pro or P is proline
Ser or S is serine
Thr or T is threonine
Trp or W is tryptophan
Tyr or Y is tyrosine
Val or V is valine
According to the present invention, the amino acids of the peptide
moiety may either be in a D- or a L- configuration. A D-configuration is
preferred if the compound of the invention is to not to be degraded
enzymatically. The D-amino acid can be at either the N-terminus or the C-
terminus. Advantageously, the D-amino acid is at the N-terminus.
In the above-mentioned formulas, x and z, independently from each
other, represent:
-H;
- OH, SH;
- a halogen atom,
- an amino group,
- an alkyl, haloalkyl or heteroalkyl group containing from 1 to 30 carbon
atoms, linear or branched,
- an alkenyl or alkynyl group containing from 2 to 30 carbon atoms, linear
or branched,
- an acyl group containing from 1 to 30 carbon atoms, saturated or
unsaturated, linear or branched,
- a cycloalkyl, cycloakenyl, cycloalkynyl or an heterocyclic group
containing from 3 to 30 carbon atoms, linear or branched,
- one or more aryl or heteroaryl groups containing from 3 to 10 carbon
atoms per cycle,
- a alkoxy, thioalkyl, sulf onylalkyl, aminoalkyl containing from 1 to 30
carbon atoms, linear or branched,
- one or more heterocyclic group, containing from 5 to 10 carbon atoms
per cycle,
said groups being optionally substituted by one or more halogen atoms, alkyl
groups, hydroxy groups, alkoxy groups, aryloxy groups, acyloxy groups,
carbamoyloxy groups, carboxy groups, mercapto groups, alkylthio groups,
acylthio groups, arylthio groups, aryl groups, heterocyclic groups, heteroaryl
groups or amino groups.
"Halogen atom" means F, CI, Br, or I. In one embodiment, the
halogen atom is F, CI, or Br, more particularly CI or F, and most particularly F.
"Alkyl group" means a chain of 1 to 40 carbon atoms, particularly 1
to 30 carbon atoms. Lower alkyl groups can include alkyl groups such as
methyl and ethyl. Monovalent hydrocarbon groups may have a straight-chain or
branched-chain structure. In one embodiment, the branched alkyl groups have
one or two branches, particularly one branch. Alkyl groups are saturated. One
to four hydrogen atoms bonded to carbon atoms in the alkyl group may have
been replaced with one or more substituents. As an example, x represents a
palmitoyl, a stearyl or a palmitoyl residue.
According to the present invention, "substituent" may include, but
are not limited to, halogen atoms, halogenated hydrocarbon groups, alkyl
groups (e.g., methyl, ethyl, propyl, and butyl), hydroxy groups, alkoxy groups
(e.g., methoxy, ethoxy, propoxy, butoxy, and pentoxy), aryloxy groups (e.g.,
phenoxy, chlorophenoxy, tolyloxy, methoxyphenoxy, benzyloxy,
alkyloxycarbonylphenoxy, and acyloxyphenoxy), acyloxy groups (e.g.,
propionyloxy, benzoyloxy, and acetoxy), carbamoyloxy groups, carboxy groups,
mercapto groups, alkylthio groups, acylthio groups, arylthio groups (e.g.,
phenylthio, chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio, and
alkyloxycarbonylphenylthio), aryl groups (e.g., phenyl, tolyl, alkoxyphenyl,
alkoxycarbonylphenyl, and halophenyl), heterocyclic groups, heteroaryl groups,
and amino groups (e.g., amino, mono- and di- alkanyl-amino groups of 1 to 3
carbon atoms, methylphenylamino, methylbenzylamino, alkanylamido groups of
1 to 3 carbon atoms, carbamamido, ureido, and guanidino).
"Alkenyl group" and "alkynyl group" mean a chain of 2 to 30 carbon
atoms, having one or more double bonds, one or more triple bonds, or
combinations thereof. Alkenyl and alkynyl groups are unsaturated. One or more
hydrogen atoms bonded to carbon atoms in the alkenyl or alkynyl group may
have been replaced with one or more substituents. As an example, x
represents a oleyl, a linolenyl, an arachidyl or a myristoyl residue
"Acyl group" maens a chain of 1 to 30 carbon atoms, saturated or
unsaturated, which may have a straight-chain or branched-chain structure. Acyl
groups usually derived from carboxylic acids. As an example, x represents a
palmitoyl, a stearyl or a palmitoyl residue.
"Cycloalkyl group", "cycloakenyl group" and "cycloalkynyl group"
mean a saturated or unsaturated carbocyclic (or hydrocarbon) ring. Carbocyclic
groups are monocyclic, or are fused, spiro, or bridged bicyclic ring, systems.
Monocyclic carbocyclic groups contain 3 to 10 carbon atoms, particularly 4 to 7
carbon atoms, and more particularly 5 to 6 carbon atoms in the ring. Bicyclic
carbocyclic groups contain 8 to 12 carbon atoms, and more particularly 9 to 10
carbon atoms in the ring. Examples of carbocyclic groups include, but are not
limited to, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
The carbocyclic groups are particularly cyclopentyl, cyclohexyl, and cyclooctyl.
According to the present invention, carbocyclic groups may be substituted or
unsubstituted.
"Heterocyclic group" means a saturated or unsaturated ring
structure containing carbon and 1 to 4 heteroatoms in the ring. The attachment
point for heterocyclic groups may be at one or more carbon atoms, one or more
nitrogen atoms (if present) or a combination of carbon and nitrogen atoms.
Heterocyclic groups are monocyclic, or are fused or bridged bicyclic ring
systems. Monocyclic heterocyclic groups contain 3 to 10 member atoms (i.e.,
including both carbon atoms and at least 1 heteroatom), particularly 4 to 7
member atoms, and more particularly 5 to 6 member atoms in the ring. Bicyclic
heterocyclic groups contain 8 to 12 member atoms, particularly, 9 or 10
member atoms in the ring. Examples of heterocyclic groups include 1,3-
dioxalane, 1,3-dioxane, piperzyl, morpholinyl, tetrahydrofuranyl,
tetrahydropyranyl, and piperdyl. According to the present invention,
heterocyclic groups may be substituted or unsubstituted.
"Aryl group" means an aromatic group having a monocyclic ring
structure or fused bicyclic ring structure. Monocyclic aromatic groups contain 3
to 10 carbon atoms, particularly 5 to 7 carbon atoms, and more particularly 5 to
6 carbon atoms in the ring. Bicyclic aromatic groups contain 8 to 12 carbon
atoms, particularly 9 or 10 carbon atoms in the ring. Bicyclic aromatic groups
include groups wherein only one ring is aromatic or where both rings are
aromatic. Aromatic groups may be substituted or unsubstituted. One suitable
aromatic group is phenyl.
"Heteroaryl group" means an aromatic ring containing carbon and 1
to 4 heteroatoms in the ring. Heteroaromatic groups are monocyclic or fused
bicyclic rings. Monocyclic heteroaromatic groups contain 3 to 10 member
atoms (i.e., carbon and heteroatoms), particularly 5 to 7 member atoms, and
more particularly 5 to 6 member atoms in the ring. Bicyclic heteroaromatic rings
contain 8 to 12 member atoms and more particularly 9 or 10 member atoms in
the ring. Examples of heteroaromatic groups include, but are not limited to,
thienyl, thiazolyl, purinyl, pyrimidyl, pyridyl, and furanyl. In one embodiment,
heteroaromatic groups include thienyl, furanyl, and pyridyl. One particularly
suitable heteroaromatic group is thienyl. According to the present invention,
heteroaryl groups may be substituted or unsubstituted.
In one embodiment, according to the present invention, x and z
may, similarly of independently, be a lipid component, a carbohydrate
component, a terpene, a terpenoid, a carotenoid, a vitamin component, a
rosmarinic acid residue or a flavonoid.
According to the present invention, the lipid component can be any
lipid-containing component, such as a lipopeptide, fatty acid, phospholipid,
steroid, or a lipidated amino acids and glycolipids such as Lipid A derivatives.
In one embodiment, the lipid component is a fatty-acid, that is a
monocarboxylic acid with an unbranched aliphatic chain, which is either
saturated or unsaturated. Examples of saturated fatty acid include, but are not
limited to, butanoic acid, hexanoic acid, octanoic acid decanoic acid,
dodecanoic acid, tetradecanoic acid, hexadecanoic acid (or palmitic acid),
octadecanoic acid (or stearic acid), eicosanoic acid, docosanoic acid and
tetracosanoic acid. Omega-3 fatty acids, omega-6 fatty acids and omega-9 fatty
acids are examples of polyunsaturated fatty acids. Examples of unsaturated
fatty acids include, but are not limited to, myristoleic acid, palmitoleic acid, oleic
acid, linoleic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid,
erucic acid and docosahexaenoic acid. Preferred lipids for use as the lipid
component of the peptide-based compound of the invention include palmitic
acid (or palmitate) and stearic acid (or stearate).
The carbohydrate component of the peptide-based compound can
be any component that contains a carbohydrate. Examples of suitable
carbohydrate components include oligosaccharides, polysaccharides and
monosaccharides, and glycosylated biomolecules (glycoconjugates) such as
glycoproteins, glycopeptides, glycolipids, glycosylated amino acids, DNA, or
RNA. Glycosylated peptides (glycopeptides) and glycosylated amino acids
contain one or more carbohydrate moieties as well as a peptide or amino acid.
The carbohydrate component of the peptide-based compound of
the invention includes a carbohydrate that contains one or more saccharide
monomers. For example, the carbohydrate can include a monosaccharide, a
disaccharide or a trisaccharide; it can include an oligosaccharide or a
polysaccharide. An oligosaccharide is an oligomeric saccharide that contains
two or more saccharides and is characterized by a well-defined structure. A
well-defined structure is characterized by the particular identity, order, linkage
positions (including branch points), and linkage stereochemistry ([alpha], [beta])
of the monomers, and as a result has a defined molecular weight and
composition. An oligosaccharide typically contains about 2 to about 20 or more
saccharide monomers. A polysaccharride, on the other hand, is a polymeric
saccharide that does not have a well defined structure; the identity, order,
linkage positions (including brand points) and/or linkage stereochemistry can
vary from molecule to molecule. Polysaccharides typically contain a larger
number of monomeric components than oligosaccharides and thus have higher
molecular weights. The term "glycan" as used herein is inclusive of both
oligosaccharides and polysaccharides, and includes both branched and unb
ranched polymers. When the carbohydrate component contains a carbohydrate
that has three or more saccharide monomers, the carbohydrate can be a linear
chain or it can be a branched chain. In a preferred embodiment, the
carbohydrate component contains less than about 15 saccharide monomers;
more preferably in contains less than about 10 saccharide monomers.
According to an embodiment of the present invention, x and z may
also be, independently from each other, a terpene. Terpenes are derived
biosynthetically from units of isoprene, which has the molecular formula C5H8.
The basic molecular formulas of terpenes are multiples of that, (C5H8)n where n
is the number of linked isoprene units. Terpenes are classified according to the
number of isoprene units: monoterpenes (n=2), sesquiterpenes (n=3),
diterpenes (n=4), triterpenes (n=6) and tetraterpenes (n=8). Examples of
monoterpenes include, but are not limited to, pinene, nerol, citral, camphor,
geraniol and limonene. Examples of sesquiterpenes include, but are not limited
to, nerolidol and farnesol. Squalene is an example of a triterpene.
According to the present invention, terpenes may be hydrocarbons,
but also oxygen-containing compounds such as alcohols, aldehydes or ketones
(terpenoids). The term "terpenoid", for purposes of the present invention, is
intended to cover terpenes and oxygen containing derivatives thereof having at
least one C5H8 unit which may have one or more points of unsaturation and/or
be part of a cyclic unit within the chemical structure.
The carotenoids belong to the category of tetraterpenoids (i.e. they
contain 40 carbon atoms). Structurally they are in the form of a polyene chain
which is sometimes terminated by rings. Carotenoids with molecules containing
oxygen, such as lutein and zeaxanthin, are known as xanthophylls. The
unoxygenated (oxygen free) carotenoids such as alpha-carotene, beta-
carotene and lycopene are known as carotenes. Carotenes typically contain
only carbon and hydrogen.
The vitamin component of the peptide-based compound can be
either water-soluble or fat-soluble. Water-soluble vitamins include B vitamins,
notably vitamin B1 (thiamin) and vitamin B6 (pyridoxine, pyridoxal, and
pyridoxamine) and vitamin C. Fat-soluble vitamins include vitamins A, D
(notably D3 vitamin, D2 vitamin or derivatives thereof), E (tocopherol and
tocotrienol) and K.
According to an embodiment of the present invention, x and z may
also be a rosmarinic acid residue. Rosmarinic acid is known as having anti-
inflammatory properties.
According to an embodiment of the present invention, x and z may
also be, independently from each other, a flavonoid. According to the present
invention, the flavonoids may derived from a 2-pheny!chromen-4-one (2-
phenyl-1,4-benzopyrone) structure, from a 3-phenylchromen-4-one (3-phenyl-
1,4-benzopyrone) structure (commonly known as isoflavonoid) or from a 4-
phenylcoumarine (4-phenyl-1,2-benzopyrone) structure (commonly known as a
neoflavonoid). Flavonoids are most commonly known for their antioxidant
activity. Examples of flavonoid according to the present invention include, but
are not limited to, quercetin and genistein.
According to the present invention, when the compound is x-
QRFSR-z and z is H, then x is different from H. Additionally, when the
compound is x-QRFSR-z and z is H, then x is different from a tyrosine amino
acid. Finally, when the compound is x-RFSR-z and z is H, then x is different
from a pyroglutamate residue. Accordingly, the peptide-based compound of the
invention is different from the QRFSR peptide, as well as the YQRFSR and
Glp-RFSR peptides/compounds.
One or more linkers are optionally used for assembly of the
compound of the invention. In on embodiment, the linker is a bifunctional linker
that has functional groups in two different places, preferably at a first and
second end, in order to covalently link the peptide moiety of the compound with
another component of the peptide. A bifunctional linker can be either
homofunctional (i.e., containing two identical functional groups) or
heterofunctional (i.e., containing two different functional groups). In another
embodiment, the linker is trifunctional (hetero- or homo-). A suitable functional
group has reactivity toward or comprises any of the following: amino, alcohol,
carboxylic acid, sulfhydryl, alkene, alkyne, azide, thioester, ketone, aldehyde,
or hydrazine. An amino acid, e.g., cysteine, can constitute a linker.
According to the invention, x and z are preferably chosen in order
to enhance the bioavailability and cutaneous barrier crossing of the
compounds, as well as their lipophilicity or lipophilic character. In this case, x
and z are preferably a lipophilic moiety.
x and z may also be chosen to promote complementary biological
properties of the compound, in addition to the property of the peptidic moiety.
The compound of the invention may also, for example, have an anti-
inflammatory activity. It may also have anti-oxidant or antiseptic properties.
According to an embodiment of the invention, the compound is
selected from the group consisting of x-SR-z, x-FSR-z, x-RFSR-z and
x-QRFSR-z, x and z having the above-mentioned definitions.
According to another embodiment of the present invention, z is H or
NH2 and x is a saturated or unsaturated, linear or branched fatty acid
containing from 2 to 24 carbon atoms and possibly substituted by one or more
heteroatoms.
Preferably, x is a palmitic acid, a stearic acid. Alternatively, x is an
omega-3 or an omega-6 fatty acid, more preferably an arachidonic acid.
According to another embodiment of the present invention, z is H
and x is an acetic acid.
Preferred compounds of the invention consist in palmitoyl-QRFSR
(Figure 1), palmitoyl-QRFSR-NH2 (Figure 2), palmitoyl-NRFSR, palmitoyl-
NRFTR, palmitoyl-FSR, acetyl-SR, quercetin-RFSR and arachidonyl-QRFSR.
The peptide moiety and the compound themselves may be
prepared in a conventional manner, by solid phase or liquid phase synthesis.
They can also be prepared enzymatic synthesis, by fermentation or by plant or
animal extraction.
Solid phase peptide synthesis has been developed by R. B.
Merrifield. An insoluble polymer support, also called resin, is used to anchor the
peptide chain as each additional alpha-amino acid is attached. A labile group
protects the alpha-amino group of the amino acid. This group is removed after
each coupling reaction so that the next alpha-amino protected amino acid may
be added. Typical labile protecting groups include t-Boc (tert-butyloxycarbonyl)
and FMOC (9-flourenylmethloxycarbonyl). t-Boc is removed with dilute
solutions of trifluoroacetic acid (TFA) and dichloromethane. FMOC is removed
by concentrated solutions of amines (usually 20-55% piperidine in N-
methylpyrrolidone). When using FMOC alpha-amino acids, an acid labile (or
base stable) resin, such as an ether resin, is used. The stable blocking group
protects the reactive functional group of an amino acid and prevents formation
of complicated secondary chains. This blocking group must remain attached
throughout the synthesis and may be removed after completion of synthesis.
After generation of the resin bound synthetic peptide, the peptide is cleaved
from the resin, purified by HPLC if necessary and characterized by HPLC or
MALDI.
Compositions
The composition of the invention is useful for delivering the peptide-
based compound to cells or across epithelial and endothelial tissues, such as
skin, mucous membranes, vasculature tissues, gastrointestinal tissues, blood
brain barrier tissues, ophthalmologic tissues, pulmonary tissues, liver tissues,
cardiac tissues, kidney tissues etc. The composition of the invention can be
used both for delivery to a particular site of administration or for systemic
delivery. Preferably, the composition of the invention is a topical composition.
The composition of the invention can increase delivery or
availability of the peptide-based compound to cells or tissues compared to
delivery of the peptides described in the prior art, especially the petides
described in EP 1 577 320.
The composition of the invention can either be a cosmetic
composition or a pharmaceutical composition.
The composition of the invention is used in a form selected from the
group consisting of emulsions (oil-in-water, water-in-oil or oil-in-water-in-oil
emulsions), dispersions, solutions, suspensions, liposomes, chylomicrons,
nanocapsules, microcapsules, macrocapsules, nanoparticles, microparticles,
macroparticles, creams, lotions, ointments, milks, gels, cleansers, foundations,
anhydrous preparations (sticks, body and bath oils), shower and bath gels,
shampoos and scalp treatment lotions, cream or lotion for care of the skin or
hair, sun-screen lotions, milks or creams, artificial suntan lotions, milks, shaving
creams or foams, aftershave lotions, make-up, mascaras or nail varnishes,
lipsticks, serums, adhesive or absorbent materials, transdermal patches,
powders, emollient lotion, emollient milk, emollient cream, sprays, sprayable
formulation, oils for the body and the bath, foundation tint bases, pomade,
colloid, compact or solid suspension, pencil, brossable, mouthwash, toothpaste
tooth liquid gel, an oral or dental care product.
"Liposomes" refer to a membrane composed of a phospholipid and
cholesterol bilayer. Liposomes can be composed of naturally-derived
phospholipids with mixed lipid chains (e.g. phosphatidylethanolamine), or of
pure surfactant components, e.g. DOPE (dioleoylphosphatidylethanolamine).
"Nanocapsules" refer to submicroscopic colloidal drug carrier
systems composed of an oily or an aqueous core surrounded by a thin polymer
membrane. Two technologies can be used to obtain such nanocapsules: the
interfacial polymerization of a monomer or the interfacial nanodeposition of a
preformed polymer.
According to the present invention, a "nanoparticle", also called a
nanopowder, a nanocluster or a nanocrystal, refers to a small particle which is
less than 100 nm. A "microparticle" refers to a particle which is less than 1 mm.
A "macroparticle" refers to a particle which is more than 1 mm.
According to one embodiment of the invention, the composition is a
pharmaceutical composition and comprises a pharmaceutically acceptable
earner. The composition of the invention may for example be a
dermopharmaceutical composition. As used herein, the phrase
"pharmaceutically acceptable" refers to molecular entities and compositions
that are "generally regarded as safe", e.g., that are physiologically tolerable and
do not typically produce an allergic or similar untoward reaction, such as gastric
upset, dizziness and the like, when administered to a human. Preferably, as
used herein, the term "pharmaceutically acceptable" means approved by a
regulatory agency, for example listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more particularly in
humans.
The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle
with which the compound is administered. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water or aqueous solution saline solutions and
aqueous dextrose and glycerol solutions are preferably employed as carriers,
particularly for injectable solutions. Suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
According to another embodiment of the invention, the composition
is a cosmetic composition, for example a topical, cosmetic composition and
comprises a cosmetically acceptable carrier or a dermatologically acceptable
carrier. The term "dermatologically acceptable", as used herein, means that the
compositions or components described are suitable for use in contact with
human skin without risk of toxicity, incompatibility, instability, allergic response,
and the like. The dermatologically acceptable carrier can be an aqueous or
hydroalcoolic solution, a water-in-oil emulsion, an-oil-in-water emulsion, a
microemulsion, an aqueous gel, an anhydrous gel, a serum or vesicle
dispersion. As examples of cosmetic composition, can be mentioned body
hygiene compositions, hair compositions, make-up compositions or care
compositions. According to this embodiment, the composition of the invention
may further comprise at least one additive and/or active agent common in
dermocosmetics. Said at least one additive and/or active agent may be chosen
from: fragrances; stabilizing agents; UV-A and UV-B screening agents;
hydrophilic and lipophilic antioxidants; chelating agents; a- and 0-hydroxy
acids; ceramides; antidandruff agents; antiacne agents; agents for combating
hair loss; antifungal and antiseptic agents; antiperspirant active substances,
bactericidal active substances, and odor absorbing active substances; hair
fixing material and conditioning material; hair care active agents and sheen-
reinforcing agents and hair dyes; fillers, dyes and dyestuffs; gelling material;
and silicone gums.
Therapeutic and cosmetic applications
The compounds of the invention demonstrate potency on at least
one enkepahlin-inactivating ectopeptidase, e.g. human neutral ecto-
endopeptidase, hNEP, and/or the human ecto-aminopeptidase, hAP-N. They
inhibit pain behavior.
One object of the invention is the use of the above-defined
compound for the preparation of a medicament for treating of preventing pain
or as an analgesic agent.
Another object of the invention is the use of the above-mentioned
compound in a topical cosmetic formulation.
The compound or composition of the invention may notably be
used to treat or prevent external aggressions such as insect stings, burns,
sunburns, erythema, itches, eczema, psoriasis, wound healing, acute pain,
chronic pain polyarthritis and other inflammation pathologies, tumors,
infections. The compound of the invention may also promote healing, skin
repair, hydro-mineralization of the skin.
Another object of the invention is a process of cosmetically treating
human skin or a mucous membrane comprising applying an effective amount of
the above-defined topical composition to said skin or mucous membrane.
Another object of the invention is a process of treating a condition
comprising applying an effective amount of the above-defined pharmaceutical
composition.
Figures
Figure 1 shows one peptide-based compound according to the invention, the
palmitoyl-QRFSR.
Figure 2 shows another peptide-based compound according to the invention,
the palmitoyl-QRFSR-NH2.
Figure 3 shows kinetics of Ala-Mca breakdown by recombinant hAP-N in
absence of inhibitor (black squares) or in the presence of 6 to 70 uM
Pal-QRFSR-NH2. Each point represents the intensity of the signal expressed in
RFU (Relative Fluorescence Unit), which was directly proportional to the
quantity of metabolites formed, as function of reaction time (min).
Figure 4 shows the concentration-dependent inhibition by Pal-QRFSR-NH2 of
Ala-Mca breakdown by pure recombinant human AP-N. Each open square
represents the percentage of intact substrate recovered after incubation and
calculated as follows: percentage of velocity without inhibitor - velocity in
presence of inhibitor / velocity without inhibitor, which was measured in the
absence or in the presence of various concentrations of Pal-QRFSR-NH2
plotted in µM (log-scale).
Figure 5 shows kinetics of Abz-dRGL-EDDnp breakdown by recombinant hNEP
in the presence of corresponding vehicle (black triangles & circles) or in the
presence of 3 to 70 µM Pal-QRFSR-NH2. Each point represents the intensity of
the signal expressed in RFU (Relative Fluorescence Unit), which was directly
proportional to the quantity of metabolites formed, as function of reaction time
(min).
Figure 6 shows the concentration-dependent inhibition by Pal-QRFSR-NH2 of
Abz-dRGL-EDDnp breakdown by pure recombinant human hNEP. Each open
square represents the percentage of intact substrate recovered and calculated
as follows: percentage of velocity without inhibitor - velocity in presence of
inhibitor / velocity without inhibitor, which was measured in the absence or in
the presence of various concentrations of Pal-QRFSR-NH2 plotted in µM (log-
scale).
Figure 7 shows kinetics of Abz-RGFK-DnpOH breakdown by recombinant
hNEP in the absence of inhibitor (black circles) or in the presence of 10 to 60
uM Pal-QRFSR-NH2. Each point represents the intensity of the signal
expressed in RFU (Relative Fluorescence Unit), which was directly proportional
to the quantity of metabolites formed, as function of time-reaction (min).
Figure 8 shows the concentration-dependent inhibition by Pal-QRFSR-NH2 of
Abz-RGFK-DnpOH breakdown by pure recombinant human hNEP. Each point
represents the percentage of intact substrate recovered and calculated as
follows: percentage of velocity without inhibitor - velocity in presence of inhibitor
/ velocity without inhibitor, which was measured in the absence or in the
presence of various concentrations of tested compound plotted in uM (log-
scale).
Figure 9 shows the kinetic of Mca-RPPGFSAFK-(Dnp)-OH breakdown by
recombinant hNEP in the absence of inhibitor (grey triangles & squares) or in
the presence of 10 to 70 µM Pal-QRFSR-NH2. Each point represents the
intensity of the signal expressed in RFU (Relative Fluorescence Unit), which
was directly proportional to the quantity of metabolites formed, as function of
time-reaction (min).
Examples
Example 1 - Synthesis of the compounds of the invention
The following compounds have been synthesized: palmitoyl-QRFSR and
palmitoyl-QRFSR-NH2.
The amino acids protected at N-terminal extremity by a group Fmoc (e.g.
Fmoc-Gln-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, FmocSer(tBu)-OH) were
purchased from SIGMA.
The N.N-dimethylformamide (DMF), the dichloromethane (DCM), methanol, the
acetonitrile, ethylic ether, trifluoracetic acid (TFA), the piperidine, were
purchased from Carlo Erba or Acros Organics.
The diisopropylcarbodiimide (DIC), the N,N-diisopropylethylamine (DIEA), the
N- hydroxybenzotriazole (HOBt), the triisopropylsilane (TIPS), the symetric
anhydre palmitic acid (MW = 494) were purchased from SIGMA and Alfa
Aesar.
a) Fmoc-deprotection
Fmoc deprotection after each amino acid coupling was accomplished using a
mixture of DMF/piperidine (80/20, v/v) under agitation during 20 minutes.
b) Washing
The resin was then washed with DMF (3x5 min), followed by methanol (2x5
min) and successively DCM (2x5 min).
c) Coupling of amino acids
Each amino acid was coupled to the deprotected N-terminal amine of the resin,
or previously coupled amino acid, using a coupling solution containing 2
equivalents of amino acid Fmoc, DIC and HOBt in DMF. The coupling solution
was added to the deprotected resin during 2 hours.
d) Monitoring the progress of amino acid couplings
The progress of amino acid couplings was followed using ninhydrin. The
ninhydrin solution turns dark blue (positive result) in the presence of a free
primary amine but is otherwise colorless (negative result).
e) Cleaving the peptide from the resin
The resin was treated with a solution of TFA/water/TIPS (95/2.5/2.5, v/v/v) for
cleavage (5ml/g, 3 hours). The resin was then filtrated and washed with TFA
(5ml/g). The filtrate was then concentrated under vacuum.
The compounds were precipitated with diethylic ether.
The samples were solubilised, frozen at-80 °C and lyophilized.
f) Coupling of palmitate
The palmitate was added by manual coupling. 3 equivalents of palmitic acid
(0.33 mmol) were mixed in DIEA (0,4 mmol) and DCM for 3 hours.
g) Washing
Washing was performed using DCM and NMP. The Kaiser test was negative.
h) Solubility of the peptides
The solubilities of the lipopeptides were good in a solution of water/acetonitrile
(50/50).
i)HPLC
The HPLC analyses were performed on Dionex Summit HPLC system,
equipped with a P580 pump, a Gina 50 autosampler and a UVD340S diode
array detector. The column used is a C18 Merck Chromolith column (100 x 4.6
mm) thermostated at 35°C. A flow rate of 1 ml/min and a gradient from 20% to
80% of acetonitrile in ammonium formate at 0.1% in 20 minutes were used.
Example 2 - In vitro activity of Palmitoyl-QRFSR-NH2
The Palmytoyl-QRFSR-NH2 compound was tested for its inhibitory potency
towards two membrane-anchored ectoenzymes that is neutral endopeptidase
NEP (EC 3.4.24.11) and aminopeptidase AP-N (EC 3.4.11.2) by using selective
fluorescence-based enzyme assays. Wisner et al. (PNAS, 2006) have
previoulsy demonstrated that the QRFSR peptide is an efficient dual inhibitor of
this two enkephalin-inactivating ectopeptidases.
A. Biochemical Assays
Formal kinetic analysis was performed for each assay using real-time
fluorescence monitoring of specific substrate hydrolysis. For each 96-well
adapted fluorimetric model, all parameters allowing the analysis of human NEP
and human AP-N enzyme activity were defined under conditions of initial
velocity measurement.
1- Sources of the human ectopeptidases, hNEP and hAP-N
Recombinant human NEP and recombinant human AP-N (devoid of their
respective N-terminal cytosol and transmembrane segment) that were
purchased from R&D Systems, were used as pure source of ectopeptidase.
2- Substrates
Aminopeptidase, NEP-carboxydipeptidase and NEP-endopeptidase activities
were assayed in vitro by measuring the breakdown of the following synthetic
selective substrates:
a) Abz-dR-GL-EDDnp FRET-peptide, an internally quenched fluorescent
substrate specific for NEP-endopeptidase activity, was synthesized by Thermo-
Fisher Scientific (Germany) (Barros et al., 2007)
b) Abz-RGFK-DnpOH FRET-peptide, an internally quenched fluorescent
substrate specific for NEP-carboxydipeptidase activity, was synthesized by
Thermo-Fisher Scientific (Germany) (Barros et al., 2007)
c) Mca-RPPGFSAFK-DnpOH FRET-peptide (Mca-BK2), an intramolecularly
quenched fluorogenic peptide structurally related to bradykinin and a selective
substrate for measuring NEP and ECE activity, was purchased from R&D
Systems. ECE, or Endothelin Converting Enzyme, is a mealloectopeptidase
enzyme belonging to the same Zn ectoenzyme family than NEP. ECE
inactivates the bradykinin and activates the endothelin from its precursor.
FRET is the distance-dependant transfer of energy from a donor fluorophore
(Abz= ortho-aminobenzoyl; Mca= 7-methoxycoumarin-4-yl-acetyl) to an
acceptor fluorophore (DnpOH= 2,4-dinitrophenyl; EDDnp= 2,4-dinitrophenyl
ethylenediamine).
d) L-alanine-Mca (or Ala-Mca), a fluorogenic substrate for measuring
aminopeptidase activity, was purchased from SIGMA.
3- Measurement of Ectopeptidase Activities using 96-well adapted
fluorimetric assays
According to conditions of initial velocity measurement: the time, pH and
temperature of incubation as well as enzyme and substrate concentrations
were defined for each assay. Hydrolysis of peptide-substrates was measured
by real-time monitoring their metabolism rate by ectopeptidases in the
presence and absence of tested inhibitory compound (concentrations ranging
from 1 to 70 µM). These were added to the preincubation medium. The
background rate of substrate autolysis representing the fluorescent signal
obtained in the absence of enzyme was subtracted to calculate the initial
velocities in Relative Fluorescent Unit/min (or RFU/min).
a) Measurement of NEP-endopeptidase activity using FRET specific peptide-
substrate, Abz-dR-GL-EDDnp
Under conditions of initial velocity measurement, the intensity of the signal was
directly proportional to the quantity of metabolites formed during the 20-40 min
time-period of the reaction. Thus, in absence of inhibitor, the initial velocity of
rhNEP-mediated specific endoproteolysis of Abz-dR-GL-EDDnp, was
calculated from the linear regression (slope = NEP activity in presence of
vehicle / incubation time) as 8218 ± 2878 RFU/min/ug rhNEP, n=3 independent
determinations.
b) Measurement of NEP-carboxydipeptidase activity using FRET specific
peptide-substrate Abz-RGFK-DnpOH
Under conditions of initial velocity measurement, human NEP-mediated specific
hydrolysis of Abz-RGFK-DnpOH was evaluated at 59796 ± 18685 RFU/min/ug
rhNEP, n= 4 independent determinations.
In addition, the intramolecularly quenched fluorogenic peptide, Mca-BK2, was
submitted to hydrolysis by rhNEP. Under conditions of initial velocity
measurement, human NEP-mediated specific hydrolysis of Mca-BK2 was
evaluated at 139263 ± 19780 RFU/min/µg rhNEP, n= 2 independent
determinations.
c) Measurement of AP-N-ectopeptidase activity using Ala-Mca substrate.
Under conditions of initial velocity measurement, the human AP-N-mediated.
aminoproteolysis of Ala-Mca was directly calculated (from the slope: AP-N
activity in absence of inhibitor in function of incubation time) as 147042 ± 44657
RFU/min/ug rhAP-N, n = 3 independent determinations.
B. In vitro functional characterization of palmitoyl-QRFSR-NH2
The palmitoyl-peptide was dissolved in ethanol at 10 mg/ml (10.7 mM) and
stored at -30 °C until it was extemporaneously diluted in the appropriate buffer
for testing in each fluorescence-based enzyme-assay. However, as Pal-
QRFSR-NH2 was not soluble in aqueous Tris-buffer conditions at concentration
higher than 70 µM, the tested concentrations ranged from 3 to 70 µM.
1- Effect of palmitoyl-QRFSR-NH2 on hAP-N activity
Pal-QRFSR-NH2 efficiently prevented hAP-N mediated-aminoproteolysis of
Ala-Mca substrate. Its inhibitory potency was strictly concentration dependent
(r2 = 0.92, n = 34 points of determination); ranging from 3 to 60 uM (Figures 3
and 4). Under these experimental conditions, its inhibitory potency was less
effective than the QRFSR peptide, but more potent than the pGlu-RFSR
peptide. pGlu is pyroglutamate.
Accordingly, the presence of palmitate substitute at the N-terminal moiety of the
peptide which is advantageous to prepare a topical formulation, has no
negative effects on the recognition by hAP-N and its inhibitory activity.
2- Effect of palmitoyl-QRFSR-NH2 on hNEP endopeptidase activity
Pal-QRFSR-NH2 prevented hNEP mediated-endoproteolysis of Abz-dRGL-
EDDnp substrate. Its inhibitory potency was strictly concentration dependent
(r2 = 0.95, n = 27 points of determination); ranging from 5 to 60 uM (Figures 5
and 6). Under these experimental conditions, the compound of the invention
demonstrated the same inhibitory potency towards hNEP endopeptidase than
the QRFSR.
Accordingly, the presence of palmitate substitute at the N-terminal moiety of the
peptide which is advantageous to prepare a topical formulation, has no
negative effects on the recognition by hNEP ectoenzyme and its inhibitory
activity.
3- Effect of palmitovl-QRFSR-NH? on hNEP carboxvdipeptidase
activity
Pal-QRFSR-NH2 displayed a weak inhibitory potency towards hNEP
carboxydipeptidase activity (Figure 8).
Under the same experimental conditions, Pal-QRFSR-NH2 displayed a better
inhibitory activity on hNEP-mediated hydrolysis of Mca-BK2 substrate (Figure
9) than on hNEP-mediated hydrolysis of Abz-RGFK-DnpOH substrate (Figure
7). The latest substrate is particularly specific for carboxydipeptidase activity of
NEP, while the Mca-BK2 substrate can be cleaved not only at Phe8 position by
carboxydipeptidase activity of NEP but also at Phe5 position by endopeptidase
activity of NEP.
References
Barros, N.M., Campos, M., Bersanetti, PA, Oliveira, V., Juliano, MA, Boileau,
G., Juliano, L. & Carmona, A.K. Neprilysin carboxydipeptidase specificity
studies and improvement in its detection with fluorescence energy transfer
peptides. Biol Chem (2007) 388,447-455
Nishimura K, Hazato T (1993) Biochem Biophys Res Commun 194:713-719.
Rougeot C, Messaoudi M, Hermitte V, Rigault AG, Blisnick T, Dugave C, Desor
D, Rougeon F. Sialorphin, a natural inhibitor of rat membrane-bound neutral
endopeptidase that displays analgesic activity. Proc Natl Acad Sci U S A
2003;100:8549-54.
Wisner, A., et al., Human opiorphin, a natural antinociceptive modulator of
opioid-dependent pathways. Proc. Natl. Acad. Sci. USA, (2006).
Yamamoto Y, Ono H, Ueda A, Shimamura M, Nishimura K, Hazato T (2002)
Curr Protein Pept Sci 3:587-599.
We Claim:
1. A compound that inhibits a metallo-ectopeptidase, selected in the
group consisting of x-aa2-aa1-z, x-aa3-aa2-aa1-z, x-aa4-aa3-aa2-aa1-z and x-
5 aa5-aa4-aa3-aa2-aa1-z, wherein:
aa1 and aa4, independently from each other, represent an arginine amino
acid, an histidine amino acid, an asparagine amino acid or a lysine amino acid,
aa2 represents a serine amino acid, a glycine amino acid, a threonine
amino acid or an alanine amino acid,
10 aa3 represents a phenylalanine amino acid, a leucine amino acid, an
isoleucine amino acid, a tyrosine amino acid or a tryptophan amino acid,
aa5 represents a glutamine amino acid, a glutamic acid amino acid, an
asparagines amino-acid or a lysine amino acid,
x and z, independently from each other, represent:
15 -H;
- OH, SH ;
- a halogen atom,
- an amino group,
- an alkyl, haloalkyl or heteroalkyl group containing from 1 to 30 carbon
20 atoms, linear or branched,
- an alkenyl or alkynyl group containing from 2 to 30 carbon atoms, linear
or branched,
- an acyl group containing from 1 to 30 carbon atoms, saturated or
unsaturated, linear or branched,
25 - a cycloalkyl, cycloakenyl, cycloalkynyl or an heterocyclic group
containing from 3 to 30 carbon atoms, linear or branched,
- one or more aryl or heteroaryl groups containing from 3 to 10 carbon
atoms per cycle,
- a alkoxy, thioalkyl, sulf onylalkyl, aminoalkyl containing from 1 to 30
30 carbon atoms, linear or branched,
- one or more heterocyclic group, containing from 5 to 10 carbon atoms
per cycle,
said groups being optionally substituted by one or more halogen atoms, alkyl
groups, hydroxy groups, alkoxy groups, aryloxy groups, acyloxy groups,
35 carbamoyloxy groups, carboxy groups, mercapto groups, alkylthio groups,
acylthio groups, arylthio groups, aryl groups, heterocyclic groups, heteroaryl
groups or amino groups,
with the proviso that when the compound is x-QRFSR-z and z is H, then x is
different from H and from a tyrosine amino acid and when the compound is
5 x-RFSR-z and z is H, then x is different a pyroglutamate residue.
2. The compound of claim 1, wherein said peptide is selected from the
group consisting of x-SR-z, x-FSR-z, x-RFSR-z and x-QRFSR-z.
10 3. The compound of claim 1 or 2, wherein z is H or NH2 and x is a
saturated or unsaturated, linear or branched fatty acid containing from 2 to 24
carbon atoms and possibly substituted by one or more heteroatoms.
4. The compound of claim 3, wherein x is a palmitic acid or a stearic acid.
15
5. The compound of claim 3, wherein x is an omega-3 or an omega-6 fatty
acid.
6. The compound of claim 5, wherein x is an arachidonic acid.
20
7. The compound of claim 1 or 2, wherein z is H and x is an acetic acid.
8. A composition comprising the compound of any one of claims 1 to 7, in
association with an acceptable carrier.
25
9. The composition of claim 8, wherein said composition is a cosmetic or
pharmaceutical topical composition.
10. The composition according to claim 9, wherein said topical
30 composition further comprises at least one additive and/or active agent
common in dermocosmetics.
11. The composition of any one of claims 8 to 10, wherein said
composition is used in a form selected from the group consisting of emulsions,
35 dispersions, solutions, suspensions, liposomes, chylomicrons, nanocapsules,
microcapsules, macrcapsules, nanoparticules, microparticules,
macroparticules, creams, lotions, ointments, milks, gels, cleansers,
foundations, anhydrous preparations (sticks, body and bath oils), shower and
bath gels, shampoos and scalp treatment lotions, cream or lotion for care of the
skin or hair, sun-screen lotions, milks or creams, artificial suntan lotions, milks,
5 shaving creams or foams, aftershave lotions, make-up, mascaras or nail
varnishes, lipsticks, serums, adhesive or absorbent materials, transdermal
patches, powders, emollient lotion, emollient milk, emollient cream, sprays,
sprayable formulation, oils for the body and the bath, foundation tint bases,
pomade, colloid, compact or solid suspension, pencil, brossable, mouthwash,
10 toothpaste tooth liquid gel, an oral or dental care product.
12. The composition of any one of claims 8 to11, wherein said compound
is present at a concentration between 0.00001% (w/w) and 100% (w/w),
preferably at a concentration between 0.0001% (w/w) and 20% (w/w), and
IS more preferably a concentration between 0.001% and 5% (w/w) by weight of
the composition.
13. Use of the compound of any one of claims 1 to 7, for the preparation
of a medicament for treating of preventing pain.
20
14. Use of the compound of any one of claims 1 to 7, in a topical cosmetic
formulation.
15. A process of cosmetically treating human skin or a mucous membrane
25 comprising applying an effective amount of the topical composition of any one
of claims 9 to 12 to said skin or mucous membrane.

The invention relates to a compound that inhibits a metallo-ectopeptidase, selected in the
group consisting of x-aa2-aal-z, x-aa3-aa2-aal-z, x-aa4-aa3- aa2-aal-z and x-aa5-aa4-aa3-
aa2-aal-z, wherein: aal and aa4, independently from each other, represent an arginine
amino acid, an histidine amino acid, an asparagine amino acid or a lysine amino acid, aa2
represents a serine amino acid, a glycine amino acid, a threonine amino acid or an alanine
amino acid, aa3 represents a phenylalanine amino acid, a leucine amino acid, an isoleucine
amino acid, a tyrosine amino acid or a tryptophan amino acid, aa5 represents a glutamine
amino acid, a glutamic acid amino acid, an asparagines amino-acid or a lysine amino acid, x
and z, independently from each other, represent: - H; - OH1 SH; - a halogen atom, - an
amino group, - an alkyl, haloalkyl or heteroalkyl group containing from 1 to 30 carbon
atoms, linear or branched, - an alkenyl or alkynyl group containing from 2 to 30 carbon
atoms, linear or branched, - a cycloalkyl, cycloakenyl, cycloalkynyl or an heterocyclic
group containing from 3 to 30 carbon atoms, linear or branched, - one or more aryl or
heteroaryl groups containing from 3 to 10 carbon atoms per cycle, - a alkoxy, thioalkyl, sulf
onylalkyl, aminoalkyl containing from 1 to 30 carbon atoms, linear or branched, - one or
more heterocyclic group, containing from 5 to 10 carbon atoms per cycle, said groups being
optionally substituted by one or more halogen atoms, alkyl groups, hydroxy groups, alkoxy
groups, aryloxy groups, acyloxy groups, carbamoyloxy groups, carboxy groups, mercapto
groups, alkylthio groups, acylthio groups, arylthio groups, aryl groups, heterocyclic groups,
heteroaryl groups or amino groups.