Note: Descriptions are shown in the official language in which they were submitted.
CA 02355874 2001-06-18
Inhibitors of the integrin a"(36
The invention describes novel peptides which, as
ligands of the integrin a~~36, are biologically active.
These peptides all have a common structural motif,
namely - Asp Leu Xaa Xaa Leu -, or in a preferred form
- Arg Xaa Asp Leu Xaa Xaa Leu Arg -, where Xaa is any
desired amino acid residue. The peptides according to
the invention can be employed as effective inhibitors
of the aV(36 integrin receptor and thus for the treatment
of various diseases and pathological findings.
Integrins belong to the Class I family of heterodimers
- transmembrane receptors which play an important part
in numerous cell-matrix and cell-cell adhesion
processes (Tuckwell et al., 1996, Symp. Soc. Exp. Biol.
47). They can be roughly divided into three classes:
the (31 integrins, which are receptors for the
extracellular matrix, the (32 integrins, which are
activatable on leucocytes and are "triggered" during
inflammatory processes, and the aV integrins, which
affect the cell response during wound healing and other
pathological processes (Marshall and Hart, 1996, Semin.
Cancer Biol. 7, 191).
The integrins a5(31, aIIbN3i aay. a~(~m a~(33 and aV(36 all
bind to the Arg-Gly-Asp (RGD) peptide sequence, e.g. in
the natural ligand fibronectin. Soluble RGD-containing
peptides are able to inhibit the interaction of each of
these integrins with fibronectin. a~~i6 is a relatively
rare integrin (Busk et al., 1992 J. Biol. Chem. 267(9),
5790), which is formed in increased amounts during
repair processes in epithelial tissue and preferably
binds the natural matrix molecules fibronectin and
tenascin (Wang et al., 1996, Am. J. Respir. Cell Mol.
Biol. 15(5), 664). The physiological and pathological
functions of a~(36 are still not precisely known;
however, it is suspected that this integrin plays an
important part in physiological processes and disorders
J a n .18 . 2 0 01 12 :10 P M ~ 02355874 2001-06-18 N o . 4 8 6 3 P . 2
. , -2_
(e. g. infections, wound healings, tumors) in which epithelial cells
are involved. Thus, a"~6 is expressed on keratinocytes in wounds
(Iiaapasalmi et al., 1996, J. Invest. Dermatol. 106(1), 42), from
which it can be assumed that, in addition to wound healing
processes and infections, other patholigcal occurrences of the
skin, for exempla, psoriasis, can be affected by agonists or
antagonists of the said integrity. Furthermore, a~(36 plays a role in
respiratory tract epithelium (Weinacker et al., 1996, Am. J.
Respir. Cell. Mol. Biol. 12(5), 547), so that appropriate
agonists/antagonists of this integrity can be used successfully in
respiratory diseases such as bronchitis, asthma, pulmonary fibrosis
and respiratory tract tumors. Finally, it is known that a"~6 also
plays a role in intestinal epithelium, so that appropriate integrity
agoniets/antagonists could also be used in the treatment of
infections, tumors and wounds of the gastro-intestinal tract.
To date, no low-molecular inhibitor has been found which
s~elactivel.y binds the a,,(~6 integrity. Thus, the vb~ect was to find,
in addition to the previously known natural high-molecular ligands
and antibodies which are difficult to handle therapeutically and
diagnostical7.y, novel, specific or selective low-molecular ligands
for a"~6, preferably peptides, which can be used for the
aforementioned therapeutic fields and also as a diagnostic or
reagent.
It was found that the peptide compounds of the formulae noted below
and their salts as soluble molecules exert action on cells which
carry the noted receptor or, if they are bound to surfaces,
represent synthetic ligands for the a"~'6 transmitted cell adhesion.
Above all, they act as a"/36 inhibitors, in particular, they inhibit
the interaction of the receptor with other ligands, such as e.g.
the
CA 02355874 2001-06-18
- 3 -
binding of fibronectin. This action can be demonstrated
by the method which is described by J.W. Smith et al.
in J. Biol. Chem. 265, 12267-12271 (1990).
The dependence of the origin of angiogenesis on the
interaction between vascular integrins and
extracellular matrix proteins is described by P.C.
Brooks, R.A. Clark and D.A. Cheresh in Science 264,
569-71 (1994).
It was furthermore found that the new substances have
very valuable pharmacological properties together with
good tolerability and can be employed as medicaments.
This is described in greater detail further below.
The peptide compounds according to the invention can
furthermore be used in vivo as diagnostics for the
detection and localization of pathological conditions
in the epithelial system if they are equipped with the
appropriate markers (e. g. the biotinyl radical)
according to the prior art. The invention also
encompasses conjugates with other active compounds,
such as cytotoxic active compounds, as well as
conjugates with radiolabels for radiotherapy or PET
diagnosis but also fusion proteins with marker proteins
such as GFP or antibodies, or therapeutic proteins such
as IL-2.
The invention thus relates to peptide compounds of the
formula I
W1-XlnArg XZ Asp Leu X3X4Leu X5X6m-WZ I
in which:
X1, X2, X3, X4, X5, X6 each independently of one another
are an amino acid residue, the amino acids
independently of one another being selected from a
group consisting of Ala, Asn, Asp, Arg, Cys, Gln, Glu,
Gly, Phe, His, Ile, Leu, Lys, Met, Nle, homo-Phe, Phg,
Pro, Ser, Thr, Trp, Tyr or Val, and the amino acids
mentioned possibly also being derivatized,
CA 02355874 2001-06-18
- 4 -
WZ is selected from a group OH, OR, NHR, NR2, NH2,
W1 is H or an acyl radical
R is alkyl having 1-6 C atoms and
n,m each independently of one another are a number
from 0-15. In the cases in which m or n assumes a value
of greater than 1, the radicals X1 and X6 can each
independently of one another be identical or different.
According to the invention, those amino acids or amino
acid residues are also encompassed which, starting from
the natural amino acids, are derivatized, or are
homologues or isomers thereof. The amino acid residues
are customarily linked to one another via their a-amino
and a-carboxyl groups (peptide bonding).
The invention furthermore preferably relates to those
peptide compounds in which XZ is an amino acid residue
which was selected from the group consisting of Thr,
Ser, Asp and glycine, furthermore those peptide
compounds in which X3 is an amino acid residue which was
selected from the group consisting of Asp, Glu, Arg,
Lys, His and Tyr, and finally those peptide compounds
in which X4 is an amino acid residue which was selected
from the group consisting of Ser, Tyr, Thr, Gly and
Val.
The preferred compounds (for meanings or abbreviations
see above and below) thus include those of the general
formula II
W1 -X1"ArgThr AspLeu X3X4Leu Arg X6m-WZ IIa,
W1 -XlnArgSer AspLeu X3X4Leu Arg X6m-WZ IIb,
-XlnArgAsp AspLeu X3X4Leu Arg X6m-W2 IIc,
W1
W1 -XlnArgSer AspLeu X3X4Leu Arg X6m-WZ IId,
W1 -XlnArgGly AspLeu X3X4Leu Arg X6m-WZ IIe,
and thegeneral formula II I
those
of
W1 -XlnArgX2As p Arg X6m-WZ IIIa,
Leu
Asp
X4Leu
CA 02355874 2001-06-18
- 5 -
W1 -XlnArgXZAsp Leu Glu X4Leu Arg X6m-WZ IIIb,
W1 -XlnArgXZAsp Leu Arg X4Leu Arg X6m-WZ IIIc,
W1 -XlnArgXZAsp Leu Lys X4Leu Arg Xm- W2 IIId,
W1 -XlnArgXZAsp Leu His X4Leu Arg X6m-W2 IIIe,
-XlnArgX2Asp Leu Tyr X4Leu Arg X6m-W2 IIIf,
W1
and he general IV
those formula
of
t
W1-XlnArg XZAsp Leu X3Ser Leu Arg X6m-W2 IVa,
W1-XlnArg X2Asp Leu X3Tyr Leu Arg X6m-W2 IVb,
W1-XlnArg X2Asp Leu X3Thr Leu Arg X6m-Wz IVc,
W1-XlnArg X2Asp Leu X3Gly Leu Arg X°m-W2 IVd,
W1-XlnArg X2Asp Leu X3Val Leu Arg X6m-WZ IVe.
Particularly preferred peptide compounds according to
the invention are those of the formula V
W1-XlnArg Thr Asp Leu Asp Ser Leu Arg X6m-WZ V
and in this context in particular those of the formula
VI
W1-XlnArg Thr Asp Leu Asp Ser Leu Arg Thr X6m-i-W2 VI.
Finally, the following individual compounds are
particularly preferred, those also being included which
are modified at the N and C termini:
(a) H-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-Thr-Tyr-Thr-Leu-
OH
(b) H-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-OH
(c) Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-Thr-OH
(d) Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-Thr-NHZ
(e) H-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-Thr-OH
(f) H-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-Thr-NHz
(g) H-Arg-Thr-Asp-Leu-Tyr-Tyr-Leu-Arg-Thr-Tyr-OH
(h) Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NHZ
The abbreviations mentioned above and below stand for
the radicals of the following amino acids:
CA 02355874 2001-06-18
- 6 -
Ala A alanine
Asn N asparagine
Asp D aspartic acid
Arg R arginine
Cys C cysteine
Gln Q glutamine
Glu E glutamic acid
G1y G glycine
His H histidine
Ile I isoleucine
Leu L leucine
Lys K lysine
Met M methionine
Nle norleucine
Orn ornithine
Phe F phenylalanine
phg phenylglycine
Pro P proline
Ser S serine
Thr T threonine
Trp W tryptophan
Tyr Y tyrosine
Val V valine
If the abovementioned amino acids can occur in a number
of enantiomeric forms, all these forms and also their
mixtures are included above and below, e.g. as a
constituent of the compounds of the formulae I-VI.
Furthermore, the amino acids for example, as a
constituent of compounds of the formulae I-VI, can be
provided with appropriate protective groups known per
se.
The compounds of the formulae I-VI can have one or more
chiral centres and therefore occur in various
stereoisomeric forms. The formulae indicated include
all these forms, in particular the D and L forms,
especially in enantiomeric and racemic mixtures.
Finally, the formulae I and II mentioned above and
below according to the invention also include the
CA 02355874 2001-06-18
_ '7 _
corresponding salts, in particular the corresponding
physiologically acceptable salts.
So-called prodrug derivatives are also included in the
compounds according to the invention, i.e. compounds of
the formula I modified with, for example, alkyl or acyl
groups, sugars or oligopeptides, which are rapidly
cleaved in the body to give the active compounds
according to the invention. Furthermore, derivatives
are also included in the compounds according to the
invention which consist of the actual peptides
according to the invention and known marker compounds
which make it possible to detect the peptides easily.
Examples of such derivatives are biotinylated or
fluorescence-labelled peptides.
In general, the peptides according to the invention are
linear, but they can also be cyclized. The invention
comprises not only the peptides of the formulae I to VI
mentioned but also mixtures and preparations which in
addition to these compounds according to the invention
also contain other pharmacological active compounds or
adjuvants which can influence the primary
pharmacological action of the peptides according to the
invention in a desired manner.
The compounds according to the invention and also the
starting substances for their preparation are otherwise
prepared by methods which are known per se and
frequently employed, such as are described in the
literature (e. g. in the standard works such as Houben-
Weyl, Methoden der organischen Chemie [Methods of
Organic Chemistry], Georg-Thieme-Verlag, Stuttgart),
namely under reaction conditions which are known and
suitable for the reactions mentioned. Use can also be
made here of variants which are known per se.
Preferably, the peptides according to the invention can
be prepared by means of solid-phase synthesis and
subsequent removal and purification, as has been
CA 02355874 2001-06-18 .
described, for example, by Jonczyk and Meienhofer
(Peptides, Proc. 8th Am. Pept. Symp., Eds. V. Hruby and
D.H. Rich, Pierce Comp. III, p. 73-77, 1983, or Angew.
Chem. 104, 1992, 375), or according to Merrifield (J.
Am. Chem. Soc. 94, 1972, 3102). Otherwise, they can be
prepared by customary methods of amino acid and peptide
synthesis, such as are known, for example, from
Novabiochem - 1999 Catalog & Peptide Synthesis Handbook
of Calbiochem-Novabiochem GmbH, D-65796 Bad Soden, from
numerous standard works and published patent
applications. Biotinylated or fluorescence-labelled
peptides/proteins can likewise be prepared by standard
methods (e.g. E.A. Bayer and M. Wilchek in Methods of
Biochemical Analysis, Vol. 26, The Use of the Avidin-
Biotin Complex as a Tool in Molecular Biology; and
Handbook of Fluorescent Probes and Research Chemicals,
6th Edition, 1996, by R.P. Haugland, Molecular Probes,
Inc.; or alternatively WO 97/14716).
Of course, the peptides of the formulae I-VI can also
be liberated by solvolysis, in particular hydrolysis,
or by hydrogenolysis of their functional derivatives.
Preferred starting substances for the solvolysis or
hydrogenolysis are those which, instead of one or more
free amino and/or hydroxyl groups, contain
corresponding protected amino and/or hydroxyl groups,
preferably those which, instead of an H atom which is
connected to an N atom, carry an amino protective group
or which, instead of the H atom of a hydroxyl group,
carry a hydroxyl protective group.
The same applies to carboxylic acids which can be
protected by substitution of their -CO-OH hydroxyl
function by means of a protective group, e.g. as an
ester.
The expression "amino protective group" is generally
known and relates to groups which are suitable for
protecting (for blocking) an amino group from chemical
reactions, but which are easily removable after the
CA 02355874 2001-06-18
- 9 -
desired chemical reaction has been carried out at other
positions in the molecule. The expression "hydroxyl
protective group" is likewise generally known and
relates to groups which are suitable for protecting a
hydroxyl group from chemical reactions, but which are
easily removable after the desired chemical reaction
has been carried out at other positions in the
molecule. The liberation of the compounds from their
functional derivatives is carried out - depending on
the protective group used - e.g. using strong acids,
expediently using TFA or perchloric acid, but also
using other strong inorganic acids such as hydrochloric
acid or sulfuric acid, strong organic carboxylic acids
such as trichloroacetic acid or sulfonic acids such as
benzene- or p-toluenesulfonic acid. Hydrogenolytically
removable protective groups (e.g. CBZ or benzyl) can be
removed by treating with hydrogen in the presence of a
catalyst (e.g. of a noble metal catalyst such as
palladium, expediently on a support such as carbon).
The procedures are generally known and are not to be
described in greater detail here.
As already mentioned, the peptides according to the
invention include their physiologically acceptable
salts, which can likewise be prepared by standard
methods. Thus, a base of the formula I can be converted
into the associated acid addition salt using an acid,
for example by reaction of equivalent amounts of the
base and of the acid in an inert solvent such as
ethanol and subsequent evaporation. For this reaction,
suitable acids are in particular those which yield
physiologically acceptable salts. Thus inorganic acids
can be used, e.g. sulfuric acid, nitric acid,
hydrohalic acids such as hydrochloric acid or
hydrobromic acid, phosphoric acids such as
orthophosphoric acid, sulfamic acid, furthermore
organic acids, in particular aliphatic, alicyclic, or
araliphatic, aromatic or heterocyclic mono- or
polybasic carboxylic, sulfonic or sulfuric acids, e.g.
CA 02355874 2001-06-18
- 10 -
formic acid, acetic acid, propionic acid, pivalic acid,
diethylacetic acid, malonic acid, succinic acid,
pimelic acid, fumaric acid, malefic acid, lactic acid,
tartaric acid, malic acid, citric acid, gluconic acid,
ascorbic acids, nicotinic acid, isonicotinic acid,
methane- or ethanesulfonic acid, ethanedisulfonic acid,
2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-
toluenesulfonic acid, naphthalenemono- and disulfonic
acids, laurylsulfuric acid. Salts with physiologically
unacceptable acids, e.g picrates, can be used for the
isolation and/or purification of the compounds
according to the invention. On the other hand, an acid
of the formula I can be converted into one of its
physiologically acceptable metal or ammonium salts by
reaction with a base. Possible salts in this case are
in particular the sodium, potassium, magnesium, calcium
and ammonium salts, furthermore substituted ammonium
salts, e.g. the dimethyl-, diethyl- or
diisopropylammonium salts, monoethanol-, diethanol- or
diisopropylammonium salts, cyclohexyl- or
dicyclohexylammonium salts, dibenzylethylenediammonium
salts, furthermore, for example, salts with arginine or
lysine.
The peptide compounds according to the invention can be
employed, as already mentioned, as pharmaceutical
active compounds in human and veterinary medicine, in
particular for the prophylaxis and/or therapy of
disorders in which epithelial cells are involved.
Particularly to be emphasized in this context are
disorders or inflammations or wound healing processes
of the skin, the respiratory organs and the stomach and
intestinal area, thus, for example, apoplexy, angina
pectoris, oncoses, osteolytic illnesses such as
osteoporosis, pathologically angiogenic illnesses such
as, for example, inflammations, pulmonary fibrosis,
ophthalmological illnesses, diabetic retinopathy,
macular degeneration, myopia, ocular histoplasmosis,
rheumatoid arthritis, osteoarthritis, rubeotic
CA 02355874 2001-06-18
- 11 -
glaucoma, ulcerative colitis, Crohn's disease,
atherosclerosis, psoriasis, restenosis after
angioplasty, in acute kidney failure or nephritis.
The invention accordingly relates to peptide compounds
of the formulae defined above and below and in the
claims including their physiologically acceptable salts
as medicaments, diagnostics or reagents.
The invention relates in particular to appropriate
medicaments as inhibitors for the control of disorders
which are based indirectly or directly on expression of
the a,~(36 integrin receptor, thus in particular on
pathologically angiogenic disorders, thromboses,
cardiac infarct, coronary heart disorders,
arteriosclerosis, tumours, osteoporosis, inflammations,
infections and for influencing wound healing processes.
The invention also relates to appropriate
pharmaceutical preparations which comprise at least one
medicament of the formulae I to VI and, if appropriate,
vehicles and/or excipients.
The invention furthermore relates to the use of the
peptide compounds and/or their physiologically
acceptable salts according to the claims and the
description for the production of a medicament for
controlling disorders which are based indirectly or
directly on expression of the a,~(36 integrin receptor,
thus in particular in pathologically angiogenic
disorders, thromboses, cardiac infarct, coronary heart
disorders, arteriosclerosis, tumours, osteoporosis,
inflammations, infections and for influencing wound
healing processes. The medicaments according to the
invention or pharmaceutical preparations comprising
them can be used in human or veterinary medicine.
Possible excipients are organic or inorganic substances
which are suitable for enteral (e.g. oral) or
parenteral administration or topical application or for
CA 02355874 2001-06-18
- 12 -
administration in the form of an inhalation spray and
do not react with the new compounds, for example water,
vegetable oils, benzyl alcohols, alkylene glycols,
polyethylene glycols, glycerol triacetate, gelatin,
carbohydrates such as lactose or starch, magnesium
stearate, talc, petroleum jelly. Tablets, pills, coated
tablets, capsules, powders, granules, syrups, juices or
drops, in particular, are used for oral administration,
suppositories are used for rectal administration,
solutions, preferably oily or aqueous solutions,
furthermore suspensions, emulsions or implants, are
used for parenteral administration, and ointments,
creams or powders are used for topical application. The
new compounds can also be lyophilized and the
lyophilizates obtained used, for example, for the
production of injection preparations. The preparations
indicated can be sterilized and/or can contain vehicles
such as lubricants, preservatives, stabilizers and/or
wetting agents, emulsifiers, salts for affecting the
osmotic pressure, buffer substances, colourants,
flavourings and/or one or more further active
compounds, e.g. one or more vitamins.
For administration as an inhalation spray, sprays can
be used which contain the active compound either
dissolved or suspended in a propellant or propellant
mixture (e. g. COZ or chlorofluorohydrocarbons).
Expediently, the active compound is used here in
micronized form, it being possible for one or more
additional physiologically tolerable solvents to be
present, e.g. ethanol. Inhalation solutions can be
administered with the aid of customary inhalers.
The substances according to the invention can as a rule
be administered in analogy to other known, commercially
available peptides (e. g. described in US-A-4 472 305),
preferably in doses between approximately 0.05 and
500 mg, in particular between 0.5 and 100 mg, per dose
unit. The daily dose is preferably between
approximately 0.01 and 20 mg/kg of body weight. The
CA 02355874 2001-06-18
- 13 -
specific dose for each patient depends, however, on all
sorts of factors, for example on the efficacy of the
specific compound employed, on the age, body weight
general state of health and sex, on the diet, on the
time and route of administration, on the excretion
rate, pharmaceutical combination and severity of the
particular disorder to which the therapy applies.
Parenteral administration is preferred.
The invention finally also comprises recombinant DNA
sequences which contain sections which code for peptide
regions which contain the peptide structural motifs of
the formulae I to VI according to the invention.
Such DNA can be transferred to cells by particles, as
is described in Ch. Andree et al. Proc. Natl. Acad.
Sci. 91, 12188-12192 (1994), or the transfer to cells
can be increased by other vehicles, such as liposomes
(A.I. Aronsohn and J.A. Hughes J. Drug Targeting, 5,
163-169 (1997)).
The transfer of such a DNA could accordingly be used in
yeasts, by means of bacculoviruses or in mammalian
cells, for the production of the peptide substances of
this invention.
If an animal or human body is infected with such a
recombinant DNA, the peptides according to the
invention finally themselves formed by the infected
cells can bind directly to the a,~(36 integrin receptor,
for example of tumour cells, and block it.
Appropriate recombinant DNA, which can be prepared by
known and customary techniques, can, for example,
however also be present in the form of virus DNA which
contains sections which code for the virus coat
protein. By infection of a host organism with
recombinant, preferably non-pathogenic viruses of this
CA 02355874 2001-06-18
- 14 -
type, host cells which express the integrin a,~~36 can
preferably be attacked (targeting).
Suitable viruses are, for example, adenovirus species
which have been used a number of times already as
vectors for foreign genes in mammalian cells. A number
of properties make them good candidates for gene
therapy, as can be inferred from S.J. Watkins et al.
Gene Therapy 4, 1004-1012 (1997) (see also J.
Engelhardt et al. Hum. Gene Ther. 4, 759-769 (1993)).
As can be found in A. Fasbender et al. J. Clin. Invest.
102, 184-193 (1998), the limited efficiency of the gene
transfer is a common problem in gene therapy by viral
and non-viral vectors. Using the above-described
additional ligand sequence for a~(36 integrin in the coat
protein of the adenoviruses, an improvement in the
transfer, for example, of cystic fibrosis transmembrane
conductance regulator (CFTR) cDNA can be achieved.
Similarly to the work of T. Tanaka et al. Cancer
Research 58, 3362-3369 (1998), instead of the DNA for
angiostatin the DNA for the sequences of this invention
can also be used for cell transfections by means of
retroviral or adenoviral vectors.
The peptides according to the invention can also be
employed for use in gene therapy in man within a
liposome complex of lipid/peptide/DNA prepared for
transfection of cell cultures together with a liposome
complex consisting of lipid/DNA (without peptide). The
preparation of a liposome complex of lipid/DNA/peptide
is described, for example, in Hart S.L., et al 1998:
Lipid-Mediated Enhancement of Transfection by a Non
Viral Integrin-Targeting Vector, Human Gene Therapy 9,
575-585.
A liposome complex of lipid/peptide/DNA can be
prepared, for example, from the following stock
solutions: 1 ~zg/~1 of lipofectin (equimolar mixture of
CA 02355874 2001-06-18
- 15 -
DOTMA (= N-[1-(2,3-dioleyloxy)propyl]-N,N,N-tri-
methylammonium chloride} and DOPE (dioleylphosphatidyl-
ethanolamine), 10 ug/ml of plasmid DNA and 100 ug/ml of
peptide. For this, both DNA and peptide are dissolved
in cell culture medium. The liposome complex is
prepared by mixing the three components in a specific
weight ratio (lipid: DNA: peptide, for example,
0.75:1:4). Liposome DNA complexes for gene therapy in
man have already been described (Caplen N.J., et al
1995: Liposome-mediated CFTR gene transfer to the nasal
epithelium of patients with cystic fibrosis, Nature
Medicine 1, 39-46).
The invention thus also relates to the use of
appropriately modified recombinant DNA of gene-
releasing systems, in particular virus DNA, for the
control of illnesses which are based indirectly or
directly on an expression of ctV(36 integrin receptors,
thus in particular in pathologically angiogenic
disorders, thromboses, cardiac infarcts, coronary heart
disorders, arteriosclerosis, tumours, osteoporosis,
inflammations, infections and for influencing wound
healing processes.
The new compounds according to the invention can also
be used as integrin ligands for the preparation of
columns for affinity chromatography for preparing
integrins in pure form. The complex of an avidin-
derivatized support material, e.g. Sepharose, and the
new compounds of the formula I is formed by methods
known per se (e. g. E.A. Bayer and M. Wilchek in Methods
of Biochemical Analysis, Vol. 26, The Use of the
Avidin-Biotin Complex as a Tool in Molecular Biology).
Suitable polymeric support materials in this case are
the polymeric solid phases known per se in peptide
chemistry and having preferably hydrophilic properties,
for example crosslinked polysugars such as cellulose,
Sepharose or Sephadex°, acrylamide, polymers based on
polyethylene glycol or Tentakel polymers°.
CA 02355874 2001-06-18
- 16 -
Example 1
Preparation and purification of peptides according to
the invention:
In principle, the preparation and purification was
carried out by means of Fmoc strategy with protection
of acid-labile side chains on acid-labile resins using
a commercially obtainable continuous flow peptide
synthesizer according to the details of Haubner et al.
(J. Am. Chem. Soc. 118, 1996, 17703).
In the following, the synthesis and purification is
described by way of example for the peptide amide
Ac-RTDLDSLR-NH2. For the synthesis of peptide acids, an
o-chlorotrityl chloride resin (Novabiochem) was coated
with the appropriate C-terminal Fmoc amino acid
according to the manufacturer's instructions and used
in the synthesis apparatus according to the
manufacturer's instructions (Milligen). The principal
steps are washing - Fmoc protective group removal -
washing - coupling with the next Fmoc amino acid -
capping (acetylation) - washing. If an N-terminal
acylation is desired after the last amino acid
coupling, this is carried out after removal of the last
Fmoc protective group using the appropriate activated
acyl radical, e.g. the acetic anhydride.
2 g of 9-Fmoc-aminoxanthenyloxy resin (Novabiochem,
0.37 mmol/g) were subjected to a coupling step, for
60 min in each case, in succession with 0.45 g each of
hydroxybenzotriazole hydrate (HOBt), 0.5 ml of
ethyldiisopropylamine, 4 equivalents each of
diisopropylcarbodiimide (DIC) and Fmoc-amino acid in
dimethylformamide (DMF), in a commercial synthesis
apparatus and a typical procedure (apparatus and
Milligen 9050 PepSynthesizer'M Handbook, 1987). Washing
steps were carried out in DMF for 10 min, removal steps
in piperidine/DMF (1:4 vol) for 5 min, N-terminal
acetylations (capping) were carried out for 15 min
using acetic anhydride/pyridine/DMF (2:3:15 vol). The
CA 02355874 2001-06-18
- 17 -
amino acids Fmoc-Arg (Pmc), then Fmoc-Leu, then Fmoc-
Ser(But}, then Fmoc-Asp(OBut), then Fmoc-Leu, then
Fmoc-Asp(OBut), then Fmoc-Thr(But), and finally Fmoc-
Arg(Pmc) were used. After washing with DMF and
isopropanol and subsequent drying in vacuo, 3.48 g of
the N-terminally acetylated peptidyl resin,
Ac-Arg(Pmc)-Thr(But)-Asp(OBut)-Leu-Asp(OBut)-Ser(But)-
Leu-Arg(Pmc)-aminoxanthenyloxy resin, were obtained.
By treatment of this peptidyl resin with trifluoro-
acetic acid/anisole/dichloromethane (74 ml/3.7 ml/
74 ml) for 4 h at room temperature, filtration,
concentration in vacuo and trituration with diethyl
ether, it was possible to obtain a precipitate of 0.6 g
of peptide, Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2.
Purification of the product was carried out by RP-HPLC
on Lichrosorb RP18 (250-25, 7 um, Merck KGaA) in 0.30
TFA using a gradient of 40 on 240 2-propanol in 2 h at
8 ml/min and assessment by means of a UV flow-through
photometer at 215 nm.
The product-containing fractions were freeze-dried.
According to FAB-MS (Fast Atom Bombardment Mass
Spectroscopy), the product obtained corresponded to the
expectations : C41H73Ni50i5M 1015 . 5 g/mol; (M+H) + is 1016.
In the analytical HPLC on SuperSpher RPl8e (250-4,
Merck KGaA) in a gradient of 0-99o A (0.08 M phosphate
pH 3.5, 15o acetonitrile) to B (0.03 M phosphate pH
3.5, 70% acetonitrile) in 50 min, at 1 ml/min, and
detection at 215 nm, the purified product Ac-Arg-Thr-
Asp-Leu-Asp-Ser-Leu-Arg-NHZ has a retention time of
7.22 min.
Further HPLC analyses were carried out in the two
following systems:
System A: 0.3% trifluoroacetic acid having a gradient
of 0-800 2-propanol in 50 min on LichroSpher 60 RP-
Select B° (250-4) (Merck KGaA, Darmstadt, Germany), at
1 ml/min, and detection at 215 nm.
CA 02355874 2001-06-18
- 18 -
System B: 0.1a trifluoroacetic acid having a gradient
of 30-70o acetonitrile in 50 min on SuperSpher 100
RPl8e° (250-4) (Merck KGaA, Darmstadt, Germany), at
1 ml/min and detection at 215 nm.
Example 2
The following peptides shown in Table 1 were prepared
and purified analogously to Example 1.
Table 1:
Structure MW FAB-MS Rt(HPLC)/min Rt(HPLC)/min
(g/mol) [M+H] (System A) (System A)
found
RTDLDSLRTYTL 1453.6 1456 21.9
DSLRTYTL 968.1 969 18.6
RTDLDSL 818.9 820 18.6 23.6
DLDSLRTY 982.1 983 16.6
RTDLDSLR 975.1 975 13.5
RTDLDSLRTY 1239.3 1239 16.6
Ac-RTDLDSLRT 1118.2 1119 16.2 15.6
RTDLDSLRT 1076.2 1076 13.9
RTDLPSLRTY 1221.4 1221 19.2
RTDLDLRT-NHS 988.1 989 13.4
Ac-RTDLDLRT-NHz 1030.2 1031 15.3
RTDLYYLMDL 1302.5 1302 28.2
RTDLDSLRT-NHz 1075.2 1076 11.1 13.8
RTDLDPLRTY 1249.4 1250 16.3
RTDLYYLRTY 1363.5 1363 11.5
Ac-RTDLDSLRT-NHS 1117.2 1118 13.2 15.0
Ac-RTDLDSLR-NHZ 1015.5 1016 See Example
1
TDLDSLRT 920.0 920 14.8
PVDLYYLMDL 1241.5 1241 36.1
The comparison compounds used were known RGD peptides
such as GRGDSPK, cyclo-(RGDfV), and the linear peptide
DLYYLMDL.
CA 02355874 2001-06-18
- 19 -
Example 3
Preparation of an aV~6 integrin preparation:
a~(36 was obtained and purified in soluble transmembrane
truncated form (Weinacker et al. 1994, J. Biol. Chem.
269, 6940) from a Baculovirus expression system
according to recombination techniques known for aV(33
(Mehta et al., 1998, Biochem. J. 330, 861) using
14D9.F8 antibody affinity chromatography (Mitjans et
al., 1995, J Cell Sci. 108, 2825) . Human a~ and (36 cDNA
clones are generally known and commonly accessible. The
transfer vector pAcUW31 (Clontech Lab. Inc., USA),
which allows simultaneous expression of two different
target cDNAs, was employed in order to express
transmembrane truncated aV(36 from recombinant
Baculovirus cells. To this end, an aV transfer vector
was prepared and transmembrane truncated (OTM)a~ was
excised from the plasmid a~OTM (pBAc9) using the
restriction enzymes EcoRI and XbaI (Mehta et al., for
reference see above) and cloned into the BamHI cleavage
site of pAcUW31 downstream of the polyhedrin promoter
by means of blunt-end ligation. Transmembrane truncated
(36 cDNA was excised from the plasmid pCDNAneo(36
(Weinacker et al., for reference see above) using the
restriction enzymes EcoRI and XbaI and likewise cloned
into the BamHI cleavage site of pAcUW31 downstream of
the polyhedrin promoter by means of blunt-end ligation.
The tandem vectors containing truncated a~ and (36 were
used in order to obtain recombinant Baculovirus (Mehta
et al., for reference see above). The recombinant
Baculoviruses were employed in order to infect High
Five insect cells. The soluble receptor was obtained
after culturing for 48-71 hours by passing the
supernatant from the cell culture through affinity
columns of the type indicated above and eluting at pH
3.1. All process steps were carried out at room
temperature and in the absence of any detergents. The
peak fractions were neutralized, concentrated and
dialysed at 40°C and finally stored at -80°C. The
CA 02355874 2001-06-18
- 20 -
recombinant soluble human receptor thus obtained is
biologically active and retains its ligand specificity.
A similar preparation method used for soluble aV(33 was
described in EP 0846 702.
Example 4:
a~/36/Fibronectin receptor binding test:
The prepared peptides according to the invention were
bonded to the immobilized aV(36 receptor in solution
together with competitively acting fibronectin and the
Q value was determined as a measure of the selectivity
of the binding of the peptide to be tested to a~(36. The
Q value is in this case calculated from the quotient of
the ICSO values of test peptide and a standard. The
standard used was the linear hepta-RGD peptide GRGDSPK
(ref./Patent cf. Pytela et al. Science 231, 1559,
(1986) ) .
In detail, the binding test was carried out as follows:
The immobilization of soluble aV(36 receptor on
microtitre plates was carried out by dilution of the
protein solution in TBS++ and subsequent incubation
overnight at 4°C (100 ~1/well). Non-specific binding
sites were blocked by incubation (2 h, 37°C) with 30
(w/v) BSA in TBS++ (200 ul/well). Excess BSA was
removed by washing three times with TBSA++. Peptides
were serially diluted (1:10) in TBSA++ and incubated
with the immobilized integrin (50 ~l of peptide + 50 ul
of ligand per well; 2 h; 37°C) together with
biotinylated fibronectin (2 ug/ml). Unbound fibronectin
and peptides were removed by washing three times with
TBSA++. The detection of the bound fibronectin was
carried out by incubation (1 h; 37°C) with an alkaline
phosphatase-coupled anti-biotin antibody (Biorad)
(1:20,000 in TBSA++; 100 ~1/well). After washing three
times with TBSA++, the colorimetric detection was
carried out by incubation (10-15 min; 25°C, in the
dark) with substrate solution (5 mg of nitrophenyl
CA 02355874 2001-06-18
- 21 -
phosphate, 1 ml of ethanolamine, 4 ml of H20;
100 u1/well). The enzyme reaction was stopped by
addition of 0.4 M NaOH (100 ~l/well). The colour
intensity was determined at 405 nm in an ELISA
measuring apparatus and made equal to the zero value.
wells which were not coated with receptor were used as
a zero value. The standard employed was GRGDSPK. The
ICSO values for the tested peptides were read off from a
graph and the Q value of the peptide according to the
invention was determined from this together with the
ICSO value of the standard peptide. The results of the
test described are summarized in the following table:
Table 2
Structure Q value =
ICSO test peptide/
ICSO standard
peptide
GRGDSPK 1.0 (ICSO= 400 nM)
cyclo-(RGDfV) 0.6
DLYYLMDL Inactive (ICSO>50 ~M)
RTDLDSLRTYTL 0.27
DSLRTYTL Inactive ( ICSO>50 ~ZM)
RRDLDSL 2.5
DLDSLRTY Inactive ( ICSO>50 ~ZM)
RTDLDSLR 0.17
RTDLDSLRTY 0.10
Ac-RTDLDSLRT 0.029
RTDLDSLRT 0.11
RTDLDLRT-NHZ 1.1
Ac-RTDLDLRT-NH2 0.5
RTDLYYLMDL 0.33
RTDLDSLRT-NH2 0.056
RTDLDPLRTY 0.50
RTDLYYLRTY 0.042
Ac-RTDLDSLRT-NH2 0.013
TDLDSLRT 66
PVDLYYLMDL Inactive (ICSO>50 uM)
CA 02355874 2001-06-18
- 22 -
Q values of less than 1 mean that they exhibit a
relatively better binding to the receptor than,
comparatively, the standard peptide, which seen in
absolute terms already has a good binding in
competition with the natural ligand fibronectin.
Example 5
Analogously to the preceding example, for comparison
purposes integrin ligand binding tests were carried out
with different integrins (e. g. aV(33, aV(35) and their
corresponding ligands (e. g. vitronectin, fibrinogen).
Example 6:
General preparation of a DNA-liposome complex and use
for gene therapy:
Lipid and DNA are mixed in the weight ratio 5:1
(lipid:DNA) in Krebs-HEPES solution (140mM NaCl, 1mM
MgCl2, 2mM CaCl2, 6mM KCl, lOmM HEPES, lOmM D-glucose;
pH 9.0). The individual dose here is 30 ~zg of
DNA/200 ul. 200 ul of this lipid-DNA complex are
applied to the nasal epithelium using a pump atomizer.
This is repeated 10 times at an interval of 15 min. The
total dose of DNA is 300 ug.
The following examples relate to pharmaceutical
preparations:
Example A: Inection vials
A solution of 100 g of an active compound of the
formula I and 5 g of disodium hydrogenphosphate are
adjusted to pH 6.5 in 3 1 of double-distilled water
using 2 N hydrochloric acid, sterile-filtered, filled
into injection vials, lyophilized under sterile
conditions and aseptically sealed. Each injection vial
contains 5 mg of active compound.
CA 02355874 2001-06-18
- 23 -
Example B: Suppositories
A mixture of 20 g of an active compound of the formula
I is fused with 100 g of Soya lecithin and 1400 g of
cocoa butter, poured into moulds and allowed to cool.
Each suppository contains 20 mg of active compound.
Example C: Solution
A solution of 1 g of an active compound of the formula
I, 9 . 38 g of NaH2P04 ~ 2H20, 28 . 48 g of Na2HP04 ~ 12H20 and
0.1 g of benzalkonium chloride in 940 ml of double-
distilled water is prepared. It is adjusted to pH 6.8,
made up to 1 1 and sterilized by irradiation. This
solution can be used in the form of eye drops.
Example D: Ointment
500 mg of an active compound of the formula I are mixed
with 99.5 g of petroleum jelly under aseptic
conditions.
Example E: Tablets
A mixture of 1 kg of active compound of the formula I;
4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of
talc and 0.1 kg of magnesium stearate is compressed to
give tablets in a customary manner such that each
tablet contains 10 mg of active compound.
Example F: Coated tablets
Analogously to Example E, tablets are pressed and are
then coated in a customary manner with a coating of
sucrose, potato starch, tragacanth and colourant.
CA 02355874 2001-06-18
- 24 -
Example G: Capsules
2 kg of active compound of the formula I are filled
into hard-gelatin capsules in a customary manner such
that each capsule contains 20 mg of the active
compound.
Example H: Ampoules
A solution of 1 kg of active compound of the formula I
in 60 1 of double-distilled water is sterile-filtered,
filled into ampoules, lyophilized under sterile
conditions and aseptically sealed. Each ampoule
contains 10 mg of active compound.
Example I: Inhalation spray
14 g of active compound of the formula I are dissolved
in 10 1 of isotonic NaC1 solution and the solution is
filled into customary spray containers having a pump
mechanism. The solution can be sprayed into the mouth
or nose. One puff of spray (approximately 0.1 ml)
corresponds to a dose of approximately 0.14 mg.
CA 02355874 2001-06-18
SEQUENZPROTOKOLh _. l ,_
~11G> MewCk Patent 5:n~a-H
<120> Inhibitors or rnregrins avl3n
<130~> P9bSe8:.7-bzrs
<140> pCT/EP99105342
<I41> 1999-12-11
<1.60> 21
<170> Patentln ;ler. 2.1
<210> 1
<22I> 12
<212> PRT
<223> Artifici~:l Se-guence
<220>
.-223> bescription o~ artifzewa? sequence: avf36
inhibitory peptide 1
.400: 1
Arg Thr Asp Leu Asp Ser Leu Arg Thr Tyr Thr Leu
1 5 10
<210> 2
<211> 8
<212> PRT
<213> Axxifi.c~.a~l Seauence
<220>
<223> pescription of artificial sequence; avi36
inhibitory pe~~tide 2
<400> 2
Asp Ser Leu Arg Thr Tyr Thr Leu
1
<210> 3
<211> ~
<212> PRT
<21.~> .L~,Z'~1Z1.C1c31 SC~~ueriCe
<220i '
<223> De~~Cription o.G art.ificial sequence: av136
inhibitory pe~~tide
<400> 3
Arg Thr Asp Leu Asp Ser Teu
1
<210> .3
<21?> g
<212> PRT
<213> Axciticial Se;xuence
<220:
<223> Dascript,~or o:F axCi~icial sequence: a~'f~6
inh~.i~i~cxy pe~:tide
CA 02355874 2001-06-18
<400> 4
Asp Leu Asp Ser Leu Arg Thr Tyr
1 S
<21Q> S
<211> 8
<212> PRT
213> ?~rti.ficial Seguence
<220>
<223> Description of artificial sequence: a~rl~6
inhibitory peptide 5
<400> 5
Arg ihr Ash Leu Asp Ser Leu Arg
1 5
<210> 6
<211> 10
<%12> PRT
<213> Frtiricial Sequence
<220>
<223> pescription of artificial sequence: avi~6
inhibitory peptide n'
<940> 6
Arg Thr Asp LEU Asp Ser Leu Arg Thr Tyr
1 5 10
<210> 7
<2L:.> 9
<212> PRT
<2i3> Artificial Seauence
<220>
<2?1> SITS
<222> tl)
<223> xaa = Rcetyl-Arg
<224>
<223> De3cription of artificial sequence: avTi6
inhibitory peptide 7
<400> 7 '
Xaa Thr Asp Leu Asp Ser Leu Arg Thx
1 5
<210> &
<2i17
<212> PRT
<2?3> Artificial Sequence
<< 2'J>
<223> Description of artificial sequence: avJ36
inhibitory peptide 8
;~o> a
Arg Thr :.sp Leu Asp Ser Lcu erg ~'hx
1 j
CA 02355874 2001-06-18
<21G> 9
<211> 10
<212> PRT _
<213> Artiricial SequencE
<220>
<223> Deser~.pLion cf artificial Sequence: avl3o
inhibitory peptiNa 9
<1G0> 9
Arg Thr Asp Leu Fro Ser Leu Arg Th= Tyr
1 5 1C
<210> la
<211> 8 '
<212> PRT
<213> Artificial Sequence
<220>
<2Z1> SITE
<222> (8)
<223> Xaa = Thr-?~TH?
<zzo>
<223> Description of artificial sequence: avf~G
inhibitory peptide 10
<400> 1G
Arg Thz~ Asp Leu Asp l,eu Arg Kaa
1 S
<210> 11
<211> 8
<212> fRT - -
<213% Artificial Sequence
<220>
<221> SITE
<222> tl)
<2?3> Xaa = Ac2ty1-Arg
<220>
<221> SITE
<222> {$)
<223> xaa = Thx'-1VH2
<220>
<223= Description of artzLicial sequence: av136
.nhiNitory pept-.~de 11
<gpG: 11
xaa Tnr Asp Leu ~.sp Leu Arg xaa
7. 5
<210> 12
<211> 10
212> Fit=
<213> rrtiLici.aw Seauence
X220:
CA 02355874 2001-06-18
<223> Description of artificial sequence: avl~6
inhibitory peptide 12
<.~00> 12 _
Arg Thx :~.sp Leu Tyr Tyr Leu t~tet ASP Leu
1 5 i0
i
<210> 13
<211> 9
<23.z> PRT
<213> Artificial Sequence
<220>
<221> SIiE
<222> {9;
<223> Xaa = Thr-NH2
<zzo>
<223> Description cf artificial sequence: avl3&
inhibitory peptide 13
<400> 13
Arg Thr Asp Lau Asp Sir T.eu ~Axg Xaa
1 S
<210> 14
<z~.l> to
<212> PRT
<213> Artificial Sequences
<220>
<223> Description of artificial sequence: avf36
inhibitory peptide 14
<400% 14
-Arg Thr 1sp Lea Asp Pro Lea Arq Thr Tyr ,
1 5 10
<210> 1S
<zll> to
<212> PRT
<213> Artificial Saquence
<220>
<223> Description of artifLczul_ sequence: av136
inhibitory pept~.de 15
<g00>. 1S
rg Thr Asp Leu Tyr Tyr Lcu Arg Thr Tyr
1 ~ 10
<210> i6
<2i2> g
<212> PRT
<213> Artificial Seauence
<220>
<2ZZ> SITE
<222> (1
<223> Xaa ~ Ace~yl-Arg
CA 02355874 2001-06-18
<220>
<221> SITE
<222> (9)
<223> Xaa = Thr-rlH2
\2207
<223> Descripcio:2 of artificial saquerce: avl~6
inhibitcry peptide 16
«00> :.6
Xaa Thr ?asp Leu Asp 5er Leu Arg Xaa
1 S
<210> 17
<211> 3
<212> PRT
<213> Artificial Sequencc-
<220>
<2z1> STTE
<222> (1)
<223> Xaa = vcetyl--Arg
<224>
<221> SITE
<222> (8)
<223> Xaa = Arg-NH2
<220>
<223> Description of artificial seauence: avl~6
inhibitory peptide 17
<400> 1?
Xaa '~hr Asp Leu Asp Ser Leu Xaa
Z J
<210> 18
<?11> 8
<212> PRT
<213> Ar~Citicial Sequence
<220>
<223> bescripticn of artificial sequence: aerl36
inhihitcry peptide 18
«OO> J.V
Tt~~r asp Leu Asp Ser Leu Arg Thr
1 S
<210> i9
<21I> 1C
<21t> PRT
<213> Artificial 5eguence
<22C>
<223> Description c~ artificial, sequence: am36
ir_hibi Cory peptide 15
<40G> i9
Pro Val Asn L2u Tyr Tyr Leu T~let Asp Leu
i0
CA 02355874 2001-06-18
<~],~> 2~
<2L1> 7
<21~> PRT
<2i3> .~rti ~icial Sc:quenC2
~c220>
<223> Description of artificial sequence: a-~f~n
inhibitory peptide 20
<400> 20
Arg Arg Asp Leu Asp Ser Leu
.1 5
<2i0> 21
<217.> 10
<212> PRT
<220>
<221> SITE
<222> ( 7. )
<22,i> Xaa = each natural aa, dnd Nle, homy-Phe,
Phg oiler H, N-terminal: H oiler Acetyl
<220>
<221> SrTE
<222> (3)
<223> Xaa = each natural aa, and hom- Phe, Phg,
I31 a
<220>
<221> SITE
<222> (6)..(7)
<223> Xaa= each natuz~al aa, and Nle, homo-PhE, Phg '
<220>
<221> SITE .
.222> (9)..110)
<223> Xaa = each natural aa, and Nie, Fhg,
homo-Phe; ac position 9 also H: C-termir~a~.: uH,
FIH2, Ok, NH-Alkyl, N-Alkyl
<400> 21
Xaa Arg Xc~a Asp Leu Xaa X.aa ~.eu Xaa Xaa
1 5 lp
