Note: Descriptions are shown in the official language in which they were submitted.
s.
CA 02377224 2001-12-21
WO 01/00660 PCT/EP00/05404
- 1 -
Inhibitors of the integrin a,"~is
The invention relates to novel peptides of the formula
I which, as ligands of the integrin oc~,~is. are
biologically active
Ac-Arg-X1-Asp-Xz-X3-X4-X5-X6-NH2 I
in which
is acetyl,
Ac
X1 is Ser, Gly, Thr, Asp, Arg, Val, Tyr, His or Ala,
Xz is Leu, Ile, Nle, Val or Phe,
X3 is Asp, Glu, Lys, Phe, Aib, Nal, Gly, Ala, Bgl or
Phg,
is Gly, Ala, Ser, (3-Ala or co-Abu,
X4
X5 is Leu, Ile, Nle, Val or Phe,
X6 is Arg, Har, Lys , Leu, Orn, Phe, Ala, Tyr, Gly,
Ser or Asp,
where the amino acids mentioned can also be
derivatized,
the amino acid residues are linked to one another in
peptide fashion via the a-amino and a-carboxyl. groups,
the D and the L forms of the optically active amino
acid residues are included,
and their physiologically acceptable salts,
and where Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2 is
excluded.
The invention is based on the object of finding novel
compounds having valuable properties, in particular
those which can be used for the production of
medicaments.
It has been found that the compounds according to the
invention and their salts have valuable pharmacological
properties together with good tolerability.
CA 02377224 2001-12-21
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The peptides according to the invention can be employed
as efficacious inhibitors of the oc~,(36 integrin receptor
and thus for the treatment of various diseases and
pathological findings.
Other inhibitors of the integrin oc~,(36 are described in
DE 19858857 and by S. Kraft et al. in J. Biol. Chem.
274, 1979-85 (1999). The compounds according to the
invention are to be considered as a selection invention
with respect to the application mentioned.
Integrins belong to the family of heterodimers of Class
I - transmembrane receptors which play an important
role in numerous cell-matrix or 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 ~iz integrins, which are
activatable on leucocytes and are "triggered" during
inflammatory processes, and the a~, integrins, which
influence the cell response during wound-healing and
other pathological processes (Marshall and Hart, 1996,
Semin. Cancer Biol. 7, 191).
The integrins x5(31, allb(33. oce(31, a."~il, oc~,(33, oc~,(35. ow(3a and
oc~,~i6 all bind to the Arg-Gly-Asp (RGD) peptide sequence
in natural ligands, such as, for example, fibronectin
or vitronectin. Soluble RGD-containing peptides are
able to inhibit the interaction of each of these
integrins with the corresponding natural ligands. oc"(36
is a relatively rare integrin (Busk et al., 1992 J.
Biol. Chem. 267(9), 5790), which is formed to an
increased extent in repair processes in epithelial
tissue and preferably binds the natural matrix
molecules fibronectin and tenascin (Want et al., 1996,
Am. J. Respir. Cell Mol. Biol. 15(5), 664). The
physiological and pathological functions of oc~,(36 are
still not precisely known; it is suspected, however,
that this integrin plays an important role in
CA 02377224 2001-12-21
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physiological processes and disorders (e. g.
inflammation, wound healing, tumours) in which
epithelial cells are involved. Thus oc~,~36 is expressed on
keratinocytes in wounds (Haapasalmi et al., 1996, J.
Invest. Dermatol. 106(1), 42), from which it is to be
assumed that in addition to wound-healing processes and
inflammation other pathological skin events, such as,
for example, psoriasis, can also be influenced by
agonists or antagonists of the said integrin, oc~,~36
furthermore plays a role in the respiratory tract
epithelium (Weinacker et al., 1995, Am. J. Respir. Cell
Mol. Biol. 12(5), 547), so that appropriate
agonists/antagonists of this integrin could be
successfully employed in respiratory tract disorders,
such as bronchitis, asthma, pulmonary fibrosis and
respiratory tract tumours. Finally, it is known that
oc"~i6 also plays a role in the intestinal epithelium, so
that appropriate integrin agonists/antagonists could be
used in the treatment of inflammation, tumours and
wounds of the stomach/intestinal tract.
The dependence of the formation of angiogenesis on the
interaction between vascular integrins and extra-
cellular matrix proteins is described by P.C. Brooks,
R.A. Clark and D.A. Cheresh in Science 264, 569-71
(1994).
The object was therefore, in addition to the previously
known natural high molecular weight ligands and
antibodies, which are therapeutically and diagnos-
tically difficult to handle, to find potent, specific
and selective low molecular weight ligands for a,~,(36,
preferably peptides, which can be used for the
therapeutic areas mentioned, but also as diagnostic or
reagent.
It has been found that the peptide compounds according
to the invention and their salts, as soluble molecules,
exert an effect on cells which carry the said receptor,
a.
CA 02377224 2001-12-21
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or if they are bound to surfaces, are artificial
ligands for a,~,(36-mediated cell adhesion. Above all, they
act as oc~,(36 integrin inhibitors, where they particularly
inhibit the interactions of the receptor with other
ligands, such as, for example, the binding of fibro-
nectin. This action can be detected, for example, by
the method which is described by J.W. Smith et al. in
J. Biol. Chem. 265, 12267-12271 (1990).
It has furthermore been found that the novel substances
have very valuable pharmacological properties together
with good tolerability and can be employed as
medicaments. This is described more precisely 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
appropriate markers (e. g. the biotinyl radical)
according to the prior art.
The invention also comprises combinations with at least
one other active compound and/or conjugates with other
active compounds, such as cytotoxic active compounds
and conjugates with radiolabels for X-ray therapy or
PET diagnosis, but also fusion proteins with marker
proteins such as GFP or antibodies, or therapeutic
proteins such as IL-2.
Some preferred groups of compounds can be expressed by
the following subformulae Ia to If, which correspond to
the formula I and in which the radicals not designated
in greater detail have the meaning indicated in the
formula I, but in which
in a) X1 is Ser, Gly or Thr;
in b) X1 is Ser, Gly or Thr,
XZ is Leu;
CA 02377224 2001-12-21
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in c) X1 is Ser, Gly or Thr,
X2 is Leu,
X3 is Asp or D-Asp;
in d) X1 is Ser, Gly or Thr,
Xz is Leu,
X3 is Asp or D-Asp,
X4 is Gly, Ala or Ser;
in e) X1 is Ser, Gly or Thr,
XZ is Leu,
X3 is Asp or D-Asp,
X4 is Gly, Ala or Ser,
X5 is Leu;
in f) X1 is Ser, Gly or Thr,
Xz is Leu,
X3 is Asp or D-Asp,
X4 is Gly, Ala or Ser,
XS is Leu,
X6 is Arg;
and their salts.
The invention relates in particular to peptide
compounds selected from the group consisting of
Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2,
Ac-Arg-Gly-Asp-Leu-Asp-Ser-Leu-Arg-NH2,
Ac-Arg-Ser-Asp-Leu-Asp-Ser-Leu-Arg-NH2,
Ac-Arg-Asp-Asp-Leu-Asp-Ser-Leu-Arg-NHz,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Ala-NHZ,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-D-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Arg-NH2,
Ac-D-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-D-Ala-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-Aib-Ser-Leu-Arg-NHZ,
Ac-Arg-Thr-Asp-Leu-D-Nal-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-Gly-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-Ala-Ser-Leu-Arg-NH2,
CA 02377224 2001-12-21
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Ac-Arg-Thr-Asp-Nle-D-Asp-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Ile-D-Asp-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-Asp-D-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-Asp-Ala-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-Asp-Gly-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Har-NH2,
Ac-Arg-Thr-Asp-Leu-Asp-Ser-Leu-Lys-NH2,
Ac-Arg-Thr-Asp-Leu-D-Asp-D-Ser-Leu-Arg-NH2,
Ac-Arg-Thr-Asp-Leu-D-Asp-Ser-Leu-Ala-NHz,
Ac-Arg-Thr-Asp-Leu-D-Asp-Gly-Leu-Arg-NHZ,
and their physiologically acceptable salts.
The abbreviations of amino acid residues mentioned
above and below stand for the radicals of the following
amino acids:
Abu 4-aminobutyric acid
Aha 6-aminohexanoic acid, 6-aminocaproic acid
Aib a-aminoisobutyric acid
Ala alanine
Asn asparagine
Asp aspartic acid
Arg arginine
Bgl C-alpha-tert-butylglycine
Cys cysteine
Dab 2,4-diaminobutyric acid
Dap 2,3-diaminopropionic acid
Gln glutamine
Glp pyroglutamic acid
Glu glutamic acid
Gly glycine
Har homoarginine
His histidine
homo-Phe homo-phenylalanine
Ile isoleucine
Leu leucine
Lys lysine
Met methionine
Nal naphth-2-ylalanine
r.
CA 02377224 2001-12-21
- '
Nle norleucine
Orn ornithine
Phe phenylalanine
Phg phenylglycine
4-Hal -Phe 4-halophenylalanine
Pro proline
Ser serine
Thr threonine
Trp tryptophan
Tyr tyrosine
Val valine.
In addition, the following have the meanings below:
Ac acetyl
BOC tert-butoxycarbonyl
BSA bovine serum albumin
CBZ or benzyloxycarbonyl
Z
DCC1 dicyclohexylcarbodiimide
DMF dimethylformamide
EDC1 N-ethyl-N, N'-(dimethylaminopropyl)-
carbodiimide
Et ethyl
FCA fluoresceincarboxylic acid
FITC fluorescein isothiocyanate
Fmoc 9-fluorenylmethoxycarbonyl
FTH fluoresceinthiourea
HOBt 1-hydroxybenzotriazole
Me methyl
MBHA 4-methylbenzhydrylamine
Mtr 4-methoxy-2,3,6-trimethylphenylsulfonyl.
HONSu N-hydroxysuccinimide
OBut tert-butyl ester
Oct octanoyl
OMe methyl ester
OEt ethyl ester
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl
Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl
CA 02377224 2001-12-21
POA phenoxyacetyl
Sal salicyloyl
TBS++ tris buffered saline with divalent cations
TBSA TBS + BSA
TBTU 2-(1H-benzotriazol-1-yl)-1,1,3-
tetramethyluronium tetrafluoroborate
TFA trifluoroacetic acid
Trt trityl (triphenylmethyl).
If the abovementioned amino acids can occur in two or
more enantiomeric forms, all these forms and their
mixtures (for example the DL forms) are included above
and below. In addition, the amino acids can be provided
with appropriate protective groups which are known per
se.
So-called prodrug derivatives are included in the
compounds according to the invention, that is compounds
modified with, for example, alkyl or acyl groups,
sugars or oligopeptides, which are cleaved rapidly in
the body to give the active compounds according to the
invention. These also include biodegradable polymer
derivatives of the compounds according to the
invention, as is described, for example, in Int. J.
Pharm. 115, 61-67 (1995).
The amino acids and amino acid residues mentioned, such
as, for example, the NH functions or alternatively the
C-terminal amide function, can also be derivatized, the
N-methyl, N-ethyl, N-propyl, N-benzyl or Ca-methyl
derivatives being preferred. Derivatives which are
additionally preferred are those of Asp and Glu, in
particular the methyl, ethyl, propyl, butyl, tert-
butyl, neopentyl or benzyl esters of the side chain
carboxyl groups, and in addition also derivatives of
Arg, which can be substituted on the -NH-C(=NH)-NHZ
group by an acetyl, benzoyl, methoxycarbonyl or
ethoxycarbonyl radical.
CA 02377224 2001-12-21
_ g _
In the compounds according to the invention, in
addition to the compounds of the formula I which carry
an acetyl group N-terminally, those compounds are also
included in which Ac is replaced by another acyl
function, such as, for example, propionyl, butyryl or
alternatively benzoyl.
Furthermore, derivatives additionally included in the
compounds according to the invention are those 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 radiolabelled, biotinylated or
fluorescence-labelled peptides.
A fluorescent dye radical is preferably 7-acetoxy-
coumarin-3-yl, fluorescein-5-(and/or 6-)yl, 2',7'-
dichlorofluorescein-5-(and 6-)yl, dihydrotetramethyl-
rosamin-4-yl, tetramethylrhodamin-5-(and 6-)yl, 4,4-
difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-
ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-
s-indacene-3-ethyl.
Suitable functionalized fluorescent dye radicals which
can serve as reagents for the preparation of the
compounds of the formula I according to the invention
are described, for example, in "Handbook of Fluorescent
Probes and Research Chemicals, 5th Edition, 1992-1994,
by R.P. Haughland, Molecular Probes, Inc.".
In general, the peptides according to the invention are
linear, but they can also be cyclized [sic]. The
invention comprises not only the peptides 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 desirably influence the primary
pharmacological action of the peptides according to the
invention.
CA 02377224 2001-12-21
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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 in this case 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 was described,
for example, by Jonczyk and Meienhofer (Peptides, Proc.
Stn Am. pept. Symp., Eds. V. Hruby and D.H. Rich, Pierce
Comp. III, pp. 73-77, 1983, or Angew. Chem. 104, 1992,
375) or according to Merrifield (J. Am. Chem. Soc. 94,
1972, 3102).
The peptides according to the invention can be prepared
on a solid phase (manually or in an automated
synthesizer) in an Fmoc strategy using acid-labile side
protective groups and purified by means of RP-HPLC. The
peak homogeneity can be measured by RP-HPLC and the
substance identity by means of FAB-MS.
Otherwise, the peptides can be prepared by customary
methods of amino acid and peptide synthesis, such as is
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.
Stepwise couplings and fragment condensations can be
utilized. Different N-terminal, C-terminal and side
protection groups can be used, which are preferably
selected to be orthogonally cleavable. Coupling steps
can be carried out using different condensing reagents
such as carbodiimides, carbodiimidazole, those of the
uronium type such as TBTU, mixed anhydride methods, and
CA 02377224 2001-12-21
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acid halide or active ester methods. Activated esters
are expediently formed in situ, for example by addition
of HOBt or N-hydroxysuccinimide.
A cyclization of a linear precursor molecule having
side protective groups can likewise be carried out
using such condensation reactions, as is described, for
example, in DE 43 10 643 or in Houben-Weyl, 1.c.,
Volume 15/II, pages 1 to 806 (1974).
Different resins and anchor functions can be utilized
in the solid-phase peptide synthesis. Resins can be
based, for example, on polystyrene or polyacrylamide,
anchor functions such as Wang, o-chlorotrityl are
utilizable for the preparation of peptide acids,
aminoxanthenoxy anchors, for example for the
preparation of paptide [sic] amides.
Biotinylated or fluorescence-labelled peptides/proteins
can likewise be prepared by standard methods. (for
example 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 according to the invention 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
CA 02377224 2001-12-21
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by substitution of their -CO-OH hydroxyl function by
means of a protective group, for example as an ester.
The expression "amino protective group" is generally
known and relates to groups which are suitable for
protecting (or blocking) an amino 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 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 takes place - depending on the
protective group utilized - for example 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
(for example CBZ or benzyl) can be removed, for
example, by treating with hydrogen in the presence of a
catalyst (for example of a noble metal catalyst such as
palladium, expediently on a support such as carbon).
Typical protective groups for N termini and for side-
position amino groups are Z, BOC, Fmoc, those for C-
termini or the Asp or Glu side chains are O-prim-alkyl
(for example OMe or OEt), 0-tert-alkyl (for example
OBut) or Obenzyl. Z, BOC, NO2, Mtr, Pmc or Pbf, for
example, is suitable for the guanidino function of the
Arg. Alcoholic functions can be protected by tert-alkyl
radicals or trityl groups.
The groups BOC, OBut and Mtr can preferably be removed,
for example, using TFA in dichloromethane or using
CA 02377224 2001-12-21
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approximately 3 to 5 N HCl in dioxane at 15-30°, the
Fmoc group using an approximately 5 to 50~ solution of
dimethylamine, diethylamine or piperidine in DMF at 15-
30°.
The trityl group is employed, for example, for the
protection of the amino acids histidine, asparagine,
glutamine and cysteine. Removal is carried out,
depending on the desired final product, using TFA/10~
thiophenol, the trityl group being removed from all
amino acids mentioned, when using TFA/anisole or
TFA/thioanisole only the trityl group of His, Asn and
Gln is removed, whereas that on the Cys side chain
remains.
Hydrogenolytically removable protective groups (for
example CBZ or benzyl) can be removed, for example, by
treating with hydrogen in the presence of a catalyst
(for example of a noble metal catalyst such as
palladium, expediently on a support such as carbon).
Suitable solvents in this case are those indicated
above, in particular, for example, alcohols such as
methanol or ethanol or amides such as DMF. As a rule,
the hydrogenolysis is carried out at temperatures
between approximately 0 and 100° and pressures between
approximately 1 and 200 bar, preferably at 20-30° and
1-10 bar. Hydrogenolysis of the CBZ group takes place
readily, for example, on 5 to 10~ Pd/C in methanol or
using ammonium formate (instead of hydrogen) on Pd/C in
methanol/DMF at 20-30°.
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 a compound according to the
invention 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
CA 02377224 2001-12-21
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evaporation. Acids which are suitable for this reaction
are in particular those which yield physiologically
acceptable salts. Thus inorganic acids can be used, for
example 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, araliphatic, aromatic or hetero-
cyclic mono- or polybasic carboxylic, sulfonic or
sulfuric acids, e.g. 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 acid,
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 compounds according to the invention 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, for
example 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.
CA 02377224 2001-12-21
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Particularly to be emphasized in this context are
disorders or inflammations or wound-healing processes
of the skin, the respiratory tract organs and the
stomach and intestinal region, thus, for example,
apoplexy, angina pectoris, oncoses, osteolytic diseases
such as osteoporosis, pathological angiogenic diseases
such as, for example, inflammation, pulmonary fibrosis,
opthalmological diseases, diabetic retinopathy, macular
degeneration, myopia, ocular histoplasmosis, rheumatoid
arthritis, osteoarthritis, rubeotic glaucoma, ulcer-
ative colitis, Crohn's disease, atherosclerosis,
psoriasis, restenosis after angioplasty, in acute
kidney failure, nephritis, microbial infections and
multiple sclerosis.
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 in particular relates to appropriate
medicaments as inhibitors for the control of disorders
which are indirectly or directly based on expression of
the oc"(36 integrin receptor, thus in particular in
pathological angiogenic disorders, thromboses, cardiac
infarct, coronary heart disorders, arteriosclerosis,
tumours, osteoporosis, inflammation, infections and for
influencing wound-healing processes.
The invention also relates to appropriate
pharmaceutical preparations, which contain at least one
medicament of the formula I 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 producing a medicament for the control
of disorders which are based indirectly or directly on
expression of the oc"(36 integrin receptor, thus in
CA 02377224 2001-12-21
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particular in pathological angiogenic disorders,
thromboses, cardiac infarct, coronary heart disorders,
arteriosclerosis, tumours, osteoporosis, inflammation,
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 vehicles are
organic or inorganic substances which are suitable for
enteral (e.g. oral) or parenteral administration, or
topical application or for administration in the form
of an inhalation spray and do not react with the novel
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. In particular, tablets, pills, coated tablets,
capsules, powders, granules, syrups, juices or drops
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 novel compounds can
also be lyophilized and the lyophilizates obtained can
be used, for example, for the production of injection
preparations. The preparations indicated can be
sterilized and/or can contain excipients such as
lubricants, preservatives, stabilizers and/or wetting
agents, emulsifiers, salts for influencing the osmotic
pressure, buffer substances, colorants, 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 in
dissolved form or suspended in a propellant or
propellant mixture (for example COZ or fluoro-
chlorohydrocarbons). The active compound is expediently
used in this case in micronized form, where one or more
additional physiologically tolerable solvents can be
CA 02377224 2001-12-21
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present, e.g. ethanol. Inhalation solutions can be
administered with the aid of customary inhalers.
As a rule, the substances according to the invention
can be administered in analogy to other known,
commercially available peptides (for example 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, 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,
and on the excretion rate, pharmaceutical combination
and severity of the particular disorder to which the
therapy relates. Parenteral administration is
preferred.
Furthermore, the novel compounds of the formula I can
be used in analytical biology and molecular biology.
The novel compounds of the formula I, where X is a
fluorescent dye radical linked via a -CONH, -C00, -NH-
C(=S)-NH, -NH-C(=O)-NH, -SOZNH or -NHCO bond, can be
used as diagnostic markers in the FACS (Fluorescence
Activated Cell Sorter) technique and fluorescence
microscopy.
The use of labelled compounds in fluorescence
microscopy is described, for example, by Y.-L. Wang and
D.L. Taylor in "Fluorescence Microscopy of Living Cells
in Culture, parts A + B, Academic Press, Inc. 1989".
The novel compounds according to the invention can also
be used as integrin ligands for the preparation of
columns for affinity chromatography for the preparation
of integrins in pure form. The complex of an avidin-
derivatized support material, e.g. Sepharose, and the
CA 02377224 2001-12-21
- 18 -
novel compounds is formed by methods known per se (for
example 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 here are the
polymeric solid phases having preferably hydrophilic
properties and known per se in peptide chemistry, for
example crosslinked polysugars such as cellulose,
Sepharose or Sephadex~, acrylamides, polymers based on
polyethylene glycol or Tentakel polymers.
The invention finally also includes recombinant DNA
sequences which contain sections which code for peptide
regions which have the peptide structural motifs
according to the invention.
DNA of this type 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 excipients,
such as liposomes (A.I. Aronsohn and J.A. Hughes J.
Drug Targeting, 5, 163-169 (1997)).
The transfer of a DNA of this type could accordingly be
utilized 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 a
recombinant DNA of this type, the peptides according to
the invention finally themselves formed by the infected
cells can immediately bind to the oc~,~i6 integrin
receptor, for example of tumour cells, and block it.
Corresponding 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 02377224 2001-12-21
- 19 -
type, host cells which express the integrin oc~,(36 can
preferably be attacked (targeting).
Suitable viruses are, for example, adenoviral types
which have been used a number. of times already as
vectors for foreign genes in mammalian cells . A number
of properties make them into good candidates for gene
therapy, as can be seen 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), a common problem in gene therapy
by means of viral and non-viral vectors is the limited
efficiency of the gene transfer. Using the additional
ligand sequence for a~,(36 integrin described above the
coat protein of the adenoviruses, an improvement in the
transfer, for example, of cystic fibrosis transmembrane
conductance regulator (CFTR) cDNA can be achieved.
In a manner similar to that in 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 utilized for cell
transfections by means of retroviral or adenoviral
vectors.
The peptides according to the invention can also be
employed within a liposome complex of lipid/peptide/DNA
for a transfection of cell cultures together with a
liposome complex consisting of lipid/DNA (without
peptide) for use in gene therapy in man. The
preparation of a liposome complex from
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 ~g/~1 of lipofectin (equimolar mixture of
CA 02377224 2001-12-21
- 20 -
DOTMA (= N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl
ammonium chloride) and DOPE (dioleyl phosphatidyl
ethanolamine)), 10 ~.g/ml of plasmid DNA and 100 ~,g/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). Liposomal 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
accordingly modified recombinant DNA of gene-releasing
systems, in particular virus DNA, for the control of
diseases which are based indirectly or directly on
expression of oc~(36 integrin receptors, thus in
particular in pathological angiogenic disorders,
thromboses, cardiac infarct, coronary heart disorders,
arteriosclerosis, tumours, osteoporosis, inflammations,
infections and for influencing wound-healing processes.
Above and below, all temperatures are indicated in °C.
The HPLC analyses (retention time Rt) were carried out
in the following systems:
Column 5 um LichroSpher 60 RP-Select B (250-4), with a
50-minute gradient from 0 to 80~ 2-propanol in
water/0.3~ trifluoroacetic acid, at 1 ml/min flow and
detection at 215 nm.
Mass spectrometry (MS): EI (electron impact
ionization) M+
FAB (fast atom bombardment)
(M+H)+
CA 02377224 2001-12-21
- 21 -
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 Haubner et al. (J. Am. Chem.
Soc. 118, 1996, 17703).
Ac-Arg-Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NHz
2 g of aminoxanthenyloxypolystyrene resin (Novabiochem
0.37 mmol/g) were subjected twice in succession to a
coupling step in a double coupling technique using 0.50
g of TBTU, 0.53 ml of ethyldiisopropylamine and Fmoc-
amino acid in DMF each time in a commercial synthesis
apparatus and a typical procedure (apparatus and
handbook Milligen 9050 PepSynthesizerTM, 1987), for 30
minutes in each case. Washing steps were carried out in
DMF for 10 minutes, cleavage steps in piperidine/DMF
(1:4 vol) for 5 minutes, and N-terminal acetylations
(capping) were carried out with acetic
anhydride/pyridine/DMF (2:3:15 vol) for 15 minutes.
The amino acids Fmoc-Arg(Pmc), then Fmoc-Leu, then
Fmoc-Ser(But), then Fmoc-D-Asp(OBut), then Fmoc-Leu,
then Fmoc-Asp(OBut), then Fmoc-Gly and finally
Fmoc-Arg(Pmc) were used. After removal of the Fmoc
protective group from the Fmoc-Arg(Pmc)-Gly-
Asp(OBut)-Leu-D-Asp(OBut)-Ser(But)-Leu-Arg(Pmc)-amino-
xanthenyloxypolystyrene resin, acetylation was again
carried out. After washing with DMF and isopropanol and
subsequent drying in vacuo at room temperature, 2.8 g
of Ac-Arg(Pmc)-Gly-Asp(OBut)-Leu-D-Asp(OBut)-Ser(But)-
Leu-Arg(Pmc)-aminoxanthenyloxypolystyrene resin were
obtained.
By treatment of this peptidyl resin with 20 ml of
trifluoroacetic acid/water/TIS (94:3:3 vol) for 2 hours
CA 02377224 2001-12-21
- 22 -
at room temperature, filtration, concentration in vacuo
and trituration with diethyl ether, 0.47 g of Ac-Arg-
Gly-Asp-Leu-D-Asp-Ser-Leu-Arg-NHZ (code EMD 272974) was
obtained.
Purification of the product was carried out by RP-HPLC
on Lichrosorb RP 18 (250 - 25, 7 ~,m, Merck KGaA) in
0.3o TFA using a gradient of 4a to 24~ 2-propanol in
one hour at 10 ml/min and assessment of the eluate by
means of a UV flow-through photometer at 215 and
254 nm. 168 mg of product were obtained; Rt [sic] 15.5
min; FAB 973.
The following products were prepared analogously:
Code Sequence MW FAB Rt
(EMD) (g/mol) [sic]
271277 Ac-RADLDSLR-NHZ 986.1 986.6 18.18
271315 Ac-RGDLdsLR-NH2 972.1 972.6 15.7
272973 Ac-RaDLDSLR-NHZ 986.1 986.6 16.39
271294 Ac-RSDLDSLR-NH2 1002.1 1002 18.28
271296 Ac-RVDLDSLR-NH2 1014.2 1014 19.68
271295 Ac-RYDLDSLR-NHZ 1078.2 1078.6 19.58
271297 Ac-RGDLDSLR-NHZ 972.1 972 16.83
271298 Ac-RHDLDSLR-NHZ 1052.2 1052 17.10
271299 Ac-RRDLDSLR-NHz 1071.2 1071 16.50
271300 Ac-RDDLDSLR-NHZ 1030.1 1030 16.95
Ac-RGDLdGLR-NHz
Ac-RGDLdALR-NHz
271293 Ac-RTDLDSLR-NHZ 1016.1 1017 17.67
271265 Ac-RTDLDSLA-NH2 931.0 931.6 18.15
271266 Ac-RTDLDSAR-NH2 974.0 974.6 14.43
271267 Ac-RTDLDALR-NH2 1000.1 1000.6 18.64
271268 Ac-RTDLASLR-NH2 972.1 972.6 18.42
271270 Ac-RTDADSLR-NHZ 974.0 974.6 14.95
271273 Ac-RTALDSLR-NH2 972.1 972.6 18.95
271277 IAc-RADLDSLR-NHz 986.1 986.6 X18.18
CA 02377224 2001-12-21
- 23 -
271283 Ac-ATDLDSLR-NH2 931.0 931.5 18.75
271285 Ac-RTDLDSLr-NHZ 1016.1 1017 17.38
271286 Ac-RTDLDS1R-NHz 1016.1 1017 17.22
271287 Ac-RTDLDsLR-NHZ 1016.1 1017 16.98
271288 Ac-RTDLdSLR-NH2 1016.1 1017 17.70
271289 Ac-RTD1LDSLR-NHZ 1016.1 1017 17.31
271290 Ac-RTdLDSLR-NHz 1016.1 1017 17.72
271291 Ac-RtDLDSLR-NH2 1016.1 1017 16.84
271292 Ac-rTDLDSLR-NH2 1016.1 1017 18.07
271577 Ac-RTELDSLR-NHZ 1030.2 1030.Fi 17.27
271309 Ac-RTDLaSLR-NH2 972.1 973 17.03
271317 Ac-RTDL-(D-Phg)-SLR-NH2 1034.2 1034.7 19.86
271318 Ac-RTDLfSLR-NH2 1048.2 1049 22.66
271319 Ac-RTDL-(D-Nal)-SLR-NHZ 1098.3 1099 26.38
271320 Ac-RTDLGSLR-NH2 958.1 958.6 16.74
271321 Ac-RTDL-Aib-SLR-NHz 986.1 986 19.63
271322 Ac-RTDLpSLR-NHZ 998.2 998.6 18.23
271575 Ac-RTDLPSLR-NH2 998.2 998.5 17.30
271576 Ac-RTDL-(D-Bgl)-SLR-NHz 1014.2 1014.6 20.06
271310 Ac-RTDLdSLA-NHZ 931.0 931.5 18.20
271311 Ac-RTDLdSAR-NHz 974.0 974.6 14.59
271547 Ac-RTDLdSPR-NHZ 1000.1 1000.4 13.26
271573 Ac-RTDLdSFR-NHZ 1050.1 1050.5 16.82
271574 Ac-RTDLdSIR-NHZ 1016.1 1016.5 15.99
249381 Ac-RTDFdSLR-NHZ 1050.1 1050.5 17.04
249383 Ac-RTDIdSLR-NHZ 1016.1 1016.5 16.22
249385 Ac-RTD-N1e-dSLR-NH2 1016.1 1016.5 17.03
271314 Ac-RTDLdsLR-NHZ 1016.1 1016.8 15.4
271303 Ac-Orn-RDLDSLR-NHZ 974.1 974 16.26
271308 Ac-Har-TDLDSLR-NHz 1030.1 1030 17.66
271302 Ac-HTDLDSLR-NHz 997.1 997 17.28
271301 Ac-KTDLDSLR-NHZ 988.1 988 16.46
271323 Ac-RTDLdLR-NHZ 929.1 929 15.95
271324 Ac-RTDLdGLR-NH2 986.1 986 16.66
271325 Ac-RTDLd-(3Ala-LR-NHZ 1000.1 1000 15.92
271327 Ac-RTDLd-Abu-LR-NHz 1014.1 1014 16.43
272975 Ac-RTDLDSLG-NHz 916.9 917 16.19
271329 IAc-RTDLDSLF-NHZ 1007.1 X1007 X24.23
CA 02377224 2001-12-21
- 24 -
271332 Ac-RTDLDSLD-NH2 975.0 975 16.40
271336 Ac-RTDLDSLL-NHZ 973.1 973 23.51
271339 Ac-RTDLDSLS-NHZ 947.0 947 16.54
271343 Ac-RTDLDSLK-NH2 988.1 988 16.36
271346 Ac-RTDLDSLY-NHZ 1023.1 1023 18.98
271350 Ac-RTDLDSL-Orn-NH2 974.1 974 15.84
271355 Ac-RTDLDSL-Har-NHz 1030.1 1030 17.55
271316 Ac-RTDLDGLR-NHZ 986.1 986.7 17.55
Ac-RTDLdALR-NHZ
272974 Ac-RGDLdSLR-NH2 972.1 973 X16.4
Nomenclature for amino acids according to Eur. J.
Biochem. 138, 9-37 (1984)
lower case letter = D-amino acid
Example 2:
x",136 / fibronectin receptor binding test:
The prepared peptides according to the invention were
bonded to the immobilized oc~,~i6 receptor together with
competitively acting fibronectin in solution 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 calculated here from the quotient of the ICSo
values of test peptide and a standard. The standard
used was the linear Ac-RTDLDSLR-NHz (Code EMD 271293)
(ref./patent cf. Pytela et al. Science 2:31, 1559,
(1986)). The binding test was carried out in detail as
follows:
The immobilization of soluble oc"~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/hollow). Non-specific binding
sites were blocked by incubation (2 h, 37°C) with 3~
(w/v) BSA in TBS++ (200 ~,1/hollow). Excess BSA was
removed by washing three times with TBSA++. Peptides
were diluted serially (1:10) in TBSA++ and incubated
with the immobilized integrin (50 ~1 of peptide + 50 ~,1
of ligand per hollow; 2 h; 37°C) together with
CA 02377224 2001-12-21
- 25 -
biotinylated fibronectin (2 ~g/ml). Unbound fibronectin
and peptides were removed by washing three times with
TBSA++. The bound fibronectin was detected by
incubation (1 h; 37°C) with an alkaline phosphatase-
coupled anti-biotin antibody (Biorad) (1:20,000 in
TBSA++; 100 ~1/hollow). After washing three times with
TBSA++, colorimetric detection was carried out by
incubation (10-15 min; 25°C, in the dark) with
substrate solution (5 mg of nitropnenyl pnospnate, 1 m1
of ethanolamine, 4 ml of H20; 100 ~.1/hollow). The
enzyme reaction was stopped by addition of 0.4 M NaOH
(100 ~1/hollow). The colour intensity was determined at
405 nm in an ELISA measuring apparatus and made equal
to the zero value. Hollows which were not coated with
receptor served as a zero value. Ac-RTDLDSLR-NH2 was
employed as a standard. The ICso values for the peptides
tested were read off from a graph and from this,
together with the ICSO value of the standard peptide,
the Q value of the peptide according to the invention
was determined.
Q value = ICSO test peptide / ICSO standard
Q values were calculated as means from repeat
experiments.
The results of the test described are summarized in the
Table 1 which follows:
Table 1
Results of the oc"(36 / fibronectin receptor binding test
Code (EMD) Sequence Q value = ICSo
test peptide /
ICSO EMD 271293
271293 Ac-RTDLDSLR-NHZ 1.00 (=75 nM)
272974 Ac-RGDLdSLR-NHZ 0.15
271297 Ac-RGDLDSLR-NHz 0.22
271294 Ac-RSDLDSLR-NH2 3.2
CA 02377224 2001-12-21
- 26 -
271300 Ac-RDDLDSLR-NHZ 14
271299 Ac-RRDLDSLR-NHZ 15
271296 Ac-RVDLDSLR-NH2 16
271291 Ac-RtDLDSLR-NH2 20
271295 Ac-RYDLDSLR-NHZ 30
271298 Ac-RHDLDSLR-NHZ 30
271277 Ac-RADLDSLR-NHZ 31
272973 Ac-RaDLDSLR-NHZ 98
271265 Ac-RTDLDSLA-NH2 7
271266 Ac-RTDLDSAR-NHZ 60
271270 Ac-RTDADSLR-NHZ 60
271285 Ac-RTDLDSLr-NH2 1
271286 Ac-RTDLDS-(D-Leu)-R-NHZ 10
271288 Ac-RTDLdSLR-NH2 0.08
271289 Ac-RTD-(D-Leu)-DSLR-NH2 40
271290 Ac-RTdLDSLR-NHZ 15
271292 Ac-rTDLDSLR-NH2 7
271308 Ac-Har-TDLDSLR-NHz 20
271302 Ac-HTDLDSLR-NH2 70
271301 Ac-KTDLDSLR-NHZ 110
271303 Ac-Orn-TDLDSLR-NH2 190
271309 Ac-RTDLaSLR-NHZ 0.16
271321 Ac-RTDL-Aib-SLR-NH2 0.4
271319 Ac-RTDL-(D-Nal)-SLR-NHZ 0.7
271320 Ac-TRDLGSLR-NHZ 1
271268 Ac-RTDLASLR-NHZ 1
271318 Ac-RTDLfSLR-NHZ 1.6
271317 Ac-RTDL-(D-Phg)-SLR-NHZ 3.6
271576 Ac-RTDL-(D-Bgl)-SLR-NHZ 71
271575 Ac-RTDLPSLR-NHZ 157
271322 AC-RTDLpSLR-NH2 182
249385 Ac-RTD-Nle-dSLR-NHZ 0.15
249383 Ac-RTDIdSLR-NHZ 0.6
249381 Ac-RTDFdSLR-NH2 7.1
271287 Ac-RTDLDsLR-NH2 0.45
271267 Ac-RTDLDALR-NHZ 0.8
271316 Ac-RTDLDGLR-NH2 0.95
CA 02377224 2001-12-21
- 27 -
271323 Ac-RTDLdLR-NHZ 273
271355 Ac-RTDLDSL-Har-NHz 1.8
271343 Ac-RTDLDSLK-NHZ 2.2
271336 Ac-RTDLDSLL-NHZ 4.4
271350 Ac-RTDLDSL-Orn-NHz 4.5
271329 Ac-RTDLDSLF-NH2 5.5
271265 Ac-RTDLDSLA-NHz 7
271346 Ac-RTDLDSLY-NHZ 8.5
272975 Ac-RTDLDSLG-NH2 12
271339 Ac-RTDLDSLS-NH2 13
271332 Ac-RTDLDSLD-NHZ 51
271314 Ac-RTDLdsLR-NH2 0.26
271310 Ac-RTDLdSLA-NH2 0.47
271324 Ac-RTDLdGLR-NHZ 1.1
271574 Ac-RTDLdSIR-NHZ 1.4
271573 Ac-RTDLdSFR-NHZ 2.4
271311 Ac-RTDLdSAR-NHz 13
271325 Ac-RTDLd-~Ala-LR-NH2 18
271327 Ac-RTDLd-Abu-LR-NHz 33
The following examples relate to pharmaceutical
preparations:
Example A: injection vials
A solution of 100 g of Ac-RGDLdSLR-NHZ and 5 g of
disodium hydrogenphosphate is adjusted to pH 5.5 in 3 1
of double-distilled water using 2 N hydrochloric acid,
sterile-filtered, dispensed into injection vials,
lyophilized under sterile conditions and aseptically
sealed. Each injection vial contains 5 mg of active
compound.
CA 02377224 2001-12-21
- 28 -
Example B: suppositories
A mixture of 20 g of Ac-RGDLdSLR-NHz 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 is prepared from 1 g of Ac-RGDLdSLR-NH2,
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. The mixture 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 Ac-RGDLdSLR-NH2 are mixed with 99.5 g of
petroleum jelly under aseptic conditions.
Example E: tablets
A mixture of 1 kg of Ac-RGDLdSLR-NH2, 4 kg of lactose,
1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of
magnesium stearate is compressed in a customary manner
to give tablets in such a way 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, talc, tragacanth and colorant.
CA 02377224 2001-12-21
- 29 -
Example G: capsules
2 kg of Ac-RGDLdSLR-NH2 are dispensed 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 Ac-RGDLdSLR-NHZ in 60 1 of
double-distilled water is sterile-filtered, dispensed
into ampoules, lyophilized under sterile conditions and
aseptically sealed. Each ampoule contains 10 mg of
active compound.
Example I: inhalation spray
14 g of Ac-RGDLdSLR-NHz are dissolved in 10 1 of
isotonic NaCl solution and the solution is dispensed
into commercially available spray containers having a
pump mechanism. The solution can be sprayed into the
mouth or nose. One burst of spray (approximately
0.1 ml) corresponds to a dose of approximately 0.14 mg.
