Note: Descriptions are shown in the official language in which they were submitted.
CA 02339537 2001-02-02
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REGULATION OF SUBSTRATIE ACTIVITY
Background of the Invention
s This invention relates to inhibitors for dipeptidyl peptidase IV which are
used to
regulate substrate activity. As used herein, the term "substrate" denotes
chemokines, cytokines
and biological peptides which are substrates for DPP-IV. 'this invention also
relates to the use
of DPP-IV inhibitors in the treatment of medical disorders which may result
from the
inactivation of substrates implicated in the medical disorder. As used herein,
dipeptidyl
to peptidase IV is alternatively described as "DPP-IV," "DP-~IV" and "CD26."
CD26 is an
ectoenzyme with activity identical to that of DPP-IV.
DPP-IV is a serine type exopeptidase with high substrate specificity cleaves N-
terminal dipeptides from proteins if the penultimate amino acid is proline, or
in some cases
alanine (Fleischer, B. Immunol. Today 15:180 (1994)).
15 A class of low molecular weight synthetic monomeric molecules with high
affinity for
CD26 have previously been developed and characterized (G.R. Flentke et aL,
Inhibition of
dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of
these inhibitors
to examine the role of DP-IV in T-cell function, PNAS (LISA) 88, IS56-1559
(1991); W.G.
Gutheil and W.W. Bachovchin, Separation of L-Pro-DL-boroPro into Its Component
2o Diastereorners and Kinetic Analysis of Their Inhibition of Dipeptidyl
Peptidase IV: A New
Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32,
8723-8731
( 1993)). These molecules have been shown to be potent and specific synthetic
inhibitors for
CD26- associated DP-IV proteinase activity.
Representative monomeric structures of these transition-state-analog-based
inhibitors,
2s Xaa-boroPro, wherein Xaa is an amino acid residue, include Pro-boroPro, Ala-
boroPro, Val-
boroPro, and Lys-boroPro. BoroPro refers to the analog of proline in which the
carhoxylate
group (COOH) is replaced with a boronyl group [B(OH}2];. Pro-boroPro, the most
thoroughly
characterized of these inhibitors, has a Ki of 16 picomolar (pM) (W.G. Gutheil
and W.W.
Bachovchin, supra). Val-boroPro has an even higher affinity, with a Ki of 1.6
pM (W.G.
3o Gutheil and W.W. Bachovchin, supra; R.J. Snow et al., Studies on Proline
Boronic Acid
Dipeptide Inhibitors of Dipeptidyl Peptidase IV: Identification of a Cyclic
Species Containing
a B-N Bond, J. Am. Chem. Soc. 116, 10860-10869 (1994}). Thus, these Xaa-
boroPro
inhibitors are about 10+6 fold more potent than the next best known
inhibitors.
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.2_
United States Patent Nos. 4,935,493 (Bachovchin '493) and 5,462,928
(Bachovchin
'928}, both of which are incorporated herein by reference, disclose protease
inhibitors and
transition state analogs (the '493 patent} and methods for treating transplant
rejection in a
patient, arthritis, or systemic lupus erythematosis (SLE) by administering a
potent inhibitor of
the catalytic activity of soluble amino peptidase activity o:f dipeptidyl
peptidase type IV (G.R.
Flentke et al., supra).
Chemokines, or chemoattractant cytokines, are a family of small proteins with
a
conserved cysteine motifs. These small proteins have been implicated in a wide
range of
disease states, such as acute and chronic inflammatory processes,
angiogenesis, leukocyte
1 o migration, regulation of cell proliferation and maturation, hematopoiesis,
viral replication, and
other immunoregulatory functions. Chemokines are expressed by a number of
different cells
and have distinct but overlapping cellular targets.
There are two groups of chemokines defined according to their structural
characteristics: the CXC and the CC groups. In addition, C-chemokines and CX3C-
~s chemokines have been identified. Members of the CXC group, which include
SDF-l and IL-8,
attract mostly neutrophils, while the CC group acts on monocytes and
granulocytes. The CC
group includes such molecules as human monocyte chemotactic protein 1 (MCP-1)
and
RANTES. MCP-1 and RANTES are potent direct mediators of the release of
histamine by
human basophils. Both groups of chemokines are involved in lymphocyte
migration to
2o inflammatory sites.
The majority of chemokine receptors are transmembrane spanning molecules which
belong to the family of G-protein-coupled receptors. Many of these receptors
couple to
guanine nucleotide binding proteins to transmit cellular signals. Chemokines
and receptor
expression is upregulated during inflammatory responses and cellular
activation. Chemokines,
25 through binding to their respective receptors, have been shown to be
involved in a number of
physiologic conditions. For instance, chemokines of the CXC group, like
Interleukin-8, can
stimulate angiogenesis, while Platelet Factor-4, growth-related oncogene-(3
(GRO-~3) and
interferon-'y induced Protein-10 (IP-10) inhibit endothelial cell
proliferation and angiogenesis.
Interleukin-8 stimulates endothelial cell proliferation and chemotaxis in
vitro, and appears to
3o be a primary inducer of macrophage induced angiogenesis~. It was shown that
the activities of
these chemokines are dependent on the NHZ terminal amino acid sequence
(Streiter et al., J.
Biol. Chem., 27fl; pages 27348-27357). SDF-1, another CXC chemokine, is active
in the
CA 02339537 2001-02-02
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recruitment and mobilization of hematopoietic cells from the bone marrow, as
well as the
attraction of monocytes and lymphocytes. In addition, it interferes with
cellular infection of
HIV-1 by blocking the interaction of HIV-1 with CXCR-4. As with other
chemokines in this
group, the amino terminal sequence regulates its activity (ahioda, T., et al.,
PNAS, 95; pages
6331-6336).
Chemokine receptors have been shown to serve not only as receptors to
chemokines,
but most recently have been identified as receptors for a vaxiety of microbes
and the HIV-I
virus. For instance, the Duffy blood group Ag, a chemokine receptor on
erythrocytes, is the
receptor for the malaria parasite Plasmodium vivax, and the platelet
activating receptor is a
7 o receptor for Streptococcus pneumonia.
A number of chemokines, such as RANTES, MIP-:l and SDF-1, or cytokines Iike IL-
2,
or peptides Iike GLP-I, GLP-2, and Substance P, are substrates for DPP-IV. DPP-
IV cleaves
peptides at the NHZ terminus if the penultimate amino acid is proline. Several
cytokines and
chemokines have the conserved sequence NHZX-Pro-X, and have been shown to be
substrates
15 for DPP-IV. DPP-IV is expressed on the surface of T cells and macrophages.
The relationship
of CD26 protease activity to its immune function is not clear, however there
are indications
that cleavage by CD26 of the NHz-dipeptide of several cytokines changes their
receptor
specificity and/or their functional activities.
Cytokines that are known to be potential substrates for DPP-IV include G-CSF,
2o erythropoietin, IL-1(3, IL-2, IL-3, IL-6, IL-1 l, TNF-(3 and GM-CSF.
In addition to cytokines and chemokines, a number of biologically active
peptides
have, on their amino termini, the arnina acid sequence Xaa-Pro-Xaa, which
serves as the
substrate for DPP-IV. Among these are the Glucagon Like Peptides, GLP-1 and
GLP-2. GLP-
I is involved in insulin release and glucose uptake, and cleavage by DPP-IV
causes
25 inactivation of its activity. Inhibition of DPP-IV will result: in the
prolonged activation state of
this peptide, and represents a therapeutic indication of DPF'-IV inhibitors.
GLP-2 peptide is
involved in intestinal growth and nutrient uptake, and increased activity of
GLP-2 will result
in an increased nutrient uptake for individuals with intestinal diseases.
In addition to the foregoing, CD26 is known to be highly expressed on
hepatosplenic T
3o cell lymphoma, and DPP-IV activity, or the ability of CD26 to bind to
collagen fibronectin on
the extracellular matrix, may be part of the pathogenic me<;hanism utilized in
by neoplastic
cells (Ruiz et al., Cytometry 199&, 34: pages 30-35).
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DPP-IV and DPP-II levels are known to be significantly increased in the
gingival
crevicular fluid of patients with periodontitis. Increased levels of these two
proteases seem to
be associated with increased attachment loss. It is believed that collagen-
CD26 interactions
are a part of the pathological observations.
PCT published application WO 95/11689 discloses the use of inhibitors of DPP-
IV to
block CD26, thereby blocking entry of HIV into CD26-bearing cells. These
inhibitors are
tetrapeptides having the general formula X-Pro-Y-boroPro where X and Y are
chosen from
any amino acid. Although the dipeptides are also disclosed, the reference
states that dipeptides
are unstable, and tetrapeptides are preferred. The inhibitors are used to
treat pre-symptomatic
to HIV-infected patients not by neutralizing the virus, but by blocking viral
entry into the cells.
The reference does not disclose the effect of DPP-IV inhibition on chemokine
activity.
Summary of the Invention
According to the present invention, a method is provided for the treatment of
a
15 medical disorder in a patient which is mediated by substrate activity.
Pursuant to this method,
a pharmaceutical composition is administered to the patient in an amount which
is effective to
inhibit DPP-IV activity. This pharmaceutical composition. contains, as an
active ingredient, a
compound represented by the general formula PR, where P is a targeting moiety
that binds to
DPP-IV, and R is a reactive group that reacts with a functional group in DPP-
IV, preferably a
2o reactive center of DPP-IV. Preferably, the active ingredient is a compound
comprising an
amino group covalently bonded to an alpha-amino boronic acid analog of proline
(the term
"boroPro" is used herein to designate such an analog having the carboxyl group
of proline
replaced with a B(OH}2 group, where (OH)2 represents two hydroxyl groups and B
represents
boron). The active ingredient can therefor be designated as Xaa-boroPro, where
Xaa is an
z5 amino acid residue. Most preferably, the active ingredient is Val-boroPro
wherein the
carboxy terminal boroProline is coupled via a peptide linkage in accordance
with standard
peptide chemistry to a valine amino acid residue.
This active ingredient acts to suppress DPP-IV activity for those substrates
which are
DPP-IV substrates, resulting in an increase in bioavailability of active
chemokine in vivo. The
3o net effect is a positive therapeutic beneft to the patient, particularly
with respect to certain
disease states, such as inflammation, angiogenesis, arteriolosclerosis,
intestinal diseases,
diabetes, anorexia, and anti-tumor activity, which could not have been
predicted on the basis
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-5
of current knowledge of DPP-IV activity or inhibition.
Substrates which are applicable to the method of this invention include those
which
share a conserved NHZ-X-Pro sequence (where X is any amino acid or a short
peptide). These
substrates for DPP-IV, and their activity is altered upon cleavage of the N-
terminal sequence.
The altered substrates can theoretically possess either enhanced or attenuated
activity, but they
will most typically be inactivated to some extent. Specific examples of
chemakines which are
inactivated by DPP-IV include, but are not limited to, SDF-1, RANTES, MIP-3.
The
inhibition of DPP-IV activity prevents the digestion of chemokines, cytokines
and growth
factors. The substrates of this invention are implicated in a variety of
disease states, including
inflammation, arteriolosclerosis, angiogenesis, and anti-tumor activity.
Inhibition of DPP-IV
by the active compounds of this invention is believed to result in the
bioavailability of the
substrates for enhancing responses to medical trauma.
The pharmaceutical composition will typically include the active component
(preferably Xaa-boroPra, and most preferably Val-boroPro), and a
pharmaceutically
acceptable carrier. The pharmaceutical composition can also include various
adjuvants,
enhancers, cytokines, etc., as are known to those skilled in the art. Patient
dosages will
generally be within the range of 0.001 mg/kg to 100 mglkg.
Brief Description of the Drawings
2o Figure i shows the bioavaiIability of PT-100 (Val--boroPro), and the HCl
and methane
suifonate salts of PT-100.
Detailed Description of the Invention
The invention involves the use of certain compounds to inhibit DPP-IV activity
when
appropriately administered to a subject. As used herein, the term "subject" is
intended to mean
a human, non-human primates, dogs, cats, sheep, goats, horses, cows, pigs and
rodents.
Preferably, the subject is a human patient undergoing medical treatment.
It has now been discovered that inhibition of DPP-IV activity results in the
alteration
of certain substrates which are implicated in various medical disorders. Since
the substrates of
3o this invention are substrates for DPP-IV, it is believed that DPP-IV acts
on the substrates in
vivo to cleave the two N-terminal sequence where the penultimate amino acid is
proline.
Alternatively, other peptidases such as peptides A or N could cleave the
terminal sequence to
CA 02339537 2001-02-02
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-6-
expose the Xaa-Pro-Xaa moiety. This cleavage may result in altering the
receptor specificity
or functionality of the substrate, and typically will result in changes of the
activity of the
substrate. This may directly effect the patient's response to a particular
medical disorder or
trauma. For instance, certain chemokines, such as SDF-l, MIP-1 and RANTES, are
known to
act as attractants for lymphocytes, monocytes, etc. Cleavage of these
chemokines by DPP-IV
alters their activity and affects the migration of the lymphocytes to the site
of an inflammation,
hematopoiesis or immune function. The DPP-IV inhibitor acts to block DPP-IV
from cleaving
the chemokine, either through competitive interaction with DPP-IV or
attachment to the active
site.
Active Compounds
Compounds useful in the invention include, but are not limited to, compounds
that
inhibit DPP-IV and are embraced by the formula PR, wherein P represents a
targeting moiety
that binds to DPP-IV and R represents a reactive group that reacts with a
functional group in
DPP-IV, preferably a reactive center of DPP-IV. P can be any molecule that
binds DPP-IV,
including DPP-IV binding molecules embraced by the formula: D~A, -A~ -A3 -AQ,
wherein D
is independently selected from the group consisting of NH and NH2, wherein N
represents any
isotope of nitrogen, wherein H represents any isotope of hydrogen; "~",
independently, is
selected from the group consisting of a single bond and a double bond; A, is
selected from the
2o group consisting of a C, a CX and an N, wherein C represents any isotope of
carbon, X
represents any atom that forms a single bond with carbon; each A2, A3, and A4,
independently,
is selected from the group consisting of a CX moiety, a CXZ moiety, a CZ
moiety, a NX
moiety, and an O, wherein X and Z, independently are selected from the group
consisting of
any atom that forms a single bond and any atom that forms a double bond with C
or N and
wherein O represent any isotope of oxygen.
Compounds useful according to the invention include the following alternative
structures designated as Group I or Group II.
Group I has the structure:
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H ht O H O H H
I I II I II I I
H N-C-C N'C'C -N-C-X
I I
R R~_ ! _Y R
q R2 P
where H represents a hydrogen; C represents a carbon; O represents an oxygen;
N represents a
nitrogen; each R, independently, is chosen from the group consisting of the R
groups of an
amino acid, including proiine; X represents any atom that :forms a single bond
with carbon,
including hydrogen and halogens; Y is
Ra ~ Ra Rs R7
Rs ~ -Ra , R3 i - f -R6 . or R3-C__C_ i - ~
and each R,, R2, R3, R4, R5, Its, R7, and R8, separately is a group which does
not significantly
interfere with site specific recognition of the inhibitory compound by DPP-IV,
and allows a
complex to be formed with DPP-IV. Preferably, R,-R8 are H each H represents a
hydrogen
o atom; and q and p are integers which are independently varied between 0 and
4 inclusive.
Alternatively, Group I has the structure:
G~ C
n
where n is between 0 and 3 inclusive,
15 each GZ and G3 independently is H or C,-C3 (one to three carbon atoms)
alkyl;
G, is NH3 (H3 represents three hydrogens);
or G, is
I
HN-C-NH2
1
NH2
(H~ represents two hydrogens),
20 or G, is NG4, where G4 is
CA 02339537 2001-02-02
WO OO/1OS49 PCT/US99/18315
_g_
-G5
G~
where GS and G6 can be NH, H, or C,-C3 alkyl or alkenyl with one or more
carbons substituted
with a nitrogen. G1 bears a charge, and G, and GZ do not form a covalently
bonded ring
structure at pH 7Ø
Group I may also have the structure:
S1
S6 a S2
/e~d'~\
S5 ~ Sa
Sa
where one or two of the a, b, c, d, e, and f group is N, and the rest are C,
and each S,-S6
independently is H or Cj-C3 alkyl. Group I may also include a five membered
unsaturated ring
o having two nitrogen atoms, e.g., an imidazole ring.
Group II has the structure:
H
X~~_~_T
m
R~- i -Y
R2
where T is a group of the formula:
1 s D2
i
where each D, and D2, independently, is a hydroxyl group or a group which is
capable of
being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH;
or T is a group of the formula:
CA 02339537 2001-02-02
- WO ~OI10549 PCT/US99/18315
_g_
-C-CF-G
fl I
O F
where G is either H, F, or an alkyl group containing 1 to 20 carbon atoms and,
optionally,
heteroatoms which can be N, S or O;
or T is a phosphonate group of the formula:
O
!I
-P-J
O-J
where each 3, independently, is any number of N, H, C, O or S atoms, in any
combination, or
O-alkyl, N-alkyl, or alkyl, each O-alkyl, N-alkyl or alkyl containing 1-20
carbon atoms and,
1 o optionally, heteroatoms which can be N, S, or O;
or T is
O O
II II
-C-C-NH2
or
O O
11 il
-C-C-OR
where R is an alkyl, or aryl group and may be substituted or unsubstituted, an
alphaketo ester;
or
Zo 0 0
!! II
-C-C-OH
T is generally able to form a complex with the catalytic side of a DPP-IV.
Y is:
CA 02339537 2001-02-02
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-10-
t
and each R,, R2, R3, R4, R5, R6, R7, and Rg, separately is a group which does
not significantly
interfere with site specific recognition of the inhibitory compound by DPP-IV,
and allows a
complex to be formed with DPP-IV. Preferably, R,-Rg are H. X is any number of
C, H, O, S,
or N atoms, in any combination, including any amino acid or organic molecule,
and m can
vary from 0 to 20.
In preferred embodiments, T is a boronate group, a phosphonate group, a cyano
group,
or a txifluaroalkyl ketone group; each R,-R8, is H; each R, and RZ is H, and
each Y is CH2-
o CH2; each R is independently chosen from the R group of proline and alanine;
the inhibitory
compound has a binding or dissociation constant to DPP-IV of at least 10-9M, I
O-8M or even
10-7M; and each D1 and D2 is, independently, F or D1 and D2 together are a
ring containing 1
to 20 carbon atoms, and optionally heteroatoms which can be N, S, or O. These
compounds
are described in U.S. Patent No. 5,462,928, hereby incorporated by reference.
15 The more preferred compounds are of the formula:
H
X ~ C-B.
H2C ~ ~C H2
C
H2
where each D, and D2, independently, is a hydroxyl group or a group which is
capable of
being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH;
and wherein X
2o is a targeting moiety that mimics the site of a substrate recognized and
cleaved by DPP-IV,
and preferably is an amino acid, an imino acid, or a peptide which mimics the
site of a
substrate recognized by a post prolyl cleaving enzyme; C is bonded to B in the
L-
configuration; and the bonds between B and C, and between Y and N are peptide
bonds. By
the expression "C is bonded to B in the L-configuration" is meant that the
absolute
25 configuration of the C is Iike that of an L-amino acid.
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The active Xaa-boroPro compound of this invention can have an open chain
{linear)
form or a cyclic form. The linear form can be converted to~ the cyclic form by
a trans to cis
isomerization of the proline, and the formation of a new N-B bond.
Accordingly, "cyclic
form" refers to the cyclized structure of the compounds described in the
foregoing formula
that are the boron analogs of diketopiperazine.
In a particularly preferred embodiment of the invention, the active compound
is an
Xaa-boroPro compound, and still more preferably is a VaI-boroPro compound. A
"Val-
boroPro compound" refers to a compound as defined in the formula above in
which the
carboxy terminal boroPro is covalently coupled via a peptide linkage in
accordance with
~o standard peptide chemistry to a valine amino acid residue. In a most
preferred embodiment,
the compound of the invention is Val-boroPro (also referred to by the
manufacturer's
designation "PT-100"}.
The preferred active compounds have targeting moieties that are peptides which
mimic
the substrate binding site of DPP-IV. Peptide analogs and nonpeptides or
peptidomimetics
also can be used as targeting moieties. Such molecules can be rationally
designed based upon
the known sequence of substrates of DPP-IV or can be identified using
combinatorial
chemistry and screening assays such as are described below.
The development of phage display libraries and chemical combinatorial
libraries
permits the selection of synthetic compounds which mimic the substrate binding
site of a
2o protease such as DPP-IV. Such libraries can be screened to identify non-
naturally occurring
putative targeting moieties by assaying protease cleavage activity in the
presence and absence
of the putative phage display library molecule or combinatorial library
molecule and
determining whether the molecule inhibits cleavage by the protease of its
natural substrate or
of a substrate analog (e.g., a chromophoric substrate analog which is easily
detectable in a
spectrophotometric assay}. Those phage library andlor combinatorial library
molecules which
exhibit inhibition of the protease then can be covalently coupled to the
reactive groups R
disclosed herein and again tested to determine whether these novel molecules
selectively bind
to the protease (e.g., by repeating the above-noted screening assay). In this
manner, a simple,
high-through-put screening assay is provided for identifying non-naturally
occurring targeting
3o moieties of the invention.
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-I2
Substrates
The substrates of this invention are those substrate;s which share a conserved
NHZ-X-
Pro sequence (where X is any amino acid or a short peptide) at the NHZ
terminus of the
molecule. Substrates having this structural configuration act as substrates
for DPP-IV. The
inhibitory effect of the present compounds on DPP-IV serves to increase the
bioavailability of
the substrate in the subject, which in turn results in a positive biological
result. For instance,
increased bioavailability of selected substrates can produce an increase in
immune and anti-
inflammatory function in the subject. Suitable substrates are known in the
art, but until now,
no one has correlated the use of DPP-IV inhibitors with a positive medical
effect on those
1o conditions which are mediated by the presence and activii;y of the
substrates.
Typical substrates which are the subject of this invention are more fully
described
below. It should of course be appreciated, however, that other unspecified
substrates having
conserved NH,-X-Pro sequences, although not specifically described, would also
be within
the scope of this invention.
SDF-l, or stromal cell-derived factor l, is a CXC chemokine containing a
proline
residue at the second position form the N-terminus of the molecule. SDF-I acts
on
lymphocytes and monocytes but not neutrophils in vitro, and is an effective
and potent
mononuclear cell attractant in vivo. SDF-1 is expressed in a broad range of
tissues, it may
assist in the treatment of arteriolosclerosis, and it also may have anti-HIV
activity.
2o MIP-1 (macrophage inflammatory protein-1 ) is an attractant for lymphocytes
which
are essential for immune and inflammatory responses.
RANTES (regulated on activation, normal T-cell expressed and secreted}
modulates
integrin adhesion and has also been implicated in inflammatory diseases.
GLP-1 (glucagon-like peptide-1 } is known to stimulate insulin secretion. This
effect is
limited in vivo due to the rapid degradation of GLP-1 by DPP-IV. DPP-iV
inhibition, using
the active compounds of this invention, would potentiate its insulinotropic
effects, and may
provide assist in the treatment of diabetes.
GLP-2 (glucagon-like peptide-2) is known to stimulate small intestinal growth
through
the induction of intestinal epithelial proliferation. GPL-2 is also
inactivated by DPP-IV in
3o vivo. DPP-IV inhibition may result in increased capacity for nutritional
digestion and
absorption in vivo, and provide a treatment for AIDS, ane~~nia and anorexia.
GLP-2 may be
therapeutically useful to enhance mucosal regeneration in patients with
intestinal diseases.
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-13
Inhibition of DPP-IV promotes the absorption of enterostatin and desanginine-
enterostatin
across rat jejunum.
G-CSF (granulocyte-colony stimulating factor) is a growth factor for
hematopoietic
cells such as neutrophils.
EPO (erythropoietic-6) is a red blood cell growth factor.
IL-6 (Interleukin-6) is a hematopoietic and lymphocyte growth factor.
IL-11 (Interleukin-I 1) is a lymphokine.
IL-8 (Interleukin-8) is a hematopoietic growth factor. angiogenesis cytokine.
Substance P is a neuropeptide and a hematopoietic growth factor.
1o Other suitable chemokines include Substance P, which has vasoactive
properties,
monomeric fibrin, which effects blood clotting, f bronectin, which promotes
binding of
hepatocytes and could enhance liver regeneration, MIP-3, a chemoattractant for
B-cells, and
collagen types I, II, III, and IV, which regulate in part the migration of a
number of effector
cells, including T cells, across the endothelial barrier.
Similarly, other medical disorders which may be treated according to the
method of
this invention include allergies, angiogenesis, cardiogenesis, anti-tumor
responses, hepatic
disease, and organ vascularization.
Substrates not specifically disclosed herein, both known and unknown, which
are
capable of acting as substrates for DPP-IV, are considered to be fully within
the scope of this
2o invention.
Formulation of Pharmaceutical Composition
The compounds of the invention or compositions thereof can be administered
alone or
in combination with one another, or in combination with other therapeutic
agents. For
example, treatment with one or more of the compounds of the invention can be
combined with
more traditional therapies for treating medical disorders, or combined with
other cytokines to
enhance treatment success.
When administered, the pharmaceutical preparations of the invention are
applied in
pharmaceutically-acceptable amounts and in pharmaceutically-acceptably
compositions. Such
3o preparations may routinely contain salt, buffering agents, preservatives,
compatible carriers,
and optionally other therapeutic agents. When used in medicine, the salts
should be
pharmaceutically acceptable, but non-pharmaceutically acceptable salts may
conveniently be
CA 02339537 2001-02-02
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-I4
used to prepare pharmaceutically-acceptable salts thereof ;and are not
excluded from the scope
of the invention. Such pharmacologically and pharmaceutically-acceptable salts
include, but
are not limited to, those prepared from the following acids: hydrochloric,
hydrobromic,
sulfuric, nitric, phosphoric, malefic, acetic, salicylic, citric, formic,
malonic, succinic, and the
like. Also, pharmaceutically-acceptable salts can be prepared as alkaline
metal or alkaline
earth salts, such as sodium, potassium or calcium salts. The pharmaceutical
compositions also
may contain, optionally, suitable preservatives, such as: benzalkonium
chloride;
chlorobutanol; parabens and thimerosal. Carrier formulation suitable for oral,
subcutaneous,
intravenous, intramuscular, etc., administrations can be found in Remington's
Pharmaceutical
to Sciences, Mack Publishing Co., Easton, PA.
Routes of Administration
A variety of administration routes are available for treating a subject. The
particular
mode of delivery selected will depend, of course, upon the particular compound
selected, the
severity of the condition being treated and the dosage required for
therapeutic efficacy. The
methods of the invention, generally speaking, may be practiced using any mode
of
administration that is medically acceptable, meaning any mode that produces
effective levels
of the active compounds without causing clinically unacceptable adverse
effects. Such modes
of administration include oral, rectal, topical, nasal, interdermal, or
parenteral routes.
2o Compositions suitable for oral administration may be presented as discrete
units, such
as capsules, tablets, lozenges, each containing a predetermined amount of the
compound of
the invention. Other compositions include suspensions in aqueous liquids or
non-aqueous
liquids such as a syrup, elixir or an emulsion. The oral preparation may
include an enteric
coating.
2s As used herein, the term "parenteral" includes subcutaneous, intravenous,
intramuscular, or infusion. Compositions suitable for parenteral
administration conveniently
comprise a sterile aqueous preparation of the compound, which is preferably
isotonic with the
blood of the recipient. This aqueous preparation may be formulated according
to known
methods using suitable dispersing or wetting agents and suspending agents. The
sterile
3o injectable preparation also may be a sterile injectable solution or
suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-
butane diol.
Among the acceptable vehicles and solvents that may he employed are water,
Ringer's
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solution, and isotonic sodium chloride solution. In addition, sterile, fixed
oils are
conventionally employed as a solvent or suspending medium. For this purpose
any bland
fixed oil may be employed including synthetic mono- or di-glycerides. In
addition, fatty acids
such as oleic acid may be used in the preparation of inject:ables. Intravenous
or intramuscular
s routes are not particularly suitable for long-term therapy and prophylaxis.
They could,
however, be preferred in emergency situations. Oral administration will be
preferred for
prophylactic and other treatment because of the convenience to the patient as
well as the
dosing schedule.
The pharmaceutical compositions may conveniently be presented in unit dosage
form
t o and may be prepared by any of the methods well-known in the art of
pharmacy. The methods
include the step of bringing the compounds of the invention into association
with a carrier
which constitutes one or more accessory ingredients. In general, the
compositions are
prepared by uniformly and intimately bringing the compounds into association
with a liquid
carrier, a finely divided solid carrier, or both, and then, if necessary,
shaping the product.
15 Other delivery systems can include time-release, delayed release or
sustained release
delivery systems. Such systems can avoid repeated administrations of the
compounds
described above, increasing convenience to the subject and the physician. Many
types of
release delivery systems are available and known to those of ordinary skill in
the art. They
include polymer base systems such as poly(lactide-glycolide), copolyoxalates,
2o polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric
acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing drugs are
described in,
for example, U.S. Patent S,07S,I09. Delivery systems also include non-polymer
systems that
are: lipids including sterols such as cholesterol, cholesterol esters and
fatty acids or neutral
fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic
systems; peptide
25 based systems; wax coatings; compressed tablets using conventional binders
and excipients;
partially fused implants; and the like. Specific examples include, but axe not
limited to: (a)
erosional systems in which the compound is contained in a form within a matrix
such as those
described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660
and (b)
diffusional systems in which an active component permeates at a controlled
rate from a
3o polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480. In
addition, pump-
based hardware delivery systems can be used, some of which are adapted for
implantation.
Use of a long-term sustained release implant may be particularly suitable for
treatment
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of chronic conditions. Long-term release, as used herein, means that the
implant is
constructed and arranged to deliver therapeutic levels of the active
ingredient for at least I O
days, and preferably 60 days. Long-term sustained release implants are well-
known to those
of ordinary skill in the art and include some of the release systems described
above.
The compounds described herein are administered in effective amounts. An
effective
amount is a dosage of the compound sufficient to provide a medically desirable
result. The
effective amount will vary with the particular condition being treated, the
age and physical
condition of the subject being treated, the severity of the condition, the
duration of the
treatment, the nature of the concurrent therapy (if any), the specific route
of administration,
1 o and like factors within the knowledge and expertise of the health
practitioner. An effective
amount for stimulating a desired immune response also can be measured, for
example, by
determining a change in the immune function in a subject (e.g., increased B
cell response,
increased cytotoxic T cell response, or an ability to slow, halt, or prevent
an infection). An
effective amount for treating an autoimmune disorder or allergic disorder
would be that
15 amount sufficient to lessen or inhibit altogether the immune or allergic
response associated
with the disorder so as to slow or halt the development of or the progression
of the disorder.
As used in the claims, "inhibit" embraces all of the foregoing. Likewise, an
effective amount
for treating an immune system disorder is that amount which can slow or halt
altogether the
symptoms associated with the immune system disorder so as to prevent the
disorder, slow its
2o progression, or halt the progression of the immune system disorder. It is
preferred generally
that a maximum dose be used, that is, the highest safe dose according to sound
medical
judgment.
Generally, doses of active compounds will be fronn about 0.001 mg/kg per day
to 1000
mglkg per day. It is expected that doses range of 0.01 to 100 mg/kg per day
will be suitable,
25 preferably orally and in one or several administrations per day. Lower
doses will result from
other forms of administration, such as intravenous administration. In the
event that a response
in a subject is insufficient at the initial doses applied, higher doses (or
effectively higher doses
by a different, more localized delivery route) may be employed to the extent
that patient
tolerance permits. Multiple doses per day are contemplated to achieve
appropriate systemic
30 levels of compounds.
All patents, references and other documents that are identified in this patent
application are incorporated in their entirety herein by reference.
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The following examples are illustrative only and a.re not intended to limit
the invention
in any way. A particularly preferred compound of the invention, Val-boroPro
("PT-100"), is
used in the examples.
Throughout this application and in particular, in each of the examples,
particular
embodiments are described and illustrated. It is to be understood that any of
the reactive
groups disclosed herein can be substituted for the particular reactive groups
(e.g., boronyl
group) as described in the examples.
EXAMPLE 1: GENERAL SYNTHESIS OF ACTIVE COMPOUNDS
l0 Synthesis of the boroPro compounds of this invention are described in
Bachovchin
'493. In general, the preparatory technique involves straightforward peptide
coupling
chemistry. The standard peptide coupling chemistry methods and procedures used
in this
invention are readily available. Examples of books using these methods
include, but are not
limited to, the following citations incorporated herein by reference: P.D.
Bailey, An
Introduction to Peptide Chemistry, Ed.: John Wiley & Sons, 1990; Miklos
Bodansky,
Peptide Chemistry, A Practical Textbook, Ed.: Springer-Verlag, 1988; Miklos
Bodansky,
Principles of Peptide Synthesis, "Reactivity and Structure Concepts in Organic
Chemistry,"
Volume 16, Ed.: Springer-Verlag, 1984; and Mikios Bodansky, Principles of
Peptide
Synthesis, "Reactivity and Structure Concepts in Organic Chemistry," Volume
21, Ed.:
2o Springer-Verlag, 1984.
The compounds of the invention can begin with the synthesis of H-boroPro as
taught
in WO 98/00439. Use of H-boroPro is for illustrative purposes only, and is not
intended to
limit the scope of this invention.
According to WO 98/00439, H-boroPro was prepared by the synthetic route
previously
developed and described (G.R. Flentke, et al., "Inhibition of dipeptidyi
aminopeptidase IV
(DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the
role of DP-IV
in T-cell function," PNAS (U.S.A.) 88, 1SS6-1559 (1991); also described in
United States
Patent No. 5,462,928). Alternatively, H-boroPro may be produced by a new
procedure {Kelly,
T.A., et al., "The efficient synthesis and simple resolution of a proiine
boronate ester suitable
3o for enzyme inhibition studies," Tetrahedron 49, 1009-1016 (1993)). Both of
these synthetic
routes reportedly yield racemic H-boroPro pinanediol.
According to WO 98/00439, stereochemically pure L, L and L, D diastereomers of
Z-
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Lys-boroPro were prepared by first resolving racemic H-boroPro through
crystallization with
optically active blocking protecting groups ((1S, 2S, 3R, SS)-+-pinanediol
isomer) followed
by coupling the isotopically pure L-boroPro and D-boroPro to the
stereochemically pure L
isomer of lysine (See United States Patent No. 5,462,928). Alternatively, the
L,L and L,D
s diastereomers of Lys-boroPro were prepared in high optical purity by
coupling racemic H-
boroPro by L-Lys and separating the resulting diastereomeric Z-Lys-boroPro-
diester into its
component L,D and L,L diastereomers using reverse phase HPLC as previously
described for
diastereomeric Pro-boroPro (W.G. Gutheil and W.W. Bachovchin, "Separation of L-
Pro-DL-
boroPro into Its Component Diastereomers and Kinetic Analysis of Their
Inhibition of
o Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-
Binding Inhibition,"
Biochemistry 32, 8723-8731 (I993)).
EXAMPLE 2: SYNTHESIS OF ACTIVE CYCLIC COMPOUNDS
15 In aqueous solution at all pH values, the inhibitors exist as a slowly
equilibrating
mixture of two conformations: an open chain structure (linear boroProline
compound) which
is inhibitory (active species), and a cyclic structure (cyclic; boroProline
compound) which is
non-inhibitory (inactive species). The open, active, inhibil;ory chain species
is favored at low
pH while the cyclized structure is favored at neutral pH. 'The reaction is
fully reversible: the
zo open chain becomes predominant at low pH. The open chain to cyclic species
reaction
involves a trans to cis isomerization of the proline and the formation of a
new N-B bond. The
cyclized structure is the boron analog of a diketopiperazine, a product often
seen in peptide
chemistry. Cyclization liberates one equivalent of H+ thereby explaining the
requirement for
base in the cyclization reaction and acid in the opening reaction. The cyclic
structure is quite
2s stable in aqueous solutions of high pH.
Prolonged incubation at high pH does not lead to the complete disappearance of
DPP-
IV inhibitory activity for any of the Xaa-boroPro compounds examined. This
observation was
the first evidence that the active inhibitor was in a conformation equilibrium
with a non-
inhibitory species rather than undergoing an irreversible inactivation. The
half life for the
3o reformation of the open chain species from the cyclic structure is
surprisingly low. Thus, it
was concluded that the loss of inhibitory activity in aqueous solution was due
to a pH
dependent conformational equilibrium rather than a degradation reaction.
The fact that the inhibitory activity does not go to zero for any of the Xaa-
boroPro
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inhibitors, even after prolonged incubation, together with i:he fact that the
reverse reaction,
i.e., cyclic to open chain is slow, suggested that it should be possible to
measure the
equilibrium constant for the conformation equilibrium by measuring the
apparent Ki at
equilibrium and comparing it with the true Ki. It has been reported that the
ratio of [cyclic]:
[open] forms, at neutral pH, is 156:1 for Pro-boroPro and 1130:1 for Val-
boroPro (W.G.
Gutheil and W.W. Bachovchin, Separation of L-Pro-DL-boroPro into Its Component
Diastereomers and Kinetic Analysis of Their Inhibition of Dipeptidyl Peptidase
IV. A New
Method for the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32,
8723-8731
(1993)). This means that less than 1% Pro-boroPro and less than 0.1% of Val-
boroPro exists
1 o as the open chain, inhibitory species, at equilibrium at pH 7Ø
Nevertheless, under these
conditions the inhibitors behave as though they had Ki's of 2.5 nM and 1.8 nM
respectively.
This apparent Ki of the "fully inactivated" species is still substantially
better than, ( ~ 1000-
fold) that of other inhibitors of DPP-IV thus far reported.
The inventors believe that the cyclic compounds of the invention have the
ability to
specifically bind to CD26. Accordingly, the inventors predict that the
biological function of
the compounds of the invention could be significantly increased (approximately
100-1000
times) by orally administering the cyclic compounds of the invention and
permitting the
conformational changes, e.g., linearization, to occur i~ vivo (e.g., under the
acidic conditions
of the stomach).
2o Thus, if Iinearization is necessary, it can be accomplished in vivo and
therefore, therapeutic
concentrations in the systemic circulation can be generated in situ and,
accordingly, it is
believed that the bioactivity of the compounds of the invention can be
increased by
approximately 100 - 1000 fold. In addition, it is believed that the cyclic
boroProline
compounds of the invention, in lyophilized or solid form, have improved shelf
life properties,
thereby contributing to the further utility of the compound s of the
invention.
Each of the compounds prepared as described above can be purified to
homogeneity
using HPLC and its identity can be confirmed by NMR spf°ctroscopy,
amino acid
composition, or mass spectroscopy as deemed necessary.
The cyclic compounds of the invention can be converted to linear form by
adjusting
3o the pH to an acidic pH {e.g., pH range: 1-3} and the potency of inhibition
of CD26 proteinase
activity by the linear boroProline compounds can be determined using
conventional enzyme
analysis (example provided below). In addition, the immunomodulatory effects
of the
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compounds of the invention are evaluated by in vivo experiments using animal
models and by
in vitro experiments using cell culture methods that are believed by those of
ordinary skill in
the art to be predictive of an in vivo activity.
EXAMPLE 3: ASSESSMENT OF FUNCTIONAL ACTIVITY
The compounds of the invention have at least the following properties: (I)
binding site
is the DPP-IV active site; and (ii) exhibit cross-species specificity.
The assays which are used to assess functional activity include: DPP-IV
activity, oral
and subcutaneous bioavailability assays, and are described below.
1o EXAMPLE 4: MEASURING STANDARD DPP-IV ACTIVITY
Assays to measure DPP-IV activity can be performed on the compounds of the
invention. Methods for quantitatively measuring the interaction of small
peptidomimetic
inhibitors with DPP- IV, as well as for the interaction of CD26 with larger
ligands, e.g., the
HIV Tat protein, have been developed (W.G. Gutheil and W.W. Bachovchin.
Separation of
L-Pro-DL-boroPro into Its Component Diastereomers and Kinetic Analysis of
Their Inhibition
of Dipeptidyl Peptidase IV. A New Method for the Analysis of Slow, Tight-
Binding
Inhibition, Biochemistry 32, 8723-8731 (1993); Gutheil,'JV.G., and W., B.W.
Kinlsq, A
Matlab Program for Fitting Kinetics Data with Numerically Integrated Rate
Equations and Its
Application to the Analysis of Slow, Tight Binding Data, Analytical
Biochemistry 223, I3-20
(1994); Gutheil, W.G., et al., HIV-1 Tat Binds to DP IV (CD26): A possible
Mechanism for
Tats Immunosuppressive Activity, Proc. Natl. Acad. Sci. U.S.A. 91, 6594-6598
(1994)).
These methods use the chromatogenic substrate Ala-Pro-p-nitroanilide {AppNA)
and
fluorescent substrate Ala-Pro-7-amino-4-trifluoromethyl coumarin {AP-AFC).
AppNA and
AP-AFC are commercially available (e.g., Enzyme Systems Products, Dublin, CA).
2s EXAMPLE 5: MEASURING ORAL BIOAVAILABILITY
An in vitro assay of serum DPP-IV activity has been developed, which can be
used as
a surrogate marker to determine the bioavailability of PT-100 in serum
following
subcutaneous or oral administration in mice. This assay is based on the
ability of PT-100 to
inhibit serum DDP-IV protease activity in a dose dependent manner.
3o As shown in Figure 1, mice received PT-100 at indicated doses by gavage.
Blood
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samples are taken two hours post administration and serum DPP-IV activity is
determined.
DPP-IV activity is measured in a fluorometric assay using the synthetic
substrate 7-amino-4-
trifluoromethyl coumarin (AFC)-AlaPro (15). Total inhibition of serum DPP-IV
activity is
achieved at doses of 2 pg or 5 beg PT-100. Doses below 2 ~g result in a dose
dependent
decrease of DPP-IV inhibition.
While the invention has been described with respect to certain embodiments, it
should
be appreciated that many modifications and changes may be made by those of
ordinary skill in
the art without departing from the spirit of the invention. It is intended
that such
modifications, changes and equivalents fall within the scope of the appended
claims.
