U.S. patent application number 10/172809 was filed with the patent office on 2003-07-10 for dipeptidyl peptidase iv inhibitors and their uses as anti-cancer agents.
Invention is credited to Demuth, Hans-Ulrich, Hoffmann, Torsten, von Hoersten, Stephan.
Application Number | 20030130199 10/172809 |
Document ID | / |
Family ID | 43706178 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030130199 |
Kind Code |
A1 |
von Hoersten, Stephan ; et
al. |
July 10, 2003 |
Dipeptidyl peptidase IV inhibitors and their uses as anti-cancer
agents
Abstract
The present invention provides new uses of DPIV-inhibitors of
the present invention, and their corresponding pharmaceutically
acceptable acid addition salt forms, for treating conditions
mediated by DPIV or DPIV-like enzymes, such as cancer and tumors.
In a more preferred embodiment, the compounds of the present
invention are useful for the treatment of metastasis and tumor
colonization.
Inventors: |
von Hoersten, Stephan;
(Wedemark, DE) ; Demuth, Hans-Ulrich;
(Halle/Saale, DE) ; Hoffmann, Torsten;
(Halle/Saale, DE) |
Correspondence
Address: |
Mark A. Hofer
Brown Rudnick Berlack Israels, LLP
One Financial Center
Boston
MA
02111
US
|
Family ID: |
43706178 |
Appl. No.: |
10/172809 |
Filed: |
June 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60301158 |
Jun 27, 2001 |
|
|
|
60360909 |
Feb 28, 2002 |
|
|
|
Current U.S.
Class: |
514/19.3 ;
514/20.1 |
Current CPC
Class: |
A61K 38/05 20130101;
A61K 31/401 20130101; A61K 38/07 20130101; A61K 38/55 20130101;
A61K 38/06 20130101; A61K 31/426 20130101 |
Class at
Publication: |
514/17 ; 514/18;
514/19 |
International
Class: |
A61K 038/06; A61K
038/05 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 2001 |
EP |
01 114 796.4 |
Oct 12, 2001 |
DE |
101 50 203.6 |
Nov 9, 2001 |
DE |
101 54 689.0 |
Claims
We claim:
1. A method for reducing cancer or a related disorder comprising
the step of administering a therapeutically effective amount of at
least one inhibitor of dipeptidyl peptidase IV (DPIV) or DPIV-like
enzyme activity, wherein said inhibitor is selected from the group
consisting of dipeptide compounds, peptide compounds comprising
tri-, tetra- and pentapeptides, peptidylketones, aminoketone
derivatives and side chain modified DP IV inhibitors.
2. The method according to claim 1, wherein the related disorder is
metastasis.
3. The method according to claim 1, wherein the related disorder is
tumor colonization.
4. The method according to claim 1, wherein the dideptidyl
peptidase IV-like enzyme is selected from the group consisting of
fibroblast activation protein .alpha., dipeptidyl peptidase IV
.beta., dipeptidyl aminopeptidase-like protein, N-acetylated
.alpha.-linked acidic dipeptidase, quiescent cell proline
dipeptidase, dipeptidyl peptidase II, attractin and dipeptidyl
peptidase IV related protein (DPP 8), dipeptidyl peptidase 9 (DPP9)
or KIAA1492.
5. The method according to claim 1, wherein the structure of the
dideptidyl peptidase IV-like enzyme is undiscovered.
6. The method according to claim 1, wherein the inhibitor is a
dipeptide compound formed from an amino acid and a thiazolidine or
pyrrolidine group, and salts thereof.
7. The method according to claim 6 wherein the dipeptide compound
is selected from the group consisting of L-threo-isoleucyl
pyrrolidine, L-allo-isoleucyl thiazolidine, 1-allo-isoleucyl
pyrrolydine, L-glutaminyl thiazolidine, L-glutaminyl pyrrolidine,
L-glutamic acid thiazolidine, L-glutamic acid pyrrolidine and salts
thereof.
8. The method according to claim 1 wherein the inhibitor is a
peptide compound useful for competetive modulation of dipeptidyl
peptidase IV catalysis represented by the general formula 15wherein
A, B, C, D and E are any amino acid residues including
proteinogenic amino acids, non-proteinogenic amino acids, L-amino
acids and D-amino acids and wherein E and/or D may be absent or B
and/or A may be absent provided that: A is any amino acid residue
except D-amino acid residues; B is any proteinogenic amino acid
residue, but If B is an amino acid selected from Pro, Ala, Ser,
Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, then C
is any amino acid residue including D-amino acids, except Pro, Ala,
Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid and
E may be unused to generate tetrapeptides of the formula A-B-C-D,
or D and E may be unused to generate tripeptides of the formula
A-B-C provided, but If B is not an amino acid selected from Pro,
Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic
acid, then C is any .alpha.-amino acid except D-amino acids; D is
Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic
acid; E is any amino acid residue including D-amino acids, except
Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic
acid, but If D is Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic
acid or pipecolic acid, then C is any .alpha.-amino acid except
D-amino acids and Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic
acid or pipecolic acid, and A may be unused to generate
tetrapeptides of the formula B-C-D-E, or A and B may be unused to
generate tripeptides of the formula C-D-E provided however, If D is
not selected from Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic
acid or pipecolic acid, then E is any amino acid residue including
D-amino acids.
9. The method according to claim 1, wherein the inhibitor is
peptidylketone represented by the general formula 16including all
stereoisomers and pharmaceutical acceptable salts thereof, wherein
A is selected from: 17 and X.sup.1 is H or a acyl or oxycarbonyl
group including all amino acids and peptide residues; X.sup.2 is H,
--(CH).sub.n--NH--C.sub.5H.sub.3N--Y with n=2-4 or
C.sub.5H.sub.3N--Y (a divalent pyridyl residue) and Y is selected
from H, Br, Cl, I, NO.sub.2 or CN; X.sup.3 is H or selected from an
alkyl, alkoxy, halogen, nitro, cyano or carboxy substituted phenyl
or pyridyl residue; X.sup.4 is H or selected from an alkyl, alkoxy,
halogen, nitro, cyano or carboxy substituted phenyl or pyridyl
residue; X.sup.5 is H or an alkyl, alkoxy or phenyl residue; and
X.sup.6 is H or a alkyl residue; provided that for n=1, X is
selected from: H, OR.sub.2, SR.sub.2, NR.sub.2 R.sup.3,
N.sup.+R.sub.2 R.sup.3R.sup.4, wherein: R.sup.2 stands for acyl
residues, which are substituted with alkyl, cycloalkyl, aryl or
heteroaryl residues, or for all amino acids and peptidic residues,
or alkyl residues, which are substituted with alkyl, cycloalkyl,
aryl and heteroaryl residues; R.sup.3 stands for alkyl and acyl
functions, wherein R.sup.2 and R.sup.3 may be embedded in ring
structures of saturated and unsaturated carbocyclic or heterocyclic
structures; R.sup.4 stands for alkyl residues, wherein R.sup.2 and
R.sup.4 or R.sup.3 and R.sup.4 may be embedded in ring structures
of saturated and unsaturated carbocyclic or heterocyclic
structures; and for n=0, X is selected from: 18 wherein B stands
for: O, S, NR.sup.5, wherein R.sup.5 is H, a alkyl or acyl, C, D,
E, F, G, H are independently selected from alkyl and substituted
alkyl residues, oxyalkyl, thioalkyl, aminoalkyl, carbonylalkyl,
acyl, carbamoyl, aryl and heteroaryl residues; and Z is selected
from H, or a branched or single chain alkyl residue from
C.sub.1-C.sub.9 or a branched or single chain alkenyl residue from
C.sub.2-C.sub.9, a cycloalkyl residue from C.sub.3-C.sub.8, a
cycloalkenyl residue from C.sub.5-C.sub.7, a aryl- or heteroaryl
residue, or a side chain selected from all side chains of all
natural amino acids or derivatives thereof.
10. The method according to claim 1 wherein the inhibitor is a
aminoketone derivative represented by the general formulas 5, 6, 7,
8, 9, 10 and 11, including all stereoisomers and pharmaceutical
acceptable salts thereof, 19wherein: R.sup.1 is H, a branched or
linear C.sub.1-C.sub.9 alkyl residue, a branched or linear
C.sub.2-C.sub.9 alkenyl residue, a C.sub.3-C.sub.8 cycloalkyl-,
C.sub.5-C.sub.7 cycloalkenyl-, aryl- or heteroaryl residue or a
side chain of a natural amino acid or a derivative thereof; R.sup.3
and R.sup.4 are selected from H, hydroxy, alkyl, alkoxy, aryloxy,
nitro, cyano or halogen, A is H or an isoster of an carbonic acid,
like a functional group selected from CN, SO.sub.3H, CONHOH,
PO.sub.3R.sup.5R.sup.6, tetrazole, amide, ester, anhydride,
thiazole and imidazole; B is selected from: 20 wherein: R.sup.5 is
H, --(CH).sub.n--NH--C.sub.5H.sub.3N--Y with n=2-4 and
C.sub.5H.sub.3N--Y (a divalent pyridyl residue) with Y=H, Br, Cl,
I, NO.sub.2 CN; R.sup.10 is H, a acyl, oxycarbonyl or a amino acid
residue; W is H or a phenyl or pyridyl residue, substituted with
alkyl, alkoxy, halogen, nitro, cyano or carboxy residue; W.sup.1 is
H, a alkyl, alkoxy or phenyl residue; Z is H or a phenyl or pyridyl
residue, substituted with alkyl, alkoxy, halogen, nitro, cyano or
carboxy residue; Z.sup.1 is H or a alkyl residue; D is a cyclic
C.sub.4-C.sub.7 alkyl, C.sub.4-C.sub.7 alkenyl residue or a alkyl
substituted derivative thereof or a cyclic 4-7-membered heteroalkyl
or 4-7-membered heteroalkenyl residue; X.sup.2 is O, NR.sup.6,
N.sup.+(R.sup.7).sub.2, or S; X.sup.3 to X.sup.12 are selected from
CH.sub.2, CR.sup.8R.sup.9, NR.sup.6, N.sup.+(R.sup.7).sub.2, O, S,
SO and SO.sub.2, including all saturated and unsaturated
structures; R.sup.6, R.sup.7, R.sup.8, R.sup.9 are selected from H,
a branched or linear C.sub.1-C.sub.9 alkyl residue, a branched or
lienar C.sub.2-C.sub.9 alkenyl residue, a C.sub.3-C.sub.8
cycloalkyl residue, a C.sub.5-C.sub.7 cycloalkenyl residue, an aryl
or heteroaryl residue; with the following provisos: Formula 6:
X.sup.6 is CH if A is not H; Formula 7: X.sup.10 is C if A is not
H; Formula 8: X.sup.7 is CH if A is not H; and Formula 9: X.sup.12
is C if A is not H.
11. The method according to claim 1, wherein the inhibitor is a
side chain modified inhibitor of DPIV or DPIV-like enzyme activity
represented by the general formula, 21including all stereoisomers
and pharmaceutical acceptable salts thereof, wherein A is an amino
acid having at least one functional group in the side chain; B is a
chemical compound covalently bound to at least one functional group
of the side chain of A, namely oligopeptides having a chain length
of up to 20 amino acids, except for homopolymers of glycine
consisting of up to 6 glycine monomers; or polyethylene glycols
having molar masses of up to 20,000 g/mol; or optionally
substituted organic amines, amides, alcohols, acids or aromatic
compounds having from 8 to 50 C atoms; and C is a thiazolidine,
pyrrolidine, cyanopyrrolidine, hydroxyproline, dehydroproline or
piperidine group amide-bonded to A.
12. The method according to claim 11, wherein A is an amino acid,
preferably an .alpha.-amino acid, especially a natural
.alpha.-amino acid having at least one functional group in the side
chain selected from the group consisting of threonine, tyrosine,
serine, arginine, lysine, aspartic acid, glutamic acid or
cysteine.
13. The method according to claim 8 wherein said inhibitor is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent and a therapeutically effective amount of a said
inhibitor or a pharmaceutically acceptable acid addition salt
thereof.
14. A pharmaceutical composition comprising: an inhibitor of DPIV
or DPIV-like enzyme activity or a pharmaceutically acceptable acid
addition salt thereof for the manufacture of a medicament for
inhibiting dipeptidyl peptidase IV or dipeptidyl peptidase IV-like
enzyme activity for the prevention or treatment of diseases or
conditions related to dipeptidyl peptidase IV or dipeptidyl
peptidase IV-like enzymes.
15. The pharmaceutical composition according to claim 14 for the
manufacture of a medicament for the treatment of cancer and related
disorders.
16. The pharmaceutical composition according to claim 14 for the
manufacture of a medicament for the treatment of metastasis.
17. The pharmaceutical composition according to claim 14 for the
manufacture of a medicament for the treatment of tumor
colonization.
18. The method according to claim 9 wherein said inhibitor is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent and a therapeutically effective amount of a said
inhibitor or a pharmaceutically acceptable acid addition salt
thereof.
19. The method according to claim 10 wherein said inhibitor is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent and a therapeutically effective amount of a said
inhibitor or a pharmaceutically acceptable acid addition salt
thereof.
20. The method according to claim 11 wherein said inhibitor is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent and a therapeutically effective amount of a said
inhibitor or a pharmaceutically acceptable acid addition salt
thereof.
Description
RELATED APPLICATIONS
[0001] This application claims the priority of U.S. provisional
application No. 60/301,158 entitled Peptide Structures Useful for
Competitive modulation of Dipeptidyl Peptidase IV Catalysis filed
on Jun. 27, 2001. Priority is also claimed from U.S. provisional
application No. 60/360,909 entitled Glutaminyl-based DPIV
Inhibitors filed on Feb. 28, 2002. This application also claims the
priority of the following foreign applications EP 01 114 796.4
entitled Peptide Structures Useful for Competitive Modulation of
Dipeptidyl Peptidase IV Catalysis having a priority date of Jun.
27, 2001, DE 101 50 203.6 entitled Peptidylketone als Inhibitoren
der DPIV having a priority date of Oct. 12, 2001 and DE 101 54
689.0 entitled Substituierte Aminoketonverbindungen having a
priority Date of Nov. 09, 2001. The above applications are
incorporated in their entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to inhibitors of dipeptidyl
peptidase IV and dipeptidyl peptidase IV-like enzyme activity and,
more particularly, pharmaceutical compositions containing said
compounds, and the use of said compounds for the treatment of
cancer and tumors. The present invention especially provides a
method for the inhibition of metastasis and tumor colonization.
BACKGROUND ART
[0003] Dipeptidyl peptidase IV (DPIV) is a serine protease which
cleaves N-terminal dipeptides from a peptide chain containing,
preferably, a proline residue in the penultimate position. Although
the biological role of DPIV in mammalian systems has not been
completely established, it is believed to play an important role in
neuropeptide metabolism, T-cell activation, attachment of cancer
cells to the endothelium and the entry of HIV into lymphoid
cells.
[0004] Likewise, it has been discovered that DPIV is responsible
for inactivating glucagon-like peptide-1 (GLP-1) and
glucose-dependent insulinotropic peptide also known as
gastric-inhibitory peptide (GIP). Since GLP-1 is a major stimulator
of pancreatic insulin secretion and has direct beneficial effects
on glucose disposal, in WO 97/40832 and U.S. Pat. No. 6,303,661
inhibition of DPIV and DPIV-like enzyme activity was shown to
represent an attractive approach for treating non-insulin-dependent
diabetes mellitus (NIDDM).
[0005] The present invention provides a new use of DPIV-inhibitors
for the treatment of conditions mediated by inhibition of DPIV and
DPIV-like enzymes, in particular the treatment of cancer and tumors
and the inhibition of metastasis and tumor colonization, and
pharmaceutical compositions e.g. useful in inhibiting DPIV and
DPIV-like enzymes and a method of inhibiting said enzyme
activity.
[0006] This invention relates to a method of treatment, in
particular to a method for the treatment of cancer, tumors,
metastasis and tumor colonization and to compositions for use in
such method. Dipeptidyl peptidase IV (DPIV) is a post-proline (to a
lesser extent post-alanine, post-serine or post-glycine) cleaving
serine protease found in various tissues of the body including
kidney, liver, and intestine.
[0007] It is known that DPIV-Inhibitors may be useful for the
treatment of impaired glucose tolerance and diabetes mellitus
(International Patent Application, Publication Number WO 99/61431,
Pederson R A et al, Diabetes. 1998 August; 47(8):1253-8 and Pauly
RP et al, Metabolism 1999 March; 48(3):385-9). In particular WO
99/61431 discloses DPIV-Inhibitors comprising an amino acid residue
and a thiazolidine or pyrrolidine group, and salts thereof,
especially L-threo-isoleucyl thiazolidine, L-allo-isoleucyl
thiazolidine, L-threo-isoleucyl pyrrolidine, L-allo-isoleucyl
thiazolidine, L-allo-isoleucyl pyrrolidine, and salts thereof.
[0008] Further examples of low molecular weight dipeptidyl
peptidase IV inhibitors are agents such as
tetrahydroisoquinolin-3-carboxamide derivatives, N-substituted
2-cyanopyroles and -pyrrolidines, N-(N'-substituted
glycyl)2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines,
N-(substituted glycyl)-4-cyanothiazolidines,
amino-acyl-borono-prolyl-inhibitors, cyclopropyl-fused pyrrolidines
and heterocyclic compounds. Inhibitors of dipeptidyl peptidase IV
are described in U.S. Pat. Nos. 6,380,398, 6,011,155; 6,107,317;
6,110,949; 6,124,305; 6,172,081; WO 95/15309, WO 99/61431, WO
99/67278, WO 99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C
2, WO 98/19998, WO 00/07617, WO 99/38501, WO 99/46272, WO 99/38501,
WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO
02/02560 and WO 02/14271, the teachings of which are herein
incorporated by reference in their entirety.
[0009] The term DPIV-like enzymes relates to structurally and/or
functionally DPIV/CD26-related enzyme proteins (Sedo & Malik,
Dipeptidyl peptidase IV-like molecules: homologous proteins or
homologous activities? Biochimica et Biophysica Acta 2001, 36506:
1-10). In essence, this small group of enzymes has evolved during
evolution to release H-Xaa-Pro-Dipeptides and H-Xaa-Ala-Dipeptides
from N-terminus of oligo- or polypeptides. They show the common
feature, that they accomotate in the Pro-position also Al, Ser, Thr
and other amino acids with small hydrophobic side-chains as, Gly or
Val. The hydrolytic efficacy is ranked Pro>AlaSer, Thr Gly, Val.
Same proteins have been only available in such small quantities,
that only the post-Pro or post-Ala cleavage could be established.
While the proteins: DPIV, DP II, FAP.alpha. (Seprase), DP 6, DP 8
and DP 9 are structurally related and show a high sequence
homology, attractin is an extraordinary functional DPIV-like
enzyme, characterized by a similar activity and inhibitory
pattern.
[0010] Further DPIV-Like enzymes are disclosed in WO 01/19866, WO
02/04610, WO 02/34900 and WO02/31134. WO 01/19866 discloses novel
human dipeptidyl aminopeptidase (DPP8) with structural and
functional similarities to DPIV and fibroblast activation protein
(FAP). WO 02/04610 provides reagents, which regulate human
dipeptidyl peptidase IV-like enzyme and reagents which bind to
human dipeptidyl peptidase IV-like enzyme gene product. These
reagents can play a role in preventing, ameliorating, or correcting
dysfunctions or diseases including, but not limited to, tumors and
peripheral and central nervous system disorders including pain and
neurodegenerative disorders. The dipeptidyl peptidase IV-like
enzyme of WO 02/04610 is well known in the art. In the Gene Bank
data base, this enzyme is registered as KIAA1492 (registration in
February 2001, submitted on Apr. 04, 2000, AB040925). In the Merops
data base, the dipeptidyl peptidase IV-like enzyme of WO 02/04610
is registered as non-protease homologue, because the active site
serine motive is GKGYGG in contrast to DPIV, which has a active
site serine motive consisting of GWSYGG. The Merops homologue of
the dipeptidyl peptidase IV-like enzyme disclosed in WO 02/04610
and the active site motive thereof was confirmed by the human
genome project. WO 02/34900 discloses a novel dipeptidyl peptidase
9 (DPP9) with significant homology with the amino acid sequences of
DPIV and DPP8. WO 02/31134 discloses three DPIV-like enzymes,
DPRP1, DPRP2 and DPRP3. Sequence analysis revealed, that DPRP1 is
identical to DPP8, as disclosed in WO 01/19866, that DPRP2 is
identical to DPP9 and that DPRP3 is identical to KIAA1492 as
disclosed in WO 02/04610.
[0011] DPIV and DPIV-like Enzymes in Immunophysiology and
Cancer
[0012] Dipeptidyl peptidase IV (DPIV; EC 3.4.14.5; CD26) CD26 is a
M r 110,000 surface glycoprotein with an array of diverse
functional properties that is expressed on a number of tissues,
including epithelial cells and leukocyte subsets (Mentlein, 1999).
Furthermore, it is a membrane-associated ectopeptidase that
possesses DPIV-like activity in its extracellular domain and is
able to cleave N-terminal dipeptides from polypeptides with either
L-proline or L-alanine in the penultimate position. In general,
DPIV is recognized as an ectopeptidase with a triple functional
role. DPIV is involved in catalyzing the release of Xaa-Pro
dipeptides from circulating hormones and chemokines (De Meester et
al, 1999; Mentlein, 1999), in T cell dependent immune responses
(Khne et al, 1999; Korom et al, 1997), and in cell adhesion
including metastasis (Mentlein, 1999).
[0013] In addition DPIV has been identified as the ADA binding
protein, thereby regulating ADA surface expression, with the
DPIV/ADA complex perhaps playing a key role in the catalytic
removal of local adenosine to regulate immune system function.
Besides being a key immunoregulatory molecule, DPIV may have a
potential role in the development of certain neoplasms (Mattern et
al., 1993; Carbone et al., 1995). In eukaryotic cells, cell cycle
progression is controlled at the G1-S checkpoint by a group of
related enzymes known as the CDKs, which are positively regulated
by their physical association with regulatory subunits called
cyclins. It has been demonstrated that binding of soluble anti-CD26
antibodies inhibits the growth of anaplastic large cell T-cell
lymphoma cell lines, both in in vitro and in vivo experiments (Ho
et al., 2001).
[0014] Cancer Pathomechanisms
[0015] Cancer is a group of over 150 diseases characterized by the
uncontrolled growth of abnormal cells in the body. Normal cells can
become abnormal when they are exposed to carcinogens such as
radiation or particular drugs or chemicals. They can also turn
malignant (cancerous) when they are attacked by certain viruses or
when some not-yet-fully-understood internal signal occurs. Once
cells become malignant, they multiply more rapidly than usual. Then
they often form masses called tumors that invade nearby tissue and
interfere with normal bodily functions. Cancer cells also have a
tendency to spread to other parts of the body, where they may form
a secondary tumor.
[0016] Mechanisms of Metastasis
[0017] The outcome of cancer metastasis depends on multiple
interactions within the target tissue and depends on the
microenvironment including cellular adhesion molecules (Carlos,
2001), chemokines (Muller et al., 2001), or hydrodynamic effects
(Haier and Nicholson, 2001) and many other factors (Fidler, 2001).
In addition, a very rapid attraction of leukocytes and specific
cellular responses at the tumor sites may play a critical role in
the early host defense against cancer (Shingu et al.; 2002). These
early changes may be of critical importance for the outcome of
metastatic disease and may extend the present understanding of the
host resistance against metastasis.
[0018] DPIV and DPIV-like Enzymes and Tumor Adhesion and
Colonization
[0019] For cancer cell or metastatic cell adhesion, a prominent
expression of DPIV on endothelia of lung capillaries accounts for
arrest of blood borne breast cancer cells (Johnson et al, 1993).
Fibronectin (FN) and probably also collagen collected on the breast
cancer cell surface were identified as the principal ligands for
DPIV (Abdel-Ghany et al, 1998; Cheng et al, 1998).
[0020] Ho and colleagues (2001) show that binding of soluble
anti-CD26 monoclonal Ab 1F7 inhibits the growth of the human CD301
anaplastic large cell T-cell lymphoma cell line Karpas 299 in both
in in vitro and in vivo experiments. In vitro experiments show that
1F7 induces cell cycle arrest at the G1-S checkpoint, associated
with enhanced p21 expression that is dependent on de novo protein
synthesis. Furthermore, experiments with a severe combined
immunodeficient mouse tumor model demonstrate that 1F7 treatment
significantly enhances survival of tumor-bearing mice by inhibiting
tumor formation.
[0021] Protease Inhibitors, Antibodies and Proteases as Anti-tumor
Agents
[0022] WO 95/29691 discloses proline phosphonate derivatives as
inhibitors of serine proteases with chymotrypsin-like,
trypsin-like, elastase-like and dipeptidyl peptidase IV specificity
and their roles as anti-inflammatory agents, anticoagulants,
anti-tumor agents and anti-AIDS agents.
[0023] WO 98/53812 and WO 97/48409 disclose novel methods of using
phosphonate derivatives, hydroxyphosphinyl derivatives, and
phosphoramidate derivatives to inhibit N-Acetylated .alpha.-Linked
Acidic Dipeptidase (NAALADase) enzyme activity, and to treat
prostate diseases, especially using these compounds for the
inhibition of prostate cancer cell growth.
[0024] WO 01/92273 discloses new benzenedicarboxylic acid
derivative compounds, pharmaceutical compositions, diagnostic
methods and diagnostic kits that include those compounds and
methods of using those compounds for inhibiting NAALADase enzyme
activity, detecting diseases where NAALADase levels are altered,
affecting neuronal activity, affecting TGF-.beta. activity,
inhibiting angiogenesis and treating glutamate abnormalities,
neuropathy, pain, compulsive disorders, prostate diseases, cancer
and glaucoma.
[0025] WO 01/34596 discloses pyrrolecarbonylimino derivatives,
pharmaceutical compounds and methods of using those compounds to
inhibit NAALADase enzyme activity, thereby affecting neuronal
activities, inhibiting angiogenesis and treating glutamate
abnormalities, compulsive disorders, prostate diseases and
cancer.
[0026] WO 00/71135 discloses a method for treating subjects with
abnormal cell proliferation. The method involves administering to
subjects in need of such treatment an effective amount of
boro-proline compounds, to inhibit cell proliferation such as that
associated with tumor growth and metastasis. A method for
inhibiting angiogenesis in an abnormal proliferative cell mass by
the administration of a boro-proline derivative is also provided.
The invention of WO 00/71135 is based, in part, on the observation,
that the boro-proline derivatives are able to inhibit the enzymatic
activity of fibroblast activation protein-alpha (FAP-.alpha.).
[0027] WO 00/71571 relates to a prodrug that is capable of being
converted into a drug by the catalytic action of human fibroblast
activation protein-alpha (FAP-.alpha.), said prodrug having a
cleavage site which is recognised by FAP-.alpha., and said drug
being cytotoxic or cytostatic under physiological conditions. These
prodrugs are converted into a drug at the site of the tumor.
[0028] WO 00/10549 discloses compounds and a method for regulation
of substrate activity in vivo useful for the treatment of medical
disorders such as arteriosclerosis, allergies, inflammation,
angiogenesis, cardiogenesis, neoplasm, tumor, cancer, a hepatic
disease, an intestinal disease, organ vascularization, and
microbial and viral infections. The compounds consist of a
targeting moiety that binds to DPIV, and a reactive group, that
reacts at a reactive center of DPIV. Said compounds are used to
prevent chemokine alteration by inhibiting DPIV activity.
[0029] WO 00/36420 discloses a method for identifying nucleotide
sequences that are differentially expressed in tumor cells,
preferably primary breast tumor cells, comprising exposing a tumor
cell containing tissue sample to an agent specific for fibroblast
activation protein (FAP) separating cells recognised by said agent
from the remaining cells in the sample and harvesting said
remaining cells. Nucleic acid molecules derived from the use of
this technique are also described, together with compositions
comprising the same and their uses in pharmaceutical compositions
for treating a disease, preferably breast cancer.
[0030] WO 99/47152 discloses a method of suppressing the malignant
phenotype or inducing apoptosis of cancer cells in a subject,
comprising introducing into the cancer cell an amount of a nucleic
acid encoding a dipeptidyl peptidase IV protein or fibroblast
activating protein-.alpha., thereby suppressing the malignant
phenotype of the cancer. WO 99/47152 also discloses a method of
inducing expression of dipeptidyl peptidase IV or fibroblast
activating protein-.alpha. in cancer cells of a subject, comprising
administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of an agent capable
of activating transcription of the dipeptidyl peptidase IV gene or
fibroblast activating protein-.alpha. gene and a pharmaceutical
acceptable carrier or diluent.
[0031] WO 01/74299 discloses antibodies that specifically bind to a
membrane protease complex, the complex consisting of two homodimers
of seprase and dipeptidyl peptidase IV (DPIV), obtained from
mammalian, preferably human cell membranes. The antibodies
specifically bind to the DPIV protease of the seprase-DPIV complex.
This membrane protease complex resides on cell surface invadopodia
at the leading edge of angiogenic endothelia, migratory
fibroblasts, and invading cancer cells.
[0032] WO 02/20825 relates to novel methods and compositions for
detection and isolation of cancer cells with metastatic potential.
WO 02/20825 further relates to assays for measuring the metastatic
potential of such cancer cells and drug screening assays for the
indentification of agents having anti-metastatic potential. Also
disclosed are methods and compositions for inhibiting the
metastatic potential of cancer cells by modulating the activity of
serine integral membrane proteases [(SIMP) consisting of seprase
and dipeptidyl peptidase IV (DPIV)] expressed on the surface of
metastasizing cancer cells, by using antibodies against SIMP.
[0033] Current Treatments of Cancer and Tumor Cell Adhesion
[0034] Current cancer treatment regimens comprise surgery,
chemotherapy, radiation therapy, and other treatment methods
including immunotherapy. Immunotherapy is composed of the usage or
the modification of intrinsic bodily mechanisms--in most cases
immune mechanisms--to fight cancer. Chemotherapy kills cancer cells
through the use of drugs or hormones. Taken either orally or
through injection, chemotherapeutic agents are used to treat a wide
variety of cancer. They may be given alone or in combination with
surgery or radiation or both. Chemotherapy is an established way to
destroy hard-to-detect cancer cells that have spread and are
circulating in the body. Anemia (low number of red blood cells) is
a frequent side effect of chemotherapy and may cause symptoms such
as extreme tiredness, dizziness, or shortness of breath. Epoetin
alfa (Procrit.RTM., Epogen.RTM.)--recombinant erythropoietin that
stimulates red blood cell production--is a prescription drug
available for the treatment of chemotherapy-related anemia.
[0035] Immunotherapy uses the body's own immune system or other
parts of the organism to destroy cancer cells. This form of
treatment is still being intensively studied in clinical trials; it
is not yet widely available to most cancer patients. The various
immunological agents used include substances produced by the body
(such as the interferons, the interleukins and tumor necrosis
factor) and laboratory-produced substances (such as monoclonal
antibodies and vaccines). Immunological agents work in different
ways and can be used independently or in combination with other
forms of treatment.
[0036] Angiogenesis Inhibitors as Anti-metastatic Drugs in
Immunotherapy
[0037] Angiogenesis inhibitors are drugs that block the development
of new blood vessels. Solid tumors cannot grow without inducing the
formation of new blood vessels. Blocking the development of new
blood vessels cuts off the tumor's supply of oxygen and
nutrients.
[0038] Several angiogenesis inhibitors are currently being tested
in human trials. In cancerous tissue, tumors cannot grow or spread
(metastasize) without the development of new blood vessels. Blood
vessels supply tissues with oxygen and nutrients necessary for
survival and growth.
SUMMARY OF THE INVENTION
[0039] The present invention provides new uses of DPIV-inhibitors
of formulas 1 to 12, and their corresponding pharmaceutically
acceptable acid addition salt forms for treating cancer and tumors.
In a more preferred embodiment, the compounds of the present
invention are useful for the prevention and inhibition of
metastasis and tumor colonization.
[0040] Reduced expression of the ectopeptidase DPIV and lack of
DPIV-like activity in lungs of mutant F344 rats lacking DPIV
enzymic activity and expression results in reduced adhesion of
cancer cells and in reduced lung metastasis. In vivo cell adhesion
and growth of the F344 rat syngeneic mammary adenocarcinoma MADB106
was studied in F344 rats after acutel and chronic treatment with
DPIV-ligands in vivo. Mutant F344 substrains lacking DPIV enzymic
activity and wild-type-like F344 were tested. Chronic intragastric
infusion of isoleucyl cyano pyrrolidine TFA and isoleucyl
thiazolidine fumarate via osmotic minipumps over two weeks
dose-dependently reduced the cancer-induced weight loss and the
number of tumor colonies on the lung surface. Thus, metastasis of
MADB106 is reduced by chronic treatment using different DPIV
Inhibitors (isoleucyl thiazolidine fumarate; isoleucyl cyano
pyrrolidine TFA) suggesting protective-like class effects by the
two different DPIV-inhibitior/ligands. Possibly, isoleucyl
thiazolidine fumarate and isoleucyl cyano pyrrolidine TFA protect
from metastasis either via interaction with cell adhesion
processes, via a modification of the cellular host defense
mechanisms, via modulation of angiogenesis, via direct effects on
cancer cells, or via increased levels of DPIV substrates, which
indirectly mediate protective-like effects.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1: Effect of single injection of isoleucyl thiazolidine
fumarate on lung metastasis in F344 rats. Vital dye
(Carboxyfluorescein; CFSE) labeled MADB106 tumor cells were
injected via the lateral tail vein and lungs were collected 30 min
after inoculation. CFSE positive tumor cells in lungs were
quantified by means of immunohistology and image analysis. Data
represent means.+-.SEM; no significant differences vs. saline
treated controls were found.
[0042] FIG. 2: Effect of single injection of isoleucyl thiazolidine
fumarate on tumor cell adhesion 30 min after injection in F344
substrains mutant for DPIV. CFSE labeled MADB106 tumor cells were
injected via the lateral tail vein and lungs were collected 30 min
after inoculation. Note the promoting effect in mutant F344GER and
F344JAP rats in contrast to the lack of effects in wild-type F344
rats. Data represent means.+-.SEM; *p<0.05 reflecting
significant differences vs. wild-type F344USA animals determined by
ANOVA and Fisher PLSD.
[0043] FIG. 3: Effect of single injection of isoleucyl
cyanopyrrolidine TFA on tumor cell adhesion 30 min after injection
in F344USA rats. CFSE labeled MADB106 cancer cells were injected
via the lateral tail vein and lungs were collected 30 min after
inoculation. CFSE positive tumor cells in lungs were quantified by
means of immunohistology and image analysis. Data represent
means.+-.SEM; significant differences vs. saline treated controls
were not found.
[0044] FIG. 4: Effect of single injection of valyl pyrrolidine
fumarate on tumor cell adhesion 30 min after injection in F344USA
rats. CFSE labeled of MADB106 adenocarcinoma cells were injected
via the lateral tail vein and lungs were collected 30 min after
inoculation. CFSE positive tumor cells in lungs were quantified by
means of immunohistology and image analysis. Data represent
means.+-.SEM; significant differences vs. saline treated controls
were not found.
[0045] FIG. 5: Effect of chronic intragastric infusion of isoleucyl
thiazolidine fumarate on body weight change in grams in F344 rats
with lung metastasis. A dose dependent reduction of the loss of
body weight after chronic infusion of different dosages of
isoleucyl thiazolidine fumarate in F344 rats 2 weeks after
injection of MADB106 tumor cells is illustrated. One factor ANOVA
revealed a significant effect on body weight, which became
significant in the post-hoc analysis at the 0.4 mg and 4 mg
dosages. Data represent means.+-.SEM; *p<0.05 reflecting
significant differences vs. saline treated SHAM controls determined
by Fisher PLSD.
[0046] FIG. 6: Effect of chronic intragastric infusion of isoleucyl
thiazolidine fumarate on the number of lung tumor colonies in F344
rats. A dose dependent reduction of lung colony numbers after
chronic infusion of different dosages of isoleucyl thiazolidine
fumarate in F344 rats 2 weeks after injection of MADB106 tumor
cells is illustrated. One factor ANOVA revealed a significant
effect, which became significant in the post-hoc analysis at the 4
mg dosage. Data represent means.+-.SEM; *p<0.05 reflecting
significant differences vs. saline treated SHAM controls determined
by Fisher PLSD.
[0047] FIG. 7: Effect of chronic intragastric infusion of isoleucyl
thiazolidine fumarate; isoleucyl cyanopyrrolidine TFA, and valyl
pyrrolidine fumarate on the number of lung tumor colonies in F344
rats. A significant reduction of lung colony numbers after chronic
infusion of isoleucyl thiazolidine fumarate and isoleucyl
cyanopyrrolidine TFA in F344 rats 2 weeks after injection of
MADB106 tumor cells is illustrated. Data represent means.+-.SEM;
*p<0.05 reflecting significant differences vs. saline treated
SHAM controls determined by ANOVA and Fisher PLSD.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention relates to the area of dipeptidyl
peptidase IV (DPIV) inhibition and, more particularly, to a new use
of inhibitors of DPIV and DPIV-like enzyme activity for the
treatment of cancer and tumors, in particular for the prevention
and inhibition of metastasis and tumor colonization, and
pharmaceutical compositions containing said compounds.
[0049] In contrast to other proposed methods in the art, the
present invention provides an orally available therapy with low
molecular weight inhibitors of dipeptidyl peptidase IV. The instant
invention represents a novel approach for the treatment of cancer
and metastatic disease. It is user friendly, commercially useful
and suitable for use in a therapeutic regime, especially concerning
human diseases.
[0050] Spontaneous mutations of the DPIV gene observed in
substrains of F344 rats provide a model for studying the role of
DPIV in tumor adhesion and colonization. The mutations in F344 rats
result in a lack of DPIV enzymatic activity and are found in
substrains from Germany (GER) and Japan (JAP) (Thompson et al,
1991; Tsuji et al, 1992), while rats from USA breeders show
significant enzyme activity. In F344JAP rats, a G633R substitution
in the DPIV protein causes markedly reduced expression of a mutant
inactive enzyme (Cheng et al, 1999; Tsuji et al, 1992;), while the
other DPIV negative F344GER substrain expresses a non-active mutant
enzyme (Thompson et al, 1991). Studies by Pauli and co-workers
(Abdel-Ghany et al, 1998; Cheng et al, 1998; Cheng et al, 1999;
Johnson et al, 1993) have demonstrated an important role of
DPIV/Fibronectin binding in lung metastasis and have discussed the
F344JAP rat as a "protein knock-out" model, although this substrain
expresses mutant, enzymatically inactive DPIV on endothelial cell
surfaces, albeit at greatly reduced levels when compared to
expression of wild type DPIV (Cheng et al, 1999).
[0051] On the basis of these findings, the investigation of the
role of DPIV expression and enzymic activity in cancer and cancer
according to the present invention revealed the oral administration
of DPIV inhibitors in results in a decrease of lung metastasis and
colonization.
[0052] The goal of the present invention is the development of
dipeptidyl peptidase IV inhibitors and/or ligands, which display a
high bioavailability. In another preferred embodiment, the present
invention provides DPIV inhibitors, which have an exactly
predictable activity time in the target tissue.
[0053] Examples for target specific, orally available low molecular
weight agents are prodrugs of stable and unstable dipeptidyl
peptidase IV inhibitors which comprise general formula A-B-C,
whereby A represents an amino acid, B represents the chemical bond
between A and C or an amino acid, and C represents an unstable or a
stable inhibitor of dipeptidyl peptidase IV respectively. They are
described in WO 99/67278, WO 99/67279 the teachings of which are
herein incorporated by reference in their entirety.
[0054] The present invention relates to a novel method, in which
the reduction of activity in the enzyme dipeptidyl peptidase (DPIV
or CD 26), or of DPIV-Like enzyme activity, or where binding of a
DPIV specific ligand exerts tumor suppressive or immunostimulating
effects in the organisms of mammals induced by effectors of the
enzyme leads as a causal consequence to a reduced growth or
adhesion of cancer cells. Such treatment will result in a reduction
or delay of cancer cell adhesion (metastasis) or the growth of
tumor. As a consequence mammals bearing cancer will benefit from
the treatment with inhibitors of DPIV a DPIV-like enzyme
activity.
[0055] The method of the present invention for treating cancer in
an animal, including humans, in need thereof, comprises anti-cancer
effects by binding or by inhibiting DPIV, or related enzyme
activities, using an inhibitor or ligand of these enzymes. Oral
administration of a DPIV inhibitor may be preferable in most
circumstances.
[0056] The present invention will now be illustrated with reference
to the following examples focusing on the anti-cancer-like and
anti-metastatic-like action of reduced DPIV-like activity and/or
binding in an in vivo cancer cell adhesion assay (example 13), and
in cancer colonization assays (example 14).
[0057] In one illustrative embodiment, the present invention
relates to dipeptide compounds and compounds analogous to dipeptide
compounds that are formed from an amino acid and a thiazolidine or
pyrrolidine group, and salts thereof, referred to hereinafter as
dipeptide compounds.
[0058] The use of such compounds as inhibitors of DPIV or of
DPIV-analogous enzyme activity is already known from DD 296 075,
PCT/DE 97/00820 and PCT/EP 99/03712.
[0059] Especially suitable for that purpose according to the
invention are dipeptide compounds in which the amino acid is
selected from a natural amino acid, such as, for example, leucine,
valine, glutamine, glutamic acid, proline, isoleucine, asparagines
and aspartic acid.
[0060] The dipeptide compounds according to the invention exhibit
at a concentration (of dipeptide compounds) of 10 .mu.M, especially
under the conditions indicated in Table 1, a reduction in the
activity of dipeptidyl peptidase IV or DPIV-analogous enzyme
activities of at least 10%, especially of at least 40%. Frequently
a reduction in activity of at least 60% or at least 70% is also
required. Preferred effectors may also exhibit a reduction in
activity of a maximum of 20% or 30%.
[0061] Preferred compounds are L-allo-isoleucyl thiazolidine,
L-threo-isoleucyl pyrrolidine and salts thereof, especially the
fumaric salts, and L-allo-isoleucyl pyrrolidine and salts thereof.
Especially preferred compounds are glutaminyl pyrrolidine and
glutaminyl thiazolidine of formulas 1 and 2: 1
[0062] Further preferred compounds are given in Table 1.
[0063] The salts of the dipeptide compounds can be present in a
molar ration of dipeptide (-analogous) component to salt component
of 1:1 or 2:1. Such a salt is, for example, (lle-Thia).sub.2
fumaric acid.
1TABLE 1 Structures of further preferred dipeptide compounds
Effector H-Asn-pyrrolidine H-Asn-thiazolidine H-Asn-pyrrolidine
H-Asn-thiazolidine H-Asp(NHOH)-pyrrolidine H-Asp(NHOH)-thiazolidine
H-Glu-pyrrolidine H-Glu-thiazolidine H-Glu(NHOH)-pyrrolidine
H-Glu(NHOH)-thiazolidine H-His-pyrrolidine H-His-thiazolidine
H-Pro-pyrrolidine H-Pro-thiazolidine H-Ile-azididine
H-Ile-pyrrolidine H-L-allo-Ile-thiazolidine H-Val-pyrrolidine
H-Val-thiazolidine
[0064] In another preferred embodiment, the present invention
provides peptide compounds of formula 3 useful for competitive
modulation of dipeptidyl peptidase IV catalysis: 2
[0065] wherein
[0066] A, B, C, D and E are any amino acid residues including
proteinogenic amino acids, non-proteinogenic amino acids, L-amino
acids and D-amino acids and wherein E and/or D may be absent or B
and/or A may be absent with additional conditions as hereinafter
detailed:
[0067] Further conditions regarding formula (3):
[0068] A is any amino acid residue except D-amino acid
residues;
[0069] B is any proteinogenic amino acid residue, but
[0070] If B is an amino acid selected from Pro, Ala, Ser, Gly, Hyp,
acetidine-(2)-carboxylic acid or pipecolic acid, then C is any
amino acid residue including D-amino acids, except Pro, Ala, Ser,
Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid and E may
be unused to generate tetrapeptides of the formula A-B-C-D, or D
and E may be unused to generate tripeptides of the formula A-B-C
provided, but
[0071] If B is not an amino acid selected from Pro, Ala, Ser, Gly,
Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, then C is any
.alpha.-amino acid except D-amino acids; D is Pro, Ala, Ser, Gly,
Hyp, acetidine-(2)-carboxylic acid or pipecolic acid; E is any
amino acid residue including D-amino acids, except Pro, Ala, Ser,
Gly, Hyp, acetidine-(2)-carboxylic acid or pipecolic acid, but
[0072] If D is Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic
acid or pipecolic acid, then C is any .alpha.-amino acid except
D-amino acids and Pro, Ala, Ser, Gly, Hyp, acetidine-(2)-carboxylic
acid or pipecolic acid, and A may be unused to generate
tetrapeptides of the formula B-C-D-E, or A and B may be unused to
generate tripeptides of the formula C-D-E provided however,
[0073] If D is not selected from Pro, Ala, Ser, Gly, Hyp,
acetidine-(2)-carboxylic acid or pipecolic acid, then E is any
amino acid residue including D-amino acids.
[0074] Amino acid residues used for the preparation of the
compounds of formula (3) can be generally subclassified into four
major subclasses as follows.
[0075] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH.
[0076] Basic: The residue has a positive charge due to association
with H ion at physiological pH and the residue is attracted by
aqueous solution so as to seek the surface positions in the
conformation of a peptide in which it is contained when the peptide
is in aqueous medium at physiological pH.
[0077] Neutral/nonpolar: The residues are not charged at
physiological pH and the residue is repelled by aqueous solution so
as to seek the inner positions in the conformation of a peptide in
which it is contained when the peptide is in aqueous medium. These
residues are also designated "hydrophobic" herein.
[0078] Neutral/polar: The residues are not charged at physiological
pH, but the residue is attracted by aqueous solution so as to seek
the outer positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium.
[0079] It is understood, of course, that in a statistical
collection of individual residue molecules some molecules will be
charged, and some not, and there will be an attraction for or
repulsion from an aqueous medium to a greater or lesser extent. To
fit the definition of "charged", a significant percentage (at least
approximately 25%) of the individual molecules are charged at
physiological pH. The degree of attraction or repulsion required
for classification as polar or nonpolar is arbitrary, and,
therefore, amino acids specifically contemplated by the invention
have been specifically classified as one or the other. Most amino
acids not specifically named can be classified on the basis of
known behavior.
[0080] Amino acid residues can be further subclassified as cyclic
or noncyclic, and aromatic or nonaromatic, self-explanatory
classifications with respect to the side chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of 4 carbon atoms or less, inclusive
of the carboxyl carbon. Small residues are, of course, always
nonaromatic.
[0081] For the naturally occurring protein amino acids,
subclassification according to the foregoing scheme is as
follows.
[0082] Acidic: Aspartic acid and Glutamic acid;
[0083] Basic/noncyclic: Arginine, Lysine;
[0084] Basic/cyclic: Histidine;
[0085] Neutral/polar/small: Glycine, Serine and Cysteine;
[0086] Neutral/polar/large/nonaromatic: Threonine, Asparagine,
Glutamine;
[0087] Neutral/polar/large/aromatic: Tyrosine;
[0088] Neutral/nonpolar/small: Alanine;
[0089] Neutral/nonpolar/large/nonaromatic: Valine, Isoleucine,
Leucine, Methionine;
[0090] Neutral/nonpolar/large/aromatic: Phenylalanine, and
Tryptophan.
[0091] The gene-encoded amino acid proline, although technically
within the group neutral/nonpolar/large/cyclic and nonaromatic, is
a special case due to its known effects on the secondary
conformation of peptide chains, and is not, therefore, included in
this specific defined group.
[0092] Certain commonly encountered amino acids, which are not
encoded by the genetic code, include, for example, beta-alanine
(beta-ala), or other omega-amino acids, such as 3-amino propionic,
4-amino butyric and so forth, alpha-aminoisobutyric acid (Aib),
sarcosine (Sar), ornithine (Orn), citrulline (Cit), homoarginine
(Har), t-butylalanine (t-butyl-Ala), t-butylglycine (t-butyl-Gly),
N-methylisoleucine (N-Melle), phenylglycine (Phg),
cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and
methionine sulfoxide (MSO). These also fall conveniently into
particular categories.
[0093] Based on the above definition,
[0094] Sar and beta-Ala are neutral/nonpolar/small;
[0095] t-butyl-Ala, t-butyl-Gly, N-Melle, Nle and Cha are
neutral/nonpolar/large/nonaromatic;
[0096] Har and Orn are basic/noncyclic;
[0097] Cya is acidic;
[0098] Cit, Acetyl-Lys, and MSO are
neutral/polar/large/nonaromatic; and
[0099] Phg is neutral/nonpolar/large/aromatic.
[0100] The various omega-amino acids are classified according to
size as neutral/nonpolar/small (beta-Ala, i.e., 3-aminopropionic,
4-aminobutyric) or large (all others).
[0101] Other amino acid substitutions for those encoded in the
genetic code can also be included in peptide compounds within the
scope of the invention and can be classified within this general
scheme.
[0102] Proteinogenic amino acids are defined as natural
protein-derived .alpha.-amino acids. Non-proteinogenic amino acids
are defined as all other amino acids, which are not building blocks
of common natural proteins.
[0103] The resulting peptides may be synthesized as the free
C-terminal acid or as the C-terminal amide form. The free acid
peptides or the amides may be varied by side chain modifications.
Such side chain modifications include for instance, but not
restricted to, homoserine formation, pyroglutamic acid formation,
disulphide bond formation, deamidation of asparagine or glutamine
residues, methylation, t-butylation, t-butyloxycarbonylation,
4-methylbenzylation, thioanysilation, thiocresylation,
bencyloxymethylation, 4-nitrophenylation, bencyloxycarbonylation,
2-nitrobencoylation, 2-nitrosulphenylation,
4-toluenesulphonylation, pentafluorophenylation,
diphenylmethylation, 2-chlorobenzyloxycarbonylation,
2,4,5-trichlorophenylation, 2-bromobenzyloxycarbonylation,
9-fluorenylmethyloxycarbonylation, triphenylmethylation,
2,2,5,7,8,-pentamethylchroman-6-sulphonylation, hydroxylation,
oxidation of methionine, formylation, acetylation, anisylation,
bencylation, bencoylation, trifluoroacetylation, carboxylation of
aspartic acid or glutamic acid, phosphorylation, sulphation,
cysteinylation, glycolysation with pentoses, deoxyhexoses,
hexosamines, hexoses or N-acetylhexosamines, farnesylation,
myristolysation, biotinylation, palmitoylation, stearoylation,
geranylgeranylation, glutathionylation, 5'-adenosylation,
ADP-ribosylation, modification with N-glycolylneuraminic acid,
N-acetylneuraminic acid, pyridoxal phosphate, lipoic acid,
4'-phosphopantetheine, or N-hydroxysuccinimide.
[0104] In the compounds of formula (3), the amino acid residues
comprising A, B, C, D, and E substituents are attached to the
adjacent moiety according to standard nomenclature so that the
amino-terminus (N-terminus) of the amino acids is drawn on the left
and the carboxyl-terminus of the amino acid is drawn to the
right.
[0105] Until the present invention by Applicants, known peptide
substrates of the proline-specific serine protease dipeptidyl
peptidase IV in vitro are the tripeptides Diprotin A (lle-Pro-lle),
Diprotin B (Val-Pro-Leu) and Diprotin C (Val-Pro-lle). Applicants
have unexpectedly discovered that the compounds disclosed here act
as substrates of dipeptidyl peptidase IV in vivo in a mammal and,
in pharmacological doses, inhibit the physiological turnover of
endogenous substrates by competitive catalysis.
[0106] Particularly preferred compounds of the present invention
that could be useful as modulators of dipeptidyl peptidase IV and
DPIV--like enzymes include those compounds which show
K.sub.i-values for DPIV-binding, effectively in DPIV-inhibition in
vivo after i.v. and/or p.o. administration to Wistar rats
[0107] Further preferred compounds according to the present
invention are peptidylketones of formula 4: 3
[0108] wherein
[0109] A is selected from: 4
[0110] X.sup.1 is H or a acyl or oxycarbonyl group incl. all amino
acids and peptide residues,
[0111] X.sup.2 is H, --(CH).sub.n--NH--C.sub.5H.sub.3N--Y with
n=2-4 or C.sub.5H.sub.3N--Y (a divalent pyridyl residue) and Y is
selected from H, Br, Cl, I, NO.sub.2 or CN,
[0112] X.sup.3 is H or selected from an alkyl, alkoxy, halogen,
nitro, cyano or carboxy substituted phenyl or pyridyl residue,
[0113] X.sup.4 is H or selected from an alkyl, alkoxy, halogen,
nitro, cyano or carboxy substituted phenyl or pyridyl residue,
[0114] X.sup.5 is H or an alkyl, alkoxy or phenyl residue,
[0115] X.sup.6 is H or a alkyl residue.
[0116] for n=1
[0117] X is selected from: H, OR.sup.2, SR.sup.2, NR.sup.2R.sup.3,
N.sup.+R.sup.2R.sup.3R.sup.4, wherein:
[0118] R.sup.2 stands for acyl residues, which are substituted with
alkyl, cycloalkyl, aryl or heteroaryl residues, or for all amino
acids and peptidic residues, or alkyl residues, which are
substituted with alkyl, cycloalkyl, aryl and heteroaryl
residues,
[0119] R.sup.3 stands for alkyl and acyl functions, wherein R.sup.2
and R.sup.3 may be embedded in ring structures of saturated and
unsaturated carbocyclic or heterocyclic structures,
[0120] R.sup.4 stands for alkyl residues, wherein R.sup.2 and
R.sup.4 or R.sup.3 and R.sup.4 may be embedded in ring structures
of saturated and unsaturated carbocyclic or heterocyclic
structures.
[0121] for n=0
[0122] X is selected from: 5
[0123] wherein
[0124] B stands for: O, S, NR.sup.5, wherein R.sup.5 is H, a alkyl
or acyl, C, D, E, F, G, H are independently selected from alkyl and
substituted alkyl residues, oxyalkyl, thioalkyl, aminoalkyl,
carbonylalkyl, acyl, carbamoyl, aryl and heteroaryl residues;
and
[0125] Z is selected from H, or a branched or single chain alkyl
residue from C.sub.1-C.sub.9 or a branched or single chain alkenyl
residue from C.sub.2-C.sub.9, a cycloalkyl residue from
C.sub.3-C.sub.8, a cycloalkenyl residue from C.sub.5-C.sub.7, a
aryl- or heteroaryl residue, or a side chain selected from all side
chains of all natural amino acids or derivatives thereof.
[0126] Further, the present invention provides compounds of
formulas 5, 6, 7,8, 9, 10 and 11, including all stereoisomers and
pharmaceutical acceptable salts thereof, 6
[0127] wherein:
[0128] R.sup.1 is H, a branched or linear C.sub.1-C.sub.9 alkyl
residue, a branched or linear C.sub.2-C.sub.9 alkyl residue, a
C.sub.3-C.sub.8 cycloalkyl-, C.sub.5-C.sub.7 cycloalkenyl-, aryl-
or heteroaryl residue or a side chain of a natural amino acid or a
derivative thereof,
[0129] R.sup.3 and R.sup.4 are selected from H, hydroxy, alkyl,
alkoxy, aryloxy, nitro, cyano or halogen.
[0130] A is H or an isoster of an carbonic acid, like a functional
group selected from CN, SO.sub.3H, CONHOH, PO.sub.3R.sup.5R.sup.6,
tetrazole, amide, ester, anhydride, thiazole and imidazole,
[0131] B is selected from: 7
[0132] wherein
[0133] R.sup.5 is H, --(CH).sub.n--NH--C.sub.5H.sub.3N--Y with
n=2-4 and C.sub.5H.sub.3N--Y (a divalent pyridyl residue) with Y=H,
Br, Cl, I, NO.sub.2 CN,
[0134] R.sup.10 is H, a acyl, oxycarbonyl or a amino acid
residue,
[0135] W is H or a phenyl or pyridyl residue, substituted with
alkyl, alkoxy, halogen, nitro, cyano or carboxy residue,
[0136] W.sup.1 is H, a alkyl, alkoxy or phenyl residue,
[0137] Z is H or a phenyl or pyridyl residue, substituted with
alkyl, alkoxy, halogen, nitro, cyano or carboxy residue,
[0138] Z.sup.1 is H or a alkyl residue,
[0139] D is a cyclic C.sub.4-C.sub.7 alkyl, C.sub.4-C.sub.7 alkenyl
residue or a alkyl substituted derivative thereof or a cyclic
4-7-membered heteroalkyl or 4-7-membered heteroalkenyl residue,
[0140] X.sup.2 is O, NR.sup.6, N.sup.+(R.sup.7).sub.2, or S,
[0141] X.sup.3 to X.sup.12 are selected from CH.sub.2,
CR.sup.8R.sup.9, NR.sup.6, N.sup.+(R.sup.7).sub.2, O, S, SO and
SO.sub.2, including all saturated and unsaturated structures,
[0142] R.sup.6, R.sup.7, R.sup.8, R.sup.9 are selected from H, a
branched or linear C.sub.1-C.sub.9 alkyl residue, a branched or
lienar C.sub.2-C.sub.9 alkenyl residue, a C.sub.3-C.sub.8
cycloalkyl residue, a C.sub.5-C.sub.7 cycloalkenyl residue, an aryl
or heteroaryl residue,
[0143] with the following provisions:
[0144] Formula 6: X.sup.6 is CH if A is not H,
[0145] Formula 7: X.sup.10 is C if A is not H,
[0146] Formula 8: X.sup.7 is CH if A is not H,
[0147] Formula 9: X.sup.12 is C if A is not H.
[0148] Because of the wide distribution of the protein in the body
and the wide variety of mechanisms involving DPIV, DPIV-activity
and DPIV-related proteins, systemic therapy (enteral or parenteral
administration) with DPIV-inhibitors can result in a series of
undesirable side-effects.
[0149] It has been possible to show that side chain-modified
substrates of the enzyme dipeptidyl peptidase IV can be recognised
by the enzyme and cleaved in the same way as unmodified substrates
(DEMUTH, H.-U., HEINS, J., 1995).
[0150] For example, it has been possible to show that
phosphorylated dipeptide-(B)-p-nitroanilides [KASPARI, A., et al.,
1996] are substrates of DPIV. DPIV-inhibitors such as, for example,
Glu(Gly)-Thia or Lys(Z-NO.sub.2)-Thia [REINHOLD, D., et al., 1998]
are transported completely.
[0151] The problem to be solved consisted in preparing compounds
that can be used for targeted influencing of locally limited
pathophysiological and physiological processes. The problem of the
invention especially consists in obtaining locally limited
inhibition of DPIV or DPIV-analogous activity for the purpose of
targeted intervention in the regulation of the activity of locally
active substrates.
[0152] This problem is solved according to the invention by
providing compounds of the general formula (12) 8
[0153] wherein
[0154] A is an amino acid having at least one functional group in
the side chain,
[0155] B is a chemical compound covalently bound to at least one
functional group of the side chain of A, namely
[0156] oligopeptides having a chain length of up to 20 amino acids,
except for homopolymers of glycine consisting of up to 6 glycine
monomers, or
[0157] polyethylene glycols having molar masses of up to 20 000
g/mol, and
[0158] C is a thiazolidine, pyrrolidine, cyanopyrrolidine,
hydroxyproline, dehydroproline or piperidine group amide-bonded to
A.
[0159] In accordance with the invention, pharmaceutical
compositions are provided comprising at least one compound of the
general formula (12) 9
[0160] wherein
[0161] A is an amino acid, preferably an .alpha.-amino acid,
especially a natural .alpha.-amino acid having at least one
functional group in the side chain, preferably threonine, tyrosine,
serine, arginine, lysine, aspartic acid, glutamic acid or
cysteine,
[0162] B is a chemical compound covalently bound to at least one
functional group in the side chain of A, namely oligopeptides
having a chain length of up to 20 amino acids, polyethylene glycols
having molar masses of up to 20 000 g/mol, optionally substituted
organic amines, amides, alcohols, acids or aromatic compounds
having from 8 to 50 C atoms,
[0163] C is a thiazolidine, pyrrolidine, cyanopyrrolidine,
hydroxyproline, dehydroproline or piperidine group amide-bonded to
A,
[0164] and at least one customary adjuvant appropriate for the site
of action.
[0165] Throughout the description and the claims for the compounds
of formula (12), the expression "alkyl" can denote a C.sub.1-50
alkyl group, preferably a C.sub.6-30 alkyl group, especially a
C.sub.8-12 alkyl group; for example, an alkyl group may be a
methyl, ethyl, propyl, isopropyl or butyl group. The expression
"alk", for example in the expression "alkoxy", and the expression
"alkan", for example in the expression "alkanoyl", are defined as
for "alkyl"; aromatic compounds are preferably substituted or
optionally unsubstituted phenyl, benzyl, naphthyl, biphenyl or
anthracene groups, which preferably have at least 8 C atoms; the
expression "alkenyl" can denote a C.sub.2-10 alkenyl group,
preferably a C.sub.2-6 alkenyl group, which has the double bond(s)
at any desired location and may be substituted or unsubstituted;
the expression "alkynyl" can denote a C.sub.2-10 alkynyl group,
preferably a C.sub.2-6 alkynyl group, which has the triple bond(s)
at any desired location and may be substituted or unsubstituted;
the expression "substituted" or substituent can denote any desired
substitution by one or more, preferably one or two, alkyl, alkenyl,
alkynyl, mono- or multi-valent acyl, alkanoyl, alkoxyalkanoyl or
alkoxyalkyl groups; the afore-mentioned substituents may in turn
have one or more (but preferably zero) alkyl, alkenyl, alkynyl,
mono- or multi-valent acyl, alkanoyl, alkoxyalkanoyl or alkoxyalkyl
groups as side groups; organic amines, amides, alcohols or acids,
each having from 8 to 50 C atoms, preferably from 10 to 20 C atoms,
can have the formulae (alkyl).sub.2N-- or alkyl-NH--,
--CO--N(alkyl).sub.2 or --CO--NH(alkyl), -alkyl-OH or
-alkyl-COOH.
[0166] Despite an extended side chain function, the compounds of
formula (12) can still bind to the active centre of the enzyme
dipeptidyl peptidase IV and analogous enzymes but are no longer
actively transported by the peptide transporter PepT1. The
resulting reduced or greatly restricted transportability of the
compounds according to the invention leads, in ideal manner, to
local or site directed inhibition of DPIV and DPIV-like enzyme
activity.
[0167] The compounds of formula (12) or compounds used in
accordance with the invention can be present or used, respectively,
in the form of racemates or in the form of enantiomerically pure
compounds, preferably in the L-threo or L-allo form with respect to
part A of formula (12).
[0168] By extending/expanding the side chain modifications, for
example beyond a number of seven carbon atoms, it is accordingly
possible to obtain a dramatic reduction in transportability (see
Example 12). The Examples in Table 12.1 clearly show that, with
increasing spatial size of the side chains, there is a reduction in
the transportability of the substances. By spatially and sterically
expanding the side chains, for example beyond the atom group size
of a monosubstituted phenyl radical, hydroxylamine radical or amino
acid residue, it is possible according to the invention to modify
or suppress the transportability of the target substances.
[0169] According to the present invention, the compounds of formula
(12) inhibit DPIV or DPIV-like enzyme activity in the body of a
mammal in a site specific manner. It is accordingly possible to
influence local physiological and pathophysiological conditions
(inflammation, psoriasis, arthritis, autoimmune diseases,
allergies) effectively and with dramatically reduced
side-effects.
[0170] Preferred compounds of formula (12) are compounds, wherein
the oligopeptides have chain lengths of from 3 to 15, especially
from 4 to 10, amino acids, and/or the polyethylene glycols have
molar masses of at least 250 g/mol, preferably of at least 1500
g/mol and up to 15 000 g/mol, and/or the optionally substituted
organic amines, amides, alcohols, acids or aromatic compounds have
at least 12 C atoms and preferably up to 30 C atoms.
[0171] The compounds of the present invention can be converted into
acid addition salts, especially pharmaceutically acceptable acid
addition salts. The pharmaceutically acceptable salt generally
takes a form in which an amino acids basic side chain is protonated
with an inorganic or organic acid. Representative organic or
inorganic acids include hydrochloric, hydrobromic, perchloric,
sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic,
succinic, maleic, fumaric, malic, tartaric, citric, benzoic,
mandelic, methanesulfonic, hydroxyethanesulfonic, benzenesulfonic,
oxalic, pamoic, 2-naphthalenesulfonic, p-toulenesulfonic,
cyclohexanesulfamic, salicylic, saccharinic or trifluoroacetic
acid. All pharmaceutically acceptable acid addition salt forms of
the compounds of formulas (1) to (12) are intended to be embraced
by the scope of this invention.
[0172] In view of the close relationship between the free compounds
and the compounds in the form of their salts, whenever a compound
is referred to in this context, a corresponding salt is also
intended, provided such is possible or appropriate under the
circumstances.
[0173] The present invention further includes within its scope
prodrugs of the compounds of this invention. In general, such
prodrugs will be functional derivatives of the compounds which are
readily convertible in vivo into the desired therapeutically active
compound. Thus, in these cases, the methods of treatment of the
present invention, the term "administering" shall encompass the
treatment of the various disorders described with prodrug versions
of one or more of the claimed compounds, but which converts to the
above specified compound in vivo after administration to the
subject. Conventional procedures for the selection and preparation
of suitable prodrug derivatives are described, for example, in
"Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985 and the
patent applications DE 198 28 113 and DE 198 28 114, which are
fully incorporated herein by reference.
[0174] Where the compounds according to this invention have at
least one chiral center, they may accordingly exist as enantiomers.
Where the compounds possess two or more chiral centers, they may
additionally exist as diastereomers. It is to be understood that
all such isomers and mixtures thereof are encompassed within the
scope of the present invention. Furthermore, some of the
crystalline forms of the compounds may exist as polymorphs and as
such are intended to be included in the present invention. In
addition, some of the compounds may form solvates with water (i.e.
hydrates) or common organic solvents, and such solvates are also
intended to be encompassed within the scope of this invention.
[0175] The compounds, including their salts, can also be obtained
in the form of their hydrates, or include other solvents used for
their crystallization.
[0176] As indicated above, the compounds of the present invention,
and their corresponding pharmaceutically acceptable acid addition
salt forms, are useful in inhibiting DPIV and DPIV-like enzyme
activity. The ability of the compounds of the present invention,
and their corresponding pharmaceutically acceptable acid addition
salt forms to inhibit DPIV and DPIV-like enzyme activity may be
demonstrated employing the DPIV activity assay for determination of
the K.sub.i-values and the IC.sub.50-values in vitro, as described
in examples 7 and 8.
[0177] The ability of the compounds of the present invention, and
their corresponding pharmaceutically acceptable acid addition salt
forms to inhibit DPIV in vivo may be demonstrated by oral or
intravasal administration to Wistar rats, as described in example
11. The compounds of the present invention inhibit DPIV activity in
vivo after both, oral and intravasal administration to Wistar
rats.
[0178] DPIV is present in a wide variety of mammalian organs and
tissues e.g. the intestinal brush-border (Gutschmidt S. et al., "In
situ" --measurements of protein contents in the brush border region
along rat jejunal villi and their correlations with four enzyme
activities. Histochemistry 1981, 72 (3), 467-79), exocrine
epithelia, hepatocytes, renal tubuli, endothelia, myofibroblasts
(Feller A. C. et al., A monoclonal antibody detecting
dipeptidylpeptidase IV in human tissue. Virchows Arch. A. Pathol.
Anat. Histopathol. 1986; 409 (2):263-73), nerve cells, lateral
membranes of certain surface epithelia, e.g. Fallopian tube, uterus
and vesicular gland, in the luminal cytoplasm of e.g., vesicular
gland epithelium, and in mucous cells of Brunner's gland (Hartel S.
et al., Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of
immunohistochemistry and activity histochemistry. Histochemistry
1988; 89 (2): 151-61), reproductive organs, e.g. cauda epididymis
and ampulla, seminal vesicles and their secretions (Agrawal &
Vanha-Perttula, Dipeptidyl peptidases in bovine reproductive organs
and secretions. Int. J. Androl. 1986, 9 (6): 435-52). In human
serum, two molecular forms of dipeptidyl peptidase are present
(Krepela E. et al., Demonstration of two molecular forms of
dipeptidyl peptidase IV in normal human serum. Physiol. Bohemoslov.
1983, 32 (6): 486-96). The serum high molecular weight form of DPIV
is expressed on the surface of activated T cells (Duke-Cohan J. S.
et al., Serum high molecular weight dipeptidyl peptidase IV (CD26)
is similar to a novel antigen DPPT-L released from activated T
cells. J. Immunol. 1996,156 (5): 1714-21).
[0179] The compounds of the present invention, and their
corresponding pharmaceutically acceptable acid addition salt forms
are able to inhibit DPIV in vivo., In one embodiment of the present
invention, all molecular forms, homologues and epitopes of DPIV
from all mammalian tissues and organs, also of those, which are
undiscovered yet, are intended to be embraced by the scope of this
invention.
[0180] Among the rare group of proline-specific proteases, DPIV was
originally believed to be the only membrane-bound enzyme specific
for proline as the penultimate residue at the amino-terminus of the
polypeptide chain. However, other molecules, even structurally
non-homologous with the DPIV but bearing corresponding enzyme
activity, have been identified recently. DPIV-like enzymes, which
are identified so far, are e.g. fibroblast activation protein
.alpha., dipeptidyl peptidase IV .beta., dipeptidyl
aminopeptidase-like protein, N-acetylated .alpha.-linked acidic
dipeptidase, quiescent cell proline dipeptidase, dipeptidyl
peptidase II, attractin and dipeptidyl peptidase IV related protein
(DPP 8), and are described in the review article by Sedo &
Malik (Sedo & Malik, Dipeptidyl peptidase IV-like molecules:
homologous proteins or homologous activities? Biochimica et
Biophysica Acta 2001, 36506: 1-10). Further DPIV like enzymes are
disclosed in WO 01/19866, WO 02/04610 and WO 02/34900. WO 01/19866
discloses novel human dipeptidyl aminopeptidase (DPP8) with
structural and functional similarities to DPIV and fibroblast
activation protein (FAP). The dipeptidyl peptidase IV-like enzyme
of WO 02/04610 is well known in the art. In the Gene Bank data
base, this enzyme is registered as KIAA1492. In another preferred
embodiment of the present invention, all molecular forms,
homologues and epitopes of proteins comprising DPIV-like enzyme
activity, from all mammalian tissues and organs, also of those,
which are undiscovered yet, are intended to be embraced by the
scope of this invention.
[0181] The ability of the compounds of the present invention, and
their corresponding pharmaceutically acceptable acid addition salt
forms to inhibit DPIV-Like enzymes may be demonstrated employing an
enzyme activity assay for determination of the K.sub.i-values in
vitro as described in example 9. The K.sub.i-values of the
compounds of the present invention against porcine dipeptidyl
peptidase II were exemplary determined as K.sub.i=8.52*10.sup.-5
M.+-.6.33*10.sup.-6 M for glutaminyl pyrrolidine and
K.sub.i=1.07*10.sup.-5 M.+-.3.81*10.sup.-7 M for glutaminyl
thiazolidine.
[0182] In another embodiment, the compounds of the present
invention, and their corresponding pharmaceutically acceptable acid
addition salt forms have only low, if no inhibitory activity
against non-DPIV and non-DPIV-like proline specific enzymes. As
described in example 10, with glutaminyl thiazolidine and
glutaminyl pyrrolidine exemplarily, no inhibition of dipeptidyl
peptidase I and prolyl oligopeptidase was found. Against prolidase,
both compounds explained a marked lower efficacy compared to DPIV.
The IC 50-values against prolidase were determined as IC 50>3 mM
for glutaminyl thiazolidine and as IC
50=3.4*10.sup.-4M.+-.5.63*10.sup.-5 for glutaminyl pyrrolidine.
[0183] The present invention provides a method of treating a
condition mediated by modulation of the DPIV or DPIV-like enzyme
activity in a subject in need thereof which comprises administering
any of the compounds of the present invention or pharmaceutical
compositions thereof in a quantity and dosing regimen
therapeutically effective to treat the condition. Additionally, the
present invention includes the use of the compounds of this
invention, and their corresponding pharmaceutically acceptable acid
addition salt forms, for the preparation of a medicament for the
treatment of a condition mediated by modulation of the DPIV
activity in a subject. The compound may be administered to a
patient by any conventional route of administration, including, but
not limited to, intravenous, oral, subcutaneous, intramuscular,
intradermal and parenteral.
[0184] In a further illustrative embodiment, the present invention
provides formulations for the compounds of formulas 1 to 12, and
their corresponding pharmaceutically acceptable acid addition salt
forms, in pharmaceutical compositions.
[0185] The term "subject" as used herein, refers to an animal,
preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment.
[0186] The term "therapeutically effective amount" as used herein,
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a tissue system,
animal or human, being sought by a researcher, veterinarian,
medical doctor or other clinician, which includes alleviation of
the symptoms of the disease or disorder being treated.
[0187] As used herein, the term "composition" is intended to
encompass a product comprising the claimed compounds in the
therapeutically effective amounts, as well as any product which
results, directly or indirectly, from combinations of the claimed
compounds.
[0188] To prepare the pharmaceutical compositions of this
invention, one or more compounds of formulas 1 to 12, and their
corresponding pharmaceutically acceptable acid addition salt forms,
as the active ingredient, is intimately admixed with a
pharmaceutical carrier according to conventional pharmaceutical
compounding techniques, which carrier may take a wide variety of
forms depending of the form of preparation desired for
administration, e.g., oral or parenteral such as intramuscular. In
preparing the compositions in oral dosage form, any of the usual
pharmaceutical media may be employed. Thus, for liquid oral
preparations, such as for example, suspensions, elixirs and
solutions, suitable carriers and additives may advantageously
include water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents and the like; for solid oral
preparations such as, for example, powders, capsules, gelcaps and
tablets, suitable carriers and additives include starches, sugars,
diluents, granulating agents, lubricants, binders, disintegrating
agents and the like. Because of their ease in administration,
tablets and capsules represent the most advantageous oral dosage
unit form, in which case solid pharmaceutical carriers are
employed. If desired, tablets may be sugar coated or enteric coated
by standard techniques. For parenterals the carrier will usually
comprise sterile water, through other ingredients, for example, for
purposes such as aiding solubility or for preservation, may be
included.
[0189] Injectable suspensions may also prepared, in which case
appropriate liquid carriers, suspending agents and the like may be
employed. The pharmaceutical compositions herein will contain, per
dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful
and the like, an amount of the active ingredient necessary to
deliver an effective dose as described above. The pharmaceutical
compositions herein will contain, per unit a dosage unit, e.g.,
tablet, capsule, powder, injection, suppository, teaspoonful and
the like, of from about 0.03 mg to 100 mg/kg (preferably 0.1-30
mg/kg) and may be given at a dosage of from about 0.1-300 mg/kg per
day (preferably 1-50 mg/kg per day). The dosages, however, may be
varied depending upon the requirement of the patients, the severity
of the condition being treated and the compound being employed. The
use of either daily administration or post-periodic dosing may be
employed. Typically the dosage will be regulated by the physician
based on the characteristics of the patient, his/her condition and
the therapeutic effect desired.
[0190] Preferably these compositions are in unit dosage forms from
such as tablets, pills, capsules, powders, granules, sterile
parenteral solutions or suspensions, metered aerosol or liquid
sprays, drops, ampoules, autoinjector devices or suppositories; for
oral parenteral, intranasal, sublingual or rectal administration,
or for administration by inhalation or insufflation. Alternatively,
the composition may be presented in a form suitable for once-weekly
or once-monthly administration; for example, an insoluble salt of
the active compound, such as the decanoate salt, may be adapted to
provide a depot preparation for intramuscular injection. For
preparing solid compositions such as tablets, the principal active
ingredient is ideally mixed with a pharmaceutical carrier, e.g.
conventional tableting ingredients such as corn starch, lactose,
sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g. water, to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention, or a
pharmaceutically acceptable salt thereof. When referring to these
preformulation compositions as homogeneous, it is meant that the
active ingredient is ideally dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective dosage forms such as tablets, pills and capsules.
This solid preformulation composition may then be subdivided into
unit dosage forms of the type described above containing from 0.1
to about 500 mg of the active ingredient of the present
invention.
[0191] The tablets or pills of the novel composition can be
advantageously coated or otherwise compounded to provide a dosage
form affording the advantage of prolonged action. For example, the
tablet or pill can comprise an inner dosage and an outer dosage
component, the latter being in the form of an envelope over the
former. The two components can be separated by an enteric layer
which serves to resist disintegration in the stomach and permits
the inner component to pass intact into the duodenum or to be
delayed in release. A variety of materials can be used for such
enteric layers or coatings, such materials including a number of
polymeric acids with such materials as shellac, cetyl alcohol and
cellulose acetate.
[0192] This liquid forms in which the novel compositions of the
present invention may be advantageously incorporated for
administration orally or by injection include, aqueous solutions,
suitably flavored syrups, aqueous or oil suspensions, and flavored
emulsions with edible oils such as cottonseed oil, sesame oil,
coconut oil or peanut oil, as well as elixirs and similar
pharmaceutical vehicles. Suitable dispersing or suspending agents
for aqueous suspensions include synthetic and natural gums such as
tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or
gelatin.
[0193] Where the processes for the preparation of the compounds
according to the invention give rise to mixture of stereoisomers,
these isomers may be separated by conventional techniques such as
preparative chromatography. The compounds may be prepared in
racemic form, or individual enantiomers may be prepared either by
enantiospecific synthesis or by resolution. The compounds may, for
example, be resolved into their components enantiomers by standard
techniques, such as the formation of diastereomeric pairs by salt
formation with an optically active acid, such as
(-)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-l-tartaric
acid followed by fractional crystallization and regeneration of the
free base. The compounds may also resolved by formation of
diastereomeric esters or amides, followed by chromatographic
separation and removal of the chiral auxiliary. Alternatively, the
compounds may be resolved using a chiral HPLC column.
[0194] During any of the processes for preparation of the compounds
of the present invention, it may be necessary and/or desirable to
protect sensitive or reactive groups on any of the molecules
concerned. This may be achieved by means of conventional protecting
groups, such as those described in Protective Groups in Organic
Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W.
Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis,
John Wiley & Sons, 1991, fully incorporated herein by
reference. The protecting groups may be removed at a convenient
subsequent stage using methods known from the art.
[0195] The method of treating conditions modulated by dipeptidyl
peptidase IV and DPIV--like enzymes described in the present
invention may also be carried out using a pharmaceutical
composition comprising any of the compounds as defined herein and a
pharmaceutically acceptable carrier. The pharmaceutical composition
may contain between about 0.01 mg and 1000 mg, preferably about 5
to 500 mg, of the compound, and may be constituted into any form
suitable for the mode of administration selected. Carriers include
necessary and inert pharmaceutical excipients, including, but not
limited to, binders, suspending agents, lubricants, flavorants,
sweeteners, preservatives, dyes, and coatings. Compositions
suitable for oral administration include solid forms, such as
pills, tablets, caplets, capsules (each including immediate
release, timed release and sustained release formulations),
granules, and powders, and liquid forms, such as solutions, syrups,
elixirs, emulsions, and suspensions. Forms useful for parenteral
administration include sterile solutions, emulsions and
suspensions.
[0196] Advantageously, compounds of the present invention may be
administered in a single daily dose, or the total daily dosage may
be administered in divided doses of two, three or four times daily.
Furthermore, compounds of the present invention can be administered
in intranasal form via topical use of suitable intranasal vehicles,
or via transdermal skin patches well known to those of ordinary
skill in that art. To be administered in the form of transdermal
delivery system, the dosage administration will, of course, be
continuous rather than intermittent throughout the dosage regimen
and dosage strength will need to be accordingly modified to obtain
the desired therapeutic effects.
[0197] More preferably, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water and the like. Moreover, when desired or
necessary, suitable binders; lubricants, disintegrating agents and
coloring agents can also be incorporated into the mixture. Suitable
binders include, without limitation, starch, gelatin, natural
sugars such as glucose or betalactose, corn sweeteners, natural and
synthetic gums such as acacia, tragacanth or sodium oleate, sodium
stearate, magnesium stearate, sodium benzoate, sodium acetate,
sodium chloride and the like. Disintegrators include, without
limitation, starch, methyl cellulose, agar, bentonite, xanthan gum
and other compounds known within the art.
[0198] The liquid forms are suitable in flavored suspending or
dispersing agents such as the synthetic and natural gums, for
example, tragacanth, acacia, methyl-cellulose and the like. For
parenteral administration, sterile suspensions and solutions are
desired. Isotonic preparations which generally contain suitable
preservatives are employed when intravenous administration is
desired.
[0199] The compound of the present invention can also be
administered in the form of liposome delivery systems, such as
small unilamellar vesicles, large unilamellar vesicles, and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as cholesterol, stearylamine or
phosphatidylcholines using processes well described in the art.
[0200] Compounds of the present invention may also be coupled with
soluble polymers as targetable drug carriers. Such polymers can
include polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropylmethacrylamidephe- nol,
polyhydroxyethylaspartamidephenol, or polyethyl eneoxidepolyllysine
substituted with palmitoyl residue. Furthermore, compounds of the
present invention may be coupled to a class of biodegradable
polymers useful in achieving controlled release of a drug, for
example, polyactic acid, polyepsilon caprolactone, polyhydroxy
butyeric acid, polyorthoesters, polyacetals, polydihydropyrans,
polycyanoacrylates and cross-linked or amphipathic block copolymers
of hydrogels.
[0201] Compounds of this invention may be administered in any of
the foregoing compositions and according to dosage regimens
established in the art whenever treatment of the addressed
disorders is required.
[0202] The daily dosage of the products may be varied over a wide
range from 0.01 to 1.000 mg per adult human per day. For oral
administration, the compositions are preferably provided in the
form of tablets containing, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0,
10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of
the active ingredient for the symptomatic adjustment of the dosage
to the patient to be treated. An effective amount of the drug is
ordinarily supplied at a dosage level of from about 0.1 mg/kg to
about 300 mg/kg of body weight per day. Preferably, the range is
from about 1 to about 50 mg/kg of body weight per day. The
compounds may be administered on a regimen of 1 to 4 times per
day.
[0203] Optimal dosages to be administered may be readily determined
by those skilled in the art, and will vary with the particular
compound used, the mode of administration, the strength of the
preparation, bioavailability due to the mode of administration, and
the advancement of disease condition. In addition, factors
associated with the particular patient being treated, including
patient age, weight, diet and time of administration, should
generally be considered in adjusting dosages.
EXAMPLES
Example 1
[0204] Synthesis of dipeptide compounds
[0205] 1.1 General synthesis of isoleucyl thiazolidine salt
[0206] The Boc-protected amino acid BOC-lle-OH is placed in ethyl
acetate and the batch is cooled to about -520 C. N-Methylmorpholine
is added dropwise, pivalic acid chloride (on a laboratory scale) or
neohexanoyl chloride (on a pilot-plant scale) is added dropwise at
constant temperature. The reaction is stirred for a few minutes for
activation. N-Methylmorpholine (laboratory scale) and thiazolidine
hydrochloride (laboratory scale) are added dropwise in succession,
thiazolidine (pilot-plant scale) is added. Working-up in the
laboratory is effected in conventional manner using salt solutions,
on a pilot-plant scale the batch is purified with NaOH and
CH.sub.3COOH solutions.
[0207] The removal of the BOC protecting group is carried out using
HCl/dioxane (laboratory scale) or H.sub.2SO.sub.4 (pilot-plant
scale). In the laboratory the hydrochloride is crystallised from
EtOH/ether.
[0208] On a pilot-plant scale the free amine is prepared by the
addition of NaOH/NH.sub.3. Fumaric acid is dissolved in hot
ethanol, the free amine is added dropwise, and (lle-Thia).sup.2
furmarate (M=520.71 gmol.sup.-1) precipitates. The analysis of
isomers and enantiomers is carried out by electrophoresis.
[0209] 1.2 Synthesis of glutaminyl pyrrolidine free base
[0210] Acylation:
[0211] N-Benzyl-oxycarbonylglutamine (2.02 g, 7.21 mmol) was
dissolved in 35 ml THF and brought to -15.degree. C. Into that
mixture CAIBE (isobutylchloroformiate) (0.937 ml, 7.21 mmol) and
4-methylmorpholine (0.795 ml, 7.21 mmol) where added and the
solution was stirred for 15 min. The formation of the mixed
anhydride was checked by TLC (eluent: CHCl.sub.3/MeOH: 9/1). After
warming to -10.degree. C. pyrrolidine (0.596 ml, 7.21 mmol) was
added. The mixture was brought to room temperature and stirred
overnight.
[0212] Workup:
[0213] The sediment formed was filtered off and the solvent was
evaporated. The resulting oil was taken up in ethylacetate (20 ml)
and washed with a saturated solution of sodiumhydrogensulfate
followed by a saturated solution of sodiumbicarbonate, water and
brine. The organic layer was separated, dried and evaporated. The
resulting product was checked for purity by TLC (eluent:
CHCl.sub.3/MeOH: 9/1)
[0214] Yield: 1.18 g, waxy solid
[0215] Cleavage:
[0216] 1.18 g of the resulting solid Z-protected compound was
dissolved in 40 ml absolute ethanol. Into the solution ca. 20 mg Pd
on charcoal (10%, FLUKA) was added and the suspension was shaken
under a hydrogen atmosphere for 3h. The progress of the reaction
was monitored by TLC (eluent: CHCl.sub.3/MeOH: 9/1). After
completion of the reaction the was removed to provide the free
base.
[0217] Yield: 99%
[0218] The purity was checked by means of TLC:
n-butanole/AcOH/water/ethyl- acetate: 1/1/1/1, R.sub.f=0.4. The
identity of the reaction product was checked by NMR analysis.
[0219] 1.3 Synthesis of glutaminyl thiazolidine hydrochloride
[0220] Acylation:
[0221] N-t-Butyl-oxycarbonylglutamine (2.0 g, 8.12 mmol) was
dissolved in, 5 ml THF and brought to -15.degree. C. Into that
mixture CAIBE (isobutylchloroformiate) (1,06 ml, 8.12 mmol) and
4-methylmorpholine (0.895 ml, 8.12 mmol) where added and the
solution was stirred for 15 min. The formation of the mixed
anhydride was checked by TLC (eluent: CHCl3/MeOH: 9/1). After
warming to -10.degree. C. another equivalent 4-methylmorpholine
(0.895 ml, 8.12 mmol) and thiazolidinehydrochloride (1.02 g, 8.12
mmol was added. The mixture was brought to room temperature and
stirred overnight.
[0222] Workup:
[0223] The sediment formed was filtered off and the solvent was
evaporated. The resulting oil was taken up in chloroform (20 ml)
and washed with a saturated solution of sodiumhydrogensulfate
followed by a saturated solution of sodiumbicarbonate, water and
brine. The organic layer was separated, dried and evaporated. The
resulting product was checked for purity by TLC (eluent:
CHCl.sub.3/MeOH: 9/1)
[0224] Yield: 1.64 g, solid
[0225] Cleavage:
[0226] 640 mg of the resulting solid Boc-protected compound was
dissolved in 3.1 ml ice cold HCl in dioxane (12.98 M, 20
equivalents) and left on ice. The progress of the reaction was
monitored by TLC (eluent: CHCl.sub.3/MeOH: 9/1). After completion
of the reaction the solvent was removed and the resulting oil was
taken up in methanole and evaporated again. After that the
resulting oil was dried over phosphorous-V-oxide and triturated two
times with diethylether. The purity was checked by HPLC.
[0227] Yield: 0.265 g
[0228] The purity was checked by HPLC. The identity of the reaction
product was checked by NMR analysis.
[0229] 1.4 Synthesis of glutaminyl pyrrolidine hydrochloride
[0230] Acylation:
[0231] N-t-Butyl-oxycarbonylglutamine (3.0 g, 12.18 mmol) was
dissolved in 7 ml THF and brought to -15.degree. C. Into that
mixture CAIBE (isobutylchloroformiate) (1,6 ml, 12.18 mmol) and
4-methylmorpholine (1.3 ml, 12.18 mmol) where added and the
solution was stirred for 15 min. The formation of the mixed
anhydride was checked by TLC (eluent: CHCl.sub.3/MeOH: 9/1). After
warming to -10.degree. C. 1 equivalent of pyrrolidine (1.0 ml,
12.18 mmol) was added. The mixture was brought to room temperature
and stirred overnight.
[0232] Workup:
[0233] The sediment formed was filtered off and the solvent was
evaporated. The resulting oil was taken up in chloroform (20 ml)
and washed with a saturated solution of sodiumhydrogensulfate
followed by a saturated solution of sodiumbicarbonate, water and
brine. The organic layer was separated, dried and evaporated. The
resulting product was checked for purity by TLC (eluent:
CHCl.sub.3/MeOH: 9/1)
[0234] Yield: 2.7 g solid
[0235] Cleavage:
[0236] 2.7g of the resulting solid was dissolved in 13.0 ml ice
cold HCl in dioxane (12.98 M, 20 equivalents) and left on ice. The
progress of the reaction was monitored by TLC (eluent:
CHCl.sub.3/MeOH: 9/1). After completion of the reaction the solvent
was removed and the resulting oil was taken up in methanole and
evaporated again. After that the resulting oil was dried over
phosphorous-V-oxide and triturated two times with diethylether.
[0237] Yield: 980 mg
[0238] The purity was checked by HPLC. The identity of the reaction
product was, checked by NMR analysis.
Example 2
[0239] Chemical characterization of selected dipeptide
compounds
[0240] 2.1 Melting Point Determination
[0241] Melting points were determined on a Kofler heating platform
microscope from Leica Aktiengesellschaft, the values are not
corrected, or on a DSC apparatus (Heumann-Pharma).
[0242] 2.2 Optical Rotation
[0243] The rotation values were recorded at different wavelengths
on a "Polarimeter 341" or higher, from the Perkin-Elmer
company.
[0244] 2.3 Measurement Conditions for the Mass Spectroscopy
[0245] The mass spectra were recorded by means of electrospray
ionisation (ESI) on an "API 165" or API 365" from the PE Sciex
company. The operation is carried out using an approximate
concentration of c=10 .mu.g/ml, the substance is taken up in
MeOH/H.sub.2O 50:50, 0.1% HCO.sub.2H, the infusion is effected
using a spray pump (20 .mu.l/min). The measurement were made in
positive mode [M+H].sup.+, the ESI voltage is U=5600V.
[0246] 2.4. Results
[0247] 2.4.1 Tests on isoleucyl thiazolidine fumarate (isomer)
2 Substance Mp (.degree. C.) CE (min) MS [.alpha.]H.sub.2O
L-threo-IT*F 150.sup.DSC 160 203 -10.7 (405 nm) D-threo-IT*F 147
158 203 not determined L-allo-IT*F 145-6 154 203 -4.58 (380 nm)
D-allo-IT*F 144-6 150 203 4.5 (380 nm) IT*F = isoleucyl
thiazolidine fumarate The NMR and HPLC data confirm the identity of
the substance in question.
[0248] 2.4.2 Tests on other isoleucyl thiazolidine salts
3 IT*salt M (gmol.sup.-1) MP (.degree. C.) succinate 522.73 116
tartrate 352.41 122 fumarate 520.71 156 hydrochloride 238.77 169
phosphate 300.32 105
Example 3
[0249] Synthesis of Xaa-Pro-Yaa tripeptides
[0250] All syntheses were carried out on a peptide synthesizer SP
650 (Labortec AG) applying Fmoc/tBu-strategy. Protected amino acids
were purchased from Novabiochem or Bachem. trifluoro acetic acid
(TFA) was purchased from Merck, triisopropyl silane (TIS) was
purchased from Fluka.
[0251] Pre-loaded Fmoc-Yaa-Wang resin (2.8 g/substitution level
0.57 mmol/g) was deprotected using 20%
piperidine/N,N-dimethylformamide (DMF). After washing with DMF a
solution of 2 eq (1.1 g) of Fmoc-Pro-OH were solved in DMF (12 ml
solvent per gram resin). 2eq (1.04 g) of 2-(1H-Benzotriazole
1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and 4 eq
(1.11 ml) of N,N-diisopropylethylamine (DIEA) were added and placed
in the reaction vessel. The mixture was shaken at room temperature
for 20 minutes. Then the coupling cycle was repeated. After
subsequent washing with DMF, dichlormethane, isopropanol and
diethyl ether the resulting Fmoc-Pro-lle-Wang resin was dried and
then divided into 6 parts before coupling the last amino acid
derivative.
[0252] Fmoc protecting group was removed as described above. After
that 0.54 mmol of the Boc-amino acid, 0.54 mmol TBTU and 0.108 mmol
DIEA in DMF were shaken for 20 min. The coupling cycle was
repeated. Finally the peptide resin was washed and dried described
above.
[0253] The peptide was cleaved from the resin using a mixture of
trifluoroacetic acid (TFA) for 2.5 h, containing the following
scavengers: TFA/H.sub.2O/triisipropylsilane (TIS)=9.5/0.25/0.25
[0254] The yields of crude peptides were 80-90% on the average. The
crude peptide was purified by HPLC on a Nucleosil C18 column (7
.mu.m, 250*21.20 mm, 100 A) using a linear gradient of 0.1%
TFA/H.sub.2O with increasing concentration of 0.1% TFA/acetonitrile
(from 5% to 65% in 40 min) at 6 ml/min.
[0255] The pure peptide was obtained by lyophilization, identified
by Electrospray mass spectrometry and HPLC analysis. 3.1
Results--Identification of Xaa-Pro-Yaa tripeptides after chemical
synthesis
4 Mass (exp.).sup.1 Peptide Mass (calc.) [M + H.sup.+] HPLC
k`.sup.2 Abu-Pro-Ile 313.4 314.0 5.7 Cha-Pro-Ile 381.52 382.0 10.4
Nva-Pro-Ile 327.43 328.2 6.82 Phg-Pro-Ile 361.44 362.2 7.9
Nle-Pro-Ile 341.45 342.2 8.09 Pip-Pro-Ile 338.56 340.0 6.5
Thr-Pro-Ile 329.4 330.0 5.12 Trp-Pro-Ile 414.51 415.2 9.85
Phe-Pro-Ile 375.47 376.2 8.96 Ser-Pro-Ile 315.37 316.3 5.24
Ser(P)-Pro-Ile 395.37 396.0 3.35 Tyr(P)-Pro-Ile 471.47 472.3 5.14
Val-Pro-Val 313.4 314.0 5.07 Ile-Pro-Val 327.43 328.5 6.41
Ile-Pro-allo-Ile 341.4 342.0 7.72 Val-Pro-allo-Ile 327.4 328.5 6.51
Tyr-Pro-allo-Ile 391.5 392.0 7.02 2-Amino octanoic acid- 369.5
370.2 10.63 Pro-Ile Ser (Bzl)-Pro-Ile 405.49 406.0 9.87 Orn-Pro-Ile
342.42 343.1 3.73 Tic-Pro-Ile 387.46 388.0 8.57 Aze-Pro-Ile 311.4
312.4 5.29 Aib-Pro-Ile 313.4 314.0 5.25 t-butyl-Gly-Pro-Ile 341.47
342.1 7.16 Ile-Hyp-Ile 356.45 358.2 6.57 t-butyl-Gly-Pro-Val 327.4
328.4 6.32 t-butyl-Gly-Pro-Gly 285.4 286.3 3.74
t-butyl-Gly-Pro-Ile-amide 340.47 341.3 7.8 t-butyl Gly-Pro-D-Val
327.4 328.6 7.27 t-butyl-Gly-Pro-t-butyl-Gly 341.24 342.5 9.09
Ile-Pro-t-butyl-Gly 341.47 342.36 6.93 Val-Pro-t-butyl-Gly 327.4
328.15 5.98 .sup.1[M + H.sup.+] were determined by Electrospray
mass spectrometry in positive ionization mode. .sup.2RP-HPLC
conditions: column: LiChrospher 100 RP 18 (5 .mu.m), 125 .times. 4
mm detection (UV): 214 nm gradient system: acetonitrile
(ACN)/H.sub.2O (0.1% TFA) from 5% ACN to 50% in 15 min, flow: 1
ml/min
[0256] t.sub.0=1.16 min
[0257] t-butyl-Gly is defined as: 10
[0258] Ser(Bzl) and Ser(P) are defined as benzylserine and
phosphorylserine, respectively. Tyr(P) is defined as
phosphoryltyrosine.
Example 4
[0259] Synthesis of peptidylketones 11
[0260] H-Val-Pro-OMe*HCl 2
[0261] Boc-Val-OH (3.00 g, 13.8 mmol) was dissolved in 10 ml of dry
THF and cooled down to -15.degree. C. To the mixture CAIBE (1.80
ml, 13.8 mmol) and NMM (1.52 ml, 13.8 mmol) where added and the
solution was stirred until the formation of the mixed anhydride was
complete. Then the mixture was brought to -10.degree. C. and NMM
(1.52 ml, 13.8 mmol) was added followed by H-Pro-OMe*HCl (2.29 g,
13.8 mmol). The mixture was allowed to reach room temperature and
left overnight. After removing the solvent and the usual workup the
resulting ester 1 was taken without further characterisation. The
ester 1 was dissolved in HCl/HOAc (5 ml, 6N) and left at 0.degree.
C. until the removal of the Boc-group was complete. The solvent was
then removed and the resulting oil was treated with diethylether to
give a white solid 2.
[0262] Yield: 2.5 g, 80%
[0263] Z-Ala-Val-Pro-OMe 3
[0264] Z-Ala OH (3.5 g, 15.7 mmol) and 2 (4.18 g, 15.7 mmol) where
treated in the same manner as above for 1, to give 3 as a white
solid.
[0265] Yield: 4.2 g, 64%
[0266] Z-Ala-Val-Pro-OH 4
[0267] 3 (4.2 g, 9.6 mmol) was dissolved in 30 ml of water/acetone
(1/5 v/v) and 11.6 ml NaOH (1N) where added. After completion of
the reaction the organic solvent was removed by evaporation and the
resulting solution was diluted by 15 ml NaHCO.sub.3 solution
(saturated). Then the mixture was extracted three times by 10 ml of
acetic acid ethyl ester. After that the solution was brought to pH2
by adding HCl (15% in water). The resulting mixture was extracted
three times by 30 ml of acetic acid ethyl ester. The organic layer
was separated and washed three times with brine, dried
(Na.sub.2SO.sub.4) and evaporated.
[0268] Yield: 3.5 g, 87%
[0269] Z-Ala-Val-Pro-CH.sub.2-Br 5
[0270] 4 (2.00 g, 4.76 mmol) was dissolved in 15 ml of dry THF and
converted into a mixed anhydride (see compound 1) using CAIBE
(0.623 ml, 4.76 mmol) and NMM (0.525 ml, 4.76 mmol). The
precipitate formed was filtered off and cooled down to -15.degree.
C. Then diazomethane (23.8 mmol in 30 ml ether) was dropped into
the solution under an argon atmosphere. After leaving the mixture
for 1 h at 0.degree. C. 1.27 ml of HBr (33% in AcOH) was added and
the solution was stirred for 30 min at room temperature. After that
70 ml of ether was added and the mixture was washed with 20 ml of
water. The organic layer was separated and dried (Na.sub.2SO.sub.4)
and evaporated.
[0271] Yield (crude): 1.8 g , 80%
[0272] Z-protected acyloxymethylene ketones
[0273] The acid (2eq) was dissolved in DMF and an equimolar amount
of KF was added. The suspension was allowed to stir at room
temperature for 1 hour. Then the brommethylene (1eq) component was
added and the solution was allowed to stir overnight. After that
the solvent was removed under vacuum and the resulting oil was
dissolved in chloroform and washed with brine. Then the organic
layer was separated dried (Na.sub.2SO.sub.4) and the solvent was
removed. The product was purified by column chromatography using
silica gel and heptane/chloroform.
[0274] Z-Ala-Val-Pro-CH.sub.2O--C(O)--CH.sub.3 6
[0275] Acetic acid (230 .mu.l, 4.02 mmol), KF (0.234 g, 4.02 mmol),
5 (1.00 g, 2.01 mmol)
[0276] Yield: 0.351 g, 36%
[0277] Z-Ala-Val-Pro-CH.sub.2O--C(O)--Ph 7
[0278] Benzoic acid (0.275 g, 2.25 mmol), KF (0.131 mg, 2.25 mmol),
5 (0.56 g. 1.13 mmol)
[0279] Yield: 0.34 g, 56%
[0280] Deprotection
[0281] The Z-protected compound was dissolved in HBr/AcOH and
stirred. When the reaction was complete ether was added, the white
precipitate formed was filtered off and dried.
[0282] H-Ala-Val-Pro-CH.sub.2O--C(O)CH.sub.3*HBr 8
[0283] 6 (0.351 g, 0,73 mmol)
[0284] Yield: 0.252 g, 98%
[0285] H-Ala-Val-Pro-CH.sub.2O--C(O)Ph*HBr 9
[0286] 7 (0.34 g, 0.63 mmol)
[0287] Yield: 0,251 g, 99%
Example 5
[0288] Synthesis of cycloalkylketones 12
[0289] Boc-isoleucinal 2
[0290] Oxalylchloride (714 .mu.l, 8.28 mmol) was dissolved in 10 ml
of dry dichlormethane and brought to -78.degree. C. Then DMSO (817
.mu.l, 8.28 mmol) was added dropwise. The solution was stirred for
20 min at -78.degree. C. Then 1 (1.00 g, 4.6 mmol) was added and
the mixture was stirred for 20 min. After that TEA (2.58 ml, 18.4
mmol) was added and the mixture was allowed to reach room
temperature. The mixture was diluted with hexane/ethylacetate (2/1
v/v) and 10 ml of HCl (10% in water) was added. The organic layer
was separated and the aqueous phase was extracted with 20 ml of
methylenechloride. All organic layers were collected and washed
with brine, followed by water, then dried. The product was purified
by column chromatography using silica gel and
heptane/chloroform.
[0291] Yield: 0.52 g, 52%
[0292] tert-butyl
N-1-[cyclopentyl(hydroxy)methyl]-2-methylbutylcarbamate 3
[0293] 2 (0.52 g, 2.42 mmol) was dissolved in 10 ml of dry THF and
cooled down to 0.degree. C. Then cyclopentylmagnesiumbromide (1.45
ml of a 2 M solution) was added. After completion of the reaction
(2 ml) of water was added and solution was neutralized by adding
aqueous HCl. Then methylenechloride was added and the organic layer
was separated and dried (Na.sub.2SO.sub.4). After evaporation the
resulting oil was used without further characterisation.
[0294] tert-butyl
N-[1-(cyclopentylcarbonyl)-2-methylbutyl]carbamate 4
[0295] 3 (0.61 g, 2.15 mmol) was treated like 1. Oxalylchloride
(333 .mu.l, 3.87 mmol), DMSO (382 .mu.l, 5.37 mmol), TEA (1.2 ml,
8.59 mmol)
[0296] Yield: 0.180 g, 30%
[0297] 1-cyclopentyl-3-methyl-1-oxo-2-pentanaminium chloride 5
[0298] 4 (0.18 g, 0.63 mmol) was dissolved in 2 ml HCl (7 N in
dioxane). After completion of the reaction the solvent was removed
and the resulting oil was purified by column chromatography on
silical gel using a chloroform/methanol/water gradient. The
resulting oil was triturated with ether.
[0299] Yield: 0.060 g, 54%
Example 6
[0300] Synthesis of Side Chain Modified DPIV-inhibitors
[0301] 6.1 Synthesis of Boc-glutamyl-thiazolidine
(Boc-Glu-Thia)
[0302] Reaction of Boc-Glu(OMe)-OH with Thia*HCl according to
Method B (see section 6.4 for methods), hydrolysis of
Boc-Glu(OMe)-Thia according to Method G
[0303] 6.1.1 Analytical Data for Boc-Glu-Thia
5 Empirical formula M.sub.r MS [M + H].sup.+ [.alpha.].sup.20D
Elemental Synthesis TLC: Concen- analysis HPLC method
R.sub.f/system tration (calc./ R.sub.t Compound Yield m.p. Solvent
found) % [min]/system Boc-Glu- C.sub.13H.sub.22N.sub.2O.sub.5S
319.5 -3.1 C:49.04/48.89 13.93/ Thia 318.38 0.52/A.sup.1 c = 1
H:6.96/6.82 A.sup.2 B + G 0.42/B.sup.1 methanol N:8.80/8.59 62%
115-118.degree. C. .sup.1Thin-layer chromatography System A:
chloroform/methanol 90:10 System B: benzene/acetone/acetic acid
25:10:0.5 System C: n-butanol/EA/acetic acid/H.sub.2O 1:1:1:1
.sup.2HPLC separation conditions Column: Nucleosil C-18, 7.mu., 250
mm .times. 21 mm Eluant: isocratic, 40% ACN/water/0.1% TFA Flow
rate: 6 ml/min .lambda. = 220 nm
[0304] 6.2 Side chain-modified Boc-glutamyl thiazolidines
[0305] Boc-Glu-Thia was modified at the .gamma.-carboxylic acid
function by introducing radicals of varying size. The radicals were
coupled by way of their amino group by forming an amide bond to the
.gamma.-carboxylic acid function, with a variety of coupling
methods being used depending on the radical. The following amino
components were attached to Boc-Glu-Thia using the method
stated:
6 Coupling methods Amino component (see section 3.4) Yields
Polyethylene glycol amine (M.sub.r .apprxeq. 8000) C 93%
H-Gly-Gly-Gly-OH D + E 49% H-Gly-Gly-Gly-Gly-Gly-OH D + E 86%
[0306] In 2 cases, purification of the reaction products differs
from the general description of synthesis.
[0307] Boc-Glu(Gly.sub.5)-Thia
[0308] The product already precipitates out from the mixture on
stirring overnight; it is subsequently filtered off and washed with
0.1N HCl and copious amounts of water and then dried over
P.sub.4O.sub.10 in vacuo.
[0309] Boc-Glu(PEG)-Thia
[0310] In contrast to the general procedure, the starting materials
for the synthesis are dissolved in a 500-fold excess of DMF. After
the reaction is complete, the DMF is completely removed in vacuo
and the residue is dissolved in a large amount of methanol. After
ether is poured on, to form an upper layer, the product
precipitates out together with the unreacted PEG. Fine purification
was carried out by preparative HPLC separation on a gel filtration
column (Pharmazia, Sephadex G-25, 90 .mu.m, 260 mm-100 mm).
[0311] Separating conditions: eluant: water; flow rate: 5 ml/min;
.lambda.=220 nm
[0312] 6.2.2 Synthesis data for side chain-modified Boc-glutamyl
thiazolidines
7 Empirical MS [M + H].sup.+ Elemental formula TLC/R.sub.f/
[.alpha.].sup.20D analysis HPLC M.sub.r system Concentration
(calc./ R.sub.t Compound Yield m.p. Solvent found) % [min]/system
Boc- C.sub.19H.sub.31N.sub.5O.sub.8S 490.5 C:46.62 Glu(Gly.sub.3)-
489.54 H:6.38 Thia 49% N:14.31 Boc-
C.sub.23H.sub.37N.sub.7O.sub.10S 604.5 n.dm. C:45.76/45.60
11.93/A.sup.2 Glu(Gly.sub.5)- 603.64 0.09/C H:6.18/6.11 Thia 86%
decomp. N:16.24/16.56 from 202.degree. C. Boc- 93% .apprxeq.8000
n.dm. n.dm. n.dm. Glu(PEG)- (mass Thia emphasis) 52-53.degree. C.
.sup.2HPLC separation conditions Column: Nucleosil C-18, 7 .mu.,
250 mm .times. 21 mm Eluant: isocratic, 40% ACN/water/0.1% TFA Flow
rate: 6 ml/min .lambda. = 220 nm
[0313] 6.3 Side chain-modified glutamyl thiazolidines
[0314] The N-terminal Boc protecting groups were cleaved off the
compounds described in Table 6.2.2 using method F. The substances
modified with Gly derivatives were purified by preparative HPLC
separation and are present as trifluoroacetates. The
H-Glu(PEG)-Thia was purified on a gel filtration column in the same
manner as the Boc-protected precursor.
[0315] 6.3.1 Synthesis data for side chain-modified glutamyl
thiazolidines
8 Empirical MS [M + H].sup.+ Elemental formula TLC/R.sub.f/
[.alpha.].sup.20D analysis HPLC M.sub.r system Concentration
(calc./ R.sub.t[min]/ Compound Yield m.p. Solvent found) % system
H- C.sub.16H.sub.24N.sub.5O.sub.8SF.sub.3 503.45 +4.1 C:38.17/37.56
7.84/C.sup.3 Glu(Gly.sub.3)- 503.45 0.32/C c = 1 H:4.80/4.78 Thia
*TFA 94% 91-94.degree. C. methanol N:13.91/13.43 H-
C.sub.20H.sub.30N.sub.7O.sub.10SF.sub.3 617.55 n.dm. C:38.90/38.82
8.22/C.sup.3 Glu(Gly.sub.5)- 0.25/C H:4.90/4.79 Thia *TFA 617.55
105-107.degree. C. N:15.88/15.39 98% H- 92% .apprxeq.8000 n.dm.
n.dm. n.dm. Glu(PEG)- (mass Thia *HCl emphasis) .sup.3HPLC
separation conditions Column: Nucleosil C-18, 7 .mu., 250 mm
.times. 21 mm Eluant: ACN/water/0.1% TFA Gradient: 20% ACN .fwdarw.
90% ACN over 30 min Flow rate: 6 ml/min .lambda. = 220 nm
n.dm.--not determined or not determinable
[0316] 6.4 General Synthesis Procedures
[0317] Method A: Peptide bond attachment by the mixed anhydride
method using CFIBE as activation reagent
[0318] 10 mmol of N-terminally protected amino acid or peptide are
dissolved in 20 ml of absolute THF. The solution is cooled to
-15.degree. C..+-.20.degree. C. With stirring in each case, 10 mmol
of N-MM and 10 mmol of chloroformic acid isobutyl ester are added
in succession, the stated temperature range being strictly adhered
to. After approximately 6 min, 10 mmol of the amino component is
added. When the amino component is a salt, a further 10 mmol of
N-MM is then added to the reaction mixture. The reaction mixture is
then stirred for 2 h in the cold state and overnight at room
temperature. The reaction mixture is concentrated using a rotary
evaporator, taken up in EA, washed with 5% KH.sub.2SO.sub.4
solution, saturated NaHCO.sub.3 solution and saturated NaCl
solution and dried over NaSO.sub.4. After removal of the solvent in
vacuo, the compound is recrystallized from EA/pentane.
[0319] Method B: Peptide bond attachment by the mixed anhydride
method using pivalic acid chloride as activation reagent
[0320] 10 mmol of N-terminally protected amino acid or peptide are
dissolved in 20 ml of absolute THF. The solution is cooled to
0.degree. C. With stirring in each case, 10 mmol of N-MM and 10
mmol of pivalic acid chloride are added in succession, the stated
temperature range being strictly adhered to. After approximately 6
min, the mixture is cooled to -15.degree. C. and, once the lower
temperature has been reached, 10 mmol of the amino component is
added. When the amino component is a salt, a further 10 mmol of
N-MM is then added to the reaction mixture. The reaction mixture is
then stirred for 2 h in the cold state and overnight at room
temperature.
[0321] Further working up is carried out as in Method A.
[0322] Method C: Peptide bond attachment using TBTU as activation
reagent
[0323] 10 mmol of the N-terminally protected amino acid or peptide
and 10 mmol of the C-terminally protected amino component are
dissolved in 20 ml of absolute DMF. The solution is cooled to
0.degree. C. With stirring in each case, 10 mmol of DIPEA and 10
mmol of TBTU are added in succession. The reaction mixture is
stirred for one hour at 0.degree. C. and then overnight at room
temperature. The DMF is completely removed in vacuo and the product
is worked up as described in Method A.
[0324] Method D: Synthesis of an active ester (N-hydroxysuccinimide
ester)
[0325] 10 mmol of N-terminally protected amino acid or peptide and
10 mmol of N-hydroxysuccinimide are dissolved in 20 ml of absolute
THF. The solution is cooled to 0.degree. C. and 10 mmol of
dicyclohexylcarbodiimid- e are added, with stirring. The reaction
mixture is stirred for a further 2 h at 0.degree. C. and then
overnight at room temperature. The resulting N,N'-dicyclohexylurea
is filtered off and the solvent is removed in vacuo and the
remaining product is recrystallized from EA/pentane.
[0326] Method E: Amide bond attachment using N-hydroxysuccinimide
esters
[0327] 10 mmol of the C-terminally unprotected amino component is
introduced into an NaHCO.sub.3 solution (20 mmol in 20 ml of
water). At room temperature and with stirring, 10 mmol of the
N-terminally protected N-hydroxysuccinimide ester dissolved in 10
ml of dioxane are slowly added dropwise. Stirring of the reaction
mixture is continued overnight and the solvent is then removed in
vacuo.
[0328] Further working up is carried out as in Method A.
[0329] Method F: Cleavage of the Boc Protecting Group
[0330] 3 ml of 1.1N HCl/glacial acetic acid (Method F1) or 3 ml of
1.1N HCl/dioxane (Method F2) or 3 ml of 50% TFA in DCM (Method F3)
are added to 1 mmol of Boc-protected amino acid pyrrolidide,
thiazolidide or peptide. The cleavage at RT is monitored by means
of TLC. After the reaction is complete (approximately 2 h), the
compound is precipitated in the form of the hydrochloride using
absolute diethyl ether and is isolated with suction and dried over
P.sub.4O.sub.10 in vacuo. Using methanol/ether, the product is
recrystallized or reprecipitated.
[0331] Method G: Hydrolysis
[0332] 1 mmol of peptide methyl ester is dissolved in 10 ml of
acetone and 11 ml of 0.1M NaOH solution and stirred at room
temperature. The course of the hydrolysis is monitored by means of
TLC. After the reaction is complete, the acetone is removed in
vacuo. The remaining aqueous solution is acidified, using
concentrated KH.sub.2SO.sub.4 solution, until a pH of 2-3 is
reached. The product is then extracted several times using EA; the
combined ethyl acetate fractions are washed with saturated NaCl
solution and dried over NaSO.sub.4, and the solvent is removed in
vacuo. Crystallization from EA/pentane is carried out.
Example 7
[0333] K.sub.i-determination
[0334] For K.sub.i determination, dipeptidyl peptidase IV from
porcine kidney with a specific activity against
glycylprolyl-4-nitroaniline of 37.5 U/mg and an enzyme
concentration of 1.41 mg/ml in the stock solution was used.
[0335] Assay Mixture:
[0336] 100 .mu.l test compound in a concentration range of
1*10.sup.-5M-1*10.sup.-8M respectively were admixed with 50 .mu.l
glycylprolyl-4-nitroaniline in different concentrations (0.4 mM,
0.2 mM, 0.1 mM, 0,05 mM) and 100 .mu.l HEPES (40 mM, pH7.6; ion
strength=0.125). The assay mixture was pre-incubated at 30.degree.
C. for 30 min. After pre-incubation, 20 .mu.l DPIV (1:600 diluted)
was added and measurement of yellow color development due to
4-nitroaniline release was performed at 30.degree. C. and
.lambda.=405 nm for 10 min. using a plate reader (HTS7000 plus,
Applied Biosystems, Weiterstadt, Germany).
[0337] The K.sub.i-values were calculated using Graphit version
4.0.13, 4.0.13 and 4.0.15 (Erithacus Software, Ltd, UK). 7.1
Results--K.sub.i Values of DPIV Inhibition
9 Compound Ki [M] H-Asn-pyrrolidine 1.20 * 10.sup.-5
H-Asn-thiazolidine 3.5 * 10.sup.-6 H-Asp-pyrrolidine 1.4 *
10.sup.-8 H-Asp-thiazolidine 2.9 * 10.sup.-6
H-Asp(NHOH)-pyrrolidine 1.3 * 10.sup.-5 H-Asp(NHOH)-thiazolidine
8.8 * 10.sup.-6 H-Glu-pyrrolidine 2.2 * 10.sup.-6
H-Glu-thiazolidine 6.1 * 10.sup.-7 H-Glu(NHOH)-pyrrolidine 2.8 *
10.sup.-6 H-Glu(NHOH)-thiazolidine 1.7 * 10.sup.-6
H-His-pyrrolidine 3.5 * 10.sup.-6 H-His-thiazolidine 1.8 *
10.sup.-6 H-Pro-pyrrolidine 4.1 * 10.sup.-6 H-Pro-thiazolidine 1.2
* 10.sup.-6 H-Ile-azididine 3.1 * 10.sup.-6 H-Ile-pyrrolidine 2.1 *
10.sup.-7 H-L-threo-Ile-thiazolidine 8.0 * 10.sup.-8
H-L-allo-Ile-thiazolidine 1.9 * 10.sup.-7
D-threo-isoleucyl-thiazolidine-fumarate no inhibition
D-allo-isoleucyl-thiazolidine-fumarate no inhibition
H-L-threo-Ile-thiazolidine-succinate 5.1 * 10.sup.-8
H-L-threo-Ile-thiazolidine-tartrate 8.3 * 10.sup.-8
H-L-threo-Ile-thiazolidine-fumarate 8.3 * 10.sup.-8
H-L-threo-Ile-thiazolidine-hydrochloride 7.2 * 10.sup.-8
H-L-threo-Ile-thiazolidine-phosphate 1.3 * 10.sup.-7
H-Val-pyrrolidine 4.8 * 10.sup.-7 H-Val-thiazolidine 2.7 *
10.sup.-7 Diprotin A 3.45 * 10.sup.-6 Diprotin B 2.24 * 10.sup.-5
Nva-Pro-Ile 6.17 * 10.sup.-6 Cha-Pro-Ile 5.99 * 10.sup.-6
Nle-Pro-Ile 9.60 * 10.sup.-6 Phe-Pro-Ile 1.47 * 10.sup.-5
Val-Pro-Val 4.45 * 10.sup.-6 Ile-Pro-Val 5.25 * 10.sup.-6
Abu-Pro-Ile 8.75 * 10.sup.-6 Ile-Pro-allo-Ile 5.22 * 10.sup.-6
Val-Pro-allo-Ile 9.54 * 10.sup.-6 Tyr-Pro-allo-Ile 1.82 * 10.sup.-5
AOA-Pro-Ile 1.26 * 10.sup.-5 t-butyl-Gly-Pro-Ile 3.10 * 10.sup.-6
Ser(Bzl)-Pro-Ile 2.16 * 10.sup.-5 Aze-Pro-Ile 2.05 * 10.sup.-5
t-butyl-Gly-Pro-Val 3.08 * 10.sup.-6 Gln-Pyrr 2.26 * 10.sup.-6
Gln-Thia 1.21 * 10.sup.-6 Val-Pro-t-butyl-Gly 1.96 * 10.sup.-5
t-butyl-Gly-Pro-Gly 1.51 * 10.sup.-5 Ile-Pro-t-butyl-Gly 1.89 *
10.sup.-5 t-butyl-Gly-Pro-IleNH.sub.2 5.60 * 10.sup.-6
t-butyl-Gly-Pro-D-Val 2.65 * 10.sup.-5 t-butyl-Gly-Pro-t-butyl-Gly
1.41 * 10.sup.-5 Ile-cyclopentyl ketone 6.29 * 10.sup.-6
t-butyl-Gly-cyclohexyl ketone 2.73 * 10.sup.-4 Ile-cyclohexyl
ketone 5.68 * 10.sup.-5 Val-cyclopentyl ketone 1.31 * 10.sup.-5
Val-Pro- methyl ketone 4.76 * 10.sup.-8 Val-Pro- acyloxy methyl
ketone 1.05 * 10.sup.-9 Val-Pro- benzoyl methyl ketone 5.36 *
10.sup.-10 Val-Pro-benzothiazol methyl ketone 3.73 * 10.sup.-8
H-Glu-Thia 6.2 * 10.sup.-7 H-Gly(NHOH)-Thia 1.7 * 10.sup.-6
H-Glu(Gly.sub.3)-Thia 1.92 * 10.sup.-8 H-Glu(Gly.sub.5)-Thia 9.93 *
10.sup.-8 H-Glu(PEG)-Thia 3.11 * 10.sup.-6
[0338] t-butyl-Gly is defined as: 13
[0339] Ser(Bzl) and Ser(P) are defined as benzyl-serine and
phosphoryl-serine, respectively. Tyr(P) is defined as
phosphoryl-tyrosine.
Example 8
[0340] Determination of IC.sub.50-Values
[0341] 100 .mu.l inhibitor stock solution were mixed with 100 .mu.l
buffer (HEPES pH7.6) and 50 .mu.l substrate (Gly-Pro-pNA, final
concentration 0.4 mM) and preincubated at 30.degree. C. Reaction
was started by addition of 20 .mu.l purified porcine DPIV.
Formation of the product pNA was measured at 405 nm over 10 min
using the HTS 7000Plus plate reader (Perkin Elmer) and slopes were
calculated. The final inhibitor concentrations ranged between 1 mM
and 30 nM. For calculation of IC50 GraFit 4.0.13 (Erithacus
Software) was used.
[0342] 8.1 Results--Determination of IC.sub.50 Values
10 Compound IC50 [M] Ile-thiazolidine fumarate 1.28 * 10.sup.-7
Diprotin A 4.69 * 10.sup.-6 Diprotin B 5.54 * 10.sup.-5 Phg-Pro-Ile
1.54 * 10.sup.-4 Nva-Pro-Ile 2.49 * 10.sup.-5 Cha-Pro-Ile 2.03 *
10.sup.-5 Nle-Pro-Ile 2.19 * 10.sup.-5 Ser(P)-Pro-Ile 0.012
Tyr(P)-Pro-Ile 0.002 Phe-Pro-Ile 6.20 * 10.sup.-5 Trp-Pro-IIe 3.17
* 10.sup.-4 Ser-Pro-Ile 2.81 * 10.sup.-4 Thr-Pro-Ile 1.00 *
10.sup.-4 Val-Pro-Val 1.64 * 10.sup.-5 Ile-Pro-Val 1.52 * 10.sup.-5
Abu-Pro-Ile 3.43 * 10.sup.-5 Pip-Pro-Ile 0.100 Ile-Pro-allo-Ile
1.54 * 10.sup.-5 Val-Pro-allo-Ile 1.80 * 10.sup.-5 Tyr-Pro-allo-Ile
6.41 * 10.sup.-5 AOA-Pro-Ile 4.21 * 10.sup.-5 t-butyl-Gly-Pro-Ile
9.34 * 10.sup.-6 Ser(Bz)-Pro-Ile 6.78 * 10.sup.-5 Tic-Pro-Ile 0.001
Orn-Pro-Ile 2.16 * 10.sup.-4 Gln-Thia 5.27 * 10.sup.-6 Aze-Pro-Ile
7.28 * 10.sup.-5 Ile-Hyp-Ile 0.006 t-butyl-Gly-Pro-Val 1.38 *
10.sup.-5 Gln-Pyrr 1.50 * 10.sup.-5 Val-Pro-t-butyl-Gly 6.75 *
10.sup.-5 t-butyl-Gly-Pro-Gly 5.63 * 10.sup.-5 Ile-Pro-t-butyl-Gly
8.23 * 10.sup.-5 t-butyl-Gly-Pro-IleNH.sub.2 2.29 * 10.sup.-5
t-butyl-Gly-Pro-D-Val 1.12 * 10.sup.-4 t-butyl-Gly-Pro-t-butyl-Gly
2.45 * 10.sup.-5 Aib-Pro-Ile no inhibition Ile-cyclopentyl ketone
3.82 * 10.sup.-5 t-butyl-Gly-cyclohexyl ketone 2.73 * 10.sup.-4
Ile-cyclohexyl ketone 2.93 * 10.sup.-4 Val-cyclopentyl ketone 4.90
* 10.sup.-5 Val-cyclohexyl ketone 0.001 Val-Pro- methyl ketone 5.79
* 10.sup.-7 Val-Pro- acyloxy methyl ketone 1.02 * 10.sup.-8
Val-Pro- benzoyl methyl ketone 1.79 * 10.sup.-8
Val-Pro-benzothiazol methyl ketone 1.38 * 10.sup.-7
[0343] t-butyl-Gly is defined as: 14
[0344] Ser(Bzl) and Ser(P) are defined as benzyl-serine and
phosphoryl-serine, respectively. Tyr(P) is defined as
phosphoryl-tyrosine.
Example 9
[0345] Inhibition of DPIV-like enzymes--dipeptidyl peptidase II
[0346] DP II (3.4.14.2) releases N-terminal dipeptides from
oligopeptides if the N-terminus is not protonated (McDonald, J. K.,
Ellis, S. & Reilly, T. J., 1966, J. Biol. Chem., 241,
1494-1501). Pro and Ala in P.sub.1-position are preferred residues.
The enzyme activity is described as DPIV-like activity, but DP II
has an acidic pH-optimum. The enzyme used was purified from porcine
kidney.
[0347] Assay:
[0348] 100 .mu.l glutaminyl pyrrolidine or glutaminyl thiazolidine
in an concentration range of 1*10.sup.-4M-5*10.sup.-8M were admixed
with 100 .mu.l .mu.l buffer solution (40 mM HEPES, pH7.6, 0.015%
Brij, 1 mM DTT), 50 .mu.l lysylalanylaminomethylcoumarine solution
(5 mM) and 20 .mu.l porcine DP II (250fold diluted in buffer
solution). Fluorescence measurement was performed at 30.degree. C.
and .lambda..sub.exiatation=38- 0 nm, .lambda..sub.emission=465 nm
for 25 min using a plate reader (HTS7000plus, Applied Biosystems,
Weiterstadt, Germany). The K.sub.i-values were calculated using
Graphit 4.0.15 (Erithacus Software, Ltd., UK) and were determined
as K.sub.i=8.52*10.sup.-5 M.+-.6.33*10.sup.-6 M for glutaminyl
pyrrolidine and K.sub.i=1.07*10.sup.-5 M.+-.3.81*10.sup.-7 M for
glutaminyl thiazolidine.
Example 10
[0349] Cross Reacting Enzymes
[0350] Glutaminyl pyrrolidine and glutaminyl thiazolidine were
tested for their cross reacting potency against dipeptidyl
peptidase I, prolyl oligopeptidase and prolidase.
[0351] Dipeptidyl peptidase I (DP I, cathepsin C):
[0352] DP I or cathepsin C is a lysosomal cysteine protease which
cleaves off dipeptides from the N-terminus of their substrates
(Gutman, H. R. & Fruton, J. S., 1948, J. Biol: Chem., 174,
851-858). It is classified as a cysteine protease. The enzyme used
was purchased from Qiagen (Qiagen GmbH, Hilden, Germany). In order
to get a fully active enzyme, the enzyme was diluted 1000fold in
MES buffer pH5,6 (40 mM MES, 4 mM DTT, 4 mM KCl, 2 mM EDTA, 0.015%
Brij) and pre-incubated for 30 min at 30.degree. C.
[0353] Assay:
[0354] 50 .mu.l glutaminyl pyrrolidine or glutaminyl thiazolidine
in a concentration range of 1*10.sup.-5M-1*10.sup.-7M were admixed
with 110 .mu.l buffer-enzyme-mixture. The assay mixture was
pre-incubated at 30.degree. C. for 15 min. After pre-incubation,
100 .mu.l histidylseryl-.beta.-nitroaniline (2*10.sup.-5M) was
added and measurement of yellow color development due to
.beta.-nitroaniline release was performed at 30.degree. C. and
.lambda..sub.excitation=380 nm, .lambda..sub.emission=465 nm for 10
min., using a plate reader (HTS7000 plus, Applied Biosystems,
Weiterstadt, Germany).
[0355] The IC.sub.50-values were calculated using Graphit 4.0.15
(Erithacus Software, Ltd., UK). No inhibition of the DP I enzyme
activity by glutaminyl pyrrolidine or glutaminyl thiazolidine was
found.
[0356] Prolyl oligopeptidase (POP)
[0357] Prolyl oligopeptidase (EC 3.4.21.26) is a serine type
endoprotease which cleaves off peptides at the N-terminal part of
the Xaa-Pro bond (Walter, R., Shlank, H., Glass, J. D., Schwartz,
I. L. & Kerenyi, T. D., 1971, Science, 173, 827-829).
Substrates are peptides with a molecular weight up to 3000 Da. The
enzyme used was a recombinant human prolyl oligopeptidase.
Recombinant expression was performed in E. coli under standard
conditions as described elsewhere in the state of the art.
[0358] Assay:
[0359] 100 .mu.l glutaminyl pyrrolidine or glutaminyl thiazolidine
in an concentration range of 1*10.sup.-4M-5*10.sup.-8M were admixed
with 100 .mu.l .mu.l buffer solution (40 mM HEPES, pH7.6, 0.015%
Brij, 1 mM DTT) and 20 .mu.l POP solution. The assay mixture was
pre-incubated at 30.degree. C. for 15 min. After pre-incubation, 50
.mu.l glycylprolylprolyl-4-nitroaniline solution (0.29 mM) was
added and measurement of yellow color development due to
4-nitroaniline release was performed at 30.degree. C. and
.lambda.=405 nm for 10 min using a plate reader (sunrise, Tecan,
Crailsheim, Germany). The IC.sub.50-values were calculated using
Graphit 4.0.15 (Erithacus Software, Ltd., UK). No inhibition of POP
activity by glutaminyl pyrrolidine or glutaminyl thiazolidine was
found.
[0360] Prolidase (X-Pro dipeptidase)
[0361] Prolidase (EC 3.4.13.9) was first described by Bergmann
& Fruton (Bergmann, M. & Fruton, J S, 1937, J. Biol Chem.
189-202). Prolidase releases the N-terminal amino acid from Xaa-Pro
dipeptides and has a pH optimum between 6 and 9.
[0362] Prolidase from porcine kidney (ICN Biomedicals, Eschwege,
Germany) was solved (1 mg/ml) in assay buffer (20 mM
NH.sub.4(CH.sub.3COO).sub.2, 3 mM MnCl.sub.2, pH 7.6). In order to
get a fully active enzyme the solution was incubated for 60 min at
room temperature.
[0363] Assay:
[0364] 450 .mu.l glutaminyl pyrrolidine or glutaminyl thiazolidine
in an concentration range of 5*10.sup.-3 M-5*10.sup.-7 M were
admixed with 500 .mu.l buffer solution (20 mM
NH.sub.4(CH.sub.3COO).sub.2, pH 7.6) and 250 .mu.l lle-Pro-OH (0.5
mM in the assay mixture). The assay mixture was pre-incubated at
30.degree. C. for 5 min. After pre-incubation, 75 .mu.l Prolidase
(1:10 diluted in assay buffer) were added and measurement was
performed at 30.degree. C. and .lambda.=220 nm for 20 min using a
UV/Vis photometer, UV1 (Thermo Spectronic, Cambridge, UK).
[0365] The IC 50-values were calculated using Graphit 4.0.15
(Erithacus Software, Ltd., UK). They were determined as
IC.sub.50>3 mM for glutaminyl thiazolidine and as
IC.sub.50=3.4*10.sup.-4M.+-.5.63*10.sup.-5 for glutaminyl
pyrrolidine.
Example 11
[0366] Determination of DPIV Inhibiting Activity after Intravasal
and Oral Administration to Wistar Rats
[0367] Animals
[0368] Male Wistar rats (Shoe: Wist(Sho)) with a body weight
ranging between 250 and 350 g were purchased from Tierzucht
Schonwalde (Schonwalde, Germany).
[0369] Housing Conditions
[0370] Animals were single-caged under conventional conditions with
controlled temperature (22.+-.2.degree. C.) on a 12/12 hours
light/dark cycle (light on at 06:00 AM). Standard pelleted chow
(ssniff.RTM. Soest, Germany) and tap water acidified with HCl were
allowed ad libitum.
[0371] Catheter Insertion into Carotid Artery
[0372] After .gtoreq.one week of adaptation at the housing
conditions, catheters were implanted into the carotid artery of
Wistar rats under general anaesthesia (i.p. injection of 0.25 ml/kg
b.w. Rompun.RTM. [2%], BayerVital, Germany and 0.5 ml/kg b.w.
Ketamin 10, Atarost GmbH & Co., Twistringen, Germany). The
animals were allowed to recover for one week. The catheters were
flushed with heparin-saline (100 IU/ml) three times per week. In
case of catheter dysfunction, a second catheter was inserted into
the contra-lateral carotid artery of the respective rat. After one
week of recovery from surgery, this animal was reintegrated into
the study. In case of dysfunction of the second catheter, the
animal was withdrawn from the study. A new animal was recruited and
the experiments were continued in the planned sequence, beginning
at least 7 days after catheter implantation.
[0373] Experimental Design
[0374] Rats with intact catheter function were administered placebo
(1 ml saline, 0.154 mol/l) or test compound via the oral and the
intra-vasal (intra-arterial) route. After overnight fasting, 100
.mu.l samples of heparinised arterial blood were collected at -30,
-5, and 0 min. The test substance was dissolved freshly in 1.0 ml
saline (0.154 mol/l) and was administered at 0 min either orally
via a feeding tube (75 mm; Fine Science Tools, Heidelberg, Germany)
or via the intra-vasal route. In the case of oral administration,
an additional volume of 1 ml saline was injected into the arterial
catheter. In the case of intra-arterial administration, the
catheter was immediately flushed with 30 .mu.l saline and an
additional 1 ml of saline was given orally via the feeding
tube.
[0375] After application of placebo or the test substances,
arterial blood samples were taken at 2.5, 5, 7.5, 10, 15, 20, 40,
60 and 120 min from the carotid catheter of the conscious
unrestrained rats. All blood samples were collected into ice cooled
Eppendorf tubes (Eppendorf-Netheler-Hinz, Hamburg, Germany) filled
with 10 .mu.l 1 M sodium citrate buffer (pH 3.0) for plasma DPIV
activity measurement. Eppendorf tubes were centrifuged immediately
(12000 rpm for 2 min, Hettich Zentrifuge EBA 12, Tuttlingen;
Germany): The plasma fractions were stored on ice until analysis or
were frozen at -20.degree. C. until analysis. All plasma samples
were labelled with the following data:
[0376] Code number
[0377] Animal Number
[0378] Date of sampling
[0379] Time of sampling
[0380] Analytical Methods
[0381] The assay mixture for determination of plasma DPIV activity
consisted of 80 .mu.l reagent and 20 .mu.l plasma sample. Kinetic
measurement of the formation of the yellow product 4-nitroaniline
from the substrate glycylprolyl-4-nitroaniline was performed at 390
nm for 1 min at 30.degree. C. after 2 min pre-incubation at the
same temperature. The DPIV activity was expressed in mU/ml.
[0382] Statistical Methods
[0383] Statistical evaluations and graphics were performed with
PRISM.RTM. 3.02 (GraphPad Software, Inc.). All parameters were
analysed in a descriptive manner including mean and SD.
[0384] 11.1 Results--in vivo DPIV-inhibition at t.sub.max
11 Dose STRUCTURE (mg/kg) i.v. (%) p.o. (%) Gln-Pyrr 100 80 67
Gln-Thia 100 88 71 Diprotin A 100 73 no inhibition Diprotin B 100
50 no inhibition Tyr(P)-Pro-Ile 100 37 no inhibition tBuGly-Pro-Ile
100 71 28 tButGly-Pro-Val 100 72 25 Ala-Val-Pro- 100 89 86
Acyloxymethylketone Ala-Val-Pro-Benzoyl- 100 97 76 methylketone
Ile-cyclopentyl-ketone 100 34 15
Example 12
[0385] Action of side chain-modified glutamyl thiazolidines as
non-readily-transportable DPIV-inhibitors
[0386] Side chain-modified glutamyl thiazolidines having a
structure H-Glu(X)-Thia were synthesised, with polyethylene glycol
or glycine oligomers of various chain lengths being used as X (see
Method A of example for description of synthesis). The binding
characteristics of those derivatives and their transportability by
the peptide transporter PepT1 were investigated.
[0387] Surprisingly, it was found that the side chain modifications
alter the binding characteristics of the compounds to DPIV only to
a slight extent. In contrast, the ability of the inhibitors to be
transported by the peptide transporter is dramatically diminished
by the side chain modification.
[0388] Side chain modified inhibitors of DPIV or DPIV-like enzymes
are therefore well suited to achieving site directed inhibition of
DPIV in the body.
[0389] 12.1 Results: Transportability of Selected
DPIV-inhibitors.
12 Compound EC50 (mM).sup.1 I.sub.max (nA).sup.2 amino acid
thiazolidides H-Ile-Thia 0.98 25 .+-. 8 H-Glu-Thia 1.1 35 .+-. 13
side chain-modified glutamylthiazolidines H-Gly(NHOH)-Thia 3.18 42
.+-. 11 H-Glu(Gly.sub.3)-Thia 8.54 n.d..sup.3 H-Glu(Gly.sub.5)-Thia
>10 n.d..sup.3 H-Glu(PEG)-Thia >10 n.d..sup.3 .sup.1Effective
concentrations of the compounds inhibiting the binding of
.sup.3H-D-Phe-Ala (80 mM) to PepT1-expressing P. pastoris cells by
50% (EC.sub.50 values) .sup.2Transport characteristics at
PepT1-expressing oocytes of X. leavis - by means of two-electrode
voltage clamp method, I = inward currents generated by the
transport
[0390] .sup.1 Effective concentrations of the compounds inhibiting
the binding of .sup.3H-D-Phe-Ala (80 mM) to PepT1-expressing P.
pastoris cells by 50% (EC.sub.50 values) .sup.2 Transport
characteristics at PepT1-expressing oocytes of X. leavis--by means
of two-electrode voltage clamp method, I=inward currents generated
by the transport
Example 13
[0391] In vivo Cancer Cell Adhesion Assay
[0392] Using a novel in vivo adhesion assay which takes advantage
of vital dye labeled tumor cells and their detection in the target
tissue in situ (von Horsten et al, 2000; Shingu et al., 2002), the
current example investigates whether the in vivo adhesion of
MADB106 tumor cells differs in DPIV in treated wild type F344 rats
and F344 substrains with a mutation of the DPIV gene.
[0393] Animals, Injection of Tumor Cells and Processing of
Lungs
[0394] F344USA, F344JAP and F344GER substrains were obtained from a
breeding colony at the Central Animal Laboratory at Hannover
Medical School, Germany. All substrains were bred for one
generation and maintained in a specific-pathogen-free facility at
25.degree. C. under a 12h light-12h dark cycle (light on at 07.00
h), with ad libitum access to food and water. The exact number of
animals used per experiment is indicated by the F values with at
least four animals per condition and time point.
[0395] Cell culture, injection of tumor cells, dissection of the
animals and immunohistochemistry were conducted as previously
described (von Horsten et al., 2000). In brief, 1.times.10.sup.6
MADB106 tumor cells derived from log phase of tumor growth were
injected via the lateral tail vein and lungs removed at different
time points thereafter. For in situ quantification of tumor cells
at early time points after injection (30 min), cells were vital dye
stained using the fluorescein derivate 5-(and
-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) before
injection. For quantification of lung surface colonies at later
time points (2 weeks after tumor cell inoculation), en-bloc
dissected lungs and the heart were injected with 8 ml Bouin's
solution (72% saturated picric acid solution, 23% formaldehyde, and
5% glacial acetic acid) and fixed in the same solution until lung
surface nodules were counted (see below).
[0396] Experiments
[0397] Four experiments were conducted:
[0398] Effect of a single injection of isoleucyl thiazolidine
fumarate (2 mg i.v.+isoleucyl thiazolidine fumarate 2 mg i.p.) on
lung tumor colonization in F344USA wild-type rats;
[0399] Effect of single injection of isoleucyl thiazolidine
fumarate 2 mg i.v.+isoleucyl thiazolidine fumarate 2 mg i.p. on
tumor cell adhesion to lungs of F344JAP, F344GER and F344USA
rats;
[0400] Effect of single injection of isoleucyl cyanopyrrolidine
(0.1 mg i.v.+0.1 mg i.p.) on tumor cell adhesion to lungs of
F344USA wild-type rats;
[0401] Effect of single injection of valyl-pyrrolidine fumarate
(0.1 mg i.v.+0.1 mg i.p.) on tumor cell adhesion to lungs of
F344USA wild-type rats;
[0402] Immunohistochemistry of CFSE-labeled tumor Cells in
Lungs
[0403] Immunostaining of CFSE-labeled MADB106 tumor cells was
achieved using mAb characterizing the intracellular CFSE antigen
(anti-CFSE; mAb DE1, Boehringer, Mannheim, Germany; mouse, 1:100).
For immunohistochemistry, one or two consecutive APAAP stainings
were performed as previously described (von Horsten et al, 2000;
Shingu et al., 2002). Control sections were included in which one
or both primary antibodies were omitted.
[0404] Quantification of Tumor Targets: In vivo/in situ Cell
Adhesion Assay
[0405] Vital dye (Carboxyfluorescein; CFSE) labeling of MADB106
tumor cells allows the quantification of tumor cells and NK cells
in thick sections of lung tissue by stereology in situ (von Horsten
et al, 2000). In the present study we produced thin sections (8
.mu.m) of the same lungs (n=10) and performed additional
microscopic counting by image analysis of DE1 positive cells. This
was done to further simplify the previously validated stereological
quantification technique. Therefore, in the present study, the
assessment of DE1 positive tumor cells in lung tissue from
different substrains 30 min after tumor inoculation was carried out
using image analysis approach. All CFSE-labeled MADB106 tumor cells
and leukocyte subsets within a grid on the ocular lens were counted
(Zeiss Kpl-W 12.5.times.; grid 0.75.times.0.75 mm=0.5625
mm.sup.2/grid, using a Zeiss Neofluar objective, .times.10,
NA=0.3). Each right upper lobe of the lungs was sectioned at 6
randomly chosen non-adjacent levels. From each level, three
sections were evaluated. On average, 30 grid numbers per section
were examined (i.e. 0.5625 mm.sup.2/grid.times.30 grids.times.3
sections.times.6 levels) resulting in an area per animal of 3.04
cm.sup.2.
[0406] Quantification of Macrometastasis on Lungs
[0407] For quantification of lung surface colonies at later time
points (2 weeks after tumor cell inoculation), en-bloc dissected
lungs and the heart were injected with 8 ml Bouin's solution (72%
saturated picric acid solution, 23% formaldehyde, and 5% glacial
acetic acid) and fixed in the same solution until lung surface
nodules were counted. Three areas per lungs were examined using a
gauge (1 cm.sup.2) and lung surface colony numbers were expressed
as mean/cm.sup.2 according to the method of Wexler (Wexler,
1966).
[0408] Statistical Analysis
[0409] Data from in vivo adhesion assay were analyzed by one-way
ANOVAs and Fisher's PLSD post hoc tests, if appropriate. An
asterisk indicates significant post hoc effects vs. saline (SHAM)
treated controls obtained by Fisher's PLSD. All data are presented
as means.+-.S.E.M.
[0410] Results
[0411] Effect of a single injection of isoleucyl thiazolidine
fumarate on lung tumor colonization in F344USA rats
[0412] The number of lung surface tumor colonies after single
isoleucyl thiazolidine fumarate administration in F344 rats 2 weeks
after injection of MADB106 tumor cells is illustrated in FIG. 1.
One factor ANOVA revealed no significant effect (F(1,12)=3.2; p=0.1
n.s.) on colony numbers. A trend toward decreased colony numbers in
experimental rats was evident.
[0413] Effect of single injection of isoleucyl thiazolidine
fumarate on tumor adhesion in F344JAP, F344GER and F344USA rats
[0414] The mean number of CFSE positive cells in lung tissue at 30
min after inoculation of MADB106 tumor cells in the three
substrains is illustrated in FIG. 2. Two-way ANOVA showed a
significant effect of "substrain" (F(2,43)=3.5; p<0.04) and
"treatment" (F(1,43)=44.1; p<0.0001), as well as a significant
interaction factors but no significant interaction (F(2,43)=26.2;
p<0.0001). Separate one factor ANOVAs split for these substrains
revealed that these effects are significant.
[0415] Effect of single injection of isoleucyl cyanopyrrolidine TFA
on tumor adhesion of F344USA rats
[0416] The mean number of CFSE positive cells in lung tissue at 30
min after inoculation of MADB106 tumor cells after isoleucyl
cyanopyrrolidine TFA treatment is illustrated in FIG. 3. ANOVA
showed no significant effect of "treatment" (F(1,18)=0.1;
p=0.8n.s.).
[0417] Effect of single injection of valyl pyrrolidine fumarate on
tumor adhesion of F344USA rats
[0418] The mean number of CFSE positive cells in lung tissue at 30
min after inoculation of MADB106 tumor cells after valyl
pyrrolidine fumarate treatment is illustrated in FIG. 4. ANOVA
showed no significant effect of "treatment" (F(1,18)=0.6;
p=0.5n.s.).
[0419] Discussion
[0420] Tumor cell adhesion and colonization is significantly
modified by single injection of isoleucyl thiazolidine fumarate
only in mutant F344 substrains suggesting an interaction of the
ligand with mutant DPIV and tumor cells. Since the ligand did not
significantly affect tumor adhesion in wild type F344USA rats, this
may indicate that compound is not interacting with the binding site
of MADB106 tumor cells.
Example 14
[0421] Cancer Colonization Assays
[0422] In the previous example it was demonstrated that MADB106
tumor cell adhesion is significantly modified by a single
administration of isoleucyl thiazolidine fumarate only in mutant
F344 substrains but not in wild type DPIV expressing F344USA rats.
DPIV inhibitors/ligands may interact with the growth of tumor
metastases exhibiting properties similar to chemotherapeutic
compounds and/or immunotherapeutical compounds. In contrats to
that, the current example investigates whether the tumor
colonization of MADB106 tumor cells differs in chronically
DPIV-inhibitor treated wild type F344 rats.
[0423] Animals, Injection of Tumor Cells and Processing of
Lungs
[0424] F344USA rats were obtained from a breeding colony at the
Central Animal Laboratory at Hannover Medical School, Germany. All
rats were bred at least for one generation and maintained in a
specific-pathogen-free facility at 25.degree. C. under a 12h
light-12h dark cycle (light on at 07.00 h), with ad libitum access
to food and water. The exact number of animals used per experiment
is indicated by the F values with at least four animals per
condition and time point.
[0425] Cell culture, injection of tumor cells, dissection of the
animals and immunohistochemistry were conducted as previously
described (von Horsten et al., 2000; Shingu et al., 2002). In
brief, 1.times.10.sup.6 MADB106 tumor cells derived from log phase
of tumor growth were injected via the lateral tail vein and lungs
removed at different time points thereafter. For in situ
quantification of tumor cells at early time points after injection
(30 min), cells were vital dye stained using the fluorescein
derivate 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester
(CFSE) before injection. For quantification of lung surface
colonies at later time points (2 weeks after tumor cell
inoculation), en-bloc dissected lungs and the heart were injected
with 8 ml Bouin's solution (72% saturated picric acid solution, 23%
formaldehyde, and 5% glacial acetic acid) and fixed in the same
solution until lung surface nodules were counted (see below).
[0426] Experiments
[0427] Two experiments were conducted:
[0428] Effect of chronic infusion of different dosages of isoleucyl
thiazolidine fumarate (0 mg, 0.04 mg, 0.4 mg, 4 mg/24h intragastral
via implanted osmotic minipumps) on body weight change and lung
tumor colonization in F344USA wild-type rats
[0429] Effect of chronic infusion of isoleucyl thiazolidine
fumarate (4 mg/24h intragastral via implanted osmotic minipumps),
lisoleucyl cyanopyrrolidine TFA (0.1 mg/24 h intragastral via
implanted osmotic minipumps) and valyl pyrrolidine fumarate (0.1
mg/24 h intragastral via implanted osmotic minipumps) on lung tumor
colonization in F344USA wild-type rats.
[0430] Implantation of Osmotic Minipumps for Chronic Intragastric
Infusion of Compounds
[0431] Osmotic minipumps (Alzet model 2ML4; flow rate, 2.5
.mu.l/hr; Alza Corporation), administering a constant supply of the
different compounds, aseptically prefilled with either saline or
DPIV inhibitor were placed subcutaneously in the abdominal area.
Minipums were attached to a cannula via polyethylene tubing. The
cannula was implanted intragastrically with a heating-induced
enlarged tip of the cannula in the lumen of the gaster.
[0432] Quantification of Macrometastasis on Lungs
[0433] For quantification of lung surface colonies at later time
points (2 weeks after tumor cell inoculation), en-bloc dissected
lungs and the heart were injected with 8 ml Bouin's solution (72%
saturated picric acid solution, 23% formaldehyde, and 5% glacial
acetic acid) and fixed in the same solution until lung surface
nodules were counted. Three areas per lungs were examined using a
gauge (1 cm.sup.2) and lung surface colony numbers were expressed
as mean/cm.sup.2 according to the method of Wexler (Wexler,
1966).
[0434] Statistical Analysis
[0435] Data from in vivo body weight gain and number of lung
surface tumor colonies were analyzed by one-way ANOVAs and Fisher's
PLSD post hoc tests, if appropriate. An asterisk indicates
significant post hoc effects vs. saline (SHAM) treated controls
obtained by Fisher's PLSD. All data are presented as
means.+-.S.E.M.
[0436] Results
[0437] Effect of chronic infusion of isoleucyl thiazolidine
fumarate (0 mg, 0.04 mg, 0.4 mg, 4 mg/24 h) on lung tumor
colonization
[0438] The change of body weight after chronic infusion of
different dosages of isoleucyl thiazolidine fumarate in F344 rats 2
weeks after injection of MADB106 tumor cells is illustrated in FIG.
5. One factor ANOVA revealed a significant effect (F(3,22)=3.5;
p=0.03) on body weight, which became significant in the post-hoc
analysis at the 0.4 mg and 4 mg dosages.
[0439] The number of lung surface tumor colonies after chronic
infusion of different dosages of isoleucyl thiazolidine fumarate in
F344 rats 2 weeks after injection of MADB106 tumor cells is
illustrated in FIG. 6. One factor ANOVA revealed a significant
effect (F(3,22)=3.8; p=0.03) on colony numbers, which became
significant in the post-hoc analysis at the 4 mg dosage.
[0440] Effect of chronic infusion of isoleucyl thiazolidine
fumarate, isoleucyl cyanopyrrolidine TFA, and valyl pyrrolidine
fumarate on lung tumor colonization
[0441] The number of lung surface tumor colonies after chronic
infusion of isoleucyl thiazolidine fumarate; isoleucyl
cyanopyrrolidine TFA, and valyl pyrrolidine fumarate in F344 rats 2
weeks after injection of MADB106 tumor cells is illustrated in FIG.
7. One factor ANOVA revealed a significant effect (F(3,20)=3.8;
p=0.03) on colony numbers, which became significant in the post-hoc
analysis for isoleucyl cyanopyrrolidine TFA and isoleucyl
thiazolidine fumarate compounds.
[0442] Discussion
[0443] Metastasis of MADB106 is reduced by chronic treatment using
different DPIV Inhibitors (isoleucyl thiazolidine fumarate;
isoleucyl cyanopyrrolidine TFA) suggesting protective-like class
effects. Possibly, isoleucyl thiazolidine fumarate and isoleucyl
cyanopyrrolidine TFA protect from metastasis either via interaction
with cell adhesion processes or via a modification of the cellular
host defense mechanisms. It is also possible that DPIV inhibitor
treatment exhibits cytostatic effects. These antimetastic effects
substantiate the biological properties of DPIV Inhibitors for the
treatment of cancer and metastatic disease.
[0444] References
[0445] Abdel-Ghany M, Cheng H, Levine R, Pauli B U (1998) Truncated
dipeptidyl peptidase IV is a potent antiadhesion and
anti-metastasis peptide for rat breast cancer calls. Invasion
Metastasis 18: 35-43
[0446] Aguirre K M, McCormik R J, Schwarzbauer J E (1994)
Fibronectin self-association is mediated by complementary sites
within the amino-terminal one-third of the molecule. J Biol Chem
269: 27863-27868
[0447] Barlozzari T, Leonhardt J, Wiltrout R H, Herberman R B,
Reynolds C W (1985) Direct evidence for the role of LGL in the
inhibition of experimental tumor metastases. J Immunol 134:
2783-2789
[0448] Buhling F, Kunz D, Reinhold D, Ulmer, A J, Ernst M, Flad H
D, Ansorge S (1994) Expression and functional role of dipeptidyl
peptidase IV (CD26) on human natural killer cells. Nat Immun 13:
270-279
[0449] Cheng H C, Abdel-Ghany M, Elble R C, Pauli B U (1998) Lung
endothelial dipeptidyl peptidase IV promotes adhesion and
metastasis of rat breast cancer cells via tumor cell
surface-associated fibronectin. J Biol Chem 273: 24207-24215
[0450] Cheng H C, Abdel-Ghany M, Zhang S, Pauli B U (1999) Is the
Fischer 344/CRJ rat a protein-knock-out model for dipeptidyl
peptidase IV-mediated lung metastasis of breast cancer?. Clin Exp
Metastasis 17: 609-615
[0451] Chernousov M A, Fogerty F J, Koteliansky V E, Mosher D F
(1991) Role of the I-9 and III-1 modules of fibronectin in
formation of an extracellular fibronectin matrix. J Biol Chem 266:
10851-10858
[0452] De Meester I, Korom S, Van Damme J, Scharp S (1999) CD26,
let it cut or cut it down. Immun today 20: 367-375
[0453] Held-Feindt J, Krisch B, Mentlein R (1999) Molecular
analysis of the somatostatin receptor subtype 2 in human glioma
cells. Mol Brain Res 64: 101-107
[0454] Hocking D C, Smith R K, McKeown-Longo P J (1996) A novel
role for the integrin-binding 111-10 module in fibronectin matrix
assembly. J Cell Biol 133: 431-444
[0455] Hoshimoto K, Ohta N, Ohkura T, Inaba N (2000) Changes in
plasma soluble CD26 and CD30 during pregnancy: markers of Th1/Th2
balance? Gynecol Obstet Inves 50: 260-263
[0456] Ingham K C, Brew S A, Huff S, Litvinovich S V (1997) Cryptic
self-association sites in type III modules of fibronectin. J Biol
Chem 272: 1718-1724
[0457] Iwata S, Yamaguchi N, Munakata Y, Ikushima H, Lee J F,
Hosono O, Schlossman S F, Morimoto C (1999) CD26/dipeptidyl
peptidase IV differentially regulates the chemotaxis of T cells and
monocytes toward RANTES: possible mechanism for the switch from
innate to acquired immune response. Int immunol 11: 417-426
[0458] Jacobs R, Stoll M, Stratmann G, Leo R, Link H, Schmidt R E
(1992) CD16- CD56+ natural killer cells after bone marrow
transplantation. Blood 79: 3239-3244
[0459] Khne T, Lendeckel U, Wrenger S, Neubert K, Ansorge S,
Reinhold D (1999) Dipeptidyl peptidase IV: a cell surface peptidase
involved in regulating T cell growth (review). Int J Mol Med 4:
3-15
[0460] Korom S, De Meester I, Stadibauer T H, Chandraker A, Schaub
M, Sayegh M H, Belyaev A, Haemers A, Scharpe S, Kupiec-Weglinski J
W (1997) Inhibition of CD26/dipeptidyl peptidase IV activity in
vivo prolongs cardic allograft survival in rat recipients.
Transplntation 63: 1495-1500
[0461] Mentlein R and Struckhoff G (1989) Purification of two
dipeptidylaminopeptidases II from rat brain and their action on
proline-containing neuropeptides. J Neurochem 52:1284-1293
[0462] Mentlein R (1999) Dipeptidyl-peptidase IV (CD26)--role in
the inactivation of regulatory peptides. Regul Pept 85: 9-24
[0463] Morla A and Ruoslahti E (1992) A fibronectin self-assembly
site involved in fibronectin matrix assembly: reconstruction in a
synthetic peptide. J Cell Biol 118: 421-429
[0464] Struyf S, Proost P, Schols D, De Clercq E, Opdenakker G,
Lenaerts J P, Detheux M, Parmentier M, De Meester I, Scharpe S, Van
Damme J (1999) CD26/dipeptidyl-peptidase IV down-regulates the
eosinophil chemotactic potency, but not the anti-HIV activity of
human eotaxin by affecting its interaction with CC chemokine
receptor 3. J Immunol 162: 4903-4909
[0465] Tayebati S K, Bronzetti E, Morra di Cella S, Mulatero P,
Ricci A, Rossodivita I, Schena M, Schiavone D, Veglio F, Amenta F
(2000) In situ hybridization and immunohistochemistry of
.alpha..sub.1-adrenoceptors in human periperal blood lymphocytes. J
Auton Pharmacol 20: 305-312
[0466] Thompson N L, Hixson D C, Callanan H, Panzica M, Flanagan D,
Faris R A, Hong W J, Hartel-Schenk S, Doyle D (1991) A Fischer rat
substrain deficient in dipeptidyl peptidase IV activity makes
normal steady-state RNA levels and an altered protein. Use as a
liver-cell transplantation model. Biochem J 273: 497-502
[0467] Tsuji E, Misumi Y, Fujiwara T, Takami N, Ogata S, Ikehara Y
(1992) An active-site mutation (Gly.sup.633.fwdarw.Arg) of
dipeptidyl peptidase IV causes its retention and rapid degradation
in the endoplasmic reticulum. Biochemistry 31: 11921-11927
[0468] von Horsten S, Ballof J, Helfritz F, Nave H, Meyer D,
Schmidt R E, Stalp M, Klemm A, Tschernig T, Pabst R (1998)
Modulation of innate immune functions by intracerebroventricularly
applied neuropeptide Y: dose and time dependent effects. Life Sci
63: 909-922
[0469] Wexler H (1966) Accurate identification of experimental
pulmonary metastases. J Natl Cancer Inst 36: 641-645.
* * * * *