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| United States Patent |
6,613,879 |
| Firestone , et al. |
September 2, 2003 |
FAP-activated anti-tumour compounds
Abstract
The invention relates to a prodrug that is capable of being converted into a
drug by the catalytic action of human fibroblast activation protein
(FAP.alpha.), said prodrug having a cleavage site which is recognised by
FAP.alpha., and said drug being cytotoxic or cytostatic under physiological
conditions.
| Inventors: |
Firestone; Raymond A. (Stamford, CT);
Rettig; Wolfgang J. (Biberach, DE); Lenter; Martin (Ulm,
DE); Peters; Stefan (Ingelheim, DE); Garin-Chesa; Pilar
(Biberach, DE); Mack; Juergen (Biberach, DE); Leipert;
Dietmar (Ingelheim, DE); Park; John E. (Biberach, DE);
Telan; Leila A. (Somerville, MA) |
| Assignee: |
Boehringer Ingelheim Pharma KG
(Ingelheim, DE); Boehringer Ingelheim Pharmaceuticals, Inc.
(Ridgefield, CT) |
| Appl. No.: |
553800 |
| Filed: |
April 21, 2000 |
| Current U.S. Class: |
530/330; 530/329; 530/330;
548/535; 514/17; 514/18; 514/19; 514/152 |
| Intern'l Class: |
C07K 005/08 |
| Field of Search: |
514/17-19,152 530/329,330,331
548/535 |
References Cited [Referenced
By]
U.S. Patent Documents
| 3915800 |
Oct., 1975 |
Kang |
435/101. |
| 4703107 |
Oct., 1987 |
Monsigny et al. |
530/330. |
| 5206221 |
Apr., 1993 |
Lipsky et al. |
514/19. |
| 5776892 |
Jul., 1998 |
Counts et al. |
514/11. |
| 6271342 |
Aug., 2001 |
Lerchen |
530/322. |
| Foreign Patent Documents |
| 0 255 341 |
Feb., 1988 |
EP. |
|
| 0 624 377 |
Nov., 1994 |
EP. |
|
| WO 97/12624 |
Apr., 1997 |
WO. |
|
| WO 97/14416 |
Apr., 1997 |
WO. |
|
| WO 97/45117 |
Dec., 1997 |
WO. |
|
| WO 98/04277 |
Feb., 1998 |
WO. |
|
| WO 98/13059 |
Apr., 1998 |
WO. |
|
| WO 00/33888 |
Jun., 2000 |
WO. |
|
| WO 00/64486 |
Nov., 2000 |
WO. |
|
Other References
Abstract--1998-231397 of Patent Application DE
19640970.
De Marre, Anne; et al, Synthesis and evaluation of
macromolecular prodrugs of mitomycin C, Journal of Controlled Release,
Special Issue 36 (1995) Sep., Nos. 112, Amsterdam, NL, p. 87-97.
Nichifor, Marieta, et al; Chemical and enzymatic hydrolysis of
dipeptide derivatives of 5-fluorouracil, Journal of Controlled Release 47
(1997) 271-281, XP 000689098.
John E. Park. et al; Fibroblast
Activation Protein, a Dual Specificity Serine Protease Expressed in
Reactive Human Tumor Stromal Fibroblasts, Journal of Biological Chemistry,
Dec. 17, 1999, pp. 36505-36512, vol. 274, No. 51.
P. Calieti,et al;
Preparation and Properties of Monomethoxy Poly(Ethylene Glycol)
Doxorubicin Conjugates Linked by an Amino Acid or a peptide as Spacer,
2193 II Farmaco, 48 (1993) Jul., No. 7, Rom, It. pp 919-932.
|
Primary Examiner: Low; Christopher S. F.
Assistant Examiner: Lukton; David
Attorney, Agent or Firm:
Raymond; Robert P., Bottino; Anthony P., Stempel; Alan R.
Parent Case Text
RELATED APPLICATIONS
This application claims priority to U.S.
Provisional Application serial No. 60/134,136 filed May 14, 1999.
Claims
What is claimed is:
1. A compound of formula (I) ##STR29##
or a pharmaceutically acceptable salt thereof, wherein
R.sup.1
represents a residue of formula Cg--A, Cg--B--A or Cg--(D).sub.m --B--A, in
which
Cg represents a capping group selected from the group consisting
of R.sup.5 --CO, R.sup.5 --O--CO--, R.sup.5 --NH--CO--, R.sup.5 --SO.sub.2 -- or
R.sup.5 --, wherein R.sup.5 is an optionally substituted C.sub.3 -C.sub.8
-cycloalkyl, aryl, aralkyl or heteroaryl ggroup;
A is an amino carboxylic
acid moiety selected from L-proline, glycine, L-norleucine, L-cyclohexylglycine,
L-5-hydroxynorleucine, L-6-hydroxynorleucine, L-5-hydroxylysine, L-arginine, and
L-lysine; and
B and D each independently represent an amino carboxylic
moiety of the formula --[NR.sup.6 --(X).sub.p --CO]-- wherein X represents
CR.sup.7 R.sup.8 and wherein R.sup.6, R.sup.7 and R.sup.8 each independently
represent a hydrogen atom, an optionally substituted C.sub.1 -C.sub.6 -alkyl,
C.sub.3 -C.sub.8 -cycloalkyl, aryl or heteroaryl group, and p is 1, 2, 3, 4 or
5; or
B and D each independently represent moieties derived from cyclic
amino carboxylic acids of formula ##STR30##
wherein
R.sup.9
represents C.sub.1 -C.sub.6 -alkyl, OH, or NH.sub.2,
m is an integer
from 1 to 10;
q is 0, 1 or 2; and
r is 0, 1 or 2;
R.sup.a and R.sup.b together with the interjacent N--C group form an
optionally substituted, optionally benzo- or cyclohexano-condensed 3- to
7-membered saturated or unsaturated heterocyclic ring, in which one or two
CH.sub.2 groups may also be replaced by NH, O or S,
and --NH--Cyt'
represents a cytotoxic compound or cytostatic compound, less a hydrogen atom.
2. The compound of formula I according to claim 1, wherein the
heterocyclic ring formed by R.sup.a, R.sup.b and the interjacent N--C is
substituted by R.sup.2 and R.sup.3, wherein R.sup.2 and R.sup.3 each
independently represent hydrogen, halogen atom, C.sub.1 -C.sub.6 -alkyl, C.sub.1
-C.sub.6 -alkylamino, di-C.sub.1 -C.sub.6 -alkylamino, C.sub.1 -C.sub.6 -alkoxy,
thiol, C.sub.1 -C.sub.6 -alkylthio, oxo, imino, fomlyl, C.sub.1 -C.sub.6 -alkoxy
carbonyl, amino carbonyl, C.sub.3 --C.sub.8 -cycloalkyl, aryl or heteroaryl
group.
3. A compound of formula IA ##STR31##
wherein R.sup.1 and
--NH--Cyt' are as defined in claimed, and R.sup.2 and R.sup.3 are as defined in
claim 2,
X--Y represents CHR.sup.2 --CH.sub.2, CR.sup.2.dbd.CH,
NH--CH.sub.2, CH.sub.2 --NH, --CR.sup.2 -- or CH.sub.2 --CHR.sup.2 --CH.sub.2.
4. A compound of formula IA1 ##STR32##
wherein R.sup.1 and
--NH--Cyt' are as defined in claim 1.
>
5. A compound selected from the
formulae IA2, IA3, IA4 and IA5 ##STR33##
wherein R.sup.1 and --NH--Cyt'
are defined in claim 1.
6. The compound according to claim 1 wherein
R.sup.1 is a group selected from
Cg-Gly,
Cg-Nle and
Cg-(Xaa).sub.m -Xaa-Gly;
Cg represents a hydrogen atom or a
capping group selected from benzoyloxycarbonyl, phenylacetyl,
phenylmethylsulfonyl and benzylaminocarbonyl;
Xaa represents an amino
carboxylic acid moiety and
m is an integer from 1 to 6.
7. The
compound according to claim 6 wherein the amino carboxylic acid moieties exist
in the (L)-configuration.
8. A compound according to claim 1 wherein
--HN--Cyt' is an anthracycline derivative..
9. A compound according to
claim 8 selected from the formulae (IIIA), (IIIB), (IIIE) and (IIIF): ##STR34##
##STR35##
10. A pharmaceutical composition comprising a compound
according to claim 1 in combination with a pharmaceutically acceptable carrier.
11. A process for the production of a compound of formula I according to
claim 1, ##STR36##
said process comprising
reacting a compound
of formula (V) ##STR37##
wherein R.sup.1, R.sup.a and R.sup.b are as
defined in claim 31, X.sup.1 is hydroxy or a leaving group
which is
suitable to be substituted by an amino group, with a cytotoxic or cytostatic
compound H.sub.2 N--Cyt', and isolating the resulting final product compound of
the formula (I).
12. A method of treatment of cancer, comprising
administering to a patient in need thereof a pharmaceutical composition
according to claim 10 for a time and under conditions effective to inhibit
proliferation of tumor cells.
13. A method of treatment of cancer
comprising administering to a patient in need thereof a compound according to
claim 1 in an amount effective to inhibit proliferation of tumor cells cells and
for a time and under conditions effective to inhibit proliferation of tumor
cells.
14. A method of treating a disease chosen from epithelial
carcinomas chosen from breast, lung, colorectal, head and neck, pancreatic,
ovarian, bladder, gastric, skin, endometrial, ovarian, testicular, esophageal,
prostatic and renal origin;
bone and soft-tissue sarcomas chosen from
osteosarcoma, chondrosarcoma,
fibrosarcorma, malignant fibrous
histiocytoma (MFH) and leiomyosarcoma;
hematopoietic malignancies chosen
from Hodgkin's and non-Hodgkin's lymphomas; neuroectodermal tumors chosen from
peripheral nerve tumors, astrocytomas and melanomas;
and mesotheliomas
cancer comprising administering to a patient in need thereof a compound
according to claim 1 in an amount effective to inhibit proliferation of tumor
cells.
Description
FIELD OF THE INVENTION
The present invention relates to the
field of tumour treatment by administration of a prodrug that is converted into
a drug at the site of the tumour. In particular, the invention relates to
prodrugs which may be converted into a drug by the catalytic action of
FAP.alpha., their manufacture and pharmaceutical use.
BACKGROUND AND
PRIOR ART
The human fibroblast activation protein (FAP.alpha.) is a M,
95,000 cell surface molecule originally identified with monoclonal antibody
(mAb) F19 (Rettig et al. (1988) Proc. Natl. Acad. Sci. USA 85, 3110-3114; Rettig
et al. (1993) Cancer Res. 53, 3327-3335). The FAP.alpha. cDNA codes for a type
II integral membrane protein with a large extracellular domain, trans-membrane
segment, and short cytoplasmic tail (Scanlan et al. (1994) Proc. Natl. Acad.
Sci. USA 91, 5657-5661; WO 97/34927). FAP.alpha. shows 48% amino acid sequence
identity to the T-cell activation antigen CD26, also known as dipeptidyl
peptidase IV (DPPIV; EC 3.4.14.5), a membrane-bound protein with dipeptidyl
peptidase activity (Scanlan et al., loc. cit.). FAP.alpha. has enzymatic
activity and is a member of the serine protease family, with serine 624 being
critical for enzymatic function (WO 97/34927). Work using a membrane overlay
assay revealed that FAP.alpha. dimers are able to cleave
Ala-Pro-7-amino-4-trifluoromethyl coumarin, Gly-Pro-7-amino-4-trifluoromethyl
coumarin, and Lys-Pro-7-amino-4-trifluoromethyl coumarin dipeptides (WO
97/34927).
FAP.alpha. is selectively expressed in reactive stromal
fibroblasts of many histological types of human epithelial cancers, granulation
tissue of healing wounds, and malignant cells of certain bone and soft tissue
sarcomas. Normal adult tissues are generally devoid of detectable FAP.alpha.,
but some foetal mesenchymal tissues transiently express the molecule. In
contrast, most of the common types of epithelial cancers, including >90% of
breast, non-small-cell lung, and colorectal carcinomas, contain
FAP.alpha.-reactive stromal fibroblasts (Scanlan et al., loc. cit.). These
FAP.alpha..sup.+ fibroblasts accompany newly formed tumour blood vessels,
forming a distinct cellular compartment interposed between the tumour capillary
endothelium and the basal aspect of malignant epithelial cell clusters (Welt et
al. (1994) J. Clin. Oncol. 12(6), 1193-1203). While FAP.alpha..sup.+ stromal
fibroblasts are found in both primary and metastatic carcinomas, the benign and
premalignant epithelial lesions tested (Welt et al., loc. cit.), such as
fibroadenomas of the breast and colorectal adenomas, only rarely contain
FAP.alpha..sup.+ stromal cells. Based on the restricted distribution pattern of
FAP.alpha. in normal tissues and its uniform expression in the supporting stroma
of many malignant tumours, clinical trials with .sup.131 I-labeled mAb F19 have
been initiated in patients with metastatic colon carcinomas (Welt et al., loc.
cit.).
For new cancer therapies based on cytotoxic or cytostatic drugs,
a major consideration is to increase the therapeutic index by improving the
efficacy of cancerous tissue killing and/or reducing the toxicity for normal
tissue of the cytotoxic or cytostatic agents. To increase specificity of tumour
tissue killing and reduce toxicity in normal tissues, trigger mechanisms can be
designed so that the toxic agents synthesised in their prodrug or inactive forms
are rendered active when and where required, notably in the cancerous tissues
(Panchal (1998) Biochem. Pharmacol. 55, 247-252). Triggering mechanisms may
include either exogenous factors such as light or chemicals or endogenous
cellular factors, such as enzymes with restricted expression in cancer tissues.
Another concept, that has been further elaborated, is called `antibody-directed
enzyme prodrug therapy` (ADEPT) or `antibody-directed catalysis` (ADC)
(Huennekens (1994) Trends Biotechnol. 12, 234-239; Bagshawe (1994) Clin.
Pharmacokinet. 27, 368-376; Wang et al. (1992) Cancer Res. 52, 4484-4491;
Sperker et al. (1997) Clin. Pharmacokinet. 33(1), 18-31). In ADEPT, an antibody
directed at a tumour-associated antigen is used to target a specific enzyme to
the tumour site. The tumour-located enzyme converts a subsequently administered
prodrug into an active cytotoxic agent. The antibody-enzyme conjugate (AEC)
binds to a target antigen on cell membranes or to free antigen in extracellular
fluid (ECF). A time interval between giving the AEC and prodrug allows for the
AEC to be cleared from normal tissues so that the prodrug is not activated in
the normal tissues or blood. However, some disadvantages of ADEPT are related to
the properties of the AEC (Bagshawe, loc. cit.). For example, in humans, only a
small fraction of the administered dose of the targeting ACE binds to tumour
tissue and the remainder is distributed through body fluids from which it is
cleared with significant time delays. Even very low concentrations of targeted
enzyme can catalyse enough prodrug to have toxic effects because plasma and
normal ECF volumes are much greater than those of tumour ECF. The AEC may also
be immunogenic, thus preventing repeat administration, in many instances.
The International patent applications WO 97/12624 and WO 97/14416
disclose oligopeptides including the following penta- and hexapeptide
(SEQ.ID.NOs.: 151 and 177: hArg-Tyr-Gln-Ser-Ser-Pro; hArg-Tyr-Gln-Ser-Pro;),
comprising amino acid sequences, which are recognized and proteolytically
cleaved by free prostate specific antigen (PSA) and therapeutic agents which
comprise conjugates of such oligopeptides and known therapeutic or cytotoxic
agents. These oligopeptide conjugates which comprise at least one
glutamineserine moiety are useful for treatment of prostate cancer only.
The problem underlying the present invention was to provide methods and
means for improving normal tissue tolerability of cytotoxic or cytostatic agents
with known efficacy against a broad range of tumour tissues.
DISCLOSURE
OF THE INVENTION
The present invention relates to enzyme-activated
anti-tumour compounds. In particular, the invention provides prodrugs that are
capable of being converted into drugs by the catalytic action of endogenous
fibroblast activating protein alpha (FAP.alpha.) shown to reside in human cancer
tissues. Preferably, a prodrug of the present invention is capable of being
converted into a drug by the catalytic action of FAP.alpha., said prodrug having
a cleavage site which is recognised by FAP.alpha., and said drug being cytotoxic
or cytostatic against cancer cells under physiological conditions.
In
the context of this invention, a "drug" shall mean a chemical compound that may
be administered to humans or animals as an aid in the treatment of disease. In
particular, a drug is an active pharmacological agent.
The term
"cytotoxic compound" shall mean a chemical compound which is toxic to living
cells, in particular a drug that destroys or kills cells. The term "cytostatic
compound" shall mean a compound that suppresses cell growth and multiplication
and thus inhibits the proliferation of cells. Examples for cytotoxic or
cytostatic compounds suitable for the present invention are anthracycline
derivatives such as doxorubicin, analogs of methotrexate such as methothrexate,
pritrexime, trimetrexate or DDMP, melphalan, analogs of cisplatin such as
cisplatin, JM216, JM335, bis(platinum) or carboplatin, analogs of purines and
pyrimidines such as cytarbine, gemcitabine, azacitidine, 6-thioguanine,
flurdarabine or 2-deoxycoformycin, and analogs of other chemotherapeutic agents
such as 9-aminocamptothecin, D,L-aminoglutethimide, trimethoprim, pyrimethamine,
mitomycin C, mitoxantrone, cyclophosphanamide, 5-fluorouracil, extramustine,
podophyllotoxin, bleomycin or taxol.
A "prodrug" shall mean a compound
that, on administration, must undergo chemical conversion by metabolic processes
before becoming an active pharmacological agent. In particular, a prodrug is a
precursor of a drug. In the context of the present invention, the prodrug is
significantly less cytotoxic or cytostatic than the drug it is converted into
upon the catalytic action of FAP.alpha.. The expert knows methods of determining
cytotoxicity of a compound, see e.g. example 6 herein, or Mosmann ((1983) J.
Immun. Meth. 65, 55-63). Preferably, the prodrug is at least three times less
cytotoxic as compared to the drug in an in vitro assay.
A "drug being
cytostatic or cytotoxic under physiological conditions" shall mean a chemical
compound which is cytostatic or cytotoxic in a living human or animal body, in
particular a compound that kills cells or inhibits proliferation of cells within
a living human or animal body.
A "prodrug having a cleavage site which
is recognised by FAP.alpha." shall mean a prodrug which can act as a substrate
for the enzymatic activity of FAP.alpha.. In particular, the enzymatic activity
of FAP.alpha. can catalyse cleavage of a covalent bond of the prodrug under
physiological conditions. By cleavage of this covalent bond, the prodrug is
converted into the drug, either directly or indirectly. Indirect activation
would be the case if the cleavage product of the FAP.alpha. catalysed step is
not the pharmacologically active agent itself but undergoes a further reaction
step, e.g. hydrolysis, to become active. More preferably, the cleavage site of
the prodrug is specifically recognised by FAP.alpha., but not by other
proteolytic enzymes present in the human or animal body. Also preferably, the
cleavage site is specifically recognised by FAP.alpha., but not by proteolytic
enzymes present in human or animal body fluids, especially plasma. In a
particularly preferred embodiment, the prodrug is stable in plasma, other body
fluids, or tissues, in which biologically active FAP.alpha. is not present or
detectable. Preferably, in an in vitro assay as carried out in Example 7 herein,
more than 50%, more preferably more than 80%, more preferably more than 90% of
the prodrug are still present in a solution containing 10% (v/v) of human plasma
after 8 h at 37.degree. C. The cleavage site should most preferably be specific
for FAP.alpha.. In a preferred embodiment, the cleavage site comprises a
L-proline residue which is linked to a cytotoxic or cytostatic drug via an amide
bond. An example of this class is a doxorubicin-peptide conjugate. FAP.alpha.
may catalyse the cleavage of a peptidic bond between the C-terminal amino acid
residue of the peptide, which is preferably L-proline, and the cytotoxic or
cytostatic compound.
Preferred compounds show at least 10% conversion to
free drug, under standard conditions listed below. More preferred are compounds
that show at least 20% conversion to free drug, under standard conditions. Even
more preferred are compounds that show at least 50% conversion to free drug,
under standard conditions. In this context, standard conditions are defined as
follows: Each compound is dissolved in 50 mM Hepes buffer, 150 mM NaCl, pH 7.2,
at a final concentration of 5 .mu.M and incubated with 100 ng CD8FAP.alpha. (see
example 4) for 24 hours at 37.degree. C. Release of free drug by CD8FAP.alpha.
is determined as described in example 5.
Preferably, the present
invention relates to a compound of formula (I) ##STR1##
or a
pharmaceutically acceptable salt thereof, wherein
R.sup.1 represents an
amino alkanoyl, an oligopeptidoyl, in particular a di- or tripeptidoyl group,
the N-terminal amino function of which may be attached to a capping group;
R.sup.1 and R.sup.b together with the interjacent N--C group form an
optionally substituted, optionally benzo- or cyclohexano-condensed 3- to
7-membered saturated or unsaturated heterocyclic ring, in which one or two
CH.sub.2 groups may also be replaced by NH, O or S,
R.sup.4 represents
H, C.sub.1 -C.sub.6 -alkyl, C.sub.3 -C.sub.8 -cycloalkyl, aryl or heteroaryl;
and
Cyt' represents the residue of a cytotoxic or cytostatic compound,
with the proviso that,
N2-acetyl-L-homoarginyl-L-tyrosyl-L-glutaminyl-L-seryl-N-[2,3,6-trideoxy-1-
O-[(1S,3S)-1,2,3,4,6,11-hexahydro-3,5,12-trihydroxy-3-(hydroxyacetyl)-10-me
thoxy-6,11-dioxo-1-naphthacenyl]-.alpha.-L-lyxo-hexopyranos-3-yl]-L-prolina
mide; and
N2-acetyl-L-homoarginyl-L-tyrosyl-L-glutaminyl-L-seryl-L-seryl-N-[2,3,6-tri
deoxy-1-O-[(1S,3S)-1,2,3,4,6,11-hexahydro-3,5,12-trihydroxy-3-(hydroxyacety
l)-10-methoxy-6,11-dioxo-1-naphthacenyl]-.alpha.-L-lyxo-hexopyranos-3-yl]-L
-prolinamide are excluded.
Particuularly preferred are those compounds of
formula I, wherein R.sup.1 is a residue of formula
Cg--A, Cg--B--A or
Cg--(D).sub.m --B--A, in which
Cg represents a hydrogen atom, or a
capping group selected from the group consisting of R.sup.5 --CO, R.sup.5
--O--CO--, R.sup.5 --NH--CO--, R.sup.5 --SSO.sub.2 -- or R.sup.5 --, wherein
R.sup.5 is an optionally substituted C.sub.1 -C.sub.6 -alkyl, C.sub.3 -C.sub.8
-cycloalkyl, aryl, aralkyl or heteroaryl ggroup;
preferably Cg is an
acetyl, benzoyl, D-alanyl, (R)--H.sub.2 NCH(CH.sub.3)--, or H.sub.2 NCOCH.sub.2
CH.sub.2 -- substituent or another capping group for the protection of the
N-terminal amino function;
A, B and D each independently represent
moieties derived from amino carboxylic acids of the formula --[NR.sup.6
--(X).sub.p --CO]-- wherein X represents CCR.sup.7 R.sup.8 and wherein R.sup.6,
R.sup.7 and R.sup.8 each independently represent a hydrogen atom, an optionally
substituted C.sub.1 -C.sub.6 -alkyl, C.sub.3 -C.sub.8 -cycloalkyl, aryl or
heteroarylgroup, and p is 1, 2, 3, 4, 5; or
A, B and D each
independently represent moieties derived from cyclic amino carboxylic acids of
formula ##STR2##
wherein
R.sup.9 represents C.sub.1 -C.sub.6
-alkyl, OH, or NH.sub.2,
m is an iinteger from 1 to 10,
q is 0, 1
or 2; and
r is 0, 1 or 2.
Furthermore preferred are those
compounds of formula I, wherein R.sup.6, R.sup.7 and R.sup.8 each independently
represent a hydrogen atom or CH.sub.3 --, CH.sub.3 CH.sub.2 --, CH.sub.3
CH.sub.2 CH.sub.2 --, (CH.sub.3).sub.2 CH--, CH.sub.3 CH.sub.2 CH.sub.2 CH.sub.2
--, (CH.sub.3).sub.2 CHCH.sub.2 --, CH.subb.3 CH.sub.2 CH(CH.sub.3)--,
(CH.sub.3).sub.3 C--, HOCH.sub.2 --, CH.sub.3 CH(OH)--, CH.sub.3 CH(OH)CH.sub.2
CH.sub.2 --, HOCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 --, H.sub.2 NCH.sub.2
CH.sub.2 CH.sub.2 --, H.sub.2 NCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 --, H.sub.2
NCH.sub.2 CH(OH)CH.sub.2 CH.sub.2 --, H.sub.2 NC(.dbd.NH)NHCH.sub.2 CH.sub.2
CH.sub.2 --, HSCH.sub.2 --, CH.sub.3 SCH.sub.2 CH.sub.2 --, HOOCCH.sub.2 --,
HOOCCH.sub.2 CH.sub.2 --, H.sub.2 NC(.dbd.O)CH.sub.2 --, H.sub.2
NC(.dbd.O)CH.sub.2 CH.sub.2 --, benzyl, para-hydroxy-benzyl, ##STR3##
cyclohexyl, phenyl, p is 1, and wherein the configuration at CR.sup.7
R.sup.8 can be R or S; if R.sup.7 is other than H, then R.sup.8 is preferably H;
R.sup.6 is preferably H; if p is greater than one, R.sup.7 and R.sup.8 are
preferably H;
Another preferred embodiment of the present invention are
those compounds of formula I, wherein the heterocyclic ring formed by R.sup.a,
R.sup.b and the interjacent N--C is substituted by R.sup.2 and R.sup.3, wherein
R.sup.2 and R.sup.3 each independently represent a hydrogen or halogen atom or a
C.sub.1 -C.sub.6 -alkyl, C.sub.1 -C.sub.6 -alkylamino, di-C.sub.1 -C.sub.6
-alkylamino, C.sub.1 -C.sub.6 -alkoxy, thiiol, C.sub.1 -C.sub.6 -alkylthio, oxo,
imino, fomyl, C.sub.1 -C.sub.6 -alkoxy carbonyl, amino carbonyl, C.sub.3
-C.sub.8 -cycloalkyl, aryl, or heteroaryl group.
Unless indicated
otherwise, the simple stereoisomers as well as mixtures or racemates of the
stereoisomers are included in the invention.
"C.sub.1 -C.sub.6 -alkyl"
generally represents a straight-chained or branched hydrocarbon radical having 1
to 6 carbon atoms.
The term "optionally substituted" as used hereinabove
or hereinbelow with respect to a group or a moiety refers to a group or moiety
which may optionally be substituted by one or several halogen atoms, hydroxyl,
amino, C.sub.1 -C.sub.6 -alkyl-amino, di- C.sub.1 -C.sub.6 -alkyl-amino, C.sub.1
-C.sub.6 -alkyl-oxy, thiol, C.sub.1 -C.subb.6 -alkyl-thio, .dbd.O, .dbd.NH,
--CHO, --COOH, --CONH.sub.2, --NHC(.dbd.NHH)NH.sub.2, C.sub.3 -C.sub.8
-cycloalkyl, aryl, or heteroaryl substitueents, which may be identical to one
another or different.
The following radicals may be mentioned by way of
example:
Methyl, ethyl, propyl, 1-methylethyl (isopropyl), butyl,
1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl,
2-methylbutyl, 3-methylbutyl, 1,1-dimethyl-propyl, 1,2-dimethylpropyl,
2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl,
3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,
1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl,
1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl,
1-ethyl-1-methylpropyl and 1-ethyl-2methyl-propyl, HOCH.sub.2 --, CH.sub.3
CH(OH)--, CH.sub.3 CH(OH)CH.sub.2 CH.sub.2 --, HOCH.sub.2 CH.sub.2 CH.sub.2
CH.sub.2 --, H.sub.2 NCH.sub.2 CH.sub.2 CH.sub.2 --, H.sub.2 NCH.sub.2 CH.sub.2
CH.sub.2 CH.sub.2 --, H.sub.2 NCH.sub.2 CH(OH)CH.sub.2 CH.sub.2 --, H.sub.2
NC(.dbd.NH)NHCH.sub.2 CH.sub.2 CH.sub.2 --, HSCH.sub.2 --, CH.sub.3 SCH.sub.2
CH.sub.2 --, HOOCCH.sub.2 --, HOOCCH.sub.2 CH.sub.2 --, H.sub.2
NC(.dbd.O)CH.sub.2 --, H.sub.2 NC(.dbd.O)CH.sub.2 CH.sub.2 --, benzyl,
para-hydroxy-benzyl, ##STR4##
If a C.sub.1 -C.sub.6 -alkyl group is
substituted, the substituents are preferably hydroxyl, amino, dimethylamino,
diethylamino, thiol, methyl-thiol, methoxy, ethoxy, .dbd.O, .dbd.NH, --CHO,
--COOH, --COOCH.sub.3, --COOCH.sub.2 CH.suub.3, --CONH.sub.2,
--NHC(.dbd.NH)NH.sub.2, cyclohexyl, phenyll, benzyl, para-hydroxy-benzyl,
##STR5##
If C.sub.1 -C.sub.6 -alkyl is substituted with aryl or
heteroaryl, C.sub.1 -C.sub.6 -alkyl is preferably C.sub.1, more preferably a
methylene group.
The terms "amino alkanoyl" and "oligopeptidoyl"
including "di- or tripeptidoyl" as used hereinabove or hereinbelow with respect
to radical R.sup.1 describe a radical in which an amino acid or an oligomer
comprising up to 12, preferably 2 or 3 amino acid moieties is attached
C-terminally to the nitrogen atom of the heterocyclic ring via an amid bond.
A person of ordinary skill in the chemistry of amino acids and
oligopeptides will readily appreciate that certain amino acids may be replaced
by other homologous, isosteric and/or isolectronic amino acids wherein the
biological activity of the original amino acid or oligopeptide has been
conserved upon modification. Certain unnatural and modified natural amino acids
may also be utilized to replace the corresponding natural amino acid. Thus, for
example, tyrosine may be replaced by 3-iodotyrosine, 2- or 3-methyltyrosine,
3-fluorotyrosine.
The term "capping group" as used hereinabove or
hereinbelow with respect to a group which is attached to the N-terminal nitrogen
atom of the amino alkanoyl or oligopeptidoyl group of radical R.sup.1 defines a
group or moiety which reduces or eliminates the enzymatic degradation of the
compounds of the present invention by the action of amino peptidases which are
present in the blood plasma of warm blooded animals. Suitable capping groups
include C.sub.1 -C.sub.10 alkanoyl, C.sub.6 -C.sub.18 -aryl-C.sub.1 -C.sub.10
-alkanoyl, C.sub.6 -C.sub.18 -aryl-C.sub.11 -C.sub.10 -alkylsulfonyl. Such
capping groups also include hydrophilic blocking groups, which are chosen upon
the presence of hydrophilic functionality. Such capping groups increase the
hydrophilicity of the compounds of the present invention and thus enhance their
solubility in aqueous media. These hydrophilicity enhancing capping groups are
preferably selected from hydroxylated alkanol, polyhydroxylated alkanoyl,
hydroxylated aroyl, hydroxylated arylalkanoyl, polyhydroxylated aroyl,
polyhydroxylated arylalkanoyl, polyethylene glycol, glycosylates, sugars, and
crown ethers.
"C.sub.3 -C.sub.8 -Cycloalkyl" generally represents cyclic
hydrocarbon radical having 3 to 8 carbon atoms which may optionally be
substituted by one or several hydroxyl, amino, C.sub.1 -C.sub.6 -alkyl-amino,
di-C.sub.1 -C.sub.6 -alkyl-amino, C.sub.1 -C.sub.6 -alkyl, C.sub.1 -C.sub.6
-alkyloxy, thiol, C.sub.1 -C.sub.6 -alkyl--thio, .dbd.O, .dbd.NH, --CHO, --COOH,
--COOCH.sub.3, --COOCH.sub.2 CH.sub.3, --CCONH.sub.2, --NHC(.dbd.NH)NH.sub.2, or
halogen substituents , which may be identical to one another or different.
"Heterocyclic ring" as used hereinabove and hereinbelow with respect to
the group formed by R.sup.a and R.sup.b together with the interjacent N--C group
generally represents a 3 to 7-membered, preferably 4-, 5- or 6-membered
non-aromatic heterocyclic ring system, containing one nitrogen atom and
optionally 1 or 2 additional heteroatoms selected from the group of nitrogen,
oxygen and sulfur, which may be substituted by one or several halogen atoms or
C.sub.1 -C.sub.6 -alkyl, C.sub.1 -C.sub.6 -alkylamino, di-C.sub.1 -C.sub.6
-alkylamino, C.sub.1 -C.sub.6 -alkoxy, thiiol, C.sub.1 -C.sub.6 -alkylthio, oxo,
imino, fomyl, C.sub.1 -C.sub.6 -alkoxy carbonyl, amino carbonyl, C.sub.3
-C.sub.8 -cycloalkyl, aryl, or heteroaryl groups, which may be identical to one
another or different, and which optionally may be benzo- or
cyclohexano-condensed. Such heterocyclic rings are preferably azetidine or are
derived from a fully or partially hydrogenated pyrrole, pyridine, thiazole,
isoxazole, pyrazole, imidazole, indole, benzimidazole, indazole, pyridazine,
pyrimidine, pyrazin group. Most preferred are azetidine, pyrrolidine,
3,4-dehydropyrrolidine, piperidine, hexahydro-1H-azepine, octahydroindole,
imidazolidine, thiazolidine.
If such heterocyclic ring is substituted,
the substituents are preferably methyl, ethyl, propyl, 1-methylethyl
(isopropyl), butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, droxyl,
amino, dimethyl-amino, diethyl-amino, thiol, methyl-thiol, methoxy, ethoxy,
--CHO, --COOH, --COOCH.sub.3, --COOCH.sub..2 CH.sub.3, or --CONH.sub.2.
"Aryl" generally represents an aromatic ring system with 6 to 10,
preferably 6 carbon atoms which may optionally be substituted by one or several
hydroxyl, amino, C.sub.1 -C.sub.6 -alkyl-amino, di-C.sub.1 -C.sub.6
-alkyl-amino, C.sub.1 -C.sub.6 -alkyl, C.ssub.1 -C.sub.6 -alkyloxy, thiol,
C.sub.1 -C.sub.6 -alkyl-thio, --CHO, --OOH, --COOCH.sub.3, --COOCH.sub.2
CH.sub.3, --CONH.sub.2, or halogen substituents, which may be idential to one
another or different, and which optionally may be benzocondensed. Aryl
subtituents may be preferably derived form benzene, preferred examples being
phenyl, 2-hyroxy-phenyl, 3-hydroxy-phenyl, 4-hydroxy-phenyl, 4-amino-phenyl,
2-amino-phenyl, 3-amino-phenyl.
If aryl is substituted, the substituents
are preferably methyl, ethyl, propyl, 1-methylethyl (isopropyl), butyl,
1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, hydroxyl, amino,
dimethyl-amino, diethyl-amino, thiol, methyl-thiol, methoxy, ethoxy, --CHO,
--COOH, --COOCH.sub.3, --COOCH.sub.2 CH.suub.3, or --CONH.sub.2.
"Heteroaryl" generally represents a 5 to 10-membered aromatic
heterocyclic ring system, containing 1 to 5 heteroatoms selected from the group
of nitrogen, oxygen, or sulfur, which may optionally be substituted by one or
several hydroxyl, amino, C.sub.1 -C.sub.6 -alkyl-amino, di-C.sub.1 -C.sub.6
-alkyl-amino, C.sub.1 -C.sub.6 -alkyl, C.ssub.1 -C.sub.6 -alkyloxy, thiol,
C.sub.1 -C.sub.6 -alkyl-thio, --CHO, --COOH, COOCH.sub.3, --COOCH.sub.2
CH.sub.3, --CONH.sub.2, or halogen substituents, which may be identical to one
another or different, and which optionally may be benzocondensed. Heteroaryl
substituents may preferably be derived from furane, pyrrole, thiophene,
pyridine, thiazole, isoxazole, pyrazole, imidazole, benzofuran, thianaphthene,
indole, benzimidazole, indazole, chinoline, pyridazine, pyrimidine, pyrazin,
chinazoline, pyrane, purine, adenine, guanine, thymine, cytosine, uracil.
If heteroaryl is substituted, the substituents are preferably methyl,
ethyl, propyl, 1-methylethyl (isopropyl), butyl, 1-methylpropyl, 2-methylpropyl,
1,1-dimethylethyl, hydroxyl, amino, dimethyl-amino, diethyl-amino, thiol,
methyl-thiol, methoxy, ethoxy, --CHO, --COOH, --COOCH.sub.3, --COOCH.sub.2
CH.sub.3, or --CONH.sub.2.
"Residue of a cytotoxic or cytostatic
compound" means that the compound H.sub.2 N--Cyt', which is released upon
cleavage of the amide bond shown in formula (I), is either cytotoxic or
cytostatic itself, or may be converted into a cytotoxic or cytostatic compound
in a subsequent step.
In the latter case, --Cyt' may be a residue of
formula --L--Cyt", wherein L is a linker residue derived from a bifunctional
molecule, for instance a diamine H.sub.2 N--L'--NH.sub.2, an amino alcohol
H.sub.2 N--L'--OH, for example p-amino-benzyl alcohol (PABOH), an amino
carbonate, for example ##STR6##
or an unnatural amino carboxylic acid.
If --Cyt' is of formula --L--Cyt", the compound H.sub.2 N--L'--Cyt" is generated
by the enzymatic cleavage of the amide bond shown in formula (I). The compound
H.sub.2 N--L'--Cyt" may be cytotoxic or cytostatic itself or the linker residue
cleaved off from Cyt" in a subsequent step releasing the cytotoxic or cytostatic
agent. For example, the compound H.sub.2 N--L'--Cyt" may be hydrolysed under
physiological conditions into a compound H.sub.2 N--L'--OH and the cytotoxic or
cytostatic compound H--Cyt", which is the active therapeutic agent (In the
following, only the term Cyt' is used for both Cyt' and Cyt", and only the term
L is used for both L and L', for simplicity).
The pharmaceutically
acceptable salts of the compounds of the present invention include the
conventional non-toxic salts formed from non-toxic inorganic or organic acids.
For example, such conventional non-toxic salts include those from inorganic
acids such as hydrochloric acid, hydrobromic, sulfuric, sulfamic, phosphoric,
nitric and the like; and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,
ascorbic, maleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
oxalictrifluoroacetic and the like.
Preferred compounds of formula I are
those of fomula IA ##STR7##
wherein R.sup.2, R.sup.3, R.sup.4, Cyt' are
as defined hereinabove,
R.sup.1 represents an amino alkanoyl or
oligopeptidoyl group, and
X--Y represents CHR.sup.2 --CH.sub.2,
CR.sup.2.dbd.CH, NH--CH.sub.2, CH.sub.2 --NH, --CR.sup.2 --, CH.sub.2
--CHR.sup.2 --CH.sub.2 ; with the proviso that R.sup.1 represents an amino
alkanoyl, di- or tripeptidoyl group or R.sup.1 represents an oligopeptidoyl
having more than three amino acid moieties which does not contain a Gln-Ser
amino acid sequence, in the event that X--Y represents a CH.sub.2 --CH.sub.2
group.
Preferably the .alpha. carbon atom of the cyclic amino acid
residue is racemic, i.e. of (R/S) configuration, most preferably of (S)
configuration; in a particularly prefererred embodiment, the .alpha. carbon atom
is of (S) configuration and R.sup.2 is H. In the event that R.sup.2 is OH, it is
preferably in trans position.
R.sup.2, R.sup.3 preferably represent a
hydrogen atom or a methyl, ethyl, propyl, isopropyl, phenyl, methoxy, ethoxy or
hydroxy group, most preferably a hydrogen atom. R.sup.4 is preferably a hydrogen
atom or a methyl, ethyl, propyl, isopropyl or phenyl group, most preferably a
hydrogen atom.
Particularly preferred compounds of formula IA are
selected from the formulae IA1, IA2, IA3, IA4 and IA5 ##STR8##
H.sub.2
N--Cyt' is preferably an anthracycline derivative of formula II ##STR9##
wherein
R.sup.c represents C.sub.1 -C.sub.6 alkyl, C.sub.1
-C.sub.6 hydroxyalkyl or C.sub.1 -C.sub.6 alkanoyloxy C.sub.1 -C.sub.6 alkyl, in
particular methyl, hydroxymethyl, diethoxyacetoxymethyl or buryryloxymethyl;
R.sup.d represents hydrogen, hydroxy or C.sub.1 -C.sub.6 alkoxy, in
particular methoxy;
one of R.sup.e and R.sup.f represents a hydrogen
atom; and the other represents a hydrogen atom or a hydroxy or
tetrahydropyrany-2-yloxy (OTHP) group.
Paricularly preferred are the
following compounds of formula II: R.sup.c R.sup.d R.sup.e R.sup.f Cyt
CH.sub.2 OH OCH.sub.3 H OH doxorubicin
CH.sub.3 OCH.sub.3 H OH daunorubicin
CH.sub.2 OH OCH.sub.3 OH H epirubicin
CH.sub.3 H H OH idarubicin
CH.sub.2 OH OCH.sub.3 H OTHP THP
CH.sub.2 OH OCH.sub.3 H H esorubicin
CH.sub.2 OCOCH(OC.sub.2 H.sub.5).sub.2 OCH.sub.3 H OH
detorubicin
CH.sub.2 OH H H OH carminorubicin
CH.sub.2 OCOC.sub.4 H.sub.9 OCH.sub.3 H OH
Most preferred is doxorubicin (Dox). Other cytotoxic or cytostatic
residues Cyt' may be derived for example from methotrexate, trimetrexate,
pyritrexim, 5,10-dideazatetrahydrofolatepyrimetamine, trimethoprim,
10-propargyl-5,8-dideazafolate-2,4-diamino-5(3',4'-dichloropheyl)-6-methyl
pyrimidine, aminoglutethimide, goreserelin, melphalan, chlorambucil, analogs of
other chemotherapeutic agents such as 9-aminocamtothecin (for examples see e.g.
Burris HA, r. d. and S. M. Fields (1994). "Topoisomerase I inhibitors. An
overview of the camptothecin analogs. [Review]." Hematol. Oncol. Clin. North Am.
8(2): 333-355; Iyer, L. and M. J. Ratain (1998). "Clinical pharmacology of
camptothecins. [Review] [137 refs]." Cancer Chemother. Pharmacol. 42 Suppl:
S31-S43.)
In formula (I), Cyt' may also be a biological effector
molecule which either directly or indirectly effects destruction of tumour
cells, like for example TNF.alpha..
Preferred examples of amino
carboxylic acids from which the A, B, and D units may be derived are glycine
(Gly), or the D- or, more preferably, the L-forms of alanine (Ala), valine
(Val), leucine (Leu), isoleucine (lle), phenylalanine (Phe), tyrosine (Tyr),
tryptophan (Trp), cysteine (Cys), methionine (Met), serine (Ser), threonine
(Thr), lysine (Lys), arginine (Arg), histidine (His), aspartatic acid (Asp),
glutamic acid (Glu), asparagine (Asn), glutamine (Gln), proline (Pro),
trans-4-hydroxy-proline (Hyp), 5-hydroxy-lysine (Hyl), norleucine (Nle),
5-hydroxynorleucine, 6-hydroxynorleucine (Hyn), omithine (Orn),
cyclohexylglycine (Chg), phenylglycine (Phg), glutamine (Gln), cyclohexylalanine
(Cha), methionine-S-oxide (Met), .beta.-cyclopropylalanine (Cpa), tert.-leucine
(Tle), or homo-serine (Hse).
Preferred compounds have the general
formula (I), wherein the A unit is derived from alanine, valine (Val), leucine
(Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp),
cysteine (Cys), methionine (Met), serine (Ser), threonine (Thr), lysine (Lys),
arginine (Arg), histidine (His), aspartatic acid (Asp), glutamic acid (Glu),
asparagine (Asn), glutamine (Gln), proline (Pro), trans-4-hydroxy-proline (Hyp),
5-hydroxy-lysine (Hyl), norleucine (Nle), 5-hydroxynorleucine,
6-hydroxynorleucine (Hyn), ornithine (Orn), or cyclohexylglycine (Chg),
phenylglycine (Phg), glutamine (Gln), cyclohexylalanine (Cha),
methionine-S-oxide (Met), .beta.-cyclopropylalanine (Cpa), tert.-leucine (Tle)
or homoserine (Hse).
Particularly preferred are those compounds of
formula (I), wherein R.sup.1 is a group selected from the formnulae (1) to (34):
H-Chg (1) H-Tle (18)
H-Lys (2) H-Hyl (19)
H-Nle (3) H-Hse (20)
H-Ala (4) Cg-Gly (21)
H-Hyn (5) Cg-Nle (22)
H-Pro (6) Cg-Val (23)
H-Phg (7) Cg-Met (24)
H-Gln (8) H-Xxx-Lys (25)
H-trans-Hyp (9) H-Xxx-Hyn (26)
H-Val (10) H-Xxx-Pro (27)
H-Cha (11) H-Xxx-His (28)
H-Met (12) H-Xxx-Met (29)
H-Nva (13) H-Xxx-Ala (30)
H-Met(O) (14) Cg-Xxx-Hyn (31)
H-.beta.-Cpa (15) Cg-Xxx-Ala-Gly (32)
H-Ile (16) Cg-(Xxx).sub.m -Xxx-Ala-Gly (33)
H-Ser (17) Cg-(Xaa).sub.m -Xaa-Gly (34)
wherein
Cg represents a hydrogen atom or a capping group
selected from benzoyloxycarbonyl, phenylacetyl, phenylmethylsulfonyl and
benzylaminocarbonyl; Xaa represents a moiety derived from an amino carboxylic
acid, preferably selected form the group natural amino acids, in particular from
the group consisting of Ala, Pro, Tyr, Phe, His, Ser, Thr, Hyp and Lys; and m is
an integer from 1 to 6.
Preferred capping groups Cg are acetyl (Ac),
succinimidyl (Suc), D-alanyl, benzyloxycarbonyl (Cbz or Z), or macromolecules
such as polyethylene glycol.
Preferred anthracycline prodrugs are the
compounds of formula III ##STR10##
wherein R.sup.a, R.sup.b, R.sup.c,
R.sup.d, R.sup.e, R.sup.f and R.sup.1 are as defined hereinabove.
Most
preferred compounds of the invention are doxorubicin derivatives of formulae
(IIIA) to (IIIF): ##STR11## ##STR12##
If the part Cg--B--A or
Cg--(D)m_B--A of formula (I) contains two or more sulfur atoms, the compound of
the invention may contain one or more disulfide bonds.
One class of
cytotoxic or cytostatic compounds which may be used for the present invention
has a primary amino function which is available for formation of an amidic bond
as shown in formula (I), like doxorubicin. In this case, a linker molecule L is
not necessary. If a cytostatic or cytotoxic compound does not have such an amino
function, such a function may be created in such a compound by way of chemical
modification, e.g. by introducing or converting a functional group or attaching
a linker molecule to the compound. A linker molecule may also be inserted
between the oligomeric part (i.e. the part comprising the amino carboxylic
residues) and the cytostatic or cytotoxic part of the compound of the invention
to ensure or optimise cleavage of the amide bond between the oligomeric part and
the cytotoxic or cytostatic part. If a linker molecule is present, i.e. in
compounds containing the structure L--Cyt', the bond between L and Cyt' is
preferably an amidic or ester bond. In a preferred embodiment, such a linker
molecule is hydrolysed off the cytostatic or cytotoxic compound under
physiological conditions after the enzymatic cleavage and thus the free
cytostatic or cytotoxic compound is generated. In any case, the compound of the
invention must have the property of being cleavable upon the catalytic action of
FAP.alpha. and, as a direct or indirect consequence of this cleavage, releasing
under physiological conditions a cytostatic or cytotoxic compound.
In a
further aspect, the present invention relates to a prodrug that is capable of
being converted into a drug by the catalytic action of FAP.alpha., said prodrug
having a cleavage site which is recognised by FAP.alpha., and said drug being
cytotoxic or cytostatic under physiological conditions. Such a prodrug
preferably comprises an oligomeric part comprising two or more amino carboxylic
residues and a cytotoxic or cytostatic part, wherein the C-terminal amino
carboxylic residue of the oligomeric part is a 3- to 7-membered natural or
unnatural cyclic amino acid, preferably D- or L-proline, or D- or
L-hydroxyproline, and the C-terminal carboxy function is linked to the cytotoxic
or cytostatic part by an amide bond which may be cleaved by the catalytic action
of FAP.alpha.. The oligomeric part is preferably a peptide. Preferably, the
oligomeric part comprises two, three, four, five, six, seven, eight, nine, ten,
eleven, or twelve amino carboxylic acid residues, more preferably two, three, or
four amino carboxylic residues. The N-terminal amino function is preferably
protected by a capping group.
The compounds of the invention may be
synthesized by processes known in the art (E. Wunsch, Synthese von Peptiden, in
"Methoden der organischen Chemie", Houben-Weyl (Eds. E. Muller, O. Bayer), Vol.
XV, Part 1 and 2, Georg Thieme Verlag, Stuttgart, 1974). For example, the
compounds could be synthesized in a block synthetic fashion by condensation of
the terminal carboxy function of the oligomeric part, wherein X may be OH or an
activation leaving group, with the amino group of the cytotoxic or cytostatic
molecule H.sub.2 N--Cyt' resulting in an amide formation. ##STR13##
If a
linker residue (L) is required between the oligomeric part and the cytotoxic or
cytostatic agent the block synthesis can be done in the same manner. ##STR14##
If the cytotoxic or cytostatic bears a carboxy function for the
attachment to the oligomeric part, the linker molecule can be an imine or an
amino alcohol and the block synthesis of such compounds can be carried out in a
similar way by reaction of the activated XOC--Cyt' with either the hydroxy or
the amino component. ##STR15##
If the cytotoxic or cytostatic reagent
has a hydroxy function which is suitable for the coupling to the oligomeric part
the linker residue may be an amino carboxylic acid and a block synthesis can be
done similarly.
If necessary, other functional groups in the units Cyt',
L, hydroxyproline, A, B and D which shall not react during the assembly of the
target molecules may be protected by suitable protecting groups. Suitable
protecting groups are well known from the state of the art (P. G. M. Wuts,
"Protective groups in organic synthesis", John Wiley and Sons Inc., New York
1991). These protecting groups are removed at the end of the synthesis.
By way of example only, useful amino-protecting groups may include, for
example, C.sub.1 -C.sub.10 alkanoyl groups such as formyl, acetyl
dichloroacetyl, propionyl, 3,3diethylhexanoyl, and the like, C.sub.1 -C.sub.10
alkoxycarbonyl and C.sub.6 -C.sub.17 aralkyloxycarbonyl groups such as
tert-butoxycarbonyl, benzyloxycarbonyl (BOC), fluorenylmethoxycarbonyl, and the
like. Most preferred is fluorenylmethoxycarbonyl (FMOC).
Suitable
carboxy-protecting groups may include, for example, C.sub.1 -C.sub.10 alkyl
groups such as methyl, tert-butyl, decyl; C.sub.6 -C.sub.17 aralkyl such as
benzyl, 4-methoxybenzyl, diphenylmethyl, triphenylmethyl, fluorenyl;
tri-(C.sub.1 -C.sub.10 alkyl)silyl or (C.sub.1 -C.sub.10 alkyl)-diarylsilyl such
as trimethylsilyl, dimethyl-tert-butylsilyl, diphenyl-tert-butylsilyl and
related groups.
To achieve such ester- or amide formations, it may be
necessary to activate the carbonyl group of the Larboxylic acid for a
nucleophilic attack of an amine or alcohol, i.e. X to be an activation group or
leaving group which is suitable to be substituted by an amino group. This
activation can be done by conversion of the carboxylic acid into an acid
chloride or acid fluoride or by conversion of the carboxylic acid into an
activated ester, for instance a N-hydroxysuccinimidyl ester or a
pentafluorophenyl ester. Another method of activation is the transformation into
a symmetrical or unsymmetrical anhydride. Alternatively, the formation of the
amide- or ester bonds can be achieved by the use of in situ coupling reagents
like benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
(PyBOP) (E. Frerot et al., Tetrahedron, 1991, 47, 259-70),
1,1'-carbonyldimidazole (CDI) (K. Akaji et al., THL, 35, 1994, 3315-18),
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)
(R. Knorr et al., THL, 30, 1989, 1927-30),
1-(mesitylene-2-sulonyl)-3-nitro-1H-1,2,4-triazole (MSNT) (B. Blankenmeyer-Menge
et al., THL, 31, 1990, 1701-04).
As an alternative to the block
synthesis the molecules in the general formula (I) can be assembled in a step by
step manner starting at the right hand side by stepwise condensation reactions
of the respective monomers Cyt', L, the cyclic amino acid group formed by
R.sup.a, R.sup.b and the inteijacent N--C group, in particular proline or
hydroxyproline, A, B and D. For the condensation reaction the same above
mentioned coupling methods can be applied. to Since the units L,
proline/hydroxyproline, A, B and D are at least bifunctional molecules
containing an amino- and (at least the units A, B, D, and the cyclic amino acid
group formed by Ra, Rb and the interhjacent N--C group, in particular
proline/hydroxyproline) a carboxy group, the amino group needs to be blocked by
a protecting group (PG) prior to the activation of the carboxylic function. For
the protection of the amino groups the group BOC or preferably the group FMOC
can be applied. After the coupling reaction the amino protecting group has to be
removed and the coupling with the next Fmoc- or Boc-protected unit can be
carried out. If necessary, other functional groups in the units Cyt', L, the
cyclic amino acid group formed by R.sup.a, R.sup.b and the interhjacent N--C
group, in particular hydroxyproline, A, B and D which shall not react during the
assembly of the target molecules may be protected by suitable protecting groups.
These protecting groups are removed at the end of the synthesis.
Capping
groups as defined in the context of formula (I) may also serve as protection
groups, in particular when the last (N-terminal) amino carboxylic acid unit is
added. In this latter case the protecting group is not removed as it is a part
of the target molecule. Alternatively, the capping group may be added after the
last amino carboxylic acid unit has been coupled and deprotected.
The
step by step synthesis is outlined in the following schemes. The second scheme
is exemplary as the linker residue as well as the Cyt' residue may contain other
functional groups as indicated in this scheme (see above): ##STR16##
Accordingly, a further aspect of the invention is a process for the
production of a compound of formula (I), characterised in that a compound of the
general formula (V) ##STR17##
wherein R.sup.1, R.sup.a and R.sup.b are
as defined hereinabove, X.sup.1 represents OH, or a leaving group which is
suitable to be substituted by a amino group,
is reacted with a compound
HN(R.sup.4)--Cyt', wherein Cyt' is the residue of a cytotoxic or cytostatic
compound, and R.sup.4 is as defined hereinabove.
Preferably, X.sup.1
within formula (V) is a leaving group, for example --Cl, --F,
N-hydroxysuccinimidyl, pentafluorophenyl, or a carboxylate. Alternatively,
X.sup.1 may be OH, and condensation is achieved by the use of an in situ
coupling reagent, for example
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP),
1,1'-carbonyldimidazole (CDI),
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU),
or 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT).
A further
aspect of the invention is a process for the production of a compound of formula
(I), characterised in that a compound of the general formula (VI) ##STR18##
wherein R.sup.1, R.sup.a and R.sup.b are as defined in claim 1, Y.sup.1
represents L--COX.sup.2, wherein L is a linker residue, and X.sup.2 represents
OH, or a leaving group which is suitable to be substituted by a amino group or a
hydroxy group,
is reacted with a compound H.sub.2 N--Cyt' or with a
compound HO--Cyt', wherein Cyt' is the residue of a cytotoxic or cytostatic
compound.
Preferably, X.sup.2 within formula (VI) is a leaving group,
for example --Cl, --F, N-hydroxysuccinimidyl, pentafluorophenyl, or a
carboxylate. Alternatively, X.sup.2 may be OH and condensation is achieved by
the use of an in situ coupling reagent, for example
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP),
1,1'-carbonyldimidazole (CDI),
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU),
or 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT).
A further
aspect of the invention is a process for the production of a compound of formula
(I), characterised in that a compound of the general formula (VII) ##STR19##
wherein R.sup.1, R.sup.a and R.sup.b are as defined hereinabove, Y.sup.2
is of formula L--OH or L--NH.sub.2,
wherein L is a linker residue,
is reacted with a compound X.sup.3 OC--Cyt', wherein X.sup.3 may be OH,
or a leaving group which is suitable-to be substituted by a amino group or a
hydroxy group, and wherein Cyt' is the residue of a-cytotoxic or cytostatic
compound.
Preferably, X.sup.3 of the compound X.sup.3 OC--Cyt' is a
leaving group, for example --Cl, --F, N-hydroxysuccinimidyl, pentafluorophenyl,
or a carboxylate. Alternatively, X may be OH and condensation is achieved by the
use of an in situ coupling reagent, for example
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP),
1,1'-carbonyldimidazole (CDI),
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU),
or 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT).
A further
aspect of the invention is a process for the production of a compound of formula
(I), characterised in that a compound H.sub.2 N--Cyt' is condensed stepwise with
the units making up the compound of formula (I). Before each coupling step, it
may be necessary to remove a protecting group PG, if present.
Accordingly, a further aspect of the invention is a process for the
production of a compound of formula (I), characterised in that a compound of the
general formula (VIII) ##STR20##
wherein PG.sup.1 is a protecting group,
and the other the substituents have the meaning as described before,
is
reacted with a compound HN(R.sup.4)--Cyt', wherein Cyt' is the residue of a
cytotoxic or cytostatic compound;
the protecting group PG.sup.1 is then
removed and the resulting compound of formula (VIIIA) ##STR21##
is
subsequently reacted with a compound PG.sup.2 --A--X.sup.4, wherein
PG.sup.2 is a protecting group, and X.sup.4 represents OH, or a leaving
group suitable to be substituted by a amino group;
and further coupling
steps are carried out, if necessary, until the complete compound is obtained.
PG.sup.1 and pG.sup.2 may be, for example BOC, or preferably FMOC.
Accordingly, a further aspect of the invention is a process for the
production of a compound of formula (I), characterised in that a compound of
formula PG.sup.3 --N(R.sup.4)--L--COX.sup.3, wherein
PG3 is a protecting
group, and the other substituents have the meaning as described before, is
reacted with a compound of formula Y.sup.4 --Cyt', wherein
Cyt' is the
residue of a cytotoxic or cytostatic compound; and Y.sup.4 represents H.sub.2 N
or HO; the protecting group PG.sup.3 is then removed; and the resulting compound
HN(R.sup.4)--L--Y.sup.4 --Cyt' is reacted with a compound of formula (VIII)
##STR22##
the protecting group PG.sup.1 is then removed and the
resulting compound of formula ##STR23##
is then reacted with a compound
PG.sup.4 --A--X.sup.4, wherein
PG.sup.4 is a protection group, and
X.sup.4 may be OH, or a leaving group suitable to be substituted by a amino
group;
and further coupling steps are carried out, if necessary, until
the complete molecule is obtained.
A further aspect of the invention is
a process for the production of a compound of formula (I), characterised in that
a compound of formula PG.sup.5 --N(R.sup.4)--L--Y.sup.5, wherein
PG.sup.5 represents a protecting group, Y.sup.5 represents OH or
NH.sub.2 and the substituents have the meaning as described before,
is
reacted with a compound of formula X.sup.5 OC--Cyt', wherein
Cyt' is the
residue of a cytotoxic or cytostatic compound and X.sup.5 is OH or a suitable
leaving group;
the protecting group PG.sup.5 is then removed; and the
resulting compound HN(R.sup.4)--L--Y.sup.5 --CO--Cyt' is reacted with a compound
of formula (VIII) ##STR24##
the protecting group is then removed and the
resulting compound ##STR25##
is then reacted with a compound PG.sup.2
--A--X.sup.4, wherein
PG.sup.2 is a protecting group, and X.sup.4
represents OH, or a leaving group suitable to be substituted by a amino group;
and further coupling steps are carried out, if necessary, until complete
molecule is obtained.
Another aspect of the present invention are the
novel intermediate compounds of formula VIIIA ##STR26##
wherein R.sup.a,
R.sup.b, R.sup.4 and Cyt' are as defined hereinabove.
The compounds of
the invention are intended for medical use. In particular, these compounds are
useful for the treatment of tumours which are associated with stromal
fibroblasts that express FAP.alpha. and which are generally not optimally
treated with available cytotoxic and/or cytostatic agents. Tumours with this
property are, for example, epithelial cancers, such as lung, breast, and colon
carcinomas. Tumours, such as bone and soft tissue sarcomas which express
FAP.alpha., may also be treated with these compounds.
Consequently,
another aspect of the present invention are pharmaceutical compositions
comprising a compound of the present invention and optionally one or more
suitable and pharmaceutically acceptable excipients, as exemplified in:
Remington: the science and practice of pharmacy. 19th ed. Easton: Mack Publ.,
1995. The pharmaceutical compositions may be formulated as solids or solutions.
Solid formulations may be for preparation of a solution before injection.
Preferably, the pharmaceutical compositions of the invention are solutions for
injection. They may be administered systemically, e.g. by intravenous injection,
or topically, e.g. by direct injection into the tumour site. The dosage will be
adjusted according to factors like body weight and health status of the patient,
nature of the underlying disease, therapeutic window of the compound to be
applied, solubility, and the like. It is within the knowledge of the expert to
adjust dosage appropriately. For doxorubicin conjugates, for example, the dose
will preferably be in the range from 10 mg/m.sup.2 to 1350 mg/m.sup.2, but also
higher or lower doses may be appropriate.
Accordingly, a further aspect
of the present invention is the use of a compound of the invention in the
preparation of a pharmaceutical composition for the treatment of cancer.
Furthermore, an aspect of the invention is a method of treatment of cancer,
comprising administering an effective amount of a pharmaceutical composition of
the invention to a patient. Indications include the treatment of cancer,
specifically,
1) The treatment of epithelial carcinomas including
breast, lung, colorectal, head and neck, pancreatic, ovarian, bladder, gastric,
skin, endometrial, ovarian, testicular, esophageal, prostatic and renal origin;
2) Bone and soft-tissue sarcomas: Osteosarcoma, chondrosarcoma,
fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyosarcoma;
3)
Hematopoietic malignancies: Hodgkin's and non-Hodgkin's lymphomas;
4)
Neuroectodermal tumors: Peripheral nerve tumors, astrocytomas, melanomas;
5) Mesotheliomas.
Also included are the treatment of chronic
inflammatory conditions such as rheumatoid arthritis, osteoarthritis, liver
cirrhosis, lung fibrosis, arteriosclerosis, and abnormal wound healing.
A further aspect of the invention is a method of treatment of cancer,
wherein a prodrug is administered to a patient wherein said prodrug is capable
of being converted into a cytotoxic or cytostatic drug by an enzymatic activity,
said enzymatic activity being the expression product of cells associated with
tunour tissue. Preferably, said enzymatic activity is the proteolytic activity
of FAP.alpha..
One method of administration of the compounds is
intravenous infusion. Other possible routes of administration include
intraperitoneal (either as a bolus or infusion), intramuscular or intratumoral
injection. Where appropriate, direct application may also be possible (for
example, lung fibrosis).
FIGURES
FIG. 1: Cleavage of
doxorubicin-peptide conjugates by FAP.alpha.. Chromatograms for ZGP-Dox
(Z-Gly-(L)-Pro-Doxorubicin) after incubation with purified FAPmCD8 fusion
protein (A), or with buffer (B). See example 5.
FIG. 2: Reduction of
doxorubicin cytotoxicity by conjugation of doxorubicin to FAP.alpha.-cleavable
peptides. See example 6.
FIG. 3: Demonstration of cytotoxicity of
doxorubicin released from FAP.alpha.-cleavable doxorubicin-peptide conjugates by
FAP.alpha.-expressing HT1080 clone 33 cells versus parental HT1080 cells. See
example 8.
FIG. 4: Plasma stability of N-Cbz-Gly-(L)-Pro-Doxorubicin and
N-Cbz-(L)-Pro-(L)-Ala-Gly-(L)-Pro-Doxorubicin in mouse and human plasma. See
example 9.
FIG. 5: Demonstration of FAP.alpha. enzyme activity and
confirmation of its apparent molecular weight in human tumour tissue samples.
See Example 12.
One skilled in the art will appreciate that although
specific reagents and reaction conditions are outlined in the following
examples, modifications can be made which are meant to be encompassed by the
scope of the invention. The following examples, therefore, are intended to
further illustrate the invention and are not limiting.
EXAMPLES
Example 1
Synthetic Procedures of Doxorubicin Conjugates
N-Cbz-Gly-Pro-Doxorubicin: N-Cbz-Gly-Pro (116.1 mg, 0.37mmol) and
N-hydroxy succinimide (44 mg, 0.37 mmol) were weighed out and placed in a 2
neck-round bottom flask under dinitrogen. Anhydrous N,N-dimethylformamide (20
ml) was added and the flask was cooled to 0.degree. C. in an ice bath.
Dicyclohexylcarbodiimide (78 mg, 0.37 mmol) was added as a 1 ml solution in
N,N-dimethylforrnamide. The solution was stirred at 0.degree. C. for 40 minutes.
Doxorubicin.HCl (100 mg, mmol) was weighed into a vial with a small stir
bar and placed under dinitrogen. N,N-Dimethylformamide (3 ml) and
N,N-diisopropylethylamine (33.1 .mu.l 0.19 mmol) were added to the vial with
stirring. The doxorubicin solution was added via syringe to the peptide
solution, and the vial was rinsed with an additional 2 ml of
N,N-dimethylformamide. The ice bath was removed and reaction mixture was stirred
for approximately 48 hours at room temperature.
The reaction solution
was extracted with ethyl acetate (500 ml). The ethyl acetate was washed with of
10% aqueous citric acid solution (250 ml), saturated aqueous sodium bicarbonate
(250 ml) and brine (250 ml) sequentially. The organic extract was dried with
anhydrous MgSO.sub.4, and the solvent was removed with a roto-evaporator. The
product, which was rich in DMF contaminant, was chromatographed on a C-18
reversed phase flash column with 8:2 methanol:water as the eluent. One orange
spot, rf.apprxeq.0.3, which fluoresced under long wave UV light was isolated.
The methanol was removed with the roto-evaporator and the last traces of solvent
were removed with the high vacuum pump overnight.
N-Cbz-Pro-Ala-Gly-Pro:
N-Cbz-Pro-Ala (5 g, 15 mmol) and carbonyldiimidazole (2.43 g, 15 mmol) were
placed in a 250 ml, 3-neck round bottom flask under an argon atmosphere.
Anhydrous tetrahydrofuran (50 ml) was added and the solution was stirred at room
temperature for approximately 45 minutes. Rigorous evolution of a gas (CO.sub.2)
was observed.
Into a separate flask was weighed Gly-Pro-OCH.sub.3.HCl
(2.9 g, 15 mmol). Tetrahydrofuran (5 ml) and N,N-diisopropylethylamine (5.23 ml,
30 mmol) were added and the solution was stirred for several minutes. The
material that dissolved was added via syringe to the activated peptide, the
remaining material was dissolved in a minimum amount of CH.sub.2 Cl.sub.2 and
also added to the activated peptide via syringe.
The reaction solution
was stirred overnight at room temperature (15 hours) and in the morning there
was copious amounts of white precipitate. The reaction mixture was washed with
10% aqueous citric acid solution (300 ml) and extracted with ethyl acetate (500
ml). The ethyl acetate extract was washed with saturated aqueous bicarbonate
solution (300 ml) and dried with brine (300 ml) and anhydrous MgSO.sub.4 The
ethyl acetate was removed with the roto-evaporator to yield 5 g of a colourless
oil, which gave satisfactory characterisation data.
The crude oil of the
N-Cbz-Pro-Ala-Gly-Pro-OCH.sub.3 (5g, 12 mmol) was dissolved in methanol (20 ml)
in a round bottom flask. The flask was placed in an ambient temperature water
bath. 1 N Sodium hydroxide solution (12 ml) was added cautiously. The solution
was stirred for 3.5 hours after which time 1 N HCl solution (12 ml) was added.
The solution was concentrated on the roto-evaporator and a few more drops of 1 N
HCl was added until the pH is approximately 1.5 with pH paper. The water was
removed with the vacuum pump to give an oil, which was recrystalized slowly from
ethanol.
N-Cbz-Pro-Ala-Gly-Pro-Doxorubicin: N-Cbz-Pro-Ala-Gly-Pro (180
mg, 0.38 mmol) was dissolved in anhydrous N,N-dimethylformamide (15 ml).
1-Hydroxybenzotriazole (51.3 mg, 38 mmol) and dicyclohexylcarbodiimide (78 mg,
38 mmol) were dissolved in 1 ml each of N,N-dimethylformamide and added to the
peptide as solutions. The reaction mixture was stirred at room temperature for
45 minutes.
Doxorubicin.HCl (116 mg, 20 mmol) was weighed into a
separate vial and dissolved in N,N-dimethylforrnamide (3 ml).
N,N-Diisopropylethylamine (34.8 .mu.l, 20 mmol) was syringed into the vial
containing the doxorubicin, and the contents were stirred for several minutes to
ensure complete dissolution. The doxorubicin solution was added via syringe to
the activated peptide. The solution was stirred for 48 hours at room
temperature.
The product was extracted with ethyl acetate (2 l) and
washed with 10% aqueous citric acid solution (500 ml). The ethyl acetate layer
was separated, dried with MgSO.sub.4 and concentrated to an oil on the
roto-evaporator. The oil was chromatographed on C-18 reversed phase silica gel,
which gave an orange, long wave UV fluorescing spot at rf=0.25. The final
product gave satisfactory characterization data.
Example 2
Preparation of FAP.alpha.-expressing Cell Lines
Mammalian cell
lines expressing recombinant FAP.alpha. were prepared. HT1080 fibrosarcoma
cells, widely known and available from the DSMZ (German Collection of
Microorganisms and Cell Cultures, Braunschweig, Germany) under the accession
number DSMZ ACC 315, were maintained in a DMEM/F12 mix 50:50 containing 10%
fetal bovine serum in an atmosphere of 95% air and 5% CO.sub.2. HT1080 cells
were transfected with FAP.38 vector (WO 97/34927, Scanlan et al., loc. cit.)
using the Lipofectin method according to the manufacturer's instructions
(Gibco/BRL). Transfectants were selected for resistance to antibiotics (200
.mu.g/ml Geneticin) and thereafter maintained in medium containing Geneticin.
Individual colonies of resistant cells were picked, grown to confluence in 10 cm
tissue culture petri dishes and tested for FAP.alpha. expression in an
immunofluorescence assay using the FAP.alpha.-specific monoclonal antibody F19,
as described (Garin-Chesa et al. (1990) Proc. Natl. Acad. Sci. USA 87(18),
7235-7239). The parental HT1080 cell line showed no detectable FAP.alpha.
expression in this immunofluorescence assay, while one clone, referred to
hereafter as HT1080 clone 33, was positive for FAP.alpha..
Similarly,
human embryonic kidney 293 cells, widely known and available from American
Tissue Type Collection (Rockville, Md.), were maintained in a DMEM containing
10% fetal bovine serum in an atmosphere of 95% air and 5% CO.sub.2. Cells were
transfected with a FAP.alpha. expression vector, pFAP.38 using calcium phosphate
transfection as described (Park, J. E., Chen, H. H., Winer, J., Houck, K. A.
& Ferrara, N. (1994). Placenta growth factor. Potentiation of vascular
endothelial growth factor bioactivity, in vitro and in vivo, and high affinity
binding to Flt-1 but not to Flk-1/KDR. J. Biol. Chem. 269(41), 25646-5654).
Transfectants were selected and analyzed as described above for FAP.alpha.
expresion. The parental 293 cell line showed no detectable FAP.alpha.
expression. One clone, referred to hereafter as 293-I/2, was FAP.alpha.
positive.
Example 3
Examination of FAP.alpha. Expression in
Transfected Cell Lines
FAP.alpha. expression was examined in the HT1080
and HT1080 clone 33 cells. Metabolic labeling, immunoprecipitations and
fluorography were performed essentially as described (Park et al. (1991) Somatic
Cell Mol. Genet. 17(2), 137-150). HT1080 and HT1080 clone 33 cells were
metabolically labelled with .sup.35 S-methionine. Detergent extracts of these
cells were immunoprecipitated with monoclonal antibody F19 or with mouse IgG1
antibody as a negative control. Precipitates were boiled in sample buffer and
separated by sodium dodecyl sulfate gel electrophoresis (as described by Laemmli
(1970) Nature 227(259), 680-685). Fluorographic analysis of the resulting gel
confirmed that the HT1080 clone 33 cells produce FAP.alpha. protein. No
FAP.alpha. protein was detectable in extracts of the parental HT1080 cells nor
in immunoprecipitates with mouse IgG1.
Example 4
Soluble
Recombinant FAP.alpha.
A soluble recombinant form of FAP.alpha. protein
was prepared as follows. A cDNA encoding the extracellular domain (ECD) of
murine CD8.alpha. (Genbank M12825), consisting of the N-terminal 189 amino acids
of CD8.alpha., was ligated to a cDNA encoding the extracellular domain of
FAP.alpha. (amino acids 27 to 760), generating a fusion protein construct,
FAPmCD8, similar in structure to the CD8.alpha.-CD40 ligand fusion protein, as
previously described (Lane et al. (1993) J. Exp. Med. 177(4), 1209-1213). The
cDNAs were verified by sequencing and inserted into the pVL1393 vector.
Transfection of Sf9 cells and amplification of the resulting recombinant
baculovirus were performed as described (O'Reilly (1994) Baculovirus Expression
Vectors: A Laboratory Manual, Oxford University Press, New York). The culture
supernatant of High Five cells infected with recombinant FAPmCD8 baculovirus for
four days was collected and cleared by ultracentrifugation. FAPmCD8 fusion
protein was purified from such supernatants using an anti-FAP.alpha. monoclonal
antibody immobilized on activated agarose beads (Pierce Chemical, Indianapolis,
Ind., USA). The culture supernatant was passed through the antibody affinity
column and eluted by pH shift using 0.1 M citrate buffer, pH 3. The samples were
immediately neutralized with a saturated Tris solution (Sigma Chemicals, St.
Louis, Mo.) and protein-containing fractions were pooled.
Example 5
Measurement of Cleavage of Doxorubicin-peptide Conjugates
Samples were separated by reversed-phase high performance liquid
chromatographic (HPLC) assay that was established to measure cleavage of
doxorubicin-peptide conjugates. The HPLC system consisted of a Waters 717
autosampler equipped with a 100 microliter (.mu.l) loop and two Waters model 510
pumps to deliver solvents. Separations were performed under isocratic conditions
at a flow rate of 0.7 ml/min on a Nucleosil C-18 column, 100 mm long.times.4 mm
I.D. with 5 .mu.m particle size (Dr. Ing. H. Knauer GmbH, Berlin). The mobile
phase consisted of methanol:water (70:30, v/v) containing 0.2 M ammonium
acetate, adjusted to pH 3.2. Free doxorubicin and doxorubicin-peptide conjugates
were detected by fluorescence (excitation, 475 nm; emission, 585 nm) using a
Waters 474 fluorescence detector. Injection, solvent delivery, data acquisition,
and data analysis were all performed using the Millennium 2010 chromatography
software package (Waters Corp., Milford, Mass., USA). Substances to be tested
were first dissolved in dimethyl sulfoxide at a concentration of 5 mM and
subsequently diluted in aqueous solution before being applied to the HPLC
column.
The ability of soluble recombinant FAP.alpha. enzyme to release
free doxorubicin from doxorubicin-peptide conjugates was examined.
Doxorubicin-peptide conjugate stock solutions (5 mM) were diluted with
Hepes-buffered saline pH 7.4 to a final concentration of 50 to 100 .mu.M. Twenty
.mu.l of the resulting solution was mixed with 50 .mu.l of purified FAPmCD8
fusion protein (approximately 20 ng) described above and 30 .mu.l Hepes-buffered
saline, pH 7.4. The mixture was allowed to incubate at 37.degree. C. for 1 day
and release of free doxorubicin was measured in the HPLC assay described. Areas
under each peak were quantified using the software package above and the initial
value was set to 100%. The rate of release of free doxorubicin was measured by
the appearance of a peak with the same retention time as free doxorubicin under
these HPLC conditions. The areas under each peak were used to calculate the
relative amounts of free doxorubicin to doxorubicin-peptide conjugate.
Integration of peak areas to determine percent cleavage was carried out using
the Millennium 2010 chromatography software package above. As seen in the
chromatograms for ZGP-Dox (N-Cbz-Gly-Pro-Doxorubicin) shown in FIG. 1, the
doxorubicin-peptide conjugate could be converted to free doxorubicin after
incubation with purified FAPmCD8 fusion protein but the retention time of the
conjugate was not altered by incubation with buffer.
Example 6
Reduction of Cytotoxicity of Doxorubicin by Conjugation to
FAP.alpha.-cleavable Peptides
The ability of FAP.alpha.-cleavable
peptides to block the cytotoxic action of doxorubicin on FAP.alpha.-negative,
doxorubicin-sensitive cells was determined. K562 cells, available from American
Type Tissue Culture Collection, Rockville, Md., USA (ATCC Number: CCL-243), were
seeded in 96 well plates (Greiner Scientific) at a density of 1000 cells/well.
Serum-free cell culture media containing various concentrations of free
doxorubicin or equivalent molar concentrations of doxorubicin-peptide conjugates
were added to the cells. Four days later, cell number was determined using an
automated CASY.TM. cell counter (Scharfe System GmbH, Reutlingen, Germany). The
results are shown in FIG. 2.
Example 7
Release of Free
Doxorubicin by Cell-bound FAP.alpha.
The ability of cell-bound
FAP.alpha. enzyme to release free doxorubicin from doxorubicin-peptide
conjugates was examined. Each conjugate was dissolved in serum-free cell culture
medium at a final concentration of 1 .mu.M. Ten milliliters of this solution was
added to confluent monolayers of HT1080 or HT1080 clone 33 cells in 10 cm tissue
culture dishes for 19 hours at 37.degree. C. The media were removed and release
of doxorubicin measured as described in Example 5. The FAP-expressing cell line,
HT1080 clone 33, converted 81% and 43% of the ZGP-Dox
(N-Cbz-Gly-Pro-Doxorubicin) and ZPAGP-Dox (N-Cbz-Pro-Ala-Gly-Pro-Doxorubicin)
conjugates to free doxorubicin, respectively. The parental HT1080 cell line
converted only 9% of ZGP-Dox to free doxorubicin under the same conditions.
Little or no detectable conversion of ZPAGP-Dox to free doxorubicin by the
parental HT1080 cell line was observed under these conditions.
Example 8
Killing of Sensitive Cells by FAP.alpha.-released Doxorubicin
The ability of FAP.alpha. to generate free doxorubicin capable of
killing doxorubicin-sensitive cells was determined. K562 cells, available from
American Type Tissue Culture Collection, Rockville, Md., USA (ATCC Number:
CCL-243), were seeded in 96 well plates (Greiner Scientific) at a density of
1000 cells/well. Serum-free cell culture media containing 1 .mu.M
doxorubicin-peptide conjugate was added to HT1080 or HT1080 clone 33 cells
dishes for 19 hours at 37.degree. C. The media were removed and release of
doxorubicin was confirmed as in Example 5. Sixty-six .mu.l of this medium was
then added per well to the K562 cells. Four days later, cell number was
determined using an automated CASY.TM. cell counter. The results are shown in
FIG. 3.
Example 9
Plasma Stability of Doxorubicin-peptide
Conjugates
The plasma stability of doxorubicin-peptide conjugates was
measured using methods described in Example 5. Samples containing
doxorubicin-peptide conjugates (at a concentration of 1 .mu.M) were incubated in
the presence of 10% (v/v) mouse or human plasma for the times indicated at
37.degree. C. The results for ZGP-Dox and ZPAGP-Dox in mouse and human plasma
are shown in FIG. 4.
Example 10
FAP.alpha.-catalyzed Cleavage of
Selected 4-Methoxy-.beta.-napthylamide-peptide Conjugates
To identify
preferred FAP.alpha. peptide substrates, oligomers composed of natural and/or
unnatural amino carboxylic acids were synthesized and coupled to
Proline-4-methoxy-.beta.-napthylamine (Pro-MNA) using methods known to the art
(E. Wunsch, Synthese von Peptiden, in Methoden der organischen Chemie,
Houben-Weyl (Eds. E. Muller, O. Bayer), Vol. XV, Part 1 and 2, Georg Thieme
Verlag, Stuttgart, 1974).
Synthesis of
Pro-Pro-4-methoxy-.beta.-naphthylamide:
Boc-Pro (32 mg, 0.15 mmol),
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluoronium tetrafluoroborate (53 mg,
0.15 mmol) and Pro-4-methoxy-.beta.-naphthylamide hydrochloride (46 mg, 0.15
mmol) were dissolved in anhydrous N,N-dimethylformamide/tetrahydrofuiran 1:1 (4
ml). N-ethyldiisopropylamine (0.26 ml, 0.44 mmol) dissolved in
N-methylpyrrolidone (1.7 molar) was added and the mixture was stirred at room
temperature overnight.
The solvent was removed with a roto-evaporator
and the residue was dissolved in ethyl acetate (10 ml) and extracted 3 times
with water. The organic layer was dried with anhydrous Na.sub.2 SO.sub.4 and the
solvent was removed with a roto-evaporator. The residue was dissolved in
trifluoroacetic acididichloromethane (1:4, 25 ml) and allowed to react for 1
hour. The solvent was removed with a roto-evaporator and the resulting oil was
dried with a stream of nitrogen. The crude product was purified by preparative
reversed phase HPLC applying a acetonitrile/water gradient. The product gave
satisfactory analytical data (NMR and mass spectra).
Release of free MNA
from the peptides was then measured in a Cytofluor fluorimeter (Per-Septive
Biosystems, Inc.) using the 355 nm excitation/405 nm emission filter set. Enzyme
kinetic parameters (Michaelis-Menten K.sub.m and k.sub.cat values) were
calculated using methods known to the art (see for example, Yun, S. L. &
Suelter, C. H. (1977). A simple method for calculating Km and V from a single
enzyme reaction progress curve. Biochim. Biophys. Acta 480(1), 1-13.) with
FAP.alpha. enzyme derived from membrane extracts of the 293-I/2 transfected
cells in Example 2.
TABLE 1
K.sub.m and k.sub.cat values for FAP.alpha.-catalyzed cleavage of selected
4-methoxy-.beta.-napthylamide (MNA)-peptide conjugates
K.sub.M K.sub.cat K.sub.cat /K.sub.M .times.
Substrates [.mu.M] [s.sup.-1 ] 10.sup.4 [M.sup.-1 s.sup.-1
]
Chg-Pro-MNA 75 53.7 71.6
Hyn-Pro-MNA 69 27.4 39.7
Pro-Pro-MNA 154 49.5 32.1
Val-Pro-MNA 95 27.7 29.2
Met-Pro-MNA 127 27.9 22.0
Arg-Pro-MNA 278 50.6 18.2
trans-Hyp-Pro-MNA 254 37.9 14.9
Gln-Pro-MNA 273 40.5 14.8
Ala-Pro-MNA 267 35.7 13.4
Lys-Pro-MNA 530 57.1 10.8
Ile-Pro-MNA 43 12.6 8.8
Met(O)-Pro-MNA 378 26.9 7.1
Ser-Pro-MNA 872 28.3 3.3
Chg = cyclohexylglycine,
Hyn = 6-hydroxynorleucine,
trans-Hyp = trans-4-hydroxyproline
Example 11
Specificity of MNA-coupled Peptides for
FAP.alpha. Versus DPP-IV
Among the-known prolyl-specific serine
oligopeptidase family members, the most closely related enzyme to FAP.alpha. is
DPP-IV. Since active DPP-IV is found in plasma and on many different cell types,
optimization of the relative (optional) selectivity of prodrug peptidics for
FAP.alpha. compared to DPP-IV is necessary to reduce undesirable conversion of
the prodrug at sites other than the tumor (e.g., in the plasma). To identify
peptides specific for FAP.alpha., cleavage of MNA-coupled peptides by FAP.alpha.
was compared to the ability of DPP-IV to cleave the same peptide conjugates.
Release of free MNA was measured as described in Example 9. Results are shown in
Table 2.
TABLE 2
Comparison of cleavage selectivity of MNA-peptide conjugates
by FAP.alpha. and DPP-IV.
Cleavage specificity
FAP DPP IV
Ala-Pro-MNA + +
Z-Gly-Pro-MNA + -
Z-Pro-Ala-Gly-Pro-MNA + -
+ indicates the enzyme was able to cleave the substrate
- indicates lack of cleavage
Example 12
FAP.alpha. Activity in Tumour Samples
Enzyme activity of FAP.alpha. measured in human tumor samples.
Ninety-six-well ELISA (enzyme-linked immunosorbent assay) plates (Costar,
Corning, N.Y.) were coated overnight at 4.degree. C. with 1 .mu.g/ml F 19
antibody or control antibody in phosphate-buffered saline (PBS), pH 7.4. Wells
were then rinsed with wash buffer (PBS, 0.1% Tween 20, pH 7.4) and excess
binding sites were blocked with blocking buffer (5% bovine serum albumin in PBS,
pH 7.4) for 1 hour at room temperature. FAP.alpha. activity was measured in
tumor tissue (closed symbols) or matched normal control tissue (corresponding
open symbols) from Concanavalin A-enriched membrane extracts (FIG. 5a). Tumor
samples included breast (.tangle-solidup.,.box-solid.), colon (.circle-solid.),
colon metastasized to the liver (.rect-solid.), and lung cancer
(.tangle-soliddn.,+). Extracts were added to F19-coated plates and incubated for
1 hour at room temperature. The unbound material was removed, wells were washed
thrice with wash buffer, and FAP.alpha. enzyme activity was assayed using 100
.mu.l Ala-Pro-AFC as described (WO 97/34927) for one hour at 37.degree. C. The
first two Concanavalin A fractions of each extract were measured and each value
individually plotted. Background fluorescence (as measured using control
antibody-coated plates) was subtracted from each value.
Independent
biochemical confirmation of FAP.alpha. enzymatic activity and its apparent
molecular mass in tumor extracts were obtained by labelling the aforementioned
tissue extracts with .sup.14 C-labelled diisopropylfluorophosphate (DFP;
NEN-DuPont, Cologne, Germany). DFP is known to bind covalently and irreversibly
to active site serines of many serine proteases, thereby preventing further
catalysis (Hayashi, R., Bai, Y. & Hata, T. (1975). Further confirmation of
carboxypeptidase Y as a metal-free enzyme having a reactive serine residue. J.
Biochem. 77(6), 1313-1318; Wahlby, S. & Engstrom, L. (1968). Studies on
Streptomyces griseus protease. II. The amino acid sequence around the reactive
serine residue of DFP-sensitive components with esterase activity. Biochim.
Biophys. Acta 151(2), 402-408). .sup.14 C-DFP labelling of FAP.alpha.
immunopurified from a tumor (T) corresponding to colon sample .circle-solid.
from FIG. 5a is shown in FIG. 5b. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (Laemmli; U. K. (1970). Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227(259), 680-685) and
subsequent autoradiography (Park, J. E., Draper, R. K. & Brown, W. J.
(1991). Biosynthesis of lysosomal enzymes in cells of the End3 complementation
group conditionally defective in endosomal acidification. Somatic Cell Mol.
Genet. 17(2), 137-150) of these samples revealed the presence of a labelled 95
kD protein, present in the colon cancer sample. No radiolabeled bands were
observed in the normal matched control immunoprecipitate (N) nor with control
antibody. The apparent molecular mass of the immunopurified, .sup.14 C-labelled
protein seen in FIG. 5b agrees with previous reports for FAP.alpha. (Rettig, W.
J., Su, S. L., Fortunato, S. R., Scanlan, M. J., Mohan Raj, B. K., Garin-Chesa,
P., Healey, J. H. & Old, L. J. (1994). Fibroblast activation protein:
Purification, epitope mapping and induction by growth factors. Int. J. Cancer
58(3), 385-392; WO 97/34927).
Example 13
Preparation of
Protected Oligopepdides
Protected oligopeptides were either prepared in
solution according to state-of-the-art protocols (e.g. M. Bodanszky and A.
Bodanszky, "The practice of Peptide Synthesis", 2.sup.nd edition, Springer, New
York, 1994) or by solid phase synthesis on an Applied Biosystems model 430A
automated peptide synthesizer. Deprotection and removal of the oligopeptide from
the resin support were achieved by treatment with mixtures of trifluoracetic
acid and frequently used scavenger additives. Purification was carried out by
preparative high pressure liquid chromatography on reverse phase C18 silica
columns using an aqueous 0.1% trifluoracetic acid/acetonitrile gradient.
Identity and homogeneity of peptides were confirmed by high pressure liquid
chromatography and mass spectral analysis. The oligopeptides that were prepared
by this method are shown in Table 3.
TABLE 3
Oligopeptides.
Z--Gly--Pro--OH
Z--Pro--Ala--Gly--Pro--OH
Fmoc--Pro--Ala--Gly--Pro--OH
Fmoc--Chg--Pro--OH
Fmoc--Nle--Pro--OH
Fmoc--Hyn--Pro--OH
Fmoc--Pro--Pro--OH
Fmoc--Ser--Ala--Hyn--Pro--OH
Fmoc--Ser--Ala--N1e--Pro--OH
Fmoc--Ser--Ala--Chg--Pro--OH
Gly--Ser--Ala--Glu--Pro--OH
Gly--Gly--Ser--Ala--Glu--Pro--OH
Gly--Gly--Gly--Ser--Ala--Glu--Pro--OH
Ser--Glu--Asn--Arg--Lys--Val--Pro--OH
Gly--Tyr--Ser--Arg--Met--Pro--OH
Gln--Gly--Tyr--Ser--Arg--Met--Pro--OH
Gly--Gly--Gly--Trp--Pro--OH
Asn--Arg--Lys--Val--Pro--OH
Glu--Asn--Arg--Lys--Val--Pro--OH
Ser--Glu--Asn--Arg--Lys--Val--Pro--OH
Ala--His--Met--His--Pro--OH
Tyr--Ala--Phe--His--Pro--OH
Ser--Tyr--Ala--Phe--His--Pro--OH
Leu--Asn--Leu--Tyr--Met--Pro--OH
Gly--Ser--Ala--Glu--Pro--OH
Gly--Gly--Ser--Ala--Glu--Pro--OH
Gly--Gly--Gly--Ser--Ala--Glu--Pro--OH
Z is benzyloxycarbonyl
Fmoc is 9-fluorenylmethoxycarbonyl
Chg is L-cyclohexylglycyl
Nle is L-norleucinyl
Hyn is L-6-hydroxynorleucinyl
Example 14
Preparation of Fmoc-Pro-Ala-Gly-Pro-Dox
Fmoc-Pro-Ala-Gly-Pro-OH (44 mg, 0.078 mmol) was dissolved in anhydrous
N,N-dimethylformamide (10 ml) and pH was adjusted to 7.5 by
N,N-diisopropylethylamine. N-hydroxysuccinimide (1 M in DMF, 78 .mu.l, 0.078
mmol) was added and the mixture was cooled in an ice bath. Under stirring,
dicyclohexylcarbodiimide (1 M in DMF, 87 .mu.l, 0.087 mmol) was added and the
solution was stirred at 0.degree. C. for 1 h.
Doxorubicin*HCl (25 mg,
0.043 mmol) was dissolved in 20 ml anhydrous DMF and N,N-diisopropylethylamine
(8.2 .mu.l, 0.048 mmol) was added. This mixture was syringed to the activated
peptide. The reaction was allowed to warm up to room temperature and was stirred
for 48 h.
The solvent was then removed and the product was purified by
preparative RP-HPLC on C18 using a gradient of water/acetonitrile with 0.1%
trifluoracetic acid.
Analytical HPLC >90%; ES-MS 1110.5 (M+Na.sup.+);
674.4
Example 15
Preparation of H-Pro-Ala-Gly-Pro-Dox
Fmoc-PAGP-Dox (prepared in Example 14, 44 mg, 0.040 mmol) was dissolved
in THF/diethylamine (2:1, 20 ml) at 0.degree. C. and stirred for 2 h. The
solvent was removed and the product was purified by preparative RP-HPLC on C18
using a gradient of water/acetonitrile with 0.1% trifluoracetic acid.
Analytical HPLC >90%; ES-MS 866.6 [M+H].sup.+, 452.4
Example
16
Preparation of H-Chg-Pro-Dox
Fmoc-Chg-Pro-OH (46 mg, 0.096
mmol) was dissolved in anhydrous N,N-dimethylformamide (10 ml) and pH was
adjusted to 7.5 by N,N-diisopropylethylamine. N-hydroxysuccinimide (1 M in DMF,
96 .mu.l, 0.096 mmol) was added and the mixture was cooled in an ice bath. Under
stirring, dicyclohexylcarbodiimide (1 M in DMF, 107 .mu.l, 0.107 mmol) was added
and the solution was stirred at 0.degree. C. for 1 h.
Doxorubicin*HCl
(31 mg, 0.053 mmol) was dissolved in 20 ml anhydrous DMF and
N,N-diisopropylethyl amine (10 .mu.l, 0.059 mmol) was added. This mixture was
syringed to the activated peptide. The reaction was allowed to warm up to room
temperature and was stirred for 48 h
The solvent was then removed and
the product was purified by preparative RP-HPLC on C18 using a gradient of
water/acetonitrile with 0.1% trifluoracetic acid.
The lyophilized
product was dissolved in THF/diethylamine (2:1, 20 ml) at 0.degree. C. and
stirred for 2 h. The solvent was removed and the product was purified by
preparative RP-HPLC on C18 using a gradient of water/acetonitrile with 0.1%
trifluoracetic acid.
Analytical HPLC >90%; ES-MS 780.2 (M+H+); 366.4
The following table shows the peptide-doxorubicin conjugates which have
been prepared analogously and includes cleavage data by FAP (after 20 h).
TABLE 4
##STR27##
Cleavage
R.sup.1 by FAP
Z-Gly- >95%
Z-Pro-Ala-Gly- >95%
Fmoc-Pro-Ala-Gly- >95%
H-Pro-Ala-Gly- ca. 11%
H-Chg- >95%
H-Nle- >95%
H-Hyn- >95%
Z is benzyloxycarbonyl
Fmoc is 9-fluorenylmethoxycarbonyl
Chg is L-cyclohexylglycyl
Nle is L-norleucinyl
Hyn is L-6-hydroxynorleucinyl
Example 17
Preparation of N-Cbz-Gly-Pro-Melphalan
##STR28##
N-Cbz-Gly-Pro-OH (22 mg, 0.072 mmol) was dissolved in
anhydrous N,N-dimethylformamide (8 ml) and pH was adjusted to 7.5 by
N,N-diisopropylethylamine. N-hydroxysuccinimide (1 M in DMF, 72 .mu.l, 0.072
mmol) was added and the mixture was cooled in an ice bath. Under stirring,
dicyclohexylcarbodiimide (1 M in DMF, 67 .mu.l, 0.67 mmol) was added and the
solution was stirred at 0.degree. C. for 2 h.
Melphalan (14.7 mg, 0.048
mmol) was dissolved in 30 ml anhydrous DMF and N,N-duisopropylethylamine (12.3
.mu.l, 0.072 mmol) was added. This mixture was syringed to the activated
peptide. The reaction was allowed to warm up to room temperature and was stirred
for 24 h.
The solvent was then removed and the product was purified by
preparative RP-HPLC on C18 using a gradient of water/acetonitrile with 0.1%
trifluoracetic acid.
Analytical data: HPLC >90%, ES-MS 593.0
([M+H].sup.+).
SEQUENCE LISTING
<100> GENERAL INFORMATION:
<160> NUMBER OF SEQ ID NOS: 28
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: FAP binding cytotoxic compound protein
conjugate
<400> SEQUENCE: 1
Pro Ala Gly Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 2
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: FAP binding cytotoxic compound protein
conjugate
<400> SEQUENCE: 2
Lys Ala Gly Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 3
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: FAP binding cytotoxic compound protein
conjugate
<400> SEQUENCE: 3
Pro Ala Gly Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 4
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor peptide to SEQ ID #1
<400> SEQUENCE: 4
Pro Ala Gly Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 5
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Artificial test peptide containing MNA to
test
for FAP activity
<400> SEQUENCE: 5
Pro Ala Gly Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 6
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Contains Fmoc
<400> SEQUENCE: 6
Pro Ala Gly Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 7
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Contains Fmoc
<400> SEQUENCE: 7
Ser Ala Xaa Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 8
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Contains Fmoc
<400> SEQUENCE: 8
Ser Ala Xaa Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 9
<211> LENGTH: 4
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Contains Fmoc
<400> SEQUENCE: 9
Ser Ala Xaa Pro
1
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 10
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 10
Gly Ser Ala Glu Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 11
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 11
Gly Gly Ser Ala Glu Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 12
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 12
Gly Gly Gly Ser Ala Glu Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 13
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 13
Ser Glu Asn Arg Lys Val Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 14
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 14
Gly Tyr Ser Arg Met Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 15
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 15
Gln Gly Tyr Ser Arg Met Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 16
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 16
Gly Gly Gly Trp Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 17
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 17
Asn Arg Lys Val Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 18
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 18
Glu Asn Arg Lys Val Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 19
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 19
Ser Glu Asn Arg Lys Val Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 20
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 20
Ala His Met His Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 21
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 21
Tyr Ala Phe His Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 22
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 22
Ser Tyr Ala Phe His Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 23
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 23
Leu Asn Leu Tyr Met Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 24
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 24
Gly Ser Ala Glu Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 25
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 25
Gly Gly Ser Ala Glu Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 26
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: Precursor
<400> SEQUENCE: 26
Gly Gly Gly Ser Ala Glu Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 27
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: SEQ ID 151 of WO 97/12624
<400> SEQUENCE: 27
Xaa Tyr Gln Ser Ser Pro
1 5
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 28
<211> LENGTH: 5
<212> TYPE: PRT
<213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: SEQ NO 177 of WO 97/14416
<400> SEQUENCE: 28
Xaa Tyr Gln Ser Pro
1 5
1-1087 US.ST25
Page 1
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