U.S. patent application number 10/475876 was filed with the patent office on 2005-10-13 for folate mimetics and folate-receptor binding conjugates thereof.
Invention is credited to Green, Mark A, Ke, Chun-Yen, Leamon, Christophe P.
Application Number | 20050227985 10/475876 |
Document ID | / |
Family ID | 23096986 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050227985 |
Kind Code |
A9 |
Green, Mark A ; et
al. |
October 13, 2005 |
Folate mimetics and folate-receptor binding conjugates thereof
Abstract
A cell population expressing folate receptors is selectively
targeted with a folate mimetic. The folate mimetic is conjugated to
a diagnostic or therapeutic agent to enable selective delivery of
the agent to the targeted cell population.
Inventors: |
Green, Mark A; (West
Lafayette, IN) ; Ke, Chun-Yen; (West Lafayette,
IN) ; Leamon, Christophe P; (West Lafayette,
IN) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55458
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0242582 A1 |
December 2, 2004 |
|
|
Family ID: |
23096986 |
Appl. No.: |
10/475876 |
Filed: |
June 21, 2004 |
PCT Filed: |
April 24, 2002 |
PCT NO: |
PCT/US02/13045 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60286082 |
Apr 24, 2001 |
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Current U.S.
Class: |
514/243 ;
514/248; 514/251; 514/262.1; 514/264.1; 544/183; 544/236; 544/256;
544/257; 544/280 |
Current CPC
Class: |
A61K 51/0497 20130101;
C07D 475/04 20130101; A61K 31/519 20130101 |
Class at
Publication: |
514/243 ;
514/248; 514/251; 514/264.1; 514/262.1; 544/183; 544/236; 544/256;
544/257; 544/280 |
International
Class: |
C07D 487/02; A61K
031/53; A61K 031/519; A61K 031/525 |
Goverment Interests
[0002] This invention was made with Government support under Grant
R01-CA70845 awarded by the National Institutes of Health--National
Cancer Institute. The Government has certain rights in the
invention.
Claims
1. A compound having the formula 8wherein X and Y are
each-independently selected from the group consisting of halo,
R.sup.2, OR.sup.2, SR.sup.3, and NR.sup.4R.sup.5; U, V, and W
represent divalent moieties each independently selected from the
group consisting of --(R.sup.6')C.dbd., --N.dbd.,
--(R.sup.6')C(R.sup.7')--, and --N(R.sup.4')--; Q is selected from
the group consisting of C and CH; T is selected from the group
consisting of S, O, N and --C.dbd.C-- such that the ring structure
of which T is a member is aromatic; A.sup.1 and A.sup.2 are each
independently selected from the group consisting of --C(Z)-,
--C(Z)O--, --OC(Z)-, --N(R.sup.4")--, --C(Z)-N(R.sup.4")--,
--N(R.sup.4")--C(Z)-, --O--C(Z)-N(R.sup.4")--,
--N(R.sup.4")--C(Z)-O--, --N(R.sup.4")--C(Z)-N(R- .sup.5")--,
--O--, --S--, --S(O)--, --S(O).sub.2--, --N(R.sup.4')S(O).sub.2--,
--C(R.sup.6")(R.sup.7")--, --N(C.ident.CH)--,
--N(CH.sub.2--C.ident.CH)--, C.sub.1-C.sub.12 alkyl and
C.sub.1-C.sub.12 alkoxy; where Z is oxygen or sulfur provided that
A.sup.2 does not represent --C(O)NH--; R.sup.1is selected-from the
group consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and
C.sub.1-C.sub.12 alkoxy; R.sup.2, R.sup.3, R.sup.4, R.sup.4',
R.sup.4", R.sup.5, R.sup.5", R.sup.6" and R.sup.7" are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12
alkanoyl, C.sub.1-C.sub.12 alkenyl, C.sub.1-C.sub.12 alkynyl,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl; R.sup.6 and R.sup.7 are each independently
selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; or, R.sup.6
and R.sup.7 are taken together to form O.dbd.; R.sup.6' and
R.sup.7' are each independently selected from the group consisting
of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12
alkoxy; or, R.sup.6' and R.sup.7' are taken together to form
O.dbd.; L is a divalent linker; n, p, r and s are each
independently either 0 or 1 provided that when n=1, then r=1; and B
is hydrogen or a leaving group; provided that the linker L does not
include a naturally occurring amino acid covalently linked to
A.sup.2 at its .alpha.-amino group through an amide bond.
2. The compound of claim 1 having binding affinity for a folate
receptor molecule.
3. The compound of claim 1 wherein X and Y are each independently
selected from the group consisting of hydrogen, halo, CH.sub.3, OH,
SH and NH.sub.2; U, V and W represent divalent moieties each
independently selected from the group consisting of --CH.dbd. and
--N.dbd.; Q represents C; A.sup.1 is selected from the group
consisting of --C(Z)-, --NH--, --N(CH.sub.3)--, --O--, --S--,
--S(O)--, --S(O).sub.2--, --CH.sub.2--, --CH(CH.sub.3)--,
--C(CH.sub.3).sub.2--, --N(CH.sub.2--C.ident.CH)-- and
--N(C.ident.CH)-- where Z is oxygen or sulfur; R.sup.1 is selected
from the group consisting of hydrogen, halo and methyl; R.sup.6 and
R.sup.7 are each independently selected from the group consisting-
of hydrogen, halo, CH.sub.3, OH, SH and NH.sub.2; or, R.sup.6 and
R.sup.7 are taken together to form O.dbd.; A.sup.2 is selected from
the group consisting of --C(Z)-, --C(Z)O--, --OC(Z)-,
--N(R.sup.4")--, --C(Z)-N(R.sup.4")--, --N(R.sup.4")--C(Z)-,
--O--C(Z)-N(R.sup.4")--, --N(R.sup.4")--C(Z)-O--,
--N(R.sup.4")--C(Z)-N(R- .sup.5")--, --O--, --S--, --S(O)--,
--S(O).sub.2--, --N(R.sup.4")S(O).sub.2--, --C(R.sup.6")(R.sup.7"),
C.sub.1-C.sub.6 alkyl; C.sub.1-C.sub.6 alkoxy; where Z is oxygen or
sulfur provided that A.sup.2 does not represent --C(O)NH--; and p
and r are each 1.
4. The compound of claim 3 wherein T is --C.dbd.C--.
5. The compound of claim 3 wherein X is OH.
6. The compound of claim 3 having binding affinity for a folate
receptor molecule.
7. The compound of claim 1 wherein X is OH; Y is NH.sub.2; U and W
are each --N.dbd.; V is --CH.dbd.; Q is C; T is --C.dbd.C--;
A.sup.1 is --NH--; R.sup.1 is hydrogen; A.sup.2 is --C(O)-- or
--C(O)O-- and is para to A.sup.1; R.sup.6 and R.sup.7 are each H;
and p, r and s are each 1.
8. The compound of claim 7 having binding affinity for a folate
receptor molecule.
9. A compound that is isosteric with the compound of claim 7 and
that has binding affinity for a folate receptor molecule.
10. A ligand-agent conjugate having the formula 9wherein X, Y, U,
V, W, Q, T, A.sup.1, A.sup.2, R.sup.1, R.sup.6, R.sup.7, L, n, p, r
and s are as defined in claim 1; q is an integer .gtoreq.1; and, D
is a diagnostic agent or a therapeutic agent.
11. The ligand-agent conjugate of claim 10 that has binding
affinity for a folate receptor molecule.
12. The ligand-agent conjugate of claim 10 comprising a
metabolically labile linker L.
13. The ligand-agent conjugate of claim 12 wherein the
metabolically labile linker L is hydrolytically or reductively
cleaved in the cell to release the diagnostic or therapeutic agent
Z.
14. The ligand-agent conjugate of claim 12 wherein the
metabolically labile linker L comprises a disulfide or an
ester.
15. A pharmaceutical composition comprising the ligand agent
conjugate of claim 10 and at least one component selected from the
group consisting of a pharmaceutically acceptable carrier,
excipient, or diluent.
16. A method for delivering a diagnostic agent or a therapeutic
agent to a target cell population comprising a folate receptor, the
method comprising: providing a ligand-agent conjugate having the
formula 10wherein X, Y, U, V, W, Q, T, A.sup.1, A.sup.2, R.sup.1,
R.sup.6, R.sup.7, L, n, p, r and s are as defined in claim 1, q is
an integer .gtoreq.1; and D is a diagnostic agent or a therapeutic
agent; and contacting the target cell population with an effective
amount of the ligand-agent conjugate to permit binding of the
ligand-conjugate to the folate receptor.
17. The method of claim 16 wherein D is a diagnostic agent
comprising a contrast agent for use in medical imaging.
18. The method of claim 16 wherein the ligand-agent conjugate binds
to the cell surface and is not internalized by the cells of the
cell population.
19. The method of claim 16 wherein the diagnostic or therapeutic
agent D is internalized by the cells of the cell population.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/286,082, filed Apr. 24, 2001, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to folate mimetics and their use in
therapeutic and diagnostic applications. More particularly, this
invention relates to using des-glutamyl folic acid analogs
recognized by and selectively bound by folate receptors and other
folate binding proteins and the use of such analogs for targeted
delivery of diagnostic or therapeutic agents to folate-receptor
bearing cell populations.
BACKGROUND OF THE INVENTION
[0004] A number of methods are known for selectively targeting
cells in a patient for delivery of diagnostic or therapeutic
agents. Selective targeting has led to the introduction of various
diagnostic agents for visualization of tissues, such as contrast
agents useful in Magnetic Resonance Imaging (MRI), radiodiagnostic
compositions, and the like. Introduction of therapeutic agents,
such as compositions for radiotherapy or for neutron capture
therapy, compositions for chemotherapy, various proteins, peptides,
and nucleic acids, protein toxins, antisense oligonucleotides,
liposomes, analgesics, antibiotics, antihypertensive agents,
antiviral agents, antihistamines, expectorants, vitamins, plasmids,
and the like, has also been demonstrated.
[0005] Folate conjugates have been used for the selective targeting
of cell populations expressing folate receptors or other folate
binding proteins to label or deliver bioactive compounds to such
cells. The relative populations of these receptors and binding
proteins have been exploited in achieving selectivity in the
targeting of certain cells and tissues, such as the selective
targeting of tumors expressing elevated levels of high-affinity
folate receptors. The following publications, the disclosures of
which are incorporated herein by reference, illustrate the nature
and use of folate conjugates for diagnosis or delivery of
biologically significant compounds to selected cell populations in
patients in need of such diagnosis or treatment:
[0006] (a) Leamon and Low, "Cytotoxicity of Momordin-folate
Conjugates in Cultured Human Cells" in J. Biol. Chem., 1992, 267,
24966-24967.
[0007] (b) Leamon et al., "Cytotoxicity of Folate-pseudomonas
Exotoxin Conjugates Towards Tumor Cells" in J. Biol. Chem., 1993,
268, 24847-24854.
[0008] (c) Lee and Low, "Delivery of Liposomes into Cultured Kb
Cells via Folate Receptor-mediated Endocytosis" in J. Biol. Chem.,
1994, 269, 3198-3204.
[0009] (d) Wang et al., "Delivery of Antisense Oligonucleotides
Against the Human Epidermal Growth Factor Receptor into Cultured Kb
Cells with Liposomes Conjugated to Folate via Polyethyleneglycol"
in Proc. Natl. Acad. Sci. USA., 1995, 92, 3318-3322.
[0010] (e) Wang et al., "Synthesis, Purification and Tumor Cell
Uptake of Ga-67-deferoxamine-folate, a Potential
Radiopharmaceutical for Tumor Imaging" in Bioconj. Chem., 1996, 7,
56-63.
[0011] (f) Leamon et al., "Delivery of Macromolecules into Living
Cells: a Method That Exploits Folate Receptor Endocytosis" in Proc.
Natl. Acad. Sci., U.S.A., 1991, 88, 5572-5576.
[0012] (g) Krantz et al., "Conjugates of Folate Anti-Effector Cell
Antibodies" in U.S. Pat. No. 5,547,668.
[0013] (h) Wedeking el al., "Metal Complexes Derivatized with
Folate for Use in Diagnostic and Therapeutic Applications" in U.S.
Pat. No. 6,093,382.
[0014] (i) Low et al., "Method for Enhancing Transmembrane
Transport of Exogenous Molecules" in U.S. Pat. No. 5,416,016.
[0015] (j) Miotti et al., "Characterization of Human Ovarian
Carcinoma-Associated Antigens Defined by Novel Monoclonal
Antibodies with Tumor-Restricted Specificity"in Int. J. Cancer,
1987, 39,297-303.
[0016] (k) Campell et al., "Folate-Binding Protein is a Marker for
Ovarian Cancer"in Cancer Res., 1991, 51, 5329-5338.
[0017] (l) Jansen et al., "Identification of a Membrane-Associated
Folate-Binding Protein in Human Leukemic CCRF-CEM Cells with
Transport-Related Methotrexate Resistance"in Cancer Res., 1989, 49,
2455-2459.
[0018] Multiple types of folate recognition sites present on cells,
such as .alpha.-folate receptors, .beta.-folate receptors, folate
binding proteins, and the like, have been shown to recognize and
bind the conjugates described above. The primary pathway for entry
of folate derivatives into cells is through a facilitated transport
mechanism mediated by a membrane transport protein. However, when
folate is covalently conjugated to certain small molecules and
macromolecules, the transport system can fail to recognize the
folate molecule.
[0019] Advantageously, in addition to the facilitated transport
protein, some cells possess a second membrane-bound receptor,
folate binding protein (FBP), that allows folate uptake via
receptor-mediated endocytosis. At physiological plasma
concentrations (nanomolar range), folic acid binds to cell surface
receptors and is internalized via an endocytic process.
Receptor-mediated endocytosis is the movement of extracellular
ligands bound to cell surface receptors into the interior of the
cells through invagination of the membrane, a process that is
initiated by the binding of a ligand to its specific receptor. The
uptake of substances by receptor-mediated endocytosis is a
characteristic ability of some normal, healthy cells such as
macrophages, hepatocytes, fibroblasts, reticulocytes, and the like,
as well as abnormal or pathogenic cells, such as tumor cells.
Notably, folate binding proteins involved in endocytosis are less
sensitive to modification of the folate molecule than the membrane
transport proteins, and often recognize folate conjugates. Both
targeting and uptake of conjugated diagnostic and therapeutic
agents are enhanced.
[0020] Following endosome acidification, the folate receptor
changes conformation near its ligand-binding domain and releases
the folic acid molecule. Folate receptors are known to recycle back
to the membrane surface for additional rounds of ligand-mediated
internalization. However, a significant fraction of the
internalized receptor-folic acid complex has been shown to return
back to the cell surface shortly after endocytosis. This suggests
that the acid-triggered ligand release mechanism does not proceed
to completion, at least after the first round of internalization
(Kamen et al., 1988, J. Biol. Chem. 263, 13602-13609).
[0021] Pteroic acid, which is essentially folic acid lacking the
distal glutamyl residue (FIG. 1), does not bind to the
high-affinity folate receptor to any appreciable extent (Kamen et
al., 1986, Proc. Natl. Acad. Sci., USA. 83, 5983-5987); in fact, 2
.mu.M pteroic acid (100-fold excess) had absolutely no effect on
the binding of folate to the folate receptor. Thus, the glutamyl
residue of folic acid, or some portion thereof, was generally
thought to be required for efficient, specific receptor
recognition. However, recent studies have revealed that the
glutamyl residue of folic acid could be replaced with a lysyl
residue without disturbing the binding affinity of the ligand
(McAlinden et al., 1991, Biochemistry 30, 5674-5681.; Wu et al.,
1997, J. Membrane Biol. 159, 137-147), that the glutamyl residue
can be replaced with a glycyl residue without substantially
altering cellular uptake, and that no selective isomeric (i.e.,
.alpha.-glutamyl vs. .gamma.-glutamyl) conjugation requirement
necessarily exists (Leamon et al., J. Drug Targeting 7:157-169
(1999); Linder et al., J. Nuclear Med. 41(5):470 Suppl. 2000).
[0022] Efforts to improve the selectivity of targeting or increase
the diversity of the agents delivered to the cell or tissue have
been hampered by a number of complications, including the complex
syntheses required for the preparation of these conjugates. Such
synthetic schemes are not only time consuming, but may also
preclude the use of certain conjugates due to synthetic
incompatibilities. A folic acid analog capable of expanding the
number or diversity of agents, via the conjugates of such agents
and these folic acid analogs, presentable to target cells would be
advantageous.
SUMMARY OF THE INVENTION
[0023] The present invention provides a compound that is capable of
binding as a ligand to a folate recognition site. The compound is
referred to as a "non-peptide folic acid analog."
[0024] The present invention also provides a ligand-agent conjugate
capable of binding to a folate recognition site, the ligand-agent
conjugate comprising a diagnostic or therapeutic agent in
association with a non-peptide folic acid analog.
[0025] The present invention also provides a ligand-agent conjugate
capable of binding to a folate recognition site with high affinity,
the ligand-agent conjugate comprising a diagnostic or therapeutic
agent in association with a plurality of non-peptide folic acid
analogs.
[0026] The present invention also provides a method for targeting a
cell or tissue with a diagnostic or therapeutic agent, comprising
the step of administering to a patient an effective amount of a
ligand-agent conjugate comprising a diagnostic or therapeutic agent
in association with a non-peptide folic acid analog.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a schematic representation of folic acid and
pteroic acid.
[0028] FIG. 2 is a schematic representation of the synthesis of a
pteroic acid conjugate, CYK4-013.
[0029] FIG. 3 is a schematic representation of the synthesis of
pteroic acid conjugate linked to the tetraazamacrocyclic DOTA
chelating ligand (CY4-036).
[0030] FIG. 4 is a schematic representation of the metabolism of a
ligand-agent conjugate of the invention involving bioreduction to
release the agent.
[0031] FIG. 5 is a schematic representation of the metabolism of a
ligand-agent conjugate of the invention involving acid hydrolysis
to release the agent.
[0032] FIG. 6 depicts the binding activity of
(S)-.alpha.-carboxybenzoyl pteroate (ACBP) and
N-pteroyl-2-amino-2-carboxymethylpyridine (Pte-AP).
[0033] FIG. 7 is a schematic representation of the synthesis of
pteroylhydrazido-benzenetetracarboxylic acid-diacetoxyscirpenol
(Pte-hydrazideo-BTCA-DAS).
[0034] FIG. 8 depicts the binding activity of pteroyl hydrazide
(Pte-hydrazide) and Pte-hydrazido-BTCA-DAS.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0035] The present invention provides a ligand capable of binding
to a folate recognition site, comprising a non-peptide folic acid
analog of general formula I: 1
[0036] wherein
[0037] X and Y are each independently selected from the group
consisting of halo, R.sup.2, OR.sup.2, SR.sup.3, and
NR.sup.4R.sup.5;
[0038] U, V, and W represent divalent moieties each independently
selected from the group consisting of --(R.sup.6')C.dbd., --N.dbd.,
--(R.sup.6')C(R.sup.7')--, and --N(R.sup.4')--;
[0039] T is selected from the group consisting of S, O, N and
--C.dbd.C-- such that the ring structure of which T is a member is
aromatic;
[0040] A.sup.1 and A.sup.2 are each independently selected from the
group consisting of --C(Z)-, --C(Z)O--, --OC(Z)-, --N(R.sup.4")--,
--C(Z)-N(R.sup.4")--, --N(R.sup.4")--C(Z)-,
--O--C(Z)-N(R.sup.4")--, --N(R.sup.4")--C(Z)-O--,
--N(R.sup.4")--C(Z)-N(R.sup.5")--, --O--, --S--, --S(O)--,
--S(O).sub.2--, --N(R.sup.4")S(O).sub.2--,
--C(R.sup.6")(R.sup.7")--, --N(C.ident.CH)--,
--N(CH.sub.2--C.ident.CH)--- , C.sub.1-C.sub.12 alkyl and
C.sub.1-C.sub.12 alkoxy; where Z is oxygen or sulfur;
[0041] R.sup.1 is selected from the group consisting of hydrogen,
halo, C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy;
[0042] R.sup.2, R.sup.3, R.sup.4, R.sup.4', R.sup.4", R.sup.5,
R.sup.5", R.sup.6" and R.sup.7" are each independently selected
from the group consisting of hydrogen, halo, C.sub.1-C.sub.12
alkyl, C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12 alkanoyl,
C.sub.1-C.sub.12 alkenyl, C.sub.1-C.sub.12 alkynyl,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl;
[0043] R.sup.6 and R.sup.7 are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and
C.sub.1-C.sub.12 alkoxy; or, R.sup.6 and R.sup.7 are taken together
to form O.dbd.;
[0044] R.sup.6' and R.sup.7' are each independently selected from
the group consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and
C.sub.1-C.sub.12 alkoxy; or, R.sup.6' and R.sup.7' are taken
together to form O.dbd.;
[0045] L is a divalent linker;
[0046] n, p, r and s are each independently either 0 or 1; and
[0047] B is hydrogen or a leaving group;
[0048] provided that the linker L does not include a naturally
occurring amino acid covalently linked to A.sup.2 at its
.alpha.-amino group through an amide bond. It should be understood
that the structure of formula I includes tautomeric structures, for
example in compounds where X is OH, SH or NH.
[0049] In the compound of the invention wherein any one or more of
A.sup.1, A.sup.2, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.4',
R.sup.4", R.sup.5, R.sup.5", R.sup.6", R.sup.7", R.sup.6, R.sup.7,
R.sup.6' and R.sup.7' comprises an alkyl, alkoxy, alkylamino,
alkanoyl, alkenyl, alkynyl, alkoxy carbonyl, or alkylamino carbonyl
group, the group preferably contains 1 to 6 carbon atoms
(C.sub.1-C.sub.6);
[0050] more preferably it contains 1 to 4 carbon atoms
(C.sub.1-C.sub.4).
[0051] Folic acid contains a glutamyl residue bound at its
.alpha.-amino group via an amide bond to the benzoate moiety of
pteroic acid (FIG. 1). This amide bond would typically not be
classified as a "peptide" bond because pteroic acid is not an amino
acid; a peptide bond is typically characterized as a bond in which
the carboxyl group of one amino acid is condensed with the amino
group of another to form a --CO.NH-- linkage.
[0052] Nonetheless, for ease of reference, the compound of formula
I, which is defined as having linker L that lacks a glutamyl or any
other naturally occurring amino acid residue covalently linked to
A.sup.2 at its .alpha.-amino group through an amide bond, is termed
herein a "non-peptide" folic acid analog. That is, the term
"non-peptide" as used herein in reference to the compound of
formula I, means that linker L does not include a naturally
occurring amino acid covalently linked to A.sup.2 through an amide
bond at its .alpha.-amino group, thereby distinguishing the
compound of formula I from, for example, folic acid,
pteroyl-.gamma.-glutamate-cysteine,
pteroyl-.alpha.-glutamate-cysteine, and pteroyl-glycine-cysteine
(Leamon et al., J. Drug Targeting 7:157-169 (1999)). In a preferred
embodiment of the non-peptide folic acid analog of the invention,
linker L does not include any amino acid (whether naturally
occurring or non-naturally occuring) covalently linked to A.sup.2
through an amide bond at its .alpha.-amino group.
[0053] It should be further understood that the compound of formula
I can contain a naturally occurring amino acid covalently linked to
A.sup.2 at a site other than its .alpha.-amino group, a
non-naturally occurring amino acid covalently linked to A.sup.2
through an amide bond or otherwise, as well as any other non-amino
acid moiety covalently linked to A.sup.2 through an amide bond or
otherwise, as defined with reference to the formula.
[0054] The general chemical terms used in the formulae above have
their usual meanings. For example, the term "alkyl" as used herein
refers to a linear or branched chain of carbon atoms, such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl,
octyl and the like.
[0055] The term "alkoxy" as used herein refers to alkyl, as defined
above, substituted with oxygen, such as methoxy, ethoxy, propoxy,
isopropoxy, butoxy, tert-butoxy and the like.
[0056] The term "alkanoyl" as used herein refers to formyl, or
alkyl, as defined above, terminally-substituted with a carbonyl
such as acetyl, propanoyl, butanoyl, pentanoyl and the like.
[0057] The term "alkenyl" as used herein refers to a linear or
branched chain of carbon atoms with one or more carbon-carbon
double bonds, such as vinyl.
[0058] The term "alkynyl" as used herein refers to a linear or
branched chain of carbon atoms with one or more carbon-carbon
triple bonds.
[0059] The term "alkylamino" as used herein refers to alkyl, as
defined above, substituted with nitrogen, including both
monoalkylamino such as methylamino, ethylamino, propylamino,
tert-butylamino, and the like, and dialkylamino such as
dimethylamino, diethylamino, methylpropylamino, and the like.
[0060] The term "halo" as used herein refers to any Group 17
element and includes fluoro, chloro, bromo, iodo, and
astatine(o).
[0061] The term "alkylenyl" as used herein refers to a divalent
linear or branched chain of carbon atoms such as methylene,
ethylene, 2-methylpropylene, and the like.
[0062] The term "leaving group" as used herein refers to a
functionality that may be replaced, such as an activated halo or
alkoxy, by an introduced substituent, such as a alkylamino, carbon
nucleophile, a different alkoxy, a different halo, and the
like.
[0063] The term "naturally occurring amino acid" as used herein
refers to the 20 coded amino acids available for endogenous protein
synthesis, such as glycine, alanine, methionine, and the like.
[0064] A preferred embodiment of the ligand is one having the
general formula I wherein p is 1, s is 1, and T, U, V, W, X, Y,
R.sup.1, R.sup.6, R.sup.7 and A.sup.1 are selected such that at
least a portion of the molecule is isosteric with pteroic acid. By
"isosteric" it is meant that the two compounds or portions of
compounds comprise isosteric substituents that occupy similar
volumes and, preferably but not necessarily, have similar
electronic character. As a nonlimiting example, hydrogen, halo,
CH.sub.3, OH, SH and NH.sub.2 may be considered for purposes of
this invention as being isosteric substituents.
[0065] Folate receptor activity is expected to be retained when
isosteric substitutions are made to that portion of the non-peptide
folic acid analog that is derived from pteroic acid. For example,
as reported in Jansen, "Receptor- and Carrier-Mediated Transport
Systems for Folates and Antifolates," in Anticancer Drug
Development Guide: Anlifolate Drugs in Cancer Therapy, Jackmian,
Ed., Humana Press Inc, Totowa N.J. (1999), ring substituents X and
Y, ring components U, V, W, T, and A.sup.1 can be substituted in
the pteroic acid reference structure while in most cases retaining
folate receptor affinity.
[0066] An example of a preferred ligand according to the invention
having a portion that is isosteric with pteroic acid is a ligand
having formula I (including tautomers thereof) wherein
[0067] X and Y are each independently selected from the group
consisting of hydrogen, halo, CH.sub.3, OH, SH and NH.sub.2, with X
more preferably being OH;
[0068] U, V and W represent divalent moieties each independently
selected from the group consisting of --CH.dbd. and --N.dbd.;
[0069] A.sup.1 is selected from the group consisting of --C(Z)-,
--NH--, --N(CH.sub.3)--, --O--, --S--, --S(O)--, --S(O).sub.2--,
--CH.sub.2--, --CH(CH.sub.3)--, --C(CH.sub.3).sub.2--,
--N(CH.sub.2--C.ident.CH)-- and --N(C.ident.CH)--; where Z is
oxygen or sulfur;
[0070] R.sup.1 is selected from the group consisting of hydrogen,
halo and methyl;
[0071] R.sup.6 and R.sup.7 are each independently selected from the
group consisting of hydrogen, halo, CH.sub.3, OH, SH and NH.sub.2;
or, R.sup.6 and R.sup.7 are taken together to form O.dbd.;
[0072] A.sup.2 is selected from the group consisting of --C(Z)-,
--C(Z)O--, --OC(Z)-, --N(R.sup.4')--, --C(Z)-N(R.sup.4")--,
--N(R.sup.4")--C(Z)-, --O--C(Z)-N(R.sup.4")--,
--N(R.sup.4")--C(Z)-O--, --N(R.sup.4")--C(Z)-N(R.sup.5")--, --O--,
--S--, --S(O)--, --S(O).sub.2--, --N(R.sup.4")S(O).sub.2--,
--C(R.sup.6")(R.sup.7")--, C.sub.1-C.sub.6 alkyl; C.sub.1-C.sub.6
alkoxy; where Z is oxygen or sulfur;
[0073] p and rare each 1; and
[0074] T, R.sup.4", R.sup.5", R.sup.6", R.sup.7", L, n, s and B are
as defined above; provided that the linker L does not include a
naturally occurring amino acid covalently linked to A.sup.2 at its
.alpha.-amino group through an amide bond. More preferably, T is
--C.dbd.C--.
[0075] A particularly preferred ligand of the invention is a
derivative of pteroic acid and has formula I (including tautomers
thereof) wherein X is OH; Y is NH.sub.2; U and W are each --N.dbd.;
V is --CH.dbd.; T is --C.dbd.C--; A.sup.1 is --NH--; R.sup.1 is
hydrogen; A.sup.2 is --C(O)--, --C(O)O--, or --C(O)NH-- and is para
to A.sup.1; R.sup.6 and R.sup.7 are hydrogen; p, r and s are each
1; and L, n and B are as defined elsewhere herein; provided that
the linker L does not include a naturally occurring amino acid
covalently linked to A.sup.2 at its .alpha.-amino group through an
amide bond.
[0076] One embodiment of the invention is a ligand capable of
binding to a folate recognition site, such as a folate binding
protein, folate receptor, and the like. Such a non-peptide folate
analog may also be described as a folate mimetic. Compounds
illustrative of this embodiment are selected from the general
formula I. Such analogs may operate as surrogates for folate in
methods utilizing folate, such as targeting molecules for cells or
tissues expressing folate recognition sites.
[0077] In a particularly preferred embodiment, the compound of the
invention has formula I and further exhibits binding affinity for a
folate receptor. A relative binding affinity assay is described in
detail in Example IV and in Westerhoffet al. (Mol. Pharm., 1995,
48:459-471). Performing this assay is straightforward. A preferred
compound exhibits a binding affinity for the folate receptor
relative to folic acid of at least about 0.01, more preferably at
least about 0.05, even more preferably at least about 0.10, even
more preferably at least about 0.25, even more preferably at least
about 0.50, and most preferably at least about 0.75, wherein the
binding affinity of folic acid for the folate receptor is defined
as 1.0. It should be understood that the binding affinity of the
compound of the invention may exceed 1.0, in cases where the
binding affinity of the compound for the folate receptor is greater
than that of folic acid itself.
[0078] The compounds of formula I may optionally include a linker,
spacer, or couple of variable length. The linker, spacer, or
couple, hereinafter collectively referred to as a "linker," is
adapted for connecting the folate analog to another molecule in
other embodiments of the invention. A divalent linker L is present
in the folate analog of formula I when the integer n is equal to 1.
Such linkers are known in the art and are often used to "associate"
one chemical entity to another. As used herein, the term
"association" refers to any manner of coexistence of two or more
molecules, such as complexation, chelation, ion-pairing, covalant
bonding, and the like, such that for a time sufficient to
administer the associated molecules, the associated molecules may
be interpreted as a single entity.
[0079] The linker may create either a permanent or a semipermanent
(i.e., labile) linkage. The inclusion of a semipermanent linkage is
especially advantageous for applications in which cellular uptake
of the drug is desired. The ability to form a bioactive conjugate
utilizing a linkage other than a peptide linkage (e.g., the
glutamyl linkage of typical folate conjugates) provides an
important degree of chemical flexibility for the linkage of the
pteroic acid moiety to the drug payload. The capacity of a target
cell for uptake of a folate-drug conjugate is expected to be
dramatically increased when a linkage is selected that promotes
drug release from the conjugate by exploiting known endosomal
hydrolytic or reductive mechanisms (i.e., molecular separation
between the drug payload and the ligand).
[0080] A preferred embodiment of the ligand-agent conjugate of the
invention therefore includes a linker whereby a cell targeting
ligand (i.e., the non-peptide folic acid analog) is chemically
coupled to a drug molecule via a linker that is designed to be
metabolized within the endosomal milieu. Following extracellular
receptor binding and endocytic entry of the drug conjugate,
endosome factors are expected to hydrolytically or reductively
cleave the linker moiety of the conjugate, thereby facilitating
release of the drug from the ligand. This process is depicted
below: 2
[0081] wherein the abbreviations are as follows: FRBL, folate
receptor binding ligand; X, endosome-cleavable linker; D, drug
moiety; X', linker fragment.
[0082] A preferred semi-permanent linker thus includes a
functionality, such as a disulfide, ester, other hydrolyzable
group, and the like, that allows separation of the ligand and the
agent once the conjugate has reached the treatment site.
[0083] Semi-permanent linkers preferably depend upon endogenous
mechanisms of cleavage, and include metabolically labile linkers,
such as a nucleotide, amide, ester, and the like subject to
cleavage by peptidases, esterases, phosphodiesterases, reductases,
and the like, which provides a stable ligand-agent conjugate prior
to delivery but allows cleavage upon reaching the target or
treatment site. Preferred linkers used to produce these drug
conjugates are biologically labile (pH sensitive, redox sensitive,
enzymatically sensitive) such that the ligand-receptor complex can
be separated from the macromolecule "payload" in a predetermined
manner (e.g., following endocytosis). The inclusion of a
metabolically labile function is advantageously chosen in an
end-use dependent manner such that following the binding of the
conjugate, or additionally subsequent uptake of the conjugate as
described below, the metabolically labile association may be
cleaved thus releasing the agent from the ligand, either locally
(extracellularly) in the case of binding of the conjugate to the
cell surface, or intracellularly, as in the case of post-uptake by
the cell.
[0084] The divalent linker L comprises a linear or branched chain
comprising a plurality of linking groups L.sub.1, L.sub.2, . . . ,
L.sub.m, wherein "m" is an integer from 0 to about 50. Preferably,
m is selected such the number of atoms in the linear backbone of
linker L is at least about 1, more preferably at least about 3,
most preferably at least about 6; and at most about 100, more
preferably at most about 50, and most preferably at most about
20.
[0085] Each linking group "L.sub.m" is also a divalent moiety
composed of atoms selected from the group consisting of carbon,
nitrogen, oxygen, and sulfur, providing that an oxygen atom is not
adjacent to another oxygen or sulfur atom, except when the sulfur
atom is oxidized, as in --S(O).sub.2--. Each individual linking
unit "L.sub.m" can be the same or different and is thus
independently selected from the group of divalent radicals.
Illustrative divalent radicals are --CR.sup.6"R.sup.7"--,
--(R.sup.6")C.dbd.C(R.sup.7")--, --CC--, --C(O)--, --O--, --S--,
--SO.sub.2--, --N(R.sup.3")--, --(R.sup.6")C.dbd.N--, --C(S)--,
--P(O)(OR.sup.3")--, --P(O)(OR.sup.3")O--, and the like.
[0086] R.sup.3" is a group suitable for nitrogen or oxygen
attachment, such as hydrogen, C.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, C.sub.3-C.sub.8 cycloalkyl, aryl,
C.sub.1-C.sub.4 alkanoyl, aryloyl, and the like. R.sup.3" attached
to nitrogen may also be hydroxy, C.sub.1-C.sub.4 alkoxy, amino,
monoalkylamino, or dialkylamino. It is appreciated that R.sup.3"
may be selected independently for each linking group L.sub.m.
[0087] R.sup.6" and R.sup.7" are each independently selected from
groups suitable for carbon attachment such as hydrogen,
C.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl, hydroxy, halo,
C.sub.1-C.sub.4 alkoxy, C.sub.3-C.sub.8 cycloalkyl, aryl,
C.sub.1-C.sub.4 alkanoyl, aryloyl, and the like. In addition,
R.sup.6" and R.sup.7" are selected independently for each linking
group L.sub.m.
[0088] The linker L may also possess one or more cyclic regions,
wherein a subset of the linking groups "L.sub.m" form one or more
rings, including, but not limited to divalent cycloalkyl, such as
cyclopent-1,3-diyl, cyclohex-1,1-diyl, and the like; divalent
heterocyclyl, such as pyrrolidin-1,3-diyl, piperidin-2,2-diyl; and
divalent aromatic groups, such as 1,3-phenylene, pyrrol-1,2-diyl,
and the like.
[0089] Illustrative linkers L are polyalkylenes, polyalkylene
glycols such as polyethylene glycol (PEG),
N-(2-hydroxypropyl)methacrylamide (HPMA), and the like. Other
examples of such linkers may be found in U.S. Pat. Nos. 6,207,157,
6,184,042, 6,177,404, 6,171,859, and 6,171,614, the disclosures of
which are incorporated herein by reference. The invention is not
intended to be limited by the nature or length of the linker L.
[0090] The term "alkenyl" as used herein refers to a linear or
branched chain of carbon atoms, such as ethenyl, propenyl,
2-methylethenyl, and the like.
[0091] The term "cycloalkyl" as used herein refers to a cyclic
chain of carbon atoms, such as cyclopropyl, cyclopentyl,
cyclohexyl, and the like.
[0092] The term "aryl" as used herein refers to an aromatic moiety,
such as phenyl, pyridinyl, pyrimidinyl, and the like. The aryl
group is optionally substituted with from 1 to 3 substituents, such
as with halo, alkyl, alkoxy, as defined above, and the like.
[0093] The term "aryloyl" as used herein refers to aryl, as defined
above, substituted with a carbonyl group, such as benzoyl,
picolinyl, and the like.
[0094] The term "polyalkylene" as used herein refers to polymers of
alkenes, such as polyethylene, polypropylene, and the like.
[0095] Synthesis of non-peptide folic acid analogs may be
accomplished by methods known to the skilled artisan. In addition,
the optional incorporation of a linker may also be accomplished by
methods known to the skilled artisan.
[0096] The present invention also provides a ligand-agent conjugate
capable of binding to a folate recognition site, comprising a
diagnostic or therapeutic agent in association with a non-peptide
folic acid analog of general formula II: 3
[0097] where X, Y, U, V, W, T, A.sup.1, A.sup.2, R.sup.1, R.sup.6,
R.sup.7, L, n, p, r and s are as defined above;
[0098] q is an integer .gtoreq.1; and,
[0099] D is a diagnostic agent or a therapeutic agent.
[0100] One embodiment of the invention is a ligand-agent conjugate
capable of binding to a folate recognition site, such as a folate
binding protein, folate receptor, and the like. Compounds
illustrative of this embodiment are selected from the general
formula II, where the integer q is equal to 1. Such analogs may
operate as a means for targeting of and delivery to cells or
tissues expressing folate recognition sites. The compounds of
formula II may optionally include a linker L, where L is as defined
above and where the integer n is equal to 1.
[0101] Another embodiment of the present invention is a
ligand-agent conjugate capable of binding to a folate recognition
site with high affinity, comprising a diagnostic or therapeutic
agent in association with a plurality of non-peptide folic acid
analogs of general formula II, where the integer q is 2 or greater.
Similarly, such ligand-agent conjugates may optionally comprise a
plurality of ligands each possessing a linker L, where L is as
defined above and where the integer n is equal to 1. Such
conjugates possessing a plurality of folate analogs in association
with the diagnostic or therapeutic agent may advantageously enhance
recognition of the conjugate by the recognition site.
[0102] The diagnostic or therapeutic agent D can be linked to the
ligand at (L).sub.n by any type of molecular interaction including
a covalent bond, and ionic bond or association, hydrogen bonding or
other type of complexation to form the ligand-agent conjugate.
[0103] Synthesis of ligand-agent conjugates may be accomplished by
methods known to the skilled artisan depending upon the nature of
the association of the ligand and the agent.
[0104] Virtually any type of molecule (small molecular weight
chemotherapeutic, peptide, protein, oligosaccharide, antisense
oligonucleotide, plasmid, ribozyme, artificial chromosome, micelle,
liposome, etc.) can be more efficiently delivered into cells using
this technology.
[0105] Diagnostic agents useful in the present invention include
compounds capable of labeling a cell or tissue with a contrast
agent for the generation or modulation of signal intensity in
biomedical imaging. Such contrast agents may be used for imaging
such cells and tissues using techniques such as Magnetic Resonance
Imaging (MRI), radio-imaging, radio-diagnosis, and the like. Such
labeling of the cell or tissue is illustratively accomplished by
incorporation of superparamagnetic, paramagnetic, ferrimagnetic, or
ferromagnetic metals, radioactive gamma-emitting, positron-emitting
or photon-emitting metals, radionuclides, other radioactive
elements such as certain halogen isotopes (radiohalogens), and the
like, in the agent. The diagnostic agent may be a chelating agent
capable of binding such metals described above, or a
radio-pharmaceutical possessing an organic fragment, such as an
aromatic ring, possessing a radiohalogen. Such chelating agents are
known to the skilled artisan.
[0106] Metals useful in the invention employing such chelating
agents for MRI include certain ions of chromium, manganese, iron,
cobalt, nickel, copper, praseodymium, neodymium, samarium,
gadolinium, terbium, dysprosium, holmium, erbium, ytterbium, and
the like, such as Cr(III), Mn(II), Fe(II), Fe(III), and Ni(II).
Metals useful in the invention employing such chelating agents for
radio-imaging include certain isotopes of gallium, indium, copper,
technetium, rhenium, and the like, such as .sup.99mTc, .sup.51Cr,
.sup.67Ga, .sup.68Ga, .sup.103Ru, .sup.211Bi, .sup.64Cu and
.sup.111In. Radiobalogens useful in the invention employing
radio-pharmaceuticals include certain isotopes of fluorine, iodine,
astatine, and the like, such as .sup.18F, .sup.123I, and
.sup.131I.
[0107] Visualization techniques suitable for radioimaging are known
in the art, such as positron emission tomography (PET), planar or
SPECT imaging, gamma cameras, scintillation, and the like.
[0108] Therapeutic agents useful in the present invention include
compounds capable of modifying, modulating, or otherwise altering
certain cellular or tissue functions. Therapies include elimination
of certain pathogenic cell populations or pathogenic tissues,
enhancing beneficial functions in host cells or host tissues,
protecting host cells or host tissues from non-selective treatment,
and the like.
[0109] One embodiment of the invention is a ligand-therapeutic
agent conjugate wherein the therapeutic agent targets pathogenic
cells or tissues, such as tumors, bacteria, and the like. Such
therapeutic agents include chemotherapeutic agents, antimicrobial
agents, or other cytotoxic agents associated with the targeting
ligand. Such cytotoxic agents, may lead to the destruction of the
pathogenic cell or tissue. The therapeutic agent may be a
radiotherapeutic agent. These agents, like the related diagnostic
agents above, may possess a chelating functionality capable of
sequestering a radionuclide, such as a radioactive metal or a
radioactive alpha or beta-emitting metal suitable for nuclear
medicine, or alternatively a suitable functionality bearing a
radiohalogen, such as an aryl group. In this context however, the
metal or halogen is used for radiotherapy rather than for
radiodiagnosis. Metals appropriate for such radiotherapeutic agents
are known in the art, including certain isotopes of gadolinium,
technetium, chromium, gallium, indium, ytterbium, lanthanum,
yttrium, samarium, holmium, dysprosium, copper, ruthenium, rhenium,
lead, bismuth, and the like, such as .sup.157Gd, .sup.64Cu,
.sup.67Cu, .sup.186Re, .sup.188Re, .sup.90Y, .sup.111In, and
.sup.177Lu. Radiohalogens are also useful in the invention for
radiotherapeutic agents, including certain isotopes of iodine,
astatine, and the like, such as .sup.125I, .sup.131I, and
.sup.211At. In another embodiment, the therapeutic may be a species
suitable for neutron capture therapy, such as an organoborane
moiety, comprising .sup.10B.
[0110] Chemotherapeutic agents useful in the present invention
include certain alkylating agents, such as busulfan, carboquone,
chlomaphazine, lomustine, tubercidin, and the like, certain
antimetabolites, such as fludarabine, doxifluridine, and the like,
certain steroids and steroid analogs, such as calusterone,
testolactone, flutamide, tamoxifen, hexestrol, melengestrol, and
the like, certain antiadrenals, such as mitolane, and the like,
certain LH--RH analogs, such as buserelin, leuprolide, and the
like, and certain anti-angiogenic agents.
[0111] Another embodiment of the invention is therapeutic
ligand-agent conjugate that targets cells or tissue, such that a
beneficial function of the targeted cell or tissue is enhanced by
the therapeutic agent, such as an inflammatory, pro-inflammatory,
or anti-inflammatory agent, antibiotic, analgesic, antiviral agent,
and the like. Still other therapeutic agents useful in the present
invention may protect a targeted cell or tissue from a subsequent
non-selective treatment targeted to a different pathogenic cell or
tissue, such as an immunosuppressant.
[0112] The present invention also provides a method for delivering
a diagnostic or therapeutic agent to a targeted cell population. An
effective amount of a ligand-agent conjugate comprising a
diagnostic or therapeutic agent in association with a non-peptide
folic acid analog of general formula II, where the integer q is 1
or greater, is delivered to the targeted cell population. The
targeted cells possess a folate receptor to which the ligand-agent
conjugate binds. The ligand thus selectively targets a certain cell
or tissue, by binding to the receptors or proteins present in such
cells or tissues that recognize the folic acid and folic acid
analogs. If desired, a plurality of non-peptide folate analog
conjugates can be administered.
[0113] In one embodiment of the method of the invention, the
diagnostic or therapeutic effect is achieved as a direct or
indirect result of binding of the ligand-agent conjugate to the
folate receptor on the cell surface (i.e., "docking"). For example,
in vivo biomedical imaging can be facilitated whether or not the
diagnostic agent is internalized, and in some instances, for
example in the case of a cytotoxic diagnostic agent, it is
preferable that the diagnostic agent remain outside the cell. As
another example, the therapeutic agent can include an immune
stimulating factor such as an antigen, which is preferably retained
on the extracellular surface on the cell.
[0114] In another embodiment of the method of the invention, the
diagnostic or therapeutic effect is achieved as a result of uptake
or internalization of the therapeutic or diagnostic agent via
binding to the folate receptor followed by internalization of the
receptor-ligand complex. It is appreciated that the method of the
invention is suitable for effecting uptake by cells or tissue of
ligand-agent conjugates, where the agent is a molecule or compound
that would otherwise exhibit poor uptake by the cell or tissue by
active transport, diffusion, or other passive transport.
[0115] The targeted cell population can be endogenous to exogenous
to the patient. For example, it can be an endogenous population
comprising a somatic or tumor cell population in a patient, a
cancerous cell population, an organ, tissue or bodily fluid, or a
virus-infected cell population. The ligand-agent conjugate can be
delivered to a patient locally or systemically. For example, the
conjugate can be delivered parenterally by intramuscular,
intraveneous or subcutaneous injection; likewise it can be
formulated for oral administration.
[0116] An exogenous population of cells can include an ex vivo or
in vitro population of cells. For example, the target cell
population can be an ex vivo population of cells such as bone
marrow cells, stem cells, or cells of an organ or tissue that have
been removed from the patient's body. The ex vivo cells are
contacted with the ligand-agent conjugate of the invention and
subsequently returned to the body of the patient. Gene therapy, for
example, can be accomplished using a ligand-agent conjugate of the
invention wherein the therapeutic agent is a nucleic acid.
[0117] Likewise the target population can be an in vitro population
of cells such as a tissue or fluid sample or biopsy that has been
removed from a patient for diagnostic purposes. The biological
sample can be contacted with the ligand-agent conjugate of the
invention comprising a diagnostic agent for detection or
characterization of the disease state of the patient.
[0118] An exogenous population of cells can also include a
population of exogenous organisms such as bacteria, mycoplasma,
yeast, or fungi, provided the organisms possess a receptor molecule
that binds the ligand-agent conjugate. See, e.g., Kumaret al.,
1987, J. Biol. Chem. 262(15):7171-9. The ligand-agent conjugate
binds to the surface of the tumor cells or pathogenic organisms and
"labels" or otherwise alters or modifies cell or tissue function;
it may or may not be internalized, depending on the intended
application.
EXAMPLES
[0119] The following examples are illustrative of certain
embodiments of the invention. The examples, methods, and conditions
presented therein are not to be construed as limiting the scope nor
the spirit of the invention.
Example I
Targeting the Tumor-Associated Folate Receptor with a
.sup.111In-DTPA Conjugate of Pteroic Acid
[0120] Objective. The present study was undertaken to evaluate the
structural requirements for folate-receptor-targeting with
low-molecular-weight radiometal chelates, specifically examining
the role of the amino acid fragment of folic acid (pteroyl-glutamic
acid) in mediating folate-receptor affinity.
[0121] Methods. The amide-linked conjugate
pteroyl-NHCH.sub.2CH.sub.2OCH.s-
ub.2CH.sub.2OCH.sub.2CH.sub.2NH-DTPA (CYK4-013), which lacks an
amino acid in the linker region, was prepared by a three-step
procedure from pteroic acid, 2,2'-(ethylenedioxy)-bis(ethylamine),
and t-Bu-protected DTPA. 4
[0122] This conjugate (CYK4-013) was prepared as outlined in FIG.
2. CY3-064 was obtained from pteroic acid (0.025 g; 0.080 mmol) and
a large excess of 2,2'-(ethylenedioxy)-bis(ethylamine) (0.237 g;
1.6 mmol) using the coupling reagents.
Benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniu- m
hexafluorophosphate (PyBOP) (0.125 g; 0.24 mmol),
N-hydroxybenzotriazole (HOBt) (0.037 g; 0.24 mmol), and
N-methylmorpholine (Nmm)(0.049 g; 0.48 mmol) in dry
dimethylsulfoxide (DMSO) (0.8 mL) at room temperature for 22 hours
under nitrogen. The excess reagent and solvent DMSO were removed
under vacuum and the resulting brown residue triturated with
diethylether, methanol, and water to produce 20 mg of CY3-064 as a
yellow solid (57% estimated yield).
[0123] This yellow solid was then coupled with t-butyl-protected
DTPA (synthesized as described by S. A. Chilefu, et al., J. Org.
Chem., 2000,65:1562-1565) using the same coupling reagents (0.012 g
CY3-064; 0.071 g t-Bu-DTPA, 0.127 mmol; 0.0848 g PyBOP, 0.16 mmol;
0.0249 g HOBt, 0.16 mmol; 0.0247 g Nmm, 0.24 mmol; 0.6 mL dry
DMSO). During coupling, the solubility of CY3-064 was very poor in
the DMSO solvent, and not improved by addition of more Nmm. 1, 3,
4, 6, 7, 8-hexahydro-1-methyl-2H-- pyrimido[1,2-a]-pyrimidine
(MTBD) (0.0083 g; 0.0542 mmol) was added after stirring overnight
at room temperature, but the solubility remained poor. After again
stirring overnight, the DMSO and excess reagents were removed under
high vacuum overnight.
[0124] The resulting brown residue was triturated first with
diethylether and then with methanol. The methanol suspension was
centrifuged to produce crude CY3-078 as a yellow solid. This yellow
solid was purified using semi-preparative HPLC (2.times.) on a C18
column (10.times.250 mm) to produce pure CY3-078. (HPLC solvent
A=5% CH.sub.3CN in 0.1% aqueous TFA; solvent B=10% water in 0.1%
TFA in CH.sub.3CN. Linear gradients established as: 5% B at
time=zero ramping to 70% B at 30 minutes, then ramping to 100% B at
32 minutes, and remaining 100% B to 40 minutes. Flow rate=2.35
mL/min). The peak with retention time of 30.5 minutes was
collected. The purified CY3-078 was treated with 70%
TFA/CH.sub.2Cl.sub.2 at 0.degree. C. for 30 minutes, and stirred at
room temperature for 5 hours, to remove the three t-Bu protecting
groups. The resulting CYK4-013 product (6 mg) was isolated by
trituration with diethylether. Both CY3-078 and CYK4-013 exhibit
the expected parent ion peaks in their positive and negative ion
electrospray mass spectra. In particular, purified CYK4-013
exhibited the expected parent ion peaks in its positive and
negative ion electrospray mass spectra (m/e=818 and 816,
respectively).
[0125] The .sup.111In complex of CYK4-013 was prepared from
.sup.111In-chloride (1.2 mCi; 44 MBq) and purified by
reversed-phase HPLC. Specifically, the .sup.111In complex of
CYK4-013 was prepared from .sup.111In-chloride via ligand exchange
in acetate buffer. Briefly, 1.2 mCi no-carrier-added
.sup.111In-chloride (Mallinckrodt, Inc., St. Louis) in 0.05 mL 0.05
N HCl was transferred to a small tube and 0.05 mL 0.1N ammonium
acetate (pH 5.5), followed by 0.02 mL 0.5N ammonium acetate (pH
7.4) was added, producing a solution with pH 7. The CYK4-013 ligand
was weighed out and diluted in water, pH adjusted with 1N NaOH to
pH 9-10. 10 .mu.L of this ligand solution containing 95 .mu.g
CYK4-013 was added to the .sup.111In-acetate solution (120 .mu.L)
and mixed. The solution was protected from light and kept at room
temperature.
[0126] The radiochemical purity of the resulting crude
.sup.111In-CYK4-013 was evaluated by radio-HPLC using a
4.6.times.250 mm Dynamax C18 column (Varian/Rainin) eluted with an
aqueous NH.sub.4O Ac:acetonitrile gradient. HPLC conditions:
Solvent A=56 mM NH.sub.4OAc in water; Solvent B=CH.sub.3CN. Flow
rate=1 mL/min on 4.6.times.250 mm C18 reverse-phase column. Linear
gradient conditions: 5% B at zero minutes, ramping to 25% B at 25
minutes, then ramping to 60% B at 27 minutes and 100% B at 30
minutes).
[0127] The major radioactive HPLC peak, eluting with a retention
time of 14.1 minutes, was collected. HPLC analysis of this isolated
peak showed it to remain stable for at least 7 days at room
temperature. HPLC conditions: Solvent A=56 mM NH.sub.4OAc in water;
Solvent B=CH.sub.3CN. Flow rate=1 mL/min on 4.6.times.250 mm C18
reverse-phase column. Linear gradient conditions: 5% B at zero
minutes, ramping to 25% B at 25 minutes, then ramping to 60% B at
27 minutes and 100% B at 30 minutes.
[0128] Radio-TLC of the HPLC-purified .sup.111In-CYK4-013 was
performed using a C18 plate eluted with 25% NH.sub.4OAc; 75%
acetonitrile. The results confirm the absence of .sup.111In-species
that irreversibly adsorb to C18 (i.e., there is no .sup.111In
remaining at origin)
[0129] The HPLC-purified .sup.111In-CYK4-013 was removed from the
HPLC solvents by solid-phase extraction. A C18 Sep-Pak Light solid
phase extraction cartridge (Millipore, Inc.) was conditioned by
washing with ethanol followed by water. The .sup.111In-CYK4-013 was
loaded onto the C18 Sep-Pak after dilution with water to 5%
acetonitrile, the Sep-Pak was washed with 3 mL water, and the
.sup.111In-CYK4-013 product was recovered by fractional elution
with ethanol. The resulting ethanol solution of .sup.111In-CYK4-013
was evaporated to dryness under a stream of N.sub.2 at room
temperature, and the .sup.111In-CYK4-013 was reconstituted in
saline for use in a biodistribution study in mice.
[0130] Our primary animal model for evaluation of the
biodistribution and pharmacokinetics of folate-receptor-targeted
radiopharmaceuticals has been athymic mice bearing subcutaneously
implanted folate-receptor-positive human KB cell tumors. Because
normal rodent chow contains a high concentration of folic acid (6
mg/kg chow), the mice used in these receptor targeting studies were
maintained on folate-free diet for 3 weeks to achieve serum folate
concentrations close to the 4-6 .mu.g/L (9-14 nM) range of normal
human serum. After 3 weeks on folate-free diet, mouse serum folate
levels drop to 25.+-.7 nM from the initial 720.+-.260 nM serum
folate level when the animals are fed normal rodent chow. This
dietary intervention is believed to be a reasonable manipulation of
the animal model, since the mice would have serum folate levels
only slightly higher than the folate concentration of normal human
serum. Thus, in these mouse biodistribution studies the radiotracer
is competing for tumor folate receptors with physiologically
relevant concentrations of endogenous unlabeled serum folate.
[0131] Thus, to demonstrate the ability of such conjugates to
selectively localize in folate-receptor-positive tissues, the
biodistribution of .sup.111In-CYK4-013 was determined following
intravenous administration to athymic mice with subcutaneous
folate-receptor-positive human KB cell tumor xenografts. The
resulting data are presented in Tables 1 and 2. The
.sup.111In-CYK4-013 agent is found to selectively localize in the
folate-receptor-positive tumors (5.4.+-.0.8 and 5.5.+-.1.1 percent
of the injected .sup.111In dose per gram of tumor at 1 hour and 4
hours post-injection, respectively) and to exhibit prolonged tumor
retention of the radiolabel (3.6.+-.0.6 percent of the injected
.sup.111In dose per gram of tumor still remaining at 24 hours
post-injection). The tumor localization of the
.sup.111In-radiolabel clearly appears to be mediated by the
cellular folate receptor, since the tumor uptake of radiotracer
drops precipitously (0.12.+-.0.07 percent of the injected
.sup.111In dose per gram at 4 hours) when .sup.111In-CYK4-013 is
co-injected with an excess of folic acid, which will compete for
folate receptor sites. Urinary excretion appears to be the primary
whole-body clearance pathway for the .sup.111In-CYK4-013. The
substantial retention of .sup.111In in the kidneys is fully
consistent with the binding of .sup.111In-CYK4-013 to tissue folate
receptors, since the renal proximal tubule is a known normal tissue
site of folate receptor expression. This interpretation is
supported by the expected and observed marked reduction in renal
.sup.111In when .sup.111In-CYK4-013 is co-administered with excess
folic acid. The behavior of the .sup.111In-CYK4-013
radiopharmaceutical in this animal model (Table 2) is very similar
to that observed for .sup.111In-DTPA-Folate (Table 3).
[0132] Results. Biodistribution of .sup.111In-CYK4-013 is shown in
Tables 1-3. Similar to .sup.111In-DTPA-Folate, .sup.111In-CYK4-013
selectively localized in the folate-receptor-positive tumor
xenografts, and afforded prolonged tumor retention of .sup.111In
(5.4.+-.0.8; 5.5.+-.1.1; and 3.6.+-.0.6 %ID/g at 1 hour, 4 hours,
and 24 hours, respectively) (Table 2). The tumor localization of
the .sup.111In-radiolabel appears to be mediated by the cellular
folate receptor, since the tumor uptake dropped precipitously
(0.12.+-.0.07 %ID/g at 4 hours) when .sup.111In-CYK4-013 was
co-injected with an excess of folic acid (Table 2). Blockable
binding was also observed in the kidneys, where the folate receptor
occurs in the proximal tubules.
1TABLE 1 Biodistribution of .sup.111In-CYK4-013 in KB Tumor-Bearing
Athymic Mice at Various Times Following Intravenous Administration
Percentage of Injected .sup.111In Dose Per Organ (Tissue) 1 Hour 4
Hours 4 Hours - Blocked** 24 Hours Tumormass (g): 0.15 .+-. 0.10
0.080 .+-. 0.031 0.077 .+-. 0.010 0.104 .+-. 0.097 Animalmass (g):
29.5 .+-. 1.2 28.6 .+-. 1.6 28.2 .+-. 1.1 28.6 .+-. 0.8 Animal
Quantity & Gender: 3M 4M 4M 4M Blood: 0.29 .+-. 0.003 0.078
.+-. 0.010 0.025 .+-. 0.015 0.055 .+-. 0.002 Heart: 0.51 .+-. 0.09
0.41 .+-. 0.08 0.0023 .+-. 0.0016 0.18 .+-. 0.06 Lungs: 0.69 .+-.
0.06 0.60 .+-. 0.01 0.0082 .+-. 0.0051 0.34 .+-. 0.03 Liver &
Gall Bladder: 6.8 .+-. 1.3 3.0 .+-. 1.0 0.074 .+-. 0.044 1.7 .+-.
0.9 Spleen: 0.063 .+-. 0.016 0.049 .+-. 0.012 0.0049 .+-. 0.0029
0.055 .+-. 0.017 Kidney (one): 15.3 .+-. 1.2 20.2 .+-. 1.6 0.23
.+-. 0.15 26.8 .+-. 2.7 Stomach, Intestines & Contents: 5.8
.+-. 0.5 5.4 .+-. 0.8 4.6 .+-. 2.8 3.3 .+-. 0.6 Muscle: 43.6 .+-.
4.1 33 .+-. 9 1.0 .+-. 0.7 23.1 .+-. 7.0 Tumor: 0.78 .+-. 0.47 0.46
.+-. 0.26 0.0094 .+-. 0.0061 0.42 .+-. 0.4 *Athymic mice (NuNu
strain) with subcutaneous tumors. Born Sep. 11, 2000. Arrived Oct.
10, 2000. Initiated folate-free diet Oct. 10, 2000. Implanted on
Oct. 20, 2000; 0.25 .times. 10.sup.6 KB cells (passage 9) per
animal subcutaneous in intrascapular region. Study date: Nov. 2,
2000. Values shown represent the mean .+-. standard deviation.
Blood was assumed to account for 5.5% of total body mass. Muscle
was assumed to account for 42% of the total body mass. **Folate
receptors blocked by co-injection of folic acid dihydrate at a dose
of 4.1 .+-. 0.4 mg/kg.
[0133]
2TABLE 2 Biodistribution of .sup.111In-CYK4-013 in KB Tumor-Bearing
Athymic Mice at Various Times Following Intravenous Administration
Percentage of Injected .sup.111In Dose Per Gram (Tissue Wet Mass) 4
Hours - 1 Hour 4 Hours Blocked** 24 Hours Tumormass 0.15 .+-. 0.10
0.080 .+-. 0.031 0.077 .+-. 0.010 0.104 .+-. 0.097 (g): Animalmass
29.5 .+-. 1.2 28.6 .+-. 1.6 28.2 .+-. 1.1 28.6 .+-. 0.8 (g): Animal
3M 4M 4M 4M Quantity & Gender: Blood: 0.18 .+-. 0.01 0.050 .+-.
0.009 0.016 .+-. 0.010 0.035 .+-. 0.002 Heart: 3.5 .+-. 0.4 2.9
.+-. 0.9 0.015 .+-. 0.010 1.2 .+-. 0.5 Lungs: 1.5 .+-. 0.2 1.4 .+-.
0.3 0.041 .+-. 0.027 0.72 .+-. 0.21 Liver & Gall 4.3 .+-. 0.7
1.9 .+-. 0.6 0.051 .+-. 0.029 1.2 .+-. 0.6 Bladder: Spleen: 0.31
.+-. 0.09 0.29 .+-. 0.07 0.028 .+-. 0.017 0.27 .+-. 0.08 Kidney
(one): 61 .+-. 5 81 .+-. 7 0.90 .+-. 0.59 105 .+-. 7 Stomach, 1.7
.+-. 0.2 1.5 .+-. 0.9 1.7 .+-. 1.1 1.2 .+-. 0.2 Intestines &
Contents: Muscle: 3.5 .+-. 0.5 2.8 .+-. 0.8 0.080 .+-. 0.057 1.9
.+-. 0.6 Tumor: 5.4 .+-. 0.8 5.6 .+-. 1.1 0.12 .+-. 0.07 3.6 .+-.
0.6 Tumor/blood 30 .+-. 5 111 .+-. 11 7.5 .+-. 2.4 105 .+-. 20
Tumor/kidney 0.088 .+-. 0.013 0.069 .+-. 0.013 0.12 .+-. 0.03 0.035
.+-. 0.008 Tumor/liver 1.3 .+-. 0.4 3.0 .+-. 0.5 2.2 .+-. 0.6 3.7
.+-. 1.4 Tumor/muscle 1.6 .+-. 0.4 2.1 .+-. 0.2 1.5 .+-. 0.7 2.0
.+-. 0.6 *Athymic mice (NuNu strain) with subcutaneous tumors.
Values shown represent the mean .+-. standard deviation. **Folate
receptors blocked by co-injection of folic acid dihydrate at a dose
of 4.1 .+-. 0.4 mg/kg.
[0134]
3TABLE 3 Biodistribution of .sup.111In-DTPA-Folate in Athymic Mice
with Subcutaneous KB Cell Tumor Xenografts Percentage of Injected
.sup.111In Dose Per Gram (mean .+-. s.d.; n = 4) 4 Hours Post 1
Hour 4 Hours Injection Post Injection Post Injection BLOCKED
Tumormass (g): 0.138 .+-. 0.051 0.202 .+-. 0.083 0.193 .+-. 0.078
Animalmass (g): 24 .+-. 2 25 .+-. 1 24 .+-. 1 Folic Acid Dose 0 0
495 .+-. 79 (.mu.g/kg): Blood: 0.14 .+-. 0.03 0.064 .+-. 0.007
0.029 .+-. 0.011 Heart: 2.3 .+-. 0.4 2.0 .+-. 0.3 0.022 .+-. 0.010
Lungs: 1.3 .+-. 0.1 1.1 .+-. 0.3 0.065 .+-. 0.021 Liver & Gall
4.0 .+-. 1.5 2.2 .+-. 0.4 0.12 .+-. 0.03 Bladder: Spleen: 0.36 .+-.
0.03 0.35 .+-. 0.11 0.060 .+-. 0.021 Kidney: 90 .+-. 9 85 .+-. 12
2.3 .+-. 1.0* Stomach, 1.0 .+-. 0.2 1.0 .+-. 0.2 0.49 .+-. 0.20
Intestines & Contents: Muscle: 3.5 .+-. 0.8 2.9 .+-. 0.7 0.023
.+-. 0.013 Tumor: 5.3 .+-. 0.4 6.8 .+-. 1.2 0.16 .+-. 0.07
Tumor/blood 38 .+-. 7 106 .+-. 15 5.5 .+-. 0.8 Tumor/kidney 0.060
.+-. 0.011 0.080 .+-. 0.012 0.050 .+-. 0.023 Tumor/liver 1.5 .+-.
0.5 3.3 .+-. 1.1 1.4 .+-. 0.4 Tumor/muscle 1.6 .+-. 0.3 2.5 .+-.
0.9 8.4 .+-. 3.8 *n = 3 (While 4 animals were studied, one gave an
unusually high value for the kidney uptake with no apparent
underlying cause for the disparity with the other animals in this
group. If that anomalous value is included, this result becomes 5.0
.+-. 5.5% ID/g, n = 4).
[0135] Conclusion. Tumor-selective drug targeting via the folate
receptor remains feasible with pteroic acid conjugates lacking
amino acid fragments, such as the glutamic acid moiety of folic
acid.
Example II
Synthesis of a DOTA Conjugate of Pteroic Acid
[0136] A pteroic acid conjugate linked to the tetraazamacrocyclic
DOTA chelating ligand was prepared for radiolabeling with
radiometals such as .sup.64Cu.sup.2+ and .sup.111In.sup.3+. This
conjugate (CY4-036) was prepared as shown in FIG. 3. The starting
material for the synthesis was pteroic acid. Due to the poor
solubility of pteroic acid in organic solvent, pteroic acid was
protected with 2-(trimethylsilyl)ethanol to produce intermediate
CY4-033 using literatural procedure (M. Nomura, et al., J. Org.
Chem., 2000, 65, 5016-5021) to increase its solubility in organic
solvent before coupling to the DOTA derivative.
[0137] DOTA was coupled to 2,2'-(ethylenedioxy)bis(ethyleneamine)
using PyBOP, HOBt, and NMM as coupling reagents in DMF to produce
CY4-032. Protected form of pteroyl-linker-DOTA (CY4-034) was
obtained through the coupling of CY4-032 and CY4-033 in DMSO using
MTBD as a base, and then purified via flash chromatography eluted
with gradient MeOH/CHCl.sub.3.
[0138] All the protecting groups (three t-butyl groups on
carboxylic acids and one 2-(trimethylsilyl)ethyloxycarbonyl on
nitrogen) were removed by the treatment with 70% TFA/CH2C12 to
produce CY4-036.
[0139] Synthesis of CY4-032. To a solution of DOTA-tri-t-butyl
ester (0.050 g, 0.087 mmol), PyBOP (0.136 g, 0.261 mmol), and HOBt
(0.052 g, 0.339 mmol) in DMF (0.9 mL) was added NMM (0.035 g, 0.348
mmol) under N.sub.2. The clear solution was stirred at room
temperature (i.e., about 25.degree. C.) for 10 minutes followed by
the addition of 2,2'-(ethylenedioxy)bis(ethyleneamine) (0.065 g,
0.436 mmol) under N.sub.2. After stirring at room temperature for
17 h, the solution was concentrated under high vacuum to remove DMF
and excess reagents. The oily residue was triturated with Et.sub.2O
(3.times.5 mL). After the removal of Et.sub.2O, EtOAc (3 mL) was
added followed by 2 mL of water. After stirring for 2 minutes, the
EtOAc was separated from aqueous layer and then more EtOAc (3 mL)
was added for the extraction of product. The extraction was
repeated one more time. All three EtOAc layers were combined,
concentrated, and then dried under high vacuum to produce 0.153 g
of oily crude product. C.sub.34H.sub.66N.sub.6O.sub.9=702;
Electrospray (+): M+H 703. This crude material was used for the
next coupling step without further purification.
[0140] Synthesis of CY4-033. To a suspension of carbonyl
diimidazole (CDI) (0.069 g, 0.425 mmol) and pteroic acid (0.028 g,
0.090 mmol) in DMSO (0.8 mL) was added triethylamine (0.032 g, 0.32
mmol) under N.sub.2. After stirring at room termperature for 3.5
hours, 2-(trimethylsilyl)ethanol (0.076 g, 0.64 mmol) was added and
stirred at room temperature for 5.5 hours. The reaction mixture was
concentrated under high vacuum overnight to remove DMSO and excess
reagents. Yellow residue was produced and triturated with
Et.sub.2O. A yellow solid (0.067 g) was produced as crude product.
This crude material was used for next reaction without further
purification.
[0141] Synthesis of CY4-034. To a solution of CY4-032 (0.153 g) and
CY4-033 (0.067 g) in DMSO (0.8 mL) was added MTBD (0.041 g, 0.27
mmol) under N.sub.2. After stirring at room temperature for 21
hour, the reaction mixture was dried under high vacuum to remove
DMSO and excess reagents to produce 0.157 g of yellow residue. This
crude product was purified via flash chromatography eluted with
gradient MeOH/CHCl.sub.3 to produce 0.073 g of pure CY4-034.
[0142] Synthesis of CY4-036. 70%TFA/CH.sub.2Cl.sub.2 (1 mL) was
added to the purified CY4-034 (0.018 g) at room temperature. After
stirring at room temperature for 4.5 hour, the reaction mixture was
concentrated under reduced pressure and dried under high vacuum
overnight to produce 19 mg of crude product. All three t-Butyl
groups and the 2-(trimethylsilyl)ethyloxycarbonyl protecting group
were removed at this step.
[0143] .sup.64Cu-complex of CY4-036. The .sup.64Cu complex of
CY4-036 was prepared from .sup.64Cu-chloride via ligand exchange in
acetate buffer. Briefly, 2.88 mCi of no-carrier-added
.sup.64Cu-chloride (Washington University, St. Louis, Mo.) in 0.005
mL of 0.01 N HCl was transferred to a small test tube and mixed
with 0.010 mL of 0.5 M ammonium acetate (pH 7.4). The CY4-036
ligand was weighed out and diluted in water and the pH adjusted
with 1 N NaOH to pH 11-12. One .mu.L of this ligand solution
containing .about.125 .mu.g CY4-036 was added to the
.sup.64Cu-acetate solution and mixed. The pH of the solution was
adjusted to pH 8-9 with the addition of 1 .mu.L of 1 N NaOH. The
solution was protected from light and incubated at 65.degree. C.
for 30 minutes.
[0144] The resulting crude .sup.64Cu-CY4-036 was diluted with water
and injected onto radio-HPLC using a 10.times.250 mm Dynamax C18
column (Varian/Rainin) eluted with an aqueous
NH.sub.4OAc:acetonitrile gradient. HPLC conditions: Solvent A=56 mM
NH.sub.4OAc in water; Solvent B=CH.sub.3CN. Flow rate=2.35 mL/min
on 10.times.250 mm C18 reverse-phase column. Linear gradient
conditions: 5% B at zero minutes, ramping to 25% B at 25 minutes,
then ramping to 60% B at 27 minutes and 100% B at 30 minutes. The
major radioactive HPLC peak, eluting with a retention time of 20.9
minutes, was collected. Radio-TLC confirmed the absence of
.sup.64Cu(II)-acetate, which was independently shown to remain at
the origin.
[0145] HPLC-purified .sup.64Cu-CY4-036 was removed from the HPLC
solvents by solid-phase extraction. A C18 Sep-Pak Light solid phase
extraction cartridge (Millipore, Inc.) was conditioned by washing
with ethanol followed by water. The HPLC-purified .sup.64Cu-CY4-036
was loaded onto the C18 Sep-Pak after dilution with water to 5%
acetonitrile, the Sep-Pak was washed with 20 mL water, and the
.sup.64Cu-CY4-036 product recovered by fractional elution with
ethanol. The resulting ethanol solution of .sup.64Cu-CY4-036 was
evaporated to dryness under a stream of N.sub.2 at room
temperature, and the .sup.64Cu-CY4-036 was reconstituted in water.
Analytical HPLC confirmed the radiochemical purity, and stability,
of the isolated .sup.64Cu-CY4-036 product. HPLC conditions: Solvent
A=53 mM NH.sub.4OAc in water; Solvent B=CH.sub.3CN. Flow rate=1
mL/min on 4.6.times.250 mm C18 reverse-phase analytical column.
Linear gradient conditions: 5% B at zero minutes, ramping to 25% B
at 25 minutes, then ramping to 60% B at 27 minutes and 100% B at 30
minutes.
Example III
Mandelic Acid Conjugate of Pteroic Acid
[0146] Biodegradation of an ester formed from pteroic acid and a
substituted derivative of mandelic acid is shown in FIGS. 4 and 5.
FIG. 4 illustrates bioreduction of the conjugate to release the
drug or drug-bearing moiety, while FIG. 5 illustrates acid
hydrolysis of the conjugate to release the drug or drug-bearing
moiety. Evidence from a recent article describing
structure-activity relationships of the folate receptor suggests
that the proton on the nitrogen which forms part of the amide bond
between pteroic acid and glutamic acid is not necessary for
high-affinity binding (Westerhof et al., 1995, Molecular
Pharmacology 48, 459-471). Accordingly, it is highly anticipated
that the mandelate esters depicted in these schemes will bind with
high affinity to the folate receptor. An example is shown in
Example IV.
Example IV
Synthesis and Activity of (S)-.alpha.-carboxybenzoyl pteroate
(ACBP) and N-pteroyl-2-amino-2-carboxymethylpyridine (Pte-AP)
[0147] 5
[0148] Synthesis of ACBP. (S)-.alpha.-carboxybenzoyl pteroate
(ACBP) is a mandelate ester (see Example III). A solution of
(S)-mandelic acid (28 mg, 0.187 mmol) in 2 mL of anhydrous
dimethylformamide was added via syringe to 30 mg of a 60%
dispersion of NaH in mineral oil (under argon). After stirring for
10 minutes at room temperature, solid pteroyl azide (64 mg, 0.187
mmol) was added and the reaction was stirred for an additional 2
hours. The reaction was quenched with a solution of 50 mg
NH.sub.4Cl in 60 mL of deionized water. The resulting solution was
washed with hexanes (to remove the mineral oil) and then
diethylether. The aqueous solution was sparged with argon while the
flask was immerged in warm water to evaporate the residual
diethylether. The solution was brought to pH 2.2 by drop-wise
addition of 1 N HCl, whereupon the product precipitated as a
yellow-orange finely-divided solid. The solid was isolated by
centrifugation and washed twice with deionized water. The material
was dissolved in 6 mL of deionized water containing
NH.sub.4HCO.sub.3 (16 mg, 0.2 mmol). The resulting solution was
filtered and then purified by HPLC: Novapak 19.times.300 mm prep
column, gradient 0-40% B in 35 minutes; A=10 mM NH.sub.4HCO.sub.3,
B=CH.sub.3CN. R, about 16.5 minutes. 6
[0149] Relative binding affinity. To determine how well these
compounds competes with 3H-folic acid for binding to the folate
receptor(FR)-positive cell line, KB (available from the American
Type Culture Collection, ATCC #CCL-17), a binding assay was
conducted. The relative affinity of various folate derivatives was
determined according to the method described by Westerhoff et al.
(Mol. Pharm., 1995, 48:459-471) with slight modification. Briefly,
folate receptor-positive KB cells were gently trypsinized in 0.25%
trypsin in phosphate-buffered saline (PBS) at room temperature for
3 minutes and then diluted in folate-free RPMI 1640 media (FFRPMI)
(Gibco) supplemented with 10% heat-inactivated fetal calf serum.
Following a 5 minute 800.times.g spin and one PBS wash, the final
cell pellet was suspended in FFRPMI (no serum). Cells were
incubated for 15 minutes on ice with 100 nM of .sup.3H-folic acid
in the absence and presence of increasing concentrations of
pteroate-containing test articles. Samples were centrifuged at
10,000.times.g for 5 minutes, cell pellets were suspended in
buffer, transferred to individual vials containing 5 mL of
scintillation cocktail, and then counted for radioactivity.
Negative control tubes contained only the .sup.3H-folic acid in
FFRPMI (no competitor). Positive control tubes contained a final
concentration of 1 mM folic acid, and counts per minute (CPM)
measured in these samples (representing non-specific binding of
label) were subtracted from all samples. Relative affinities were
defined as the inverse molar ratio of compound required to displace
50% of .sup.3H-folic acid bound to folate receptor on KB cells, and
the relative affinity of folic acid for the folate receptor was set
to 1.
[0150] Results. The result of the binding assay are shown in FIG.
6. The ester ACBP showed a relative binding activity of 0.46
compared to folic acid, and an EC50 of 204 nM compared to 93.4 nM
for folic acid. The folate analog containing an amide bond, Pte-AP,
showed a relative binding activity of 0.48 compared to folic acid,
and an EC50 of 193 nM compared to 93.4 nM for folic acid.
Example V
Pteroyl Hydrazide and Derivative
[0151] 7
[0152] Synthesis of pteroyl hydrazide (Pte-hydrazide).
N.sup.10-trifluoroacetylpteroic acid (40 mg, 0.098 mmol) and
carbonyldiimidazole (25 mg, 0.154 mmol) were dissolved in 2 mL of
dimethylformamide and stirred under argon at room temperature for
40 minutes. Hydrazine (40 .mu.L; 1.28 mmol) was added to the
reaction vessel via syringe. A precipitate immediately formed.
Following 15 minutes of stirring, several mL of deionized water
were added, and the product was isolated by centrifugation. No
further purification was needed.
[0153] Synthesis of pteroylhydrazido-BTCA-DAS. The synthesis of
pteroylhydrazido BTCA-DAS is shown in FIG. 7. To a solution of
diacetoxyscirpenol (DAS) (50 mg, 0.137 mmol) in 2.5 mL CH3CN was
added 30 mg (0.137 mmol) benzenetetracarboxylic dianhydride
followed by 24 .mu.L Hunig's base (17.7 mg, 0.137 mmol, also known
as DIPEA, diisopropylethylamine). The reaction mixture was stirred
1.5 hour under argon at room temperature. Some DAS remained, so an
additional 6 mg anhydride was added and stirring was continued an
additional 1 hour and 10 minutes. Pteroyl hydrazide (59 mg, 0.17
mmol) in 2.5 mL anhydrous dimethylsulfoxide (DMSO) was added,
followed by an additional 24 .mu.L (0.137 mmol) of Hunig's base.
The reaction was stirred for 1 hour and 10 minutes and precipitated
by the addition of ethanol.
[0154] Binding activity. The binding activity of pteroyl hydrazide
and pteroylhydrazido-BTCA-DAS was determined using the assay
described in Example IV.
[0155] Results. The results of the binding assay are shown in FIG.
8. Pteroyl hydrazide showed a relative binding activity of 0.74
compared to folic acid, and an EC50 of 94 nM compared to 70 nM for
folic acid. Pteroylhydrazido-BTCA-DAS showed a relative binding
activity of 0.60 compared to folic acid, and an EC50 of 116 nM
compared to 70 nM for folic acid.
[0156] The complete disclosures of all patents, patent applications
including provisional patent applications, and publications, and
electronically available material (e.g., GenBank amino acid and
nucleotide sequence submissions) cited herein are incorporated by
reference. The foregoing detailed description and examples have
been provided for clarity of understanding only. No unnecessary
limitations are to be understood therefrom. The invention is not
limited to the exact details shown and described; many variations
will be apparent to one skilled in the art and are intended to be
included within the invention defined by the claims.
* * * * *